[go: up one dir, main page]

US20030124689A1 - Mitomycin biosynthetic gene cluster - Google Patents

Mitomycin biosynthetic gene cluster Download PDF

Info

Publication number
US20030124689A1
US20030124689A1 US10/267,255 US26725502A US2003124689A1 US 20030124689 A1 US20030124689 A1 US 20030124689A1 US 26725502 A US26725502 A US 26725502A US 2003124689 A1 US2003124689 A1 US 2003124689A1
Authority
US
United States
Prior art keywords
nucleic acid
mitomycin
seq
host cell
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/267,255
Inventor
David Sherman
Yingqing Mao
Mustafa Varoglu
Min He
Paul Sheldon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Minnesota System
Original Assignee
University of Minnesota System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Minnesota System filed Critical University of Minnesota System
Priority to US10/267,255 priority Critical patent/US20030124689A1/en
Publication of US20030124689A1 publication Critical patent/US20030124689A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • Streptomyces are filamentous Gram-positive soil bacteria with a nucleotide base composition greater than 70 mole % G+C (Stackebrandt and Woese, 1981). They produce a wide array of biologically active compounds including over two thirds of the commercially important natural product metabolites (Alderson et al., 1993; Bevax, 1998). Genetic information accumulated over the past 15 years has demonstrated that genes encoding enzymes for natural product assembly are clustered on the Streptomyces genome (Martin, 1992). In addition, one or more pathway-specific transcriptional regulatory genes, and at least one resistance gene are typically found within the antibiotic biosynthetic gene cluster (Chater, 1992).
  • the mitomycins are a group of natural products that contain a variety of functional groups, including aminobenzoquinone and aziridine ring systems.
  • One representative of the family, mitomycin C (MC) was the first recognized bioreductive alkylating agent.
  • MC mitomycin C
  • MC has become one of the most effective antitumor drugs against non-small cell lung carcinoma and other soft tumors, as well as a clinically important component of combination cancer chemotherapy and radiotherapy of solid tumors (Henderson, 1993).
  • MC derivatives have been synthesized and tested for enhanced activities, including the recently identified selective protein tyrosine kinase inhibitor, 1 a-docosahexaenoyl MC (Kasai and Arai, 1995; Shikano et al., 1998).
  • Streptomyces lavendulae produces MC.
  • the molecule has an unusual structure comprised of aziridine, pyrrolizidine, pyrrolo-(1,2a)-indole, and amino-methylbenzoquinone rings to give the mitosane nucleus (Webb et al., 1962).
  • the mitosane core of MC was shown to be derived from the junction of an amino-methylbenzoquinone (mC 7 N unit) and hexosamine (C 6 N unit) (Hornemann, 1981).
  • the C 6 N unit consists of carbons 1, 2, 3, 9, 9a, 10, with the aziridine nitrogen derived intact from D-glucosamine (Homemann et al., 1974).
  • Floss (1997) provided strong support for this new variant of the shikimate pathway by showing that aminoDAHP, 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) could be efficiently converted into AHBA by a cell-free extract of Amycolatopsis mediterranei (rifamycin producer), in contrast to the normal shikimate pathway intermediate DAHP which was not converted (Kim et al., 1992; Kim et al., 1996). Recently, the AHBA synthase (rifk) gene from A. mediterranei has been cloned, sequenced and functionally characterized (Kim et al., 1998).
  • the biosynthetic portion of the gene cluster includes genes that encode polypeptides involved in the generation of biosynthetic precursors, mitosane ring system assembly and functionalization (e.g., methylation, hydroxylation, aminotransfer, carbamoylation, and carbonyl reduction), a mitomycin resistance gene which is different than mrd and the unlinked mcr, as well as several regulatory genes.
  • Gene disruption was employed to further characterize some of the genes. Fourteen of 22 gene disruption mutants affected mitomycin biosynthesis, resulting in abrogation or overexpression of drug production, e.g., targeted genetic disruption of a mitomycin pathway regulator (e.g., mmcW) led to a substantial increase in drug production.
  • a mitomycin pathway regulator e.g., mmcW
  • the isolated and purified nucleic acid molecule of the invention is nucleic acid from Streptomyces spp., such as Streptomyces lavendulae (e.g., B19/ATCC 27422, NRRL 2564, KY681, ATCC 27423, or PB1000), Streptomyces caespitosus, Streptomyces verticillatus, and Streptomyces sandaensis (FERM-P7654), although isolated and purified nucleic acid molecules from other organisms which produce mitomycin or biological or functional equivalents thereof are also within the scope of the invention.
  • the nucleic acid molecules of the invention are double-stranded or single-stranded.
  • ORFs open reading frames
  • the cloned mitomycin biosynthetic genes are useful to elucidate the molecular basis for the biosynthesis of the mitosane ring system, as well as to engineer the biosynthesis of novel natural products. Moreover, genetic engineering or overexpression of the transport, resistance and regulatory proteins may lead to higher titers of mitomycin compounds from production cultures.
  • the isolated nucleic acid molecule comprising the gene cluster includes a nucleic acid sequence comprising SEQ ID NO:96 or SEQ ID NO:76, a variant or a fragment thereof, e.g., a nucleic acid molecule that hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:96, SEQ ID NO:76 or a fragment thereof.
  • Moderate and stringent hybridization conditions are well known to the art, see, for example sections 9.47-9.51 of Sambrook et al. ( Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).
  • Another example is use of 50% formamide, 5 ⁇ SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 ⁇ Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2 ⁇ SSC and 0.1% SDS.
  • a preferred nucleic molecule of the invention comprises a nucleic acid sequence encoding a polypeptide including, but not limited to, MitA (e.g., SEQ ID NO:10 encoded by SEQ ID NO:97), MitB (e.g., SEQ ID NO:11 encoded by SEQ ID NO:98), MitC (e.g., SEQ ID NO:12 encoded by SEQ ID NO:99), MitD (e.g., SEQ ID NO:100 encoded by SEQ ID NO:45), MitE (e.g., SEQ ID NO:101 encoded by SEQ ID NO:44), MitF (e.g., SEQ ID NO:102 encoded by SEQ ID NO:43), MitG (e.g., SEQ ID NO:103 encoded by SEQ ID NO:42), MitH (e.g., SEQ ID NO:104 encoded by SEQ ID NO:41), MitI (e.g., SEQ ID NO:105 encoded by SEQ ID NO:40), MitJ
  • the invention further provides an isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, a variant or a fragment thereof.
  • the nucleic acid molecule of this embodiment of the invention comprises at least one, preferably at least five, and more preferably at least nine, open reading frames. More preferably, the nucleic acid molecule hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:74, or a portion thereof.
  • the invention also provides probes and primers comprising at least a portion of the nucleic acid molecules of the invention.
  • the probes or primers of the invention are preferably detectably labeled or have a binding site for a detectable label.
  • the probes or primers of the invention are at least about 7, more preferably at least about 15, contiguous nucleotides bases having at least about 80% identity, more preferably at least about 90% identity, to the isolated nucleic acid molecules of the invention.
  • Host cells that have been modified genetically include host cells comprising an expression cassette, e.g., an expression cassette of the invention, or host cells in which the genome has been genetically manipulated, e.g., by deletion of a portion of, replacement of a portion of, or by disruption of, the host chromosome, so as to reduce or eliminate the expression of a particular mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention.
  • an expression cassette e.g., an expression cassette of the invention
  • host cells in which the genome has been genetically manipulated e.g., by deletion of a portion of, replacement of a portion of, or by disruption of, the host chromosome, so as to reduce or eliminate the expression of a particular mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention.
  • One embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which a portion of a nucleic acid sequence comprising the mitomycin gene cluster, i.e., the endogenous or native genomic sequence, is disrupted or replaced, for example, by an insertion with heterologous sequences or substituted with a variant nucleic acid sequence of the invention, preferably so as to result in altered mitomycin synthesis, such as an increase in mitomycin synthesis, and/or production of a novel compound.
  • the invention includes a recombinant host cell in which the mmcW gene is disrupted, for example, by replacement with a selectable marker gene, so as to yield a recombinant host cell having an increase in mitomycin production.
  • Another embodiment of the invention is a recombinant host cell, the genome of which is augmented by an expression cassette, e.g., via an extrachromosomal element such as a plasmid or by stable integration of the cassette into the host chromosome.
  • the genome of the recombinant host cell is augmented with at least one mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention so as to yield an altered level of mitomycin and/or a novel compound(s) relative to the corresponding non-recombinant host cell.
  • the genome of a recombinant host cell is augmented with a non-mitomycin biosynthetic gene and, optionally, at least one mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention so as to yield an altered level of mitomycin and/or a novel compound(s) relative to the corresponding non-recombinant host cell.
  • the recombinant host cell may be augmented with pikA (see U.S. application Ser. No. 09/105,537, filed Jun. 26, 1998, the disclosure of which is incorporated by reference herein) and pikA expressed in an amount effective to yield a novel compound(s).
  • the invention also provides isolated and purified polypeptides encoded by a nucleic acid molecule of the invention.
  • the polypeptide of the invention is obtained from recombinant host cells, e.g., the genome of which is augmented by a nucleic acid molecule of the invention.
  • expression cassettes and host cells comprising antisense sequences of at least a portion of the mitomycin biosynthetic gene cluster of the invention are envisioned.
  • the present invention provides a method of preparing a polyhydroxyalkanoate synthase.
  • the method comprises introducing an expression cassette into a host cell.
  • the expression cassette comprises a DNA molecule encoding a polyketide synthase, operably linked to a promoter functional in the host cell.
  • the DNA molecule is preferably obtained from a mitomycin-producing organism, e.g., a Streptomyces spp. such as S. lavendulae.
  • the DNA molecule encoding the polyketide synthase is then expressed in the cell.
  • another embodiment of the invention provides a purified recombinant polyketide isolated from a host cell which expresses the synthase.
  • Another embodiment of the invention is a method of preparing a polyhydroxyalkanoate polymer.
  • the method comprises introducing a first expression cassette and a second expression cassette into a host cell.
  • the first expression cassette comprises a DNA segment encoding a fatty acid synthase in which the dehydrase activity has been inactivated that is operably linked to a promoter functional in the host cell, e.g., an insect cell.
  • the inactivation preferably is via a mutation in the catalytic site of the dehydrase.
  • the second expression cassette comprises a DNA segment encoding a polyketide synthase that is preferably obtained from a mitomycin-producing organism operably linked to a promoter functional in the host cell.
  • the expression cassettes may be on the same or separate molecules.
  • the DNA segments in the expression cassettes are expressed in the cell so as to yield a polyhydroxyalkanoate polymer.
  • the present invention also provides an expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.
  • the nucleic acid molecule comprises a plurality of DNA segments.
  • the nucleic acid molecule comprises at least a first and a second DNA segment.
  • the first DNA segment encodes a first module
  • the second DNA segment encodes a second module, wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase.
  • No more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea.
  • the first DNA segment comprises a module from a mitomycin-producing organism, e.g., Streptomyces spp.
  • the nucleic acid molecule may optionally further comprise a third DNA segment encoding a polyhydroxyalkanoate synthase.
  • a second nucleic acid molecule encoding a polyhydroxyalkanoate synthase may be introduced into the host cell.
  • the DNA molecule comprises a plurality of DNA segments.
  • the DNA molecule comprises at least a first and a second DNA segment.
  • the first DNA segment encodes a first module and the second DNA segment encodes a second module.
  • the DNA segments encode a recombinant polyhydroxyalkanoate monomer synthase.
  • no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea.
  • no more than one module is derived from the gene cluster from Streptomyces hygroscopicus that encodes rapamycin or the gene cluster that encodes spiramycin.
  • a preferred embodiment of the invention employs a first DNA segment comprising a module from a mitomycin-producing organism.
  • a further preferred embodiment of the isolated DNA molecule of the invention includes a DNA segment encoding a polyhydroxyalkanoate synthase.
  • the method comprises introducing a first DNA molecule and a second DNA molecule into a host cell.
  • the first DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase.
  • the recombinant polyhydroxyalkanoate monomer synthase comprises a plurality of modules.
  • the monomer synthase comprises at least a first module and a second module.
  • the first DNA molecule is operably linked to a promoter functional in a host cell.
  • the second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell.
  • At least one module is from a mitomycin-producing organism.
  • the DNAs encoding the recombinant polyhydroxyalkanoate monomer synthase and polyhydroxyalkanoate synthase are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
  • the DNA molecule comprises a plurality of DNA segments. That is, the DNA molecule comprises at least a first and a second DNA segment.
  • the first DNA segment encodes a fatty acid synthase and the second DNA segment encodes a module of a polyketide synthase.
  • a preferred embodiment of the invention employs a second DNA segment comprising a module of a polyketide synthase from a mitomycin-producing organism such as Streptomyces.
  • the method comprises introducing an expression cassette into a host cell.
  • the expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell.
  • the monomer synthase comprises a plurality of modules.
  • the monomer synthase comprises at least a first and second module which together encode the monomer synthase.
  • a preferred embodiment of the invention employs a module from a mitomycin-producing organism.
  • the expression cassette further comprises a second DNA molecule encoding a polyhydroxyalkanoate synthase.
  • the invention also provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase.
  • at least one DNA segment is derived from DNA which is linked to the mitomycin gene cluster of S. lavendulae.
  • no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea.
  • the 3′ most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II.
  • an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.
  • Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer.
  • the method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell.
  • the second DNA molecule is derived from DNA which is linked to the mitomycin gene cluster.
  • the recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from DNA which is linked to a mitomycin gene cluster, e.g., the mitomycin gene cluster of S. lavendulae.
  • the DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
  • a second DNA molecule may be introduced into the host cell.
  • the second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell.
  • the two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
  • Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the DNA which is linked to the mitomycin gene cluster of S. lavendulae.
  • a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer.
  • a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell.
  • the first DNA segment is 5′ to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell.
  • the first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein.
  • the DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
  • the method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell.
  • the DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase.
  • At least one DNA segment is derived from DNA which is linked to the mitomycin gene cluster of S. lavendulae.
  • the DNA molecule is expressed in the host cell.
  • the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase.
  • a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.
  • Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer.
  • the method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell.
  • the recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from the mit/mmc gene cluster of S. lavendulae.
  • the DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
  • Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the mit/mmc gene cluster of S. lavendulae.
  • a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer.
  • a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell.
  • the first DNA segment is 5′ to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell.
  • the first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein.
  • the DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
  • the method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell.
  • the DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase.
  • At least one DNA segment is derived from the mit/mmc gene cluster of S. lavendulae.
  • the DNA molecule is expressed in the host cell.
  • the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase.
  • a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.
  • modules encoded by the nucleic acid segments of the invention may be employed in the methods described hereinabove to prepare polyhydroxyalkanoates of varied chain length or having various side chain substitutions.
  • the compounds produced by the recombinant host cells of the invention are preferably biologically active agents such as antibiotics, anti-inflammatory agents, anti-cancer agents, antibiotics, immune-enhancers, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, or cholesterol-lowering agents; or as crop protection agents (e.g., fungicides or insecticides), as well as biopolymers, e.g., in packaging or biomedical applications, or to engineer PHA monomer synthases.
  • biologically active agents such as antibiotics, anti-inflammatory agents, anti-cancer agents, antibiotics, immune-enhancers, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, or cholesterol-lowering agents; or as crop protection agents (e.g., fungicides or insecticides), as well as biopolymers, e.g., in packaging or biomedical applications, or to engineer PHA monomer
  • FIG. 1 The biosynthetic pathway for mitomycin antibiotics.
  • FIG. 2 Organization of the mitomycin gene cluster.
  • the deduced ORFs are drawn to scale, and their corresponding genes are marked in italics. The filled bars indicate the location of the mitomycin cluster.
  • Abbreviations of the restriction enzymes B: BamHI, S: SphI, P: PstI, E: EcoRI, X: XhoI, K: KpnI.
  • FIG. 3 The three SAM dependent methyltransferase conserved motifs can be found in MitM (SEQ ID NO:1), MitN (SEQ ID NO:2), and MmcR (SEQ ID NO:3).
  • DmpM SEQ ID NO:4; Kim et al., 1998)
  • TcmN SEQ ID NO:5; Shikano et al., 1998)
  • ORF14 SEQ ID NO:6; August et al., 1998)
  • EryG SEQ ID NO:7; Hardwick and Pelham, 1994
  • Consen consensus sequence (SEQ ID NO:8).
  • FIG. 4 Sequence similarity of MitM, MitN, and MmcR with other O-methyltransferases: DmpM (Kim et al., 1998), TcmN (Shikano et al., 1998), ORF14 (August et al., 1998), EryG (Hardwick and Pelham, 1994), RdmB (Mazodier et al., 1989), DnrK (Lee and Stock, 1996), and DauK (Devereux et al.
  • FIG. 5 MC genes and deduced enzyme functions.
  • FIG. 6 Bacterial strains and plasmids. Strains DH5 ⁇ and DH5 ⁇ F′ are available from Gibco BRL (Gaithersburg, Md.), ATCC 27643 and NRRL 2564 are available from the American Type Culture Collection, and strain S17-1 is described in Hidaka et al. (1995). Plasmids pNJ1, pUC119, pKC 1139, pDHS3001, pKN108, and pFD666 are described in Kuzuyama et al. (1995), Madduri et al. (1993), Boxer (1997), Kagan and Clarke (1994), Kim et al. (1998), and Coque et al. (1995), respectively.
  • FIG. 7 Biosynthetic pathway leading to mitomycin C.
  • FIG. 8 Southern hybridization and restriction-enzyme map of the mrd and rifK hybridizing regions from S. lavendulae.
  • A) Southern hybridization with the rifK gene probe (Kim et al., 1998). Lane 1, A. mediterranei ATCC 27643 genomic DNA digested with BamHI; Lane 2, S. lavendulae NRRL 2564 genomic DNA digested with BamHI; B) Physical map showing the mitABC genes. The location of mrd and rifK hybridizing genes in cosmid pDHS7529 are indicated by solid bars.
  • FIG. 9 Nucleotide sequence of the 3.8 kb DNA fragment containing mitABC (SEQ ID NO:9).
  • the deduced gene products are indicated in the one-letter code under the DNA sequence (SEQ ID NO:10, MitA; SEQ ID NO:11, MitB; SEQ ID NO:12, MitC).
  • Possible ribosome binding sites are marked in the boxed regions.
  • the presumed translational start site and direction of transcription for each ORF is indicated by an arrow and marked accordingly.
  • FIG. 10 Alignment of MitA with three other AHBA synthases.
  • the deduced amino acid sequence comparison from AHBAS genes derived from Streptomyces lavendulae (SEQ ID NO:10). Streptomyces collinus (Z54208; SEQ ID NO:13), Actinosynnema pretiosum (I39657; SEQ ID NO:14), and Amycolatopsis mediterranei (I39657; SEQ ID NO:15) is shown with the conserved lysine in the PLP-binding motif underlined.
  • FIG. 11 Southern blot analysis of the mitA mutant strain.
  • FIG. 12 Southern blot analysis of mitB mutant MM101.
  • FIG. 13 Chemical analysis and biological activity of extracts from S. lavendulae wild-type and mutant strains.
  • Filter discs 1) 100 ⁇ g injection of wild-type—collected 12.5-13.5 minutes; 2) 100 ⁇ g injection of mitA (ahbas) disruption mutant—collected 12.5-13.5 minutes; 3) 100 ⁇ g injection of W. T. containing vector—collected 12.5-13.5 minutes; 4) One ⁇ g of mitomycin C collected from HPLC from 12.5-13.5 minutes; 5) Tris buffer negative control; 6) methanol solvent negative control.
  • FIG. 14 Strains and plasmids employed in Example 3. BL21 (DE3) and pET17b are available from Novagen (Madison, Wis.). pDH57006 is described in Sheldon et al. (1997).
  • FIG. 15 Genetic map showing the physical linkage of the mct and mrd genes within the MC biosynthetic gene cluster. The expanded box shows the line plot of the met ORF.
  • FIG. 16 The nucleotide sequence of mct (SEQ ID NO:16). The deduced amino acid sequence of mct is indicated under the nucleotide sequence with the one letter designation (SEQ ID NO:17). A conserved motif characteristic of 14 TMS proteins is boxed while the invariant beta-turn motif is denoted with a dashed underline. The putative ribosome binding site is marked with a solid underline.
  • FIG. 17 Dot matrix alignment of the deduced amino acid sequence of mct with other actinomycete antibiotic efflux proteins. Comparable parameters were utilized in generating the alignments.
  • FIG. 18 Hydropathy analysis of the deduced amino acid sequence of MC-translocase.
  • FIG. 19 Creation of the mct disruption mutant.
  • Lane 1 and 5 wild-type genomic DNA digested with BamHI.
  • Lane 2, 3, 4, and 6 Four double crossover colonies genomic DNA digested with BamHI.
  • Lane 7 wild-type genomic DNA digested with SstI.
  • Lane 8 double crossover clone 6 genomic DNA digested with SstI.
  • FIG. 20 MC uptake analysis of strains PJS100, PJS102, and PJS103.
  • FIG. 21 Complete nucleotide sequence of the mitomycin gene cluster (SEQ ID NO:96).
  • FIG. 22 Complete nucleotide sequence of ORFs 1-9 (SEQ ID NO:74).
  • FIG. 23 Complete nucleotide sequence of ORFs 11-22 (SEQ ID NO:75).
  • FIG. 24 Codons for various amino acids.
  • FIG. 25 Exemplary amino acid substitutions.
  • FIG. 26 Complete nucleotide sequence of the mitomycin biosynthetic genes (SEQ ID NO:76).
  • a “Type I polyketide synthase” is a single polypeptide with a single set of iteratively used active sites. This is in contrast to a Type II polyketide synthase which employs active sites on a series of polypeptides.
  • a “linker region” is an amino acid sequence present in a multifunctional protein which is less well conserved in an amino acid sequence than an amino acid sequence with catalytic activity.
  • an “extender unit” catalytic or enzymatic domain is an acyl transferase in a module that catalyzes chain elongation by adding 2-4 carbon units to an acyl chain and is located carboxy-terminal to another acyl transferase.
  • an extender unit with methylmalonylCoA specificity adds acyl groups to a methylmalonylCoA molecule.
  • a “polyhydroxyalkanoate” or “PHA” polymer includes, but is not limited to, linked units of related, preferably heterologous, hydroxyalkanoates such as 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxycaproate, 3-hydroxyheptanoate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundecanoate, and 3-hydroxydodecanoate, and their 4-hydroxy and 5-hydroxy counterparts.
  • a “recombinant” nucleic acid or protein molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been modified.
  • a “multifunctional protein” is one where two or more enzymatic activities are present on a single polypeptide.
  • a “module” is one of a series of repeated units in a multifunctional protein, such as a Type I polyketide synthase or a fatty acid synthase.
  • a “premature termination product” is a product which is produced by a recombinant multifunctional protein which is different than the product produced by the non-recombinant multifunctional protein.
  • the product produced by the recombinant multifunctional protein has fewer acyl groups.
  • an “antibiotic” as used herein is a substance produced by a microorganism which, either naturally or with limited chemical modification, will inhibit the growth of or kill another microorganism or eukaryotic cell.
  • An “antibiotic biosynthetic gene” is a nucleic acid, e.g., DNA, segrnent or sequence that encodes an enzymatic activity which is necessary for an enzymatic reaction in the process of converting primary metabolites into antibiotics.
  • An “antibiotic biosynthetic pathway” includes the entire set of antibiotic biosynthetic genes necessary for the process of converting primary metabolites into antibiotics. These genes can be isolated by methods well known to the art, e.g., see U.S. Pat. No. 4,935,340.
  • Antibiotic-producing organisms include any organism, including, but not limited to, Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicilliurn, Nocardia, and Streptomyces, which either produces an antibiotic or contains genes which, if expressed, would produce an antibiotic.
  • polyketide refers to a large and diverse class of natural products, including but not limited to antibiotic, antifungal, anticancer, and anti-helminthic compounds.
  • polyketide-associated sugar refers to a sugar that is known to attach to polyketides or that can be attached to polyketides by the processes described herein.
  • sugar derivative refers to a sugar which is naturally associated with a polyketide but which is altered relative to the unmodified or native.
  • sugar intermediate refers to an intermediate compound produced in a sugar biosynthesis pathway.
  • a “recombinant” host cell of the invention has a genome that has been manipulated in vitro so as to alter, e.g., decrease or disrupt, or, alternatively, increase, the function or activity of at least one gene, e.g., in the mitomycin biosynthetic gene cluster, of the invention.
  • the “mit/mmc” or “mitomycin” gene cluster includes sequences encoding enzymes for mitosane precursor formation, mitosane ring assembly, regulation of mitomycin biosynthesis, functionalization, and resistance to mitomycin, as well as closely linked sequences encoding polyketide and sugar biosynthetic enzyes.
  • isolated and/or purified refer to in vitro isolation of a RNA, DNA or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, such as nucleic acid or polypeptide, so that is can be sequenced, replicated and/or expressed.
  • the nucleic acid may encode more than one polypeptide.
  • an isolated DNA molecule encoding an AUBA synthase is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more sequential nucleotide bases that preferably encode a biologically active polypeptide, or a fragment or variant thereof, that is complementary to the non-coding, or complementary to the coding strand, of an AHBA synthase RNA, or hybridizes to the RNA or DNA encoding the AHBA synthase and remains stably bound under low, moderate, or stringent conditions, as defined by methods well known to the art, e.g., in Sambrook et al., supra.
  • polyketide-producing microorganism includes any microorganism that can produce a polyketide naturally or after being suitably engineered (i.e., genetically).
  • actinomycetes that naturally produce polyketides include but are not limited to Micromonospora rosaria, Micromonospora megalomicea, Saccharopolyspora erythraea, Streptomyces antibioticus, Streptomyces albereticuli, Streptomyces ambofaciens, Streptomyces avermitilis, Streptomycesfradiae, Streptomyces griseus, Streptomyces hydroscopicus, Streptomyces tsukulubaensis, Streptomyces mycarofasciens, Streptomyces platenesis, Streptomyces violaceoniger, Streptomyces violaceoniger, Streptomyces thermotolerans, Streptomyces rim
  • polyketide-producing microorganisms that produce polyketides naturally include various Actinomadura, Dactylosporangium and Nocardia strains.
  • glycosylated polyketide refers to any polyketide that contains one or more sugar residues.
  • glycosylation-modified polyketide refers to a polyketide having a changed glycosylation pattern or configuration relative to that particular polyketide's unmodified or native state.
  • sugar biosynthesis genes refers to nucleic acid sequences from organisms such as S. lavendulae that encode sugar biosynthesis enzymes and is intended to include sequences of DNA from other polyketide-producing microorganisms which are identical or analogous to those obtained from S. lavendulae.
  • sugar biosynthesis enzymes refers to polypeptides which are involved in the biosynthesis and/or attachment of polyketide-associated sugars and their derivatives and intermediates.
  • An antibiotic resistance-conferring gene is a nucleic acid segment that encodes an enzymatic or other activity which alone or in combination with other gene products, confers resistance to an antibiotic.
  • mitomycin includes, but is not limited to, mitomycin A, mitomycin B, mitomycin C, porfiromycin, mitiromycin, mitomycin D, mitomycin E, mitomycin F, mitomycin G, mitomycin H, mitomycin I, mitomycin J, mitomycin L, mitomycin M, mitomycin K, albomitomycin A, isomitomycin A, KW2149, KW2149 metabolites such as M-16 and M-18, FR66979, FK973, FK317, and FR900482, as well as structural or functional equivalents thereof (“analogs”), or derivatives thereof.
  • the term “derivative” means that a particular compound produced by a host cell of the invention or prepared in vitro using polypeptides encoded by the nucleic acid molecules of the invention, is modified so that it comprises other moieties, e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.g., gamma emitters, small chemicals, metals, salts, synthetic polymers, e.g., polylactide and polyglycolide, surfactants and glycosaminoglycans, which are covalently or non-covalently attached or linked to the compound.
  • moieties e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.g.,
  • each atom of the compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically active, polymorphic or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art.
  • sequence homology or “sequence identity” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences.
  • sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
  • the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and more preferably not less than 19 matches out of 20 possible base pair matches (95%).
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M.
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
  • two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I
  • substantially identical denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 80 percent sequence identity, preferably at least about 90 percent sequence identity, more preferably at least about 95 percent sequence identity, and most preferably at least about 99 percent sequence identity.
  • nucleic acid molecule which encodes the entire pathway for the biosynthesis of mitomycin, as well as polyketide biosynthetic and sugar biosynthetic genes that are linked to the mitomycin biosynthetic genes.
  • the nucleic acid molecule is a DNA isolated from Streptomyces spp.
  • the present invention further includes isolated and purified DNA sequences which hybridize under standard or stringent conditions to the the nucleic acid molecules of the invention. It should be understood to those skilled in the art that the invention also encompasses the purified and isolated polypeptides which may be encoded by the sequences of the nucleic acid molecules of this invention.
  • the invention described herein can be used for the production of mitomycin, analogs or derivatives thereof, or novel compounds. Commercial chemical syntheses of mitomycin are not feasible.
  • the gene cluster described herein contains all the genes required for the production of the mitosane group of antibiotics, compounds which are clinically prescribed antitumor compounds employed in the treatment of a wide variety of cancers including non-small cell lung cancer, metastatic breast cancer, esophageal, gastric, pancreatic, and anal canal carcinomas.
  • this gene cluster allows for the selective production of mitomycin antibiotics, the overproduction or under production of particular compounds, e.g., overproduction of certain mitomycin antibiotics, and the production of novel compounds, e.g., mitomycin-derived compounds as well as the production of novel non-mitomycin related compounds.
  • combinational biosynthetic-based modification of mitomycin antibiotics may be accomplished by selective activation or disruption of specific genes within the cluster or incorporation of the genes into biased biosynthetic libraries which are assayed for a wide range of biological activities, to derive greater chemical diversity in the mitomycins.
  • a further example includes the introduction of a mitomycin biosynthetic gene(s) into a particular host cell so as to result in the production of a novel non-mitomycin related compound due to the activity of the mitomycin biosynthetic gene(s) on other metabolites, intermediates or components of the host cells.
  • the in vitro expression of polypeptides from this gene cluster also provides an enzymatic route to the production of known mitomycin compounds that are produced in low quantities, or conversion of currently available mitomycins to other known or novel mitomycins, e.g., the bioconversion of mitomycin C to porfiromycin.
  • the mitomycin resistance genes may also be used to provide higher mitomycin resistance to cancer patients undergoing treatment and for clonal selection purposes (e.g., using mrd).
  • the resistance gene(s) may be inserted into human bone marrow cell lines to confer higher resistance to non-cancerous cells, thus allowing higher doses of mitomycins to be administered to cancer patients.
  • mitomycin acts directly upon DNA itself, its toxicity is extremely broad, and therefore the resistance genes could be used for efficient selection in prokaryotes, fungi, plants, mammalian cell culture, and insect cell culture.
  • the regulatory resistance and transport genes may be used to create higher producing strains capable of synthesizing more mitomycin than can currently be obtained through traditional fermentation strategies.
  • the invention described herein can be used for the production of novel compounds which include a diverse range of biodegradable PHA polymers through genetic redesign of DNA such as that found in Streptomyces spp.
  • Different PHA synthases can then be tested for their ability to polymerize the monomers produced by the recombinant PHA synthase into a biodegradable polymer.
  • PHA synthases can be tested for their specificity with respect to different monomer substrates by methods well known to the art.
  • PHAs produced by PHA monomer synthases and PHA synthases include both medical and industrial applications.
  • Medical applications of PHAs include surgical pins, sutures, staples, swabs, wound dressings, blood vessel replacements, bone replacements and plates, stimulation of bone growth by piezoelectric properties, and biodegradable carrier for long-term dosage of pharmaceuticals.
  • Industrial applications of PHAs include disposable items such as baby diapers, packaging containers, bottles, wrappings, bags, and films, and biodegradable carriers for long-term dosage of herbicides, fungicides, insecticides, or fertilizers.
  • the biosynthesis of fatty acids de novo from malonyl-CoA is catalyzed by FAS.
  • the rat FAS is a homodimer with a subunit structure consisting of 2505 amino acid residues having a molecular weight of 272,340 Da. Each subunit consists of seven catalytic activities in separate physical domains (Amy et al., Proc. Natl. Acad. Sci. USA, 86, 3114 (1989)).
  • ketoacyl synthase KS
  • malonyl/acetyltransferase M/AT
  • ER enoyl reductase
  • KR ketoreductase
  • ACP acyl carrier protein
  • TE thioesterase
  • DH dehydrase
  • PKS eryA polyketide synthase
  • One embodiment of the invention employs a FAS in which the DH is inactivated (FAS DH-).
  • the FAS DH-employed in this embodiment of the invention is preferably a eukaryotic FAS DH- and, more preferably, a mammalian FAS DH-.
  • the most preferred embodiment of the invention is a FAS where the active site in the DH has been inactivated by mutation.
  • Joshi et al. J. Biol. Chem., 268, 22508 (1993)
  • ratFAS206 produced 3-hydroxybutyrylCoA as a premature termination product from acetyl-CoA, malonyl-CoA and NADPH.
  • Another embodiment of the invention employs a recombinant Streptomyces spp. PKS to produce a variety of ⁇ -hydroxyCoA esters that can serve as monomers for a PHA synthase.
  • a DNA encoding a Type I PKS is the eryA gene cluster, which governs the synthesis of erythromycin aglycone deoxyerythronolide B (DEB).
  • the gene cluster encodes six repeated units, termed modules or synthase units (SUs). Each module or SU, which comprises a series of putative FAS-like activities, is responsible for one of the six elongation cycles required for DEB formation.
  • Two other Type I PKS are encoded by the tyl (tylosin) and met (methymycin) gene clusters (see U.S. application Ser. No. 09/108,537, the disclosure of which is incorporated by reference herein).
  • the macrolide multifunctional synthases encoded by tyl and met provide a greater degree of metabolic diversity than that found in the eryA gene cluster.
  • the PKSs encoded by the eryA gene cluster only catalyze chain elongation with methylmalonylCoA, as opposed to tyl and met PKSs, which catalyze chain elongation with malonylCoA, methylmalonylCoA and ethylmalonylCoA.
  • the tyl PKS includes two malonylCoA extender units and one ethylmalonylCoA extender unit
  • the met PKS includes one malonylCoA extender unit.
  • Linker regions amino acid sequences of related modules, preferably those encoded by more than one gene cluster, are compared. Linker regions are amino acid sequences which are less well conserved than amino acid sequences with catalytic activity. Witkowski et al., Eur. J. Biochem., 198, 571 (1991).
  • a DNA encoding a module F containing KS, MT, KR, ACP, and TE catalytic activities, is introduced at the 3′ end of a DNA encoding a first module.
  • Module F introduces the final (R)-3-hydroxyl acyl group at the final step of PHA monomer synthesis, as a result of the presence of a TE domain.
  • DNA encoding a module F is not present in the eryA PKS gene cluster (Donadio et al., supra, 1991).
  • a DNA encoding a recombinant monomer synthase is inserted into an expression vector.
  • the expression vector employed varies depending on the host cell to be transformed with the expression vector. That is, vectors are employed with transcription, translation and/or post-translational signals, such as targeting signals, necessary for efficient expression of the genes in various host cells into which the vectors are introduced. Such vectors are constructed and transformed into host cells by methods well known in the art. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor (1989).
  • Preferred host cells for the vectors of the invention include insect, bacterial, and plant cells.
  • Preferred insect cells include Spodoptera frugiperda cells such as Sf21, and Trichoplusia ni cells.
  • Preferred bacterial cells include Escherichia coli, Streptomyces and Pseudomonas.
  • Preferred plant cells include monocot and dicot cells, such as maize, rice, wheat, tobacco, legumes, carrot, squash, canola, soybean, potato, and the like.
  • the appropriate subcellular compartment in which to locate the enzyme in eukaryotic cells must be considered when constructing eukaryotic expression vectors. Two factors are important: the site of production of the acetyl-CoA substrate, and the available space for storage of the PHA polymer. To direct the enzyme to a particular subcellular location, targeting sequences may be added to the sequences encoding the recombinant molecules.
  • the baculovirus system is particularly amenable to the introduction of DNA encoding a recombinant FAS or a PKS monomer synthase because an increasing variety of transfer plasmids are becoming available which can accommodate a large insert, and the virus can be propagated to high titers.
  • insect cells are adapted readily to suspension culture, facilitating relatively large-scale recombinant protein production.
  • recombinant proteins tend to be produced exclusively as soluble proteins in insect cells, thus, obviating the need for refolding, a task that might be particularly daunting in the case of a large multifunctional protein.
  • the Sf21 /baculovirus system has routinely expressed milligram quantities of catalytically active recombinant fatty acid synthase.
  • the baculovirus/insect cell system provides the ability to construct and analyze different synthase proteins for the ability to polymerize monomers into unique biodegradable polymers.
  • a further embodiment of the invention is the introduction of at least one DNA encoding a PHA synthase and a DNA encoding a PHA monomer synthase into a host cell.
  • Such synthases include, but are not limited to, A. eutrophus 3-hydroxy, 4-hydroxy, and 5-hydroxy alkanoate synthases, Rhodococcus ruber C 3 -C 5 hydroxyalkanoate synthases, Pseudomonas oleororans C 6 -C 14 hydroxyalkanoate synthases, P. putida C 6 -C 14 hydroxyalkanoate synthases, P. aeruginosa C 5 -C 10 hydroxyalkanoate synthases, P.
  • eutrophus PHB synthase utilizes only C4 and C5 compounds as substrates, it appears to be a good prototype synthase for initial studies since it is known to be capable of producing copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate (Kunioka et al., Macromolecules, 22, 694 (1989)) as well as copolymers of 3-hydroxyvalerate, 3-hydroxybutyrate, and 5-hydroxyvalerate (Doi et al., Macromolecules, 19, 2860 (1986)).
  • Other synthases especially those of Pseudomonas aeruginosa (Timm et al., Eur. J.
  • Rhodococcus ruber Pieris et al., FEMS Microbiol. Lett., 96, 73 (1992)
  • Synthase specificity may be alterable through molecular biological methods.
  • a DNA encoding a FAS and a PHA synthase can be introduced into a single expression vector, obviating the need to introduce the genes into a host cell individually.
  • a further embodiment of the invention is the generation of a DNA encoding a recombinant multifunctional protein, which comprises a FAS, of either eukaryotic or prokaryotic origin, and a PKS module F.
  • Module F will carry out the final chain extension to include two additional carbons and the reduction of the ⁇ -keto group, which results in a (R)-3-hydroxy acyl CoA moiety.
  • DNA encoding the FAS TE is replaced with a DNA encoding a linker region which is normally found in the ACP-KS interdomain region of bimodular ORFs.
  • DNA encoding a module F is then inserted 3′ to the DNA encoding the linker region.
  • Different linker regions such as those described below which vary in length and amino acid composition, can be tested to determine which linker most efficiently mediates or allows the required transfer of the nascent saturated fatty acid intermediate to module F for the final chain elongation and keto reduction steps.
  • the resulting DNA encoding the protein can then be tested for expression of long-chain ⁇ -hydroxy fatty acids in insect cells, such as Sf21 cells, or Streptomyces, or Pseudomonas.
  • the expected 3-hydroxy C-18 fatty acid can serve as a potential substrate for PHA synthases which are able to accept long-chain alkyl groups.
  • a preferred embodiment of the invention is a FAS that has a chain length specificity between 4-22 carbons.
  • linker regions that can be employed in this embodiment of the invention include, but are not limited to, the ACP-KS linker regions encoded by the tyl ORFI (ACP 1 -KS 2 ; ACP 2 -KS 3 ), and ORF3 (ACP 5 -KS 6 ), and eryA ORFI (ACP 1 -KS 1 ; ACP 2 -KS 2 ), ORF2 (ACP 3 -KS 4 ) and ORF3 (ACP 5 -KS 6 ).
  • This approach can also be used to produce shorter chain fatty acid groups by limiting the ability of the FAS unit to generate long-chain fatty acids. Mutagenesis of DNA encoding various FAS catalytic activities, starting with the KS, may result in the synthesis of short-chain (R)-3-hydroxy fatty acids.
  • PHA polymers are then recovered from the biomass. Large-scale solvent extraction can be used, but is expensive. An alternative method involving heat shock with subsequent enzymatic and detergent digestive processes is also available (Byron, Trends Biotechnical, 5, 246 (1987); Holmes, In: Developments in Crystalline Polymers, D. C. Bassett (ed.), pp. 1-65 (1988)). PHB and other PHAs are readily extracted from microorganisms by chlorinated hydrocarbons. Refluxing with chloroform has been extensively used; the resulting solution is filtered to remove debris and concentrated, and the polymer is precipitated with methanol or ethanol, leaving low-molecular-weight lipids in solution.
  • the present invention contemplates nucleic acid sequences which hybridize under low, medium or high stringency hybridization conditions to the exemplified nucleic acid sequences set forth herein. Hybridization conditions are well known in the art. Thus, nucleic acid sequences encoding variant polypeptides, i.e., those having at least one amino acid substitution, insertion, addition or deletion, or nucleic acid sequences having conservative (e.g., silent) nucleotide substitutions (see FIGS. 24 - 25 ), are within the scope of the invention. Preferably, variant polypeptides encoded by the nucleic acid sequences of the invention are biologically active. The present invention also contemplates naturally occurring allelic variations and mutations of the nucleic acid sequences described herein.
  • DNA and RNA molecules that can code for the same polypeptides as those encoded by the exemplified biosynthetic genes and fragments thereof.
  • the present invention contemplates those other DNA and RNA molecules which, on expression, encode the polypeptides of, for example, portions of SEQ ID NO:96. Having identified the amino acid residue sequence encoded by a mitomycin, sugar or polyketide biosynthetic gene, and with knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein and, which molecules are characterized simply by a change in a codon for a particular amino acid, are within the scope of this invention.
  • a TCA codon for serine exists at nucleotide positions 146-148.
  • serine can be encoded by a TCT codon, and a TCC codon. Substitution of the latter codons for serine with the TCA codon for serine or vice versa, does not substantially alter the DNA sequence of SEQ ID NO:16 and results in production of the same polypeptide.
  • substitutions of the recited codons with other equivalent codons can be made in a like manner without departing from the scope of the present invention.
  • a nucleic acid molecule, segment or sequence of the present invention can also be an RNA molecule, segment or sequence.
  • An RNA molecule contemplated by the present invention corresponds to, is complementary to or hybridizes under low, medium or high stringency conditions to, any of the DNA sequences set forth herein.
  • Exemplary and preferred RNA molecules are mRNA molecules that comprise at least one mitomycin, sugar or polyketide biosynthetic gene of this invention.
  • Mutations can be made to the native nucleic acid sequences of the invention and such mutants used in place of the native sequence, so long as the mutants are able to function with other sequences to collectively catalyze the synthesis of an identifiable sugar, polyketide or mitomycin.
  • Such mutations can be made to the native sequences using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene using restriction endonuclease digestion. (See, e.g., Kunkel, T. A. Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al.
  • the mutations can be effected using a mismatched primer (generally 10-30 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex.
  • the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. Zoller and Smith, Methods Enzymol., (1983) 100:468.
  • Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected.
  • Selection can be accomplished using the mutant primer as a hybridization probe.
  • the technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409. PCR mutagenesis will also find use for effecting the desired mutations.
  • Random mutagenesis of the nucleotide sequence can be accomplished by several different techniques known in the art, such as by altering sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants or by damaging plasmid DNA in vitro with chemicals.
  • Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formnic acid, analogues of nucleotide precursors such as nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like.
  • plasmid DNA or DNA fragments are treated with chemicals, transformed into E. coli and propagated as a pool or library of mutant plasmids.
  • chimeric means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the “native” or wild type of the species.
  • the recombinant DNA sequence or segment, used for transformation herein may be circular or linear, double-stranded or single-stranded.
  • the DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the DNA present in the resultant transformed (recombinant) host cell.
  • a portion of the DNA may be untranscribed, serving a regulatory or a structural function.
  • the preselected DNA may itself comprise a promoter that is active in a particular host cell.
  • elements functional in the host cells such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the DNA. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell.
  • Control sequences is defined to mean DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotic cells include a promoter, and optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Other regulatory sequences may also be desirable which allow for regulation of expression of the genes relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • “Operably linked” is defined to mean that the nucleic acids are placed in a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.
  • the DNA to be introduced into the cells further will generally contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure.
  • Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroA, dapA and the like. See also, the genes listed on Table 1 of Lundquist et al. (U.S. Pat. No. 5,848,956).
  • Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Prokaryotic expression systems are preferred, and in particular, systems compatible with Streptomyces spp. are of particular interest.
  • Control elements for use in such systems include promoters, optionally containing operator sequences, and ribosome binding sites.
  • Particularly useful promoters include control sequences derived from the gene clusters of the invention.
  • other bacterial promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, will also find use in the expression cassettes encoding desosamine.
  • Preferred promoters are Streptomyces promoters, including but not limited to the ermE*,pikA and tipA promoters.
  • promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the ⁇ -lactamase (bla) promoter system, bacteriophage lambda PL, and T5.
  • synthetic promoters such as the tac promoter (U.S. Pat. No. 4,551,433), which do not occur in nature, also function in bacterial host cells.
  • the various nucleic acid molecules of interest can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter.
  • the nucleic acid molecules can include flanking restriction sites to allow for the easy deletion and insertion of other sequences. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques, such as site-directed mutagenesis and PCR.
  • the choice of vector depends on the pool of mutant sequences, i.e., donor or recipient, with which they are to be employed. Furthermore, the choice of vector determines the host cell to be employed in subsequent steps of the claimed method. Any transducible cloning vector can be used as a cloning vector for the donor pool of mutants. It is preferred, however, that phagemids, cosmids, or similar cloning vectors be used for cloning the donor pool of mutant encoding nucleotide sequences into the host cell.
  • Phagemids and cosmids are advantageous vectors due to the ability to insert and stably propagate therein larger fragments of DNA than in M13 phage and ⁇ phage, respectively.
  • Phagemids which will find use in this method generally include hybrids between plasmids and filamentous phage cloning vehicles.
  • Cosmids which will find use in this method generally include ⁇ phage-based vectors into which cos sites have been inserted.
  • Recipient pool cloning vectors can be any suitable plasmid.
  • the cloning vectors into which pools of mutants are inserted may be identical or may be constructed to harbor and express different genetic markers (see, e.g., Sambrook et al., supra). The utility of employing such vectors having different marker genes may be exploited to facilitate a determination of successful transduction.
  • the cloning vector employed may be an E. coli/ Streptomyces shuttle vector (see, for example, U.S. Pat. Nos. 4,416,994, 4,343,906, 4,477,571, 4,362,816, and 4,340,674), a cosmid, a plasmid, an artificial bacterial chromosome (see, e.g., Zhang and Wing, Plant Mol. Biol., 35, 115 (1997); Schalkwyk et al., Curr. Op. Biotech., 6, 37 91995); and Monaco and Lavin, Trends in Biotech., 12, 280 (1994), or a phagemid, and the host cell may be a bacterial cell such as E.
  • coli Penicillium patulum, and Streptomyces spp. such as S. lividans, S. venezuelae, or S. lavendulae, or a eukaryotic cell such as fungi, yeast or a plant cell, e.g., monocot and dicot cells, preferably cells that are regenerable.
  • a eukaryotic cell such as fungi, yeast or a plant cell, e.g., monocot and dicot cells, preferably cells that are regenerable.
  • the recombinant DNA can be readily introduced into the host cells by any procedure useful for the introduction into a particular cell, e.g., calcium phosphate precipitation, protoplast fusion, conjugation, lipofection, electroporation, and the like.
  • the term “cell line” or “host cell” is intended to refer to well-characterized homogenous, biologically pure populations of cells. These cells may be eukaryotic cells that are neoplastic or which have been “immortalized” in vitro by methods known in the art, as well as primary cells, or prokaryotic cells. In particular, the cell line or host cell may be of mammalian, plant, insect, yeast, fungal or bacterial origin.
  • Transfected or “transformed” is used herein to include any host cell or cell line, the genome of which has been altered or augmented by the presence of at least one DNA sequence, which DNA is also referred to in the art of genetic engineering as “heterologous DNA,” “recombinant DNA,” “exogenous DNA,” “genetically engineered,” “non-native,” or “foreign DNA,” wherein said DNA was isolated and introduced into the genome of the host cell or cell line by the process of genetic engineering.
  • the transfected DNA may be maintained as an extrachromosomal element or as an element which is stably integrated into the host chromosome.
  • recombinant polypeptides having a particular activity may be prepared via “gene-shuffling”. See, for example, Crameri et al., Nature, 391, 288 (1998); Patten et al., Curr. Op. Biotech., 8, 724 (1997), U.S. Pat. Nos. 5,837,458, 5,834,252, 5,830,727, 5,811,238, 5,605,793).
  • phagemids upon infection of the host cell which contains a phagemid, single-stranded phagemid DNA is produced, packaged and extruded from the cell in the form of a transducing phage in a manner similar to other phage vectors.
  • clonal amplification of mutant encoding nucleotide sequences carried by phagemids is accomplished by propagating the phagemids in a suitable host cell.
  • the cloned donor pool of mutants is infected with a helper phage to obtain a mixture of phage particles containing either the helper phage genome or phagemids mutant alleles of the wild-type encoding nucleotide sequence.
  • Infection, or transfection, of host cells with helper phage is generally accomplished by methods well known in the art (see., e.g., Sambrook et al., supra; and Russell et al. (1986) Gene 45:333-338).
  • the helper phage may be any phage which can be used in combination with the cloning phage to produce an infective transducing phage.
  • the helper phage will necessarily be a ⁇ phage.
  • the cloning vector is a phagemid and the helper phage is a filamentous phage, and preferably phage M13.
  • the transducing phage can be separated from helper phage based on size difference (Barnes et al. (1983) Methods Enzymol. 101:98-122), or other similarly effective technique.
  • Recipient cells which may be employed in the method disclosed and claimed herein may be, for example, E. coli, or other bacterial expression systems which are not recombination deficient.
  • a recombination deficient cell is a cell in which recombinatorial events is greatly reduced, such as recv mutants of E. coli (see, Clark et al. (1965) Proc. Natl. Acad. Sci. USA 53:451-459).
  • transductants can now be selected for the desired expressed protein property or characteristic and, if necessary or desirable, amplified.
  • transductants may be selected by way of their expression of both donor and recipient plasmid markers.
  • the recombinants generated by the above-described methods can then be subjected to selection or screening by any appropriate method, for example, enzymatic or other biological activity.
  • the above cycle of amplification, infection, transduction, and recombination may be repeated any number of times using additional donor pools cloned on phagemids.
  • the phagemids into which each pool of mutants is cloned may be constructed to express a different marker gene.
  • Each cycle could increase the number of distinct mutants by up to a factor of 10 6 .
  • the probability of occurrence of an inter-allelic recombination event in any individual cell is f (a parameter that is actually a function of the distance between the recombining mutations)
  • the transduced culture from two pools of 10 6 allelic mutants will express up to 10 12 distinct mutants in a population of 10 12 /f cells.
  • the present isolated, purified polypeptides, variants or fragments thereof can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by recombinant DNA approaches (see above).
  • the solid phase peptide synthetic method is an established and widely used method, which is described in the following references: Stewart et al., Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc., 85 2149 (1963); Meienhofer in “Hormonal Proteins and Peptides,” ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp.
  • polypeptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.
  • fusion polypeptides are prepared which comprise an amino acid sequence useful in purification, e.g., a His tag is useful to purify fusion polypeptides on nickel columns.
  • derivatives e.g., chemically derived derivatives, of a given polypeptide can be readily prepared.
  • amides of the polypeptides of the present invention may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide.
  • a preferred method for amide formation at the C-terminal carboxyl group is to cleave the polypeptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.
  • Salts of carboxyl groups of a polypeptide or polypeptide variant of the invention may be prepared in the usual manner by contacting the polypeptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide
  • a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate
  • an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • N-acyl derivatives of an amino group of the polypeptide or polypeptide variants may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected polypeptide.
  • O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired.
  • One or more of the residues of the polypeptide can be altered, so long as the polypeptide variant is biologically active.
  • the variant has at least about 1% of the biological activity of the corresponding non-variant polypeptide, e.g.
  • Conservative amino acid substitutions are preferred—that is, for example, aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids.
  • Conservative amino acid substitution also includes groupings based on side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • the invention also envisions polypeptide variants with non-conservative substitutions.
  • Non-conservative substitutions entail exchanging a member of one of the classes described above for another.
  • Acid addition salts of the polypeptide or variant polypeptide or of amino residues of the polypeptide or variant polypeptide may be prepared by contacting the polypeptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid.
  • Esters of carboxyl groups of the polypeptides may also be prepared by any of the usual methods known in the art.
  • the antibodies of the invention are prepared by using standard techniques.
  • an animal is inoculated with an antigen that is an isolated and purified polypeptide of the invention, and immunoglobulins are recovered from a fluid, such as blood serum, that contains the immunoglobulins, after the animal has had an immune response.
  • the antigen is preferably bound to a carrier peptide and emulsified using a biologically suitable emulsifying agent, such as Freund's incomplete adjuvant.
  • a biologically suitable emulsifying agent such as Freund's incomplete adjuvant.
  • a variety of mammalian or avian host organisms may be used to prepare polyclonal antibodies
  • Ig is purified from the immunized bird or mammal, e.g., goat, rabbit, mouse, rat, or donkey and the like.
  • a composition in which the antibodies are essentially free of antibodies that do not react with the immunogen is composed virtually entirely of the high titer, monospecific, purified polyclonal antibodies to the antigen.
  • Antibodies can be purified by affinity chromatography. Purification of antibodies by affinity chromatography is generally known to those skilled in the art (see, for example, U.S. Pat. No. 4,533,630).
  • the purified antibody is contacted with the purified polypeptide, or a peptide thereof, bound to a solid support for a sufficient time and under appropriate conditions for the antibody to bind to the polypeptide or peptide. Such time and conditions are readily determinable by those skilled in the art.
  • the unbound, unreacted antibody is then removed, such as by washing.
  • the bound antibody is then recovered from the column by eluting the antibodies, so as to yield purified, monospecific polyclonal antibodies.
  • Monoclonal antibodies can be also prepared, using known hybridoma cell culture techniques. In general, this method involves preparing an antibody-producing fused cell line, e.g., of primary spleen cells fused with a compatible continuous line of myeloma cells, and growing the fused cells either in mass culture or in an animal species, such as a murine species, from which the myeloma cell line used was derived or is compatible.
  • fused cell line e.g., of primary spleen cells fused with a compatible continuous line of myeloma cells
  • an animal species such as a murine species
  • Such antibodies offer many advantages in comparison to those produced by inoculation of animals, as they are highly specific and sensitive and relatively “pure” immunochemically.
  • Inmunologically active fragments of the present antibodies are also within the scope of the present invention, e.g., the F(ab) fragment, scFv antibodies, as are partially humanized monoclonal antibodies.
  • hybridomas herein referred to may be subject to genetic mutation or other changes while still retaining the ability to produce monoclonal antibody of the same desired specificity.
  • the present invention encompasses mutants, other derivatives and descendants of the hybridomas.
  • a monoclonal antibody may be subjected to the techniques of recombinant DNA technology to produce other derivative antibodies, humanized or chimeric molecules or antibody fragments which retain the specificity of the original monoclonal antibody.
  • Such techniques may involve combining DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of the monoclonal antibody with DNA coding the constant regions, or constant regions plus framework regions, of a different immunoglobulin, for example, to convert a mouse-derived monoclonal antibody into one having largely human immunoglobulin characteristics (see EP 184187A, 2188638A, herein incorporated by reference).
  • the antibodies of the invention are useful for detecting or determining the presence or amount of a polypeptide of the invention in a sample.
  • the antibodies are contacted with the sample for a period of time and under conditions sufficient for antibodies to bind to the polypeptide so as to form a binary complex between at least a portion of said antibodies and said polypeptide.
  • Such times, conditions and reaction media can be readily determined by persons skilled in the art.
  • the cells are lysed to yield an extract which comprises cellular proteins.
  • intact cells are permeabilized in a manner which permits macromolecules, i.e., antibodies, to enter the cell.
  • the antibodies of the invention are then incubated with the protein extract, e.g., in a Western blot, or permeabilized cells, e.g., prior to flow cytometry, so as to form a complex.
  • the presence or amount of the complex is then determined or detected.
  • the antibodies of the invention may also be coupled to an insoluble or soluble substrate.
  • Soluble substrates include proteins such as bovine serum albumin.
  • the antibodies are bound to an insoluble substrate, i.e., a solid support.
  • the antibodies are bound to the support in an amount and manner that allows the antibodies to bind the polypeptide (ligand).
  • the amount of the antibodies used relative to a given substrate depends upon the particular antibody being used, the particular substrate, and the binding efficiency of the antibody to the ligand.
  • the antibodies may be bound to the substrate in any suitable manner. Covalent, noncovalent, or ionic binding may be used. Covalent bonding can be accomplished by attaching the antibodies to reactive groups on the substrate directly or through a linking moiety.
  • the solid support may be any insoluble material to which the antibodies can be bound and which may be conveniently used in an assay of the invention.
  • Such solid supports include permeable and semipermeable membranes, glass beads, plastic beads, latex beads, plastic microtiter wells or tubes, agarose or dextran particles, sepharose, and diatomaceous earth.
  • the antibodies may be bound to any porous or liquid permeable material, such as a fibrous (paper, felt etc.) strip or sheet, or a screen or net.
  • a binder may be used as long as it does not interfere with the ability of the antibodies to bind the ligands.
  • S. lavendulae NRRL 2564 was used as the source strain for cosmid library construction and the creation of gene disruption mutants.
  • E. coli DH5 ⁇ was used as the host strain for constructing the library and subsequent DNA manipulation.
  • E. coli strain S17-1 (Mazodier et al., 1989) served as the conjugative host for introducing foreign DNA into S. lavendulae.
  • the cosmid library was constructed with the E. coli/ Streptomyces shuttle vector pNJ1 (Tuan et al., 1990), and pUC119 was routinely used as a vector for subcloning and sequencing.
  • the conjugative E. coli/ Streptomyces shuttle vector pKC 1139 (Bierman et al., 1992) was used for gene disruption in S. lavendulae.
  • a library of size-fractionated genomic DNA in pNJ1 was screened with the rifamycin AHBA synthase (rifK) gene probe from Amycolatopsis mediterranei (Kim et al., 1998).
  • rifK rifamycin AHBA synthase
  • M13 forward and reverse primers were used for sequencing (Gibco BRL, Gaithersburg, Md.).
  • individual fragments of less than 5 kb were subcloned into pUC 119 and serial deletion subdlones were generated using the exonuclease III Erase-a Base System (Promega, Madison, Wis.).
  • a 1.4 kb ApaL1-HindIII fragment from pFD666 (Denis and Brzezinski, 1998) containing the aphII gene for kanamycin resistance was routinely used as the selection marker for the creation of gene disruption constructs.
  • the target genes were subcloned into pUC 119, cut at a unique internal restriction site, blunt-ended, and ligated with the end-blunted selection marker. The inserts were then transferred from pUC119 to pKC1139, and conjugated into wild-type S. lavendulae.
  • Disruption mutants were selected based on the phenotype changing from apramycin and kanamycin resistant to apramycin sensitive and kanamycin resistant. Replacement of the chromosomal copy of the target gene with the disrupted plasmid-born copy was confirmed by Southern blot hybridization.
  • MC production was evaluated using 3-day cultures in Nishikohri media (Nishikohri and Fukui, 1978).
  • the culture broth was extracted twice with equal volumes of ethyl acetate.
  • the crude broth extract was dissolved in 50% methanol and 50% 50 mM pH 7.2 Tris buffer and monitored by HPLC (C 18 reverse phase column) at 363 nm.
  • HPLC C 18 reverse phase column
  • a continuous methanol gradient from 20% to 60% in methanol/50 mM pH 7.2 Tris buffer system over 24 minutes was employed to resolve MC from other crude extract components.
  • a 90% CHCl 3 /10% MeOH solvent system was used to resolve and detect MC on TLC plates.
  • the mitomycin cluster was identified by linkage of a cosmid clone containing mrd and a gene (mitA) that hybridized with the rifK gene encoding the rifamycin AHBA synthase (Kim et al., 1998) from Amycolatopsis mediterranei. mitA was subsequently shown to be essential for mitomycin biosynthesis since genetic disruption of the chromosomal copy blocked MC production, and could be complemented with exogenous AHBA (Example 2). Linkage of mitA with one of the mitomycin resistance genes (mrd) implied that the corresponding biosynthetic genes were adjacent to mitA.
  • Cosmid walking was used to obtain overlapping DNA fragments spanning more than 120 kb of the S. lavendulae chromosome adjacent to mitA. Subsequent nucleotide sequence analysis included 55 kb of contiguous DNA, revealing 47 genes involved in mitomycin assembly, regulation and resistance (FIGS. 2 and 5). TABLE 1 MC production in wild-type S. lavendulae and gene disruption mutants MC No.
  • mitT Defines the Left-hand Boundary of the Mitomycin Cluster
  • Nucleotide sequence analysis extended 30 kb downstream of mitA and revealed a set of genes corresponding to a type I polyketide synthase (PKS, orf9, SEQ ID NO:18; orf8, SEQ ID NO:19) and thioesterase (TEII, orf7, SEQ ID NO:20).
  • MC is not derived from the polyketide pathway, and thus an orf8 disruption mutant showed normal MC production as expected (Table 1).
  • two genes (mitT, SEQ ID NO:29 and mitS, SEQ ID NO:30) encoding a putative aminoquinate dehydrogenase and glucose kinase, respectively, were located.
  • mmcY Defines the Right-hand Boundary of the Mitomycin Cluster
  • Nucleotide sequence analysis of the mitomycin biosynthetic gene cluster extended 30 kb upstream of mitA and several orfs corresponding to genes involved in sugar metabolism were identified. They included an acid trehalase (orf12, SEQ ID NO:28), one ABC type transporter (orf16, SEQ ID NO:79), and four adjacent ⁇ -amylases (orf19, SEQ ID NO:82; orf20, SEQ ID NO:83; orf21, SEQ ID NO:84; orf22, SEQ ID NO:85) for starch degradation spanning more than 18 kb (FIG. 2).
  • an acid trehalase orf12, SEQ ID NO:28
  • one ABC type transporter orf16, SEQ ID NO:79
  • four adjacent ⁇ -amylases orf19, SEQ ID NO:82; orf20, SEQ ID NO:83; orf21, SEQ ID NO:84; orf22, SEQ ID NO:85
  • mct encodes 484 amino-acid protein with 14 predicted transmembrane domains. Disruption of mct resulted in a mutant S. lavendulae strain substantially more sensitive to MC, while coexpression of mct with mrd in E. coli dramatically increased MC resistance levels compared to individual expression of the genes (Example 3).
  • MmcW showed high sequence similarity with the MarR groups of repressors.
  • EmrR the negative regulator of the E. coli multidrug resistance pump EmrAB (Lomovskaya et al., 1995)
  • Pacs a repressor for pectinase, cellulase, and blue pigment production in Erwinia chrysanthemi (Praillet et al., 1996).
  • the mmcW disruption mutant displayed a several-fold increase in MC production (Table 1).
  • AHBA is an intermediate for both the ansamycin and mitomycin natural products (Becker et al., 1983; Example 2). Combining the biochemical, enzymatic and molecular genetic results on the biosynthesis of the ansamycin antibiotic rifamycin, Floss has proposed that AHBA is derived from the ammoniated shikimate pathway via phosphenolpyruvate (PEP) and erythose 4-phosphate (E4P) by the early incorporation of nitrogen (Kim et al., 1996).
  • PEP phosphenolpyruvate
  • E4P erythose 4-phosphate
  • DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate
  • DHQ 3-dehydroquinate
  • DHS 3-dehydroshikimate
  • shikimate catalyzed by DAHP synthase, DHQ synthase, DHQ dehydratase, and shikimate dehydrogenase, respectively (Dewick, 1998).
  • Quinate can also enter the pathway by the action of quinate dehydrogenase to generate DHQ.
  • rifamycin biosynthetic gene cluster (rif cluster) has been sequenced, and all of the genes encoding early shikimate pathway enzymes are found within the cluster (August et al., 1998).
  • RifK rifamycin AHBA synthase
  • mitA AHBA synthase gene
  • AHBA biosynthetic genes similar to those described for rif have been identified in the mitomycin cluster.
  • six gene products in the cluster showed high sequence similarity (over 43% identity) with their rifamycin AHBA biosynthetic gene homologs.
  • These gene products include aminoDHQ synthase (MitP, RifG equivalent), aminoquinate dehydrogenase (MitT, Rift equivalent), oxidoreductase (MitG, RifL equivalent), phosphatase (MitJ, RifM equivalent), kinase (MitS, RifN equivalent), and aminoDHQ dehydratase (MmcF, RifJ equivalent).
  • MitS most similar to RifN (53% identity, 63% similarity), also showed significant similarity with the glucose kinase (involved in glucose repression) from S. coelicolor and Bacillus megaterium (Angell et al., 1992; Spath et al., 1997).
  • mitG the third non-shikimate pathway-related AHBA biosynthetic gene in this cluster is also worthy of note since it shows exclusive similarity (46% identity, 61% similarity) with oxidoreductase RifL and its equivalent in Actinosynnema pretiosum auranticum.
  • mitosane core is assembled form the condensation of AHBA and D-glucosamine. Although no specific gene products can be assigned for forming the three bonds bridging AHBA and D-glucosamine, two genes downstream of mitA (SEQ ID NO:97), mitb (SEQ ID NO:99), and mitE (SEQ ID NO:44) likely encode enzymes that mediate one of these reactions. MitB shows local sequence similarity with a group of glycosyltransferases involved in glycopeptide antibiotic and polysaccharide biosynthesis, the typical function of which is to attach an activated sugar residue to a core compound (Yamazaki et al., 1996; Example 2).
  • MitE showed weak similarity (22% identity and 45% similarity) to the two cloned 4-hydroxybenzoate-CoA ligases from Rhodopseudomonas palustris in the anaerobic degradation of aromatic compounds (Gibson et al., 1994). It also showed similarity to a group of long chain fatty acid CoA ligases, as well as to the O-succinylbenzoic acid CoA synthetase in Vitamin K2 biosynthesis (Kwon et al., 1996). mitB and mitE disruption mutants both had a MC deficient phenotype (Table 1).
  • the condensation of AHBA with D-glucosamine may be initiated in two different ways. This includes either initial formation of a C 8a -C 9 bond by an acylation or alkylation reaction, or formation of a Schiff base between the AHBA nitrogen and D-glucosamine C1 aldehyde, followed by the ring closure at C 8a -C 9 .mitR (SEQ ID NO:31), one of the two McrA homologues may be involved in one of the ring closure reactions.
  • MitR showed high sequence homology with the plant berberine bridge enzyme (BBE) (30% identity, 37% similarity) in benzophenanthridine alkaloid formation, where it catalyzes an unusual C—C bond formation of the berberine bridgehead carbon of (S)-scoulerine from the N-methyl carbon of (S)-reticuline (Dittrich and Kutchan, 1991).
  • BBE plant berberine bridge enzyme
  • MitB is involved in C 8a -C 9 bond formation.
  • the decreased MC production in the mitR disruption mutant may be due to the existence of isoenzymes (e.g., MmcM) that could catalyze the reaction in the absence of a functional MitR.
  • mitomycin A and mitomycin B also contain a C-7 O-methyl group, while mitomycin B, mitomycin D and porfiromycin have an N-methyl on the aziridine ring (FIG. 1).
  • Radio-labeled precursor incorporation studies showed that all of the O- and N-methyl (but not the C-methyl) groups in the mitomycin molecules are derived from L-methionine (Bezanson and Vining, 1971).
  • the methyl donor for most C1 reactions is S-adenosyl-L-methionine (SAM), which can be formed through activation of L-methionine by ATP.
  • SAM S-adenosyl-L-methionine
  • MmcR may be involved in the O-methylation of the phenol ring of MC before oxidation to the quinone.
  • mmcR SEQ ID NO:67
  • mitM SEQ ID NO:36
  • MmcD SAM-independent methyltransferase
  • this group of methyltransferases uses methylcobalamine or a structurally related protoporphyrin as the direct methyl donor. While the greatest number of matches were made to protoporphyrin methyltransferases, it is expected that this enzyme has another function in the mitomycin C biosynthetic pathway as all the O- and N-methyl groups of MC have been shown to be derived from SAM-dependent methyltransferases.
  • Steps 4 to 6 from CH-H 4 MPT to CH 2 -H 4 MPT, and CH 3 -H 4 MPT to CH 3 -CoM are catalyzed by N 5 , N 10 -methylene-H 4 MPT dehydrogenase, N 5 , N 10 -methylene-H 4 MPT reductase, and N 5 -methyl-H 4 MPT:CoM methyltransferase, respectively (Deppenmeier et al., 1996; Thauer et al., 1993).
  • MmcE is notable since the deduced protein sequence contains two domains showing significant alignment (33% identity, 56% similarity) to the N-terminus of H 4 MPT:CoM methyltransferase from Methanobacterium thermoautotrophicum (Stupperich et al., 1993), while the remaining C-terminus is related to fatty acid biosynthetic acyl carrier proteins (ACP) (Morbidoni et al., 1996; Platt et al., 1990).
  • ACP fatty acid biosynthetic acyl carrier proteins
  • the potential function of this ACP-like domain in MC biosynthesis remains unknown, as does the role of a distinct gene (mmcB, SEQ ID NO:50) encoding a putative ACP identified just upstream of mmcE (SEQ ID NO:53).
  • mmcB SEQ ID NO:50
  • MmcN belongs to the cytochrome P450 family of monooxygenases, with greatest homology (37% identity, 56% similarity) to the two herbicide-inducible cytochrome P450s (P450-SU1 and P450-SU2) from S. griseolus, as well as to RapJ and RapN in the rapamycin biosynthetic gene cluster from S.
  • MmcT showed highest similarity to the tetracenomycin C hydroxylase (TcmG) in Streptomyces glaucescens (38% identity, 55% similarity), with lower but significant sequence similarity to a group of phenol or hydroxybenzoate hydroxylases (Decker et al., 1993). Genetic disruption of mmcT completely blocked MC biosynthesis (Table 1).
  • carbamoyl group of MC is derived intact from L-citrulline or L-arginine with carbamoyl phosphate as the incorporated precursor (Homemann, 1981).
  • carbamoyl phosphate can be generated from L-glutamine, HCO 3 , and ATP by the enzyme carbamoyl phosphate synthetase, which is indispensable for pyrimidine biosynthesis.
  • One candidate carbamoyl transferase gene (mmcS, SEQ ID NO:68) was identified directly upstream of mmcT.
  • MmcS belongs to the NodU/CmcH family of O-carbamoylation enzymes, with the greatest similarity (35% identity, 44% similarity) to No1O from Rhizobium sp. (Jabbouri et al., 1998).
  • Other members with significant alignment in this family include No1O from Bradyrhizobium japonicum (Luka et al., 1993) and NodU from Rhizobium sp. for 6-O-carbamoylation of Nod-factors (Jabbouri et al., 1995) and CmcH from Nocardia lactamdurans and S. clavuligerus for 3′-hydroxymethylcephem O-carbamoylation in cephamycin biosynthesis (Coque et al., 1995).
  • the shikimate pathway is an essential metabolic route in microorganisms and plants for aromatic amino acid biosynthesis.
  • Genes encoding the early shikimate pathway enzymes from various organisms have been well studied and are often dispersed along the chromosome as revealed by genome sequencing projects (Blattner et al., 1997; Bult et al., 1996; Cole et al., 1998).
  • the finding that the ansamycin and mitomycin natural products are derived in part from an ammoniated shikimate pathway whose genes are clustered on the bacterial chromosome is a significant difference to the primary metabolic network, and may suggest an important evolutionary bridge leading to secondary metabolism.
  • the organization of the AHBA biosynthetic genes in the MC cluster is quite different compared to the rif cluster.
  • rif (with the exception rifJ)
  • all AHBA biosynthetic genes are found within a defined sub-cluster that are organized into a single apparent operon.
  • almost all of the mit/mmc encoded AHBA genes are scattered within the 55 kb MC cluster.
  • the MC cluster is biochemically less transparent based on a similar primary analysis.
  • the MC cluster provides a good model for analyzing genetic evolution both vertically, from the primary metabolic shikimate pathway to the secondary shikimate pathway related route, and horizontally by comparing different groups of secondary metabolic biosynthetic clusters.
  • MC production initiates 24 hours after inoculating the seed culture, reaches maximum production in two days, and maintains drug synthesis during stationary phase for another two days.
  • MC production is relatively low ( ⁇ 5 ⁇ g/ml MC).
  • the significant gap between the self-resistance and production levels makes it possible to improve drug production through genetic engineering.
  • disruption of the candidate repressor gene (mmcW) and downstream mmcX (encoding a putative membrane protein) in the mitomycin cluster resulted in a several-fold increase in MC production.
  • a repressor gene(s) is not uncommon in Streptomyces antibiotic biosynthetic gene clusters.
  • Previous examples include, mmyR from the methylenomycin cluster (Chater and Briton, 1985), actII-orfI in the actinorhodin cluster (Caballero et al., 1991), jadR (Anderson et al., 1980) in jadomycin biosynthesis (Yang et al., 1995), and dnrO in the daunorubicin cluster (Otten, 1995). Disruption of jadR and mmyR also resulted in increased levels of the corresponding antibiotic (Chater and Bruton, 1985; Yang et al., 1995).
  • mitR is also followed by a cotranscribed regulatory gene (mitQ). Meanwhile, the putative mitomycin translocase gene, mct is followed by the repressor gene, mmcW.
  • the cloned genes presented here are useful to study mitomycin biosynthesis and natural product assembly.
  • the advantage of having this information has already been demonstrated through genetic disruption of the candidate repressor gene (mmcW) that provided a several-fold increase in MC production.
  • expression and genetic disruption of selected genes should be useful for engineering the biosynthesis of clinically valuable mitomycin analogues, as well as more complex hybrid natural product systems.
  • the MC resistance and regulatory genes identified in this cluster provide important insight into the mitomycin biosynthetic and regulatory network in the S. lavendulae.
  • E. coli DH5 ⁇ was grown in either Luria broth (LB) or tryptic soy broth (TSB) (Difco) as liquid medium or agar plates.
  • E. coli DH5 ⁇ F′ the host for harvesting single-stranded DNA, was grown at 37° C. on TBG (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH 2 PO 4 , 55 mM K 2 HPO 4 , and 20 mM glucose).
  • E. coli S17-1 (Mazodier et al., 1989) used for conjugation was grown in TSB with 10 ug/ml of streptomycin.
  • lavendulae was grown in TSB or on R5T plates.
  • S. lavendulae was grown in Nishikohri media (g/L: glucose 15, soluble starch 5, NaCl 5, CaCO 3 3, yeast extract 5) for 72 hours from a 1% v/v inoculum of frozen mycelia.
  • Pulse feeding of AHBA to the disruption mutant, MV100, and the site-directed mutant, MV102 occurred with feedings of 2.5 mg of a 20 mg/mL solution of the sodium salt of AHBA at pH 7.1 in three pulses at 24, 43, and 57 hours of a culture that was harvested at 76 hours.
  • DNA preparation and amplification Isolation and purification of DNA was performed using standard methods (Sambrook et al., 1989). S. lavendulae NRRL 2564 genomic DNA was isolated by using the modified Chater protocol (Hopwood et al., 1988). Plasmid DNA was isolated from E. coli by using the alkaline-sodium dodecyl sulfate method.
  • pDHS2002 was constructed as follows: The 1.1 kb thiostrepton resistance gene (tsr) fragment was removed from pDHS5000 with a SmaI-BamHI digestion, blunt-ended with the large fragment of DNA polymerase (Gibco BRL), and ligated to MscI restriction enzyme digested pDHS7601 to yield pDHS20001. MscI digestion of pDHS7601 resulted in the removal of 155 nucleotides at the C-terminus of the mitA gene, and ligation of the blunt-ended BamHI site of the tsr adjacent to the MscI site of pDHS7601 resulted in regeneration of the BamHI site in pDHS2001. The 4.9 kb EcoRI-HindIII fragment from pDHS2001 containing the tsr disrupted mitA gene was removed and ligated into EcoRI-HindIII digested pKC1139 to yield pDHS2002.
  • Primer-mediated site-directed mutagenesis was employed to construct pDHS2015 containing a K191A mutation in mitA.
  • Primer 1 5′-GGCAAGGCATGCGAGGGTCGC-3′ (SEQ ID NO:46) and primer 2: 5′-TTCCAGAACGGCGCCCTGATGACCGCCGGC-3′ (SEQ ID NO:47) were used to amplify the 691 bp fragment of the 5′ end of mitA.
  • the 3′ end of mitA was amplified with primer 3: 5′-GCCGGCGGTCATCAGGGCGCCGTTCTGGAA-3′ (SEQ ID NO:48) and primer 4: 5′-TCAGAATTCGGATCCGAGGGCCGGAGT-3′ (SEQ ID NO:86) to generate a 1151 bp band (see amplification reaction conditions in Example 3).
  • a second round of PCR was performed using the overlapping 691 and 1151 bp units as the initial templates with primer 1 and primer 4 to yield a 1.8 kb fragment.
  • the final product containing mutagenized mitA was digested with EcoRI-Sph1, ligated to the 2.1 kb HindlII-SphI fragment from pDHS7601 and the EcoRI-HindIII digested pKC1139 to yield pDSH2015.
  • the site-directed mutation of MitA K191A in pDHS2015 was confirmed by sequencing with forward primer: 5′-ACCTACTGCCTCGATGCC-3′ (SEQ ID NO:87) and reverse primer: 5′-CTGATCCTTCAAGCG-3′. (SEQ ID NO:88)
  • the mitB disruption vector pDHS7702 was constructed as follows. pDHS7601 was digested with BstBI, blunt-ended, and ligated with the 1.4 kb neomycin-resistant gene fragment from pFD666 (Denis and Brzezinski et al., 1992) (ApaL1-HindIII digestion, blunt-ended). The 5.2 kb EcoRI-HindIII fragment from the resulting construct pDHS7701 was subdloned into pKC1139 to create pDHS7702.
  • the cosmids from the positive colonies were confirmed by Southern blot hybridization, and a 1.7 kb AflIII-BamHI fragment from pDHS3001 containing the mitomycin resistance determinant (mrd) (Sheldon et al., 1997) was used as a probe to establish genetic linkage.
  • This culture was grown for 3 hours at 37° C., and the cells were washed twice with TSB and resuspended in 2 ml of TSB to provide the donor E. coli culture.
  • the recipient S. lavendulae culture was generated by inoculating 9 ml of TSB with 1 ml of frozen wild-type culture. Following overnight (16 hour) incubation at 29° C., the culture was homogenized by sonication and 2 ml of this culture was used to inoculate 18 ml of TSB. Following overnight growth at 29° C. and sonication treatment to homogenize the culture, a 1 ml inoculum was placed in 9 ml of TSB. This culture was grown for 3 hours, the mycelia were washed with TSB and resuspended in 2 ml of TSB to provide the stock recipient culture.
  • pKC 1139 has a temperature-sensitive Streptomyces replication origin, which is unable to replicate at temperatures above 34° C. (Muth et al., 1989), while the S. lavendulae host grows well at 42° C. Thus, after propagating the conjugants at 39° C. for several generations, double crossover mutants were readily generated. Presence of plasmid was determined by transformation of E. coli DH5 ⁇ with plasmid extracts from S. lavendulae transconjugants.
  • Double-crossover selection procedure A single colony of S. lavendulae/ pDHS2002 grown on R5T plates (50 ⁇ g/ml of thiostrepton and apramycin) was used to inoculate TSB broth containing 20 ⁇ g/ml of thiostrepton. After 72 hours of incubation at 39° C., 10 ⁇ 4 , 10 ⁇ 5 and 10 ⁇ 6 diluted aliquots were used to inoculate R5T plates containing 50 ⁇ g/ml of thiostrepton.
  • MitA K191A site-directed mutants were selected by propagating MV100/pDHS2015 on R5T plates for two generations at 37° C. Colonies were replicated to plates containing 50 ⁇ g/ml of thiostrepton and plates without antibiotics. Of the 108 colonies replicated in the first round, one had the correct (thiostrepton sensitive) phenotype.
  • the mitA gene was amplified from the chromosome with primers 1 and 4. Mutation of the conserved lysine codon (AAG) to an alanine codon (GCC) was verified with the same sequencing primers employed to confirm the correct construction of pDHS2015. The alanine codon was observed in both the forward and reverse sequence data.
  • Mutants for mitB were selected as follows: S. lavendulae/ pDHS7702 was propagated on R5T plates for five generations at 39° C. before single colonies were replicated on R5T plates as described above. Of the 300 colonies tested, 12 clones displayed the correct phenotype (kanamycin resistance and apramycin sensitivity). The genotype of selected mitB mutants was confirmed by Southern blot hybridization of S. lavendulae genomic DNA.
  • the ethyl acetate extracts were pooled and solvent was removed by vacuum to provide the crude broth extract.
  • the preliminary screen for MC production involved thin layer chromatography (TLC) on silica gel plates (Whatman K6) eluted with 9:1 chloroform:methanol. Production of MC was monitored by HPLC (C 18 reverse phase column) using a gradient of 80% 50 mM Tris buffer (pH 7.1)/20% methanol to 40% 50 mM Tris buffer (pH 7.2)/60% methanol with the UV detector set to 363 nm.
  • Bioassay detection of MC was performed by loading a 1 cm disk with fractions eluting at the mitomycin retention time from HPLC injections of wild-type, MV100, pKC1139 vector control crude extracts and MC standards.
  • the disks were placed on antibiotic media number 2 agar plates (Difco) with Bacillus subtilis spores added directly to the media. The plates were incubated overnight at 29° C. and examined for zones of inhibition.
  • mitA comprises 1164 nucleotides and starts from ATG (position 579 of the sequenced fragment) that is preceded by a potential ribosome binding site (RBS), GAAAGG (SEQ ID NO:91).
  • RBS potential ribosome binding site
  • GAAAGG SEQ ID NO:91.
  • the deduced product of the mitA gene encodes a hydrophilic protein of 388 amino acids with a predicted M r of 41,949 Da and a calculated pI of 5.62.
  • a conserved pyridoxal phosphate (PLP) coenzyme binding motif (GX 3 DX 7 AX 8 EDX 14 GX 13 KX 4-5 geGGX 19 G) (SEQ ID NO:92) including the conserved lysine residue can also be found in these four proteins (Piepersberg, 1994).
  • the mitB gene is predicted to start at a GTG (position 1879) that is preceded by a presumed RBS (GGAACG) (SEQ ID NO:93). This gene encodes a 272 amino acid protein with a deduced M r or 28,648 Da and a deduced pI of 6.06. Database sequence homology searches revealed that the product of mitB shows local sequence similarity with a group of O-glycosyltransferases involved in polysaccharide biosynthesis.
  • the third ORF, mitC starts from the ATG at position 2694, which is coupled to the stop codon TGA of mitB and encodes a putative protein of 260 amino acids with a molecular mass of 27,817 Da and a pI of 10.45.
  • Database searches with the deduced protein product showed significant similarity over the first 90 amino acids (38% identity, 40% similarity) with the ImbE gene product (unknown function) from Mycobacterium leprae (U15183).
  • the mitA disruption construct was made by replacing a 155 bp fragment between the two MscI sites (located at the C-terminus of the mitA gene in pDHS7601) with the 1.1 kb SmaI-BamHI fragment containing a thiostrepton resistance gene from pDHS5000 (FIG. 11A). This replacement regenerated a BamHI site at the junction and the resulting construct was then subcloned into the E. coli -Streptomyces conjugative shuttle plasmid pKC1139, followed by conjugation into S. lavendulae.
  • a double crossover mutant strain (MV100) was selected based on the expected phenotype (thiostrepton resistant, apramycin sensitive), and further confirmed by Southern blot hybridization. Genomic DNA from wild-type S. lavendulae and MV100 was digested with BamHI and SphI, and hybridized with the 4.9 kb EcoRI-HindIII tsr-disrupted mitA fragment from pDHS2001. As expected, the 4.0 kb SphI hybridized band in the wild-type strain was shifted to 4.9 kb in MV100, whereas the 3.8 kb BamHI hybridization and in the wild-type was converted to two bands (2.2 kb and 2.5 kb) in the mutant (FIG. 11B).
  • the mitB gene was disrupted by inserting a neomycin resistance gene (aphII) into the BstBI site (located at the 5′-end of mitB) (FIG. 12A).
  • aphII neomycin resistance gene
  • Transconjugants were selected on kanamycin/apramycin plates, and a double crossover mutant strain (MM101) was identified with a kanamycin-resistant, apramycin-sensitive phenotype and subsequently confirmed by Southern blot hybridization.
  • MM101 double crossover mutant strain
  • the 3.8 kb BamHI hybridization band in wild-type S. lavendulae was shifted to 5.2 kb in MM101, whereas a 5.2 kb SacI hybridization band was shifted to 6.6 kb (FIG. 12B).
  • mitA and mitB disrupted strains are blocked in MC biosynthesis.
  • the growth characteristics and morphology of MV100 and MM1001 in liquid media and on agar plates was identical to wild-type S. lavendulae.
  • HPLC was used to quantify production of MC in MV100 and MM101 (FIG. 13A), and culture extracts were used in a biological assay to test for presence of the drug (FIG. 13B). Injection of one mg of wild-type S. lavendulae culture extract gave a peak in the HPLC that eluted with the same retention time as the MC standard.
  • Exogenous AHBA can restore MC production in the MC-deficient MitA K191A mutant.
  • complementation of MV100 mitA insertional disruptant
  • MC production was not restored as measured by HPLC or biological assay.
  • a polar effect on genes downstream of tsr-disrupted mitA in MV100 appeared likely since supplying mitA in trans on a medium copy number plasmid (MV103) also failed to restore MC production. Therefore, site-directed mutagenesis was employed to generate a MitA K191A mutant resulting in strain MV102. Kim et al. (1998) had demonstrated that the AHBA synthase from A.
  • the mitosane core is formed through the condensation of AHBA and D-glucosamine (Hornemann, 1981).
  • AHBA is thought to be derived from the ammoniated shikimate pathway from PEP and E4P, in which the last step from aminoDHS to AHBA is catalyzed by AHBA synthase (FIG. 7) (Kim et al., 1996; Kim et al., 1998).
  • a second possibility would be formation of a Schiff base between the nitrogen of AHBA and the D-glucosamine C1 aldehyde, followed by ring closure at C 8a -C 9 .
  • a C- or N- instead of O-glycosyltransferase is expected.
  • glycosyltransferases display a high degree of sequence divergence (Yamazaki et al., 1996), the mechanistic similarity with O-glycosyl transfer may suggest that mitB encodes a N-glycosyltransferase that initiates the formation of the mitosane system by linking glucosamine to AHBA.
  • the mitA and mitB genes and their corresponding products are likely candidates to mediate formation of AHBA and the mitosane ring system, respectively.
  • the possible function of the lmbE-like protein remains unclear, since its current role within lincomycin biosynthetic pathway of S. lincolnensis is not known (Peschke, 1995).
  • mitA The function of mitA was probed by providing strains MV100 and MV102 with exogenous 3-amino-5-hydroxybenzoic acid in the culture medium. Despite repeated attempts to complement MV100, MC production was not restored as measured by HPLC or biological assay. It is believed that insertion of the tsr gene into mitA resulted in disruption of biosynthetic genes immediately downstream, since supplying mitA in trans on a medium copy number plasmid also failed to restore MC production to MV100. This putative polar effect was eliminated by generating the MitA K191A mutant strain MV102. Providing exogenous 3-amino-5-hydroxybenzoic acid to this mutant strain of S.
  • Mitomycin Resistance in Streptomyces lavendulae Includes a Drug-Binding Protein-Dependent Export System
  • MC is unreactive until chemical or enzymatic reduction renders the molecule a highly effective alkylating agent (Iyer and Szybalski, 1964).
  • the molecular basis of MC bioactivity derives mainly from its propensity to covalently interact with DNA at 5′-CpG sequences, causing lethal intra- and inter-strand crosslinks as well as monofunctional alkylation (Tomasz, 1995).
  • the second locus encodes MRD that functions to sequester the prodrug by a specific mitomycin-binding protein (Sheldon et al., 1997).
  • mitomycin resistance has been the lack of a clear mechanism for drug transport. Indeed, the observed stoichiometry suggests that it would be ineffective for S. lavendulae to utilize MRD as a solo mechanism for cellular self-protection.
  • Pathogenic bacteria Naikaido, 1994
  • antibiotic-producing microorganisms (Cundliffe, 1992; Mendez and Salas, 1998), employ export of toxic compounds as a means of resistance.
  • E. coli DH5 ⁇ used as a host for generation of double-stranded plasmid DNA, was grown at 37° C. on LB medium.
  • E. coli BL21 (DE3) used as host for protein expression, was grown at 37° C. in NZCYM medium (Sambrook et al., 1989).
  • S. lavendulae NRRL 2564 was grown on YEME medium (Hopwood et al., 1985) at 30° C. for preparation of genomic DNA.
  • the mct disruption vector pDHS7704 was constructed as follows. pDHS7661 was digested with EcoRI, blunt-ended, and ligated with the 1.4 kb neomycin resistance gene fragment from pFD666 (ApaLI-HindIII digestion, blunt-ended) (Ames, 1986). The 5.4 kb EcoRI-HindIII fragment from the resulting construct (pDHS7703) was subcloned into pKC1139 to create pDHS7704, and conjugated into S. lavendulae according to Bierman et al. (1992). A met double crossover mutant was selected after propagating transconjugants on R5T plates for five generations at 39° C. Kanamycin-resistant and apramycin-sensitive colonies were further tested by Southern blot to confirm the desired double crossover genotype.
  • the PCR was carried out using 2.5 U of Taq polymerase, 0.4 ⁇ g of each primer, 1 ⁇ g of pDHS7661 DNA as template, 10 mM each of dATP-dGTP-dCTP-dTTP, 1.5 mM MgCl 2 , and 10 ⁇ l of 10 ⁇ Promega PCR buffer in a total volume of 100 ⁇ l.
  • Amplification was achieved with 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 37° C. for 1 minute, and extension at 70° C. for 2 minutes.
  • pDHS7023 was introduced by transformation into E. coli BL21(DE3) to provide strain PJS102.
  • MC resistance phenotype of E. coli To analyze resistance conferred by the expression of the MC-translocase in E. coli, 10 ⁇ l of strain PJS102 was spread on LB agar medium containing 100 ⁇ g/ml of ampicillin, IPTG to a final concentration of 1.0 mM, and various concentrations of MC. The cultures were grown overnight at 37° C. and colony-forming units (CFUs) were deternined. Similarly, the MC resistance phenotype of strain PJS103 (mcr-mrd co-expression strain) was quantified.
  • [ 3 H]-MC was obtained from Kyowa Hakko Kogyo, Ltd. Uptake studies were performed for whole cells of PJS100, PJS102, PJS103 and E. coli BL21(DE3)::pT7SC and pET17b.
  • PJS100, PJS102, and PJS103 as well as vector-only cultures were cultured (37° C.) in 5 ml of NCZYM medium with IPTG added to a final concentration of 1 mM (at approximately 3 hours growth). At 9 hours (late exponential phase), cells were harvested by centrifugation and resuspended in 1 ml NCZYM broth (5 ⁇ concentration).
  • the concentrated suspension of late-exponential growth phase cells was exposed to [ 3 H]-MC (59 Ci/mmol) at a final concentration of 0.022 ⁇ g/ml (0.0655 nmol). Aliquots (100 ⁇ l) were removed at frequent intervals, placed on 1.2 ⁇ M GF/C filters (Whatman International, Maidstone, U.K.) and washed once with 6 ml of 0.85% NaCl poured over the filters under vacuum pressure. Additional aliquots were simultaneously removed for determination of protein content (protein assay kit, Bio-Rad Laboratories, Richmond, Calif.). Radioactivity on the filters was quantified using a Beckman LS7000 scintillation counter. Results were expressed as nanograms of mitomycin per milligram of cell protein.
  • a gene encoding a transmembrane protein is physically linked to mrd.
  • the gene (mct, SEQ ID NO:72) encoding the putative mitomycin exporter (MC-translocase; MCT) protein is located within 5 kb of mrd (SEQ ID NO:64) and is physically linked to the mitomycin biosynthetic gene cluster (FIG. 15).
  • Transmembrane proteins that mediate resistance to antibiotics and antiseptics by active efflux are highly related, usually containing 12 or 14 transmembrane regions.
  • the actinomycete drug transport proteins that share homology with MCT appear to contain 14 transmembrane spanning regions and constitute a family of drug resistance translocases.
  • MEMSAT Membrane structure and topology program
  • hydropathy analyses based on the algorithm of Kyte and Doolittle (1982), a prediction of 14 transmembrane spanning domains was made for the deduced amino acid sequence of MCT (FIG. 18).
  • IPTG-induced cultures of PJS102 exhibited resistance to MC at drug concentrations 5-fold greater than those for E. coli BL21(DE3) containing vector alone (Table 4). TABLE 4 MC resistance of mct, mrd expressing E.
  • genes coding for drug export proteins are physically linked to the corresponding biosynthetic genes within the genome of the antibiotic-producing microorganism. Presumably, the tight linkage of antibiotic export and biosynthetic genes ensures coordinate gene regulation. Interestingly, the presence of back-to-back and overlapping divergent promoters of antibiotic export and regulatory genes has been observed within the tetracenomycin (Guilfoile and Hutchinson, 1992) and actinorhodin (Caballero et al, 1991) biosynthetic gene clusters. Conforming to this example, S. lavendulae possesses a gene coding for an integral membrane drug export protein within the mitomycin biosynthetic gene cluster.
  • MRD MRD-dependent import systems
  • MCT cellular protection afforded by MCT is a function of drug transport from the cytoplasm.
  • co-expression of mrd and met in E. coli led to cultures that are dramatically more resistant to exogenously added drug.
  • binding proteins While normally required for the transport systems with which they are associated, in many instances binding proteins are not integral to the process of solute translocation (Higgins et al., 1990).
  • MRD binding protein
  • the binding protein may be considered an accessory component, a rather specific adaptation required for optimal drug resistance.
  • MRD and MCT are components of a novel drug transport system.
  • Such a resistance mechanism, sequestering the intact drug for efficient excretion to the environment, represents a unique cellular strategy for self-preservation by the MC-producing organism.
  • QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA, J. Biol. Chem., 273, 18665-73 (1998).
  • EmrR is a negative regulator of the Escherichia coli multidrug resistance pump EmrAB, J. Bacteriol., 177, 2328-2334 (1995).
  • ⁇ 400> SEQUENCE: 144 Val Xaa Gly Xaa Leu Xaa Asp Xaa Xaa Gly Arg Lys Xaa Xaa Xaa Leu 1 5 10 15 ⁇ 210> SEQ ID NO 145 ⁇ 211> LENGTH: 11 ⁇ 212> TYPE: PRT ⁇ 213>
  • ORGANISM Artificial Sequence ⁇ 220> FEATURE: ⁇ 223> OTHER INFORMATION: A motif ⁇ 220> FEATURE: ⁇ 221> NAME/KEY: SITE ⁇ 222> LOCATION: (1)...(11) ⁇ 223> OTHER INFORMATION: Where present in this sequence, Xaa represents an amino acid that varied in this conserved motif.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention provides a biosynthetic gene cluster for mitomycin, as well as methods of using gene(s) within the cluster to alter biosynthesis.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a divisional of U.S. application Ser. No. 09/266,965, filed Mar. 12, 1999.[0001]
    • BACKGROUND OF THE INVENTION
  • Streptomyces are filamentous Gram-positive soil bacteria with a nucleotide base composition greater than 70 mole % G+C (Stackebrandt and Woese, 1981). They produce a wide array of biologically active compounds including over two thirds of the commercially important natural product metabolites (Alderson et al., 1993; Bevax, 1998). Genetic information accumulated over the past 15 years has demonstrated that genes encoding enzymes for natural product assembly are clustered on the Streptomyces genome (Martin, 1992). In addition, one or more pathway-specific transcriptional regulatory genes, and at least one resistance gene are typically found within the antibiotic biosynthetic gene cluster (Chater, 1992). Heterologous hybridization with gene probes based on highly conserved biosynthetic enzyme amino acid sequences has been useful to clone antibiotic biosynthetic genes (Hopwood, 1997; Seno and Baltz, 1989; Turgay and Marahiel, 1994). [0002]
  • The mitomycins are a group of natural products that contain a variety of functional groups, including aminobenzoquinone and aziridine ring systems. One representative of the family, mitomycin C (MC), was the first recognized bioreductive alkylating agent. In particular, since its discovery and demonstration of anticancer activity in the 1960s, many aspects of the chemistry and biology of MC have been investigated. This has provided detailed information on its unprecedented molecular mechanism, unique biological and pharmacological properties, drug resistance, and bioactive analogues (Hata et al., 1956; Verweij, 1997). MC is regarded as the prototype natural product alkylating agent whose activity is dependent on the reductive activation (either chemically, such as low pH, or enzymatically, such as DT-diaphorase, NADH cytochrome c reductase) (Boxer, 1997; Cummings et al., 1998). Activated MC crosslinks double-stranded DNA, which in turn induces diverse biological effects including selective inhibition of DNA synthesis, mutagenesis, induction of DNA repair (SOS response), sister chromatid exchange, signal transduction, and induction of apoptosis (Tomasz and Palem, 1997). Tumor hypoxia and the increased expression of bioreductive enzymes in malignant cells create a selective environment for drug activation and make MC an attractive agent for anti-tumor therapy (Spanswick et al., 1998). MC has become one of the most effective antitumor drugs against non-small cell lung carcinoma and other soft tumors, as well as a clinically important component of combination cancer chemotherapy and radiotherapy of solid tumors (Henderson, 1993). [0003]
  • In addition to its biological and pharmacological importance, MC is prominent because its molecular mechanism represents a model for structurally related antitumor antibiotics such as porfiromycin (Pan and Iracki, 1988), mitiromycin (Wakaki et al., 1958), FR66979 (Paz and Hopkins, 1997), FR900482 (Williams et al., 1997), FK973 (Hirai et al., 1994), and FK317 (Naoe et al., 1998), as well as structurally unrelated bioreductive agents such as EO9 (Smitskampwilms et al., 1996), and tirapazamine (Evans et al., 1998). Numerous MC derivatives have been synthesized and tested for enhanced activities, including the recently identified selective protein tyrosine kinase inhibitor, 1 a-docosahexaenoyl MC (Kasai and Arai, 1995; Shikano et al., 1998). [0004]
  • [0005] Streptomyces lavendulae produces MC. The molecule has an unusual structure comprised of aziridine, pyrrolizidine, pyrrolo-(1,2a)-indole, and amino-methylbenzoquinone rings to give the mitosane nucleus (Webb et al., 1962). The mitosane core of MC was shown to be derived from the junction of an amino-methylbenzoquinone (mC7N unit) and hexosamine (C6N unit) (Hornemann, 1981). The C6N unit consists of carbons 1, 2, 3, 9, 9a, 10, with the aziridine nitrogen derived intact from D-glucosamine (Homemann et al., 1974).
  • The mC[0006] 7N unit in MC and the ansamycins is derived from 3-amino-5-hydroxybenzoic acid (AHBA) (Becker et al., 1983; Kibby and Richards, 1981). AHBA was first shown to be incorporated into the ansamycin antibiotic actamycin (Kibby et al., 1980). Subsequently, it was confirmed as an efficient precursor for rifamycin (Becker et al., 1983; Kibby and Rickards, 1981; Ghilsalba and Neuesch, 1981), geldanamycin (Potgieter, 1983), ansamitocin (Hatano et al., 1982), ansatrienin (Wu et al., 1987), streptovaricin (Staley and Rinehart, 1991) and naphthomycin A (Lee et al., 1994). Anderson et al. (1980) demonstrated that [carboxy-13C] AHBA could be efficiently and specifically incorporated into the C-6 methyl group of porfiromycin, which contains the same mitosane core as MC. Incorporation experiments with radiolabeled precursors have demonstrated that the mitosane core of MC was derived from the junction of AHBA and D-glucosamine (Anderson et al., 1980; Homemann, 1981).
  • Meanwhile the O- and N- (but not C-) methyl groups were shown to be derived from L-methionine, while the C-10 carbamoyl group came from L-arginine or L-citrulline (Bezanson and Vining, 1971; Homemann and Eggert, 1975; Homemann et al., 1974). [[0007] 14C]-labeled precursor feeding studies with D-glucose, pyruvate and D-erythrose indicated that de novo biosynthesis of AHBA resulted directly from the shikimate pathway. However, no incorporation into the mC7N unit of either MC (Homemann, 1981) or the ansamycin antibiotics (Chiao et al., 1998) was found from labeling studies with shikimic acid, the shikimate precursor 3-dehydroquinic acid, or the shikimate derived amino acids. These results led to the hypothesis of a modified shikimate pathway, in which a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase-like enzyme catalyzes the conversion to 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid-7-phosphate (amino-DAHP), to give the ammoniated shikimate pathway (Kim et al., 1992). Floss (1997) provided strong support for this new variant of the shikimate pathway by showing that aminoDAHP, 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) could be efficiently converted into AHBA by a cell-free extract of Amycolatopsis mediterranei (rifamycin producer), in contrast to the normal shikimate pathway intermediate DAHP which was not converted (Kim et al., 1992; Kim et al., 1996). Recently, the AHBA synthase (rifk) gene from A. mediterranei has been cloned, sequenced and functionally characterized (Kim et al., 1998).
  • Little is known regarding the details of the convergent assembly of MC from AHBA and D-glucosamine in [0008] S. lavendulae, i.e., whether its de novo biosynthesis is related to the primary metabolic shikimate pathway, an important route in microorganisms and plants for aromatic amino acid biosynthesis (Floss, 1997). In addition, it is unclear how S. lavendulae resists the activity of MC since the preferred MC alkylation sites in DNA are guanine and cytosine, and MC-induced cell death can result from a single crosslink per genome (Tomasz, 1995).
  • Thus, there is a continuing need for the identification and isolation of antibiotic biosynthetic genes, including genes which confer resistance to antibiotics or result in enhanced production of antibiotics. [0009]
    • SUMMARY OF THE INVENTION
  • The present invention provides an isolated and purified nucleic acid molecule, e.g., DNA, comprising a gene cluster for mitomycin, a variant or a fragment thereof (the mit/mmc gene cluster). As described hereinbelow, the [0010] S. lavendulae mitomycin gene cluster includes the mitomycin biosynthetic gene cluster comprising 47 mitomycin biosynthetic genes spanning 55 kb of contiguous DNA. The biosynthetic portion of the gene cluster includes genes that encode polypeptides involved in the generation of biosynthetic precursors, mitosane ring system assembly and functionalization (e.g., methylation, hydroxylation, aminotransfer, carbamoylation, and carbonyl reduction), a mitomycin resistance gene which is different than mrd and the unlinked mcr, as well as several regulatory genes. Gene disruption was employed to further characterize some of the genes. Fourteen of 22 gene disruption mutants affected mitomycin biosynthesis, resulting in abrogation or overexpression of drug production, e.g., targeted genetic disruption of a mitomycin pathway regulator (e.g., mmcW) led to a substantial increase in drug production. It is preferred that the isolated and purified nucleic acid molecule of the invention is nucleic acid from Streptomyces spp., such as Streptomyces lavendulae (e.g., B19/ATCC 27422, NRRL 2564, KY681, ATCC 27423, or PB1000), Streptomyces caespitosus, Streptomyces verticillatus, and Streptomyces sandaensis (FERM-P7654), although isolated and purified nucleic acid molecules from other organisms which produce mitomycin or biological or functional equivalents thereof are also within the scope of the invention. The nucleic acid molecules of the invention are double-stranded or single-stranded.
  • As described hereinbelow, a 3.8 kb BamHI fragment from the [0011] S. lavendulae genome was isolated which comprises three open reading frames (ORFs). One of the ORFs (mitA) showed high similarity to previously identified AHBA synthase genes (Kim et al., 1998), while another (mitB) showed sequence similarity to several prokaryotic and eukaryotic glycosyltransferases. Nucleotide sequence analysis showed that mitA encodes a 388 amino acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediterranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of the S. lavendulae chromosomal copy of mitA completely blocked the production of MC. The function of mitA was confirmed by complementation of a S. lavendulae strain containing a K191A mutation in MitA with 3-amino-5-hydroxybenzoic acid, i.e., MC production was restored when the mitA mutant strain was cultured in the presence of exogenous 3-amino-5-hydroxybenzoic acid. mitB encodes a 272 amino acid protein.
  • Seven gene products (aminoDHQ synthase (MitP), aminoquinate dehydrogenase (MitT), aminoDHQ dehydratase (MmcF), AHBA synthase (MitA), oxidoreductase (MitG), phosphatase (MitJ), and kinase (MitS)) are likely responsible for assembly of the intermediate 3-amino-5-hydroxybenzoic acid (AHBA) through a variant of the shikimate pathway. However, the gene encoding aminoDAHP synthase, the first presumed enzyme involved in AHBA biosynthesis from phosphoenol pyruvate (PEP) and erythrose 4-phosphate (E4P), is not linked within the mitomycin biosynthetic gene cluster. [0012]
  • A mitomycin resistance determinant (mct) encodes a membrane-associated protein involved in excretion of mitomycin from cells. Disruption of met by insertional inactivation resulted in a [0013] S. lavendulae mutant strain that was considerably more sensitive to MC. Expression of mct in E. coli conferred a 5-fold increase in cellular resistance to MC, led to the synthesis of a membrane associated protein, and correlated with reduced intracellular accumulation of drug. Co-expression of mct and mrd in E. coli resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. The results establish that MRD maintains a high affinity for MC and may serve as the primary receptor (participating as an accessory component in a drug export system) for subsequent transport by MCT.
  • The cloned mitomycin biosynthetic genes are useful to elucidate the molecular basis for the biosynthesis of the mitosane ring system, as well as to engineer the biosynthesis of novel natural products. Moreover, genetic engineering or overexpression of the transport, resistance and regulatory proteins may lead to higher titers of mitomycin compounds from production cultures. [0014]
  • Preferably, the isolated nucleic acid molecule comprising the gene cluster includes a nucleic acid sequence comprising SEQ ID NO:96 or SEQ ID NO:76, a variant or a fragment thereof, e.g., a nucleic acid molecule that hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:96, SEQ ID NO:76 or a fragment thereof. Moderate and stringent hybridization conditions are well known to the art, see, for example sections 9.47-9.51 of Sambrook et al. ([0015] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). For example, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50° C., or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.
  • A preferred nucleic molecule of the invention comprises a nucleic acid sequence encoding a polypeptide including, but not limited to, MitA (e.g., SEQ ID NO:10 encoded by SEQ ID NO:97), MitB (e.g., SEQ ID NO:11 encoded by SEQ ID NO:98), MitC (e.g., SEQ ID NO:12 encoded by SEQ ID NO:99), MitD (e.g., SEQ ID NO:100 encoded by SEQ ID NO:45), MitE (e.g., SEQ ID NO:101 encoded by SEQ ID NO:44), MitF (e.g., SEQ ID NO:102 encoded by SEQ ID NO:43), MitG (e.g., SEQ ID NO:103 encoded by SEQ ID NO:42), MitH (e.g., SEQ ID NO:104 encoded by SEQ ID NO:41), MitI (e.g., SEQ ID NO:105 encoded by SEQ ID NO:40), MitJ (e.g., SEQ ID NO:106 encoded by SEQ ID NO:39), MitK (e.g., SEQ ID NO:107 encoded by SEQ ID NO:38), MitL (e.g., SEQ ID NO:108 encoded by SEQ ID NO:37), MitM (e.g., SEQ ID NO:109 encoded by SEQ ID NO:36), MitN (e.g., SEQ ID NO:108 encoded by SEQ ID NO:35), MitO (e.g., SEQ ID NO:111 encoded by SEQ ID NO:34), MitP (e.g., SEQ ID NO:112 encoded by SEQ ID NO:33), MitQ (e.g., SEQ ID NO:113 encoded by SEQ ID NO:32), MitR (e.g., SEQ ID NO:114 encoded by SEQ ID NO:31), MitS (e.g., SEQ ID NO:115 encoded by SEQ ID NO:30), MitT (e.g., SEQ ID NO:140 encoded by SEQ ID NO:29), MmcA (SEQ ID NO:116 encoded by SEQ ID NO:49), MmcB (SEQ ID NO:117 encoded by SEQ ID NO:50), MmcC (SEQ ID NO:118 encoded by SEQ ID NO:51), MmcD (SEQ ID NO:119 encoded by SEQ ID NO:52), MmcE (SEQ ID NO:120 encoded by SEQ ID NO:53), MmcF (SEQ ID NO:121 encoded by SEQ ID NO:54), MmcG (SEQ ID NO:122 encoded by SEQ ID NO:55), MmcH (SEQ ID NO:123 encoded by SEQ ID NO:56), Mmcl (SEQ ID NO:124 encoded by SEQ ID NO:57), MmcJ (SEQ ID NO:125 encoded by SEQ ID NO:58), MmcK (SEQ ID NO:126 encoded by SEQ ID NO:59), MmcL (SEQ ID NO:127 encoded by SEQ ID NO:60), MmcM (SEQ ID NO:128 encoded by SEQ ID NO:61), MmcN (SEQ ID NO:129 encoded by SEQ ID NO:62), MmcO (SEQ ID NO:130 encoded by SEQ ID NO:63), MmcP (SEQ ID NO:131 encoded by SEQ ID NO:64), MmcQ (SEQ ID NO:132 encoded by SEQ ID NO:65), MmcR (SEQ ID NO:133 encoded by SEQ ID NO:66), MmcS (SEQ ID NO:134 encoded by SEQ ID NO:67), MmcT (SEQ ID NO:135 encoded by SEQ ID NO:68), MmcU (SEQ ID NO:136 encoded by SEQ ID NO:69), MmcV (SEQ ID NO:137 encoded by SEQ ID NO:70), MmcW (SEQ ID NO:138 encoded by SEQ ID NO:71), MmcX (SEQ ID NO:139 encoded by SEQ ID NO:72), MmcY (SEQ ID NO:141 encoded by SEQ ID NO:73), Mct (SEQ ID NO:117 encoded by SEQ ID NO:16), a variant or a fragment thereof, e.g., a nucleic acid molecule that hybridizes under moderate, or more preferably stringent, hybridization conditions to at least one of the nucleic acid sequences identified hereinabove. [0016]
  • The invention further provides an isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, a variant or a fragment thereof. Preferably, the nucleic acid molecule of this embodiment of the invention comprises at least one, preferably at least five, and more preferably at least nine, open reading frames. More preferably, the nucleic acid molecule hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:74, or a portion thereof. [0017]
  • The invention also provides an isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes sugar biosynthetic enzymes, a variant or a fragment thereof. Preferably, the nucleic acid molecule of this embodiment of the invention comprises at least one, preferably at least five, more preferably at least nine, and even more preferably at least twelve, open reading frames. Preferably, the nucleic acid molecule of this embodiment of the invention hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:75, or a portion thereof. [0018]
  • The invention also provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to a polypeptide having an amino acid sequence encoded by SEQ ID NO:76, or a fragment thereof. A preferred variant polypeptide includes a variant polypeptide or fragment thereof having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO: 10-12, 17 or 100-141. Thus, for example, the activity of a polypeptide having SEQ ID NO:98 can be compared to a variant of SEQ ID NO:98 having at least one amino acid substitution, insertion, or deletion relative to SEQ ID NO:98. [0019]
  • A variant nucleic acid sequence of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising SEQ ID NO:76, or a fragment thereof. The amino acid and/or nucleic acid similarity (or homology) of two sequences may be determined manually or using algorithms well known to the art. [0020]
  • The invention also provides probes and primers comprising at least a portion of the nucleic acid molecules of the invention. The probes or primers of the invention are preferably detectably labeled or have a binding site for a detectable label. Preferably, the probes or primers of the invention are at least about 7, more preferably at least about 15, contiguous nucleotides bases having at least about 80% identity, more preferably at least about 90% identity, to the isolated nucleic acid molecules of the invention. Such probes or primers are useful to detect, quantify, isolate and/or amplify DNA strands with complementary to sequences related to the mitomycin biosynthetic gene cluster, sequences related to those encoding the polyketide biosynthetic enzymes linked to the mitomycin biosynthetic gene cluster, sequences related to those encoding sugar biosynthetic enzymes linked to the mitomycin biosynthetic gene cluster, a variant or a fragment thereof. [0021]
  • Also provided is an expression cassette comprising a nucleic acid molecule comprising at least a portion of a mitomycin biosynthetic gene cluster, a nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, a nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes sugar biosynthetic enzymes, a variant or fragment thereof, operably linked to a promoter functional in a host cell. Host cells that have been modified genetically, i.e., recombinant host cells, include host cells comprising an expression cassette, e.g., an expression cassette of the invention, or host cells in which the genome has been genetically manipulated, e.g., by deletion of a portion of, replacement of a portion of, or by disruption of, the host chromosome, so as to reduce or eliminate the expression of a particular mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention. [0022]
  • One embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which a portion of a nucleic acid sequence comprising the mitomycin gene cluster, i.e., the endogenous or native genomic sequence, is disrupted or replaced, for example, by an insertion with heterologous sequences or substituted with a variant nucleic acid sequence of the invention, preferably so as to result in altered mitomycin synthesis, such as an increase in mitomycin synthesis, and/or production of a novel compound. For example, the invention includes a recombinant host cell in which the mmcW gene is disrupted, for example, by replacement with a selectable marker gene, so as to yield a recombinant host cell having an increase in mitomycin production. [0023]
  • Another embodiment of the invention is a recombinant host cell, the genome of which is augmented by an expression cassette, e.g., via an extrachromosomal element such as a plasmid or by stable integration of the cassette into the host chromosome. Thus, the genome of the recombinant host cell is augmented with at least one mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention so as to yield an altered level of mitomycin and/or a novel compound(s) relative to the corresponding non-recombinant host cell. [0024]
  • Alternatively, the genome of a recombinant host cell is augmented with a non-mitomycin biosynthetic gene and, optionally, at least one mitomycin biosynthetic gene, polyketide biosynthetic gene or a sugar biosynthetic gene of the invention so as to yield an altered level of mitomycin and/or a novel compound(s) relative to the corresponding non-recombinant host cell. For example, the recombinant host cell may be augmented with pikA (see U.S. application Ser. No. 09/105,537, filed Jun. 26, 1998, the disclosure of which is incorporated by reference herein) and pikA expressed in an amount effective to yield a novel compound(s). [0025]
  • Host cells useful to prepare the recombinant host cells of the invention include cells which do not express or do not comprise nucleic acid corresponding to the nucleic acid molecules of the invention, e.g., mitomycin biosynthetic genes, as well as cells which naturally produce mitomycin. [0026]
  • Thus, the invention also provides isolated and purified polypeptides encoded by a nucleic acid molecule of the invention. Preferably, the polypeptide of the invention is obtained from recombinant host cells, e.g., the genome of which is augmented by a nucleic acid molecule of the invention. In addition, expression cassettes and host cells comprising antisense sequences of at least a portion of the mitomycin biosynthetic gene cluster of the invention are envisioned. [0027]
  • In another embodiment of the invention, the isolated and purified nucleic acid molecule which is linked to a mitomycin biosynthetic gene cluster and which encodes polyketide biosynthetic enzymes, e.g., a polyketide synthase, is useful in methods to prepare recombinant polyhydroxyalkanoate monomer synthases and polymers. [0028]
  • Thus, the present invention provides a method of preparing a polyhydroxyalkanoate synthase. The method comprises introducing an expression cassette into a host cell. The expression cassette comprises a DNA molecule encoding a polyketide synthase, operably linked to a promoter functional in the host cell. The DNA molecule is preferably obtained from a mitomycin-producing organism, e.g., a Streptomyces spp. such as [0029] S. lavendulae. The DNA molecule encoding the polyketide synthase is then expressed in the cell. Thus, another embodiment of the invention provides a purified recombinant polyketide isolated from a host cell which expresses the synthase.
  • Another embodiment of the invention is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first expression cassette and a second expression cassette into a host cell. The first expression cassette comprises a DNA segment encoding a fatty acid synthase in which the dehydrase activity has been inactivated that is operably linked to a promoter functional in the host cell, e.g., an insect cell. The inactivation preferably is via a mutation in the catalytic site of the dehydrase. The second expression cassette comprises a DNA segment encoding a polyketide synthase that is preferably obtained from a mitomycin-producing organism operably linked to a promoter functional in the host cell. The expression cassettes may be on the same or separate molecules. The DNA segments in the expression cassettes are expressed in the cell so as to yield a polyhydroxyalkanoate polymer. [0030]
  • The present invention also provides an expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell. The nucleic acid molecule comprises a plurality of DNA segments. Thus, the nucleic acid molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module, wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. No more than one DNA segment is derived from the eryA gene cluster of [0031] Saccharopolyspora erythraea. It is also preferred that the first DNA segment comprises a module from a mitomycin-producing organism, e.g., Streptomyces spp. The nucleic acid molecule may optionally further comprise a third DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second nucleic acid molecule encoding a polyhydroxyalkanoate synthase may be introduced into the host cell.
  • Also provided is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. Thus, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module. Together the DNA segments encode a recombinant polyhydroxyalkanoate monomer synthase. It is preferred that no more than one DNA segment is derived from the eryA gene cluster of [0032] Saccharopolyspora erythraea. Also, it is preferred that no more than one module is derived from the gene cluster from Streptomyces hygroscopicus that encodes rapamycin or the gene cluster that encodes spiramycin. A preferred embodiment of the invention employs a first DNA segment comprising a module from a mitomycin-producing organism. A further preferred embodiment of the isolated DNA molecule of the invention includes a DNA segment encoding a polyhydroxyalkanoate synthase.
  • Further provided is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first DNA molecule and a second DNA molecule into a host cell. The first DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase. The recombinant polyhydroxyalkanoate monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first module and a second module. The first DNA molecule is operably linked to a promoter functional in a host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. It is preferred that at least one module is from a mitomycin-producing organism. The DNAs encoding the recombinant polyhydroxyalkanoate monomer synthase and polyhydroxyalkanoate synthase are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer. [0033]
  • Yet another embodiment of the invention is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. That is, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a fatty acid synthase and the second DNA segment encodes a module of a polyketide synthase. A preferred embodiment of the invention employs a second DNA segment comprising a module of a polyketide synthase from a mitomycin-producing organism such as Streptomyces. [0034]
  • Also provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette into a host cell. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first and second module which together encode the monomer synthase. A preferred embodiment of the invention employs a module from a mitomycin-producing organism. Optionally, the expression cassette further comprises a second DNA molecule encoding a polyhydroxyalkanoate synthase. [0035]
  • The invention also provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase. Preferably, at least one DNA segment is derived from DNA which is linked to the mitomycin gene cluster of [0036] S. lavendulae. Also preferably, no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. In one embodiment of the invention, the 3′ most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II. Also provided is an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.
  • Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. Preferably, the second DNA molecule is derived from DNA which is linked to the mitomycin gene cluster. The recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from DNA which is linked to a mitomycin gene cluster, e.g., the mitomycin gene cluster of [0037] S. lavendulae. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer. Optionally, a second DNA molecule may be introduced into the host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
  • Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the DNA which is linked to the mitomycin gene cluster of [0038] S. lavendulae. Such a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer. Thus, a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell. The first DNA segment is 5′ to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell. The first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein. The DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
  • Further provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell. The DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. At least one DNA segment is derived from DNA which is linked to the mitomycin gene cluster of [0039] S. lavendulae. The DNA molecule is expressed in the host cell. Optionally, the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.
  • Thus, the invention provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein at least one DNA segment is derived from the mit/mmc gene cluster of [0040] S. lavendulae. Preferably, no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. In one embodiment of the invention, the 3′ most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II. Also provided is an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.
  • Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from the mit/mmc gene cluster of [0041] S. lavendulae. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer. Optionally, a a second DNA molecule may be introduced into the host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.
  • Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the mit/mmc gene cluster of [0042] S. lavendulae. Such a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer. Thus, a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell. The first DNA segment is 5′ to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell. The first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein. The DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.
  • Further provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell. The DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. At least one DNA segment is derived from the mit/mmc gene cluster of [0043] S. lavendulae. The DNA molecule is expressed in the host cell. Optionally, the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.
  • Also provided is a method for directing the biosynthesis of specific sugar-modified polyketides by genetic manipulation of a polyketide-producing microorganism. The method comprises introducing into a polyketide-producing microorganism a DNA sequence encoding enzymes in sugar biosynthesis, e.g., a DNA sequence comprising SEQ ID NO:75, a variant or fragment thereof, so as to yield a microorganism that produces specific sugar-modified polyketides. Alternatively, an anti-sense DNA sequence of the invention may be employed. Then the sugar-modified polyketides are isolated from the microorganism. It is preferred that the DNA sequence is modified so as to result in the inactivation of at least one enzymatic activity in sugar biosynthesis or in the attachment of the sugar to a polyketide [0044]
  • Thus, the modules encoded by the nucleic acid segments of the invention may be employed in the methods described hereinabove to prepare polyhydroxyalkanoates of varied chain length or having various side chain substitutions. [0045]
  • The compounds produced by the recombinant host cells of the invention are preferably biologically active agents such as antibiotics, anti-inflammatory agents, anti-cancer agents, antibiotics, immune-enhancers, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, or cholesterol-lowering agents; or as crop protection agents (e.g., fungicides or insecticides), as well as biopolymers, e.g., in packaging or biomedical applications, or to engineer PHA monomer synthases. Methods employing these compounds, e.g., to treat a mammal, e.g., a human, bird or fish in need of such therapy, are also envisioned. [0046]
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1. The biosynthetic pathway for mitomycin antibiotics. [0047]
  • FIG. 2. Organization of the mitomycin gene cluster. The deduced ORFs are drawn to scale, and their corresponding genes are marked in italics. The filled bars indicate the location of the mitomycin cluster. Abbreviations of the restriction enzymes: B: BamHI, S: SphI, P: PstI, E: EcoRI, X: XhoI, K: KpnI. [0048]
  • FIG. 3. The three SAM dependent methyltransferase conserved motifs can be found in MitM (SEQ ID NO:1), MitN (SEQ ID NO:2), and MmcR (SEQ ID NO:3). DmpM (SEQ ID NO:4; Kim et al., 1998), TcmN (SEQ ID NO:5; Shikano et al., 1998), ORF14 (SEQ ID NO:6; August et al., 1998), EryG (SEQ ID NO:7; Hardwick and Pelham, 1994) are O-methyltransferases for puromycin, tetracenomycin C, rifamycin, and erythromycin biosynthesis, respectively. Consen=consensus sequence (SEQ ID NO:8). [0049]
  • FIG. 4. Sequence similarity of MitM, MitN, and MmcR with other O-methyltransferases: DmpM (Kim et al., 1998), TcmN (Shikano et al., 1998), ORF14 (August et al., 1998), EryG (Hardwick and Pelham, 1994), RdmB (Mazodier et al., 1989), DnrK (Lee and Stock, 1996), and DauK (Devereux et al. 1984)); and C-methyltransferases: SMT (Schaferjohann et al., 1993), ESMT1 (Floss, 1997), SMT1 (Blattner et al., 1997), and SED6 (Guilfoile and Hutchinson, 1992)). The dendrogram was constructed with the program PILEUP (Denis and Brzezinki, 1992). [0050]
  • FIG. 5. MC genes and deduced enzyme functions. [0051]
  • FIG. 6. Bacterial strains and plasmids. Strains DH5α and DH5αF′ are available from Gibco BRL (Gaithersburg, Md.), ATCC 27643 and NRRL 2564 are available from the American Type Culture Collection, and strain S17-1 is described in Hidaka et al. (1995). Plasmids pNJ1, pUC119, pKC 1139, pDHS3001, pKN108, and pFD666 are described in Kuzuyama et al. (1995), Madduri et al. (1993), Boxer (1997), Kagan and Clarke (1994), Kim et al. (1998), and Coque et al. (1995), respectively. [0052]
  • FIG. 7. Biosynthetic pathway leading to mitomycin C. [0053]
  • FIG. 8. Southern hybridization and restriction-enzyme map of the mrd and rifK hybridizing regions from [0054] S. lavendulae. A) Southern hybridization with the rifK gene probe (Kim et al., 1998). Lane 1, A. mediterranei ATCC 27643 genomic DNA digested with BamHI; Lane 2, S. lavendulae NRRL 2564 genomic DNA digested with BamHI; B) Physical map showing the mitABC genes. The location of mrd and rifK hybridizing genes in cosmid pDHS7529 are indicated by solid bars. Enzymes: E, EcoRI; B, BamHI. The sequenced 3.8 kb BamHI fragment containing mitA, mitB, mitC is enlarged (wide arrows). Thin arrows below show sites of resistance gene integration for disruption experiments.
  • FIG. 9. Nucleotide sequence of the 3.8 kb DNA fragment containing mitABC (SEQ ID NO:9). The deduced gene products are indicated in the one-letter code under the DNA sequence (SEQ ID NO:10, MitA; SEQ ID NO:11, MitB; SEQ ID NO:12, MitC). Possible ribosome binding sites are marked in the boxed regions. The presumed translational start site and direction of transcription for each ORF is indicated by an arrow and marked accordingly. [0055]
  • FIG. 10. Alignment of MitA with three other AHBA synthases. The deduced amino acid sequence comparison from AHBAS genes derived from [0056] Streptomyces lavendulae (SEQ ID NO:10). Streptomyces collinus (Z54208; SEQ ID NO:13), Actinosynnema pretiosum (I39657; SEQ ID NO:14), and Amycolatopsis mediterranei (I39657; SEQ ID NO:15) is shown with the conserved lysine in the PLP-binding motif underlined.
  • FIG. 11. Southern blot analysis of the mitA mutant strain. A) Construction of mitA disruption mutant and restriction map of the wild-type and mitA disruption mutant showing expected band sizes in the Southern blot. Maps are not drawn to scale. B) [0057] S. lavendulae genomic DNA from wild-type (lanes 1 and 2) and double crossover mutant (lanes 3 and 4) were digested with BamHI (lane 1 and 3) and SphI (lane 2 and lane 4), respectively. The 4.9 kb EcoRI-HindIII fragment from pDHS2001 containing tsr-disrupted mitA was used as the probe.
  • FIG. 12. Southern blot analysis of mitB mutant MM101. A) Construction of mitB disruption mutant and restriction map of the wild-type and mitB disruption mutant showing the expected sites in the Southern blot. B) [0058] S. lavendulae genomic DNA from wild-type (lane 1 and 3) and mitB mutant (lane 2 and 4) were digested with BamHI (lane 1 and 2) and SacI (lane 3 and 4). DNA probe: 3.8 kb BamHI fragment insert from pDHS7601.
  • FIG. 13. Chemical analysis and biological activity of extracts from [0059] S. lavendulae wild-type and mutant strains. A) HPLC analysis of authentic mitomycin C standard, mitomycin C production in the wild-type S. lavendulae, mitA (AHBAS) and mitB (gtf) disruption mutants of S. lavendulae. One mg of crude extract injected, 1 μg of MC injected as standard. B) Bacillus subtilis bioassay of mitomycin C production in mitA disruption mutant strain of S. lavendulae. Filter discs: 1) 100 μg injection of wild-type—collected 12.5-13.5 minutes; 2) 100 μg injection of mitA (ahbas) disruption mutant—collected 12.5-13.5 minutes; 3) 100 μg injection of W. T. containing vector—collected 12.5-13.5 minutes; 4) One μg of mitomycin C collected from HPLC from 12.5-13.5 minutes; 5) Tris buffer negative control; 6) methanol solvent negative control.
  • FIG. 14. Strains and plasmids employed in Example 3. BL21 (DE3) and pET17b are available from Novagen (Madison, Wis.). pDH57006 is described in Sheldon et al. (1997). [0060]
  • FIG. 15. Genetic map showing the physical linkage of the mct and mrd genes within the MC biosynthetic gene cluster. The expanded box shows the line plot of the met ORF. [0061]
  • FIG. 16. The nucleotide sequence of mct (SEQ ID NO:16). The deduced amino acid sequence of mct is indicated under the nucleotide sequence with the one letter designation (SEQ ID NO:17). A conserved motif characteristic of 14 TMS proteins is boxed while the invariant beta-turn motif is denoted with a dashed underline. The putative ribosome binding site is marked with a solid underline. [0062]
  • FIG. 17. Dot matrix alignment of the deduced amino acid sequence of mct with other actinomycete antibiotic efflux proteins. Comparable parameters were utilized in generating the alignments. [0063]
  • FIG. 18. Hydropathy analysis of the deduced amino acid sequence of MC-translocase. A) Hydropathy plot obtained from prediction of Kyte and Doolittle (1982). B) Schematic representation of MC-translocase protein topology. The transmembrane spanning regions are marked (1-14). The initial and final amino acid positions of each transmembrane domain are indicated by small numbers. The relative position of positively (H, R, K) and negatively (D, Q) charged amino acids are indicated by a plus and minus, respectively. [0064]
  • FIG. 19. Creation of the mct disruption mutant. A) The chromosomal mct gene (black bar) was disrupted by inserting a neomycin resistance marker (shaded) within the gene. Following double crossover recombination, specific restriction bands are predicted to be shifted in the mct mutant genome compared to the wild-type strain. B) Southern blot analysis of the mct mutant. As expected, when probed with the 4.0 kb BamHI insert from pDHS7661, the 4.0 kb BamHI hybridization band in wild-type [0065] S. lavendulae was shifted to 5.4 kb in mct knockouts, while a 1.65 kb SacI hybridization band was shifted to 3.0 kb in size. Lane 1 and 5: wild-type genomic DNA digested with BamHI. Lane 2, 3, 4, and 6: Four double crossover colonies genomic DNA digested with BamHI. Lane 7:wild-type genomic DNA digested with SstI. Lane 8: double crossover clone 6 genomic DNA digested with SstI.
  • FIG. 20. MC uptake analysis of strains PJS100, PJS102, and PJS103. BL21(DE3)::pET17b vector control strain, (); strain PJS100, (▪); strain PJS102, (♦); strain PJS103, (×). [0066]
  • FIG. 21. Complete nucleotide sequence of the mitomycin gene cluster (SEQ ID NO:96). [0067]
  • FIG. 22. Complete nucleotide sequence of ORFs 1-9 (SEQ ID NO:74). [0068]
  • FIG. 23. Complete nucleotide sequence of ORFs 11-22 (SEQ ID NO:75). [0069]
  • FIG. 24. Codons for various amino acids. [0070]
  • FIG. 25. Exemplary amino acid substitutions. [0071]
  • FIG. 26. Complete nucleotide sequence of the mitomycin biosynthetic genes (SEQ ID NO:76).[0072]
    • DETAILED DESCRIPTION OF THE INVENTION
  • Definitions [0073]
  • As used herein, a “Type I polyketide synthase” is a single polypeptide with a single set of iteratively used active sites. This is in contrast to a Type II polyketide synthase which employs active sites on a series of polypeptides. [0074]
  • As used herein, a “linker region” is an amino acid sequence present in a multifunctional protein which is less well conserved in an amino acid sequence than an amino acid sequence with catalytic activity. [0075]
  • As used herein, an “extender unit” catalytic or enzymatic domain is an acyl transferase in a module that catalyzes chain elongation by adding 2-4 carbon units to an acyl chain and is located carboxy-terminal to another acyl transferase. For example, an extender unit with methylmalonylCoA specificity adds acyl groups to a methylmalonylCoA molecule. [0076]
  • As used herein, a “polyhydroxyalkanoate” or “PHA” polymer includes, but is not limited to, linked units of related, preferably heterologous, hydroxyalkanoates such as 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxycaproate, 3-hydroxyheptanoate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundecanoate, and 3-hydroxydodecanoate, and their 4-hydroxy and 5-hydroxy counterparts. [0077]
  • As used herein, a “recombinant” nucleic acid or protein molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been modified. [0078]
  • As used herein, a “multifunctional protein” is one where two or more enzymatic activities are present on a single polypeptide. [0079]
  • As used herein, a “module” is one of a series of repeated units in a multifunctional protein, such as a Type I polyketide synthase or a fatty acid synthase. [0080]
  • As used herein, a “premature termination product” is a product which is produced by a recombinant multifunctional protein which is different than the product produced by the non-recombinant multifunctional protein. In general, the product produced by the recombinant multifunctional protein has fewer acyl groups. [0081]
  • As used herein, a DNA that is “derived from” a gene cluster is a DNA that has been isolated and purified in vitro from genomic DNA, or synthetically prepared on the basis of the sequence of genomic DNA. [0082]
  • An “antibiotic” as used herein is a substance produced by a microorganism which, either naturally or with limited chemical modification, will inhibit the growth of or kill another microorganism or eukaryotic cell. [0083]
  • An “antibiotic biosynthetic gene” is a nucleic acid, e.g., DNA, segrnent or sequence that encodes an enzymatic activity which is necessary for an enzymatic reaction in the process of converting primary metabolites into antibiotics. [0084]
  • An “antibiotic biosynthetic pathway” includes the entire set of antibiotic biosynthetic genes necessary for the process of converting primary metabolites into antibiotics. These genes can be isolated by methods well known to the art, e.g., see U.S. Pat. No. 4,935,340. [0085]
  • Antibiotic-producing organisms include any organism, including, but not limited to, Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicilliurn, Nocardia, and Streptomyces, which either produces an antibiotic or contains genes which, if expressed, would produce an antibiotic. [0086]
  • The term “polyketide” as used herein refers to a large and diverse class of natural products, including but not limited to antibiotic, antifungal, anticancer, and anti-helminthic compounds. [0087]
  • The term “polyketide-associated sugar” refers to a sugar that is known to attach to polyketides or that can be attached to polyketides by the processes described herein. [0088]
  • The term “sugar derivative” refers to a sugar which is naturally associated with a polyketide but which is altered relative to the unmodified or native. [0089]
  • The term “sugar intermediate” refers to an intermediate compound produced in a sugar biosynthesis pathway. [0090]
  • A “recombinant” host cell of the invention has a genome that has been manipulated in vitro so as to alter, e.g., decrease or disrupt, or, alternatively, increase, the function or activity of at least one gene, e.g., in the mitomycin biosynthetic gene cluster, of the invention. [0091]
  • As used herein, the “mit/mmc” or “mitomycin” gene cluster includes sequences encoding enzymes for mitosane precursor formation, mitosane ring assembly, regulation of mitomycin biosynthesis, functionalization, and resistance to mitomycin, as well as closely linked sequences encoding polyketide and sugar biosynthetic enzyes. [0092]
  • As used herein, the terms “isolated and/or purified” refer to in vitro isolation of a RNA, DNA or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, such as nucleic acid or polypeptide, so that is can be sequenced, replicated and/or expressed. Moreover, the nucleic acid may encode more than one polypeptide. For example, “an isolated DNA molecule encoding an AUBA synthase” is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more sequential nucleotide bases that preferably encode a biologically active polypeptide, or a fragment or variant thereof, that is complementary to the non-coding, or complementary to the coding strand, of an AHBA synthase RNA, or hybridizes to the RNA or DNA encoding the AHBA synthase and remains stably bound under low, moderate, or stringent conditions, as defined by methods well known to the art, e.g., in Sambrook et al., supra. [0093]
  • The term “polyketide-producing microorganism” as used herein includes any microorganism that can produce a polyketide naturally or after being suitably engineered (i.e., genetically). Examples of actinomycetes that naturally produce polyketides include but are not limited to [0094] Micromonospora rosaria, Micromonospora megalomicea, Saccharopolyspora erythraea, Streptomyces antibioticus, Streptomyces albereticuli, Streptomyces ambofaciens, Streptomyces avermitilis, Streptomycesfradiae, Streptomyces griseus, Streptomyces hydroscopicus, Streptomyces tsukulubaensis, Streptomyces mycarofasciens, Streptomyces platenesis, Streptomyces violaceoniger, Streptomyces violaceoniger, Streptomyces thermotolerans, Streptomyces rimosus, Streptomyces peucetius, Streptomyces coelicolor, Streptomyces glaucescens, Streptomyces roseofulvus, Streptomyces cinnamonensis, Streptomyces curacoi, and Amycolatopsis mediterranei (see Hopwood, D. A. and Sherman, D. H., Annu. Rev. Genet., 24:37-66 (1990), incorporated herein by reference). Other examples of polyketide-producing microorganisms that produce polyketides naturally include various Actinomadura, Dactylosporangium and Nocardia strains.
  • The term “glycosylated polyketide” refers to any polyketide that contains one or more sugar residues. [0095]
  • The term “glycosylation-modified polyketide” refers to a polyketide having a changed glycosylation pattern or configuration relative to that particular polyketide's unmodified or native state. [0096]
  • The term “sugar biosynthesis genes” as used herein refers to nucleic acid sequences from organisms such as [0097] S. lavendulae that encode sugar biosynthesis enzymes and is intended to include sequences of DNA from other polyketide-producing microorganisms which are identical or analogous to those obtained from S. lavendulae.
  • The term “sugar biosynthesis enzymes” as used herein refers to polypeptides which are involved in the biosynthesis and/or attachment of polyketide-associated sugars and their derivatives and intermediates. [0098]
  • An antibiotic resistance-conferring gene is a nucleic acid segment that encodes an enzymatic or other activity which alone or in combination with other gene products, confers resistance to an antibiotic. [0099]
  • As used herein, “mitomycin” includes, but is not limited to, mitomycin A, mitomycin B, mitomycin C, porfiromycin, mitiromycin, mitomycin D, mitomycin E, mitomycin F, mitomycin G, mitomycin H, mitomycin I, mitomycin J, mitomycin L, mitomycin M, mitomycin K, albomitomycin A, isomitomycin A, KW2149, KW2149 metabolites such as M-16 and M-18, FR66979, FK973, FK317, and FR900482, as well as structural or functional equivalents thereof (“analogs”), or derivatives thereof. [0100]
  • As used herein, the term “derivative” means that a particular compound produced by a host cell of the invention or prepared in vitro using polypeptides encoded by the nucleic acid molecules of the invention, is modified so that it comprises other moieties, e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.g., gamma emitters, small chemicals, metals, salts, synthetic polymers, e.g., polylactide and polyglycolide, surfactants and glycosaminoglycans, which are covalently or non-covalently attached or linked to the compound. [0101]
  • It will be appreciated by those skilled in the art that each atom of the compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically active, polymorphic or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art. [0102]
  • The term “sequence homology” or “sequence identity” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred. When using oligonucleotides as probes, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and more preferably not less than 19 matches out of 20 possible base pair matches (95%). [0103]
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, 1972, [0104] volume 5, National Biomedical Research Foundation, pp. 101-101, and Supplement 2 to this volume, pp. 1-10. The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
  • The following terms are used to describe the sequence relationships between two or more polynucleotides: “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity”, and “substantial identity”. A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. [0105]
  • A “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) [0106] Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
  • The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. [0107]
  • As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 80 percent sequence identity, preferably at least about 90 percent sequence identity, more preferably at least about 95 percent sequence identity, and most preferably at least about 99 percent sequence identity. [0108]
  • In accordance with the present invention, there is provided a purified and isolated nucleic acid molecule which encodes the entire pathway for the biosynthesis of mitomycin, as well as polyketide biosynthetic and sugar biosynthetic genes that are linked to the mitomycin biosynthetic genes. Desirably, the nucleic acid molecule is a DNA isolated from Streptomyces spp. The present invention further includes isolated and purified DNA sequences which hybridize under standard or stringent conditions to the the nucleic acid molecules of the invention. It should be understood to those skilled in the art that the invention also encompasses the purified and isolated polypeptides which may be encoded by the sequences of the nucleic acid molecules of this invention. [0109]
  • The invention described herein can be used for the production of mitomycin, analogs or derivatives thereof, or novel compounds. Commercial chemical syntheses of mitomycin are not feasible. The gene cluster described herein contains all the genes required for the production of the mitosane group of antibiotics, compounds which are clinically prescribed antitumor compounds employed in the treatment of a wide variety of cancers including non-small cell lung cancer, metastatic breast cancer, esophageal, gastric, pancreatic, and anal canal carcinomas. Thus, the isolation and characterization of this gene cluster allows for the selective production of mitomycin antibiotics, the overproduction or under production of particular compounds, e.g., overproduction of certain mitomycin antibiotics, and the production of novel compounds, e.g., mitomycin-derived compounds as well as the production of novel non-mitomycin related compounds. For example, combinational biosynthetic-based modification of mitomycin antibiotics may be accomplished by selective activation or disruption of specific genes within the cluster or incorporation of the genes into biased biosynthetic libraries which are assayed for a wide range of biological activities, to derive greater chemical diversity in the mitomycins. A further example includes the introduction of a mitomycin biosynthetic gene(s) into a particular host cell so as to result in the production of a novel non-mitomycin related compound due to the activity of the mitomycin biosynthetic gene(s) on other metabolites, intermediates or components of the host cells. The in vitro expression of polypeptides from this gene cluster also provides an enzymatic route to the production of known mitomycin compounds that are produced in low quantities, or conversion of currently available mitomycins to other known or novel mitomycins, e.g., the bioconversion of mitomycin C to porfiromycin. [0110]
  • The mitomycin resistance genes may also be used to provide higher mitomycin resistance to cancer patients undergoing treatment and for clonal selection purposes (e.g., using mrd). For example, the resistance gene(s) may be inserted into human bone marrow cell lines to confer higher resistance to non-cancerous cells, thus allowing higher doses of mitomycins to be administered to cancer patients. Moreover, because mitomycin acts directly upon DNA itself, its toxicity is extremely broad, and therefore the resistance genes could be used for efficient selection in prokaryotes, fungi, plants, mammalian cell culture, and insect cell culture. Further, the regulatory resistance and transport genes may be used to create higher producing strains capable of synthesizing more mitomycin than can currently be obtained through traditional fermentation strategies. [0111]
  • In addition, the invention described herein can be used for the production of novel compounds which include a diverse range of biodegradable PHA polymers through genetic redesign of DNA such as that found in Streptomyces spp. Different PHA synthases can then be tested for their ability to polymerize the monomers produced by the recombinant PHA synthase into a biodegradable polymer. PHA synthases can be tested for their specificity with respect to different monomer substrates by methods well known to the art. [0112]
  • The potential uses and applications of PHAs produced by PHA monomer synthases and PHA synthases include both medical and industrial applications. Medical applications of PHAs include surgical pins, sutures, staples, swabs, wound dressings, blood vessel replacements, bone replacements and plates, stimulation of bone growth by piezoelectric properties, and biodegradable carrier for long-term dosage of pharmaceuticals. Industrial applications of PHAs include disposable items such as baby diapers, packaging containers, bottles, wrappings, bags, and films, and biodegradable carriers for long-term dosage of herbicides, fungicides, insecticides, or fertilizers. [0113]
  • In animals, the biosynthesis of fatty acids de novo from malonyl-CoA is catalyzed by FAS. For example, the rat FAS is a homodimer with a subunit structure consisting of 2505 amino acid residues having a molecular weight of 272,340 Da. Each subunit consists of seven catalytic activities in separate physical domains (Amy et al., [0114] Proc. Natl. Acad. Sci. USA, 86, 3114 (1989)). The physical location of six of the catalytic activities, ketoacyl synthase (KS), malonyl/acetyltransferase (M/AT), enoyl reductase (ER), ketoreductase (KR), acyl carrier protein (ACP), and thioesterase (TE), has been established by (1) the identification of the various active site residues within the overall amino acid sequence by isolation of catalytically active fragments from limited proteolytic digests of the whole FAS, (2) the identification of regions within the FAS that exhibit sequence similarity with various monofunctional proteins, (3) expression of DNA encoding an amino acid sequence with catalytic activity to produce recombinant proteins, and (4) the identification of DNA that does not encode catalytic activity, i.e., DNA encoding a linker region. (Smith et al., Proc. Natl. Acad. Sci. USA, 73, 1184 (1976); Tsukamoto et al., J. Biol. Chem., 263, 16225 (1988); Rangan et al., J. Biol. Chem., 266, 19180 (1991)).
  • The seventh catalytic activity, dehydrase (DH), was identified as physically residing between AT and ER by an amino acid comparison of FAS with the amino acid sequences encoded by the three open reading frames of the eryA polyketide synthase (PKS) gene cluster of [0115] Saccharopolyspora erythraea. The three polypeptides that comprise this PKS are constructed from “modules” which resemble animal FAS, both in terms of their amino acid sequence and in the ordering of the constituent domains (Donadio et al., Gene, 111, 51 (1992); Benh et al., Eur. J. Biochem., 204, 39 (1992)).
  • One embodiment of the invention employs a FAS in which the DH is inactivated (FAS DH-). The FAS DH-employed in this embodiment of the invention is preferably a eukaryotic FAS DH- and, more preferably, a mammalian FAS DH-. The most preferred embodiment of the invention is a FAS where the active site in the DH has been inactivated by mutation. For example, Joshi et al. ([0116] J. Biol. Chem., 268, 22508 (1993)) changed the His878 residue in the rat FAS to an alanine residue by site-directed mutagenesis. In vitro studies showed that a FAS with this change (ratFAS206) produced 3-hydroxybutyrylCoA as a premature termination product from acetyl-CoA, malonyl-CoA and NADPH.
  • A FAS DH-effectively replaces the β-ketothiolase and acetoacetyl-CoA reductase activities of the natural pathway by producing D(−)-3-hydroxybutyrate as a premature termination product, rather than the usual 16-carbon product, palmitic acid. This premature termination product can then be incorporated into PHB by a PHB synthase. [0117]
  • Another embodiment of the invention employs a recombinant Streptomyces spp. PKS to produce a variety of β-hydroxyCoA esters that can serve as monomers for a PHA synthase. One example of a DNA encoding a Type I PKS is the eryA gene cluster, which governs the synthesis of erythromycin aglycone deoxyerythronolide B (DEB). The gene cluster encodes six repeated units, termed modules or synthase units (SUs). Each module or SU, which comprises a series of putative FAS-like activities, is responsible for one of the six elongation cycles required for DEB formation. Thus, the processive synthesis of asymmetric acyl chains found in complex polyketides is accomplished through the use of a programmed protein template, where the nature of the chemical reactions occurring at each point is determined by the specificities in each SU. [0118]
  • Two other Type I PKS are encoded by the tyl (tylosin) and met (methymycin) gene clusters (see U.S. application Ser. No. 09/108,537, the disclosure of which is incorporated by reference herein). The macrolide multifunctional synthases encoded by tyl and met provide a greater degree of metabolic diversity than that found in the eryA gene cluster. The PKSs encoded by the eryA gene cluster only catalyze chain elongation with methylmalonylCoA, as opposed to tyl and met PKSs, which catalyze chain elongation with malonylCoA, methylmalonylCoA and ethylmalonylCoA. Specifically, the tyl PKS includes two malonylCoA extender units and one ethylmalonylCoA extender unit, and the met PKS includes one malonylCoA extender unit. [0119]
  • In order to manipulate the catalytic specificities within each module, DNA encoding a catalytic activity must remain undisturbed. To identify the amino acid sequences between the amino acid sequences with catalytic activity, the “linker regions,” amino acid sequences of related modules, preferably those encoded by more than one gene cluster, are compared. Linker regions are amino acid sequences which are less well conserved than amino acid sequences with catalytic activity. Witkowski et al., [0120] Eur. J. Biochem., 198, 571 (1991).
  • In an alternative embodiment of the invention, to provide a DNA encoding a Type I PKS module with a TE and lacking a functional DH, a DNA encoding a module F, containing KS, MT, KR, ACP, and TE catalytic activities, is introduced at the 3′ end of a DNA encoding a first module. Module F introduces the final (R)-3-hydroxyl acyl group at the final step of PHA monomer synthesis, as a result of the presence of a TE domain. DNA encoding a module F is not present in the eryA PKS gene cluster (Donadio et al., supra, 1991). [0121]
  • A DNA encoding a recombinant monomer synthase is inserted into an expression vector. The expression vector employed varies depending on the host cell to be transformed with the expression vector. That is, vectors are employed with transcription, translation and/or post-translational signals, such as targeting signals, necessary for efficient expression of the genes in various host cells into which the vectors are introduced. Such vectors are constructed and transformed into host cells by methods well known in the art. See Sambrook et al., [0122] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor (1989). Preferred host cells for the vectors of the invention include insect, bacterial, and plant cells. Preferred insect cells include Spodoptera frugiperda cells such as Sf21, and Trichoplusia ni cells. Preferred bacterial cells include Escherichia coli, Streptomyces and Pseudomonas. Preferred plant cells include monocot and dicot cells, such as maize, rice, wheat, tobacco, legumes, carrot, squash, canola, soybean, potato, and the like.
  • Moreover, the appropriate subcellular compartment in which to locate the enzyme in eukaryotic cells must be considered when constructing eukaryotic expression vectors. Two factors are important: the site of production of the acetyl-CoA substrate, and the available space for storage of the PHA polymer. To direct the enzyme to a particular subcellular location, targeting sequences may be added to the sequences encoding the recombinant molecules. [0123]
  • The baculovirus system is particularly amenable to the introduction of DNA encoding a recombinant FAS or a PKS monomer synthase because an increasing variety of transfer plasmids are becoming available which can accommodate a large insert, and the virus can be propagated to high titers. Moreover, insect cells are adapted readily to suspension culture, facilitating relatively large-scale recombinant protein production. Further, recombinant proteins tend to be produced exclusively as soluble proteins in insect cells, thus, obviating the need for refolding, a task that might be particularly daunting in the case of a large multifunctional protein. The Sf21 /baculovirus system has routinely expressed milligram quantities of catalytically active recombinant fatty acid synthase. Finally, the baculovirus/insect cell system provides the ability to construct and analyze different synthase proteins for the ability to polymerize monomers into unique biodegradable polymers. [0124]
  • A further embodiment of the invention is the introduction of at least one DNA encoding a PHA synthase and a DNA encoding a PHA monomer synthase into a host cell. Such synthases include, but are not limited to, [0125] A. eutrophus 3-hydroxy, 4-hydroxy, and 5-hydroxy alkanoate synthases, Rhodococcus ruber C3-C5 hydroxyalkanoate synthases, Pseudomonas oleororans C6-C14 hydroxyalkanoate synthases, P. putida C6-C14 hydroxyalkanoate synthases, P. aeruginosa C5-C10 hydroxyalkanoate synthases, P. resinovorans C4-C10 hydroxyalkanoate synthases, Rhodospirillum rubrum C4-C7 hydroxyalkanoate syntheses, R. gelatinorus C4-C7 , Thiocapsa pfennigii C4-C8 hydroxyalkanoate synthases, and Bacillus megaterium C4-C5 hydroxyalkanoate synthases.
  • The introduction of DNA(s) encoding more than one PHA synthase may be necessary to produce a particular PHA polymer due to the specificities exhibited by different PHA synthases. As multifunctional proteins are altered to produce unusual monomeric structures, synthase specificity may be problematic for particular substrates. Although the [0126] A. eutrophus PHB synthase utilizes only C4 and C5 compounds as substrates, it appears to be a good prototype synthase for initial studies since it is known to be capable of producing copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate (Kunioka et al., Macromolecules, 22, 694 (1989)) as well as copolymers of 3-hydroxyvalerate, 3-hydroxybutyrate, and 5-hydroxyvalerate (Doi et al., Macromolecules, 19, 2860 (1986)). Other synthases, especially those of Pseudomonas aeruginosa (Timm et al., Eur. J. Biochem., 209, 15 (1992)) and Rhodococcus ruber (Pieper et al., FEMS Microbiol. Lett., 96, 73 (1992)), can also be employed in the practice of the invention. Synthase specificity may be alterable through molecular biological methods.
  • In yet another embodiment of the invention, a DNA encoding a FAS and a PHA synthase can be introduced into a single expression vector, obviating the need to introduce the genes into a host cell individually. [0127]
  • A further embodiment of the invention is the generation of a DNA encoding a recombinant multifunctional protein, which comprises a FAS, of either eukaryotic or prokaryotic origin, and a PKS module F. Module F will carry out the final chain extension to include two additional carbons and the reduction of the β-keto group, which results in a (R)-3-hydroxy acyl CoA moiety. [0128]
  • To produce this recombinant protein, DNA encoding the FAS TE is replaced with a DNA encoding a linker region which is normally found in the ACP-KS interdomain region of bimodular ORFs. DNA encoding a module F is then inserted 3′ to the DNA encoding the linker region. Different linker regions, such as those described below which vary in length and amino acid composition, can be tested to determine which linker most efficiently mediates or allows the required transfer of the nascent saturated fatty acid intermediate to module F for the final chain elongation and keto reduction steps. The resulting DNA encoding the protein can then be tested for expression of long-chain β-hydroxy fatty acids in insect cells, such as Sf21 cells, or Streptomyces, or Pseudomonas. The expected 3-hydroxy C-18 fatty acid can serve as a potential substrate for PHA synthases which are able to accept long-chain alkyl groups. A preferred embodiment of the invention is a FAS that has a chain length specificity between 4-22 carbons. [0129]
  • Examples of linker regions that can be employed in this embodiment of the invention include, but are not limited to, the ACP-KS linker regions encoded by the tyl ORFI (ACP[0130] 1-KS2; ACP2-KS3), and ORF3 (ACP5-KS6), and eryA ORFI (ACP1-KS1; ACP2-KS2), ORF2 (ACP3-KS4) and ORF3 (ACP5-KS6).
  • This approach can also be used to produce shorter chain fatty acid groups by limiting the ability of the FAS unit to generate long-chain fatty acids. Mutagenesis of DNA encoding various FAS catalytic activities, starting with the KS, may result in the synthesis of short-chain (R)-3-hydroxy fatty acids. [0131]
  • The PHA polymers are then recovered from the biomass. Large-scale solvent extraction can be used, but is expensive. An alternative method involving heat shock with subsequent enzymatic and detergent digestive processes is also available (Byron, [0132] Trends Biotechnical, 5, 246 (1987); Holmes, In: Developments in Crystalline Polymers, D. C. Bassett (ed.), pp. 1-65 (1988)). PHB and other PHAs are readily extracted from microorganisms by chlorinated hydrocarbons. Refluxing with chloroform has been extensively used; the resulting solution is filtered to remove debris and concentrated, and the polymer is precipitated with methanol or ethanol, leaving low-molecular-weight lipids in solution. Longer side-chain PHAs show a less restricted solubility than PHB and are, for example, soluble in acetone. Other strategies adopted include the use of ethylene carbonate and propylene carbonate as disclosed by Lafferty et al. (Chem. Rundschau, 30, 14 (1977)) to extract PHB from biomass. Scandola et al. (Int. J. Biol. Microbiol., 10, 373 (1988)) reported that 1 M HCl-chloroform extraction of Rhizobium meliloti yielded PHB of Mw=6×104 compared with 1.4×106 when acetone was used.
  • Methods are well known in the art for the determination of the PHB or PHA content of microorganisms, the composition of PHAs, and the distribution of the monomer units in the polymer. Gas chromatography and high-pressure liquid chromatography are widely used for quantitative PHB analysis. See Anderson et al., [0133] Microbiol. Rev., 54, 450 (1990) for a review of such methods. NMR techniques can also be used to determine polymer composition, and the distribution of monomer units.
  • Variants of the Nucleic Acid Molecules of the Invention [0134]
  • The present invention contemplates nucleic acid sequences which hybridize under low, medium or high stringency hybridization conditions to the exemplified nucleic acid sequences set forth herein. Hybridization conditions are well known in the art. Thus, nucleic acid sequences encoding variant polypeptides, i.e., those having at least one amino acid substitution, insertion, addition or deletion, or nucleic acid sequences having conservative (e.g., silent) nucleotide substitutions (see FIGS. [0135] 24-25), are within the scope of the invention. Preferably, variant polypeptides encoded by the nucleic acid sequences of the invention are biologically active. The present invention also contemplates naturally occurring allelic variations and mutations of the nucleic acid sequences described herein.
  • As is well known in the art, because of the degeneracy of the genetic code, there are numerous other DNA and RNA molecules that can code for the same polypeptides as those encoded by the exemplified biosynthetic genes and fragments thereof. The present invention, therefore, contemplates those other DNA and RNA molecules which, on expression, encode the polypeptides of, for example, portions of SEQ ID NO:96. Having identified the amino acid residue sequence encoded by a mitomycin, sugar or polyketide biosynthetic gene, and with knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein and, which molecules are characterized simply by a change in a codon for a particular amino acid, are within the scope of this invention. [0136]
  • The 20 common amino acids and their representative abbreviations, symbols and codons are well known in the art (see, for example, [0137] Molecular Biology of the Cell, Second Edition, B. Alberts et al., Garland Publishing Inc., New York and London, 1989). As is also well known in the art, codons constitute triplet sequences of nucleotides in mRNA molecules and as such, are characterized by the base uracil (U) in place of base thymidine (T) which is present in DNA molecules. A simple change in a codon for the same amino acid residue within a polynucleotide will not change the structure of the encoded polypeptide. By way of example, it can be seen from SEQ ID NO:16 that a TCA codon for serine exists at nucleotide positions 146-148. However, serine can be encoded by a TCT codon, and a TCC codon. Substitution of the latter codons for serine with the TCA codon for serine or vice versa, does not substantially alter the DNA sequence of SEQ ID NO:16 and results in production of the same polypeptide. In a similar manner, substitutions of the recited codons with other equivalent codons can be made in a like manner without departing from the scope of the present invention.
  • A nucleic acid molecule, segment or sequence of the present invention can also be an RNA molecule, segment or sequence. An RNA molecule contemplated by the present invention corresponds to, is complementary to or hybridizes under low, medium or high stringency conditions to, any of the DNA sequences set forth herein. Exemplary and preferred RNA molecules are mRNA molecules that comprise at least one mitomycin, sugar or polyketide biosynthetic gene of this invention. [0138]
  • Mutations can be made to the native nucleic acid sequences of the invention and such mutants used in place of the native sequence, so long as the mutants are able to function with other sequences to collectively catalyze the synthesis of an identifiable sugar, polyketide or mitomycin. Such mutations can be made to the native sequences using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene using restriction endonuclease digestion. (See, e.g., Kunkel, T. A. [0139] Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. BioTechniques (1987) 5:786.) Alternatively, the mutations can be effected using a mismatched primer (generally 10-30 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. Zoller and Smith, Methods Enzymol., (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409. PCR mutagenesis will also find use for effecting the desired mutations.
  • Random mutagenesis of the nucleotide sequence can be accomplished by several different techniques known in the art, such as by altering sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants or by damaging plasmid DNA in vitro with chemicals. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formnic acid, analogues of nucleotide precursors such as nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNA fragments are treated with chemicals, transformed into [0140] E. coli and propagated as a pool or library of mutant plasmids.
  • Large populations of random enzyme variants can be constructed in vivo using “recombination-enhanced mutagenesis.” This method employs two or more pools of, for example, 10[0141] 6 mutants each of the wild-type encoding nucleotide sequence that are generated using any convenient mutagenesis technique and then inserted into cloning vectors.
  • Chimeric Expression Cassettes, Vectors and Host Cells of the Invention [0142]
  • As used herein, “chimeric” means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the “native” or wild type of the species. The recombinant DNA sequence or segment, used for transformation herein, may be circular or linear, double-stranded or single-stranded. Generally, the DNA sequence or segment is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by control sequences which promote the expression of the DNA present in the resultant transformed (recombinant) host cell. Aside from DNA sequences that serve as transcription units for the nucleic acid molecules of the invention or portions thereof, a portion of the DNA may be untranscribed, serving a regulatory or a structural function. For example, the preselected DNA may itself comprise a promoter that is active in a particular host cell. [0143]
  • Other elements functional in the host cells, such as introns, enhancers, polyadenylation sequences and the like, may also be a part of the DNA. Such elements may or may not be necessary for the function of the DNA, but may provide improved expression of the DNA by affecting transcription, stability of the mRNA, or the like. Such elements may be included in the DNA as desired to obtain the optimal performance of the transforming DNA in the cell. [0144]
  • “Control sequences” is defined to mean DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotic cells, for example, include a promoter, and optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Other regulatory sequences may also be desirable which allow for regulation of expression of the genes relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences. [0145]
  • “Operably linked” is defined to mean that the nucleic acids are placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice. [0146]
  • The DNA to be introduced into the cells further will generally contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of transformed cells from the population of cells sought to be transformed. Alternatively, the selectable marker may be carried on a separate piece of DNA and used in a co-transformation procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are well known in the art and include, for example, antibiotic and herbicide-resistance genes, such as neo, hpt, dhfr, bar, aroA, dapA and the like. See also, the genes listed on Table 1 of Lundquist et al. (U.S. Pat. No. 5,848,956). [0147]
  • Reporter genes are used for identifying potentially transformed cells and for evaluating the functionality of regulatory sequences. Reporter genes which encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene which is not present in or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. [0148]
  • Prokaryotic expression systems are preferred, and in particular, systems compatible with Streptomyces spp. are of particular interest. Control elements for use in such systems include promoters, optionally containing operator sequences, and ribosome binding sites. Particularly useful promoters include control sequences derived from the gene clusters of the invention. However, other bacterial promoters, such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, will also find use in the expression cassettes encoding desosamine. Preferred promoters are Streptomyces promoters, including but not limited to the ermE*,pikA and tipA promoters. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the β-lactamase (bla) promoter system, bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), which do not occur in nature, also function in bacterial host cells. [0149]
  • The various nucleic acid molecules of interest can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter. The nucleic acid molecules can include flanking restriction sites to allow for the easy deletion and insertion of other sequences. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques, such as site-directed mutagenesis and PCR. [0150]
  • For sequences generated by random mutagenesis, the choice of vector depends on the pool of mutant sequences, i.e., donor or recipient, with which they are to be employed. Furthermore, the choice of vector determines the host cell to be employed in subsequent steps of the claimed method. Any transducible cloning vector can be used as a cloning vector for the donor pool of mutants. It is preferred, however, that phagemids, cosmids, or similar cloning vectors be used for cloning the donor pool of mutant encoding nucleotide sequences into the host cell. Phagemids and cosmids, for example, are advantageous vectors due to the ability to insert and stably propagate therein larger fragments of DNA than in M13 phage and λ phage, respectively. Phagemids which will find use in this method generally include hybrids between plasmids and filamentous phage cloning vehicles. Cosmids which will find use in this method generally include λ phage-based vectors into which cos sites have been inserted. Recipient pool cloning vectors can be any suitable plasmid. The cloning vectors into which pools of mutants are inserted may be identical or may be constructed to harbor and express different genetic markers (see, e.g., Sambrook et al., supra). The utility of employing such vectors having different marker genes may be exploited to facilitate a determination of successful transduction. [0151]
  • Thus, for example, the cloning vector employed may be an [0152] E. coli/Streptomyces shuttle vector (see, for example, U.S. Pat. Nos. 4,416,994, 4,343,906, 4,477,571, 4,362,816, and 4,340,674), a cosmid, a plasmid, an artificial bacterial chromosome (see, e.g., Zhang and Wing, Plant Mol. Biol., 35, 115 (1997); Schalkwyk et al., Curr. Op. Biotech., 6, 37 91995); and Monaco and Lavin, Trends in Biotech., 12, 280 (1994), or a phagemid, and the host cell may be a bacterial cell such as E. coli, Penicillium patulum, and Streptomyces spp. such as S. lividans, S. venezuelae, or S. lavendulae, or a eukaryotic cell such as fungi, yeast or a plant cell, e.g., monocot and dicot cells, preferably cells that are regenerable.
  • The general methods for constructing recombinant DNA which can transform target cells are well known to those skilled in the art, and the same compositions and methods of construction may be utilized to produce the DNA useful herein. For example, J. Sambrook et al., [0153] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2d ed., 1989), provides suitable methods of construction.
  • The recombinant DNA can be readily introduced into the host cells by any procedure useful for the introduction into a particular cell, e.g., calcium phosphate precipitation, protoplast fusion, conjugation, lipofection, electroporation, and the like. [0154]
  • As used herein, the term “cell line” or “host cell” is intended to refer to well-characterized homogenous, biologically pure populations of cells. These cells may be eukaryotic cells that are neoplastic or which have been “immortalized” in vitro by methods known in the art, as well as primary cells, or prokaryotic cells. In particular, the cell line or host cell may be of mammalian, plant, insect, yeast, fungal or bacterial origin. [0155]
  • “Transfected” or “transformed” is used herein to include any host cell or cell line, the genome of which has been altered or augmented by the presence of at least one DNA sequence, which DNA is also referred to in the art of genetic engineering as “heterologous DNA,” “recombinant DNA,” “exogenous DNA,” “genetically engineered,” “non-native,” or “foreign DNA,” wherein said DNA was isolated and introduced into the genome of the host cell or cell line by the process of genetic engineering. The transfected DNA may be maintained as an extrachromosomal element or as an element which is stably integrated into the host chromosome. [0156]
  • Moreover, recombinant polypeptides having a particular activity may be prepared via “gene-shuffling”. See, for example, Crameri et al., [0157] Nature, 391, 288 (1998); Patten et al., Curr. Op. Biotech., 8, 724 (1997), U.S. Pat. Nos. 5,837,458, 5,834,252, 5,830,727, 5,811,238, 5,605,793).
  • For phagemids, upon infection of the host cell which contains a phagemid, single-stranded phagemid DNA is produced, packaged and extruded from the cell in the form of a transducing phage in a manner similar to other phage vectors. Thus, clonal amplification of mutant encoding nucleotide sequences carried by phagemids is accomplished by propagating the phagemids in a suitable host cell. [0158]
  • Following clonal amplification, the cloned donor pool of mutants is infected with a helper phage to obtain a mixture of phage particles containing either the helper phage genome or phagemids mutant alleles of the wild-type encoding nucleotide sequence. [0159]
  • Infection, or transfection, of host cells with helper phage is generally accomplished by methods well known in the art (see., e.g., Sambrook et al., supra; and Russell et al. (1986) [0160] Gene 45:333-338).
  • The helper phage may be any phage which can be used in combination with the cloning phage to produce an infective transducing phage. For example, if the cloning vector is a cosmid, the helper phage will necessarily be a λ phage. Preferably, the cloning vector is a phagemid and the helper phage is a filamentous phage, and preferably phage M13. [0161]
  • If desired after infecting the phagemid with helper phage and obtaining a mixture of phage particles, the transducing phage can be separated from helper phage based on size difference (Barnes et al. (1983) [0162] Methods Enzymol. 101:98-122), or other similarly effective technique.
  • The entire spectrum of cloned donor mutations can now be transduced into clonally amplified recipient cells into which has been transduced or transformed a pool of mutant encoding nucleotide sequences. Recipient cells which may be employed in the method disclosed and claimed herein may be, for example, [0163] E. coli, or other bacterial expression systems which are not recombination deficient. A recombination deficient cell is a cell in which recombinatorial events is greatly reduced, such as recv mutants of E. coli (see, Clark et al. (1965) Proc. Natl. Acad. Sci. USA 53:451-459).
  • These transductants can now be selected for the desired expressed protein property or characteristic and, if necessary or desirable, amplified. Optionally, if the phagemids into which each pool of mutants is cloned are constructed to express different genetic markers, as described above, transductants may be selected by way of their expression of both donor and recipient plasmid markers. [0164]
  • The recombinants generated by the above-described methods can then be subjected to selection or screening by any appropriate method, for example, enzymatic or other biological activity. [0165]
  • The above cycle of amplification, infection, transduction, and recombination may be repeated any number of times using additional donor pools cloned on phagemids. As above, the phagemids into which each pool of mutants is cloned may be constructed to express a different marker gene. Each cycle could increase the number of distinct mutants by up to a factor of 10[0166] 6. Thus, if the probability of occurrence of an inter-allelic recombination event in any individual cell is f (a parameter that is actually a function of the distance between the recombining mutations), the transduced culture from two pools of 106 allelic mutants will express up to 1012 distinct mutants in a population of 1012/f cells.
  • Preparation, Isolation and Modification of the Polypeptides of the Invention [0167]
  • The present isolated, purified polypeptides, variants or fragments thereof, can be synthesized in vitro, e.g., by the solid phase peptide synthetic method or by recombinant DNA approaches (see above). The solid phase peptide synthetic method is an established and widely used method, which is described in the following references: Stewart et al., [0168] Solid Phase Peptide Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J. Am. Chem. Soc., 85 2149 (1963); Meienhofer in “Hormonal Proteins and Peptides,” ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp. 48-267; Bavaay and Merrifield, “The Peptides,” eds. E. Gross and F. Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285; and Clark-Lewis et al., Meth. Enzymol., 287, 233 (1997). These polypeptides can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.
  • In particular, fusion polypeptides are prepared which comprise an amino acid sequence useful in purification, e.g., a His tag is useful to purify fusion polypeptides on nickel columns. Once isolated and characterized, derivatives, e.g., chemically derived derivatives, of a given polypeptide can be readily prepared. For example, amides of the polypeptides of the present invention may also be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. A preferred method for amide formation at the C-terminal carboxyl group is to cleave the polypeptide from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine. [0169]
  • Salts of carboxyl groups of a polypeptide or polypeptide variant of the invention may be prepared in the usual manner by contacting the polypeptide with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like. [0170]
  • N-acyl derivatives of an amino group of the polypeptide or polypeptide variants may be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected polypeptide. O-acyl derivatives may be prepared, for example, by acylation of a free hydroxy peptide or peptide resin. Either acylation may be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation may be carried out together, if desired. [0171]
  • One or more of the residues of the polypeptide can be altered, so long as the polypeptide variant is biologically active. For example, it is preferred that the variant has at least about 1% of the biological activity of the corresponding non-variant polypeptide, e.g. Conservative amino acid substitutions are preferred—that is, for example, aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the polypeptide variant. [0172]
  • Conservative substitutions are shown in FIG. 25 under the heading of exemplary substitutions. More preferred substitutions are under the heading of preferred substitutions. After the substitutions are introduced, the variants are screened for biological activity. [0173]
  • Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: [0174]
  • (1) hydrophobic: norleucine, met, ala, val, leu, ile; [0175]
  • (2) neutral hydrophilic: cys, ser, thr; [0176]
  • (3) acidic: asp, glu; [0177]
  • (4) basic: asn, gln, his, lys, arg; [0178]
  • (5) residues that influence chain orientation: gly, pro; and [0179]
  • (6) aromatic; trp, tyr, phe. [0180]
  • The invention also envisions polypeptide variants with non-conservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another. [0181]
  • Acid addition salts of the polypeptide or variant polypeptide or of amino residues of the polypeptide or variant polypeptide may be prepared by contacting the polypeptide or amine with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric acid. Esters of carboxyl groups of the polypeptides may also be prepared by any of the usual methods known in the art. [0182]
  • Antibodies of the Invention [0183]
  • The antibodies of the invention are prepared by using standard techniques. To prepare polyclonal antibodies or “antisera,” an animal is inoculated with an antigen that is an isolated and purified polypeptide of the invention, and immunoglobulins are recovered from a fluid, such as blood serum, that contains the immunoglobulins, after the animal has had an immune response. For inoculation, the antigen is preferably bound to a carrier peptide and emulsified using a biologically suitable emulsifying agent, such as Freund's incomplete adjuvant. A variety of mammalian or avian host organisms may be used to prepare polyclonal antibodies [0184]
  • Following immunization, Ig is purified from the immunized bird or mammal, e.g., goat, rabbit, mouse, rat, or donkey and the like. For certain applications, it is preferable to obtain a composition in which the antibodies are essentially free of antibodies that do not react with the immunogen. This composition is composed virtually entirely of the high titer, monospecific, purified polyclonal antibodies to the antigen. Antibodies can be purified by affinity chromatography. Purification of antibodies by affinity chromatography is generally known to those skilled in the art (see, for example, U.S. Pat. No. 4,533,630). Briefly, the purified antibody is contacted with the purified polypeptide, or a peptide thereof, bound to a solid support for a sufficient time and under appropriate conditions for the antibody to bind to the polypeptide or peptide. Such time and conditions are readily determinable by those skilled in the art. The unbound, unreacted antibody is then removed, such as by washing. The bound antibody is then recovered from the column by eluting the antibodies, so as to yield purified, monospecific polyclonal antibodies. [0185]
  • Monoclonal antibodies can be also prepared, using known hybridoma cell culture techniques. In general, this method involves preparing an antibody-producing fused cell line, e.g., of primary spleen cells fused with a compatible continuous line of myeloma cells, and growing the fused cells either in mass culture or in an animal species, such as a murine species, from which the myeloma cell line used was derived or is compatible. Such antibodies offer many advantages in comparison to those produced by inoculation of animals, as they are highly specific and sensitive and relatively “pure” immunochemically. Inmunologically active fragments of the present antibodies are also within the scope of the present invention, e.g., the F(ab) fragment, scFv antibodies, as are partially humanized monoclonal antibodies. [0186]
  • Thus, it will be understood by those skilled in the art that the hybridomas herein referred to may be subject to genetic mutation or other changes while still retaining the ability to produce monoclonal antibody of the same desired specificity. The present invention encompasses mutants, other derivatives and descendants of the hybridomas. [0187]
  • It will be further understood by those skilled in the art that a monoclonal antibody may be subjected to the techniques of recombinant DNA technology to produce other derivative antibodies, humanized or chimeric molecules or antibody fragments which retain the specificity of the original monoclonal antibody. Such techniques may involve combining DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of the monoclonal antibody with DNA coding the constant regions, or constant regions plus framework regions, of a different immunoglobulin, for example, to convert a mouse-derived monoclonal antibody into one having largely human immunoglobulin characteristics (see EP 184187A, 2188638A, herein incorporated by reference). [0188]
  • The antibodies of the invention are useful for detecting or determining the presence or amount of a polypeptide of the invention in a sample. The antibodies are contacted with the sample for a period of time and under conditions sufficient for antibodies to bind to the polypeptide so as to form a binary complex between at least a portion of said antibodies and said polypeptide. Such times, conditions and reaction media can be readily determined by persons skilled in the art. [0189]
  • For example, the cells are lysed to yield an extract which comprises cellular proteins. Alternatively, intact cells are permeabilized in a manner which permits macromolecules, i.e., antibodies, to enter the cell. The antibodies of the invention are then incubated with the protein extract, e.g., in a Western blot, or permeabilized cells, e.g., prior to flow cytometry, so as to form a complex. The presence or amount of the complex is then determined or detected. [0190]
  • The antibodies of the invention may also be coupled to an insoluble or soluble substrate. Soluble substrates include proteins such as bovine serum albumin. Preferably, the antibodies are bound to an insoluble substrate, i.e., a solid support. The antibodies are bound to the support in an amount and manner that allows the antibodies to bind the polypeptide (ligand). The amount of the antibodies used relative to a given substrate depends upon the particular antibody being used, the particular substrate, and the binding efficiency of the antibody to the ligand. The antibodies may be bound to the substrate in any suitable manner. Covalent, noncovalent, or ionic binding may be used. Covalent bonding can be accomplished by attaching the antibodies to reactive groups on the substrate directly or through a linking moiety. [0191]
  • The solid support may be any insoluble material to which the antibodies can be bound and which may be conveniently used in an assay of the invention. Such solid supports include permeable and semipermeable membranes, glass beads, plastic beads, latex beads, plastic microtiter wells or tubes, agarose or dextran particles, sepharose, and diatomaceous earth. Alternatively, the antibodies may be bound to any porous or liquid permeable material, such as a fibrous (paper, felt etc.) strip or sheet, or a screen or net. A binder may be used as long as it does not interfere with the ability of the antibodies to bind the ligands. [0192]
  • The invention will be further described by the following examples. [0193]
    • EXAMPLE 1 Molecular Characterization and Analysis of the mit/mmc Biosynthetic Gene Cluster
  • Materials and Methods [0194]
  • Bacterial Strains and Cloning Vectors [0195]
  • [0196] S. lavendulae NRRL 2564 was used as the source strain for cosmid library construction and the creation of gene disruption mutants. E. coli DH5α was used as the host strain for constructing the library and subsequent DNA manipulation. E. coli strain S17-1 (Mazodier et al., 1989) served as the conjugative host for introducing foreign DNA into S. lavendulae. The cosmid library was constructed with the E. coli/Streptomyces shuttle vector pNJ1 (Tuan et al., 1990), and pUC119 was routinely used as a vector for subcloning and sequencing. The conjugative E. coli/Streptomyces shuttle vector pKC 1139 (Bierman et al., 1992) was used for gene disruption in S. lavendulae.
  • DNA Manipulation [0197]
  • Standard in vitro techniques were used for DNA manipulation (Sambrook et al., 1989). [0198] S. lavendulae genomic DNA was harvested by standard procedures (Hopwood et al., 1985).
  • A library of size-fractionated genomic DNA in pNJ1 (Tuan et al., 1990) was screened with the rifamycin AHBA synthase (rifK) gene probe from [0199] Amycolatopsis mediterranei (Kim et al., 1998). Through subsequent cosmid walking, a contiguous 120 kb region of S. lavendulae chromosomal DNA containing the putative mitomycin biosynthetic genes was mapped. M13 forward and reverse primers were used for sequencing (Gibco BRL, Gaithersburg, Md.). To accomplish this, individual fragments of less than 5 kb were subcloned into pUC 119 and serial deletion subdlones were generated using the exonuclease III Erase-a Base System (Promega, Madison, Wis.).
  • DNA Sequencing and Analysis [0200]
  • Automatic DNA sequencing was done with the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Warrington, U.K.), and analyzed on an Applied Biosystems mode 377 DNA Sequencer at the University of Minnesota Advanced Genetic Analysis Center. Both DNA strands were sequenced redundantly a minimum of three times. Sequence compilation was performed with MacVector (Oxford Molecular Group, Mountain View, Calif.) and GeneWorks (Oxford Molecular Group) software, and sequence homology analysis was accomplished with Blast (Altschul et al., 1990) and GCG programs (Devereux et al., 1984). [0201]
  • Disruption Mutants Construction [0202]
  • A 1.4 kb ApaL1-HindIII fragment from pFD666 (Denis and Brzezinski, 1998) containing the aphII gene for kanamycin resistance was routinely used as the selection marker for the creation of gene disruption constructs. The target genes were subcloned into pUC 119, cut at a unique internal restriction site, blunt-ended, and ligated with the end-blunted selection marker. The inserts were then transferred from pUC119 to pKC1139, and conjugated into wild-type [0203] S. lavendulae. Transconjugants were selected on AS1 plates (Baltz, 1980), overlaid with apramycin, kanamycin, and nalidixic acid followed by propagation on R5T plates (g/L: sucrose 121.1, K2SO40.3, MgCl2. 6H2O 11.92, glucose 11.8, yeast extract 5.89, casamino acids 0.12, trace elements 2.35 ml (Hopwood et al., 1985), agar 25.9, after autoclaving the following solutions were added: 0.5% KH2PO4 11.8 ml, 5 M CaCl2 4.71 ml, 1 N NaOH 8.25 ml) at 37° C. for several generations. Disruption mutants were selected based on the phenotype changing from apramycin and kanamycin resistant to apramycin sensitive and kanamycin resistant. Replacement of the chromosomal copy of the target gene with the disrupted plasmid-born copy was confirmed by Southern blot hybridization.
  • Mitomycin C Analysis [0204]
  • MC production was evaluated using 3-day cultures in Nishikohri media (Nishikohri and Fukui, 1978). The culture broth was extracted twice with equal volumes of ethyl acetate. After removing the chemical solvent by vacuum, the crude broth extract was dissolved in 50% methanol and 50% 50 mM pH 7.2 Tris buffer and monitored by HPLC (C[0205] 18 reverse phase column) at 363 nm. A continuous methanol gradient from 20% to 60% in methanol/50 mM pH 7.2 Tris buffer system over 24 minutes was employed to resolve MC from other crude extract components. A 90% CHCl3/10% MeOH solvent system was used to resolve and detect MC on TLC plates.
  • Results [0206]
  • Identification of the Mitomycin Biosynthetic Gene Cluster [0207]
  • The mitomycin cluster was identified by linkage of a cosmid clone containing mrd and a gene (mitA) that hybridized with the rifK gene encoding the rifamycin AHBA synthase (Kim et al., 1998) from [0208] Amycolatopsis mediterranei. mitA was subsequently shown to be essential for mitomycin biosynthesis since genetic disruption of the chromosomal copy blocked MC production, and could be complemented with exogenous AHBA (Example 2). Linkage of mitA with one of the mitomycin resistance genes (mrd) implied that the corresponding biosynthetic genes were adjacent to mitA. Cosmid walking was used to obtain overlapping DNA fragments spanning more than 120 kb of the S. lavendulae chromosome adjacent to mitA. Subsequent nucleotide sequence analysis included 55 kb of contiguous DNA, revealing 47 genes involved in mitomycin assembly, regulation and resistance (FIGS. 2 and 5).
    TABLE 1
    MC production in wild-type S. lavendulae and gene disruption mutants
    MC
    No. gene disrupted production
    0.0 Wild-type control ++
    0.1 additional copy ++
    of orf1 in
    wild-type
    1 orf8 ++
    2 orf4 ++
    3 orf1 ++
    4 mitR +
    5 mitM −
    6 mitI −
    7 mitH −
    8 mitE −
    9 mitB −
    10 mitA −
    11 mmcA −
    12 mmcB −
    13 mmcM ++
    14 mmcP −
    15 mmcR −
    16 mmcT −
    17 mmcW ++++
    18 mmcX ++++
    19 orf11 ++
    20 orf12 ++
    21 orf16 ++
    22 orf19 ++
  • mitT Defines the Left-hand Boundary of the Mitomycin Cluster [0209]
  • Nucleotide sequence analysis extended 30 kb downstream of mitA and revealed a set of genes corresponding to a type I polyketide synthase (PKS, orf9, SEQ ID NO:18; orf8, SEQ ID NO:19) and thioesterase (TEII, orf7, SEQ ID NO:20). MC is not derived from the polyketide pathway, and thus an orf8 disruption mutant showed normal MC production as expected (Table 1). Approximately 20 kb downstream of mitA, two genes (mitT, SEQ ID NO:29 and mitS, SEQ ID NO:30) encoding a putative aminoquinate dehydrogenase and glucose kinase, respectively, were located. Both are believed to be involved in AHBA biosynthesis since their equivalents are also present in the rifamycin biosynthetic gene cluster (rif cluster) (August et al., 1998). However, whether the six genes between orf7 and mitT are involved in MC biosynthesis remained unclear, since the two putative hydroxylases (orf3, SEQ ID NO:24 and orf4, SEQ ID NO:22) and the candidate activator gene (orf1, SEQ ID NO:26) could play a role in MC production. Both orf3 and orf4 are predicted to encode cytochrome P450 monooxygenases with Orf4 most similar to OleP and RapN (50% identity, 63% similarity) for oleandomycin and rapamycin biosynthesis, respectively (Rodriguez et al., 1995; Schwecke et al., 1995). Orf3 shows a high degree of similarity to cytochrome P450 105C1(49% identity, 64% similarity) in Streptomyces sp. and cytochrome P450-SU2 in [0210] Streptomyces griseolus (Horii et al., 1990; Omer et al., 1990).
  • Database analysis revealed that Orf1 belonged to the ActII-ORF4, RedD, DnrI and CcaR family of Streptomyces antibiotic pathway specific activators regulating the production of actinorhodin, undecylprodigiosin, daunorubicin, and cephamycin, respectively (Fernandez-Moreno et al., 1991; Perez-Laraine et al., 1997; Takano et al., 1992; Tang et al., 1996; Wietzorrek and Bibb; 1997). A common feature of this group of activators is that disruption of the corresponding gene abolishes the production of the corresponding antibiotic while overexpression results in a several-fold increase in metabolite production. However, when orf1 was disrupted, the mutant strain showed normal MC production (Table 1). Moreover, the wild-type MC producer containing an additional copy of orf1 in pKC1139 also had a normal MC production profile (Table 1). Interestingly, orf4, one of the cytochrome P450 monooxygenase encoding genes adjacent to orf1 also showed normal MC production when disrupted (Table 1). Thus, mitT appears to map to the left-hand end of the mitomycin cluster, while orf1 to orf9 presumably specify biosynthesis of a polyketide product. [0211]
  • mmcY Defines the Right-hand Boundary of the Mitomycin Cluster [0212]
  • Nucleotide sequence analysis of the mitomycin biosynthetic gene cluster extended 30 kb upstream of mitA and several orfs corresponding to genes involved in sugar metabolism were identified. They included an acid trehalase (orf12, SEQ ID NO:28), one ABC type transporter (orf16, SEQ ID NO:79), and four adjacent α-amylases (orf19, SEQ ID NO:82; orf20, SEQ ID NO:83; orf21, SEQ ID NO:84; orf22, SEQ ID NO:85) for starch degradation spanning more than 18 kb (FIG. 2). Disruption of four genes (orf19, SEQ ID NO:27; orf12, SEQ ID NO:28; orf16, SEQ ID NO:79; orf19, SEQ ID NO:82) within this region resulted in mutants with wild-type level MC production profiles, indicating that they fall outside of the mitomycin cluster (Table 1). At the beginning of this group of sugar metabolism genes, a gene (mmcY, SEQ ID NO:75) encoding a presumed chitinase is proposed to be the upstream terminus of the mitomycin cluster. This is evident because mitomycin requires D-glucosamine as a biosynthetic precursor, and MmcY shows 75% identity (85% similarity) with the chitinase C gene (chiC) product from [0213] S. griseus that generates N-acetylglucosamine from chitin (Ohno et al., 1996). In addition, mutants with disrupted orf11 and orf12 genes had no effect on MC production, while disruption of mmcW (SEQ ID NO:71) and mmcX (SEQ ID NO:72) both affected MC production significantly (Table 1).
  • Mitomycin Resistance Genes [0214]
  • Antibiotic biosynthetic gene clusters typically include one or more genes for cellular self-protection (Seno and Baltz, 1989). Previous work has identified two mitomycin C resistance genes (mcr and mrd) with mrd linked to mitA (August et al., 1994; Sheldon et al., 1997; Example 2). Subsequent analysis showed that MRD is a resistance protein that binds mitomycin C with 1:1 stoichiometry (Sheldon et al., 1997). However, this resistance mechanism would be extremely inefficient unless the bound drug is transported out of the cell. Indeed, 5 kb upstream of mrd, the mct gene (SEQ ID NO:16, putative mitomycin translocase) encoding a presumed antibiotic transporter was found and shown to be a third resistance component (Example 3). mct encodes 484 amino-acid protein with 14 predicted transmembrane domains. Disruption of mct resulted in a mutant [0215] S. lavendulae strain substantially more sensitive to MC, while coexpression of mct with mrd in E. coli dramatically increased MC resistance levels compared to individual expression of the genes (Example 3). In contrast, the high-level MC resistance gene (mcrA) that encodes an MC oxidase (MCRA) capable of re-oxidizing activated MC (Johnson et al., 1997) is not linked with this cluster (August et al., 1990; Example 2). Interestingly, database searches identified two McrA homologues (MitR, MmcM) within the MC cluster, both of which encode putative flavoproteins conserved in the FMN/FAD binding motif. MitR displayed weak similarity with McrA (26% identity, 33% similarity), while MmcM showed end-to-end (54% identity, 69% similarity) alignment with the protein. mitR (SEQ ID NO:31) and mmcM (SEQ ID NO:61) were genetically disrupted giving substantially decreased MC production in the mitR mutant strain, in contrast to the mmcM mutant which displayed wild type MC production levels (Table 1).
  • Regulatory Genes [0216]
  • Two genes, mitQ (SEQ ID NO:32) and mmcW (SEQ ID NO:71), were identified in the mitomycin cluster and are presumed to be pathway-specific regulators. MitQ belongs to the OmpR-PhoB subfamily of DNA binding regulators in the two-component regulatory system, with the greatest similarity to members of the phosphate assimilation pathway (PhoR-PhoB) (Makino et al., 1986), ferric enterobactin response pair (PfeR-PfeS) (Dean et al., 1996), and one histidine protein kinase—response regulator system (HpkA-DrrA) from [0217] Thermotoga maritima (Lee and Stock, 1996). In contrast to the MitQ group of regulators that typically serve as transcriptional activators (Mizuno and Tanaka, 1997), MmcW showed high sequence similarity with the MarR groups of repressors. The most significant similarity corresponds to EmrR, the negative regulator of the E. coli multidrug resistance pump EmrAB (Lomovskaya et al., 1995), and Pacs, a repressor for pectinase, cellulase, and blue pigment production in Erwinia chrysanthemi (Praillet et al., 1996). Significantly, the mmcW disruption mutant displayed a several-fold increase in MC production (Table 1).
  • AHBA Biosynthetic Genes [0218]
  • Precursor incorporation studies previously demonstrated that AHBA is an intermediate for both the ansamycin and mitomycin natural products (Becker et al., 1983; Example 2). Combining the biochemical, enzymatic and molecular genetic results on the biosynthesis of the ansamycin antibiotic rifamycin, Floss has proposed that AHBA is derived from the ammoniated shikimate pathway via phosphenolpyruvate (PEP) and erythose 4-phosphate (E4P) by the early incorporation of nitrogen (Kim et al., 1996). In the shikimate pathway, PEP and E4P is first converted to 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) then stepwise transformed to 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS) and shikimate, catalyzed by DAHP synthase, DHQ synthase, DHQ dehydratase, and shikimate dehydrogenase, respectively (Dewick, 1998). Quinate can also enter the pathway by the action of quinate dehydrogenase to generate DHQ. [0219]
  • Evidence to support this new variant of the shikimate pathway includes the following experimental observations. First, all proposed ammoniated shikimate pathway compounds including PEP, E4P, 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (aminoDAHP), 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) can be readily converted into AHBA by cell-free extracts from the ansamycin producers, while none of the early shikimate pathway intermediates, DAHP, DHQ, DHS, quinic acid, shikimic acid can be incorporated into AHBA under the same conditions (Homemann, 1981; Kim et al., 1996). Second, the rifamycin biosynthetic gene cluster (rif cluster) has been sequenced, and all of the genes encoding early shikimate pathway enzymes are found within the cluster (August et al., 1998). Finally, the ability of the rifamycin AHBA synthase (RifK) to catalyze dehydration of aminoDHS to AHBA has been previously demonstrated (Kim et al., 1998). As described in Example 2, the AHBA synthase gene (mitA) in [0220] S. lavendulae is required for AHBA biosynthesis.
  • A group of AHBA biosynthetic genes similar to those described for rif have been identified in the mitomycin cluster. In addition to AHBA synthase, six gene products in the cluster showed high sequence similarity (over 43% identity) with their rifamycin AHBA biosynthetic gene homologs. These gene products include aminoDHQ synthase (MitP, RifG equivalent), aminoquinate dehydrogenase (MitT, Rift equivalent), oxidoreductase (MitG, RifL equivalent), phosphatase (MitJ, RifM equivalent), kinase (MitS, RifN equivalent), and aminoDHQ dehydratase (MmcF, RifJ equivalent). In addition to the significant sequence similarity to rifamycin counterparts, all three putative mitomycin shikimate pathway enzymes displayed significant alignment with microbial primary shikimate metabolic enzymes including MitT with the quinate dehydrogenase (AroE) from [0221] Methanococcus jannaschii (28% identity, 46% similarity) (Bult et al., 1996), MitP with the DHQ synthase (AroB) from Mycobacterium tuberculosis (46% identity, 61% similarity) (Cole et al., 1998), and MmcF with the DHQ dehydratase from S. coelicolor (50% identity, 62% similarity) (White et al., 1990). Despite extensive sequencing of 15 kb on either side of the mapped right- and left-hand ends of the mitomycin cluster, an aminoDAHP synthase gene corresponding to RifH (the proposed first enzyme in the de novo biosynthesis from PEP and E4P in the rif cluster), was not found (FIG. 2). Interestingly, a rifH homologue has been cloned from S. lavendulae genomic DNA through Southern hybridization and shown to be unlinked to the mitomycin cluster.
  • The existence of non-shikimate pathway-related phosphatase/kinase pair in the mitomycin cluster (MitJ/MitS) and the rif cluster (RifM/RifN) further support the finding that these two genes are required for AHBA biosynthesis (Floss, 1997). In addition to the strong homology to RifM, MitJ also showed 56% identity (69% similarity) with ORF8 from the ansamycin antibiotic ansamitocin producer [0222] Actinosynnema pretiosum auranticum. Other polypeptides with considerable sequence similarity belong to the CBBY family of phosphoglycolate phosphatases in glycolate oxidation (Schafejohann et al., 1993). MitS, most similar to RifN (53% identity, 63% similarity), also showed significant similarity with the glucose kinase (involved in glucose repression) from S. coelicolor and Bacillus megaterium (Angell et al., 1992; Spath et al., 1997). mitG, the third non-shikimate pathway-related AHBA biosynthetic gene in this cluster is also worthy of note since it shows exclusive similarity (46% identity, 61% similarity) with oxidoreductase RifL and its equivalent in Actinosynnema pretiosum auranticum.
  • Mitosane Formation Genes [0223]
  • Precursor incorporation studies established that the mitosane core is assembled form the condensation of AHBA and D-glucosamine. Although no specific gene products can be assigned for forming the three bonds bridging AHBA and D-glucosamine, two genes downstream of mitA (SEQ ID NO:97), mitb (SEQ ID NO:99), and mitE (SEQ ID NO:44) likely encode enzymes that mediate one of these reactions. MitB shows local sequence similarity with a group of glycosyltransferases involved in glycopeptide antibiotic and polysaccharide biosynthesis, the typical function of which is to attach an activated sugar residue to a core compound (Yamazaki et al., 1996; Example 2). Meanwhile, MitE showed weak similarity (22% identity and 45% similarity) to the two cloned 4-hydroxybenzoate-CoA ligases from [0224] Rhodopseudomonas palustris in the anaerobic degradation of aromatic compounds (Gibson et al., 1994). It also showed similarity to a group of long chain fatty acid CoA ligases, as well as to the O-succinylbenzoic acid CoA synthetase in Vitamin K2 biosynthesis (Kwon et al., 1996). mitB and mitE disruption mutants both had a MC deficient phenotype (Table 1).
  • The condensation of AHBA with D-glucosamine may be initiated in two different ways. This includes either initial formation of a C[0225] 8a-C9 bond by an acylation or alkylation reaction, or formation of a Schiff base between the AHBA nitrogen and D-glucosamine C1 aldehyde, followed by the ring closure at C8a-C9.mitR (SEQ ID NO:31), one of the two McrA homologues may be involved in one of the ring closure reactions. Interesting, MitR showed high sequence homology with the plant berberine bridge enzyme (BBE) (30% identity, 37% similarity) in benzophenanthridine alkaloid formation, where it catalyzes an unusual C—C bond formation of the berberine bridgehead carbon of (S)-scoulerine from the N-methyl carbon of (S)-reticuline (Dittrich and Kutchan, 1991). Using a mechanism similar to BBE, it is possible that MitB is involved in C8a-C9 bond formation. The decreased MC production in the mitR disruption mutant may be due to the existence of isoenzymes (e.g., MmcM) that could catalyze the reaction in the absence of a functional MitR.
  • Side Group Modification Genes [0226]
  • Complete assembly of MC requires functionalization of several sites on the core mitosane ring system. First, complete reduction of the carbonyl group at C-6 must occur. Second, hydroxylation at C-5 and C9a must proceed followed by methylation at C-9a. Third, amination at C-7 must occur presumably through initial hydroxylation followed by transamination. Fourth, oxidation of the hydroxyl groups at C-5 and C-8 to form the benzoquinone are required. Fifth, intramolecular amination of C-1 by N-1a to form the aziridine ring must be completed and finally, carbamoylation at C-10 completes assembly of the molecule. Several enzymes found in this cluster likely catalyze these modifications and are discussed below. [0227]
  • Methylation [0228]
  • In contrast to MC which has an O-methyl group at C-9a, mitomycin A and mitomycin B also contain a C-7 O-methyl group, while mitomycin B, mitomycin D and porfiromycin have an N-methyl on the aziridine ring (FIG. 1). Radio-labeled precursor incorporation studies showed that all of the O- and N-methyl (but not the C-methyl) groups in the mitomycin molecules are derived from L-methionine (Bezanson and Vining, 1971). Typically, the methyl donor for most C1 reactions is S-adenosyl-L-methionine (SAM), which can be formed through activation of L-methionine by ATP. Three SAM dependent methyltransferase genes were identified in this cluster (encoding MitM, MitN, and MmcR), all of which have three conserved S-adenosylmethionine or S-adenosylhomocysteine binding motifs (Kagan and Clarke, 1994) (FIG. 3). Interestingly, database searches of MitM and MitN (likely responsible for the MC C-9a side chain methylation) revealed a group of plant δ-(24)-sterol C-methyltransferases that have a closer phylogenetic relationship with the rifamycin O-methyltransferase (ORF14) and erythromycin O-methyltransferase (EryG) (5, 86) (FIG. 4). In contrast, protein database searches revealed that MmcR is most related to other Streptomyces antibiotic biosynthetic O-methyltransferases with greatest similarity to O-demethylpuromycin O-methyltransferase (44% identity, 60% similarity) from [0229] S. anulatus and carminomycin 4-O-methyltransferase from S. peucetius (Lacalle et al., 1991; Madduri et al., 1963). MmcR may be involved in the O-methylation of the phenol ring of MC before oxidation to the quinone. Both mmcR (SEQ ID NO:67) and mitM (SEQ ID NO:36) were shown to be essential for MC biosynthesis since disruption of each one completely abolished MC production (Table 1).
  • A SAM-independent methyltransferase, MmcD, was also identified in the mitomycin cluster. MmcD revealed strong sequence homology with the magnesium-protoporphyrin IX monomethyl ester oxidative cyclase (34% identity, 53% similarity) from [0230] Methanobacterium thermoautotrophicum (Accession Number 2622915), as well as the phosphonoacetaldehyde methyltransferase from Streptomyces wedmorensis (Hidaka et al., 1995), the P-methyltransferase from Streptomyces hygroscopicus (Hidaka et al., 1995) and the fortimicin KL methyltransferase from Micromonospora olivasterospora (Kuzuyama et al., 1995). Instead of SAM, this group of methyltransferases uses methylcobalamine or a structurally related protoporphyrin as the direct methyl donor. While the greatest number of matches were made to protoporphyrin methyltransferases, it is expected that this enzyme has another function in the mitomycin C biosynthetic pathway as all the O- and N-methyl groups of MC have been shown to be derived from SAM-dependent methyltransferases.
  • C-6 Carbonyl Reduction [0231]
  • The C-6 methyl group was previously shown to be derived from the reduction of the carboxylic acid of AHBA, since [carboxy-[0232] 13C] AHBA can be efficiently, and specifically incorporated into the C-6 methyl group of porfiromycin (Anderson et al., 1980). In the mitomycin cluster, four F420-dependent tetrahydromethanopterin (H4MPT) reductase genes (encoding MitH, MitK, Mmcl, MmcJ) and one H4MPT:CoM methyltransferase gene (encoding MmcE) are candidates for the C-6 carbonyl reduction. In the methanogenesis pathway of Methanobacterium thermoautotrophicum, two cofactor F420-dependent H4MPT reductases, and one cofactor CoM dependent methyltransferase are required in the seven step reduction from CO2 to CH4. Steps 4 to 6 from CH-H4MPT to CH2-H4MPT, and CH3-H4MPT to CH3-CoM are catalyzed by N5, N10-methylene-H4MPT dehydrogenase, N5, N10-methylene-H4MPT reductase, and N5-methyl-H4MPT:CoM methyltransferase, respectively (Deppenmeier et al., 1996; Thauer et al., 1993). All four enzymes (MitH, MitK, Mmcl, MmcJ) in this cluster showed local sequence similarity with the cloned F420 dependent H4MPT reductase (42% identity, 62% similarity in several 50 amino-acid fragments) (Nolling et al., 1995; Vaupel and Thauer 1995). One of these genes, mitH (SEQ ID NO:41) was disrupted, and the mutant strain displayed a MC deficient phenotype (Table 1). MmcE is notable since the deduced protein sequence contains two domains showing significant alignment (33% identity, 56% similarity) to the N-terminus of H4MPT:CoM methyltransferase from Methanobacterium thermoautotrophicum (Stupperich et al., 1993), while the remaining C-terminus is related to fatty acid biosynthetic acyl carrier proteins (ACP) (Morbidoni et al., 1996; Platt et al., 1990). The potential function of this ACP-like domain in MC biosynthesis remains unknown, as does the role of a distinct gene (mmcB, SEQ ID NO:50) encoding a putative ACP identified just upstream of mmcE (SEQ ID NO:53). Significantly, the disruption of mmcB resulted in total abrogation of MC production (Table 1).
  • Hydroxylation [0233]
  • The two putative hydroxylases (encoded by mmcN, SEQ ID NO:62; and mmcT, SEQ ID NO:69) identified in the mitomycin cluster are candidates for catalyzing hydroxylation at the C-5, C-7, and C-9a positions on the mitosane system. MmcN belongs to the cytochrome P450 family of monooxygenases, with greatest homology (37% identity, 56% similarity) to the two herbicide-inducible cytochrome P450s (P450-SU1 and P450-SU2) from [0234] S. griseolus, as well as to RapJ and RapN in the rapamycin biosynthetic gene cluster from S. hygroscopicus (Omer et al., 1990; Schwecke et al., 1995). MmcT showed highest similarity to the tetracenomycin C hydroxylase (TcmG) in Streptomyces glaucescens (38% identity, 55% similarity), with lower but significant sequence similarity to a group of phenol or hydroxybenzoate hydroxylases (Decker et al., 1993). Genetic disruption of mmcT completely blocked MC biosynthesis (Table 1).
  • Carbamoylation [0235]
  • The carbamoyl group of MC is derived intact from L-citrulline or L-arginine with carbamoyl phosphate as the incorporated precursor (Homemann, 1981). In eubacteria, carbamoyl phosphate can be generated from L-glutamine, HCO[0236] 3, and ATP by the enzyme carbamoyl phosphate synthetase, which is indispensable for pyrimidine biosynthesis. One candidate carbamoyl transferase gene (mmcS, SEQ ID NO:68) was identified directly upstream of mmcT. MmcS belongs to the NodU/CmcH family of O-carbamoylation enzymes, with the greatest similarity (35% identity, 44% similarity) to No1O from Rhizobium sp. (Jabbouri et al., 1998). Other members with significant alignment in this family include No1O from Bradyrhizobium japonicum (Luka et al., 1993) and NodU from Rhizobium sp. for 6-O-carbamoylation of Nod-factors (Jabbouri et al., 1995) and CmcH from Nocardia lactamdurans and S. clavuligerus for 3′-hydroxymethylcephem O-carbamoylation in cephamycin biosynthesis (Coque et al., 1995).
  • Discussion [0237]
  • Bridging Primary and Secondary Metabolism [0238]
  • The shikimate pathway is an essential metabolic route in microorganisms and plants for aromatic amino acid biosynthesis. Genes encoding the early shikimate pathway enzymes from various organisms have been well studied and are often dispersed along the chromosome as revealed by genome sequencing projects (Blattner et al., 1997; Bult et al., 1996; Cole et al., 1998). The finding that the ansamycin and mitomycin natural products are derived in part from an ammoniated shikimate pathway whose genes are clustered on the bacterial chromosome is a significant difference to the primary metabolic network, and may suggest an important evolutionary bridge leading to secondary metabolism. The lack of incorporation of early shikimate pathway intermediates into mitomycin and ansamycin metabolites indicated the existence and ultimate substrate specificity of the alternate ammoniated shikimate pathway enzymes. However, the conversion of aminoDA-HP and aminoshikimic acid by the corresponding primary shikimate pathway enzymes to aminoDHQ and aminoDHS, respectively (Kim et al., 1996), suggested that the substrates specificity in primary metabolic shikimate pathway is mainly determined by the initial reaction step. This notion is further supported by the disruption results for rifG and rifI mutants showing only slightly affected rifamycin production (Floss, 1997). [0239]
  • In addition to the absence of an aminoDAHP synthase gene, the organization of the AHBA biosynthetic genes in the MC cluster is quite different compared to the rif cluster. In rif (with the exception rifJ), all AHBA biosynthetic genes are found within a defined sub-cluster that are organized into a single apparent operon. In contrast, almost all of the mit/mmc encoded AHBA genes are scattered within the 55 kb MC cluster. Thus, as opposed to the multifunctional polyketide gene clusters whose linearity of architecture reflects a precise pattern of biosynthesis, the MC cluster is biochemically less transparent based on a similar primary analysis. In addition, the MC cluster provides a good model for analyzing genetic evolution both vertically, from the primary metabolic shikimate pathway to the secondary shikimate pathway related route, and horizontally by comparing different groups of secondary metabolic biosynthetic clusters. [0240]
  • The MC Biosynthetic Network [0241]
  • In a typical liquid culture of [0242] S. lavendulae, MC production initiates 24 hours after inoculating the seed culture, reaches maximum production in two days, and maintains drug synthesis during stationary phase for another two days. Compared to high level MC resistance of the wild-type S. lavendulae (>150 μg/ml), MC production is relatively low (<5 μg/ml MC). The significant gap between the self-resistance and production levels makes it possible to improve drug production through genetic engineering. As described herein, disruption of the candidate repressor gene (mmcW) and downstream mmcX (encoding a putative membrane protein) in the mitomycin cluster resulted in a several-fold increase in MC production. The existence of a repressor gene(s) is not uncommon in Streptomyces antibiotic biosynthetic gene clusters. Previous examples include, mmyR from the methylenomycin cluster (Chater and Briton, 1985), actII-orfI in the actinorhodin cluster (Caballero et al., 1991), jadR (Anderson et al., 1980) in jadomycin biosynthesis (Yang et al., 1995), and dnrO in the daunorubicin cluster (Otten, 1995). Disruption of jadR and mmyR also resulted in increased levels of the corresponding antibiotic (Chater and Bruton, 1985; Yang et al., 1995).
  • In order to avoid auto-toxicity, drug-producing microorganisms must evolve self-protection systems. Currently, three types of self-protection mechanisms have been identified in [0243] S. lavendulae for mitomycin resistance including, MC binding (MRD), efflux (MCT), and reversing MC reductive activation (MCRA). In principle, resistance genes must be expressed before drug formation. In this respect, it is interesting to note the linkage of the mitomycin resistance genes with the regulatory genes. Expression of the high-level resistance gene mcrA has been demonstrated to be regulated by the downstream gene mcrB which is presumably cotranscribed with mcrA (August et al., 1994). Though the function of the McrA homolog MitR in the mitomycin cluster remains unknown, mitR is also followed by a cotranscribed regulatory gene (mitQ). Meanwhile, the putative mitomycin translocase gene, mct is followed by the repressor gene, mmcW. Genetic linkage of membrane transporter/resistance and repressor genes have been described in a number of cases, including tetA/tetR in tetracycline resistance (Guilfoile and Hutchinson, 1992), tcmA/tcmR in tetracenomycin C resistance (Guilfoile and Hutchinson, 1992), actII-orf2/actII-orf1 in actinorhodin resistance (Caballero et al., 1991), and the qacA/qacR pair for multidrug resistance in S. aureus (Grkovic et al., 1998).
  • Conclusion [0244]
  • Although MC was first isolated more than 40 years ago and has been used in anti-cancer chemotherapy since the 1960s, the mechanistic details and order of its biosynthesis has remained unclear. The results described herein are clearly consistent with precursor incorporation studies gathered in the 1970s, showing that MC is biosynthetically derived from D-glucosamine, L-methionine, carbamoyl phosphate, and AHBA, and also support the use of the variant de novo shikimate pathway leading to AHBA (Homemann, 1981; Kim et al., 1996). Many, if not all, of the genes responsible for the formation of the mitosane and aziridine rings are evidently located within the boundary of the 55 kb mitomycin cluster. These genes are of special interest since they may be useful as probes for identification of related natural product biosynthetic genes from other microorganisms and plants. [0245]
  • The cloned genes presented here are useful to study mitomycin biosynthesis and natural product assembly. The advantage of having this information has already been demonstrated through genetic disruption of the candidate repressor gene (mmcW) that provided a several-fold increase in MC production. In addition, expression and genetic disruption of selected genes should be useful for engineering the biosynthesis of clinically valuable mitomycin analogues, as well as more complex hybrid natural product systems. Finally, the MC resistance and regulatory genes identified in this cluster provide important insight into the mitomycin biosynthetic and regulatory network in the [0246] S. lavendulae.
    • EXAMPLE 2 Genetic Localization and Molecular Characterization of Two Genes Required for MC Biosynthesis
  • Materials and Methods [0247]
  • Strains and culture conditions. [0248] E. coli DH5α was grown in either Luria broth (LB) or tryptic soy broth (TSB) (Difco) as liquid medium or agar plates. E. coli DH5αF′, the host for harvesting single-stranded DNA, was grown at 37° C. on TBG (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM KH2PO4, 55 mM K2HPO4, and 20 mM glucose). E. coli S17-1 (Mazodier et al., 1989) used for conjugation was grown in TSB with 10 ug/ml of streptomycin. S. lavendulae was grown in TSB or on R5T plates. For MC production, S. lavendulae was grown in Nishikohri media (g/L: glucose 15, soluble starch 5, NaCl 5, CaCO 3 3, yeast extract 5) for 72 hours from a 1% v/v inoculum of frozen mycelia. Pulse feeding of AHBA to the disruption mutant, MV100, and the site-directed mutant, MV102, occurred with feedings of 2.5 mg of a 20 mg/mL solution of the sodium salt of AHBA at pH 7.1 in three pulses at 24, 43, and 57 hours of a culture that was harvested at 76 hours.
  • DNA preparation and amplification. Isolation and purification of DNA was performed using standard methods (Sambrook et al., 1989). [0249] S. lavendulae NRRL 2564 genomic DNA was isolated by using the modified Chater protocol (Hopwood et al., 1988). Plasmid DNA was isolated from E. coli by using the alkaline-sodium dodecyl sulfate method.
  • pDHS2002 was constructed as follows: The 1.1 kb thiostrepton resistance gene (tsr) fragment was removed from pDHS5000 with a SmaI-BamHI digestion, blunt-ended with the large fragment of DNA polymerase (Gibco BRL), and ligated to MscI restriction enzyme digested pDHS7601 to yield pDHS20001. MscI digestion of pDHS7601 resulted in the removal of 155 nucleotides at the C-terminus of the mitA gene, and ligation of the blunt-ended BamHI site of the tsr adjacent to the MscI site of pDHS7601 resulted in regeneration of the BamHI site in pDHS2001. The 4.9 kb EcoRI-HindIII fragment from pDHS2001 containing the tsr disrupted mitA gene was removed and ligated into EcoRI-HindIII digested pKC1139 to yield pDHS2002. [0250]
  • Primer-mediated site-directed mutagenesis (SDM) was employed to construct pDHS2015 containing a K191A mutation in mitA. Primer 1: 5′-GGCAAGGCATGCGAGGGTCGC-3′ (SEQ ID NO:46) and primer 2: 5′-TTCCAGAACGGCGCCCTGATGACCGCCGGC-3′ (SEQ ID NO:47) were used to amplify the 691 bp fragment of the 5′ end of mitA. The 3′ end of mitA was amplified with primer 3: 5′-GCCGGCGGTCATCAGGGCGCCGTTCTGGAA-3′ (SEQ ID NO:48) and primer 4: 5′-TCAGAATTCGGATCCGAGGGCCGGAGT-3′ (SEQ ID NO:86) to generate a 1151 bp band (see amplification reaction conditions in Example 3). A second round of PCR was performed using the overlapping 691 and 1151 bp units as the initial templates with [0251] primer 1 and primer 4 to yield a 1.8 kb fragment. The final product containing mutagenized mitA was digested with EcoRI-Sph1, ligated to the 2.1 kb HindlII-SphI fragment from pDHS7601 and the EcoRI-HindIII digested pKC1139 to yield pDSH2015. The site-directed mutation of MitA K191A in pDHS2015 was confirmed by sequencing with forward primer:
    5′-ACCTACTGCCTCGATGCC-3′ (SEQ ID NO:87)
    and reverse primer:
    5′-CTGATCCTTCAAGCG-3′. (SEQ ID NO:88)
  • The mitB disruption vector pDHS7702 was constructed as follows. pDHS7601 was digested with BstBI, blunt-ended, and ligated with the 1.4 kb neomycin-resistant gene fragment from pFD666 (Denis and Brzezinski et al., 1992) (ApaL1-HindIII digestion, blunt-ended). The 5.2 kb EcoRI-HindIII fragment from the resulting construct pDHS7701 was subdloned into pKC1139 to create pDHS7702. [0252]
  • DNA library construction and screening. [0253] S. lavendulae NRRL 2564 genomic DNA was partially digested with Sau3AI, and a fraction containing 30-50 kb fragments was recovered by sucrose gradient centrifugation and ligated into the calf intestinal alkaline phosphatase (CIP) treated BglII site of the E. coli-Streptomyces shuttle vector pNJ1 (Tuan et al., 1990), then packaged with the Packagene Lambda DNA Packaging System (Promega). The cosmid library was constructed by transfecting E. coli DH5α, and colonies that appeared on the LB plates containing 100 ug/ml of ampicillin were transferred to a BioTrace NT nitrocellulose blotting membrane (Gelman Sciences, Ann Arbor, Mich.). Colony hybridization was performed as specified by the manufacturer. A PCR-amplified 0.7 kb DNA fragment from plasmid pKN108 (FIG. 6) was used to screen the library. The primers used for PCR were: 5′-GCGTCCGTGCTGCGCGCGCA-3′ (SEQ ID NO:89), and 5′-TGCGCGCGCAGCACGGACGC-3′ (SEQ ID NO:90). The cosmids from the positive colonies were confirmed by Southern blot hybridization, and a 1.7 kb AflIII-BamHI fragment from pDHS3001 containing the mitomycin resistance determinant (mrd) (Sheldon et al., 1997) was used as a probe to establish genetic linkage.
  • DNA sequencing and analysis. Deletion subdlones from pDHS7601 were made with exonuclease III Erase-a-Base System (Promega). Sequencing was accomplished with the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and analyzed on an Applied Biosystems 377 DNA Sequencer at the University of Minnesota Advanced Genetic Analysis Center. For generating single-stranded DNA, deletion subclones in pUC119 were transformed into [0254] E. coli DH5αF′, and M13K07 Helper Phage was used (GIBCO BRL). Nucleotide sequence data were analyzed using Wisconsin Genetics Computer Group software (version 9.0) (Devereux et al., 1984), and GeneWorks software version 2.51 (Oxford Molecular Group). The GenBank accession number for mitABC is AF 115779.
  • Conjugation from [0255] E. coli S17-1 to S. lavendulae. The procedure of Bierman et al. (Bierinan et al., 1992) was used with the following modification. A single colony of E. coli S17-1 /pDHS2002 was used to inoculate 2 ml of TSB containing 100 μg/ml of apramycin and 10 μg/ml of streptomycin. Following overnight incubation at 37° C. a 1:100 inoculation was made into TSB broth with 100 μg/ml of apramycin and 10 μg/ml of streptomycin. This culture was grown for 3 hours at 37° C., and the cells were washed twice with TSB and resuspended in 2 ml of TSB to provide the donor E. coli culture. The recipient S. lavendulae culture was generated by inoculating 9 ml of TSB with 1 ml of frozen wild-type culture. Following overnight (16 hour) incubation at 29° C., the culture was homogenized by sonication and 2 ml of this culture was used to inoculate 18 ml of TSB. Following overnight growth at 29° C. and sonication treatment to homogenize the culture, a 1 ml inoculum was placed in 9 ml of TSB. This culture was grown for 3 hours, the mycelia were washed with TSB and resuspended in 2 ml of TSB to provide the stock recipient culture.
  • The donor and recipient cultures were mixed together in 9:1, 1:1, and 1:1/10 donor:recipient ratios, and 100 μl of the cell mixture was spread on AS1 plates (Baltz, 1980). The plates were incubated overnight at 29° C. and overlaid with 1 ml of water containing a suspension of 500 μg/ml each of thiostrepton, apramycin and nalidixic acid. For the pKC1139 control, only apramycin and nalidixic acid were overlaid, while for pDHS7702, 500 μg/ml of kanamycin was used instead of thiostrepton. [0256] S. lavendulae exconjugates appeared in approximately 11-13 days at a frequency ranging from 10−7-10−5. pKC 1139 has a temperature-sensitive Streptomyces replication origin, which is unable to replicate at temperatures above 34° C. (Muth et al., 1989), while the S. lavendulae host grows well at 42° C. Thus, after propagating the conjugants at 39° C. for several generations, double crossover mutants were readily generated. Presence of plasmid was determined by transformation of E. coli DH5α with plasmid extracts from S. lavendulae transconjugants.
  • Double-crossover selection procedure. A single colony of [0257] S. lavendulae/pDHS2002 grown on R5T plates (50 μg/ml of thiostrepton and apramycin) was used to inoculate TSB broth containing 20 μg/ml of thiostrepton. After 72 hours of incubation at 39° C., 10−4, 10−5 and 10−6 diluted aliquots were used to inoculate R5T plates containing 50 μg/ml of thiostrepton. Following 48 hours of growth at 39° C., 84 colonies were picked randomly and each colony was patched out on separate 50 μg/ml of thiostrepton and 50 μg/ml of apramycin containing R5T plates. One of the 84 colonies displayed the double crossover phenotype of thiostrepton resistance and apramycin sensitivity. Integration of the tsr disrupted mitA gene and loss of plasmid pDHS2002 was confirmed by Southern hybridization analysis.
  • MitA K191A site-directed mutants (MV102) were selected by propagating MV100/pDHS2015 on R5T plates for two generations at 37° C. Colonies were replicated to plates containing 50 μg/ml of thiostrepton and plates without antibiotics. Of the 108 colonies replicated in the first round, one had the correct (thiostrepton sensitive) phenotype. To confirm the K191A mutation, the mitA gene was amplified from the chromosome with [0258] primers 1 and 4. Mutation of the conserved lysine codon (AAG) to an alanine codon (GCC) was verified with the same sequencing primers employed to confirm the correct construction of pDHS2015. The alanine codon was observed in both the forward and reverse sequence data.
  • Mutants for mitB (MM101) were selected as follows: [0259] S. lavendulae/pDHS7702 was propagated on R5T plates for five generations at 39° C. before single colonies were replicated on R5T plates as described above. Of the 300 colonies tested, 12 clones displayed the correct phenotype (kanamycin resistance and apramycin sensitivity). The genotype of selected mitB mutants was confirmed by Southern blot hybridization of S. lavendulae genomic DNA.
  • Analysis of MC production. All cultures intended for MC extraction were grown in Nishikohri media (Nishikohri and Fukui, 1975) for a period of 72 hours. In all cases a wild-type [0260] S. lavendulae culture was grown concurrently with the mutant cultures to provide a MC production reference point. A 72 hours, 50 ml culture (250 ml flask) of the MitA K191A MV102 mutant strain was supplemented with 125 μl of a 20 mg/ml solution of the sodium salt of AHBA (pH 7.05) at 24, 43 and 55 hours. In each case, the culture broth was separated from mycelia by centrifugation and then extracted three times with equal volumes of ethyl acetate. The ethyl acetate extracts were pooled and solvent was removed by vacuum to provide the crude broth extract. The preliminary screen for MC production involved thin layer chromatography (TLC) on silica gel plates (Whatman K6) eluted with 9:1 chloroform:methanol. Production of MC was monitored by HPLC (C18 reverse phase column) using a gradient of 80% 50 mM Tris buffer (pH 7.1)/20% methanol to 40% 50 mM Tris buffer (pH 7.2)/60% methanol with the UV detector set to 363 nm.
  • Bioassay detection of MC was performed by loading a 1 cm disk with fractions eluting at the mitomycin retention time from HPLC injections of wild-type, MV100, pKC1139 vector control crude extracts and MC standards. The disks were placed on [0261] antibiotic media number 2 agar plates (Difco) with Bacillus subtilis spores added directly to the media. The plates were incubated overnight at 29° C. and examined for zones of inhibition. To confirm the production of MC by MV102 in the presence of exogenous AHBA the fraction eluting at the MC retention time was collected, dried down, desalted and submitted for desorption ionization mass spectrometric analysis on a Bio-Ion 20R DS-MS instrument (Applied Biosystems). The MC (M.W.=334)-sodium (M.W.=23) adduct peak, [M+Na]+=357, was diagnostic for the presence of MC in the AHBA supplemented culture.
  • Results [0262]
  • The mrd and ahbas genes are linked in the [0263] S. lavendulae genome. Southern blot analysis with the A. mediterranei AHBA synthase (rifK) gene probe (Kim et al., 1998) showed a single 3.8 kb band that hybridized with BamHI digested S. lavendulae genomic DNA (FIG. 8). Subsequently, a S. lavendulae genomic DNA library was constructed using the E. coli-Streptomyces shuttle cosmid pNJ1. Of the 5,000 colonies screened, 21 positive clones were identified with six of these hybridizing with the mrd gene probe (none hybridized with the mcr gene probe described in August et al., 1994). Restriction-enzyme mapping and reciprocal hybridization of the cosmid clones established that the mrd and S. mediterranei AHBA synthase homologous genes were about 20 kb apart in the S. lavendulae genome. The 3.8 kb BamHI fragment bearing a putative S. lavendulae AHBA synthase gene was subdloned and its nucleotide sequence determined.
  • Three ORFs are identified within the 3.8 kb BamHI fragment. Three ORFs (mitA, mitB, mitC) were identified within the sequenced 3.8 kb BamHI fragment (FIGS. 8 and 9). mitA comprises 1164 nucleotides and starts from ATG (position 579 of the sequenced fragment) that is preceded by a potential ribosome binding site (RBS), GAAAGG (SEQ ID NO:91). The deduced product of the mitA gene encodes a hydrophilic protein of 388 amino acids with a predicted M[0264] r of 41,949 Da and a calculated pI of 5.62. A BLAST (Altschul et al., 1990) search showed that the predicted MitA protein has high sequence similarity (about 71% identity, 80% similarity) with AHBA synthases (AHBASs), both from the rifamycin producer A. mediterranei (Kim et al., 1998) and other ansamycin-producing actinomycetes, including Actinosynnema pretiosum (ansamitocin) and Streptomyces collinus (naphthomycin A and ansatrienin) (FIG. 10). A conserved pyridoxal phosphate (PLP) coenzyme binding motif (GX3DX7AX8EDX14GX13KX4-5geGGX19G) (SEQ ID NO:92) including the conserved lysine residue can also be found in these four proteins (Piepersberg, 1994).
  • The mitB gene is predicted to start at a GTG (position 1879) that is preceded by a presumed RBS (GGAACG) (SEQ ID NO:93). This gene encodes a 272 amino acid protein with a deduced M[0265] r or 28,648 Da and a deduced pI of 6.06. Database sequence homology searches revealed that the product of mitB shows local sequence similarity with a group of O-glycosyltransferases involved in polysaccharide biosynthesis. One segment of 70 amino acid residues at the N-terminus of MitB has 43% similarity (36% identity) with the two glycosyltransferases SpsL and SpsQ from Sphingomonas S88, and ExoO form Rhizobium meliloti involved in polysaccharide (S88) and succinoglycan biosynthesis, respectively (Becker et al., 1963). Another 60 amino acid residues located at the C-terminus displayed 30% identity with UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from Mus musculus and Homo sapiens (Bennett et al., 1996).
  • The third ORF, mitC, starts from the ATG at position 2694, which is coupled to the stop codon TGA of mitB and encodes a putative protein of 260 amino acids with a molecular mass of 27,817 Da and a pI of 10.45. Database searches with the deduced protein product showed significant similarity over the first 90 amino acids (38% identity, 40% similarity) with the ImbE gene product (unknown function) from [0266] Mycobacterium leprae (U15183).
  • Insertional disruption of the mitA and mitB genes in [0267] Streptomyces lavendulae. To test the dependence of functional mitA and mitB genes for MC biosynthesis, gene disruption constructs were generated for subsequent isolation of the corresponding S. lavendulae isogenic mutant strains.
  • The mitA disruption construct was made by replacing a 155 bp fragment between the two MscI sites (located at the C-terminus of the mitA gene in pDHS7601) with the 1.1 kb SmaI-BamHI fragment containing a thiostrepton resistance gene from pDHS5000 (FIG. 11A). This replacement regenerated a BamHI site at the junction and the resulting construct was then subcloned into the [0268] E. coli-Streptomyces conjugative shuttle plasmid pKC1139, followed by conjugation into S. lavendulae. A double crossover mutant strain (MV100) was selected based on the expected phenotype (thiostrepton resistant, apramycin sensitive), and further confirmed by Southern blot hybridization. Genomic DNA from wild-type S. lavendulae and MV100 was digested with BamHI and SphI, and hybridized with the 4.9 kb EcoRI-HindIII tsr-disrupted mitA fragment from pDHS2001. As expected, the 4.0 kb SphI hybridized band in the wild-type strain was shifted to 4.9 kb in MV100, whereas the 3.8 kb BamHI hybridization and in the wild-type was converted to two bands (2.2 kb and 2.5 kb) in the mutant (FIG. 11B).
  • The mitB gene was disrupted by inserting a neomycin resistance gene (aphII) into the BstBI site (located at the 5′-end of mitB) (FIG. 12A). Transconjugants were selected on kanamycin/apramycin plates, and a double crossover mutant strain (MM101) was identified with a kanamycin-resistant, apramycin-sensitive phenotype and subsequently confirmed by Southern blot hybridization. As expected, the 3.8 kb BamHI hybridization band in wild-type [0269] S. lavendulae was shifted to 5.2 kb in MM101, whereas a 5.2 kb SacI hybridization band was shifted to 6.6 kb (FIG. 12B).
  • mitA and mitB disrupted strains (MV100 MM101) are blocked in MC biosynthesis. The growth characteristics and morphology of MV100 and MM1001 in liquid media and on agar plates was identical to wild-type [0270] S. lavendulae. HPLC was used to quantify production of MC in MV100 and MM101 (FIG. 13A), and culture extracts were used in a biological assay to test for presence of the drug (FIG. 13B). Injection of one mg of wild-type S. lavendulae culture extract gave a peak in the HPLC that eluted with the same retention time as the MC standard. Upon injection of one mg of culture extract from the mitA or mitB disrupted strains (MV100, MM101) no MC peak was observed. To corroborate the lack of production of MC, the HPLC eluant obtained from the MV100 culture extracts was collected over the retention time range determined for MC. This eluant completely lacked biological activity against Bacillus subtilis (the MC target strain) while the fraction collected from the same retention time region of wild-type S. lavendulae and the vector control strain culture extracts showed substantial levels of biological activity (FIG. 13B).
  • It is important to note that the presence of the vector pKC1139 in [0271] S. lavendulae reduced the percentage of MC in the total crude extract while simultaneously increasing the total amount of material extractable by ethyl acetate. The combination of these two effects reduces the absolute amount of MC by approximately 25% in the vector control culture crude extract compared to the wild-type crude extract.
  • Exogenous AHBA can restore MC production in the MC-deficient MitA K191A mutant. Although complementation of MV100 (mitA insertional disruptant) was attempted by providing exogenous 3-amino-5-hydroxybenzoic acid in the culture medium, MC production was not restored as measured by HPLC or biological assay. A polar effect on genes downstream of tsr-disrupted mitA in MV100 appeared likely since supplying mitA in trans on a medium copy number plasmid (MV103) also failed to restore MC production. Therefore, site-directed mutagenesis was employed to generate a MitA K191A mutant resulting in strain MV102. Kim et al. (1998) had demonstrated that the AHBA synthase from [0272] A. mediterranei is PLP dependent and catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS). Thus, the nitrogen of the conserved lysine 191 is supposed to form a Schiff base with the PLP cofactor. Replacement of lysine 191 with alanine prevents binding of the cofactor and eliminates enzymatic activity. Replacement of the AGG encoding lysine 191 in wild-type S. lavendulae with a GCC codon in MV102 was confirmed by nucleotide sequence analysis. As expected, MV102 did not produce MC, however, when the culture medium was supplemented with exogenous AHBA, MC production was restored as determined by MS ([M+Na]+=357), HPLC and TLC analysis (Table 2).
    TABLE 2
    Complementation results with (+) or without (−) AHBA.
    S. lavendulae MC production
    strains −AHBA +AHBA
    Wild-type + +
    MV100 − −
    MV103 − −
    MV102 − +
  • Discussion [0273]
  • An effective strategy for the identification of natural product biosynthetic gene clusters in actinomycetes has included cloning of antibiotic resistance genes followed by investigation of adjacent DNA for the presence of structural and regulatory genes (Butler et al., 1989, Donadio et al., 1991; Motamedi and Hutchinson, 1987; Vara et al., 1985). Although linkage of antibiotic resistance and biosynthetic genes appears to be a general feature in prokaryotes, a growing number of examples involve the existence of multiple resistance loci that may be linked or unlinked to the biosynthetic gene cluster (Vara et al., 1985; Seno and Baltz, 1989; Smith et al., 1995). The identification and characterization of two genetically unlinked resistance loci (August et al, 1994; Sheldon et al., 1997) for MC created a dilemma for mounting an effective search for the MC biosynthetic gene cluster. However, the use of the AHBA synthase gene from [0274] A. mediterranei provided an effective probe to identify cosmid clones bearing a linked MC resistance gene. Thus, the isolation of several cosmid clones form an S. lavendulae genomic DNA library that hybridized to both the A. mediterranei AHBA synthase gene and the S. lavendulae mrd gene indicated that the MC biosynthetic gene cluster resided on DNA adjacent to mrd. DNA sequence analysis of the 3.8 kb BamHI fragment revealed three ORFs whose deduced protein sequences corresponded to an AHBA synthase, a glycosyltransferase, and a ImbE-like product.
  • As determined by precursor feeding experiments, the mitosane core is formed through the condensation of AHBA and D-glucosamine (Hornemann, 1981). AHBA is thought to be derived from the ammoniated shikimate pathway from PEP and E4P, in which the last step from aminoDHS to AHBA is catalyzed by AHBA synthase (FIG. 7) (Kim et al., 1996; Kim et al., 1998). Meanwhile, the reaction of attaching an activated sugar residue to a core compound is usually catalyzed by a group of enzymes called glycosyltransferases as specified by macrolide, glycopeptide antibiotic and polysaccharide biosynthesis (Kahler et al., 1996; Otten et al., 1995b; Solenberg et al., 1997; Yamazaki et al., 1996). In principle, the condensation of AHBA with D-glucosamine can be initiated in two different ways (FIG. 7). One would involve the formation of the C[0275] 8a-C9 bond by an electrophilic aromatic alkylation or acylation. A second possibility would be formation of a Schiff base between the nitrogen of AHBA and the D-glucosamine C1 aldehyde, followed by ring closure at C8a-C9. In either case, a C- or N- instead of O-glycosyltransferase is expected. Although previously described glycosyltransferases display a high degree of sequence divergence (Yamazaki et al., 1996), the mechanistic similarity with O-glycosyl transfer may suggest that mitB encodes a N-glycosyltransferase that initiates the formation of the mitosane system by linking glucosamine to AHBA. The mitA and mitB genes and their corresponding products are likely candidates to mediate formation of AHBA and the mitosane ring system, respectively. However, the possible function of the lmbE-like protein remains unclear, since its current role within lincomycin biosynthetic pathway of S. lincolnensis is not known (Peschke, 1995).
  • The involvement of AHBA synthase (mitA) and the putative glycosyltransferase (mitB) in MC biosynthesis was established by gene disruption to create mutants blocked in MC biosynthesis. This required development of a method to introduce DNA into [0276] S. lavendulae NRRL 2564 since the strain remains refractory to traditional Streptomyces protoplast and electroporation-mediated transformation procedures. Other such refractory strains include, but are not limited to, ATCC 27422. The modified Bierman protocol (Bierman et al., 1992) was used to affect efficient conjugative transfer into S. lavendulae using the E. coli-Streptomyces shuttle plasmid pKC1139. This result is significant because it permits the development of an effective system for analyzing in detail the genes involved in mitomycin biosynthesis.
  • The function of mitA was probed by providing strains MV100 and MV102 with exogenous 3-amino-5-hydroxybenzoic acid in the culture medium. Despite repeated attempts to complement MV100, MC production was not restored as measured by HPLC or biological assay. It is believed that insertion of the tsr gene into mitA resulted in disruption of biosynthetic genes immediately downstream, since supplying mitA in trans on a medium copy number plasmid also failed to restore MC production to MV100. This putative polar effect was eliminated by generating the MitA K191A mutant strain MV102. Providing exogenous 3-amino-5-hydroxybenzoic acid to this mutant strain of [0277] S. lavendulae restored production of MC as shown by TLC, HPLC and mass spectrometry. When MV102 was grown in the absence of AHBA, there was no detectable production of MC. The ability of 3-amino-5-hydroxybenzoic acid to complement the mutant MitA protein further supports the function of MitA as an AHBA synthase as indicated by the database protein sequence alignment and previous studies on rifK (Kim et al., 1998).
    • EXAMPE 3 Mitomycin Resistance in Streptomyces lavendulae Includes a Drug-Binding Protein-Dependent Export System
  • As a prodrug, MC is unreactive until chemical or enzymatic reduction renders the molecule a highly effective alkylating agent (Iyer and Szybalski, 1964). The molecular basis of MC bioactivity derives mainly from its propensity to covalently interact with DNA at 5′-CpG sequences, causing lethal intra- and inter-strand crosslinks as well as monofunctional alkylation (Tomasz, 1995). [0278]
  • [0279] S. lavendulae encounters a daunting challenge in avoiding potentially lethal MC-mediated crosslinks since it has a chromosomal G+C content of over 70%, which translates into at least one million potential drug target sites per cell. Indeed, two genetic loci that mediate mitomycin resistance have been reported in this organism. One locus (mcr) encodes a protein (MCRA) that catalyzes oxidation of the reduced, bioactivated species of MC via a redox relay mechanism (August et al., 1994; Johnson et al., 1997). The second locus (mnrd) encodes MRD that functions to sequester the prodrug by a specific mitomycin-binding protein (Sheldon et al., 1997). A paradox of current knowledge regarding mitomycin resistance has been the lack of a clear mechanism for drug transport. Indeed, the observed stoichiometry suggests that it would be ineffective for S. lavendulae to utilize MRD as a solo mechanism for cellular self-protection. Pathogenic bacteria (Nikaido, 1994), and antibiotic-producing microorganisms (Cundliffe, 1992; Mendez and Salas, 1998), employ export of toxic compounds as a means of resistance.
  • Materials and Methods [0280]
  • Bacterial strains, culture conditions, and media. [0281] E. coli DH5α used as a host for generation of double-stranded plasmid DNA, was grown at 37° C. on LB medium. E. coli BL21 (DE3), used as host for protein expression, was grown at 37° C. in NZCYM medium (Sambrook et al., 1989). S. lavendulae NRRL 2564 was grown on YEME medium (Hopwood et al., 1985) at 30° C. for preparation of genomic DNA.
  • DNA preparation and amplification. [0282] S. lavendulae genomic DNA was isolated by the lysozyyne-2X Kirby mix method (Hopwood et al., 1988). General DNA manipulation was performed as described previously (August et al., 1994). Oligonucleotides for PCR and sequencing were obtained from Gibco BRL. PCR amplifications were carried out using a Hybaid thermal cycler (Hybaid Ltd., Teddington, U.K.).
  • Cloning and sequencing of mct. A [0283] S. lavendulae NRRL 2564 genomic DNA library was constructed in the cosmid vector pNJ1 (Tuan et al., 1990) as previously described (August et al., 1994). The insert DNA of a cosmid clone containing sequences flanking mrd was digested with BamHI and subcloned into the BamHI site of pUC119. Using exonuclease III (Erase-A-Base kit, Promega, Madison, Wis.), a set of nested deletion clones was generated and both strands of the insert DNA were sequenced by the dideoxy chain termination method using the ABI Prism kit (PE Applied Biosystems) in coordination with an ABI 373 automated sequencer. 10% DMSO was added to the reactions to reduce compressions. Sequence data was analyzed using the GeneWorks (Oxford Molecular) software package. Deduced amino acid sequence data were compared to the available databases using the BLAST program of the Genetics Computer Group version 9.0 software (Oxford Molecular Group). The met gene has been deposited in the GenBank database under Accession No. AF120930.
  • Construction of the met mutant strain of [0284] S. lavendulae. The mct disruption vector pDHS7704 was constructed as follows. pDHS7661 was digested with EcoRI, blunt-ended, and ligated with the 1.4 kb neomycin resistance gene fragment from pFD666 (ApaLI-HindIII digestion, blunt-ended) (Ames, 1986). The 5.4 kb EcoRI-HindIII fragment from the resulting construct (pDHS7703) was subcloned into pKC1139 to create pDHS7704, and conjugated into S. lavendulae according to Bierman et al. (1992). A met double crossover mutant was selected after propagating transconjugants on R5T plates for five generations at 39° C. Kanamycin-resistant and apramycin-sensitive colonies were further tested by Southern blot to confirm the desired double crossover genotype.
  • Construction of mct expression plasmid. For the construction of the [0285] E. coli expression plasmid NdeI and HindIII sites were introduced at the translational start codon and downstream of the translational stop codon of mct, respectively. The primers used for PCR were 5′-GGGAATTCCATATGATGCAGTCCATGTCAC-3′ (SEQ ID NO:94) and 5′-GGGAATTCAAGCTTTCATTCCGCCGGGGTC-3′ (SEQ ID NO:95). The PCR was carried out using 2.5 U of Taq polymerase, 0.4 μg of each primer, 1 μg of pDHS7661 DNA as template, 10 mM each of dATP-dGTP-dCTP-dTTP, 1.5 mM MgCl2, and 10 μl of 10×Promega PCR buffer in a total volume of 100 μl. Amplification was achieved with 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 37° C. for 1 minute, and extension at 70° C. for 2 minutes. The 1.45 kb PCR product was recovered by 0.8% agarose gel electrophoresis, digested with NdeI-HindIII and ligated into the T7 expression plasmid pET17b (Novagen), which had been similarly cut with EcoRI-HindIII, to give pDHS7023. pDHS7023 was introduced by transformation into E. coli BL21(DE3) to provide strain PJS102.
  • Construction of mct-mrd co-expression plasmid. From plasmid pDHS7006 (Sheldon et al., 1997), a 2.1 kb SspI fragment was isolated. The fragment contained the mrd gene under the control of the T7 promoter, including transcriptional terminator sequences (rrnB T1) upstream and downstream of mrd. The fragment was ligated into the MC-translocase construct pDHS7023, which had been cut with MscI, to give pDHS7024. pDHS7024 was introduced by transformation into [0286] E. coli BL21 (DE3) to result in strain PJS103.
  • MC resistance phenotype of [0287] E. coli. To analyze resistance conferred by the expression of the MC-translocase in E. coli, 10 μl of strain PJS102 was spread on LB agar medium containing 100 ∞g/ml of ampicillin, IPTG to a final concentration of 1.0 mM, and various concentrations of MC. The cultures were grown overnight at 37° C. and colony-forming units (CFUs) were deternined. Similarly, the MC resistance phenotype of strain PJS103 (mcr-mrd co-expression strain) was quantified.
  • [[0288] 3H]-MC uptake assay of strains PJS102 and PJS103. [3H]-MC was obtained from Kyowa Hakko Kogyo, Ltd. Uptake studies were performed for whole cells of PJS100, PJS102, PJS103 and E. coli BL21(DE3)::pT7SC and pET17b. PJS100, PJS102, and PJS103 as well as vector-only cultures were cultured (37° C.) in 5 ml of NCZYM medium with IPTG added to a final concentration of 1 mM (at approximately 3 hours growth). At 9 hours (late exponential phase), cells were harvested by centrifugation and resuspended in 1 ml NCZYM broth (5×concentration). The concentrated suspension of late-exponential growth phase cells was exposed to [3H]-MC (59 Ci/mmol) at a final concentration of 0.022 μg/ml (0.0655 nmol). Aliquots (100 μl) were removed at frequent intervals, placed on 1.2 μM GF/C filters (Whatman International, Maidstone, U.K.) and washed once with 6 ml of 0.85% NaCl poured over the filters under vacuum pressure. Additional aliquots were simultaneously removed for determination of protein content (protein assay kit, Bio-Rad Laboratories, Richmond, Calif.). Radioactivity on the filters was quantified using a Beckman LS7000 scintillation counter. Results were expressed as nanograms of mitomycin per milligram of cell protein.
  • Results [0289]
  • A gene encoding a transmembrane protein is physically linked to mrd. DNA sequence analysis of a cosmid clone containing the mrd locus, a previously characterized MC resistance determinant (Sheldon et al., 1997), identified an open reading frame (ORF) encoding a polypeptide predicted to be highly hydrophobic that shows similarity to a variety of antibiotic export proteins in drug-producing actinomycetes. Significantly, the gene (mct, SEQ ID NO:72) encoding the putative mitomycin exporter (MC-translocase; MCT) protein is located within 5 kb of mrd (SEQ ID NO:64) and is physically linked to the mitomycin biosynthetic gene cluster (FIG. 15). [0290]
  • Sequence analysis of the mct locus. Nucleotide sequence analysis of cosmid clone pDHS7547 revealed an ORF predicted to start with the ATG codon at position 132 and end with the TGA codon at nucleotide 1587 (FIG. 16), resulting in a 484 amino acid polypeptide with a predicted molecular weight of 50,023 daltons. Comparison of the deduced amino acid sequence of the mct gene with proteins in the available databases revealed significant similarity to several integral membrane proteins that confer drug resistance. These include the CmcT protein from the cephamycin producer, [0291] Nocardia lactamdurans (Coque et al., 1993), the Pur8 protein from the puromycin producer, Streptomyces alboniger (Tercero et al, 1993), the Mmr protein from the methylenomycin producer, Streptomyces coelicolor (Neal and Chater, 1987), and the LmrA protein from the lincomycin producer, Streptomyces lincolnensis (Zhang et al., 1992). The similarities of the mct gene product and related proteins extend over the entire sequences, with the highest levels of similarity found within the amino-terninal regions (FIG. 17).
  • Within the N-terminal regions of several antibiotic efflux proteins, including Mmr and LmrA, several highly conserved structural motifs have been noted. The β-turn motif (VxGxLxDxxGRKxxxL), found within the highly conserved cytoplasmic loop sequence separating transmembrane domains two and three of most eukaryotic and prokaryotic transport proteins, is clearly evident in MCT at positions 79-95 (FIG. 16). A motif (LDxTVxNVALP) found at the end of transmembrane domain one, specific to the 14 transmembrane segment family (Paulson and Skurray (1993)) is present in MCT at positions 41-51 (FIG. 16). In addition, several other invariant motifs are apparent in the MCT sequence. [0292]
  • Transmembrane proteins that mediate resistance to antibiotics and antiseptics by active efflux are highly related, usually containing 12 or 14 transmembrane regions. Notably, the actinomycete drug transport proteins that share homology with MCT appear to contain 14 transmembrane spanning regions and constitute a family of drug resistance translocases. Utilizing the membrane structure and topology program MEMSAT (University College, London), and hydropathy analyses based on the algorithm of Kyte and Doolittle (1982), a prediction of 14 transmembrane spanning domains was made for the deduced amino acid sequence of MCT (FIG. 18). [0293]
  • Inactivation of met results in greater sensitivity to MC. To establish a physiological role for MCT in [0294] S. lavendulae, the corresponding gene (mct) was inactivated by insertion of the aphII gene from transposon Tn5 to give pDHS7704. After conjugal transfer of pDHS7704 from E. coli to S. lavendulae and growth of the transconjugants under selective conditions, targeted replacement of native met was achieved by double crossover homologous recombination. Gene disruption was confirmed by Southern blot hybridization of total DNA from the S. lavendulae wild-type and mutant with a DNA probe that included the mct locus. Analytical digests of the genomic DNA resulted in detection of the predicted band shifts in the mutant and wild-type strains (FIG. 19). The S. lavendulae met disruption mutant strain (MM105) exhibited an approximately 25-fold reduction in resistance to MC when exposed to 100 μg of MC per ml of medium (Table 3). In media lacking MC, the growth kinetics of the strain MM105 was comparable to the wild-type S. lavendulae strain.
    TABLE 3
    Resistance of S. lavendulae strains to varying concentrations of MC
    Plate count CFU/ml
    Strain
    Concentration S. lavendulae mct mutant
    of MC (μg/ml) S. lavendulae wild-type (MM 105)
    10 >107 >107
    20 >107 >107
    40 5.3 × 103 2.6 × 103
    80 2.6 × 103 2.4 × 102
    100  2.0 × 103 8.0 × 101
  • Expression of mct in [0295] E. coli. To investigate further the function of mct, heterologous expression of the gene in E. coli was pursued. mct was amplified by PCR and cloned into the protein expression vector pET17b to give pDHS7023. pDHS7023 was then introduced into E. coli BL21(DE3) to give strain PJS102. After disruption of the cells by sonication, MCT was found to be associated mainly with the membrane fraction of the cell lysate, as expected for an integral membrane protein. To determine if strain PJS102 was resistant to MC, cultures were grown up and plated on agar medium containing various concentrations of MC. Significantly, IPTG-induced cultures of PJS102 exhibited resistance to MC at drug concentrations 5-fold greater than those for E. coli BL21(DE3) containing vector alone (Table 4).
    TABLE 4
    MC resistance of mct, mrd expressing E. coli strains
    Plate count CFU/ml
    Strain
    Concentration BL21(DE3)::
    of MC(μg/ml) pET17b PJS100 PJS102 PJS103
    0.0 >107 >107 >107 >107
    0.01 >107 >107 >107 >107
    0.1 7.3 × 103 >107 >107 >107
    0.5 3.2 × 102 >107 2.1 × 105 >107
    1.0 0.0 3.3 × 106 5.9 × 104 >107
    2.5 — NAa 2.0 × 102 >107
    5.0 — 2.7 × 106 0.0 >107
    10 — 6.1 × 105 — >107
    20 — 2.5 × 105 — >107
    30 — 5.0 × 102 — >107
    60 — 0.0 — >107
    80 — — — 1.4 × 105
    100 — — — 9.6 × 103
    150 — — — 3.0 × 101
    Mitomycin B — >107b NAc >107d
  • Co-expression of mct and mrd in [0296] E. coli. To address the notion that MRD and MCT proteins participate as components of a binding protein-dependent drug export system, the mct and mrd genes were co-expressed in E. coli. From plasmid pDHS7006 (mrd expression construct) (Sheldon et al., 1997), a DNA fragment containing the mrd gene under the control of the T7 promoter was ligated into pDHS7023 to give pDHS7024. pDHS7024 was then introduced into E. coli BL21(DE3) to give strain PJS103. To determine if strain PJS103 was resistant to MC, cultures were grown up and plated on agar medium containing various concentrations of MC. Significantly, EPTG-induced cultures of PJS103 exhibited resistance to MC at drug concentrations 300-fold greater than those for E. coli BL21(DE3) containing vector alone (150 μg/ml vs. 0.5 μg/ml of MC; Table 4). In addition to PJS103 maintaining levels of resistance over that of the vector control strain, co-expression of mct and mrd confers MC resistance at drug concentrations 5 and 60-fold greater compared to PJS100 (containing the mrd gene alone) (Sheldon et al., 1997) or strain PJS102 (containing the mct gene alone), respectively. Strain PJS103 also displayed high-level resistance to mitomycin B (Table 4), a mitomycin also produced by S. lavendulae.
  • MC uptake by [0297] E. coli cells expressing mct, mrd or mct/mrd. Since the deduced amino acid sequence of the mct gene was similar to antibiotic export proteins, reduced accumulation of MC in MCT-expressing cells would be expected. An assay, modeled after experiments used to study tetracycline efflux-mediated resistance in E. coli (Levy and McMurry, 1978), was designed to study the uptake of [3H]-MC by the susceptible vector control and resistant mct, mrd and mct/mrd expressing E. coli strains.
  • MC accumulation by the susceptible vector control strain (BL21(DE3)::pET17b) was found to reach a maximum level at 5 minutes and thereafter maintained at constant concentrations. In contrast, the quantity of MC accumulation in the resistant, mct-expressing strain (PJS102) was only 25% of the susceptible control at 5 minutes, and thereafter remained at reduced concentrations (FIG. 21). Reduced accumulation of drug in PJS102 suggests that mct encodes a protein that facilitates MC export from the cell. To determine if the co-expression of mct and mrd in [0298] E. coli also resulted in reduced accumulation of MC, strain PJS103 was analyzed using the [3H]-MC uptake assay. Analyses of drug uptake by cultures of strain PJS100 (Sheldon et al., 1997) were also performed to determine drug accumulation levels in this MC resistant E. coli strain.
  • The results show a clear difference in MC accumulation between the MC sensitive and resistant strains. Compared to [0299] E. coli cells bearing vector alone, MC accumulation in PJS103 was only 35% at 5 minutes and thereafter remained at reduced concentrations. The accumulation of drug in strain PJS103 was found to parallel that of strain PJS102, albeit at slightly higher levels (about 23% greater) of drug over the course of the experiment. Interestingly, strain PJS100, although resistant to significant concentrations of MC, accumulated drug to levels 42% higher than the drug-sensitive vector control at 30 minutes (FIG. 20).
  • Discussion [0300]
  • Most antibiotics inhibit bacterial growth by binding to proteins or other macromolecular components that involve essential metabolic processes of the cell (Cundliffe, 1992). For instance, DNA alkylation by MC results in disruption of chromosomal replication leading to cell death (Iyer and Szybalski, 1964). In many antibiotic-producing streptomycetes, macromolecular target site(s) are likewise susceptible to endogenous cytotoxic compounds (that is certainly the case in [0301] S. lavendulae). Thus, pumping the antibiotic out of the cell at a rate equal to its production and/or re-uptake would prevent drug access to intracellular target sites. Based on the levels of drug found in most antibiotic fermentation broths (concentrations of intracellular drug being low), it is apparent that drug-producing organisms often depend on efficient antibiotic transport mechanisms. Indeed, a growing number of membrane systems implicated in transport (and therefore resistance) of a variety of antibiotics have been discovered in drug-producing streptomycetes (Mendez and Salas, 1998; Paulsen et al., 1996).
  • In general, genes coding for drug export proteins are physically linked to the corresponding biosynthetic genes within the genome of the antibiotic-producing microorganism. Presumably, the tight linkage of antibiotic export and biosynthetic genes ensures coordinate gene regulation. Interestingly, the presence of back-to-back and overlapping divergent promoters of antibiotic export and regulatory genes has been observed within the tetracenomycin (Guilfoile and Hutchinson, 1992) and actinorhodin (Caballero et al, 1991) biosynthetic gene clusters. Conforming to this example, [0302] S. lavendulae possesses a gene coding for an integral membrane drug export protein within the mitomycin biosynthetic gene cluster. Analysis of the deduced amino acid sequence of MCT revealed several similarities with actinomycete proteins predicted to function as drug exporters. By virtue of homology to tetracycline resistance proteins, which have been shown to use proton motive force to energize transport (Littlejohn et al., 1992), the actinomycete drug resistance translocases cited in this study are predicted to power excretion by a proton-dependent electrochemical gradient. It has been suggested that highly conserved sequences within the amino-terminal regions of these proteins play a role in proton translocation (Rouch et al., 1990), while the less well-conserved C-terminal regions may be involved in drug binding (Paulsen et al., 1996; and references therein) or recognition of a protein-drug complex.
  • Disruption of met in [0303] S. lavendulae resulted in a 25-fold increase in sensitivity to exogenously added MC, providing evidence that MCT maintains a role in providing drug resistance in S. lavendulae. Although the effect is significant, alternative mechanisms of cellular self-protection clearly continue to operate. This evidently includes MCRA, the novel redox-relay protein that re-oxidizes activated MC in S. lavendulae. It is also likely that unidentified xenobiotic transporters provide an alternative mode of drug transport in the absence of MCT, albeit with lower efficiency.
  • In order to probe the ability of MCT to transport drug in the presence and absence of the MC binding protein, accumulation of [[0304] 3H]-MC in E. coli was analyzed. Expression of met in E. coli resulted in MC-resistant cultures that accumulated lower levels of drug than strains bearing vector control (FIG. 20). Interestingly, strain PJS102 (expressing mct only) accumulates less drug intracellularly than strain PJS103 (expressing mrd and met) (FIG. 20). Increased drug accumulation in strain PJS100 may lend support to the model of equimolar binding between MRD and MC (Sheldon et al., 1997). Significantly higher levels of drug accumulation in strain PJS100 may be the result of intracellular sequestration of MC by MRD. Accordingly, the presence of MRD could also account for the slightly greater levels of MC accumulation in strain PJS103 (co-expressing mct-mrd) as compared to strain PJS102 (expressing met alone). Comparable to binding protein-dependent import systems (Miller et al., 1983), the binding of MC by MRD may be rate-limiting in the drug excretion process.
  • Taken together, these results suggest that cellular protection afforded by MCT is a function of drug transport from the cytoplasm. Interestingly, co-expression of mrd and met in [0305] E. coli led to cultures that are dramatically more resistant to exogenously added drug. While normally required for the transport systems with which they are associated, in many instances binding proteins are not integral to the process of solute translocation (Higgins et al., 1990). Similarly, the presence of MRD is not required for MC translocation but dramatically enhances drug tolerance. Hence, the binding protein (MRD) may be considered an accessory component, a rather specific adaptation required for optimal drug resistance. The drug-resistance phenotype of E. coli strains expressing met alone and in combination with mrd along with the MC uptake analysis of these strains provides evidence that MRD and MCT are components of a novel drug transport system. Such a resistance mechanism, sequestering the intact drug for efficient excretion to the environment, represents a unique cellular strategy for self-preservation by the MC-producing organism.
  • References [0306]
  • Alderson, G., D. A. Ritchie, C. Caballero, R. H. Cool, N. M. Ivanova, A. S. Huddleston, C. S. Flaxman, V. Kristufek, and A. Lounes. Physiology and genetics of antibiotic production and resistance. [0307] Res. Microbiol., 144, 665-672 (1993).
  • Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and D. J. Lipman. Basic local alignment search tool, [0308] J. Mol. Biol., 215, 403-410 (1990).
  • Ames, G. Bacterial periplasmic transport systems: structure, mechanism, and evolution. [0309] Ann. Rev. Biochem., 55, 397-425 (1986).
  • Anderson, M. G., Kibby, J. J., Rickards, R. W. and J. M. Rothschild. Biosynthesis of the mitomycin antibiotics from 3-amino-5-hydroxybenzoic acid, [0310] J. Chem. Soc. Chem. Commun., 1277-1278 (1980).
  • Angell, S., Schwarz, A., and M. J. Bibb. The glucose kinase gene of [0311] Streptomyces coelicolor A3(2): its nucleotide sequence, transcriptional analysis and role in glucose repression, Mol. Microbiol., 6, 2833-2844 (1992).
  • August, P. R., Flickinger, M. C. and D. H. Sherman. Cloning and analysis of a locus (mcr) involved in mitomycin C resistance in [0312] Streptomyces lavendulae, J. Bacteriol., 176, 4448-4454 (1994).
  • August, P. R., Tang, L., Yoon, Y. J., Ning, S., Muller, R., Yu, T. W., Taylor, M., Hoffhann, D., Kim, C. G., Zhang, X., Hutchinson, C. R. and H. G. Floss. Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of [0313] Amycolatopsis mediterranei S699, Chem. Biol., 5, 69-70 (1998).
  • Baltz, R. H. Genetic recombination in [0314] Streptomyces fradiae by protoplast fusion and cell regeneration, Dev. Ind. Microbiol., 21, 43-54 (1980).
  • Baltz, R. H., and T. J. Hosted. Molecular genetic methods for improving secondary-metabolite production in actinomycetes, [0315] Trends Biotech., 14:245-250 (1996).
  • Beck, A., A. Kleickmann, M. Keller, W. Arnold, and A. Puhler. Identification and analysis of the [0316] Rhizobium meliloti exoAMONP genes involved in exopolysaccharide biosynthesis and mapping of promoters located on the exoHKLAMONP fragment, Mol. Gen. Genet., 241, 367-379 (1993).
  • Becker, A. M., Herlt, A. J., Hilton, G. L., Kibby, J. J. and R. W. Rickards. 3-Amino-5-hydroxybenzoic acid in antibiotic biosynthesis, VI. Directed biosynthesis studies with ansamycin antibiotics, [0317] J. Antibiot., 36, 1323-1328 (1983).
  • Bennett, E. P., H. Hassan, and H. Clausen. cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-D-galactosamine. Polypeptide N-acetylgalatosaminyltransferase, GalNAc-t3. [0318] J. Biol. Chem., 271, 17006-17012 (1996).
  • Berdy, J. Are actinomycetes exhausted as a source of secondary metabolites?, p. 13-14. In V. Debabov, Dudnik, Y. And Danlienko, V. (eds.), Ninth International Symposium on the Biology of Actinomycetes. All-Russia Scientific Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow (1995). [0319]
  • Bezanson, G. S. and L. C. Vining. Studies on the biosynthesis of mitomycin C by [0320] Streptomyces verticillatus, Can. J. Biochem., 49, 911-918 (1971).
  • Biermnan, M., Logan, R., O'Brien, K., Seno, E. T., Rao, R. N. and B. E. Schoner. Plasmid cloning vectors for the conjugal transfer of DNA from [0321] Escherichia coli to Streptomyces spp., Gene, 116, 43-49 (1992).
  • Blattner, F. R., Plunkett, G. R., Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B. and Shao, Y., the complete genome sequence of [0322] Escherichia coli K-12, Science, 277, 1453-74 (1997).
  • Bouvier-Nave, P., Husselstein, T., Desprez, T. and Benveniste, P., Identification of cDNAs encoding sterol methyl-transferases involved in the second methylation step of plant sterol biosynthesis, [0323] Euro. J. Biochem., 246, 518-29 (1997).
  • Boyer, M. J., Bioreductive agents: a clinical update, [0324] Oncol. Res., 9, 391-395 (1997).
  • Brown, W. C., and J. L. Campbell. A new cloning vector and expression strategy for genes encoding proteins toxic to [0325] Escherichia coli, Gene, 127:99-103 (1993).
  • Bult, C. J., White, O., Olsen, G. J., Zhou, L., Flesichmann, R. D., Sutton, G. G., Blake, J. A., FitzGerald, L. M., Clayton, R. A., Gocayne, J. D., Kerlavage, A. R., Dougherty, B. A., Tomb, J. F., Adams, M. D., Reich, C. I., Overbeek, R., Kirkness, E. F., Weinstock, K. G., Merrick, J. M., Glodek, A., Scott, J. L., Geoghagen, N. and J. C. Venter. Complete genome sequence of the methanogenic archaeon, [0326] Methanococcus jannaschii, Science, 273, 1058-1073 (1996).
  • Butler, M. J., E. J. Friend, I. S. Hunter, F. S. Kaczmarek, D. A. Sugden, and M. Warren. Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by [0327] Streptomyces rimosus. Mol. Gen. Genet., 215, 231-238 (1989).
  • Caballero, J. L., Malpartida, F. and D. A. Hopwood. Transcriptional organization and regulation of an antibiotic export complex in the producing Streptomyces culture, [0328] Mol. Gen. Genet., 228, 372-380 (1991).
  • Chater, K. F. Genetic regulation of secondary metabolic pathways in Streptomyces. [0329] Ciba Foundation Symposium, 171, 144-156 (1992).
  • Chater, K. F. and C. J. Bruton. Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered, [0330] Embo Journal, 4, 1893-7 (1985).
  • Chiao, J. S., T. H. Xia, B. G. Mei, Z. K. Jin, and W. L. Gu. Rifamycin SV and related ansamycins, p. 477-498. In L. C. Vining and Stuttard, C. (Eds.), Genetics and biochemistry and antibiotic production. Butterworth-Heinemann, Newton, Mass. (1995). [0331]
  • Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. R., Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Barrell, B. G. and et al., Deciphering the biology of [0332] Mycobacterium tuberculosis from the complete genome sequence, Nature, 393, 537-544 (1998).
  • Coque, J., P. Liras, and J. Martin. Genes for a β-lactamase, a penicillin-binding protein and a transmembrane protein are clustered with the cephamycin biosynthetic genes in [0333] Nocardia lactamdurans. EMBO. J., 12, 631-639 (1993).
  • Coque, J. J., Perez-Laraine, F. J., Enguita, F. J., Fuente, J. L., Martin, J. F. and P. Liras. Characterization of the cmcH genes of [0334] Nocardia lactamdurans and Streptomyces clavuligerus encoding a functional 3′-hydroxymethylcephem O-carbamoyltransferase for cephamycin biosynthesis, Gene, 162, 21-27 (1995).
  • Cummings, J., Spanswick, V. J., Tomasz, M. and J .F. Smyth. Enzymology of mitomycin C metabolic activation in tumor tissue—implications for enzyme-directed bioreductive drug development, [0335] Biochemical Pharmacology, 56, 405-414 (1998).
  • Cundliffe, E. Self-protection mechanisms in antibiotic producers. [0336] Ciba Found. Symp., 171, 199-208 (1992).
  • Cundliffe, E., L. A. Merson-Davies, and G. H. Keleman. Aspects of tylosin production and resistance in [0337] Streptomyces fradiae, p. 235-243, Industrial microorganisms: basic and applied molecular genetics. American Society for Microbiology, Washington, D.C. (1993).
  • Dean, C. R., Neshat, S. and K. Poole. PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in [0338] Pseudomonas aeruginosa, J. Bacteriol., 178, 5361-5369 (1996).
  • Decker, H., Motamedi, H. and C. R. Hutchinson, Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in [0339] Streptomyces glaucescens, J. Bacteriol., 175, 3876-3886 (1993).
  • Denis, F. and R. Brzezinski, A versatile shuttle cosmid vector for use in [0340] Escherichia coli and Actinomycetes, Gene, 111, 115-118 (1992).
  • Deppenmeier, U., Muller, V. and G. Gottschalk. Pathways of energy conservation in methanogenic archaea, [0341] Arch. Microbiol., 165, 149-163 (1996).
  • Devereux, J., Haeberli, P. and O. Smithies. A comprehensive set of sequence analysis programs for the VAX, [0342] Nucleic Acids Res., 12, 387-395 (1984).
  • Dewick, P. M., The biosynthesis of shikimate metabolites, [0343] Nat. Prod. Rep., 15, 17-58 (1995).
  • Dickens, M. L., Ye, J. and W. R. Strohl. Analysis of clustered genes encoding both early and late steps in daunomycin biosynthesis by Streptomyces sp. strain C5. [0344] J. Bacteriol., 177, 536-543 (1995).
  • Dittrich, H. and T. M. Kutchan. Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attach, [0345] Proc. Natl. Acad. Sci. USA, 88, 9969-9973 (1991).
  • Donadio, S., M. J. Staver, J. B. McAlpine, S. J. Swanson, and L. Katz. Modular organization of genes required for complex polyketide biosynthesis. [0346] Science, 252, 675-679 (1991).
  • Evans, J. W., Yudoh, K., Delahoussaye, Y. M. and J. M. Brown. Tirpazamine is metabolized to its DNA-damaging radical by intranuclear enzymes, [0347] Cancer Research, 58, 2098-2101 (1998).
  • Femandez-Moreno, M. A., Caballero, J. L., Hopwood, D. A. and F. Malpartida. The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces, [0348] Cell, 66, 769-80 (1991).
  • Floss, H. G. Natural products derived from unusual variants of the shikimate pathway, [0349] Nat. Prod. Rep., 14, 433-52 (1997).
  • Ghisalba, O., and N. Nuesch. A genetic approach to the biosynthesis of the rifamycin-chromophore in [0350] Nocardia mediterraniae. IV. Identification of 3-amino-5-hydroxybenzoic acid as a direct precursor of the seven-carbon amino starter-unit. J. Antibiot., 34, 64-71 (1981).
  • Gibson, J., Dispensa, M., Fogg, G. C., Evans, D. T. and C. S. Harwood. 4-Hydroxybenzoate-coenzyme A ligase from [0351] Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation, J. Bacteriol., 176, 634-641 (1994).
  • Grebenok, R. J., Galbraith, D. W. and D. D. Penna. Characterization of [0352] Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants, Plant Mol. Biol., 34, 891-6 (1997).
  • Grkovic, S., Brown, M. H., Roberts, N. J., Paulsen, I. T. and R. A. Skurray. QacR is a repressor protein that regulates expression of the [0353] Staphylococcus aureus multidrug efflux pump QacA, J. Biol. Chem., 273, 18665-73 (1998).
  • Guilfoile, P. G. and C. R. Hutchinson. Sequence and transcriptional analysis of the [0354] Streptomyces glaucescens tcmAR tetracenomycin C resistance and repressor gene loci, Journal of Bacteriology, 174, 3651-3658 (1992).
  • Guilfoile, P. G. and C. R. Hutchinson. The [0355] Streptomyces glaucescens TcmR protein represses transcription of the divergently oriented tcmR and tcmA genes by binding to an intergenic operator region, Journal of Bacteriology, 174, 3659-66 (1992).
  • Hardwick, K. G. and H. R. Pelham. SED6 is identical to ERG6, and encodes a putative methyltransferase required for ergosterol synthesis, [0356] Yeast, 10, 265-269 (1994).
  • Hata, T., Sano, Y., Sugawara, R., Matsumae, A., Kanamori, K., Shima, T. and T. Hoshi. Mitomycin, a new antibiotic from Streptomyces, [0357] J. Antibiot. Ser. A, 9, 141-146 (1956).
  • Hatano, K., S. Akiyama, M. Asai, and R. W. Richards. Biosynthetic origin of amino benzenoid nucleus (C[0358] 7N-unit) of ansamitocin, a group of novel maytansinoid antibiotics. J. Antibiot., 35, 1415-1417 (1982).
  • Haydock, S. F., Dowson, J. A., Dhillon, N., Roberts, G. A., Cortes, J. and P. F. Leadlay. Cloning and sequence analysis of genes involved in erythromycin biosynthesis in [0359] Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases, Mol. Gen. Genet., 230, 120-128 (1991).
  • Henderson, I. C., Recent Advances in the Usage of Mitomycin, Proceedings of a symposium, Hawaii, March 21-24, [0360] Oncology, 1, 1-83 (1993).
  • Henderson, C. I., Recent advances in the usage of mitomycin, [0361] Oncology, 50:(Suppl. 1), 1-84 (1993).
  • Hidaka, T., Goda, M., Kuzuyama, T., Takei, N., Hidaka, M. and H. Seto. Cloning and nucleotide sequence of fosfomycin biosynthetic genes of [0362] Streptomyces wedmorensis, Mol. Gen. Genet., 249, 274-280 (1995).
  • Hidaka, T., Hidaka, M., Kuzuyama, T. and H. Seto. Sequence of a P-methyltransferase-encoding gene isolated from a bialaphos-producing [0363] Streptomyces hygroscopicus, Gene, 158, 149-150 (1995).
  • Higgins, C., S. Hyde, M. Mimmack, U. Gileadi, D. Gill, and M. Gallagher. Binding protein-dependent transport systems. [0364] J. Bioenerg. Biomem., 22, 571-592 (1990).
  • Hirai, O., Miyamae, Y., Hattori, Y., Takashima, M., Miyamoto, A., Zaizen, K. and Y. Mine. Microbial mutagenicity an in vitro chromosome aberration induction by fk973, a new antitumor agent, [0365] Mutation Res., 324, 43-50 (1994).
  • Hopwood, D. A. Genetic contributions to understanding polyketide synthases. [0366] Chem. Rev., 97, 2465-2497 (1997).
  • Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M. and H. S. Schrempf. [0367] Genetic manipulation of Streptomvces: a laboratory manual, John Innes Institute, Norwich, United Kingdom, 1985.
  • Horii, M., Ishizaki, T., Paik, S. Y., Manome, T. and Y. Murooka. An operon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from a Streptomyces sp., [0368] J. Bacteriol., 172, 3644-3653 (1990).
  • Hornemann, U., [0369] Biosynthesis of the mitomycins, 1981.
  • Hornemann, U. and J. H. Eggert. Utilization of the intact carbamoyl group of L-(NH[0370] 2CO-13C,15N) citrulline in mitomycin biosynthesis by Streptomyces verticillatus, Journal of Antibiotics, 28, 841-843 (1975).
  • Hornemann, Y., Kehrer, J. P., Nunez, C. S. and R. L. Ranieri. D-glucosamine and L-citrulline, precursors in mitomycin biosynthesis by [0371] Streptomyces verticillatus, Journal of the American Chemical Society, 96, 320-322 (1974).
  • Iyer, N., and W. Szybalski. Mitomycin or porfiromycin: chemical mechanism of activation and cross-linking of DNA. [0372] Science, 145, 55-56 (1964).
  • Jabbouri, S., Fellay, R., Talmont, F., Kamalaprija, P., Burger, U., Relic, B., Prome, J. C. and W. J. Broughton. Involvement of nodS in N-methylation and nodU in 6-O-carbamoylation of Rhizobium sp. NGR234 nod factors, [0373] J. Biol. Chem., 270, 22968-22073 (1995).
  • Jabbouri, S., Relic, B., Hanin, M., Kamalaprija, P., Burger, U., Prome, D., Prome, J. C. and W. J. Broughton. nolO and noeI (HsnIII) of Rhizobium sp. NGR234 are involved in 3-O-carbamoylation and 2-O-methylation of Nod factors, [0374] J. Biol. Chem., 273, 12047-12055 (1998).
  • Johnson, D. A., August, P. R., Shackleton, C., Liu, H. W. and D. H. Sherman. Microbial resistance to mitomycins involves a redox relay mechanism, [0375] J. Am. Chem. Soc., 119, 2576-2577 (1997).
  • Kagan, R. M. and S. Clarke. Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes, [0376] Arch. Biochem. Biophy., 310, 417-27 (1994).
  • Kahler, C. M., R. W. Carlson, M. M. Rahman, L. E. Martin, D. S. Stephens. Two glycosyltransferase genes, IgtF and rfaK, constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of [0377] Neisseria meningitidis. J. Bacteriol., 178, 6677-6684 (1996).
  • Kasai, M. and H. Arai. Novel mitomycin derivatives, [0378] Exp. Opin. Ther. Patents, 5, 757-770 (1995).
  • Kibby, J. J., I. A. McDonald, and R. W. Rickards. 3-amino-5-hydroxybenzoic acid as a key intermediate in ansamycin and maytansinoid biosynthesis. [0379] J. Chem. Soc. Chem. Comm., 1980, 768-769 (1980).
  • Kibby, J. J. and R. W. Rickards. The identification of 3-amino-5-hydroxybenzoic acid as a new natural aromatic amino acid, [0380] J. Antibiot., 34, 605-607 (1981).
  • Kim, C. G., Kirschning, A., Bergon, P., Zhou, P., Su, E., Sauerbrei, B., Ning, S., Ahn, Y., Breuer, M., Leistner, E. and H. G. Floss. Biosynthesis of 3-amino-5-hydroxybenzoic acid, the precursor of mC[0381] 7N units in ansamycin antibiotics, J. Am. Chem. Soc., 188, 7486-7491 (1996).
  • Kim, C. G., A. Kirschning, P. Bergon, Y. Ahn, J. J. Wang, M. Shibuya, and H. G. Floss. Formation of 3-amino-5-hydroxybenzoic acid, the precursor of mC[0382] 7N units in ansamycin antibiotics, by a new variant of the shikimate pathway. J. Am. Chem. Soc., 114, 4941-4943 (1992).
  • Kim, C. G., Yu, T. W., Fryhle, C. B., Handa, S. and H. G. Floss. 3-Amino-5-hydroxybenzoic acid synthase, the terminal enzyme in the formation of the precursor of mC[0383] 7N units in rifamycin and related antibiotics, J. Biol. Chem., 273, 6030-6040 (1998).
  • Kuzuyama, T., Seki, T., Dairi, T., Hidaka, T. and H. Seto. Nucleotide sequence of fortimicin KL1 methyltransferase gene isolated from [0384] Micromonospora olivasterospora and comparison of its deduced amino acid sequence with those of methyltransferases involved in the biosynthesis of bialaphos and fosfomycin, J. Antibiot., 48, 1191-3 (1995).
  • Kwon, O., Bhattacharyya, D. K. and R. Meganathan. Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and properties of o-succinylbenzoyl-coenzyme A synthetase from [0385] Escherichia coli, J. Bacteriol., 178, 6778-6781 (1996).
  • Kyte, J., and R. F. Doolittle. A simple method for displaying the hydropathic character of a protein. [0386] J. Mol. Biol., 157, 105-132 (1982).
  • Lacalle, R. A., Ruiz, D. and A. Jimenez. Molecular analysis of the dmpM gene encoding an O-dimethyl puromycin O-methyltransferase from [0387] Streptomyces alboniger, Gene, 109, 55-61 (1991).
  • Lee, J. P., S. W. Tsao, C. J. Chang, X. G. He, and H. G. Floss. Biosynthesis of naphthomycin A in [0388] Streptomyces collinus. Can. J. Chem., 72, 182-187 (1994).
  • Lee, P. J. and A. M. Stock. Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium [0389] Thermotoga maritima, J. Bacteriol., 178, 5579-5585 (1996).
  • Levy, S., and L. McMurry. Plasmid-mediated tetracycline resistance involves alternative transport systems for tetracycline. [0390] Nature, 276, 90-92 (1978).
  • Littlejohn, T., I. Paulsen, M. Gillespie, J. Tennant, M. Midgley, T. Jones, A. Purewal, and R. Skurray. Substrate specificity and energetics of antiseptic and disinfectant resistance in [0391] Staphylococcus aureus. FEMS Microbiol. Lett., 95, 259-266 (1992).
  • Lomovskaya, O., Lewis, K. and A. Matin. EmrR is a negative regulator of the [0392] Escherichia coli multidrug resistance pump EmrAB, J. Bacteriol., 177, 2328-2334 (1995).
  • Luka, S., Sanjuan, J., Carlson, R. W. and G. Stacey. nolMNO genes of [0393] Bradyrhizobium japonicum are co-transcribed with nodYABCSUIJ, and nolO is involved in the synthesis of the lipo-oligosaccharide nodulation signals, J. Biol. Chem., 268, 27053-27059 (1993).
  • Madduri, K., Torti, F., Colombo, A. L. and C. R. Hutchinson. Cloning and sequencing of a gene encoding carminomycin 4-O-methyltransferase from [0394] Streptomyces peucetius and its expression in Escherichia coli, J. Bacteriol., 175, 3900-3904 (1993).
  • Makino, K., Shinagawa, H., Amemura, M. and A. Nakata. Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of [0395] Escherichia coli K-12, J. Mol. Biol., 190, 37-44 (1986).
  • Martin, J. F. Clusters of genes for the biosynthesis of antibiotics: regulatory genes and overproduction of pharmaceuticals. [0396] J. Ind. Microbiol., 9, 73-90 (1992).
  • Mazodier, P., Petter, R. and C. Thomson. Intergeneric conjugation between [0397] Escherichia coli and Streptomyces species, J. Bacteriol., 171, 3583-3585 (1989).
  • Mendez, C., and J. A. Salas. ABC transporters in antibiotic-producing actinomycetes. [0398] FEMS Microb. Lett., 158, 1-8 (1998).
  • Miller, J., J. Olson, J. Plfugrath, and F. Quiocho. Rates of ligand binding to periplasmic proteins involved in bacterial transport and chemotaxis. [0399] J. Biol. Chem., 238, 13665-13672 (1983).
  • Mizuno, T. and I. Tanaka. Structure of the DNA-binding domain of the OmpR family of response regulators, [0400] Mol. Microbiol., 24, 665-667 (1997).
  • Morbidoni, H. R., de Mondoza, D. and J. Cronan Jr. [0401] Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes, J. Bacteriol., 178, 4794-800 (1996).
  • Motamedi, H., and C. R. Hutchinson. Cloning and heterologous expression of a gene cluster for the biosynthesis of tetracenomycin C, the anthracycline antitumor antibiotic of [0402] Streptomyces glaucescens. Proc. Natl. Acad. Sci. USA, 84, 4445-4449 (1987).
  • Muth, G., B. Nussbaumer, W. Wohileben, and A. Publer. A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes. [0403] Mol. Gen. Genet., 219, 341-348 (1989).
  • Naoe, Y., Inami, M., Matsumoto, S., Nishigaki, F., Tsujimoto, S., Kawamura, I., Miyayasu, K., Manda, T. and K. Shimomura. Fk317—a novel substituted dihydrobenzoxazine with potent antitumor activity which does not induce vascular leak syndrome, [0404] Cancer Chemo. Pharmacol., 42, 31-36 (1998).
  • Neal, R. J., and K. F. Chater. Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria. [0405] Gene, 58 229-241 (1987).
  • Niemi, J. and Mantsala, P., Nucleotide sequences and expression of genes from [0406] Streptomyces purpurascens that cause the production of new anthracyclines in Streptomyces galilaeus, J. Bacteriol., 177, 2942-2945 (1995).
  • Nikaido, H. Prevention of drug access to bacterial targets: Permeability barriers and active efflux. [0407] Science, 264, 382-388 (1994).
  • Nishikohri, K. and S, Fukui. Biosynthesis of mitomycin in [0408] Streptomyces caespitosus. Relationship of intracellular vitamin B12 level to mitomycin synthesis and enzymatic methylation of a demethylated derivative of mitomycin, Eur. J. Appl. Microbiol., 2, 129-145 (1975).
  • Nolling, J., Pihl, T. D. and J. N. Reeve. Cloning, sequencing, and growth phase-dependent transcription of the coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductase-encoding genes from [0409] Methanobacterium thermoautotrophicum delta H and Methanopyrus kandleri, J. Bacteriol., 177, 7238-7244 (1995).
  • Ohno, T., Armand, S., Hata, T., Nikaidou, N., Henrissat, B., Mitsutomi, M. and T. Watanabe. A [0410] modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037, J. Bacteriol., 178, 5065-5070 (1996).
  • Omer, C. A., Lenstra, R., Little, P. J., Dean, C., Tepperman, J. M., Leto, K. J., Romesser, J. A. and D. P. O'Keefe. Genes for two herbicide-inducible cytochromes P-450 from [0411] Streptomyces griseolus, J. Bacteriol., 172, 3335-3345 (1990).
  • Otten, S. L., X. Liu, J. Ferguson, and C. R. Hutchinson. Cloning and characterization of the [0412] Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis. J. Bacteriol., 177, 6688-6692 (1995b).
  • Otten, S. L., Ferguson, J. and C. R. Hutchinson. Regulation of daunorubicin production in [0413] Streptomyces peucetius by the dnrR2 locus, J. Bacteriol., 177, 1216-1224 (1995a).
  • Pan, S. S. and T. Iracki. Metabolites and DNA adduct formation from flavoenzyme-activated porfiromycin, [0414] Molecular Pharmacology, 34, 223-228 (1988).
  • Paulsen, I., and R. Skurray. Topology, structure and evolution of two families of proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes—an analysis. [0415] Gene, 124:1-11 (1993).
  • Paulsen, I., M. Brown, and R. Skurray. Proton-dependent multidrug efflux pumps. [0416] Microbiol. Rev., 60, 575-608 (1996).
  • Paz, M. M. and P. B. Hopkins. DNA-DNA interstrand cross-linking by FR66979-intermediates in the activation cascade, [0417] J. Am. Chem. Soc., 119, 5999-6005 (1997).
  • Perez-Laraine, F. J., Liras, P., Rodriguez-Garcia, A. and J. F. Martin. A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in [0418] Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds, J. Bacteriol., 179, 2053-2059 (1997).
  • Peschke, U., H. Schmidt, H. Z. Zhang, and W. Piepersberg. Molecular characterization of the lincomycin-production gene cluster of [0419] Streptomyces lincolnensis. 78-11. Mol. Microbiol., 16, 1137-1156 (1995).
  • Piepersberg, W. Pathway engineering in secondary metabolite-producing actinomycetes, [0420] Crit. Rev. Biotechnol., 14:251-285 (1994).
  • Platt, M. W., Miller, K. J., Lane, W. S. and E. P. Kennedy. Isolation and characterization of the constitutive acyl carrier protein from [0421] Rhozobium meliloti, J. Bacteriol., 172, 5440-4 (1990).
  • Potgieter, M. Biosynthetic studies on geldanamycin and pactamycin. Ph.D. thesis. Univ. Illinois (1983). [0422]
  • Praillet, T., Nasser, W., Robert-Baudouy, J. and S. Reverchon. Purification and functional characterization of Pacs, a regulator of virulence-factor synthesis in [0423] Erwinia chrysanthemi, Molecular Microbiology, 20, 391-402 (1996).
  • Rodriguez, A. M., Olano, C., Mendez, C., Hutchinson, C. R. and J. A. Salas. A cytochrome P450-like gene possibly involved in oleandomycin biosynthesis by [0424] Streptomyces antibioticus, FEMS Microbiol. Lett., 127, 117-20 (1995).
  • Rouch, D., D. Cram, D. DiBerardino, T. Littlejohn and R. Skurray. Efflux-mediated antiseptic resistance gene qacA from [0425] Staphylococcus aureus: common ancestry with tetracycline and sugar-transport proteins. Mol. Microbiol., 4, 2051-2062 (1990).
  • Sambrook, J., Fritsch, E. F. and Maniatis, T., [0426] Molecular cloning: a laboratory manual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • Sartorelli, A. C., W. F. Hodnick, M. F. Belcourt, M. Tomasz, B. Haffty, J. J. Fischer, and S. Rockwell. Mitomycin C: a prototype bioreductive agent, [0427] Oncol. Res., 6:501-508 (1994).
  • Schaferjohann, J., Yoo, J. G., Kusian, B. and B. Bowien. The cbb operons of the facultative chemoautotroph [0428] Alcaligenes eutrophus encode phosphoglycolate phosphatase, J. Bacteriol., 175, 7329-40 (1993).
  • Schwecke, T., Aparicio, J. F., Molnar, I., Konig, A., Khaw, L. E., Haydock, S. F., Oliynyk, M., Caffrey, P., Cortes, J., Lester, J. B. and et al., The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin, [0429] Proc. Natl. Acad. Sci. USA, 92, 7839-43 (1995).
  • Seno, E. T. and R. H. Baltz. [0430] Structural organization and regulation of antibiotic biosynthesis and resistance genes in actinomycetes, CRC Press, Boca Raton, Fla. (1989).
  • Sheldon, P. J., Johnson, D. A., August, P. J., Liu, H. W. and D. H. Sherman. Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, [0431] Streptomyces lavendulae, J. Bacteriol., 179, 1796-1804 (1997).
  • Shi, J., Gonzales, R. A. and Bhattacharyya, M. K., Identification and characterization of an S-adenosyl-L-methionine: delta 24-sterol-C-methyltransferase cDNA from soybean, [0432] J. Biol. Chem., 271, 9384-9389 (1996).
  • Shikano, M., Onimura, K., Fukai, Y., Hori, M., Fukazawa, H., Mizuno, S., Yazawa, K. and Y. Uehara. 1a-docosahexaenoyl mitomycin C: a novel inhibitor of protein tyrosine kinase, [0433] Biochem. Biophys. Res. Commun., 248, 858-863 (1998).
  • Simon, R., U. Priefer, and A. Puhler, A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram negative bacteria, [0434] Bio/Technology, 1:784-791 (1983).
  • Smith, T. M., Y. F. Jiang, P. Shipley, and H. G. Floss. The thiostrepton-resistance encoding gene in [0435] Streptomyces laurentii is located within a cluster of ribosomal protein operons. Gene, 164, 137-142 (1995).
  • Smitskampwilms, E., Hendriks, H. R. and Peters, G. J., Development, pharmacology, role of DT-diaphorase and prospects of the indoloquinone EO9, [0436] Gen. Pharmacol., 27, 421-429 (1996).
  • Solenberg, P. J., P. Matsushima, D. R. Stack, S. C. Wilkie, R. C. Thompson, and R. H. Baltz. Production of hybrid glycopeptide antibiotics in vitro and in [0437] Streptomyces toyocaensis. Chem. Biol., 4, 195-202 (1997).
  • Spanswick, V. J., Cummings, J. and J. F. Smyth. Current issues in the enzymology of mitomycin C metabolic activation, [0438] Gen. Pharmacol., 31, 539-544 (1998).
  • Spath, C., Kraus, A. and W. Hillen. Contribution of glucose kinase to glucose repression of xylose utilization in [0439] Bacillus megaterium, J. Bacteriol., 179, 7603-7605 (1997).
  • Stackebrandt, E., and C. R. Woese. Towards a phylogeny of the actinomycetes and related organisms. [0440] Curr. Microbiol., 5, 197-202 (1981).
  • Staley, A. L., and K. L. Rinehart. Biosynthesis of the streptovaricins: 3-amino-5-hydroxybenzoic acid as a precursor to the meta-C[0441] 7N unit. J. Antibiot., 44, 218-224 (1991).
  • Stupperich, E., Juza, A., Hoppert, M. and F. Mayer. Cloning, sequencing and immunological characterization of the corrinoid-containing subunit of the N5-methyltetrahydromethanopterin: coenzyme-M methyltransferase from [0442] Methanobacterium thermoautotrophicum, Euro. J Biochem., 217, 115-121 (1993).
  • Summers, R. G., Wendt-Pienkowski, E., Motamedi, H. and C. R. Hutchinson. Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of [0443] Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase, J. Bacteriol., 174, 1810-1820 (1992).
  • Takano, E., Gramajo, H. C., Strauch, E., Andres, N., White, J. and M. J. Bibb. Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in [0444] Streptomyces coelicolor A3(2), Molecular Microbiology, 6, 2797-2804 (1992).
  • Tang, L., Grimm, A., Zhang, Y. X. and C. R. Hutchinson. Purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in [0445] Streptomyces peucetius, Molecular Microbiology, 22, 801-13 (1996).
  • Tercero, J., R. Lacalle, and A. Jimenez. The pur8 gene from the pur cluster of [0446] Streptomyces alboniger encodes a highly hydrophobic polypeptide which confers resistance to puromycin. Eur. J. Biochem., 218, 963-971 (1993).
  • Thauer, R. K., Hedderich, R. and R. Fischer. [0447] Reactions and enzymes involved in methanogenesis from CO2 and H2, Chapman and Hall, New York, N.Y., 1993.
  • Tomasz, M. Mitomycin C: small fast and deadly (but very selective), [0448] Chemistry and Biology, 2, 575-579 (1995).
  • Tomasz, M. and Y. Palom. The mitomycin bioreductive antitumor agents: cross-linking and alkylation of DNA as the molecular basis of their activity, [0449] Pharmacol. Therap., 76, 73-87 (1997).
  • Tuan, J. S., Weber, J. M., Staver, M. J., Leung, J. O., Donadio, S. and L. Katz. Cloning of the genes involved in erythromycin biosynthesis from [0450] Saccaropolyspora erythraea using a novel Actinomycete-Escherichia coli cosmid, Gene, 90, 21-29 (1990).
  • Turgay, K., and M. A. Marahiel. A general approach for identifying and cloning peptide synthetase genes. [0451] Peptide Res., 7, 238-241 (1994).
  • Vara, J., F. Malpartida, D. A. Hopwood, and A. Jimenez. Cloning and expression of a puromycin N-acetyl transferase gene from [0452] Streptomyces alboniger in Streptomyces lividans and Escherichia coli Gene. 33, 197-206 (1985).
  • Vaupel, M. and R. K. Thauer. Coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductase (Mer) from [0453] Methanobacterium thermoautotrophicum strain Marburg. Cloning, sequencing, transcriptional analysis, and functional expression in Escherichia coli of the mer gene, Euro. J. Biochem., 231, 773-8 (1995).
  • Verweij, J. Mitomycins, [0454] Cancer Chemotherapy and Biological Response Modifiers, 17, 46-58 (1997).
  • Wakaki, K., Harumo, H., Tomioka, K., Shimizu, G., Kato, E., Kamada, H., Kudo, S. and Y. Fujimoto. Isolation of new fractions of antitumor mitomycins, [0455] Antibiot. Chemother., 8, 228-240 (1958).
  • Webb, J. S., D. B. Cosalich, T. H. Mowat, J. B. Patrick, R. W. Broschard, W. E. Meyor, R. P. Williams, C. F. Wolf, W. Fulmore, C. Pidacks, and J. E. Lancaster. The structure of Mitomycins A, B, and C and Porfiromycin-[0456] Part 1. J. Am. Chem. Soc., 84, 3185-3188 (1962).
  • White, P. J., Young, J., Hunter, I. S., Nimmo, H. G. and J. R. Coggins. The purification and characterization of 3-dehydroquinase from [0457] Streptomyces coelicolor, Biochem. J., 265, 735-8 (1990).
  • Wietzorrek, A. and M. Bibb. A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA-binding fold, [0458] Molecular Microbiology, 25, 1181-4 (1997).
  • Williams, R. M., Raj ski, S. R. and S. B. Rollins. FR900482, a close cousin of mitomycin C that exploits mitosene-based DNA cross-linking, [0459] Chemistry and Biology, 4, 127-137 (1997).
  • Wu, T. S., J. Duncan, S. W. Tsao, C. J. Chang, P. J. Keller, and H. G. Floss. Biosynthesis of the ansamycin antibiotic assatrienin (mycotrienin) by [0460] Streptomyces collinus. J. Nat. Prod., 50, 108-118 (1987).
  • Yamazaki, M., Thome, L., Mikolajczak, M., Armentrout, R. W. and T. J. Pollock. Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88, [0461] J. Bacteriol., 178, 2676-87 (1996).
  • Yang, K., Han, L. and L. C. Vining. Regulation of jadomycin B production in [0462] Streptomyces venezuelae ISP5230: involvement of a repressor gene, jadR2, Journal of Bacteriology, 177, 6111-7 (1995).
  • Yanisch-Perron, C., J. Vieira, and J. Messing. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors, [0463] Gene, 33:103-119 (1985).
  • Zhang, H. Z., H. Schmidt, and W. Piepersberg. Molecular cloning and characterization of two lincomycin-resistance genes, ImrA and ImrB, from [0464] Streptomyces lincolnensis 78-11. Mol. Microbiol., 6, 2147-2157 (1992).
  • While the present invention has been described in connection with the preferred embodiment thereof, it will be understood many modifications will be readily apparent to those skilled in the art, and this application is intended to cover any adaptations or variations thereof. It is manifestly intended this invention be limited only by the claims and equivalents thereof. [0465]
  • 0
    SEQUENCE LISTING
    <160> NUMBER OF SEQ ID NOS: 145
    <210> SEQ ID NO 1
    <211> LENGTH: 115
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 1
    Arg Ile Gly Ala Gly Ser Arg Val Leu Asp Leu Gly Cys Gly Val Gly
    1 5 10 15
    Thr Pro Gly Val Arg Ile Ala Arg Leu Ser Gly Ala His Val Thr Gly
    20 25 30
    Ile Ser Val Ser His Glu Gln Val Val Arg Ala Asn Ala Leu Ala Glu
    35 40 45
    Glu Ala Gly Leu Ala Asp Arg Ala Arg Phe Gln Arg Ala Asp Ala Met
    50 55 60
    Asp Leu Pro Phe Glu Asp Glu Ser Phe Asp Ala Val Ile Ala Leu Glu
    65 70 75 80
    Ser Ile Ile His Met Pro Asp Arg Ala Gln Val Leu Ala Gln Val Gly
    85 90 95
    Arg Val Leu Arg Pro Gly Gly Arg Leu Val Leu Thr Asp Phe Phe Glu
    100 105 110
    Arg Ala Pro
    115
    <210> SEQ ID NO 2
    <211> LENGTH: 114
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 2
    Arg Leu Ala Pro Gly Glu Arg Val Leu Asp Val Gly Ser Gly Asn Gly
    1 5 10 15
    Lys Ala Thr Leu Arg Ile Ala Ala Arg His Gly Val Arg Ala Thr Gly
    20 25 30
    Val Ser Ile Asn Pro Tyr Gln Val Gly Leu Ser Arg Gln Leu Ala Glu
    35 40 45
    Lys Glu Gly Asp Glu Ala Thr Glu Phe Arg Ile Gly Asp Met Leu Ala
    50 55 60
    Leu Pro Phe Pro Asp Gly Ser Phe Asp Ala Cys Tyr Ala Ile Glu Ser
    65 70 75 80
    Ile Cys His Ala Leu Glu Arg Ala Asp Val Phe Thr Glu Ile Ala Arg
    85 90 95
    Val Leu Arg Pro Gly Gly Arg Val Thr Val Thr Asp Phe Val Leu Arg
    100 105 110
    Arg Pro
    <210> SEQ ID NO 3
    <211> LENGTH: 115
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 3
    Asp Phe Ser Gly Ala Ala Thr Ala Val Asp Ile Gly Gly Gly Arg Gly
    1 5 10 15
    Ser Leu Met Ala Ala Val Leu Asp Ala Phe Pro Gly Leu Arg Gly Thr
    20 25 30
    Leu Leu Glu Arg Pro Pro Val Ala Glu Glu Ala Arg Glu Leu Leu Thr
    35 40 45
    Gly Arg Gly Leu Ala Asp Arg Cys Glu Ile Leu Pro Gly Asp Phe Phe
    50 55 60
    Glu Thr Ile Pro Asp Gly Ala Asp Val Tyr Leu Ile Lys His Val Leu
    65 70 75 80
    His Asp Trp Asp Asp Asp Asp Val Val Arg Ile Leu Arg Arg Ile Ala
    85 90 95
    Thr Ala Met Lys Pro Asp Ser Arg Leu Leu Val Ile Asp Asn Leu Ile
    100 105 110
    Asp Glu Arg
    115
    <210> SEQ ID NO 4
    <211> LENGTH: 115
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces anulatus
    <400> SEQUENCE: 4
    Asp Phe Ser Ser Tyr Gly Thr Val Val Asp Ile Gly Gly Ala Asp Gly
    1 5 10 15
    Ser Leu Leu Ala Ala Val Leu Ser Ala His Pro Gly Val Glu Gly Val
    20 25 30
    Val Phe Asp Ser Pro Glu Gly Ala Arg Asp Ala Ala Ala Thr Leu Asp
    35 40 45
    Ala Ala Gly Val Gly Glu Arg Gly Arg Val Glu Thr Gly Asp Phe Phe
    50 55 60
    Thr Arg Val Pro Gly Gly Gly Asp Leu Tyr Val Leu Lys Ser Ile Leu
    65 70 75 80
    His Asp Trp Ser Asp Ala Arg Ser Ala Asp Ile Leu Arg Thr Val Arg
    85 90 95
    Ala Ala Met Pro Ala His Ala Arg Leu Leu Val Val Glu Val Leu Leu
    100 105 110
    Pro Asp Thr
    115
    <210> SEQ ID NO 5
    <211> LENGTH: 117
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces glaucescens
    <400> SEQUENCE: 5
    Gly Met Glu Arg Phe Ser Arg Ile Ala Asp Leu Gly Gly Gly Asp Gly
    1 5 10 15
    Trp Phe Leu Ala Gln Ile Leu Arg Arg His Pro His Ala Thr Gly Leu
    20 25 30
    Leu Met Asp Leu Pro Arg Val Ala Ala Ser Ala Gly Pro Val Leu Glu
    35 40 45
    Glu Ala Lys Val Ala Asp Arg Val Thr Val Leu Pro Gly Asp Phe Phe
    50 55 60
    Thr Asp Pro Val Pro Thr Gly Tyr Asp Ala Tyr Leu Phe Lys Gly Val
    65 70 75 80
    Leu His Asn Trp Ser Asp Glu Arg Ala Val Thr Val Leu Arg Arg Val
    85 90 95
    Arg Glu Ala Ile Gly Asp Asp Asp Ala Arg Leu Leu Ile Phe Asp Gln
    100 105 110
    Val Met Ala Pro Glu
    115
    <210> SEQ ID NO 6
    <211> LENGTH: 115
    <212> TYPE: PRT
    <213> ORGANISM: Amycolatopsis mediterranei
    <400> SEQUENCE: 6
    Pro Leu Arg Ala Gly Asp Arg Leu Leu Asp Ile Gly Cys Gly Asn Gly
    1 5 10 15
    Glu Pro Ala Ile Arg Met Ala Thr Ala Asn Asp Val Met Val Thr Gly
    20 25 30
    Ile Ser Ile Ser Glu Lys Gln Val Glu Arg Ala Asn Asp Arg Ala Tyr
    35 40 45
    Lys Ala Asp Val Asp Asp Arg Val Val Phe Glu Tyr Ala Asp Ala Met
    50 55 60
    Glu Leu Pro Tyr Pro Asp Ala Ser Phe Asp Val Val Trp Ala Leu Glu
    65 70 75 80
    Ser Leu His His Met Pro Asp Arg Trp His Val Ile Arg Gln Ala Ala
    85 90 95
    Arg Val Leu Arg Pro Gly Gly Arg Leu Ala Leu Gly Asp Phe Leu Leu
    100 105 110
    Val Pro Ser
    115
    <210> SEQ ID NO 7
    <211> LENGTH: 116
    <212> TYPE: PRT
    <213> ORGANISM: Saccharopolyspora erythraea
    <400> SEQUENCE: 7
    Gly Ile Ser Glu Gly Asp Glu Val Leu Asp Val Gly Phe Gly Leu Gly
    1 5 10 15
    Ala Gln Asp Phe Phe Trp Leu Glu Thr Arg Lys Pro Ala Arg Ile Val
    20 25 30
    Gly Val Asp Leu Thr Pro Ser His Val Arg Ile Ala Ser Glu Arg Ala
    35 40 45
    Glu Arg Glu Asn Val Gln Asp Arg Leu Gln Phe Lys Glu Gly Ser Ala
    50 55 60
    Thr Asp Leu Pro Phe Gly Ala Glu Thr Phe Asp Arg Val Thr Ser Leu
    65 70 75 80
    Glu Ser Ala Leu His Tyr Glu Pro Arg Thr Asp Phe Phe Lys Gly Ala
    85 90 95
    Phe Glu Val Leu Lys Pro Gly Gly Val Leu Ala Ile Gly Asp Ile Ile
    100 105 110
    Pro Leu Asp Leu
    115
    <210> SEQ ID NO 8
    <211> LENGTH: 120
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A consensus sequence
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)...(120)
    <223> OTHER INFORMATION: Where present in this sequence, Xaa represents
    an amino acid that varied between the sequences used
    to determine this consensus sequence.
    <400> SEQUENCE: 8
    Xaa Xaa Xaa Xaa Gly Xaa Arg Xaa Leu Asp Xaa Gly Xaa Gly Xaa Gly
    1 5 10 15
    Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Thr
    20 25 30
    Gly Xaa Xaa Xaa Xaa Pro Xaa Xaa Val Xaa Xaa Ala Xaa Xaa Xaa Ala
    35 40 45
    Glu Xaa Ala Gly Val Xaa Asp Arg Xaa Xaa Phe Xaa Xaa Gly Asp Xaa
    50 55 60
    Xaa Xaa Leu Pro Xaa Pro Asp Gly Xaa Phe Asp Xaa Val Tyr Xaa Leu
    65 70 75 80
    Glu Ser Xaa Leu His Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Xaa Val Xaa
    85 90 95
    Arg Xaa Xaa Xaa Xaa Val Leu Xaa Pro Gly Xaa Gly Arg Leu Xaa Xaa
    100 105 110
    Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa
    115 120
    <210> SEQ ID NO 9
    <211> LENGTH: 3765
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 9
    ggatccgagg gccggagtgg gattcggctc aatgaaccat gcacacagca cataccagga 60
    cggtgtcgcg cccaccatac gcgacgcttc ccgctccctc cagccgtgcg gtttgagcca 120
    cttcgacgcc ggataacgtt gccgacaggc ccgccgagca gcccctgaac tggatcaatt 180
    cccttgggaa taaggcagtt tcactgctca accaccctgc tgacgagaat ccaccgccga 240
    ccggcggtcg gggcagacct tcccggcaag ggtgttgact ccggcaactg ccctatggag 300
    gctcgtgtct ggcatccgat cccggcctat gaccgggggc cggatcacat gcccgctccg 360
    gccacccctc acaccgcggg ccggatttcc cgccgccccc gaggaacggc gtttcccgtc 420
    gggtcacgca ccacccttcc cgacgcgggg cgaacacaac ggaaccgggc cgtgaagcca 480
    cggccaccga aggcaaaggc ctcgacaccc gccctcccgc cgtacagcgc cccgaagtcg 540
    accgtgccgc cgcacccgca ggaccgaaag gctgctcaat gacacctacg tccggtgatg 600
    acgtcctgtc ctttccctca tggccgcaac acggcgcgga ggagcgcgcc ggactcctgc 660
    gggccctgga ccagaagggg tggtggcgcg acgcggggca ggaggtcgat ctcttcgagc 720
    gggagttcgc cgaccaccac ggcgccccgc acgcgatcgc cacgacgaac ggcacccacg 780
    ccctggaact cgccctgggg gtcatgggga tcggccccgg tgacgaggtc atcgtccccg 840
    cgttcacctt catctcgtcg tcgctggccg tgcagcgcat gggcgcggtg ccggtgccgg 900
    cggacgtacg gcccgacacc tactgcctcg atgccgacgc ggcggcggcg ctggtgacgc 960
    cacgcaccaa agcgatcatg ccggtccaca tggcgggcca gttcgccgac atggacgccc 1020
    tggagaagct ctccgtcgcg acgggcgtgc cggtcctcca ggacgccgcg cacgcccacg 1080
    gcgcgcagtg gcagggccgc cgggtcgggg agctcggctc gatcgccgcc ttcagcttcc 1140
    agaacggcaa gctgatgacc gccggcgagg gcggcgccct gctcctgccg gacgacgagt 1200
    ccttccacga ggcgttcctc cagcactgct gcggccgccc gcccggggac cgcgtctacc 1260
    gccatctgac gcagggctcc aactaccgca tgaacgagtt ctccgcgagc gtcctgcgtg 1320
    ctcaactgaa gcgcttgaag gatcagttgc gcatcaggga ggagcgctgg gcccagctgc 1380
    gtacggcact ggccgccatc gacggcgtgg tgccgcaggg gcgcgacgag cgcggcgacc 1440
    tccactccca ctacatggcc atggtccggc tgcccggcat ctcggcccgg cgccgcctcg 1500
    cgctggtgga cgcgctggtc gagcggggag tgcccgcgtt cgtcggcttc ccgccggtct 1560
    accgcaccga gggtttcgcg cgcggcccgg cgccggcgga cgccgaggag ctggccaaga 1620
    gctgtcccgt ggcggaggag atcggcagcg actgcctctg gctgcaccat cgcgtcctgc 1680
    tcgccgacgt gaccacgctg gaccggctgg cggaggtctt ctccggcctc gtcggcgcgc 1740
    tctgacccga tgcgggcccc caacggcacc accgcccccc ggctgagcgt cgtcgtcccc 1800
    agccgggggc ccgcggcacg cctgcgcgcg accctcgcat gccttgccgg cccctccccg 1860
    ggaacgccgc ccttcgaagt ggtcgtcgtc gacgacaacg acgggggcga cgccggtgat 1920
    caactgatcg ccgtgacagg cgagatgagc ggccttctcc cgctgcgcgt ggtgcgggga 1980
    ccgctgcggg ggcgggccgc cgcccggaac gccggggcgg ccgcggccct cgcgccccgg 2040
    ctggtcttcc tcgacgacga cgtcctggtg gggcccggct tcctcgccgc acacgccgcg 2100
    gccgcggaac cggacgcctt cacccacggc cggctgcgcg aactccccac cgcggcgcgg 2160
    ttcctcgccg ctgtcgagaa ggccgccccg accgaggtcc gccgcgcccg cgccggactc 2220
    gaacccgctg ccccggccgc ctccgagcgg cgccaaccgc accggcggct cgtcgccaac 2280
    gccctggagc gggccgtgga ggccatggcc ggcggctccc tgccggacgt cgccccctgg 2340
    ctcggcttca tcggcgcgaa caccgccctc gacaaggccg catgggagca taccggcgga 2400
    ttcgacgagg agttcgggct cacctggggg tgcgaggacc tggagttcgg cttccgcctg 2460
    cacgccgccg ggctgcgcag gaccctcgcc cccgacgccc tcggtgtgca cctcagccac 2520
    gcccgccccg gccgctggga gcagcaccac cgcaacctca cgcacttctc cgccggccac 2580
    ccgcacccgt cggtacgcgc cttggaggcc ctgctcgggc ccgacggcac gccggaggcg 2640
    tatgtgcgcg ccgtcctggc cgaagaggcc gcaccggcac gggacgcggc gcgatgagcg 2700
    gcacaccggc caccgcgccg tacggtcccg tggtgctctc cccgcacgcg gacgacgccg 2760
    tgtggtccct gggcgggcgg ctggcgcgct gggccgccga gggcccgcgg ccgaccgtcg 2820
    tcacggtctt cgccgggccc gcggccggga agcccgagtc gtggcggagc gccgccgatc 2880
    ccgcggtgcg ccgggccgag gaccgggcgg catgtgccga actgggcgtg cgccacgtgc 2940
    cgctgggctt caccgacgcg gcactgcgta cggcctcggg cgcctatctc tacgcttccc 3000
    cgcgccggct cttcggcccc tggcacccgg ccgacctccc gctgctggag gaggtgcggg 3060
    cggctctgct gccgctgtgc gcgggggcgt cgagcgtcca cgttcccctg gcggcgggcc 3120
    ggcacgtcga ccaccgcctg gtccgcggcg cggtggagcc cctgtccccc gcccgtaccg 3180
    tcttctacga ggacttcccc taccggctgc gcgaacgtga ccacacgaac ctgcggccgc 3240
    gcacggaacg gctgccgtcc gaggcggtgg accgctggct gaccgccgcc ggtcactact 3300
    ccagccaggc gagcgcccac ttcggcggtg cggccgccct gcgcgaggcc ctgttcgccc 3360
    gcgcccgcgc acacggcggg cccggccggc ccggccacgc cgaccgccac tgggtgcccg 3420
    tcggccagga cgaccggggc gaggcccggc cggcacccgt ggaaaggggg ccgtgaccca 3480
    cgccgtgcgc agccccacca cgagagaggc cactcatgtc ccgtagcacc cacccgccga 3540
    cagccacccc cgacgcgggc accaggcgac gcctgccgct gatcggcaac gacctggtca 3600
    tcaacgagga ctcctgcaac ctcagctgca cctactgcct caccggacag agcaacctca 3660
    aggagggcca ctcccttcaa ctgatcttcg agcccccgcg gcgcgacagc tacgccaagg 3720
    acagcgggct ggggcagcgc atggacaagg tcgccgaccg gatcc 3765
    <210> SEQ ID NO 10
    <211> LENGTH: 388
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 10
    Met Thr Pro Thr Ser Gly Asp Asp Val Leu Ser Phe Pro Ser Trp Pro
    1 5 10 15
    Gln His Gly Ala Glu Glu Arg Ala Gly Leu Leu Arg Ala Leu Asp Gln
    20 25 30
    Lys Gly Trp Trp Arg Asp Ala Gly Gln Glu Val Asp Leu Phe Glu Arg
    35 40 45
    Glu Phe Ala Asp His His Gly Ala Pro His Ala Ile Ala Thr Thr Asn
    50 55 60
    Gly Thr His Ala Leu Glu Leu Ala Leu Gly Val Met Gly Ile Gly Pro
    65 70 75 80
    Gly Asp Glu Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Leu
    85 90 95
    Ala Val Gln Arg Met Gly Ala Val Pro Val Pro Ala Asp Val Arg Pro
    100 105 110
    Asp Thr Tyr Cys Leu Asp Ala Asp Ala Ala Ala Ala Leu Val Thr Pro
    115 120 125
    Arg Thr Lys Ala Ile Met Pro Val His Met Ala Gly Gln Phe Ala Asp
    130 135 140
    Met Asp Ala Leu Glu Lys Leu Ser Val Ala Thr Gly Val Pro Val Leu
    145 150 155 160
    Gln Asp Ala Ala His Ala His Gly Ala Gln Trp Gln Gly Arg Arg Val
    165 170 175
    Gly Glu Leu Gly Ser Ile Ala Ala Phe Ser Phe Gln Asn Gly Lys Leu
    180 185 190
    Met Thr Ala Gly Glu Gly Gly Ala Leu Leu Leu Pro Asp Asp Glu Ser
    195 200 205
    Phe His Glu Ala Phe Leu Gln His Cys Cys Gly Arg Pro Pro Gly Asp
    210 215 220
    Arg Val Tyr Arg His Leu Thr Gln Gly Ser Asn Tyr Arg Met Asn Glu
    225 230 235 240
    Phe Ser Ala Ser Val Leu Arg Ala Gln Leu Lys Arg Leu Lys Asp Gln
    245 250 255
    Leu Arg Ile Arg Glu Glu Arg Trp Ala Gln Leu Arg Thr Ala Leu Ala
    260 265 270
    Ala Ile Asp Gly Val Val Pro Gln Gly Arg Asp Glu Arg Gly Asp Leu
    275 280 285
    His Ser His Tyr Met Ala Met Val Arg Leu Pro Gly Ile Ser Ala Arg
    290 295 300
    Arg Arg Leu Ala Leu Val Asp Ala Leu Val Glu Arg Gly Val Pro Ala
    305 310 315 320
    Phe Val Gly Phe Pro Pro Val Tyr Arg Thr Glu Gly Phe Ala Arg Gly
    325 330 335
    Pro Ala Pro Ala Asp Ala Glu Glu Leu Ala Lys Ser Cys Pro Val Ala
    340 345 350
    Glu Glu Ile Gly Ser Asp Cys Leu Trp Leu His His Arg Val Leu Leu
    355 360 365
    Ala Asp Val Thr Thr Leu Asp Arg Leu Ala Glu Val Phe Ser Gly Leu
    370 375 380
    Val Gly Ala Leu
    385
    <210> SEQ ID NO 11
    <211> LENGTH: 272
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 11
    Met Val Val Val Asp Asp Asn Asp Gly Gly Asp Ala Gly Asp Gln Leu
    1 5 10 15
    Ile Ala Val Thr Gly Glu Met Ser Gly Leu Leu Pro Leu Arg Val Val
    20 25 30
    Arg Gly Pro Leu Arg Gly Arg Ala Ala Ala Arg Asn Ala Gly Ala Ala
    35 40 45
    Ala Ala Leu Ala Pro Arg Leu Val Phe Leu Asp Asp Asp Val Leu Val
    50 55 60
    Gly Pro Gly Phe Leu Ala Ala His Ala Ala Ala Ala Glu Pro Asp Ala
    65 70 75 80
    Phe Thr His Gly Arg Leu Arg Glu Leu Pro Thr Ala Ala Arg Phe Leu
    85 90 95
    Ala Ala Val Glu Lys Ala Ala Pro Thr Glu Val Arg Arg Ala Arg Ala
    100 105 110
    Gly Leu Glu Pro Ala Ala Pro Ala Ala Ser Glu Arg Arg Gln Pro His
    115 120 125
    Arg Arg Leu Val Ala Asn Ala Leu Glu Arg Ala Val Glu Ala Met Ala
    130 135 140
    Gly Gly Ser Leu Pro Asp Val Ala Pro Trp Leu Gly Phe Ile Gly Ala
    145 150 155 160
    Asn Thr Ala Leu Asp Lys Ala Ala Trp Glu His Thr Gly Gly Phe Asp
    165 170 175
    Glu Glu Phe Gly Leu Thr Trp Gly Cys Glu Asp Leu Glu Phe Gly Phe
    180 185 190
    Arg Leu His Ala Ala Gly Leu Arg Arg Thr Leu Ala Pro Asp Ala Leu
    195 200 205
    Gly Val His Leu Ser His Ala Arg Pro Gly Arg Trp Glu Gln His His
    210 215 220
    Arg Asn Leu Thr His Phe Ser Ala Gly His Pro His Pro Ser Val Arg
    225 230 235 240
    Ala Leu Glu Ala Leu Leu Gly Pro Asp Gly Thr Pro Glu Ala Tyr Val
    245 250 255
    Arg Ala Val Leu Ala Glu Glu Ala Ala Pro Ala Arg Asp Ala Ala Arg
    260 265 270
    <210> SEQ ID NO 12
    <211> LENGTH: 260
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 12
    Met Ser Gly Thr Pro Ala Thr Ala Pro Tyr Gly Pro Val Val Leu Ser
    1 5 10 15
    Pro His Ala Asp Asp Ala Val Trp Ser Leu Gly Gly Arg Leu Ala Arg
    20 25 30
    Trp Ala Ala Glu Gly Pro Arg Pro Thr Val Val Thr Val Phe Ala Gly
    35 40 45
    Pro Ala Ala Gly Lys Pro Glu Ser Trp Arg Ser Ala Ala Asp Pro Ala
    50 55 60
    Val Arg Arg Ala Glu Asp Arg Ala Ala Cys Ala Glu Leu Gly Val Arg
    65 70 75 80
    His Val Pro Leu Gly Phe Thr Asp Ala Ala Leu Arg Thr Ala Ser Gly
    85 90 95
    Ala Tyr Leu Tyr Ala Ser Pro Arg Arg Leu Phe Gly Pro Trp His Pro
    100 105 110
    Ala Asp Leu Pro Leu Leu Glu Glu Val Arg Ala Ala Leu Leu Pro Leu
    115 120 125
    Cys Ala Gly Ala Ser Ser Val His Val Pro Leu Ala Ala Gly Arg His
    130 135 140
    Val Asp His Arg Leu Val Arg Gly Ala Val Glu Pro Leu Ser Pro Ala
    145 150 155 160
    Arg Thr Val Phe Tyr Glu Asp Phe Pro Tyr Arg Leu Arg Glu Arg Asp
    165 170 175
    His Thr Asn Leu Arg Pro Arg Thr Glu Arg Leu Pro Ser Glu Ala Val
    180 185 190
    Asp Arg Trp Leu Thr Ala Ala Gly His Tyr Ser Ser Gln Ala Ser Ala
    195 200 205
    His Phe Gly Gly Ala Ala Ala Leu Arg Glu Ala Leu Phe Ala Arg Ala
    210 215 220
    Arg Ala His Gly Gly Pro Gly Arg Pro Gly His Ala Asp Arg His Trp
    225 230 235 240
    Val Pro Val Gly Gln Asp Asp Arg Gly Glu Ala Arg Pro Ala Pro Val
    245 250 255
    Glu Arg Gly Pro
    260
    <210> SEQ ID NO 13
    <211> LENGTH: 386
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces collinus
    <400> SEQUENCE: 13
    Met Ser Ser Gly Val Gln Leu Gly Ser Ala Phe Arg Val Trp Pro Gln
    1 5 10 15
    Tyr Asp Asp Ala Glu Arg Thr Gly Leu Ile Arg Ala Leu Glu Gln Gly
    20 25 30
    Gln Trp Trp Arg Met Gly Gly Gly Glu Val Glu Arg Phe Glu Arg Glu
    35 40 45
    Phe Ala Glu Tyr His Gly Gly Glu His Ala Leu Ala Val Thr Asn Gly
    50 55 60
    Thr His Ala Leu Glu Leu Ala Leu Glu Val Met Gly Val Gly Pro Gly
    65 70 75 80
    Thr Glu Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Gln Ala
    85 90 95
    Ala Gln Arg Leu Gly Ala Val Val Val Pro Val Asp Val Asp Pro Glu
    100 105 110
    Thr Tyr Cys Ile Asp Pro Ala Glu Ala Ala Lys Ala Ile Thr Pro Arg
    115 120 125
    Thr Arg Ala Ile Met Pro Val His Met Ala Gly Gln Leu Ala Asp Met
    130 135 140
    Asp Ala Leu Glu Lys Val Ala Ala Asp Ser Gly Val Pro Leu Ile Gln
    145 150 155 160
    Asp Ala Ala His Ala Gln Gly Ala Thr Trp Asn Gly Arg Arg Leu Gly
    165 170 175
    Glu Leu Gly Ser Val Ala Ala Phe Ser Phe Gln Asn Gly Lys Leu Met
    180 185 190
    Thr Ala Gly Glu Gly Gly Ala Val Leu Phe Pro Thr Ala Glu Met Ala
    195 200 205
    Glu His Ala Phe Leu Arg His Ser Cys Gly Arg Pro Arg Asn Asp Arg
    210 215 220
    Gly Tyr Phe His Arg Thr Ser Gly Ser Asn Phe Arg Leu Asn Glu Phe
    225 230 235 240
    Ser Ala Ser Val Leu Arg Ala Gln Leu Ala Arg Leu Asp Gly Gln Ile
    245 250 255
    Arg Thr Arg Glu Glu Arg Trp Pro Leu Leu Ser Ser Leu Leu Ala Glu
    260 265 270
    Ile Pro Gly Val Val Pro Gln Arg Leu Asp Arg Arg Pro Asp Arg Asn
    275 280 285
    Pro His Tyr Met Ala Met Phe Arg Val Pro Arg Ile Thr Glu Glu Arg
    290 295 300
    Arg Ala Arg Val Val Asp Thr Leu Val Glu Arg Gly Val Pro Ala Phe
    305 310 315 320
    Val Ala Phe Arg Ser Val Tyr Arg Thr Asp Ala Phe Trp Glu Met Gly
    325 330 335
    Ala Pro Asp Leu Ser Val Asp Glu Leu Ala Arg Leu Pro Pro Leu Arg
    340 345 350
    Gly Leu Thr Thr Asp Cys Leu Trp Leu His His Arg Thr Leu Leu Gly
    355 360 365
    Thr Glu Glu Gln Met His Glu Val Ala Ala Val Ile Ala Asp Val Leu
    370 375 380
    Gly Ser
    385
    <210> SEQ ID NO 14
    <211> LENGTH: 388
    <212> TYPE: PRT
    <213> ORGANISM: Actinosynnema pretiosum
    <400> SEQUENCE: 14
    Met Gly Ser Ser Pro Asp Ala Gly Ile Asp Phe Pro Ala Trp Pro Gln
    1 5 10 15
    His Asp Asp Ala Glu Arg Ala Ala Leu Leu Arg Ala Leu Asp Gln Gly
    20 25 30
    Gln Trp Trp Arg Val Gly Gly Ser Glu Val Asp Glu Phe Glu Arg Glu
    35 40 45
    Phe Ala Glu Tyr His Gly Ala Gly His Ala Leu Ala Val Thr Asn Gly
    50 55 60
    Thr His Ala Leu Glu Leu Ala Leu Gln Val Leu Asp Val Gly Pro Gly
    65 70 75 80
    Thr Glu Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Gln Ala
    85 90 95
    Val Gln Arg Leu Gly Ala Val Ala Val Pro Val Asp Val Asp Pro Asp
    100 105 110
    Thr Tyr Cys Leu Asp Val Ala Ala Ala Glu Asp Ala Val Thr Ser Arg
    115 120 125
    Thr Ser Ala Ile Met Pro Val His Met Ala Gly Gln Phe Ala Asp Met
    130 135 140
    Asp Arg Leu Asp Lys Leu Ser Ala Ser Thr Gly Val Pro Val Val Gln
    145 150 155 160
    Asp Ala Ala His Ala His Gly Ala His Trp Arg Gly Lys Arg Val Gly
    165 170 175
    Glu Leu Gly Ser Ile Ala Thr Phe Ser Phe Gln Asn Gly Lys Leu Met
    180 185 190
    Thr Ala Gly Glu Gly Gly Ala Val Leu Phe Ala Asp Gln Ala Gln Trp
    195 200 205
    Glu Lys Ala Phe Val Leu His Ser Cys Gly Arg Pro Lys Gly Asp Arg
    210 215 220
    Gly Tyr Phe His Leu Thr Ser Gly Ser Asn Phe Arg Met Asn Glu Phe
    225 230 235 240
    Ser Ala Ala Val Leu Arg Ala Gln Leu Gly Arg Leu Asp Ser Gln Ile
    245 250 255
    Ala Thr Arg Gln Ala Arg Trp Pro Val Leu Ser Ala Leu Leu Ala Gly
    260 265 270
    Ile Asp Gly Val Val Pro Gln Thr Val Asp Pro Arg Ser Asp Arg Asn
    275 280 285
    Pro Ser Tyr Met Ala Met Phe Arg Met Pro Gly Val Thr Glu Glu Arg
    290 295 300
    Arg Asn Ala Val Val Asp Glu Leu Val Arg Arg Gly Ile Pro Ala Phe
    305 310 315 320
    Met Ala Phe Arg Ala Val Tyr Arg Thr Gln Ala Phe Trp Glu Thr Gly
    325 330 335
    Ala Pro Asp Leu Thr Pro Glu Glu Leu Ala Ala Arg Cys Pro Val Ser
    340 345 350
    Glu Glu Ile Thr Arg Asp Cys Val Trp Leu His His Arg Val Leu Leu
    355 360 365
    Gly Ala Glu Glu Gln Val Arg Arg Leu Ala Ala Val Val Ala Asp Val
    370 375 380
    Val Ala Gly Ala
    385
    <210> SEQ ID NO 15
    <211> LENGTH: 388
    <212> TYPE: PRT
    <213> ORGANISM: Amycolatopsis mediterranei
    <400> SEQUENCE: 15
    Met Asn Ala Arg Lys Ala Pro Glu Phe Pro Ala Trp Pro Gln Tyr Asp
    1 5 10 15
    Asp Ala Glu Arg Asn Gly Leu Val Arg Ala Leu Glu Gln Gly Gln Trp
    20 25 30
    Trp Arg Met Gly Gly Asp Glu Val Asn Ser Phe Glu Arg Glu Phe Ala
    35 40 45
    Ala His His Gly Ala Ala His Ala Leu Ala Val Thr Asn Gly Thr His
    50 55 60
    Ala Leu Glu Leu Ala Leu Gln Val Met Gly Val Gly Pro Gly Thr Glu
    65 70 75 80
    Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Gln Ala Ala Gln
    85 90 95
    Arg Leu Gly Ala Val Thr Val Pro Val Asp Val Asp Ala Ala Thr Tyr
    100 105 110
    Asn Leu Asp Pro Glu Ala Val Ala Ala Ala Val Thr Pro Arg Thr Lys
    115 120 125
    Val Ile Met Pro Val His Met Ala Gly Leu Met Ala Asp Met Asp Ala
    130 135 140
    Leu Ala Lys Ile Ser Ala Asp Thr Gly Val Pro Leu Leu Gln Asp Ala
    145 150 155 160
    Ala His Ala His Gly Ala Arg Trp Gln Gly Lys Arg Val Gly Glu Leu
    165 170 175
    Asp Ser Ile Ala Thr Phe Ser Phe Gln Asn Gly Lys Leu Met Thr Ala
    180 185 190
    Gly Glu Gly Gly Ala Val Val Phe Pro Asp Gly Glu Thr Glu Lys Tyr
    195 200 205
    Glu Thr Ala Phe Leu Arg His Ser Cys Gly Arg Pro Arg Asp Asp Arg
    210 215 220
    Arg Tyr Phe His Lys Ile Ala Gly Ser Asn Met Arg Leu Asn Glu Phe
    225 230 235 240
    Ser Ala Ser Val Leu Arg Ala Gln Leu Ala Arg Leu Asp Glu Gln Ile
    245 250 255
    Ala Val Arg Asp Glu Pro Trp Thr Leu Leu Ser Arg Leu Leu Gly Ala
    260 265 270
    Ile Asp Gly Val Val Pro Gln Gly Gly Asp Val Arg Ala Asp Arg Asn
    275 280 285
    Ser His Tyr Met Ala Met Phe Arg Ile Pro Gly Leu Thr Glu Glu Arg
    290 295 300
    Arg Asn Ala Leu Val Asp Arg Leu Val Glu Ala Gly Leu Pro Ala Phe
    305 310 315 320
    Ala Ala Phe Arg Ala Ile Tyr Arg Thr Asp Ala Phe Trp Glu Leu Gly
    325 330 335
    Ala Pro Asp Glu Ser Val Asp Ala Ile Ala Arg Arg Cys Pro Asn Thr
    340 345 350
    Asp Ala Ile Ser Ser Asp Cys Val Trp Leu His His Arg Val Leu Leu
    355 360 365
    Ala Gly Glu Pro Glu Leu His Ala Thr Ala Glu Ile Ile Ala Asp Ala
    370 375 380
    Val Gly Arg Ala
    385
    <210> SEQ ID NO 16
    <211> LENGTH: 1800
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 16
    atggaggacc gcaagcgcga ggggtatttc tagcgcggcg gggccggtgc ggcccacaag 60
    cggaggacta gtccctaagt atgaagtccc ctactccgtt tgtctgttga gggcaggggc 120
    gccgtctgag gatgatgcag tccatgtcac agttactttc cgggaaggac ggcgcccagg 180
    aggcgccaag tcgcggcggg tccacgtggg tggcggtcct cgccgcgtgc gtggggcagt 240
    tcgtggtggt cctcgacgtg tccgtcatca atgtcgcgct gccgtcgatc cgttccggcc 300
    tcgacatcgg cgagacgggc ctgcagtggg tggtcaacgc ctacgtcatc gccttcgcgg 360
    gcttcctgct gctcggcggc cgggcctccg acctcttcgg ccgcaaggcc gtgttcgtct 420
    tcggcctcgg ggtgttcacc gccgcgagcc tgctcggcgg cctcgcgcag gcgccgtgga 480
    tgctcatcgt cgcccgcgcc ctgcaaggca tcggggcggc cgtgctctca cccgccaccc 540
    tcgcgatcct caccaccacg ttccccgagg gtccggcgcg catcaaagcc gtcgcgatct 600
    ggacggccgt gggcacgggc ggcggcgcgg ccggcggcct catcggcggc ctgctcaccg 660
    actacctctc gtggcgctgg gtgttgctga tcaacgtgcc gctgggcctt gtcgtgatcg 720
    tcgcgaccgt cgcctggctg gccgagagcc gcagcgacca ggcacaccga cgccggctgg 780
    acctcccggg agcggtgctg gtgaccctgg gcgtcggcag cctggcctac ggcatctcgc 840
    agagcgaggg ccacggctgg ggctcgccgc ggacgctcac cttcctgatc gtcggtgtcg 900
    tggcgctcct cgccttcgtc gccgtggagc agcgcacgcg cgagccgttg atgccgctcg 960
    gtgtcttccg ggtgcgctcg gtgtcggcgg ccaacgccat caccatcgtc agtggcatgg 1020
    gcttctacgc gatgtggtac ttcctctcgc tctacatgca gaacgtgctg aaatactccg 1080
    ccgtacagac cggcctggcc ctgcttcccc acaccgccac catcatcctc tccgcgcagt 1140
    tcgcaccccg cctgatgcgg tggatcaagg ggcgcaccct cctcgtgatc gcgggactgc 1200
    tgaccgccgc gggcttcatc tggcagggga acatggacgc cgacggctcc ttcctggcga 1260
    ccctgctcgg cccgggaatc gtcttctcct tcggcgcggg cctgatgatg acgctcctcg 1320
    cggtctccgc cacgacgggc gtggagctct ccgaatcggg cctggtggcc ggcctcgcca 1380
    acacctcgcg caccatgggc ggcgcgctcg gcctgtcggt cctcgcgtcc gtcgccgccc 1440
    gccgcacggc cgacgtgggg cccggcgcgg agggcctggc ctccggctac ggtcgggcgt 1500
    tcgtcgtgtc cggggccatc atcctcgtga gcatgctgat gatccccttc ctgcccaagc 1560
    cccagcccca gaccccggcg gaatgacctg tgagcacgga catacgagga ggcttcgtgg 1620
    ggcaggacag ccggccgcgg tggctcaccg acgaggaaca acgcgtgtgg cgcggctatc 1680
    tgcgggccac caggctggtg gaggaccacc tggaccgccg cctccagcgg gaagcggaca 1740
    tgccgcacct ctattacggt cttctcgtcc agctctccga ggccccgcgc cgggggatcc 1800
    <210> SEQ ID NO 17
    <211> LENGTH: 484
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 17
    Met Met Gln Ser Met Ser Gln Leu Leu Ser Gly Lys Asp Gly Ala Gln
    1 5 10 15
    Glu Ala Pro Ser Arg Gly Gly Ser Thr Trp Val Ala Val Leu Ala Ala
    20 25 30
    Cys Val Gly Gln Phe Val Val Val Leu Asp Val Ser Val Ile Asn Val
    35 40 45
    Ala Leu Pro Ser Ile Arg Ser Gly Leu Asp Ile Gly Glu Thr Gly Leu
    50 55 60
    Gln Trp Val Val Asn Ala Tyr Val Ile Ala Phe Ala Gly Phe Leu Leu
    65 70 75 80
    Leu Gly Gly Arg Ala Ser Asp Leu Phe Gly Arg Lys Ala Val Phe Val
    85 90 95
    Phe Gly Leu Gly Val Phe Thr Ala Ala Ser Leu Leu Gly Gly Leu Ala
    100 105 110
    Gln Ala Pro Trp Met Leu Ile Val Ala Arg Ala Leu Gln Gly Ile Gly
    115 120 125
    Ala Ala Val Leu Ser Pro Ala Thr Leu Ala Ile Leu Thr Thr Thr Phe
    130 135 140
    Pro Glu Gly Pro Ala Arg Ile Lys Ala Val Ala Ile Trp Thr Ala Val
    145 150 155 160
    Gly Thr Gly Gly Gly Ala Ala Gly Gly Leu Ile Gly Gly Leu Leu Thr
    165 170 175
    Asp Tyr Leu Ser Trp Arg Trp Val Leu Leu Ile Asn Val Pro Leu Gly
    180 185 190
    Leu Val Val Ile Val Ala Thr Val Ala Trp Leu Ala Glu Ser Arg Ser
    195 200 205
    Asp Gln Ala His Arg Arg Arg Leu Asp Leu Pro Gly Ala Val Leu Val
    210 215 220
    Thr Leu Gly Val Gly Ser Leu Ala Tyr Gly Ile Ser Gln Ser Glu Gly
    225 230 235 240
    His Gly Trp Gly Ser Pro Arg Thr Leu Thr Phe Leu Ile Val Gly Val
    245 250 255
    Val Ala Leu Leu Ala Phe Val Ala Val Glu Gln Arg Thr Arg Glu Pro
    260 265 270
    Leu Met Pro Leu Gly Val Phe Arg Val Arg Ser Val Ser Ala Ala Asn
    275 280 285
    Ala Ile Thr Ile Val Ser Gly Met Gly Phe Tyr Ala Met Trp Tyr Phe
    290 295 300
    Leu Ser Leu Tyr Met Gln Asn Val Leu Lys Tyr Ser Ala Val Gln Thr
    305 310 315 320
    Gly Leu Ala Leu Leu Pro His Thr Ala Thr Ile Ile Leu Ser Ala Gln
    325 330 335
    Phe Ala Pro Arg Leu Met Arg Trp Ile Lys Gly Arg Thr Leu Leu Val
    340 345 350
    Ile Ala Gly Leu Leu Thr Ala Ala Gly Phe Ile Trp Gln Gly Asn Met
    355 360 365
    Asp Ala Asp Gly Ser Phe Leu Ala Thr Leu Leu Gly Pro Gly Ile Val
    370 375 380
    Phe Ser Phe Gly Ala Gly Leu Met Met Thr Leu Leu Ala Val Ser Ala
    385 390 395 400
    Thr Thr Gly Val Glu Leu Ser Glu Ser Gly Leu Val Ala Gly Leu Ala
    405 410 415
    Asn Thr Ser Arg Thr Met Gly Gly Ala Leu Gly Leu Ser Val Leu Ala
    420 425 430
    Ser Val Ala Ala Arg Arg Thr Ala Asp Val Gly Pro Gly Ala Glu Gly
    435 440 445
    Leu Ala Ser Gly Tyr Gly Arg Ala Phe Val Val Ser Gly Ala Ile Ile
    450 455 460
    Leu Val Ser Met Leu Met Ile Pro Phe Leu Pro Lys Pro Gln Pro Gln
    465 470 475 480
    Thr Pro Ala Glu
    <210> SEQ ID NO 18
    <211> LENGTH: 990
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 18
    atcccgatcg tctcggacat gaccggcgac cttctcggcg cgcgggaggc ccaggacccc 60
    gcctactggg tgtcccacat ccgccgcgcg gtgcgcttcc acgaccagat ccgccgtctg 120
    cagcgctacg gggccggggc cttcgtcgag gtcggcccgg acacggtgct cagctcggcc 180
    ggccaggcgt gcctgacgga ccaggcgggc aggagcgcgc ccgtcctggt gtccctcgcg 240
    cacgccgagc gcgcggaggt gcccgcgctc ctgaccgctc tggccaccct gcacacccgt 300
    ggcgtggccg tggactggcg ggcgtggttc ggcgacgggc cgcgcgcggc cggcctgccc 360
    acatacgcgt tccagaagca gcactactgg ccgtcgggcc ccaccggttg gcggtccggg 420
    cccgcccccg tacccctgcc ccaggccgga acggaggacg ccgaaaggcc cggtcgcgcc 480
    gcggagtggc gggcgctgcc gcccggtgag cggtacgacg cgctgctgcg gatggtgcgc 540
    ggcgaagccg ccgccgtgat ggggcacgcc gggccggagg cggtggagcc ggagcgcggc 600
    ttcctcgacc acggcttcga ctcggtgatg gccgtgaagc tgcgcgaccg tctcgtggcc 660
    gggacggggc gggagctgcc gacgaccctg ctgttcgacc accccacgcc cgcggccgtc 720
    gccgactacc tgctggcggg gacgggcgag gccgagacgg cgccgtccgt gtccctgtcg 780
    gaccagctcg accgcctgga ggccgacctc gcgcggctgc cggccgacga ccggcagcgc 840
    gcccgcgtcg ccgagcggct caagggcctg ctcgcggtcc acgcgccgga ccggggcgcc 900
    gggagcgagg acgcgccgga ccaggacgcg ctggacacgg cgaccgacga cgagatgttc 960
    gagctgatcg agaaggaact ccgccgtgga 990
    <210> SEQ ID NO 19
    <211> LENGTH: 3978
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 19
    gtggatgaga ccaacgagac caaactccgc gagtacctgc ggctggtcac ggccgatctg 60
    cggcgaaccc gcaggcagtt ggaggaggcc gaggacgcgg cccgcgagcc cgtcgcgatc 120
    gtgggcatgg cgtgccgctt ccccggggac gtggcatcgc cggacgacct gtggcagctg 180
    gtcgccgagg gccgggacgc cgtcaccgag ttccccgccg accggggctg ggacgtcgac 240
    gccgtctacg accccgagcc gggcaccccg ggcaggacgt acgcgcgcca cggcggcttc 300
    ctcaaggacg ccgccggatt cgacgccgcc ttcttcggca tcacgccgcg cgaggcgctc 360
    gccatggacc cgcagcagcg catgatcatg gaggtctcct gggaggcgtt cgagcaggcg 420
    ggcctcgacg cgaccaccct gcggggcgag gacgtcggcg tcttcgtcgg ctccaacagc 480
    aacgactacc tgatcaacgt gctcgacgcg cgggacgtcg ccgagggctt catcgggacc 540
    ggcaactccg ccagcatcct ctccggccgc gtcgcctaca ccttcggctt cgagggcccg 600
    gccgtgtccg tcgacaccgc ctgctcctcc tcgctggtcg cgctgcacct ggccgcgcag 660
    tccctgcggc agggggagtg ctccctggcg ctggcgggcg gcgcgacggt gatggccacg 720
    ccgaccgcct tcatcgagtt cagccgccag cggggcctgg cccccgacgg ccgctgcaag 780
    tccttctcgg cgaccgccga cggcaccacc tggtccgagg gcgcggccgt gctgctgctg 840
    gcccggctct cggacgcccg ccgcctgggc taccccgtgc acgcggtcat ccggggcagc 900
    gccgtcaacc aggacggcgc gagcgcgggc ctgaccgcgc ccaacggacc ggcgcaacag 960
    cgggtgatcc ggcaggcact ggccaacgca cggctgacgg ccgacagcgt cgacgcggtc 1020
    gaggcacacg gcaccggcac cccgctgggc gacccgatcg aggcccaggc cctcctcgcc 1080
    acctacgggc gggcccgcgg cgagggcagg ccgctgtggc tgggctcgct gaagtcgaac 1140
    ctgggccaca cccagtccgc ggccggcgcg ggcggcgtca tcaagatggt gatggccatg 1200
    cggcacggga cgctgccccg cacgctgcac ctcacggagc ccaccccgcg cgtcgactgg 1260
    tccgccggtg acgtacggct gctgaccgag gcccaggact ggccggacac cggacagccg 1320
    cgccgtgcgg ccgtctcgtc cttcggcgtc agcggcacca acgcccatgt gatcctggag 1380
    ggcccgcccg ccgaggaggc accggacgcg ccgctgccgg acgtctcctc gcagccgcgg 1440
    ggcccgctgc cgtgggtcgt ctccggccgc agcgaggcgg ccgtccgagc gcaggccgag 1500
    cgcctggcgg cccacctgac cgcgcgcccg cacctggcac cggccgacgt ggccaccgcg 1560
    ctggccacca cgcgggcggc cttcgaccac cgggccgccg tcgtcggccg ggaccgtgag 1620
    gaactgctcg ccggcctcgc ggccctggcc accggaaccc gcgcgcccgg cctggtcacc 1680
    ggccggaccc cgccgtccgg cggcaaggcc gccttcctct tcaccggaca gggcagccag 1740
    cagcccggca tgggccgcga actggcggct cacagcaccg tgttcgccga cgccctggac 1800
    gaggtctgcg cccagctcga ccggcacctc gaccggccgc tgcgcgaggt gctgttcgcc 1860
    gcggacggca cgcccgaggc cgccctgctc gacacgacgg cctacaccca gcccgcgctg 1920
    ttcgccgtcg aggtcgcgct gctgcggctg ctggaggact ggggcttgcg gcccggcatg 1980
    gtcgcgggcc actcggtcgg cgaactgacc gccgcctacg ccgccggggt ctggtcgctc 2040
    gccgacgcct gcgccctggt cgccgcccgc ggccggctga cccaggcact gcccgcgggc 2100
    ggcgccatgg tcgccgtgca ggcgaccgag gacgaggtgc gcgcccaact cgccgacggc 2160
    cgccccggcg tggacatcgc cgccgtcaac ggaccggaag cggtggtgct gtccggcgac 2220
    gaggccgccg tcacggacct ggcgcgcgag tgggccgccc gcggccggga gaccaggagg 2280
    ctgcgggtca gccacgcctt ccactccgcc cacctggacg ccatgaccga ggcgttcgcc 2340
    gaggtcgcac gaggggtgtc ctacagcgcg ccgtccctcc cggtggtctc cacgctcacc 2400
    ggggcccccg tcaccgacga gctccgcagg ccggaacact gggtgcggca cgtccgggag 2460
    acggtgcgct tccacgacgc ggtccgcgcc ctgcgcgacc gcggggccac cgcgttcctg 2520
    gaggtcgggc ccggcggcgt gctgacggcc gcggcacgcc gatgcctgcc cgacgccgcc 2580
    cccgagacgt tcgtccccgt gctgcggcgc cgcaggcccg aacccgagtc cgtgctgacg 2640
    gccgtcgcgc aggcccacac gatcggcctc tcgccggcgt gggaccgcct gctgcccaag 2700
    gcccggacgc gcgtggacct gcccacgtac gccttccagc gcggccacta ctggctggcg 2760
    ggcatggccg gagcgggcac cgcgcggccg gtgcggccgg aagtgcagga gcccaccgcc 2820
    ccctccggta cgccgccgct gtcgcgacgg ctggccgacg cgtcggagga ggagcgcggc 2880
    cacctgctgc tgacgctggt acgcgagcag tcggccaccg tgatgggcgg cgtcgacccc 2940
    gcgcaggtcg aacccgaccg ccccttcctg gagctcggct tcgactccct gatgggcgtc 3000
    gagctgcgca ccgcgctcgc cgccgactgc gcactgcccc tgccgcccgg cctgatcttc 3060
    gaccacccca cgcccgccgc cctggccgcc ttcctcggcg agcagctcgc ggcggcggcc 3120
    tccggcaccc ccacggcggc ggcaccctcg ccgtactccc tggaggcgct gtaccgcaac 3180
    gccaacaccc tcgaccggcc cgaggacgcg ctcgccctca ccaaggccgc ctcccggctg 3240
    cgcccggtct tcgccagcgt ggccgaggcg gggcaggacc cggtcacggt ggagctggca 3300
    caggccaccg gccttccggg cctgatctgc tgcccggcac ccgtgccgct gtacggggca 3360
    cagcagtaca gccggctcgc agccgccttc cgcggcacgc gcggagtctc ggccctgctc 3420
    gcccccggct tctccccggg cgaactgctg cccgccgact tcgaggtgat gcaggacttc 3480
    ctcgccgagg gggtccggcg gcagaccgac ggcgcgccct tcgtcctcct gggccactcc 3540
    tccgggggct ggttcgccta cagcctggcg gcccacctgg cgcgcaccgg gccgcgcccg 3600
    gaggccgtcg tgctgctgga cacctatcag ctgcacgacc cggcgctgca ccgcatgcag 3660
    cgcgaactcg cccagggcgt cctggaccgc gaggaggact tcggggcgat gacggacgta 3720
    cggctgagtg ccatgggcaa atacttcgac ttcttcaccg actgggtggc cgaggacgcc 3780
    ggtgtcccga cgctgctgct gcgggcctcc gagcctctgg gcgaggtcgt cgagggccag 3840
    gagtggcgct ccacctggcc gttcgacagc acggtcctcg acacggaagg cgaccacttc 3900
    gccatggtca acgaccacgc gccgcggacg gcccaggccg tgaacggctg gctgtcgggc 3960
    ctcaccggcg gaaggggc 3978
    <210> SEQ ID NO 20
    <211> LENGTH: 570
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 20
    gtggagacac gcaacgccga acggccgtgg atacgcagct tccaccccgc tccccaggcc 60
    cctgtgcggc tgctgtgcct gccgcacgcc gggggctccg cgagcgccta cttcgcgctg 120
    tcgagggaac tggcgccccg ggtggaggtg ctcgccgtgc agtaccccgg gcggcaggac 180
    cggcgcgacg agccgctgct ggactcgatc gaggccctgc gcgacggggt cgccgaggcc 240
    ctgacgccct ggctggaccg gccggtcgcc ctcttcggcc acagcatggg cgccgtggtg 300
    gcctacgagc tggcgcggct gctgtgccag gacgcgggcg tgccgctcac ccacctcttc 360
    gtctccggac gccggggatc cgaccgaagt ctccgtcctt gccgccgtgt tccggaattc 420
    accgtgacac cgccgcgcgg ctcttcttcc gaagtcctcc agatccggca cgagtttgta 480
    tccgaacggg gttctgcgtg cgaaatactc tcttcgaatt gggtgacata cccccgatcg 540
    gcaccgtacc cgagcagatg tacgcctcgg 570
    <210> SEQ ID NO 21
    <211> LENGTH: 1245
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 21
    gtgcgaaata ctctcttcga attgggtgac atacccccga tcggcaccgt acccgagcag 60
    atgtacgcct cggtgatccg acgggagcgc tacggacagc cccaccaggc gttccgcagc 120
    gaggtcgtgg acgtgccgaa ggtggggccc ggtcaggcgc tggtcctcgt gatggccgcg 180
    ggcatcaact acaacaacgt ctgggcctcc ctggggcagc cggtcgacgt gatctccgcg 240
    cggcagaagc agggccacag cgaggacttc cacatcggcg ggtccgaggg ctccggcgtg 300
    gtgtgggcgg tgggggaggg cgtcacccag gtcgcggtgg gcgacgaagt gatcctctcc 360
    ggctgccagt ggacggagac ggccgccgac atccggctcg gcgccgaccc catgacctcc 420
    ggctcgcagt cggtgtgggg atacgagggc aactacggct ccttcgccca gttcgccctc 480
    gtcgacgact atcagtgcca ccccaagccg cccggcctga cctgggagga agccgcctgc 540
    ttcctgctca ccggggccac cgcctaccgc cagctgtgcg gctggcagcc gcacgacgtg 600
    cggccgggcg acccggtcct catctggggc ggggccggcg ggctcggctc catggccatc 660
    cagatcaccc gggcgcgggg cggcatcccc gtcgccgtgg tctccgacga ggagcgggcc 720
    cgctactgcc gggagctcgg cgcccagggc accatcaacc gcctggactt cgaccactgg 780
    ggacggctgc ccgacatcgg cgaccacgag gcgatgggcc gctggaccga gggtgtacgg 840
    gccttcggcc ggcgcttctg ggaggtgctg ggcgagcgca ggtccccgcg catcgtcctg 900
    gagcacagcg gccaggccac catccccacc tcgatgtacc tgtgcgacaa cgcgggcatg 960
    gtcgtcatct gcggcggcac caccggctac aacgccgaca tcgacctgcg cttcctgtgg 1020
    atgcgtcaga agcgcttgca gggctcgcac ttcgccaacc tgcggcagtg ccgcgacgtc 1080
    atccacatgg tcgcgaacgg ccagctcgac ccgtgcctgt cgtggaccgg cggcttcgac 1140
    gacatcggca aggcacacca gctgatgcac gacaaccagc acccccaggg caaccaggcc 1200
    gtcctggtca acgcgccgcg gaccggcctg accaccttcg cctga 1245
    <210> SEQ ID NO 22
    <211> LENGTH: 1224
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 22
    gtgtccgaca ccgagcagca cgcgcccacg ctgccgcggc agcgcacctg ccccttctcg 60
    ccgccgcccg agctcgagga gctgcggcgc accgatccca tcagcaggat gcggttcgcc 120
    gacgactccc cgggatggct gctgacccgc cacgccgacg tccgcgccgc gctggccgac 180
    cccggcgtca gctcgcaccc cggcaaggca ccccagccct ggcgcaacct cgcccccgag 240
    atgcgcgccg agcactacct gccgggcttc ctgatcttca tggacccgcc ggaccacacc 300
    cgctaccgcc gcctgctcac caagtggttc accatgcggg ccatccgcaa gctcgaaccc 360
    aggatcgagc agatcgtcac cgagaccctc gacgccatgg aggcccaggg cggcaccgtc 420
    gacctggtgc agtccttcgc gctgccgatc ccgctgctgg tcatctgcga gctgatgggc 480
    atccgctacg aggagcgcga ggagttcatg gacatggtcc tgcgactcca ggccctggac 540
    gccacgcccg aggaactcgg ggccctcggc gccaggatga acgagttcat gatgaagctc 600
    gccgccgcca agcgcgcgaa ccccggcgac gacctgctca gccacctcgc ccacgacccc 660
    gacgccgacc cggcgctcac ggatctggag atcgccggca tcggcgtgct gatgctcatc 720
    gcggggcacg agacctcggc caacatgctg ggcgtcggca cctacaccct gctggagaac 780
    gccgaccagt gggccctgct ccgtgacgac atcagcctga tcgaccgggc cgtcgaggag 840
    ctgctgcgcc accagaccat cgtccagcag ggcctgccgc gcggcgtcac ccgggacatg 900
    gagatcgccg ggcaccaggt gaagaccggg gagtccctgc tggcctcgct gcccgccgcc 960
    aaccgcgacc ccgccgtctt ccccgacccc gaccgcctcg acatcacgcg cgagcacaac 1020
    ccgcacctcg ccttcggcca cggcatccac ctctgcctgg gcatggagct cgcccgggtg 1080
    gagatgcgcc aggcgtggcg cggcctcgtc acgcgcttcc ccggcctgcg catggccgcc 1140
    gcgcccgagg acatccgctg gcgcgacgac cagatcgtct acggcgtgta caacctcccg 1200
    gtgacctggg acgaggccaa gtga 1224
    <210> SEQ ID NO 23
    <211> LENGTH: 531
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 23
    atggacaagc tcgacatcct ctggagcgag cgcgagatcc gtgccgtgct gcagcgctac 60
    tgccgcgggc tcgaccgcct cgacgaggaa ctggtcaagt ccgcctacca cgaggacgcg 120
    cacgacgacc gcggcgtcat ccgcggcaac gcacacgact tcgtcaagca gatcgtcccg 180
    ctcctgcgcg acgcctacac cggcaccctg cacaccctgc acggcagcac gatcgagatc 240
    gacggggatg ccgcgggcgt ggagtcctac tgcaccgcct accactaccg cgagagcgac 300
    ggcatcaagc gggtggagca gttcgccggg cgctacgtcg accgcttcga gcggcgcgac 360
    ggcgtctgga agatcgcccg ccggctcgtg ctgaacgact tcagcctcgc ccaggaggtg 420
    ccgctcgacc ccgccgaggc ccaggccggc ttcaacccct cccaccgcga cctcaccgac 480
    gccagctacc aggtgctgcc gctgcgcggc ccggacgccc ccaccctctg a 531
    <210> SEQ ID NO 24
    <211> LENGTH: 1233
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 24
    gtgaccggcc ccgaggccgc ggtgcgcggg tgccccttcg gcgccggcga ggcgcccgcg 60
    taccccttcc acgcccccga ccggctggag cccgacccgt actgggagcc gctgcgccgc 120
    gagcggccgc tgcaacgcgt cacgctgccg tacggcggcg aggcgtggct cgccacccgc 180
    tatcaggacg tgcgcgcggt cttcgccgac cgcaggttct cccggcagct cgccgtcgcg 240
    cccggcgctc cgcgcttcct cccgcaccag ccgccgccgg acgccgtcct gagcgtcgag 300
    ggccccgacc acgcgcggct gcgccggctg gtcgggaagg tcttcacgcc gcgccgcgtg 360
    gaggacatgc gtccgctcat ccagcgcacc gccgacggac tcctcgacgc gatggaggag 420
    atggggccgc ccgcggacct ggtcgaggac ttctccctgc ccttcgccgt gtccatgatc 480
    tgcgagctgc tcggcgtgcc gcccgaggac cgcaagcggt tctgcgtctg gtcggacgcg 540
    ctgctgacga ccaccgcgca cacccccgcc caggtgcgcg actacatgat gcagatgcac 600
    gactacctcg gcgggctcgt cgcgcagcgc cgggtgcggc ccaccgcgga cctgatcggc 660
    tccctcgtga ccgcgcgcga cgaggaggac aagctcaccg agggcgagct ggtgcggctg 720
    gccgaggcca tcctcatcgc cggctacgag acctcggcga gccagatccc caacttcctc 780
    tacgtcctct tccgccaccc gcagctgctg gagcggatca ggaacgacca cgacctcatc 840
    cccgacgccg tcgaggaact gctgcgcttc gtgcccatcg gcaccgtgga cggctttccc 900
    cgtacggcca ccgaggacgt cgagctcggg ggagtcctgg tcagggccgg ggagacggtc 960
    gtgccgtcga tgggcgccgc caaccgcgac cccgagctgt tcacggaccc cgacgagctg 1020
    gacctcgcgc ggcggccgaa tccgcacctg ggcttcggcg cgggaccgca ccactgcctg 1080
    ggcgcccaac tggcccgggt ggagctccag atcacgctca cgacgctgtt ccgcagatac 1140
    ccccgcctgc ggctggccgt gccggaggag agcctctcgt ggaaggaggg gctgatggtc 1200
    cgcggcatgc acaccatgcc ggtcacctgg tga 1233
    <210> SEQ ID NO 25
    <211> LENGTH: 1107
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 25
    atgagcacca tcgacgaatg ggaacacagc acgaaggagg cgggcatgga ccccgcggcc 60
    ctcagacgcc tgaccgatgt ggtgcgggcg aggggcggcg cggcgcagct gtgcgtcatg 120
    cggcggggca ccgtggtcct ggaccgctcg ttcggctgct cctccgactc cctcttcctc 180
    gtctacgcgg ccaccaagcc cgtcgccgcc ctcgccgtgc acgcgctcgc cgagcggggc 240
    ctgatcgggc tggaccggcc ggtggccgaa tactggccgc agttcgcccg gcacggcaag 300
    ggtgacgtga ccgtccgtca tgtcctccag caccgggccg gggtgccggt cggccggggc 360
    atcgtgcgca cgatgcgcac cgccggcgac tgggagcgct ccgtgcgcga ccttgagcag 420
    tcccggccca agtggcccgg cggcgaggtc gccgcctacc acttcatgag tttcggattc 480
    attctcggcg aactggtgca gcgcgtcacc gggcggtcgt tccgagattt cgtgacttcc 540
    gagctcttcg ccccacttgg gctgaatgat ttgcacatgg gattgcccgg cagtgcctgg 600
    ccccggcatg tgcccgcgcg ggccgcccac ccctccgaat ggcccaatca gtggatgagc 660
    aaccgccgcg gctaccgcca ggccgtcatt ccgtccgccg gtctttccgg aaccgccgca 720
    caaatggccc gcttttacca gatgcttatg gagggcggct cgctcgacgg catccgcgtg 780
    ctgcggcccg aaactgtgga ggaagccaga aaaccgtcca atgacggcgg aatcgacgct 840
    tccctcaagc gtccggtccg ctggtcccac ggattcatgc tcggtggtcc gggcccggac 900
    ccgcgggggc tgtccaatgt gctgggccgc acgagcgacc cgagcgcctt cgggcacgcg 960
    ggcaccacgt ccagcgtcgt gtgggccgac cccacgcgcg agctggtcct cgcctacctc 1020
    tccaacatcc agcccgagtt cggagcgggt atcgagcggc tccgcgaggt cagtgacctc 1080
    gcgctcggtg cctgcgaggc aggctga 1107
    <210> SEQ ID NO 26
    <211> LENGTH: 858
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 26
    gtgctgaatc tgcccaaagg aatggagcgc gcgcatccgc attctccgcc acaggtggga 60
    atactcggac ccttggaagt ccgctcggcc ggaggtgccg gaacgggagc cgcggtaagc 120
    ggtattcgcg tacgcacatt gcttgccgcg ttgactgccc gcctggggca ggcgatgtcg 180
    accgagcgca tcctcaaaga ggtctgggcc gacaacccgc ccgcgaccga tcgcaaggcg 240
    gtggccgtcg ccgtcctgcg gctgcggcgg gtcctcggcg acaacgaagg acggtggctg 300
    ctcacccgcc cctccggtta cgtcctggac atccccccgg accacctcga cgccgtacgc 360
    gcggagaccc tggtgcggga aggccgggcc gccctggccg ccggcgaccc acgcgtcgcg 420
    gcccgccacc tcacgcgcgc cctcgaccag tggcggggcg agccctacgc ggacgccaac 480
    gccatctcga ccgtgtccca gcgcatcacg gagctggaga acctcaggtc cgaggccgtc 540
    caggcgcaca tcgacgccag gctcgaactg ggtcaccacc aggaactggt cggcgaactc 600
    cgctcgctga ccgccgcgaa ccccctgcac gagccgcact ggctgcagct gatgctcgcc 660
    ctctaccgct ccggcaagca ggccgaggct ctcgccgcct atatgcagct gcggcaggcg 720
    ctggccgaga acctgggcat cgacccgggt cgtcagctcc aggaactgca cctgcggatc 780
    ctgcgcgccg acgcgggcct gctgacgggg tccgggccgg cggcaccggc cgagccactg 840
    ctcgtacggc agtcctga 858
    <210> SEQ ID NO 27
    <211> LENGTH: 420
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 27
    atgcgtggat cgaaggccct ccgatacgcg gcccccgtcc tggtcgccgc cgcaaccggc 60
    atcgccctcg ccgcgggacc ggcggccgcc gtcccgatcg gtcagtccgt gaacggcaag 120
    atgacctact acaccgacca gggctacggc gcctgcggca cccccatcga cgcgaactcc 180
    caggacctcg tcgcggtccc ggccgcgtgg tggacctccg ccaaccccaa caacgaccag 240
    ctctgccagg gcatatcggt ggaggtcagc tacaacggca ggaccatcag agtgccggtg 300
    cgggacaagt gcccttcgtg cgaccggacc cacatcgacc tcagcaggac ggccttccag 360
    aagctggcgc cgctcgacag gggtgtggtc aacggcatca cctggaagtt cgtccgctga 420
    <210> SEQ ID NO 28
    <211> LENGTH: 2811
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 28
    atgagttcat caaatttaag gtcgcgggac tcttggaaca gatcaagacg acggagaaca 60
    atgacgtact cccccggcgc gcggccgcgc ccggcccggc tgtccgcact gctgctcgca 120
    ggcgcgctcg tcgcctcggt gccgcccgcg gccgccgcgc gagcgccgca accccccacc 180
    gccgaccgcc cccgcaccgc cgcctccccc acaggcggct gccgtacggg tgacggctgg 240
    acactcgact ccacccgcat cgaccccgac gacacccacc acgcctatgt cggcaacggc 300
    tacctggggc agcgcgtacc gcccaacggc gccggctaca ccgacagcga caccaagacc 360
    ggctggccgc tcttcgctcc ggcctacgac ggctcgttcg tgtccgggct ctacgcgcac 420
    aacaagcaga ccgccgccga ccggcaggtg atcgccgctc tgcccacctg gaccggactg 480
    gccgtcggca ccggcggcga gcacggcgat atcttcaact cttcgacgaa gtcgggccgg 540
    atttccggat atcaccagac cctcttccag agctgcggca tcgtccgtac cgccctgacc 600
    tggaccgccg ccgacggccg caggaccgac ctggtctacg aggtgctggc cgaccgcgac 660
    gacccgcaca cgggcgccgt acggctgagc atgacgccgc gctggagcgg cgaggccacc 720
    gtcaccgacc agctggacgg acgcggcgcg cggcgcatgc ggcagaccgg cggcggcgac 780
    cgcaccggtg ggaccggccg ggacggccgc accatggacg tggccttccg caccgacggc 840
    acggacaccg acggcgccgt cgcctccacc ctgagggccg ggcgcggtgt gcacacgacc 900
    ggggaccgac gcgccgcggc cgcgaaggac ttgagcgtga accagtccct cacgttcccc 960
    gtccgtgcgg gccacgcgta cgaactcacc aaatacgtgg gtgtcgacac cgcgctcacc 1020
    tcgcacgcgc cccgcgagga cgccaccacc gcctccctgc gcgccgcccg ccgcggctgg 1080
    gacgggctgc tgcgtgccca caccgccgcc tgggcccggc tgtggcgctc cgacatcgag 1140
    ctgccgggac agcgcgacct ccaggcgtgg gtgcgttccg cccagtacgg gctgctgtcc 1200
    agcacccggc agggggcatc caacagcatc gccccggccg ggctgaccag cgacaactac 1260
    gcgggcctgg tgttctggga cgccgagacc tggatgtacc cggccctgct ggccaccgcg 1320
    ccccaactcg ccaggaccgt cgtcgactac cgctaccgca ccctcgccgg agcgcgcgag 1380
    aacgcccaca agctcggcta ccaagggctc ttctacccct ggaacagcgg cagcgagggc 1440
    gacctggccc aggagtgcca cagcgtcgac ccgccccact gccgcaccca gatccacctc 1500
    cagtcggaca tctccctcgc cacctggcag ttctacctcg ccaccggcga caccgcctgg 1560
    ctgcgcgagc gcggctggcc ggtgatggag ggcatcgccg aattctgggc cgggcgggtc 1620
    acccccaacg ccgacggcag ctactccatc aaggacaccg ccggccccga cgaatacagc 1680
    aacggcgtcg acgacgcggt cttcaccaac gccggtgccg ccaccgccct gcgcgacgcc 1740
    gcccgtgccg cgcggctgct gggcgagcgc gccccggcgg agtggacgac gatcgccgac 1800
    cggatccgca tcccgtacga cgcgcggcac aaggtcttcg agcagtacga cggctacccg 1860
    ggcagcaaga tcaagcaggc cgacacggtg ctgctgatgt accccctgga gtggccgatg 1920
    tcccaggccg acgcggcgcg caccctcgac tactacgccc ggcgcaccga ccccgacggc 1980
    cccgccatga cggactcggt ccacgccatc gacgccgcgg ccacgggcga gccgggctgc 2040
    tcggcgtaca cctatctcca gcgttccgtc cggcccttcg tgcgcggtcc tttcgaccag 2100
    ttctcggaag cccgcggcac caaggccggc gccgacgacc ccctggccgg ctcgcccgcc 2160
    cacgacttcc tcaccggcaa gggcggcttc ctccagatct tcaccaacgg cctgaccggc 2220
    atgcggatgc gcgaggaccg gctgcacctc gacccgatgc tgcccccgca gctcggccgc 2280
    ggcgtcaccc tgcgcggcct gcactggcag ggccgcacgt acgacatcgc catcggcgcc 2340
    cacgagacca ccgtgcggct caccgggggt gcgcccatga ccctctacac cccgcagggc 2400
    gagcacgtgc tgaccaaggc ggcaccggcc gtgctcaaga cccgccgccc cgacctcgct 2460
    cccaccgaca acgtggcccg ctgcaccacc gccggtgcct cctccgagga acccggtatg 2520
    tacgcggcag ccgcggtcga cggcaacccc gccaccgcct gggtccccga cgggccgaac 2580
    ggtgaactga ccaccgacct cggcaagtcc gtacgcgtca ccaaggccac ccccgtctgg 2640
    agcggcccgg caccggcctc gtacagcgtc cagctctccc tcgacggccg gcactggcac 2700
    gacgcggtcg cgggcggcgc tccggtgtcc gcgcggtacg tacgcgtcgc gctacgcggt 2760
    caggccgatg ccaagtcccg tacgggcatc gccgagctga ccgttacgta g 2811
    <210> SEQ ID NO 29
    <211> LENGTH: 813
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 29
    atggaattcc tcgggccggc ggccggtgtc tcgggcgcca cgcggctgta cgcggtgctg 60
    ggtgatcccg tcgcccaggt caaggcgccc ggtctgctca accccctgct gagcgaaagc 120
    ggtctggacg ccgtggtggt gccggtgcac gtccgggcgc gggatctcgc cgaggtggtc 180
    gaggggctca agcggatcgg caatctggac ggtctgctgg tcaccgtgcc gcacaaggcg 240
    gccctgtgcg ggctcgcgga cgggctcggg ccggcggccg ccctcatcgg gacggccaac 300
    gcgatgcggc gcgaacccga cggccgctgg tacgccgaga acttcgacgg gctcgggttc 360
    gtccagggtc ttcaggcggc cgggcacacg gtgcgcgaca ggcatgtggc actggtcggc 420
    gccggagggg cgggcagcgc gatcgccacg gcgctgctga tggccgacgc cgcgcgggtg 480
    tccgtgcacg acaccgaccg cgcccagctc gacgcgctgc tgctgcggct cgggtcccgc 540
    cggccggacg ggatccgggc gctggggccc ggcgatctgg aggcggccga tttcgccgtc 600
    aacgcgacgc ctctgggcat gcgttccgag gacccgctgc ccttcgaccc cgcgagggtg 660
    cgaccggatg ccgtggtggt cgacgtcgtc atgaagccgc acgagacggc gctgctgagc 720
    gcggccgcca ccgccgggcg ccgtgtgcac cacggcatcc atatgctgga gcagcaggtt 780
    ccgtgctacc gcgcgttctt cgggtggccg tga 813
    <210> SEQ ID NO 30
    <211> LENGTH: 948
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 30
    gtgacggggg acaccgacgg tgcgggcggc ggcgacgtga cgttccgctg gcccgccgcc 60
    ggcgacgtca ccgcggatct ggacctgctc gccgcgcggg tccgcggtct tctgggacac 120
    cgcgaggacc ccctcgccgg ggtcggcgtg gccatgcccg cgatctgcga cgcggccggg 180
    acggtccgca cgtggccggg acggccgagc tgggcgggcc tgaacctgac ggccgccttc 240
    gggcagttgc tgcccggcac cccggtcgcc tgcgccgacg acggtgacct ggccgcgctg 300
    gcggagtccc gcgccgccgg ctgccggcat ctgctgtacg tgggggtcgg cacgggcatc 360
    ggcggcggca tcgtccatga gggccgcgcc tggccgggcc ccggacgcgg ctcgtgcgag 420
    gtcggccatg tcgtcgtcga ccgctcgggc ccacgctgcg actgcgggcg cgccggctgc 480
    gtccaggcgg tcgcgtcggg accggcgacc ctccggcggg ccgccgaacg gcgcggccgg 540
    gagaccggct tcgacgaact ggcctccggg gcgcgcttgc acgccccgtg ggcggaagcg 600
    gccgtcgacg agagcgccgc ggccctggcc accgccgtga ccggcatctg cgagctggcc 660
    caccccgaac tcgtcctcgt cggcggcggg ttcgcggcgg gcgtgccggg atacgtggcc 720
    tcggtggcgg cgcacgtcga gcggctgacc cgcccgggaa cggatcccgt gcgggtgcgc 780
    ccggcggtgc tcggcgggcg gtcctccctg cacggcgcac tgctgctcgc gcgggaggca 840
    cacgggcggg gaaaccggcc gccggagagt gaccgtgttt cttccgatgt ttcttccgat 900
    gtttctttcg ggggagtgac agacagggcc gttggccggt ccgactga 948
    <210> SEQ ID NO 31
    <211> LENGTH: 1545
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 31
    atgctcgaca ggcggagcgt cattcgcgtc ggcgccgggg tggcggcggc cgccgccgtg 60
    gccggtacgg ccgccaccgg tgcggcggcc gtggggctgc cgggtgtacg gggacgcgcg 120
    gcgtcgcgcg gggtcgactg ggcctcctta cgccgtcatc tgtcgggcga gctcgtcctg 180
    ccggcggaca ccggatacga gcgggccagg aagctctaca gcggccagtt cgacggcatc 240
    cgcccgcagg ccgtcgccta ctgccggacc gaggaggacg tgcggacgac cctcgcgttc 300
    gcccaggacc acgcgctgcc cctcaccccg cgcagtggcg ggcacagctt cggcggctac 360
    tccacgaccg acggaatcgt cctggacgtc tccggcttcc acgcggtgag cctcacccgg 420
    aacaccgtcg tcatgggcgc gggcacccag caggtggacg ccctcaccgc cctgtcgccg 480
    cgcggtgtcg ccgtggcgag cggcaactgc gcgggcgtct gtcccggcgg cttcgtccag 540
    ggcggcggac tgggctggca gagccgcaag ttcggcatgg cgtgcgaccg gctcgtctcc 600
    gcccgggtcg tgctcgccga cggccgcgcc gtgaccgcct ccgccaccga acaccccgac 660
    cttttctggg cgatgcgcgg cggaggcggc ggcaacttcg gcgtcgtcac cggcttcgag 720
    ctgcgcccca ccgacgtccc ctccgtcgtc agctacaacc tcacctggcc gtgggagtcg 780
    gcgcggcgcg tcatcgaggc gtggcagcac tggatcatcg acggcccccg cgacctcggt 840
    gccgcgatgg ccgtgcagtg gcccgacgcc gggaccggca cgccggtcgt ggtcgtcacc 900
    ggcgcctggc tgggcgcggc cgacgcgctc acccccgtgc tggactccct ggtggcctcc 960
    gtgggcagcg cgcccgccac ccgctcggcc aaggcgctct cccagcacga cgcgatgatg 1020
    gcgcagtacg gctgcgccga cctcacgccc gagcagtgcc acacggtcgg ctactcgccc 1080
    gaggccgcgc tgccccggca gaacttctcc atggaccgca accggctctt ctcccgggcc 1140
    atcgggcaag gaggcgtcga gcggatcctg gaggcgttcg ccgccgaccc gcgcgccgga 1200
    cagttccgct tcctgagctt cttcgccctc ggcggcgccg ccaaccgccc cgaccgcacc 1260
    accaccgcct acgttcaccg cgacaccgag ttctacctcg gtttctcgat cgggctgaac 1320
    gacccggagt acacggcgga ggacgagagg ctcggccgcg cctgggccgc gcgaggactg 1380
    cgcacgctcg atccccactc caacggcgag agctaccaga acttcatcga cccggagctc 1440
    gacgactgga agtcggccta ctacgccgag aactacgtgc gcctggccgc cgtcaaggcg 1500
    gcctacgacc cgcaccggct cttctccttc gcgcaggccg tctga 1545
    <210> SEQ ID NO 32
    <211> LENGTH: 495
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 32
    gtggagaggg tggagctgat ccgctggccg gtggagtccg agcggcggga gcgctgccgc 60
    gaccggggcg tcatgcggat cctggtgctg gaggcggggg ccgaggcacc cttgtgcgtg 120
    gaccccaagg aggactgggt ccgcgctccc gtcagcaccg acgacctgcg ggcccgcgtc 180
    gaggccctgc gccttcgggg agccgccgcc gagtcccggc ccgaggtcga cccgaacgga 240
    gtgctgcgtt tccggtggcg ctccgccctg ctctcgccca ccgaggcccg gctcgtcgcc 300
    cggctcgccg agtcctatgc cgaggtcgtc gcccgcgacg acctgctccg cccgcccccg 360
    ggccgtaccg tgccgagccg taacgcgctc gacctccaca tcatgcggat ccgacggcgc 420
    ctcgccgcgc tgggcctgag ggtgcgcacc gtccgggggc gtggctacgt cctggagagc 480
    gcggaaggag tctga 495
    <210> SEQ ID NO 33
    <211> LENGTH: 1032
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 33
    gtgcagcagc ctcatcacag ccgcgtcgac gtggaactgg gcgagaggtc ctaccccgtc 60
    cacgtcggac cgggggtccg ccacctcctg cccggcatcg tcgcctccct cggcgcgcac 120
    cgcgccgccg tcgtgaccgc acggcccccc gacctggtgc ccgatcccgg cgtgcccgcg 180
    ctgatcgtgc gggcacgtga cggcgagcgg cacaagacgc tcgccaccgt cgaggacctg 240
    tgccgcaagt tcaccacctt cggcatcacg cgccacgacg tcgtcgtctc ctgcggagga 300
    ggctcgacga ccgacaccgt cggcctggcg gcggcgctgc accaccgtgg ggtgccggtg 360
    gtgcacctgc cgaccaccct cctggcccag gtggacgcga gcgtcggcgg caagacggcg 420
    gtcaacctgc ccgagggcaa gaacctcgtc ggcgcctact ggcagcccaa ggccgtgctg 480
    tgcgacacca cgtatctcca gacgctgccc gccgaggagt gggtcaacgg ctacggcgag 540
    atagcgcgct gccacttcat cggtgccggc gacctccgcg gcctcgccgt ccacgaccag 600
    gtcaccgcga gcctgcggct gaaggcgtcc gtcgtcgcgg ccgacgagcg ggacaccggc 660
    ctgcggcaca tcctcaacta cggccatacg ctgggccacg cactggagac cgccaccggc 720
    ttcgggctgc ggcacggact cggcgtggcg atcgggacgg tcttcgcggg ccggctcgcg 780
    gaggcgctgg gccgcatcgg cgccgaccgc gcgcgggagc acaccgaggt cgtccgccac 840
    tacggacttc ccgacagcct cccgggaaac accgacatca ccgagctcgt cgcgctgatg 900
    aggcacgaca agaaggccac gtcgggactg accttcgtgc tcgacgggcc ttccggcgtg 960
    gagctggtgt ccgggatccc ggaggacgtc gtcctgcgta cgctcgcggc gatgccgcga 1020
    ggaacggcct ga 1032
    <210> SEQ ID NO 34
    <211> LENGTH: 441
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 34
    gtgttccgtc ttccgagggg aagtgaccgt ttcgtgtcgg cagagctgtc agaaccgctg 60
    aagaaggccc tggactccct ggtgttcggc gtcgtggcga cgaccgaccc cgacggccgc 120
    ccgcaccagt cggtggtgtg ggtccggcgc gagggctccg acgtgctgtt ctcgatcacg 180
    cgcggcagcc gcaaggagag gaacatcctg cgcgacccgc gtgtgagcgt gctgatcagc 240
    ccggcggact cgccgtacac ctacgccgcg atccggggca ccgcgcactt cgaggacgtg 300
    ccggacccgg gcgcgtacct cgacacgttc tccataaagt accacggcgt gccctaccgg 360
    gagtcgttcc ccgagccgcc ggaggtgagc accattctcg ccgtccggct cgttccgacg 420
    tcggtctacg agcagtggtg a 441
    <210> SEQ ID NO 35
    <211> LENGTH: 828
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 35
    atgacggaaa ccgcgtccgc ctccgaccgg atggtcgaac tctacaaccg cgtcaccgac 60
    ttgatggtgc acgcggaagg cggctacatg cacggtggct actgggcggg acccgacgtc 120
    cccacgacgg tggaagaggc aggcgaccgg ctgaccgact acgtctcgga gcgcctgcgc 180
    ctcgcccccg gggagcgggt gctcgacgtg gggtcgggca acggcaaggc caccttgcgc 240
    atcgccgccc ggcacggggt gcgggccacc ggggtctcca tcaaccccta ccaggtgggt 300
    ctgtcgcggc agctcgccga gaaggagggc gacgaggcga ccgagttccg catcggtgac 360
    atgctcgcgc tcccctttcc cgacggctcg ttcgacgcct gttacgcgat cgagagcatc 420
    tgccacgccc tggaacgggc cgacgtcttc accgagatcg cccgggtgct gcgcccgggc 480
    ggccgggtga cggtgacgga cttcgtgctg cgccggcccc tgagcgacgc gtccaggacg 540
    atcgtcgaca ccgccaacga caacttccag cagggccccg tcctcacccg cgaggcgtac 600
    gaggactgca tgcggtcggt ggggctggag gtggtggagt tcctcgacat cggggacgag 660
    gtgcggccct cctacgaggc ggtggcggcg aagatgcgtg cggccaggga cgagctcggc 720
    tcccacatgg acgacgaggc gttccaccgc atggtcgacg gcatcgaccg catgggctcg 780
    gtggaggagg tcggctactc ggtggtcacc gcgcggaaac cggcgtag 828
    <210> SEQ ID NO 36
    <211> LENGTH: 852
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 36
    atgccgcact ccgagctgtc cgaactcccc atgccctcac ccgcctccga ggaagtgggc 60
    gcgctctacg accggttcac cgcgctggga gccgcctccc tcggcgagaa cctgcacttc 120
    ggctactggg actcccccga cagccaggtg ccgctggccg aggccaccga ccggctcacc 180
    gacatgatgg ccgagcggct gcgcatcggc gccggctccc gcgtcctgga cctcggctgc 240
    ggcgtgggga ccccgggcgt acgcatcgcc cggctcagcg gagcgcatgt cacgggcatc 300
    tcggtgagcc atgagcaggt cgtccgggcc aacgcgctgg ccgaggaggc cgggctcgcc 360
    gaccgggcgc gcttccagcg ggccgacgcg atggacctcc ccttcgagga cgagagcttc 420
    gacgccgtca tcgccctcga atcgatcatc cacatgcccg accgcgccca ggtgctcgcc 480
    caggtcggcc gggtgctgcg gcccgggggc cgtctggtgc tcaccgactt cttcgagcgg 540
    gcccccctcg cccccgaggg gcgggccgcc gtccagcgct acctccacga cttcatgatg 600
    accatggtca gcgccgaggc gtaccctccc ctgctgcggg gggcgggcct gtggctggag 660
    gagttcctcg acatcagcga ccagaccctg gagaagacct tcaggctgct ctcggagcgc 720
    atcaactcct cgaagcagag gctggagacg cagttcggcg aggagatggt gaaccagttc 780
    gaccccggcg acctcgtcgg cgtcaaggag ttcggctatc tgctgctggt cgcccagcgc 840
    ccgggaaagt ga 852
    <210> SEQ ID NO 37
    <211> LENGTH: 1563
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 37
    gtgctgaaca ccctgtccac cgcgccgttc ctgtccacgg cctggctcgc cggggccgcg 60
    aggctcgaac gcccgcccgt gggcgaacgc ggcacggtcg cgctccgcct ggagctcacc 120
    gacccaccgc ccggcgaacc cccggccgtc gacgtccagg tggacctcgt cgccgggcgg 180
    ctcggcctcg cggccgcggc cggtgagagt ccgggactgc ggatccggct tcccctggag 240
    gccgcccgcg ccctgctgct cggccccgcg cgggatcgga ccggcgtatt cgagcggggc 300
    gacgtacggg ccgagggcaa tttcagcctg ctgttcttca tcgacgccgc actggagcgg 360
    gacgcctcgg gccatgtggc cgcgctcagg ggcacgcccg gtaccacggc gcgggaagcg 420
    gccccgccgc ccggcaccga ggacgcggcc gaggccgtcc ggcgcgcccg tgcggcgctt 480
    cccggcacca tgcgggagct ggagcgcgag gtcggcacct cgaccccggg ggcgcagatc 540
    tacgtctccc gcgacggagt ccctctggcg gacgccgggt tggggctggc ccgccccggg 600
    gtggcgatga cccaccggtc gctgcccctg tggtactgct gcgccaagcc actgctgtcg 660
    gtcgccctgg gccggctgtg ggaggcggga gcgtacgacc cgtatctgcc cgtcgcgcac 720
    tatctgccgg agttcggcaa ccggggcaag gagtccatca cctcgatgga actgctgacg 780
    catacgggcc cgctgcccac cggcgacgac ccgctgcacg gcatcgtggc cggcccggac 840
    gaggagcgtg tgcgccgtgc cttcgaggtg ccggtggcac cgcgtccggg gggcacgccc 900
    ggcatcaact acagccagtg gtgggcctgg ttcgtcctgg cgcgcatcct tccggtcgtc 960
    gacggcaggg agtaccgcgc gtacgtccag gaggagatcc tcgggccgtg cggcatgtcc 1020
    ggcacccgtg tccacctgga tcgcgaggag ttcgccgcgc tcgggggcga gctgccgctg 1080
    atccatgtga gcaaccccga gggcggcccg ctgcccaccc actggtggtc gacggaggcg 1140
    gccaccacac gctgcatccc gggggtcaac acccgtggcc cgctgcggga catgggcagg 1200
    ctcttcgaga tgctgctgcg cggcggggac gctcccggcg ggcgcgtcct ggcgccgccc 1260
    accgtcgccg ccctcacggc ccggcaccgc accggcctcc aggaccgcta cggcaacgcc 1320
    gactggggca tggggttccg cctcgaatgc cgtcagctgg atccgcggtt caccagcttc 1380
    ggctcgtacg cctccccccg gtccttcggg cacgacgggc tgtggaccgc cgtggtcttc 1440
    gccgacccgg acgccgctct cgtcgtcgcc ctccacctca acgggaaggt ggagcacgaa 1500
    cggcaccgcg agcgcatcgt ccgcctcgcc gacgccgtct accaggacct ccgtctctcc 1560
    tga 1563
    <210> SEQ ID NO 38
    <211> LENGTH: 1041
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 38
    atgacgcccg cgaccccgcg ctggagcgtc gtcgccccgc agggcaccaa cctcgaactg 60
    gccggtacgg gcggccgcga gggctggcgg ctcctcctgg agaccgcccg caccgtccac 120
    cggcacggcc gtggcgccct ctggctgctg gaccgcaccg acaccctgcc ccggcgcgag 180
    cccgagccgg tctgggaggg ctggacggcg ctggcggccc tcgcgggcgc ggtgcccggt 240
    ctggatctgg gactgctctc ctcggccccg ccgttccgca acgccgcgct gatcgccaag 300
    cgggccgcga ccctggacgt cgtctgcgac ggccggctca ccctcggctt cccggcccgc 360
    gagtacctgc cggagcacca ctcgacgggg cgcgaggtgc ccacgggcct ggaggcggac 420
    gaggaggagg ccgccggcca ccgggctctc ggcgagacgg tcgaggccct gcgcgcgctg 480
    tggggcggac agcccgtcac cttcaccggg gaacacatcc gcctcacttc ggcgcactgc 540
    gtgcccgccc cacggcagca gcccctcccc ctcgcgctgc gcaccccggc cggggacgcc 600
    gggagcggcg cgctgcggcc cgccgacgcc accgtgcggg agtgcgctca tgtccagtgg 660
    accggtgagc ccgctcaggt cgccgcggcc gtcaccgcgt tccgccgccg tcgcacggag 720
    ctcgggctcg atccggacgg cgtccggcac gcctgggccg cggagtgccg gatcttcgac 780
    tccgtcctgg aacgcgaccg ctggctctcc accccgcacg aggtgctgtt ctggagccac 840
    catcccgacc tgctggcgcg gcgcagcctg tacgggacgc cggaacagct caccgagcgc 900
    gcccggcgcc tggtcgccgc gggcgtggcg gagttcgtgc tgtggttccg cgactacccg 960
    gccaccacca gcctggagcg gctgttccag gaggtcgtcc cccaggtggc gccgggggcc 1020
    gccaaggaag cggaggagtg a 1041
    <210> SEQ ID NO 39
    <211> LENGTH: 708
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 39
    atgcccctga accccccgcc cgcctcgcgg gccgcggcgg acgcgcccgc caccgctctc 60
    ccgtgccggt tcaccaccgt ggtcttcgac ctcgacggcg tcctcatcga cagtttcgcg 120
    gtgatgcgcg aggcgttcgc cgtggcctac cgcgaagtgg tggggccggg cgagccgccc 180
    ttcgaggagt accgcacgca ccagggccgc tacttcccgg acatcatgcg gctgatgggc 240
    ctgcccggcg agatggagga accgttcgtc cgggagagcc accggctgat ggaccgcgtc 300
    gaggtgtacc cggacgtgcc gcagttgctg gcggagctgc gcgcggacgg cgtcggcacc 360
    gcgatcgcca ccggcaagtc cggctcccgg gcgcgcgccg tgctggaggc ggtcgggctg 420
    ctgcccctgc tggacgaggt ggtgggcagc gacgaggtgc cgaggcccaa gccgcacccc 480
    gacatcgtgc gggaggcact gcgccggctg gacgcggcgc ccgaggacgc ggtcatggtc 540
    ggcgacgcgg tgatcgacat ccgcagcggc cgcgccgccg ggaccgccac cgtgggcgcg 600
    acctggggcg agggcgcggc cggccaactg cgcgccgagc ggcccgactt cctgctggac 660
    aagccgcaga gcctgctcgc gctggtccgc agcggcggcc acgcatga 708
    <210> SEQ ID NO 40
    <211> LENGTH: 873
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 40
    gtggagcgtc tgaagctcgt gcccgacgag caccgccgtt tcaccgtcga cgagcagagc 60
    gcgcgccggc tgcaccggat cggaccggag ctgctgtccg cgctgtgcga ggcgggcgtg 120
    cccttcgtgg gaagcggcgc gggacgcctc ttcgacggct acgacctggg caacgcggcc 180
    ctgcaccttg gcctgtcctc ggtgcagcgc cgggccatcc gctcgtgggc cggttccctg 240
    cggaccgcct cggccgcgga gagcccgcgc tggcgcgtcg acgtcacggc gtcctgcccg 300
    gtgcccggcc acgcgggccc gtgccgctac ggagtgctgc tgcccggcgc ccgccgcccg 360
    gtggaggcgg cttcgccgcg ggagaccacg ctggcgcggc tgtacacacg gtcgcgcggc 420
    cactggccgg acttcccccc ggccgtcctc gacctgctgc gcaccctgga gccggtcggc 480
    ttcttcctgc tgcccgaagc gatccgctgg gacccggggt tcctgtggag cacgcacatg 540
    gccgactgcg gcggcgccgc ggcctggctg gtggcggagg gccggcggcg cgggctcgac 600
    gtgcggttct ccttcgggct gctggtggcc aagccgtact ccacaccgca ctgctgggcc 660
    gagttcctgg tgggcggccg ctgggtgccg gccgatccgc tgctgctgag ggccatggcc 720
    gcctggggcg ggctggacgc ggcggcccac ccgccgcaca gctcgccggg ggccgtctac 780
    caccggctcg cgggccgctt cacgaaagtc gtcagccacg ccggggtctg ggccccgacg 840
    tccctaccca cggagctcct gccatgcccc tga 873
    <210> SEQ ID NO 41
    <211> LENGTH: 1149
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 41
    atgaaattcg cttatttctc ccatgtctgg ggacgtcccg gtatcacgcc gggcgagcgc 60
    tacgaagagc tgtggcgcga ggtcgaggac gccgaccggc tcggcttcga ctacgcgttc 120
    tcggtggagc accactgcac gccgcacgag agctggatgc cctcgcccgc ggtcttctgc 180
    acgggcgccg cgctgcgcac cgagcgcatc cgggtcggcc cgatggggtg ggtgccgccg 240
    ctgcgccacc cgctgcacct ggtcgaggag gtcgcgaccc tggaccagct cctgggcggg 300
    cggctggagg tggggctcgc ctcgggcgtc agccgtgacc ccttcctgcc cttcgacgcc 360
    gatttcgaca accgtcacct cctgacccgg gaggccctgg agctgctgcg tgccgcgttc 420
    gccgcgcggg gcgccttcga cttcgacggg cccgcgcacc ggctgcgcga catcgccctg 480
    tccttcccgc cggtgcagcg cccgcacccg ccgatgtggg tgcccaccac caaccgcaac 540
    accttgcgct atctcagcga ggccggtgcc cacaccagtt ccacgatgat cgtgccgcgc 600
    gcctccatgg cgctggtcta ccggcactac ctcgactggt ggcgcggcca cggccacgcg 660
    agcgacccgc gcatcggcta ctggacgctg gtccacgtgg cccggacgga cgccgaggcg 720
    gaggagcggg cggccgcgca catcaccgag acgttcacca agacgctgcg gtacggctcg 780
    gtgtcccgtt cccgcgatca gcacgcccca cccagcaggc tcagcacgac ggacatcctg 840
    gcgggctccg gcgacctgcg cttcctgctg gagaacaacc tcgtcttcgt cggctcgccg 900
    gcgaccgtgg ccgaccggat cagggccgcg tccctggagg gccatttcga cacgctgctg 960
    ggcgagttca ccttcggcga gctggcggac cggcaccgca tcgagtccat ggaactgttc 1020
    gcgcacgagg tggccccggc actgcgcgcc ttctccccct acgcgccgcg cccgcaggag 1080
    ccggcgtaca ccgcgagcga cgagcagcag gtggcggccc gcctccaggc tctgggctac 1140
    atcgactga 1149
    <210> SEQ ID NO 42
    <211> LENGTH: 1215
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 42
    gtgacaccgc cgacgacagc tcgcgaaccc ctccggatgg cagtgctggg cgcgggatgg 60
    gtctcgcgca aggtgtggct gccgctgctg gcggaacacc cggcgttccg ggtcgacttc 120
    ctcgtggacg acgaccccgt ggcggccagg tcggccctgc cggagggcgc gcggacccgc 180
    gtcctgagca gaccggaaga gctcgccccc agaagcgtgg acgcggccat catcgccctg 240
    cccaaccacc tccatctccc cgtggccaag gccctcctgg agcgggacgt gccggtgttc 300
    gtcgagaagc cggtgtgccg cacgctcttc gaggcccagg cgctcgccct ggaccaccag 360
    gcgcggggcg acagcatcgg ggacatcacc ctctacgcct ggagcgccgc ccggcaccgc 420
    accgatgtct gccgcctggc ggagctgctg ccctcgctgg gcaccgtgcg cagtgtcggg 480
    ctgagctgga tccgggccac cggcatcccg cagcgcaccg ggtggttcgt cgaccgccgg 540
    ctcgccgggg gcggcgcgct gctcgacctg ggctggcacc tgctggacgt gggcctgcac 600
    ctgctggggt ggccgcgcgt ggtccgggcg gcgagcacga tgtccgcgga ctggatgagc 660
    cggggcgagg ccacggccga ctggagccgg cgctcctccg gcacggcgcg gccaggcccc 720
    ggggagacgg tggaggacac cgcccgcggc ttcctcgtca ccgacaccga cgtgggcatc 780
    tccctggaga cacgctgggc ctcccaccag gcgctggacg tcaccacgat caccgtggag 840
    ggcaccgagg gggtggcgac gctgcgcggc accttcggct tcagccccca ccggctacag 900
    aagtcgagcc tcgtggtcct gcgccagggg gtggaggaga ccgtcgcgct gcccgacgag 960
    cccgtgggcg tggagtaccg gcggcaggtg gacgaactcg cccgcaggct cggcggctcg 1020
    gccgacgggc agggcccggt gtcgggcctg ggcgaggggt cgatggccga agtgaccatc 1080
    ctggcctcct gcatcgacca catctattcg gccgccggcg tcgaccctcc ctcgcccctg 1140
    caccggccgc agagcgacgc ggcgcccagc acgtccagtt gtccacgtgt cctgcccacc 1200
    cggggaagcc aatga 1215
    <210> SEQ ID NO 43
    <211> LENGTH: 774
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 43
    atgagcaccg tcaccgaccg ggccacggag cgcctgggac agagcggccg cgtggtcgtg 60
    gtctcgggcg cgtccgggca gataggcggc gcgtgcgcgc tggagctcgc cgcgctcggc 120
    gccaccgtcg tcgccggcta ccacagcggc gagcaggcga tccgcaagct gcgggagcag 180
    gtggagggcc agggcggcac cctcgtgccc gtggcggcgg acctgagcga acccgagggc 240
    gccgacgcgc tggtggcggc ggccgtcgaa cggttcgggc gggtggacgg ctgtgtggct 300
    gctgcgggct tgcgtacgcg ccggctcgcg atggccacgg acgcccggag cctggagaag 360
    ctgctgcggg tcaacctggc cggttccgtg ggtctcgcca aggcgtgcct gaagccgatg 420
    atgcgcgcca ggtacgggcg gatcgtgctc ttcggctccc gggccgggac cagcgggctg 480
    cccggccaca gcgcgtacgc cgccaccaag ggggcgctcc agccgtgggc ggcgtcggtg 540
    gcgggtgagg tcggcaagca cggcatcacc gtcaacgtcg tcgcgcccgg ggcgatccgc 600
    gccgaggtga tggacttctc ggaggccgag cgcgatctgg tcctgcagtt catcggggcg 660
    gggcggctcg gtgagccgga ggaggtcgcg gcggcggtgt cgttcctgct gtcgccgtcg 720
    gcctcgtacg tcaacggcaa tacgctcgtc gtcgacggtg gtgcccgctt ctga 774
    <210> SEQ ID NO 44
    <211> LENGTH: 2124
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 44
    gtggccctgc gcgcgcccaa cagcccgcgg tgggtcgtcg ccttcctctc cctgctggca 60
    tccggcgcca ggcccctgct gctcgaaccc gacacccccg gccccgagac cgcgcggctg 120
    ctgcgggctg ccgggggcgg caggtccctg gtcgtccccg ggaccggcga cggcctccgc 180
    ctgacgttga ccggctcgcc cggagaaccc tccggcgccc cgcccgccgt gctgctcccg 240
    acctcggggt cgaccggtgc gagcaagctc gtcgcccgca gcgaggagag cctgctcgcg 300
    gagggccgcc gctaccgcga cggggtcggg ctgacgggag aggacaccct gctgctgccg 360
    gtgccgctgt cccacgcgta cgcgctgggc tggctgttcg gcggactgct gacgggtgcc 420
    gcgctgcgcc ccgtaccgcc gaccgccctc ggccgcatcg ccgcggagct gtccggtggt 480
    gcgaccgtgg tggccctggt gcccagtgtg gcccggctgc tggcgacccg gcggctgcgg 540
    ggagcagcgg ccgggcgggc gcccgccgct cccggtctcc ggctggccat ggtgggtgcg 600
    gggccggtgg acgagcagct ggaccgcgcg ttcaccgagg cgttcgggac cggtctcgcc 660
    cgcaactacg gttccacgga gacgggcgcc gtgctcgccg gaccggcggg gctggagccc 720
    ttgtgcgccg gtgctcccct gccgggggtg gagtgcgaac tgaccggccc ggagggcgtg 780
    gtgccgcccg ccggcacccc ggggctgctg agcgtacggg tcgacggccg gccgtacgcc 840
    atgggcgatc tcgccgtggc cgtgcccggg ggcctgcgca tcctgggacg cgaggaccgg 900
    gcgatccgcc ggggcgggcg ctgggtctcc ccgctggaga tcgaggaggt gctgcgcggt 960
    catccggacg tggtgaatgt gcgggtgggc gcccggcggg ggcggcaccg gggcgaggac 1020
    gggatcgtcg cggaggtctc ggcggcgggg ccggggctca cccccgaggc gctgcgcgag 1080
    cacgcccgcc gggagctggc cccgcacaag gtgcccgacg agttcgtcct gcgggagagc 1140
    ctgccggtca acgccgcggg caaggtgcgg gcggcgtccg tctaccgcct cacccggagc 1200
    gcggcggagg ccgcccgggc gtacaaggca tccgaagtgc tcttcgcgct gcacgacttg 1260
    ggcgccctgg aggcactcgc ccagggtgcc ggcacggctc tcctcgccgg ggagctgggg 1320
    tgcgacgcgg atgccctgga gtggctgctg cgcacggcca ccgctctggg ggtgctgacc 1380
    accggggcgc aagagcccgg ggaccgggtc cgggccgggg agctggccgc gttcgtggcg 1440
    ctggaggagc acctctcccg tgggctggtc acgcgcgagg agctcgtcgc ggtggcccgg 1500
    agcgggacgg cgcggcgtcc cttcgaggag cgtccccccg agagcctcgg tccgctcgtc 1560
    gccctgtacc agggcgcgat ggacggcccc ggcgcacggg cccgggccgc gctcggcctg 1620
    cggctcctgc ggcccgggcc gggagcccgg gtggtggagg tgaccgcggg cccgggccgc 1680
    tatctggaac gcctgctcgc ctcggacccc ggggcgagcg gccatctggt caccgtcggc 1740
    cggctgagcg ggccgctctc ctcggccgtc gccgcggcgg tcgaggaggg cagggtgacc 1800
    gtggggacgg aactgcccgt cggctacgcc gacttctgcg tggtcgccaa cgccgtgcac 1860
    ggcccggggc cgggcagcgc tctcggtgcc ctgctcggct ccctgcggcc gggcgggcgg 1920
    ctgctggtcg acgacgtctt cctgccggcg tccgggccgg ggagcgaact ggctctggac 1980
    tggctcacgc acggcgggac cgcgtggccg gccaccggcg agctgatcgc cgggctgctg 2040
    caagaggggg cggaggtcgc acggcacgtg ccgctggacg cgtccccctg tcatctgatc 2100
    atcgccaagg aggccggttc atga 2124
    <210> SEQ ID NO 45
    <211> LENGTH: 1152
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 45
    atgtcccgta gcacccaccc gccgacagcc acccccgacg cgggcaccag gcgacgcctg 60
    ccgctgatcg gcaacgacct ggtcatcaac gaggactcct gcaacctcag ctgcacctac 120
    tgcctcaccg gacagagcaa cctcaaggag ggccactccc ttcaactgat cttcgagccc 180
    ccgcggcgcg acagctacgc caaggacagc gggctggggc agcgcatgga caaggtcgcc 240
    gaccggatcc gggaccgctt cggcctgccg ctgctcaagg tgaccggagg cgagatcttc 300
    ctggtccggg ggatcatgga cttcctggag caggaggccc gtaaatacga cgtgctggtc 360
    atccagacca acggtgtcct ggtgcgcgag gagcacctgg agcggttccg ctcgtggggc 420
    aacgtcgtgc tccaggtctc cctcgacagc cacctccacc acggcaacag ccatcgtgtg 480
    ccgtccggga gcctgcacga gaaggtcgtc gccgccatcg cccggatcct ggactcgggg 540
    ctgccggtgg agatctattc agtgctcaac gaccggagcg tcacggaggt ctgcgcgttc 600
    gccgagtggc tgtcgggatt ctcccggcct cccgtctact tccccttccc ggtgcggggc 660
    ccggactcgg aggacttcaa ggtgcggccc gggcagttcg gccacatcca ggaactcgtc 720
    gaccgctacg acgagttcgc gcgggtcctc ccgccgcggc cctacttcga ccggctgacg 780
    agcttctacc gcgagggccg ccgcaccttc cgctgccatc tgccgcggct ggtcgtctcc 840
    agcttcagcg acggcgtcgt cacgccctgc cccaacatct ggttctccga catgggcaac 900
    gccctggagg acgactggag cgagatgctg gacacggtgg gcaccagcgg cctctaccgt 960
    gccctgctcg cccccaagcc ccggctcaag gcgtgccacg gctgcttcac gccctgggac 1020
    acgctctcga tgtacttcga ggacgagatc accctcgacg agctgtgcgc cgctcccacc 1080
    tactccccgc cccgcatccg gcagatgctc agcgacgcga aggccgacta cctccagggc 1140
    ggccatgact ga 1152
    <210> SEQ ID NO 46
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 46
    ggcaaggcat gcgagggtcg c 21
    <210> SEQ ID NO 47
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A primer
    <400> SEQUENCE: 47
    ttccagaacg gcgccctgat gaccgccggc 30
    <210> SEQ ID NO 48
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A primer
    <400> SEQUENCE: 48
    gccggcggtc atcagggcgc cgttctggaa 30
    <210> SEQ ID NO 49
    <211> LENGTH: 1545
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 49
    gtgccaccct ctccccgcgc cctcgtcatc ggaatcgacg gaggcacatt cgatacggtc 60
    gacccgctga tcgagtgcgg tctgctgccc catatggcga agttgctgcg cgagagcgcc 120
    agtgccgcca cggactgcac ctggcccgcc cacacggcgc cggggtggag cacgttcgtc 180
    tccgccagcg atcccggcgg tcacgggatc tatcagttct acgacaccca ggacccggcc 240
    tacggggccc gcgtcacgcg ctccggcgac ctgggccggt cctgcgcctg ggactggctc 300
    gccgcgcagg aatattcgct gggcctcatc aacatcccga tgtcgcaccc gccggccgac 360
    ctccccggct atcaggtcac ctggccgctg gagcggacac tcaagcactg ccgcccggat 420
    tccctgctgc gcgaactcgc cgcggccaag gcccatttcc agtcggacct cgcgaccatg 480
    ttccggggcg acatggccta tctggaggag gccgagcgca atgtggcggc gcgggtccgc 540
    tccgtacggc atctgatgag cacccggccc accgatgtcg tgatggtcgt gctcaccgag 600
    gccgaccggg tcggccacca ctactggcac tacggcgacc ccggtcaccc gggccaccgg 660
    cccgccccgg agggcagcgg ctgggacgtc gccatgcccc ggatctacca ggccatcgac 720
    cacgcggtgg gcgagctcct ggagctcgtg gacgaggaca cctccgtcgt gctcgtctcc 780
    gaccacggcc tgggcaccgg gcgccacggc ctgtcggtgc acaccctcct ggaggaggcc 840
    gggctgctgg ccaccgcacc gggggaggag ccgcaggacg cggcggcgag ctggttcgcg 900
    ggcaacggcc ggcacgtcga cttccgccgc accagcgtct acatgcccgt ccccggcagc 960
    tacggcctca acatcaacgt acgcggacgc cagcagcgcg gcaccgtcgc accccgcgac 1020
    cgcgaacgcg tcatggacga ggtcacgggc ctgctctccg ggctgaccgg ccccgaggga 1080
    cagcaggtct tccgggccgt ccgcccgcgc gaagaggcgt acccagggcc gcacaccggc 1140
    cgggcacccg acctcctcct cgtcccgcgg gacgagaccg tcctgcccgt ccccgacctc 1200
    ggcggtgacg tgtggcggcc gagcgcgcag accggcctgc accgctaccg cggcctgtgg 1260
    gcgcaccgct cgccccgcgt ccgccccggc cgcctgcccg gcaccgtcgc gctcaccgac 1320
    accctgccca ccctgctcac cgacctcggg gccgcatggc ccagcgacat ccacggccgc 1380
    cccgtgaccg ccgtcctcga cgacggcgta cgcgtcccgc cctccgaccc ccgggtcgag 1440
    gccaccggca ccccggccac cacgatcccg gccgccgctt cggccgctga tgccgccgag 1500
    gacgcgtaca ccagcgaccg cttgcgcgaa atgggctacc tgtaa 1545
    <210> SEQ ID NO 50
    <211> LENGTH: 282
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 50
    atggagaccc tgacgaccga caagatcaag gaccggctgc gcaaggtgct cgtcgattcc 60
    ctcgaactgt ccctggaccc ctcggccgta cccgacgagg gactcgtgga gaagctgggc 120
    ctggactcga tcaacaccat cgaattcctc atctgggtcg agagcgaatt cggcatagag 180
    atcgccgacg aggacctgtc gatcaagctc atcgacagtc tcgacctcct cgccggctat 240
    gtgtccgagc gcgtgaacgg cgtcaccgca cccgccgaat ga 282
    <210> SEQ ID NO 51
    <211> LENGTH: 1413
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 51
    atggaccggc acgccctggt gatcgggctc gacggcatgc cgaggaccct gctgacccgc 60
    ctggccggcg acgggaccat gccgcacacc gcggcgctgc tcgccgaggg ccactgcgcg 120
    gaactgctgg cacccgtacc ggagatcagc tccacctcct gggccacctt cctcaccggc 180
    accaacccgg gccggcacgg catctacggc ttcaccgacc tcgcccccgg cgacggctac 240
    cgcatcacct tccccggtgt gcggcagctg cgcgaacccc cgctgtggga actcgccgcc 300
    cgcgccggcc gcaggaccgt gtgcctgaac gtgccgggca cctaccccgc ccccgccatc 360
    gacggcgtgc tggtctccgg cttcgtcgcg cccgaactgg agcgcgccgt cagcccgcca 420
    cggctgctgc cgctgctgcg cggcctcgac tacgaactcg acgtcgaggt cggcgacgtc 480
    gccgccgacc cggccgcctt cctcgggcgg gccgtccggg ccctgcgcgc ccgcacccgg 540
    gcgatggaac acctgctgcg ccaggagacc tgggacctcg cggtcgccgt gctcaccgag 600
    accgaccgcg tccaccactt cctgtggcgc gcggtcgccg accccgccga ccccctccac 660
    ggggacgtcc tcgccttcta ccgcctcgtg gacgactgcg tcgccaccct ggtgagcacc 720
    ctcccaccgg gcggcgaact cttcctgatg agcgaccacg gcttcggacc cgccgcctgt 780
    caggtctatc tgaacgcgtg gctcagggag tccggctggc tggccgggct cgacgtctgt 840
    ccggacctca ccgcggtcga cgctcgcagc accgccttcg cgctcgaccc cgcccgcatc 900
    cacctcaacc gcaagagccg cttccccggc ggcggcctga ccgacgcgga ggcggacgag 960
    gccgcccacg agatcgcgcg cgagctgtcc gccctgcgct gcgacggcac ccgcctgggc 1020
    cccgacgtcg acggacccct gctcgtccgc gacctctacc gcgctcagga gatctaccac 1080
    ggcccgctgt tgggcaacgc ccccgacctg gtggccgtac cggcccccgg ggtgcagctg 1140
    cgcggcggct ggggcggcac gcacaccgta cgcaacgaca tcctcaccgg cacccacacc 1200
    cgcgacgacg cggtcttcta ccggcgcggc gcgcccgcgc ccgcccccgg ggcggacgac 1260
    ggccccctcg acatgacgga cgtcgccccg accgtcctcg cctccctggg catccacccc 1320
    ggcgggctcg acggcgcggc cgtactcggc accacgggac ccgcgtccgg tcacggccgc 1380
    acggaccccc ctctcgacat cagggagctc tga 1413
    <210> SEQ ID NO 52
    <211> LENGTH: 1836
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 52
    atgaagcacg acctcggtct ggcaccatcg gcacccaaac cgggaacact cgacctgagc 60
    ctggacccac gcatcacgga ccccgcttcc ttccgggtca gtttcctgat cctcctcgac 120
    ggcgacctcg tgatgtcccc cgaacacctc ggcgtcgcct acatggccgg tgtgctgcgc 180
    catacgggct tcaccgcgga gatccgggag gtggagcacg gcgacgacca ggcggccgcc 240
    accgtcgagg cgctcaagga gtaccggccc gacctcgtct gcttcaccct gatgagcctg 300
    aacctgggca gctgtctgac cctgtgccgg atgctgcggg aggagctgcc ggggacgacg 360
    atcgcctgcg gcggcccagc cgggaccttc gcgggcctgg acgtcctgcg gaacaacccc 420
    tggaccgacg tcgtcgccgt gggggagggc gagcccacca tcctcgacct cgtccaacgg 480
    ctctacctca aggagccgtt gtccgcctgc aaggggatct gctaccgcga cgaggacggc 540
    acaccgcgcc agaaccccgc ccgccccctg atccacaacc tggaggacct ccccttcccc 600
    gcccgggacc agctgcgcca gcacggcgac aagctggagt acgtccgggt cagcaccagc 660
    cggggctgcg tcgccaactg cgccttctgc tccgccccgc acctgaagaa ccgcgtccag 720
    gcgggcaagg cgtggcgcgg ccgcgggccg gaacagatcg tggacgaggt cgccgagatc 780
    gtcgaacgcc accagttccg gaccttcgac ttcgtcgact ccaccttcga ggaccccgac 840
    ggcggccggg tcggcaagaa acgggtcgcc gccatcgcga acggcatcct ggagcgcggc 900
    ctcgacatct actacaacgt ctgcatgcgg gccgagaact ggcacgacac ccccgaggac 960
    cacgccctgc tcgacctgct ggtcgcctcg ggcctggaga aggtcaacgt cggcatcgag 1020
    gccggcaccg ccgaggaact gctcctctgg gagaagcgcg ccaccgtcga ggacaacgtc 1080
    accatcatca ggatgctgcg ggaacacggc atctatctcg ccatgggatt cattcccttc 1140
    cacccctacg cgaccctgga gaccatcgtc accaacgcgg ccttcctgcg cgacaattcc 1200
    ggccacaacc tccggcgcat gaccgaacgc ctggagatct accccggaac ggccatcgtc 1260
    agccgcatgc gggccgacgg actcctcggc gagagctatc tcgaagggct cgacccctac 1320
    ggctacgcat tcaaggatcc ccgcgtcgga cggctcgcca agcatttcgc ccagctctac 1380
    aacaacgacg actaccaccg gcacggcgtc atcaccgagc agtcctccgt cttcgccttc 1440
    gagacctaca acgtcgtact ccagaccttc atctcccggc tgcaccgccg gttcaccacc 1500
    ctgccggggg tggacgaggt gatggaggca ttcaaggccc gggtgcacga gatccgccag 1560
    gagatgggcc ggcacaacta cggcttcttc atgtccaatg tcgaggcggt catgaacgac 1620
    accctcgacc cggagaagca gcgccggcag gtggtggacg tcgagcactt cttccgcgac 1680
    cgcctcgatg tgttgcgcag cgagcaattg cgcgtcggca aggccctcac ccggctcggc 1740
    gcccgggtga cggaggtcag ctcgaccatt cccaaggagc gccccggcgg actgccgcgc 1800
    cagtacacgg gagagggcag cggtgccacg tggtga 1836
    <210> SEQ ID NO 53
    <211> LENGTH: 1080
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 53
    gtgccacgtg gtgagacggg aaccgccgcg gcgcgggtgg cggtctgcac gctgagcagc 60
    agggaactgg tcggcccgct ggcccggttg cccggtgtgg cggccgcggg cacgctgatg 120
    accgccaacc tgggcatcga gcaggtgatc aaggccctgc ggtgcgaccg gacggtccgc 180
    ggcctgctcg tgtgcggccg cgactcaccc cgcttccgcg ccggccagag cctgatcgcc 240
    ctcttccgcc acggcctgcg ccccgaggac gggcacatcc ggggagccac cggctatctc 300
    cccgtcctga ggtcggtgac ggcgcgggag accgaggagg tacgcgcccg cgtcgagctg 360
    gtggacgccc gtggcgagcg cgacgtcgag acgctgcgcg ccgaggtcgc ggcactcctc 420
    gcccgcgtac ggcgcacccc ggccctcccc tcccgcgagc acgacggcgg ccaacccagc 480
    ttcgtggagc cggacttcgg acggctgcat cctgtcggcc gccgccgctc cctggacgcg 540
    ggcatcggcg ggttcgtgct catcagcgtc gaccgtgagc accggcggat cctgctgcgc 600
    cactacacct ccgatgtgcg gccccggcac gagatgtggg gcacccgcgg ggaggcgatg 660
    ctgctcgggc tgctggaggc cggcgtcatc gaggaccccg cccacgccgg atacctcggc 720
    gccgaactgg ccaaggccga gacggcgctg cggctcggcc tgcactacga acaggacctg 780
    cccctgcgcc cgccgggcag gccgcccggc cctgtgcggc gccggaccgc gaaggagcga 840
    acgaccatgg cgcaagcacc cgcgctggag gacttcctgc gtctcgtgac gaggacgctg 900
    ggggccgagg acgccgtcct ggacctgcac acgccgctcg gcgagcaact ggcggtggac 960
    tccgcccggc tcatcgaact caccgtcgtc ctggaggagg agctcggcgc ggacctcccc 1020
    gacgacgccg acctcgccag ggccaccccc gcggaactcc acaaagcact cgtgggctga 1080
    <210> SEQ ID NO 54
    <211> LENGTH: 438
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 54
    atgcgcagcg tgctgttgct caacggaccc aacctgggga cgctcggcaa gcggcaaccg 60
    gagatctacg gaaccgacac cctggccgag atcgaggccg ccgtggccga ggaggtggga 120
    gcgcgcggct gggaggtggt ctccgaacag cgcaacggcg agggggaact ggtcgatgtg 180
    ctccagcgcc acgacgacgt ggtgggcgcc gtggtcaacc ccggcgccct gatgatcgcc 240
    ggctggtcac tgcgcgacgc gctcgccgac ttcgccccgc cctgggtgga ggtgcacctg 300
    agcaacgtgt ggggacgcga ggcattccgg cacacctccg tcacggcccc gctggcctcc 360
    ggcgtcgtga tggggatggg ggcgctgggc taccggctgg cagcgcgcgc cctcacccgg 420
    ctggtccccg aggactga 438
    <210> SEQ ID NO 55
    <211> LENGTH: 534
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 55
    gtgggacggt acggaagaga gggtctgggg atgtcgcgta cggctgaggg gaacgccgga 60
    ggcgtggtgg tgccggtggt ccggctggtc gccgtgacgg acgggccgga cgcggagggc 120
    tggcggcagg cgctcgcccc cgaactggtg gtggagcacg gcgtcgaggc gatcgcggag 180
    gcggccgggg acggcgggcc gtgggcgctg gtctgtgccg gtgccgggct gggcgcggcg 240
    ctgcgggccg ccgagcgggc cgcgcgcccg ccggtgcatg tgctgctgtg gctcggcagc 300
    cgcgggcccg gcgaaggggt gggcggggag gtctccggtc aatttccctg tccggtcacg 360
    gccttggtgt ccgcggaggt ggaccgcggt cgcgccgtgg tccccgcctg gcgcggcctg 420
    accgaggggc cgttcaccgt gcggatcctc ccggcggcct gcccgctgcc cggggcgtgc 480
    gaccaggccg gcgctcaggt gatcaaggag gagctgcggg tgtggcccgc ctga 534
    <210> SEQ ID NO 56
    <211> LENGTH: 765
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 56
    atggatgcga ctttgacgaa tgacgtcgag aaagcctccc gggatctggt cgaagccgga 60
    tactgcctga tcgagtgccc cttgccggcc gcggtcttcg aaaagctcag agggcggctg 120
    ctggaggtcg ccgagcagga gcgtgagaac ggctcggcct ttctctacga cggcggcaac 180
    caacgcgtct tcagcctgct gaacaagggc gaggaattcg agcagaacgt gcaggatccc 240
    accgtcatgc tcctgatgga ggagatcctg ggcttcggct tcctgctctc cagcacgcac 300
    gccaatatcg cgggccccgg cggttcccgg atgcatctgc acgcggacca gaccttcgcc 360
    cgcccgccgt ggcccccgta tccgctggtg gccaacagca tgtggatgct ggacgacttc 420
    accgaggaca acggcgcgac ccgcctggtg cccggctccc atctgctggg ccggcagccg 480
    gactacgacc ggggcgaggg gaacaccgag acggtcgccg tgtgcgcgcc ggccgggagc 540
    gtgatggtct tcgacgggcg cctgtggcac cagacgggcg ccaacaccac cgaccggccg 600
    cggcacggca tcctcaacta ctactgccgc ggctacgtcc ggcagcagca gaacttcttc 660
    tcgggtctgc gggaggacgt cgccacccgc gcgacgcccg aactgcgccg gctgctgggg 720
    tacgagaact acttctccct cgggatgacc gacggcctgc cgtag 765
    <210> SEQ ID NO 57
    <211> LENGTH: 795
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 57
    atggcacact caccgcggcg gccggacggc cccctccgca tcggggtctg gctggccccc 60
    cagcacacct cggtggccga actgcgcgcc gcctggcgcg cggccgactc cctgggcgtg 120
    gactcgctgt ggctgtggga ccacttcttc ccgctcaccg gggaccccga cggcagccac 180
    ttcgaggcct ggaccctgct ggcggccatg gccgccgaca cccgcgccgc ccgcctgggc 240
    accctggtgt ccaactacgc ctaccgcaac cccgacctcc tggccgacat ggcccgcacg 300
    gtcgaccaca tcggcgacgg ccgcctgatc ctcggcatgg gcgccggctg ggtcgaacgc 360
    gacctgaagg agtacggcta ccccacgccc ggcgcggggg agcgggtgga cgggctcatc 420
    gaggcggtgg agcgcgtcga ccgcagactc ggccggctgc gccccgggcc gctcggcgac 480
    ctccccctgc tcatcggcgg ggacgggcag cggcgcctgc tgcgcttcgc cgccgaacgg 540
    gccgccatct ggaacaccat ggcctggcgc ttcgccgagg gcaatcgcgt gctggacgag 600
    tggtgcgcgc gggtcggccg cgacccggcg gagatcgagc gcagcgcctt cgtcacccgc 660
    gaccagaccg acgaggagct gcgctgcctg gtggcgacgg gcgtccagca cctgatcttc 720
    caggtcgggc accccttccg cttcgacggc gtggagcggg ccctgcgctt cgcgggcggc 780
    tggagcaagg ggtaa 795
    <210> SEQ ID NO 58
    <211> LENGTH: 825
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 58
    atgaagatca gcattgctct gccgaacacc gtgcccggcg cggacgggcg cctgataacc 60
    gattgggcgc ggcgggccga ggagcgggga ttcgcctcgc tcgcggccac cgagcgcctg 120
    gtgtatccgg gccacgatcc gctgctggcg ctggcggcgg cggccggggc gacctcccgg 180
    atcgggctgc tcaccaatgt cctgatcggc ccgctgcgca ccgcgcctgt gctggcgaag 240
    gcggtcgcga gtctggactc gctgtcgggc gggcggttca ccctgggggt cgggcccggc 300
    gtgcgcgagg acgacttcga ggccgccggc cgcgccttcg acgaccggcg cgcggcgttc 360
    gaggagcagc tggagctgct cggccggggc gcccggccgg gcgcggaggg ccccggtgtg 420
    ccggtcctcg tcggcggggt cagcgcggcg gccgtgcgcc gcgtggcgcg ctgggccgac 480
    ggctggacgg cgcccggcct ggagccggag cggatcgtgc cggtcgcgga acgggtgcgc 540
    cgcgcctgga gcgaggcggg acgcgccggg gcgccgcatg tggtggcgct ggcgcgctac 600
    accctgggcg aggacgtggc ccaggagtcg gcggccttcg tccgggacta cttcgcggtg 660
    ctgggcgagg aggcggagga gttcgtggcg aagaccccgc gcaccgcggg gcagctccgc 720
    gcggcggtct cggcgctcgc cgacggcggg gtggacgagg tcgtcctcca ccccacggcg 780
    gcggcgctgt cccaagtgga ccggctggcg gacgcgttgc tctag 825
    <210> SEQ ID NO 59
    <211> LENGTH: 1383
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 59
    atgcccgctg ccggaaaagt cgccgtgata ggactcgact ccgcgactcc gcagtacatg 60
    ttcgaccggt tcgccgagga catgccggtg ttcaccgccc tcaggcgcaa gtccctgtgg 120
    ggtccgatgc gcagcatcga cccgcccatc accatgcccg cctggtcctg catgatgtcc 180
    ggccgctcgc ccggcgaact cggcgtctac ggattccgcg accgcggcgc ctacgactac 240
    gggccgttga agttcgccac ctcccacagc atccaagccc cccggatctg ggacgagatg 300
    acggccgccg ggcgctccag cgtggtcctg ggcgtccccg gcacctatcc tcccgccccc 360
    atccgcgggg ccatggtctc ctgcttcctg gctccctcca cacagtcgcg ctacacctcc 420
    ccgcccggcc tcgccgacga gctggagaag ctcaccggcg gctacgccct ggacgtggag 480
    gacttccgct ccaccgacct ggaacgcgta tcccagcgcg tcttcgacat gagcgagcag 540
    cgcttcgagg tcgcgcgcca cctggcgacc acccaggagt gggacttcct ctccttcgtg 600
    gacatgggcc ccgaccgcct ccaccacggc ttctggaaat actgcgaccc cgaccacccg 660
    cgccacgagc cgggcaacgc ctacgccggt ctcttccgcg actactaccg cgccctcgac 720
    cggcacctcg gccgcttcct ggagagcctg cccgagaaca cgaccgtcct ggtcgtctcc 780
    gaccacggcg cccagccgat ggtgggcggg ctcttcgtca acgagtggct gcgcaaggag 840
    ggttacctcg tcctgaccga ggagcccgcc ggacccaccc ccgtcgccca ggccgccgtc 900
    gactggaagc ggaccaccgc ctgggccgaa ggcggctact acggacggat cttcctcaac 960
    gtcgagggcc gggagccgca gggcaccatc ccggccgcgg agtacgagag cacccgcgac 1020
    ctcatcgcct ccgccctgga agcgctgccc gacgaccagg ggcagccgat gggcacccgc 1080
    gccctgcgcc ccggcgagct ctacggagag gtcaacggca tcgcccccga cctcctggtc 1140
    tacgtcggca acctgcgctg gcgggccctg gccaccctcg gcatgggcaa gggcctctac 1200
    acgacggaga acgacaccgg ccctgaccac gccaaccacg gggacaccgg catcttcgcc 1260
    ctcagcgccc ccggcatcac ccccggccgc gcggacggcc tgtcgctgta cgacgtggcc 1320
    cccaccctgc gggaactgct gggtctcgcg ccgcagggct cccgcggctc cctcctcggc 1380
    tga 1383
    <210> SEQ ID NO 60
    <211> LENGTH: 1536
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 60
    gtgaaggcga tggaccgggt ggacagagcg gtcgagcggt tcccgatgta catcgacggg 60
    caggccgtgc aggcccacga cggcgccgtc ctgcgcacct tcgagccggc cacgcggcgc 120
    cacctggccg accttcccag cggcggcgcg gaggacgtcc gccgggcggt gtccgccgcc 180
    cggcgggcct tcgacgaggg cccgtggccg cggatggcgc cgggcgagcg ggcgggcctg 240
    ctgcgcaagg ccgcacagcg cttgcgtgaa gaagcggagc cgctggccga gttggaggcc 300
    cgcgacaacg gctcgacgct gcgcaaggct ctcggggccg atgtgccggg ggccgcggca 360
    gccttcgagt ggagcgcgtg gtgggcggag cacgtgcccg aacggcagcc ggaggcgccc 420
    ggttcgggtt cctacgtcgt gtggcggccg gtgggggtcg tcgccgcgat cgtgccgtgg 480
    aatctgccgc tgctgctggc ggcctggcgc atcgcgcccg ccatcgccgc gggcaacacc 540
    tgtgtgatca aaccggcttc gttcgcctcg ctctccacgc tgcgactggt ggagctgctc 600
    cacgagtgcg gcctgccgcc gggcgtggtc aacgtggtca cggggccggg cggggtcgcc 660
    ggggagcagc tggtgcgctc gcccggcgtc gacctggtgg cgttcaccgg ctcggacgag 720
    accggggccg ccgtacggga gggtgccgcc gcggcgggga cgagcgcccg gctggacctg 780
    gggggcaagt cccccaacat cgtgctcgcg gacgccgatc tggaccgggc ggtcaccggc 840
    gtcacgtggg gagcgttcct gcacaacggg caggtgtgca tggccggtac ccgcgcggtg 900
    gtgcacgccg acgtccacga cgacttcctg cggctgctga gcgaacgggt gggccggctg 960
    cgcgtcggtg atccgctgga cccggccacc gacctggggc cgctggtctc gcgcaaccag 1020
    gcgcgtacgg ccaggcgctt caccgaactc gggctctccc agggcgcgga gctcgtgtgc 1080
    ggcggccggg cgcccgcggc ggacgagctg ccgcccgggc tggacgccgg ggcgtatttc 1140
    ctgcccacgg tgctggcgtc ggtcggcgcg gacgacgccg tcgcgcagga ggagatcttc 1200
    ggcccggtgc tcgcggtcgt ccgggccggg tccgacgacg acgcggtgcg catcgccaac 1260
    ggctcccgct accggctcag cgccggggtg tggtccgccg atcccgcgcg ggcccgcgcg 1320
    gtggccgagc ggctgcgcgc ggaccgggta tggatcaacg actaccggct ggtcgacctg 1380
    gagctgcccg gcacagccgg gccccgctcc gccgtctggg accggctcac caacgagctg 1440
    gacgcctacc gccacaagca cgtggtgcac ggtggcggtg cgggagcggg cggggtgccg 1500
    gcgccgccca ctccctacgc gctgctgggc gggtga 1536
    <210> SEQ ID NO 61
    <211> LENGTH: 1419
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 61
    gtgaaaccag ccagccactc cgtgacggac acgtccgcgg ccctcggcgc cgcggccgcc 60
    gaagagctcg cggcgcaggt cgccggatcc gtcctcctgc ccggggacga ggggtacgac 120
    gaggagcgct ccggcttcga actgtccgtg gaacaccgcc ccgccctcgt cgtcgtcgcc 180
    accggtgccg cggatgtcat cgccgccgtg cgcttcgcca gggcccgggg ccttgggatc 240
    gccgtccagg ccaccggtca cgggaagtcc tcggcggcca ccgacgtcct catcagcacc 300
    cggcggatga ccggcgtcag ggtcgacccg cgggcccgga ccgcccggat cgaggcgggc 360
    gtgcgctggg agcaggtgat ccacgaggcg gcggcgcacg gtcttgcacc gctgagcggc 420
    tcggcgccgt tcgtcggcgc ggtctcctac ctcctcggcg gcgggctcgg gcttctgtcg 480
    cggaagtacg ggttcgccgg cgaccatgtc gtctcgctcg acctggtgac ggccgacggg 540
    cggtttctcc aggtctccgc cgaggaacac cccgatctct tctggggcgt gcgcggcagc 600
    agggggaacc tcggcatcgt cacctccgtc gaggtcgggc tgttccccgt cacccaggtg 660
    tacggcggag ggctgttctt cgacgccggc tccacgcgcg ccgtgctgaa cacctatctc 720
    cagtgggcgc cccggatgcc cgaggacatg gcgtcgtcgg tgttcctggc cgcgtatccc 780
    gatgccgagg gggtgcccgg accgctgcgc ggccggttcg tcacccacat ccggctggcc 840
    tggctgggag accccgagga gggtgagcgc cggttcgccg agctccgggc cgccggcacg 900
    gtcgtcatgg atacggtgga cacgctcccg tacacgcggg ccgggatcat ccacaacgat 960
    ccgccggccc cggtgtcgag tcacagcaaa acggtcatgt tcgggcagct ggacgagatc 1020
    gccgtcgacg agatcctcag gctcgcgggg ccgggcacgg acgcgctgtt cggggtggag 1080
    ctgcggcacc tgggcggcgc cctcgcccgg ccgccccggc acccgagcgc ggtgggccac 1140
    ttcccggagg cggtgttcaa cgcctacgtg ggctcgctgg tcgacccgga caccctggcg 1200
    gccgtggacg cggcgcagca ggagttcgtc gacagcatgc ggccgtggac gacgcccggg 1260
    gtgtgcctga acttcctcgc gggtcacaac acatcgaggg agacgacccg cagcgcctac 1320
    acgccggagg actacgcgcg gctccaggcc ctgaagtcgc agtacgaccc gggcaacgtc 1380
    ttccggttca accccaacat cccgcccctg ccggcctga 1419
    <210> SEQ ID NO 62
    <211> LENGTH: 1188
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 62
    atgacctcag ccgccccgcc cgcctttccc ttcccgcccg gccccggcgg cacggtgccg 60
    cccgagtacg cgcggctgct caccgatgac ccggtcgccg aggtgcgcct ggcggacggc 120
    tcgcgcatct ggctggtgac ccggcacgag gacgtgcgca cggtgctcac cgacggccgc 180
    ttcagccgcc atcgcgccgc catgctgccg ggctcgggct tcggccggtc ccagggctcg 240
    ggcatcgtgg acctcgaccc gccggagcac ggccggctgc gcggtccggt ggtggccgcg 300
    ttcggtgcct cgcgcacggc gcggttcgca ccccgcatcg aggcggccgc cgaggcggcc 360
    ctggaccggc tgcccgccgg cagcggcacg gtggacctcg tcgcggcgta caccgcgccc 420
    ttcgccggcc gcgtcacagc cgagttcctc gggctgcccg gggaccggtg gcaggacgtc 480
    acctccgacg tcgagctgct gctgcttccg cgcggtgcca ccgagcaggc gctgaaggag 540
    gcccgcggca ggctcggcca ggtgctggac gaactgctcg cggcccgcag ggccgagccg 600
    ggcgacagcg tcaccgacac gctgctggac gcggaggagc tcaccgacga cgaccggcgc 660
    ctgctgctcc acggcctgat catctcgggc ttcatcacca tccgcgacct gctggcccgg 720
    cacctcttcg gcgtgctctc ctcccccggc ctcgcggccc ggctgcgcga ggacccctcc 780
    gtactgccct ctgccgtaca ggagttgctg cgctactacc cctccagcaa cgacggcctg 840
    ctgcgggtcg ccaccgagga cgtggtgctc tccggcaggc gcgtcgccgc cggggacgcc 900
    gtgctgccac tggtctcggc ggcctcccgc gaccccgagg tcttcgccga tccccacgtg 960
    ctcgacatcg agcgggtggc cgaccgcggc atcgcgttcg gcgccgggca gcacgcctgc 1020
    cccgcgaccg ggctggccgt gaccgaactg accgtcggca tcggccgcct gctggcggcc 1080
    ttcccccgca tcgccctggc cgtgcctccc gaagaggtcg agcacagctc cgaactcctg 1140
    cccctgggcg tccggtcact gccggtggtc cccggcccgc gcaactga 1188
    <210> SEQ ID NO 63
    <211> LENGTH: 1425
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 63
    atgcttcctg agttccaatt gcagtggaat tggctcgacg ccccggccgg cggcggaggc 60
    gagctgcaag cgacctgggc ccggctgcgc atcgccgtgg gcgccgagac cgtcacactc 120
    gtccaggagc ccgggcaggg gaccttccgg gagcacacga ccggctcgct ctaccccctg 180
    gccgagtgga tcgccttcaa ctggtggtcg ctggtggccg acgcgcggcc cggcacccag 240
    atatcccagc tgcgcttcgc ctaccgccac ggtgtgggcg acaaccgcgg ttcgtggtgg 300
    atgcgttcgc gccgtcacat cctgcgcgcc gcctgcgacg gcttccgctg gccggacatg 360
    ctcttcgtgc ccgagggccg ggagacccgg atcgtatgga tgccggacat gggccccgac 420
    gtacgacccg ggaaccgctt cgcgagccgg ggcaactcct gtgtggagag cgccgcgttc 480
    accgccacac tggcctcgtt cgtcgacgcg gtgaccgagc gcctcacgga ccagggcatc 540
    accggcaccc cgctccagga ggagtgggcc gccgtccgcg ccaccgacga ggacgaggcc 600
    gccttctgcc gcatcgcggc acggctgggc ctggacccct acgccgaggc cgagccgtac 660
    gaggcggaca tcctcaaggc cgccgagcag ttggcggaac cgctcgccag tgacttcttc 720
    aacggggtgc ggcctgagcg gatagccgac cagctccagt ggatcgcgcg cgtccgcacc 780
    ctgatgggca ccgcgcccgc ggataccccg ctccctcccg ccttggtgga actgcgcaag 840
    gactgcgccg acttgagcga gaagttcttc gctccggggc gactcgacaa cccctgggac 900
    ctcggctacg aggtggcgca ccgggtgcgc gcgtgggcgg gtctggacga caccgcgccc 960
    ttcgacccgg cccccctgat gggctaccgc accgagcagg tcccctatat ggaccggggc 1020
    ctggtcgccc tcggcacccg caggggcgcg gacgggccgg tcctggtctc ctcccggcgc 1080
    ttcaccgacc gcccgcgccg cttcctccag gcccgcgcgc tgtggcatct gatctgcgac 1140
    cccgacgaca ccttcctgat cgcggcggcg cacacccacc gccagcacgt ggcccgcggc 1200
    ttcgccctgg aggtcctggc ccccgccaag ggcgtggcga ccctgctggc cgaccccgga 1260
    cacctggtgt ccgccgagga cgtcgaggtc atcgccgacg actacggctg cggcaacatc 1320
    gtcgtggaac accagctgga caaccgcgtc ctggcgaagg acttcacctg gccgggccac 1380
    gcccgcgccg gcgcgccggc cggtgagagg agccggggcg catga 1425
    <210> SEQ ID NO 64
    <211> LENGTH: 1332
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 64
    gtgacaatcc gccagcgtgt cgtcgtcgtc atcaccgagg gagcggcacc cgagctgctc 60
    gaccgctggt gtgcccaggg gctgctgccc ggcttcgccg ccctgcgctc gcagggggct 120
    tccgggccgc tccacgccga gggcaccccc tacgaaccgc cgggcctgct gagcgtcctg 180
    accggccggc gcgccgcgga ccacggcttc tactcctact ggacctgtca cgacccggag 240
    tacgcgccgc aggtcctcac ccccgagcac cgccgccacc cactgctgtg gcagcacgag 300
    gtgttccagg gcgtcaggtt cgcctcgata ggcctcttcg gcacccatcc cccggagccc 360
    ttcgacggtt ccctgatcac ctatccgatg tatgccaccc tccacgcctg ccacccgcgc 420
    agcctccagc gcaccctggc gaagaagggc atccgtccgg tccacgacgt gtcgatcttc 480
    tggaccgggc aggaccgcga cgagctgctg ccttccctgc tggaggcgga cgtgcagcgc 540
    gggcgcgcgg cattggctct gctggaggag tccgatgtcg tgatcgtcaa cctcacgagc 600
    atcgaccgct gttcgcacat ctactggcag gagctggagc acggccccga gcacgagcgg 660
    gagagcgccg tcttcgccgc ctaccgcacc tgcgaccagg tcatccagga cgccctgcgg 720
    gcggccgacg accgcaccag tgtcgtggcc ttctcggaga taggcttcgg gccgctgcgc 780
    aactactgtt ccatcaacga cgagatggag caggcgggtt tcctggccac cgccgaggac 840
    ggccgcgtcg agtgggccgg cagcgcggcc ttcgaggcgg tgcagggcac gcacggggtg 900
    aacatcaacc tgcgcgaccg ctacaagcac ggcctggtcc cggagcgcga ctacgagaag 960
    gtccgcaccg acgtcgcggc cgcgctgctg gagcggcgca acccccgtac cggcaggctg 1020
    ttcttcgacg cggtgcgccg ccgggaggag gtctatcccg gcgaggccac ccagcacgcc 1080
    cccgacctca tcctggagcc ggcggactgg cgctatcttc cgctgggcga cccgcactgg 1140
    gcctcgcacg tccaccgcga ctggcagagc ggctggcacc gccgggagtc ctactggtcg 1200
    gccgtcggcc ccggcttcac cggtggggcg cggcagaccc gcaccgccgc ccccgtcgat 1260
    attcccgcga ccgtatgcgc tctgctcggg cgtgacgtgc cgaacgactg ggacggcgtg 1320
    ccgctgtcct ga 1332
    <210> SEQ ID NO 65
    <211> LENGTH: 372
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 65
    atgacaccag aggaactctc cgacttcgcg ctggagctgc cggaggcggt ggacgacgag 60
    gcgttcggcc ccggagccgc ggtcttcaag gtggagaaga aggtcttcgc cattctccag 120
    gacgcctccg aggaccgccc gccgcaggtc acgctgaagt gcgaaccgga tctggcgctg 180
    cacctgcgcg agcagtacgc ggcggtggtg cccggctacc acgtcaacaa gcgccactgg 240
    aacacggttg tcctgaacgg cacggttccc gtggaggagc tgcgggagat ggtggagcat 300
    tcgtacgatc gcgtggtggc ggggctgccc aaggcggtac gggaacgtct gcgcctcctg 360
    cgcaccgtgt ga 372
    <210> SEQ ID NO 66
    <211> LENGTH: 1056
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 66
    atgaccgtgg agcagacccc cgagaatccc gggaccgcgg cccgcgccgc cgcggaagag 60
    accgtgaacg acatcctgca aggggcgtgg aaggcccgcg ccatccacgt ggccgtcgaa 120
    ctcggcgtcc cggaactgct ccaggagggc ccccgcaccg cgaccgccct cgccgaggcc 180
    accggcgccc acgagcagac cctgcgcaga ctgctccgac tgctcgccac ggtgggcgtc 240
    ttcgacgacc tcggccacga cgacctgttc gcccagaacg ccctctccgc cgtcctgctg 300
    cccgaccccg cgagcccggt cgccaccgac gcgcgcttcc aggcggcccc ctggcactgg 360
    cgggcctggg aacagctcac gcacagcgtc cgcaccggtg aggcgtcctt tccttcgacg 420
    tggccaacgg cacctcgttc tggcagctca cccacgaggg accccaaggc gcgcgaactg 480
    ttcaaccgcg ccatggggtc ggtctccctc accgaggccg gacaggtcgc cgcggcctac 540
    gacttctccg gcgccgcgac cgccgtggac atcggcggcg gccgcggcag cctcatggcg 600
    gccgtcctcg acgccttccc cggcctgcgc ggaaccctgc tggagcgccc gcccgtcgcc 660
    gaggaggccc gtgagctcct caccggccgc ggcctcgcgg accggtgcga gatcctgccc 720
    ggcgacttct tcgagaccat ccccgacggc gccgacgtct acctcatcaa gcacgtgctg 780
    cacgactggg acgacgacga cgtcgtacgc atcctccgcc ggatcgccac cgccatgaag 840
    ccggactccc ggctcctggt catcgacaac ctcatcgacg agcggcccgc cgcatcgacg 900
    ctcttcgtcg acctgctgct gctcgtcctc gtcggcggcg ccgaacgctc ggagagcgaa 960
    ttcgccgcgc tgctggagaa gtcgggcctg agggtggagc gctcgctgcc ctgcggcgcc 1020
    ggcccggtgc gcatcgtcga gatccgcagg gcctga 1056
    <210> SEQ ID NO 67
    <211> LENGTH: 1641
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 67
    atgacggtgc tgggtctggg tggatccgga catgactggg cctcctgtgc caccgacggc 60
    cgacggctgg tggcgatcga cgaggagcgg ctggtccgca gcaagtacgg cctgggagcg 120
    gacctcctgg cgggccacag ccggcgcgcc gtcctcgacg ccctcggcac gagtgccgag 180
    gccgtggaac acgtggtggc ctgcgagctc gtaccacgcc ccttctacca ctcgttccgc 240
    aggcgcgtga cggtcgtcaa ccaccatctc gcccacgcct acagcgcgtt cggggcctcc 300
    gggatgaccc gcgccgccgt actggtctgc gacaactccg gcagcctggt gacgggcctg 360
    aagtccggcc cagggccgcg cgaggcggag acgatcagct gctacaccgc cgacgcctcc 420
    gggctgcgcc tggtcaaccg ggtcgccggg acacacgccg tggacgcctc ctccgagagc 480
    gcctactacc agcccggcga gaccgacaat tccctcggcc acttctaccg ctcggccagc 540
    ctcgcactcg gcctcgccta ctccggtccc aagacccgct accccgtcag cgaggacggc 600
    aagaccatgg gcctcgcgcc ctacggcgac gaccgcttcg tcgacgaggt cgcggagctg 660
    gtcaccctgc tgcccgaggg cggcgtgcag atctcggcga gcaaggtgaa ccacctcttc 720
    gaacgcctcg tggaatcggg tgagttcgag gaccgggcgg ccttggccta cgccgcccag 780
    gagacgctgg aacgcgccct gctgcactgc gcccgcgacc tgcaccgccg caccggcctg 840
    acggacctgt gcatcgccgg cggcgtcggc ctcaacagcg tcgccaacgg ccggatcctg 900
    cgcgagaccc ccttcgagcg ggtcttcgtc gtcccggccg cgggcgacaa cgggatcagc 960
    ctcggctgcg cctactacgg cctccacgag ctggaggggc gcgcgccgtc ggagctcccc 1020
    gccctcgaca ccgcctacct cgggcccgac taccccgccg agcgcgtcga cgcggcgctg 1080
    gccggctcgg gcttcaccgt ggagaccccc gacgacctgc ccggcagggt cgccggcctg 1140
    ctcgccgaag ggaagatcat cggctggttc gacggccgct ccgaattcgg cccgcgcgca 1200
    ctgggacacc gcagcatcct cgccgcaccc ttccccgcct ccgtgcggga ccacctcaac 1260
    gacaacgtca aacaccgcga gtggttccgc ccctacgccc ccatcgtccg cgaggaccgg 1320
    gcggcggact acttcgacct cgtccagccc tccccgttca tgctggtcgt cgcgcgcgtg 1380
    acccggcagg acgccatccc cgccgccacc cacgtggacg gcaccgcccg gctccagacg 1440
    ctgaacgccg cacagaaccc gaaggtctac gagctgctcg gcaggttcga ggcgctcacc 1500
    ggctgcgccg tgctgctcaa cacctccttc aacgtcgccg gccagcccat cgtcgagacc 1560
    ccggaggacg ccgtcgaggc gttcgcgggc atgcgcctgg accacctcgt cgtgggggac 1620
    cggctggcga ccaagccctg a 1641
    <210> SEQ ID NO 68
    <211> LENGTH: 1707
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 68
    gtggacgtcc ccgtgctcgt ggtcggagga ggaccgacgg gcttggcgat ggcgctcttc 60
    ctcgcacgcc acggcgtcgg ctgcctgctg gtcgaacggc ggacgaccac ctcgcccgtc 120
    ccgcgcgcca cccacgtcag ccgccgctcc atggaactct tccgcgaggc gggcctggag 180
    gaggagatcc gccgggccgg gttcgaggtc gtgcgcgagg acgacccacg gctgcggacc 240
    cggcccgaac gccacctgcc ccgggtggtc ctgcaagccg cctcgctcgc cggccccggc 300
    ccggtggggg tcctggagac cggtgacgag gaactggccg tacccggccc ctgcgcaccc 360
    ttctggtgcg gccaggaccg gatggaaccc ctgctcgcca aggccgcggc gcgccacggc 420
    gccgatgtgc gcttcggcca cgaactgacc ggcctgtggc cgggggagga cagcacacgg 480
    gcccgcgtcc gggcagcggg aacgggacgg acctacaccg tcgacgcccg cttcgtcatc 540
    gccgccgacg gggcgcgcgg cgagatcgcc gagcgcgtgg gcatcgcgcg ggagggcctg 600
    ggcacggtcg cccaccgggt gagcatcctc ttccgcgccg acccggggcg ctgggcccgc 660
    gaccggcggt tcttcatgtg catgatccag aacccggggt tcgacggggc ggtgatggag 720
    ctcaacaccc cgggccgctg gtgcgccgcg gtggactacg acccggcccg cgccgaaccc 780
    gacggcacct actccgcacg cacctgcctc gacctggtcc gggccgccgt cggtgacgac 840
    cggagcgacg cggcggtcga caccgtcttc cactggaagg cccggcaccg catagcggcc 900
    gcctaccgca gtggggcggt gttcctcatc ggcgacgccg cccacctcca cccgccctcc 960
    ggcggctacg gatccaacgt cggcttccag gacgcgcaca acctcgcctg gaagatcgcc 1020
    gccgtgctcg gcggctgggc cggaccgcgg ctgctggaca cctacgacga agagcgccgc 1080
    cccgtgggaa aggcgacggc ggagcagtcg atgctcctcg acggcgtgcc accggaacca 1140
    ctggggggaa gcgtcgtccg ctgcgatccc cgcaccctga tcatgggata ccgctaccac 1200
    tccgccgccg tcctcggccc cccgcacggc cccgccttcc ccgcggcctt caccctgcgc 1260
    ggagacccgg gcacccggct gccgcacgta tggctgcgta cggacgcggg ggaacgcgtc 1320
    tccacgctcg acctgtgcca cgggcacttc gtcctgctct ccgccgaccc ggtctgggcg 1380
    gcggccgcgg cgcgctcggc gaaggagacg ggcgtaccgc tgcggggcca ccacctggcg 1440
    gccaccggaa gcgaactcgc cgacccctcc ggcgagttcc cgcggagctg cgggaccggg 1500
    cccgcggggg ccgtgctcgt acggccggac ggcatggtcg cctggcgcac ggcccgcgcc 1560
    gtgcccccgg acccggacag cgcgcaggac ctggtcacgg cagcggtgag acgtgtcctc 1620
    gcactgccgg agcgcgcggc gccaccggtg ctcggtccgc cgcggttgtc acgcggttcc 1680
    tatcggcgag tcgggagcga cgggtga 1707
    <210> SEQ ID NO 69
    <211> LENGTH: 483
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 69
    gtgaagcctc attccttctg cacgtgctgg ccgggcgcca ccgtatggct gacgggccca 60
    ccgggcgcgg gcaagacgac gatcgcccgc gcactggcgg agcggctgcg cgaacggggc 120
    cggcgcgtgg aggtgctcga cggcgacgcg acccgcgcgc tcctgaccgc gggctcctcg 180
    tgggaggacc gtggcaccgg cctccagcgg gtcggcctga tggccgaggt cctggcgcgc 240
    aacggcatcg tcgtcctcgt cccggtgacc gcggcccgcg cggacagccg cgaagccgta 300
    cgcagacgcc acgagcggtc cggcaccgcg cacctggaag tgcgggtggt ccgggacgca 360
    gtgcctccga gcgggctccc cgcgccgccc ggcccagatc tgcggatcgc ggcgcacgag 420
    cagagcgccg aggagtcggc gcgggcactg caccggctcc tggcggagag ggagctggcg 480
    tga 483
    <210> SEQ ID NO 70
    <211> LENGTH: 960
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 70
    gtgaaccccg ggcgcggtgg agcgtacgcc gcggggcgcg acgggacccg cgggacgcga 60
    cgccctcacg gtctgtcgca cctggatctg ctggagtcgg agtcggtcca catcttccgt 120
    gaggtggcgg gcgagttcga gcggccggtg atcctcttct ccggcggcaa ggactcgatc 180
    gtcatgctgc acctggcgct gaagtccttc gctcccgcac ccgtgccgtt cgcgctgctg 240
    cacgtggaca ccggccacaa cttccccgag gtgatcgcct accgggaccg cgtcgtggcg 300
    gcgctcggtc tgcggctgga agtggcctcc gtgcaggact tcatcgacaa cggcaccttg 360
    cgcgaacgcc cggacggcac ccgcaatccg ctgcagacgg tgccactgct ggacgcgatc 420
    gggcgccacc gcttcgacgc cgtcttcggc ggcggccgcc gcgacgagga gaaggcccgc 480
    gcgaaggagc gggtgttctc cctgcgcgac gagttcggcg gctgggaccc gcgccgccag 540
    cgccccgaac tgtggcggct ctacaacggc cgccacgcac ccggcgagca cgtccgcgtc 600
    ttccccctct ccaactggac cgagctcgac gtgtggcagt acgtcgcccg cgaggagatc 660
    gaactcccca ccatctacta cgcccacgag cgcgaggtct tccgccgcgg cggcatgtgg 720
    ctggcaccgg gggagtgggg cggcccacgc gagggggaag cggtggagaa gcgacgggtg 780
    cgctaccgca cggtggggga catgtcctgc accggcgcgg tggactcggc ggcggccacc 840
    gtggccgacg tcgtcgccga gatcgccacg tcccgcctca cggaacgggg cgcgacccgg 900
    gccgacgaca agctgtcgga agccgcgatg gaggaccgca agcgcgaggg gtatttctag 960
    <210> SEQ ID NO 71
    <211> LENGTH: 492
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 71
    gtggggcagg acagccggcc gcggtggctc accgacgagg aacaacgcgt gtggcgcggc 60
    tatctgcggg ccaccaggct ggtggaggac cacctggacc gccgcctcca gcgggaagcg 120
    gacatgccgc acctctatta cggtcttctc gtccagctct ccgaggcccc gcgccggggg 180
    atccggatga ccgaccttgc ccgcaacgcg aagatcaccc gcccgcggct ctcgcacgcg 240
    atcacccgcc tggagaagct cggctgggtg cgccgggaat cgtgccacgg cgacaggcgc 300
    ggccagaacg ccgtcctcac ggaagagggc cgcgaggttc tggagaagtc ggcgccgggc 360
    catgtcgccg ctgtgcgcgc ggccgtcttc gacagcctca ccccggaaca ggtcgggcaa 420
    ctgggccgga tctgccaggc gatagagaag gggctggacc gggaaggcgc ggacctgccg 480
    tggctgcgct ga 492
    <210> SEQ ID NO 72
    <211> LENGTH: 1242
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 72
    gtggaacgac acgacggggc accgggctgg ggcttcaccc atacccagta cagcgcggac 60
    cacggtgaac gcggcgccac ccgcagggcc ggggccctgc tctccgcgcg gcccctgccg 120
    cagaaccagc acatcatggg ctggggcgcg gagaatcccg aaccggcgcc cggacgctac 180
    gacttcgagg tcctcgacga gcgcgtcgcc ctgatgcgcg cgacgggggc cacgcccgtc 240
    ctgaccctgt gtgccgcccc cgactggatg aagggcggcc ggcccggccg caccgactgg 300
    tcgcgactgg agaccgcccc cgacccccgg cactacgcgg acttcgcccg gctcgcgggc 360
    gtgatcgccc aacgctaccc ggacatcagg cacttcctcg tgtggaacga gctgaagggc 420
    ttctacgacg aggacaggcg gcgctgggat tatgagggat acacccggct gtacaacctc 480
    gtccacgccg agctgaagcg gcggaacccg cgcaatctgg tgggcggccc ctatgcggtg 540
    gtcgaccacg acccgcccgc cgaggacgcg gcggaccgct cgcgcgaact gcgcggtccc 600
    tggggcgagc tggaccagcg ctccgccgac gtcatccgct attggaacgc ccacaaggcg 660
    ggcgcggact tcgtcgtcgt cgacgggtcc agctacaccc gcgagggcca ccgggcgatt 720
    ccggacgagt tcgccgccac cgagaagttc gccgacgtca cccgctgggt caggagcgtg 780
    accggactcc cggtgtggtg ggccgagtgg tacgtcgagc cgcccgccga ggacgaccgg 840
    ccgggcggcc gggacggctg gggcgagggg caccgcaccg ccgtgcaggc caccgcgatg 900
    atgcggctgg cggagagcgg cgcgtcggcc gccttctact ggaacccgca gcggaccggg 960
    aaggcgtgcc ccggctgcct gtggcggagc acccacttgc gcgacggggg aggggagttg 1020
    cccatggcgg gtctcctgag ccggttcgct cgcgaattcc ctccgggcac cgccttccgg 1080
    ccggtcgccg tcacctgcgg gagcggtgac agggtcgagg ccctcgccga cgaggccgcc 1140
    gtgctcgtcg tcaacaccga gtgccggccg gtggccgcca gggtggacgg gcaggcgctg 1200
    tccctcgcgc cgtacgaggt gcgctggctg acccgcccgt aa 1242
    <210> SEQ ID NO 73
    <211> LENGTH: 816
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 73
    atgacgcgaa ggcgcccaac gggcccgatt caccgtcggc gggcgtcact caccctttcc 60
    cccacgggag ccgccatgag aagaaatcgc atcgccgccc tgctgccggc cgctctggca 120
    ctggtcggca tatccgtcct cgcccccgcc accacggcga gcgcggccgc accgcacggc 180
    ggcacctcgc aggccgccgc attccccgtg agcgaggccc agttcaagca gatgttcccg 240
    aagcggaacg cgttctatac gtacaagggc ctggtcgccg cgctcaaggc gtacccgggc 300
    ttcgcgggca ccggcagcgc cgaggtccgg aagcaggagg ccgccgcctt cctcgccaac 360
    gtcgcccacg agaccggcgg actggtctat gtcgtggagc agaacaccgc caactacccc 420
    cactactgcg accggagccg gccctacggc tgtccggcag gccaggccgc ctactacggt 480
    cgcggcccgc tccagatcag ctggaacttc aactacaagg cggcgggtga cgccctcggc 540
    atcgacctgc tccacaaccc ctcgctggtg cagaaggacg cggccgtctc ctggaagacc 600
    ggcctgtggt actggaacac ccagcgcggc cccggcacca tgaccccgca cgaggccatg 660
    gtcaaccacc gcggcttcgg gcagaccatc cgcagcatca acggcgccct ggagtgcgac 720
    ggccacaacc ccgcccaggt ccagagccgc gtcgcgaact accagcgatt caccaagatc 780
    ctcggcgtgg cgccgggcgg caatctctcc tgctga 816
    <210> SEQ ID NO 74
    <211> LENGTH: 12249
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 74
    gatcccgat cgtctcggac atgaccggcg accttctcgg cgcgcgggag gcccaggacc 60
    cgcctactg ggtgtcccac atccgccgcg cggtgcgctt ccacgaccag atccgccgtc 120
    gcagcgcta cggggccggg gccttcgtcg aggtcggccc ggacacggtg ctcagctcgg 180
    cggccaggc gtgcctgacg gaccaggcgg gcaggagcgc gcccgtcctg gtgtccctcg 240
    gcacgccga gcgcgcggag gtgcccgcgc tcctgaccgc tctggccacc ctgcacaccc 300
    tggcgtggc cgtggactgg cgggcgtggt tcggcgacgg gccgcgcgcg gccggcctgc 360
    cacatacgc gttccagaag cagcactact ggccgtcggg ccccaccggt tggcggtccg 420
    gcccgcccc cgtacccctg ccccaggccg gaacggagga cgccgaaagg cccggtcgcg 480
    cgcggagtg gcgggcgctg ccgcccggtg agcggtacga cgcgctgctg cggatggtgc 540
    cggcgaagc cgccgccgtg atggggcacg ccgggccgga ggcggtggag ccggagcgcg 600
    cttcctcga ccacggcttc gactcggtga tggccgtgaa gctgcgcgac cgtctcgtgg 660
    cgggacggg gcgggagctg ccgacgaccc tgctgttcga ccaccccacg cccgcggccg 720
    cgccgacta cctgctggcg gggacgggcg aggccgagac ggcgccgtcc gtgtccctgt 780
    ggaccagct cgaccgcctg gaggccgacc tcgcgcggct gccggccgac gaccggcagc 840
    cgcccgcgt cgccgagcgg ctcaagggcc tgctcgcggt ccacgcgccg gaccggggcg 900
    cgggagcga ggacgcgccg gaccaggacg cgctggacac ggcgaccgac gacgagatgt 960
    cgagctgat cgagaaggaa ctccgccgtg gatgagacca acgagaccaa actccgcgag 1020
    acctgcggc tggtcacggc cgatctgcgg cgaacccgca ggcagttgga ggaggccgag 1080
    acgcggccc gcgagcccgt cgcgatcgtg ggcatggcgt gccgcttccc cggggacgtg 1140
    catcgccgg acgacctgtg gcagctggtc gccgagggcc gggacgccgt caccgagttc 1200
    ccgccgacc ggggctggga cgtcgacgcc gtctacgacc ccgagccggg caccccgggc 1260
    ggacgtacg cgcgccacgg cggcttcctc aaggacgccg ccggattcga cgccgccttc 1320
    tcggcatca cgccgcgcga ggcgctcgcc atggacccgc agcagcgcat gatcatggag 1380
    tctcctggg aggcgttcga gcaggcgggc ctcgacgcga ccaccctgcg gggcgaggac 1440
    tcggcgtct tcgtcggctc caacagcaac gactacctga tcaacgtgct cgacgcgcgg 1500
    acgtcgccg agggcttcat cgggaccggc aactccgcca gcatcctctc cggccgcgtc 1560
    cctacacct tcggcttcga gggcccggcc gtgtccgtcg acaccgcctg ctcctcctcg 1620
    tggtcgcgc tgcacctggc cgcgcagtcc ctgcggcagg gggagtgctc cctggcgctg 1680
    cgggcggcg cgacggtgat ggccacgccg accgccttca tcgagttcag ccgccagcgg 1740
    gcctggccc ccgacggccg ctgcaagtcc ttctcggcga ccgccgacgg caccacctgg 1800
    ccgagggcg cggccgtgct gctgctggcc cggctctcgg acgcccgccg cctgggctac 1860
    ccgtgcacg cggtcatccg gggcagcgcc gtcaaccagg acggcgcgag cgcgggcctg 1920
    ccgcgccca acggaccggc gcaacagcgg gtgatccggc aggcactggc caacgcacgg 1980
    tgacggccg acagcgtcga cgcggtcgag gcacacggca ccggcacccc gctgggcgac 2040
    cgatcgagg cccaggccct cctcgccacc tacgggcggg cccgcggcga gggcaggccg 2100
    tgtggctgg gctcgctgaa gtcgaacctg ggccacaccc agtccgcggc cggcgcgggc 2160
    gcgtcatca agatggtgat ggccatgcgg cacgggacgc tgccccgcac gctgcacctc 2220
    cggagccca ccccgcgcgt cgactggtcc gccggtgacg tacggctgct gaccgaggcc 2280
    aggactggc cggacaccgg acagccgcgc cgtgcggccg tctcgtcctt cggcgtcagc 2340
    gcaccaacg cccatgtgat cctggagggc ccgcccgccg aggaggcacc ggacgcgccg 2400
    tgccggacg tctcctcgca gccgcggggc ccgctgccgt gggtcgtctc cggccgcagc 2460
    aggcggccg tccgagcgca ggccgagcgc ctggcggccc acctgaccgc gcgcccgcac 2520
    tggcaccgg ccgacgtggc caccgcgctg gccaccacgc gggcggcctt cgaccaccgg 2580
    ccgccgtcg tcggccggga ccgtgaggaa ctgctcgccg gcctcgcggc cctggccacc 2640
    gaacccgcg cgcccggcct ggtcaccggc cggaccccgc cgtccggcgg caaggccgcc 2700
    tcctcttca ccggacaggg cagccagcag cccggcatgg gccgcgaact ggcggctcac 2760
    gcaccgtgt tcgccgacgc cctggacgag gtctgcgccc agctcgaccg gcacctcgac 2820
    ggccgctgc gcgaggtgct gttcgccgcg gacggcacgc ccgaggccgc cctgctcgac 2880
    cgacggcct acacccagcc cgcgctgttc gccgtcgagg tcgcgctgct gcggctgctg 2940
    aggactggg gcttgcggcc cggcatggtc gcgggccact cggtcggcga actgaccgcc 3000
    cctacgccg ccggggtctg gtcgctcgcc gacgcctgcg ccctggtcgc cgcccgcggc 3060
    ggctgaccc aggcactgcc cgcgggcggc gccatggtcg ccgtgcaggc gaccgaggac 3120
    aggtgcgcg cccaactcgc cgacggccgc cccggcgtgg acatcgccgc cgtcaacgga 3180
    cggaagcgg tggtgctgtc cggcgacgag gccgccgtca cggacctggc gcgcgagtgg 3240
    ccgcccgcg gccgggagac caggaggctg cgggtcagcc acgccttcca ctccgcccac 3300
    tggacgcca tgaccgaggc gttcgccgag gtcgcacgag gggtgtccta cagcgcgccg 3360
    ccctcccgg tggtctccac gctcaccggg gcccccgtca ccgacgagct ccgcaggccg 3420
    aacactggg tgcggcacgt ccgggagacg gtgcgcttcc acgacgcggt ccgcgccctg 3480
    gcgaccgcg gggccaccgc gttcctggag gtcgggcccg gcggcgtgct gacggccgcg 3540
    cacgccgat gcctgcccga cgccgccccc gagacgttcg tccccgtgct gcggcgccgc 3600
    ggcccgaac ccgagtccgt gctgacggcc gtcgcgcagg cccacacgat cggcctctcg 3660
    cggcgtggg accgcctgct gcccaaggcc cggacgcgcg tggacctgcc cacgtacgcc 3720
    tccagcgcg gccactactg gctggcgggc atggccggag cgggcaccgc gcggccggtg 3780
    ggccggaag tgcaggagcc caccgccccc tccggtacgc cgccgctgtc gcgacggctg 3840
    ccgacgcgt cggaggagga gcgcggccac ctgctgctga cgctggtacg cgagcagtcg 3900
    ccaccgtga tgggcggcgt cgaccccgcg caggtcgaac ccgaccgccc cttcctggag 3960
    tcggcttcg actccctgat gggcgtcgag ctgcgcaccg cgctcgccgc cgactgcgca 4020
    tgcccctgc cgcccggcct gatcttcgac caccccacgc ccgccgccct ggccgccttc 4080
    tcggcgagc agctcgcggc ggcggcctcc ggcaccccca cggcggcggc accctcgccg 4140
    actccctgg aggcgctgta ccgcaacgcc aacaccctcg accggcccga ggacgcgctc 4200
    gccctcacca aggccgcctc ccggctgcgc ccggtcttcg ccagcgtggc cgaggcgggg 4260
    caggacccgg tcacggtgga gctggcacag gccaccggcc ttccgggcct gatctgctgc 4320
    ccggcacccg tgccgctgta cggggcacag cagtacagcc ggctcgcagc cgccttccgc 4380
    ggcacgcgcg gagtctcggc cctgctcgcc cccggcttct ccccgggcga actgctgccc 4440
    gccgacttcg aggtgatgca ggacttcctc gccgaggggg tccggcggca gaccgacggc 4500
    gcgcccttcg tcctcctggg ccactcctcc gggggctggt tcgcctacag cctggcggcc 4560
    cacctggcgc gcaccgggcc gcgcccggag gccgtcgtgc tgctggacac ctatcagctg 4620
    cacgacccgg cgctgcaccg catgcagcgc gaactcgccc agggcgtcct ggaccgcgag 4680
    gaggacttcg gggcgatgac ggacgtacgg ctgagtgcca tgggcaaata cttcgacttc 4740
    ttcaccgact gggtggccga ggacgccggt gtcccgacgc tgctgctgcg ggcctccgag 4800
    cctctgggcg aggtcgtcga gggccaggag tggcgctcca cctggccgtt cgacagcacg 4860
    gtcctcgaca cggaaggcga ccacttcgcc atggtcaacg accacgcgcc gcggacggcc 4920
    caggccgtga acggctggct gtcgggcctc accggcggaa ggggctgagc gccggtggag 4980
    acacgcaacg ccgaacggcc gtggatacgc agcttccacc ccgctcccca ggcccctgtg 5040
    cggctgctgt gcctgccgca cgccgggggc tccgcgagcg cctacttcgc gctgtcgagg 5100
    gaactggcgc cccgggtgga ggtgctcgcc gtgcagtacc ccgggcggca ggaccggcgc 5160
    gacgagccgc tgctggactc gatcgaggcc ctgcgcgacg gggtcgccga ggccctgacg 5220
    ccctggctgg accggccggt cgccctcttc ggccacagca tgggcgccgt ggtggcctac 5280
    gagctggcgc ggctgctgtg ccaggacgcg ggcgtgccgc tcacccacct cttcgtctcc 5340
    ggacgccggg gatccgaccg aagtctccgt ccttgccgcc gtgttccgga attcaccgtg 5400
    acaccgccgc gcggctcttc ttccgaagtc ctccagatcc ggcacgagtt tgtatccgaa 5460
    cggggttctg cgtgcgaaat actctcttcg aattgggtga catacccccg atcggcaccg 5520
    tacccgagca gatgtacgcc tcggtgatcc gacgggagcg ctacggacag ccccaccagg 5580
    cgttccgcag cgaggtcgtg gacgtgccga aggtggggcc cggtcaggcg ctggtcctcg 5640
    tgatggccgc gggcatcaac tacaacaacg tctgggcctc cctggggcag ccggtcgacg 5700
    tgatctccgc gcggcagaag cagggccaca gcgaggactt ccacatcggc gggtccgagg 5760
    gctccggcgt ggtgtgggcg gtgggggagg gcgtcaccca ggtcgcggtg ggcgacgaag 5820
    tgatcctctc cggctgccag tggacggaga cggccgccga catccggctc ggcgccgacc 5880
    ccatgacctc cggctcgcag tcggtgtggg gatacgaggg caactacggc tccttcgccc 5940
    agttcgccct cgtcgacgac tatcagtgcc accccaagcc gcccggcctg acctgggagg 6000
    aagccgcctg cttcctgctc accggggcca ccgcctaccg ccagctgtgc ggctggcagc 6060
    cgcacgacgt gcggccgggc gacccggtcc tcatctgggg cggggccggc gggctcggct 6120
    ccatggccat ccagatcacc cgggcgcggg gcggcatccc cgtcgccgtg gtctccgacg 6180
    aggagcgggc ccgctactgc cgggagctcg gcgcccaggg caccatcaac cgcctggact 6240
    tcgaccactg gggacggctg cccgacatcg gcgaccacga ggcgatgggc cgctggaccg 6300
    agggtgtacg ggccttcggc cggcgcttct gggaggtgct gggcgagcgc aggtccccgc 6360
    gcatcgtcct ggagcacagc ggccaggcca ccatccccac ctcgatgtac ctgtgcgaca 6420
    acgcgggcat ggtcgtcatc tgcggcggca ccaccggcta caacgccgac atcgacctgc 6480
    gcttcctgtg gatgcgtcag aagcgcttgc agggctcgca cttcgccaac ctgcggcagt 6540
    gccgcgacgt catccacatg gtcgcgaacg gccagctcga cccgtgcctg tcgtggaccg 6600
    gcggcttcga cgacatcggc aaggcacacc agctgatgca cgacaaccag cacccccagg 6660
    gcaaccaggc cgtcctggtc aacgcgccgc ggaccggcct gaccaccttc gcctgaacca 6720
    ccgccccggt gttccgacgt cttcccccca cacttaccga ccaaggagag atcaccatgg 6780
    acaagctcga catcctctgg agcgagcgcg agatccgtgc cgtgctgcag cgctactgcc 6840
    gcgggctcga ccgcctcgac gaggaactgg tcaagtccgc ctaccacgag gacgcgcacg 6900
    acgaccgcgg cgtcatccgc ggcaacgcac acgacttcgt caagcagatc gtcccgctcc 6960
    tgcgcgacgc ctacaccggc accctgcaca ccctgcacgg cagcacgatc gagatcgacg 7020
    gggatgccgc gggcgtggag tcctactgca ccgcctacca ctaccgcgag agcgacggca 7080
    tcaagcgggt ggagcagttc gccgggcgct acgtcgaccg cttcgagcgg cgcgacggcg 7140
    tctggaagat cgcccgccgg ctcgtgctga acgacttcag cctcgcccag gaggtgccgc 7200
    tcgaccccgc cgaggcccag gccggcttca acccctccca ccgcgacctc accgacgcca 7260
    gctaccaggt gctgccgctg cgcggcccgg acgcccccac cctctgagcc gtccggccgc 7320
    cccaactcgc cccacctcac caggagtcac caccgtgtcc gacaccgagc agcacgcgcc 7380
    cacgctgccg cggcagcgca cctgcccctt ctcgccgccg cccgagctcg aggagctgcg 7440
    gcgcaccgat cccatcagca ggatgcggtt cgccgacgac tccccgggat ggctgctgac 7500
    ccgccacgcc gacgtccgcg ccgcgctggc cgaccccggc gtcagctcgc accccggcaa 7560
    ggcaccccag ccctggcgca acctcgcccc cgagatgcgc gccgagcact acctgccggg 7620
    cttcctgatc ttcatggacc cgccggacca cacccgctac cgccgcctgc tcaccaagtg 7680
    gttcaccatg cgggccatcc gcaagctcga acccaggatc gagcagatcg tcaccgagac 7740
    cctcgacgcc atggaggccc agggcggcac cgtcgacctg gtgcagtcct tcgcgctgcc 7800
    gatcccgctg ctggtcatct gcgagctgat gggcatccgc tacgaggagc gcgaggagtt 7860
    catggacatg gtcctgcgac tccaggccct ggacgccacg cccgaggaac tcggggccct 7920
    cggcgccagg atgaacgagt tcatgatgaa gctcgccgcc gccaagcgcg cgaaccccgg 7980
    cgacgacctg ctcagccacc tcgcccacga ccccgacgcc gacccggcgc tcacggatct 8040
    ggagatcgcc ggcatcggcg tgctgatgct catcgcgggg cacgagacct cggccaacat 8100
    gctgggcgtc ggcacctaca ccctgctgga gaacgccgac cagtgggccc tgctccgtga 8160
    cgacatcagc ctgatcgacc gggccgtcga ggagctgctg cgccaccaga ccatcgtcca 8220
    gcagggcctg ccgcgcggcg tcacccggga catggagatc gccgggcacc aggtgaagac 8280
    cggggagtcc ctgctggcct cgctgcccgc cgccaaccgc gaccccgccg tcttccccga 8340
    ccccgaccgc ctcgacatca cgcgcgagca caacccgcac ctcgccttcg gccacggcat 8400
    ccacctctgc ctgggcatgg agctcgcccg ggtggagatg cgccaggcgt ggcgcggcct 8460
    cgtcacgcgc ttccccggcc tgcgcatggc cgccgcgccc gaggacatcc gctggcgcga 8520
    cgaccagatc gtctacggcg tgtacaacct cccggtgacc tgggacgagg ccaagtgacc 8580
    ggccccgagg ccgcggtgcg cgggtgcccc ttcggcgccg gcgaggcgcc cgcgtacccc 8640
    ttccacgccc ccgaccggct ggagcccgac ccgtactggg agccgctgcg ccgcgagcgg 8700
    ccgctgcaac gcgtcacgct gccgtacggc ggcgaggcgt ggctcgccac ccgctatcag 8760
    gacgtgcgcg cggtcttcgc cgaccgcagg ttctcccggc agctcgccgt cgcgcccggc 8820
    gctccgcgct tcctcccgca ccagccgccg ccggacgccg tcctgagcgt cgagggcccc 8880
    gaccacgcgc ggctgcgccg gctggtcggg aaggtcttca cgccgcgccg cgtggaggac 8940
    atgcgtccgc tcatccagcg caccgccgac ggactcctcg acgcgatgga ggagatgggg 9000
    ccgcccgcgg acctggtcga ggacttctcc ctgcccttcg ccgtgtccat gatctgcgag 9060
    ctgctcggcg tgccgcccga ggaccgcaag cggttctgcg tctggtcgga cgcgctgctg 9120
    acgaccaccg cgcacacccc cgcccaggtg cgcgactaca tgatgcagat gcacgactac 9180
    ctcggcgggc tcgtcgcgca gcgccgggtg cggcccaccg cggacctgat cggctccctc 9240
    gtgaccgcgc gcgacgagga ggacaagctc accgagggcg agctggtgcg gctggccgag 9300
    gccatcctca tcgccggcta cgagacctcg gcgagccaga tccccaactt cctctacgtc 9360
    ctcttccgcc acccgcagct gctggagcgg atcaggaacg accacgacct catccccgac 9420
    gccgtcgagg aactgctgcg cttcgtgccc atcggcaccg tggacggctt tccccgtacg 9480
    gccaccgagg acgtcgagct cgggggagtc ctggtcaggg ccggggagac ggtcgtgccg 9540
    tcgatgggcg ccgccaaccg cgaccccgag ctgttcacgg accccgacga gctggacctc 9600
    gcgcggcggc cgaatccgca cctgggcttc ggcgcgggac cgcaccactg cctgggcgcc 9660
    caactggccc gggtggagct ccagatcacg ctcacgacgc tgttccgcag atacccccgc 9720
    ctgcggctgg ccgtgccgga ggagagcctc tcgtggaagg aggggctgat ggtccgcggc 9780
    atgcacacca tgccggtcac ctggtgagga caccggcgtc ctcctgacct tcccggcgtt 9840
    ctcacgccgt cccggcagcc ttccttccga cacgagcgca cagagggtga agcgaccgca 9900
    atgagcacca tcgacgaatg ggaacacagc acgaaggagg cgggcatgga ccccgcggcc 9960
    ctcagacgcc tgaccgatgt ggtgcgggcg aggggcggcg cggcgcagct gtgcgtcatg 10020
    cggcggggca ccgtggtcct ggaccgctcg ttcggctgct cctccgactc cctcttcctc 10080
    gtctacgcgg ccaccaagcc cgtcgccgcc ctcgccgtgc acgcgctcgc cgagcggggc 10140
    ctgatcgggc tggaccggcc ggtggccgaa tactggccgc agttcgcccg gcacggcaag 10200
    ggtgacgtga ccgtccgtca tgtcctccag caccgggccg gggtgccggt cggccggggc 10260
    atcgtgcgca cgatgcgcac cgccggcgac tgggagcgct ccgtgcgcga ccttgagcag 10320
    tcccggccca agtggcccgg cggcgaggtc gccgcctacc acttcatgag tttcggattc 10380
    attctcggcg aactggtgca gcgcgtcacc gggcggtcgt tccgagattt cgtgacttcc 10440
    gagctcttcg ccccacttgg gctgaatgat ttgcacatgg gattgcccgg cagtgcctgg 10500
    ccccggcatg tgcccgcgcg ggccgcccac ccctccgaat ggcccaatca gtggatgagc 10560
    aaccgccgcg gctaccgcca ggccgtcatt ccgtccgccg gtctttccgg aaccgccgca 10620
    caaatggccc gcttttacca gatgcttatg gagggcggct cgctcgacgg catccgcgtg 10680
    ctgcggcccg aaactgtgga ggaagccaga aaaccgtcca atgacggcgg aatcgacgct 10740
    tccctcaagc gtccggtccg ctggtcccac ggattcatgc tcggtggtcc gggcccggac 10800
    ccgcgggggc tgtccaatgt gctgggccgc acgagcgacc cgagcgcctt cgggcacgcg 10860
    ggcaccacgt ccagcgtcgt gtgggccgac cccacgcgcg agctggtcct cgcctacctc 10920
    tccaacatcc agcccgagtt cggagcgggt atcgagcggc tccgcgaggt cagtgacctc 10980
    gcgctcggtg cctgcgaggc aggctgaccc gagccgtgcc gccacggccc ggcgcccgcc 11040
    cgatccgatc gggtccggtg ggggccggcc gggtccgggc ggggacgcac ttcccccggc 11100
    gtccccgccc gggccccggt gcgaaccggg cgcaaaggcg gccgatcgcc cggcgcggcc 11160
    ggatgccccc gaacggtgtg aaacgttctt atcagcctct gaccagcacc gagtgatcta 11220
    ctgcacagcc cgaggccgcg attccggcag tatcttgatc ttgacggggc accaatgcga 11280
    gcgggctatt cgccgcggtt ttccctgacg tcggatgcag atgacaccgg aggagggcca 11340
    gtgctgaatc tgcccaaagg aatggagcgc gcgcatccgc attctccgcc acaggtggga 11400
    atactcggac ccttggaagt ccgctcggcc ggaggtgccg gaacgggagc cgcggtaagc 11460
    ggtattcgcg tacgcacatt gcttgccgcg ttgactgccc gcctggggca ggcgatgtcg 11520
    accgagcgca tcctcaaaga ggtctgggcc gacaacccgc ccgcgaccga tcgcaaggcg 11580
    gtggccgtcg ccgtcctgcg gctgcggcgg gtcctcggcg acaacgaagg acggtggctg 11640
    ctcacccgcc cctccggtta cgtcctggac atccccccgg accacctcga cgccgtacgc 11700
    gcggagaccc tggtgcggga aggccgggcc gccctggccg ccggcgaccc acgcgtcgcg 11760
    gcccgccacc tcacgcgcgc cctcgaccag tggcggggcg agccctacgc ggacgccaac 11820
    gccatctcga ccgtgtccca gcgcatcacg gagctggaga acctcaggtc cgaggccgtc 11880
    caggcgcaca tcgacgccag gctcgaactg ggtcaccacc aggaactggt cggcgaactc 11940
    cgctcgctga ccgccgcgaa ccccctgcac gagccgcact ggctgcagct gatgctcgcc 12000
    ctctaccgct ccggcaagca ggccgaggct ctcgccgcct atatgcagct gcggcaggcg 12060
    ctggccgaga acctgggcat cgacccgggt cgtcagctcc aggaactgca cctgcggatc 12120
    ctgcgcgccg acgcgggcct gctgacgggg tccgggccgg cggcaccggc cgagccactg 12180
    ctcgtacggc agtcctgagg gctcacggcc acccgaagaa cgcgcggtag cacggaacct 12240
    gctgctcca 12249
    <210> SEQ ID NO 75
    <211> LENGTH: 18034
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: (302)...(302)
    <223> OTHER INFORMATION: n is a or t or g or c
    <400> SEQUENCE: 75
    cccaggacct cgtcgcggtc ccggccgcgt ggtggacctc cgccaacccc aacaacgacc 60
    agctctgcca gggcatatcg gtggaggtca gctacaacgg caggaccatc agagtgccgg 120
    tgcgggacaa gtgcccttcg tgcgaccgga cccacatcga cctcagcagg acggccttcc 180
    agaagctggc gccgctcgac aggggtgtgg tcaacggcat cacctggaag ttcgtccgct 240
    gacgccacgc cggggtcccc aaagcccggg accccggcgc tccgcgcccg gcacgccggg 300
    gnccgcccgg cgtgtcggcg tgaggttcgt cgccttcaag agtcataaag acaatcgcga 360
    ctgttgacgt tatgagttca tcaaatttaa ggtcgcggga ctcttggaac agatcaagac 420
    gacggagaac aatgacgtac tcccccggcg cgcggccgcg cccggcccgg ctgtccgcac 480
    tgctgctcgc aggcgcgctc gtcgcctcgg tgccgcccgc ggccgccgcg cgagcgccgc 540
    aaccccccac cgccgaccgc ccccgcaccg ccgcctcccc cacaggcggc tgccgtacgg 600
    gtgacggctg gacactcgac tccacccgca tcgaccccga cgacacccac cacgcctatg 660
    tcggcaacgg ctacctgggg cagcgcgtac cgcccaacgg cgccggctac accgacagcg 720
    acaccaagac cggctggccg ctcttcgctc cggcctacga cggctcgttc gtgtccgggc 780
    tctacgcgca caacaagcag accgccgccg accggcaggt gatcgccgct ctgcccacct 840
    ggaccggact ggccgtcggc accggcggcg agcacggcga tatcttcaac tcttcgacga 900
    agtcgggccg gatttccgga tatcaccaga ccctcttcca gagctgcggc atcgtccgta 960
    ccgccctgac ctggaccgcc gccgacggcc gcaggaccga cctggtctac gaggtgctgg 1020
    ccgaccgcga cgacccgcac acgggcgccg tacggctgag catgacgccg cgctggagcg 1080
    gcgaggccac cgtcaccgac cagctggacg gacgcggcgc gcggcgcatg cggcagaccg 1140
    gcggcggcga ccgcaccggt gggaccggcc gggacggccg caccatggac gtggccttcc 1200
    gcaccgacgg cacggacacc gacggcgccg tcgcctccac cctgagggcc gggcgcggtg 1260
    tgcacacgac cggggaccga cgcgccgcgg ccgcgaagga cttgagcgtg aaccagtccc 1320
    tcacgttccc cgtccgtgcg ggccacgcgt acgaactcac caaatacgtg ggtgtcgaca 1380
    ccgcgctcac ctcgcacgcg ccccgcgagg acgccaccac cgcctccctg cgcgccgccc 1440
    gccgcggctg ggacgggctg ctgcgtgccc acaccgccgc ctgggcccgg ctgtggcgct 1500
    ccgacatcga gctgccggga cagcgcgacc tccaggcgtg ggtgcgttcc gcccagtacg 1560
    ggctgctgtc cagcacccgg cagggggcat ccaacagcat cgccccggcc gggctgacca 1620
    gcgacaacta cgcgggcctg gtgttctggg acgccgagac ctggatgtac ccggccctgc 1680
    tggccaccgc gccccaactc gccaggaccg tcgtcgacta ccgctaccgc accctcgccg 1740
    gagcgcgcga gaacgcccac aagctcggct accaagggct cttctacccc tggaacagcg 1800
    gcagcgaggg cgacctggcc caggagtgcc acagcgtcga cccgccccac tgccgcaccc 1860
    agatccacct ccagtcggac atctccctcg ccacctggca gttctacctc gccaccggcg 1920
    acaccgcctg gctgcgcgag cgcggctggc cggtgatgga gggcatcgcc gaattctggg 1980
    ccgggcgggt cacccccaac gccgacggca gctactccat caaggacacc gccggccccg 2040
    acgaatacag caacggcgtc gacgacgcgg tcttcaccaa cgccggtgcc gccaccgccc 2100
    tgcgcgacgc cgcccgtgcc gcgcggctgc tgggcgagcg cgccccggcg gagtggacga 2160
    cgatcgccga ccggatccgc atcccgtacg acgcgcggca caaggtcttc gagcagtacg 2220
    acggctaccc gggcagcaag atcaagcagg ccgacacggt gctgctgatg taccccctgg 2280
    agtggccgat gtcccaggcc gacgcggcgc gcaccctcga ctactacgcc cggcgcaccg 2340
    accccgacgg ccccgccatg acggactcgg tccacgccat cgacgccgcg gccacgggcg 2400
    agccgggctg ctcggcgtac acctatctcc agcgttccgt ccggcccttc gtgcgcggtc 2460
    ctttcgacca gttctcggaa gcccgcggca ccaaggccgg cgccgacgac cccctggccg 2520
    gctcgcccgc ccacgacttc ctcaccggca agggcggctt cctccagatc ttcaccaacg 2580
    gcctgaccgg catgcggatg cgcgaggacc ggctgcacct cgacccgatg ctgcccccgc 2640
    agctcggccg cggcgtcacc ctgcgcggcc tgcactggca gggccgcacg tacgacatcg 2700
    ccatcggcgc ccacgagacc accgtgcggc tcaccggggg tgcgcccatg accctctaca 2760
    ccccgcaggg cgagcacgtg ctgaccaagg cggcaccggc cgtgctcaag acccgccgcc 2820
    ccgacctcgc tcccaccgac aacgtggccc gctgcaccac cgccggtgcc tcctccgagg 2880
    aacccggtat gtacgcggca gccgcggtcg acggcaaccc cgccaccgcc tgggtccccg 2940
    acgggccgaa cggtgaactg accaccgacc tcggcaagtc cgtacgcgtc accaaggcca 3000
    cccccgtctg gagcggcccg gcaccggcct cgtacagcgt ccagctctcc ctcgacggcc 3060
    ggcactggca cgacgcggtc gcgggcggcg ctccggtgtc cgcgcggtac gtacgcgtcg 3120
    cgctacgcgg tcaggccgat gccaagtccc gtacgggcat cgccgagctg accgttacgt 3180
    agggcaccag cagcccgcgc gcccgggctg gatgacgacg aggatccgcg ggacttcacc 3240
    cgccctcggc cgacagggac gtcctgacga gagcgagcac gtcgtcgtcg ctcagcccca 3300
    gcgcgcgggc gtcggcgacc aggcgccggg cctgcacggt gagccgggcg cgctcggcgg 3360
    tcgaccctcc ggtgacgacc gccccgcgcc cccggcgcag ttcgaggagg ccctcctcgc 3420
    gcagccgttg gtagccacgg agcacggtgt gcatgttgac cccgagcgac gcggcgagat 3480
    cgcgggcgga cggcaggcgc tcgccggagc gtacggtgcc gtcggcgatg gcaccacgga 3540
    cgcatgcggc gatctggtcg cccaggggga ggggggacgc ggggtcgacg cggaagagca 3600
    cggtcacccg cccgcggagg tgcggcgttc gcggtcggcg agggtgttga gcagcgcggc 3660
    ggccgtggcg gcgtcgtcga cggtgacgac gaactcgctg ccggtggtca gacggacgct 3720
    gatggcgtcg ccggaacgca gcacgacgcc gctcgccccg gaacggaccc ggtagcccca 3780
    gccaccgaag tcccgcagag gcttgaccgg acggtgaccg gcttcggcga tccgctggag 3840
    cggcacgttg atgcgcggcc aggggacggt cgagggcgtg acggtgagcc cgcgccggtc 3900
    ggcggtcacc cggacacccg tcagcagggt catggcggct ccgatgagga acagcgacag 3960
    cgcggacagc catccggcgg cgaccccgac gacgacgccg gaggcgaaga ccaggacacc 4020
    ggtgaggggc agcacccggg agcccgccac ccttgaccag ccggcgatct cggagtcgcc 4080
    gagcgcgaga cgcgaggcat cggcggacgg cccggaatcg ctgtcggctc cctggtcctt 4140
    gccacaggcc gcccagccca ccgccgcgta gagcgcagca gccccgaaag cgagcgcggc 4200
    ctgcgccccg ggcaaggtga cggaagaggc gtcgtggaca ccggtgttgg ccagcagcac 4260
    ggcggtggcc agccatccgg tcagcaccgc gacggcgccg ccgatgacgg ccagcacgcg 4320
    ctgtccgccg ttgcccggcc gcgtgaagta gacgagcgcg ccgaagagga caccgtcgcc 4380
    cagcagcact ccccacgcga cgccgaggaa ggagccctgc cccgagaagc cgtcggcatt 4440
    ccctcccggc ccgatgtgcg aggcgatccg cccgggcagc cggtcccgca ccgagaggaa 4500
    cacccacagg acgacggcag cgcagaccag aggcggcacg acggagacgg caaggcgacg 4560
    gacgaggacg gacgaggacc gtgacagcgg catgagagca aacctccact tgtttgcaca 4620
    ctagtagaac aagtggaggt gaactcggcg aaggcggctg cctcttcctg acgcgttccg 4680
    aacgccaccg gagccgccac gactgaccca gtgtcacccc gcgggaggcg gaacgcttca 4740
    gtccgtgccg ggagcggcgg ccgcttcccg tggtgcgact ggtggtgtct gcgtggggcg 4800
    gcgcatgagc ggcaggcgga gccgccggat gcccggcccc gagggaggtg ccagtctgcg 4860
    cgtggacctg tggaagacga gcaggctgac ggcggcggct ccggcgaacc aggccagttg 4920
    gacggcgagg tagccggaga cgggagcggt cgagaagccg gccgcggcac cggtctgcag 4980
    cgagccgtag gaggggagga gcgtcacgag gccgccgttg gcggcgccgc cggtggtgac 5040
    ggggttctgc aggcccgcgt cgagaccgct ggtcatcacg atggcgaaca tcccctcgac 5100
    gtcccggcgc agcagggagc cgaagacgat gccgatggcg ccgtaggtca gggacgcgca 5160
    gaacagggcg gcgacgaaga ggaccggccg gcggggcgac cagaaggcgt aggtgatggc 5220
    ggtggcgtag acggcgacgg tggcggagat gagggtgagg gcggtgagct tggcgagaag 5280
    gaggtggacg cgccggtagc ccgccatgga caggcgtcgg tcgaaggggc cgctggtgaa 5340
    ggtcgcggcg aacatcatga agccgacgat cagggtgatg gagttcagcg ccccgacgat 5400
    cgacgtgagt tcgttgcccc gcgggtggag gatctgcccg gtctcgtgca gcctgaaggg 5460
    gatgggcggg tcttcgatga cgcagtaggc cagcgtgatc cacaccggga tgaacgcggc 5520
    gatcaggccc atggcgagcc ggttgcgcag gtgctcgacc aaggcgaacc gggtggcggt 5580
    gacgtagagg gttgtgtggt tcataccggt gccgtcgtcc ttgagtgcag cagtccgccg 5640
    tcgaggtgcc ggagttcgtc gagccgttcg gcgtcgtagg ccaggtggga gacgaccagc 5700
    acggaccgcc cgcgttcgcg cagaccggcg gccaggctcc agaaccgctg gtgggtgtcc 5760
    cagtcgaagc cctggtaggg ctcgtccagg aggagcaggt cagggtcgtg catcagggcc 5820
    agtgtgaggt tgagcttctg tttcgtgccg ccgctgagca cgctgacccg ctcgtccccg 5880
    tagtcggaca gccggagcac gtccatgatt ctctcggcat ggctgagggt ggcgaggccg 5940
    tacgccaccc ggaaatactc caggtgctgg cggacggtga gagcctggtt caggacgaga 6000
    tgctgcgggc agtaaccgaa ccggccgccg tagtggacct gcccgcgctg cggccgcagc 6060
    tcaccggcga ggatcttcag gagcgtcgac ttccccgcgc cgttctcgcc cacgacgccg 6120
    gccagcgttc cggggcgcag ggacaagtcg atgccgcgca gcaccctgcg ggtgccgtag 6180
    gtgtggtgga cgtccctgac gtccaggaga ttccggggca cggcttcctc tcctcaggcg 6240
    acctggtcgc gccggacgac cgagagggcc agctcgtaca acaagtggct gtggcctgcc 6300
    gacggcagca ggggctcgag cttgttccac gcgtcgctca ccaactggtc ggcttcctcc 6360
    aggcaggctc tcaccgctcc gcaggcgttc aggtcccggc acacctcggc caccgccgtc 6420
    gcgctgcccg agccgtcctt gacctgctgc cagagctggt tcagccgggc tctgcggcag 6480
    ccggaccacg gcgtgggcca gtggcatggt gaccttgccg ctccgcaggt cctcggtggc 6540
    ctgcttcgtc ggtgcccccg cccgtgtgac accgctcagg tcggcgacgt cgtcggcgat 6600
    ctggtaggcg gtgcccaccg ctgaaccgaa agcccccagt gctctccgca gttccggctc 6660
    ggcacccgtg acgacccctg ctgcctccat ggccgccgag accggggccc cggacttcaa 6720
    ccggtgtgtc agacggacca gttccagcac agtgtgccgg tcgtcgccgg ccacggcctg 6780
    gtccatctct tcccggtgac cttggagatc cagtgcctga ccggcgtgag ccgctcgcag 6840
    cgcggccaga cccagtgccc gcaactcccc gcaccgcgag gcgtcgtcgg gaaaggtgag 6900
    ctgaacggcc cgctcccaga ggaaataggc ggccgtaccc gcgttcaccg cagtcggcat 6960
    gccgaacatg gtgtgcacgg ccggttgtcc gcggcgcagc ggtgaggcgt cctggacgtc 7020
    gtcgacgatg agggatccgg tgtggagcag ctcactcgcc gcgatcagca ggccgcagga 7080
    ttcgctgtca cctcccatca gaccgatggc ctcccaggcc agcaccggcc gccagcgctg 7140
    tcctccggca tcggtcagat ggcggacggg agaggtcagg gcccggtgca gccgctgggc 7200
    gacgaccggc ggcgtgccag gcccggtcgt cccggtgatg tggcccggtg gcagccacgt 7260
    ggagcaagca tctgccgacg cgttcgggca caggcggtcg atgtgatggt tgatgcgttc 7320
    ggccgtgcgg tcgatgcgct gccggatgaa gtccgcgttc gccgcgaaag tcggggagat 7380
    gtccctggga cagaggaggg cgccggtcat ggagtggtca gctttcggtc aggggcgggc 7440
    gatggacgag gctccctcat ggggtcgccg gcccgatgcc gcggagggac tgctcgggca 7500
    cggctcgcgg agtgcggcga tcatggtcag gccgcgtggc atcgcggcga gttcgaagcc 7560
    ggagcgcacc gtccggcccg gacgcgccgt gcgtacggtc cgcgtggcga ccagagtggc 7620
    gagcgcgacg ggcaccatga cgtcggccac cgcggcacca gggcagtagc ggggcccgag 7680
    cgcataggga agccaggcag caggggagac cgagggctga gcgtccggca tccagcgcgt 7740
    cggatcgaag acatcaggcc gttcgaagtg ggcggggtca cggctcatcg ccccgaggtg 7800
    gaggaacacc gtcgccttcg cgggcagccg gtggccgccc agccacgtct cgcggcgcgt 7860
    gcaacgcaca aggaccggga ggccgtgaag ccggatgact tccttgacga aggcggctgt 7920
    ccggggcaac cggtttattc cgacgccggc ctgcgagtgc cccaggccga gtgccgcgtc 7980
    ggcctcctcc tgcaaggcct gttgatggtg ggggtggcgg cccagttcgt agcaggccca 8040
    cgcgagtgtc gaggcggtgg cctccccgcc ggtgaacagc agcgtgcgca cgtcctgcac 8100
    cgccgcttcc gagggctcgc gccacgcttg cttcagcagg gagaccacat cacacccgtc 8160
    ctcagccggc cgatggcgcg cgaggacttc ccgggtggcc tcgtccagca ccgcgagggc 8220
    acggcggagc gcgcgttgtc ttggcacggg tacccaaggc cacggggaca ggagaaaccg 8280
    caaggccccg acctgcgaca gcgtggcatg cgccgcggcc agcgcggtca gcgttccggg 8340
    cgagacctcg ctgcgcaaga cgcacctgac ggcgagatgg aaagtgagcc gggtcatctc 8400
    gtggctcatg tcgaccggac ggtccgcggg aagggtggcg agcagacttc gggtctcggt 8460
    ccgcacggac gccccgaggt ccgccggccg gggcacggcg aacgcgggcc tcatcaccgc 8520
    ccgccggtca cggtggaccg ttccctcggt gctcagcagt ccctctctta cgatcacccg 8580
    cacatggggt tccctgcccc agaacatgaa ggtgtcctcg tcgcgagcag cctgccggat 8640
    cagcgaggag tcattgagca ggtacccgac gaacgggccc gccttcaccc tgaccacagg 8700
    ccccgccttg ccgagccggg ccgcgatctc cctcagatcc atcaagaacc gcacggagcg 8760
    ggcatgcccg agcaaccgac tgcagtcagg agccatcggc acaacggagt ccgccttcga 8820
    atctccgttc atcaggcgtc ctcccgcccg catgtcaccc tctgtcctcc tgtgaacgac 8880
    caggagtgag gagtgtcacg cagagcatca cctgctgtat cggcagcaat gccgacccgc 8940
    accgacggct gggcggggcg accgggaacc gccttgcggc tacgccgtgc tcgcgtgcct 9000
    gaagcgcacc gtcacattca ccggtaccca cgacagaggc gggttcgcgg ccacctgtat 9060
    ggacgcccgc tgaatgggga cggagtgcag ggggtgctcg atggcggtgc cttcgcggct 9120
    cagcaggccc tgcctcgcgg tttgagcgca acgggcggca gttcgggagc aggcagtcgg 9180
    gccccctcgt agccgtgcac ctcgccgaag cgggagccgg tcgcccgagc cgtcgccgcc 9240
    gccggcccga ttcgccatcc ggatggaaga ggacaccgcg cagggaccgc cgcccacttc 9300
    accggaatcc tctccaccgg aaaatttatc cgcaaacctg tcacatcttc gacacatgaa 9360
    gcgtcagggc ggtgacggca gttgaagccg ttgcccgacg acgccgaagg agaccgtggg 9420
    acagctacgc acgtgcgggc cctggagcgg tcggccacgc cgacaggaat cggccttccg 9480
    ccgctgatcc ggcctcccag cgttcgcgaa cacctcttgc cacacctccc gcgcggcccc 9540
    cgcattcgag cggtcggctg acgacgccct gcgacgccgc gcacaccacc cacgcacctc 9600
    gccacgccga aggctgcccg aaaacaagaa gaccgaggaa agcacacatg aagatctctc 9660
    gaataggccg cgcgtcatcc atcgccgccc tggtgacaac cgcactcgct ttcacggcag 9720
    ttggcaccgt cgctcccacg gccgtcgccg actcccgcgc ggccgccgct tccgggacgc 9780
    agaatgacca cccgagctcg gggcagggca cctccacctc tgagctccgg cgcaagggcc 9840
    tggtcccgtc gagtctcgtg gccaagccca tcacccgcag cgagaccctc agacgcgccg 9900
    ccagctggtt cggcaagggt ctccactaca gcggggacaa cacctatcag ggctggcgca 9960
    cggactgctc cggcttcgtc tccatggcct ggggactgcc cggcccgggt gagaccaccg 10020
    attcgttcat tcccgggggc gtggcccacg aaatctccaa ggacgaactg aagcccggcg 10080
    acgcgctcaa caacaaggcg ctcggcaacg acggtcacgt cgtcctgttc gagaagtggg 10140
    ccgattcctc ccagtcctcc tactggggtt atgagttcag cagcagcggt ctgcaccacc 10200
    gtgtgatccc gtacgcctac ttctccaggt ccgagcagta ccgcccgatc cgcttcaaca 10260
    ccatcgtgga cgacgacacg gccgcagggc ccgccgagga caacgcccgg gtccagggtg 10320
    acttcgacgg cgacggccgc gacgacgtgg cggtgctcta cgactacggc aggaaggacg 10380
    accgcagtcg ctcggccctg tggacgttca acagcaacgg cagcggtttc aacagtccca 10440
    agcaggtgtg ggacagcggg acgtcggaga gctggaactg ggcctccagc aagttgacgg 10500
    tcggtgactt caacggcgac ggcaaggccg acatcggcgt cctctacaac atgggcgcga 10560
    ccgaggacgg ccgcaaccgc accaagctgt tcgtgttcac cagcaccggc agcggattcg 10620
    ccgccccggt caaggtctgg gacagcaacg acgaccccgt gaagagctgg aactggaacg 10680
    ccagcaagct caccgtcggc gacttcaacg gcgacggcaa ggccgacatc ggggtgctgt 10740
    acgactacgg caaggacgac gaccacaacc ggacagggct ctggacgttc accagcaccg 10800
    gcagcgggtt caacagcccg aagcaggtgt gggacagcaa caacgacccc gtgaagagct 10860
    ggaactggga agccagcaag cccgtctccg gggacttcaa cggcgacggc aaggccgaca 10920
    tcggcgtcct ctacgactac ggcaagaccg actccggcag ccgcaccgga ctctggacgt 10980
    tcaacggcaa tggcaacggg ttcaacagcc cgaagcaggt gtgggacagc aacaacgacc 11040
    ccgtgaagag ctggaactgg gaagccggca agcccgtttc cggcgacttc aacggcgacg 11100
    gcaagagcga catcggcgtc ctctacgaca tgggtcgcac cgaggacggc cgcaaccgca 11160
    ccaagctgtt caccttcacc ggcacggcga ccggtttcaa cagcccggtc aaggtgtggg 11220
    acagcaacga cgaccccgtg aagagctgga acgcgtccgc gagcaagccc gtcgcaggtg 11280
    acttcaacgg cgacggcaag gcggacatcg gcgtcctcta cgactacggc aagaccgact 11340
    ccggcaaccg cagcggactg tggaccttca ccagcaacgg cagcggcagc gacagcccca 11400
    agcttggctg ggacagcagc gcggaccccg tcaagagctg gaactggagc gcgagcaagc 11460
    tcggctgacc ggcttcgccc ctcctcacct caccgttcgg gagagtcacc gcacatgcga 11520
    accatacgaa tacgaagaac gaacggcgtg gccttcgccg ccgctgccgc cctgatggcc 11580
    ctcgtcgcct ccggcaccgc cacggtccag gccgcgccct cgcacgccgg accctccggc 11640
    accactccga tcacctaccg tggcctcacc ctcgacatac cctccgggtg gccggtcgtg 11700
    gacctggaga aagacccgca cacgtgtgtg cggttcgacc gccacacggt gtacttgggc 11760
    caccccggca ccgaacagtc ctgcccctcc catctggtcg cggacaagac ggacgccctg 11820
    atattggagc cgatcaccgg agcgggcggc caggacgcct cccacgcgct gcgcatccct 11880
    gccggggccc cgatgccgca cgagctgccg gtgacgtacg accacgagac gaaggtcgcc 11940
    gtcgaaggcg ccggagtcat ggtcacgtcc tcctacggca cgtccagtac aacggtcgcc 12000
    gccgtcctcg gctcggcccg cacggacgcg acagccaagc cgacccccct gcccggcaag 12060
    gcgggcaggg gcctcgcggc tccaccggtt gccgccgtcg cggccgacaa gggatacaca 12120
    gggctgggct tcgagtcctg caccgcccct tcgtccgccg cgatgaaggc atggaaggcc 12180
    tcgtcgccct acggggccgt cggcatctac atcggcggtc gcaagcgggg ctgtgcgcaa 12240
    ccgcagctca ccggcgactg ggtgcgtcag cagaccgccg acggctggca cctgctgcct 12300
    ctcttcgtgg acctccaggc cggcgacatc tctccggcca ccgcggacgc gcagggccgc 12360
    gagtccgcgg acgccgccgt ggccaaggcg gcggacctgg gcctgggccc cgggacggtc 12420
    atctacagcg acatggagca ctacgacagc cgctcgtacc gggcccgggt catcgactac 12480
    gtgtcggggt ggaccagccg cctccacgaa catggctacc gctccggtgt gtacgcgggt 12540
    gaaacgagcg gcatcccgga cctcgcctcg gtggccgacg acaccaacta cgcatcaccc 12600
    gacgtgctgt ggtcggcgaa ctggaacctc aaggccgatg tgtcggacgc gtcgatggga 12660
    cttccgggcc ccggctactg gcccaatggg cggcgcatcc accagtaccg cggccaggtg 12720
    aacgacacct acggcggtgt caccctcgcc atcgaccgcg actacgtcga tgtcgccgcg 12780
    gactcggccc tgcccgcacc cggcggagag gacggttcct cgcgcgtcaa gggcgacttc 12840
    gacggcgacg gccgcgacga cgtggccgtg ctgtacgact acggcaagga gggcggcgtc 12900
    agccggtccg cgctgtggac gttcgcgggg accggcagcg gcttcggcgc cccgaagaag 12960
    gtgtgggaca gcggatcgga cagctggagt tggtcggccg ccaagctgac ggccggcgat 13020
    ttcaacggag acggcaaggc cgacatcgcg gtcctgtacg acatgggtcg cactgaggac 13080
    ggccgcaacc gcaccaagtt gtacgagttc accagcaccg gcagcggatt caacagcccg 13140
    gtcaaggtct gggacagcaa cgacgacccc gtcaagagct ggaactgggc ctccagcaag 13200
    ctgaccgtcg gcgacttcga cggcgacggc aaggccgaca tcgcggttct gtacgactac 13260
    ggcagggacg gcgaccgcag ccgtacgggc ctgtggacct tcaccagcac cggtgccgcc 13320
    ttcaccggcc ccaagctggt gtgggacagc aacaacgacc cggtcaagag ctggaactgg 13380
    aacgccagca agcccaccgt cggcgacttc aacggcgacg gcaaggccga catcggcgtc 13440
    ctctacgaca tgggtcgcac cgaggacggc cgcaaccgca ccaagctgtt caccttcacc 13500
    ggcacggcga ccggtttcaa cagcccggtc aaggtgtggg acagcaacga cgaccccgtg 13560
    aagagctgga actgggacgc cgtcaaggta gtgggaggcg acttcaacgg cgacggcaag 13620
    agcgacatcg gggtgttgta cgactacggc aaggacggcg accgcagccg caccggactg 13680
    tggaccttca ccagcaacgg cagcgggttc aacagcccga agcaggtgtg ggacagcagc 13740
    aacgacccgg tgaagagctg gaactgggcc gcgagcaagc cggtcgcagg ggacttcaac 13800
    ggcgacggaa aaacggatat cggcgtgctc tacgactacg gcaggaccga ttccggcaat 13860
    cgcaccggac tgtggacctt caccagcgac ggcaccggat tcggtacacc cctcctgggc 13920
    tgggacagcg tgacggatgc cgtgaagagc tggaactggc gtgccagcaa ggtgagttga 13980
    cacccctcct gtgagacatg gggcactcct cgacgcccgt ccggcccggc tgcggcccgg 14040
    ccggacgggc ccgtcattca atggaaggaa gaagtggatc ccttgacgcg caagacccgc 14100
    accccccgca agaagggcag acgcgcgagc gcggcggcga tgtcggcctc cggcatgctg 14160
    ctcgccttgg tggccaccgc cgcccccgtc cccgcccagg cggcatcact cgccacctgg 14220
    gaaaagatgg cccagtgcga gagcagcggg gactggggat acaaccagcc accgtactac 14280
    ggcggcctgc aattcctgga gagtacgtgg gtggcgtacc acggaacgga ctatgcgcca 14340
    tacccctatc aggccaccaa ggaacagcag atccgggtcg cgcagcggct cctcgacaat 14400
    gagggcgcgg ctccctggcc gtactgcgga aagaaggtgg ggctggctga cgacgacgca 14460
    cgccccttcc ccgacgcgcc ggacgacgac gcctccgccc ggatcaacgg tgacttcgac 14520
    ggcgacggat gcgacgacgt ggccgtgctc tatgactacg gcaaggaggg cggcgtcagc 14580
    cggtccgggc tgtggacgtt ctccgggagc ggtaccggcc tcggcagccc gaagaaggtg 14640
    tgggacagcg gatcggccag ctggagttgg tcggccgcca aactggccgt cggcgatttc 14700
    aacggcgacg gcaaggccga catcgcggtc ctgtacgaca tgggccgcac tgaggacggc 14760
    cgcaaccgca ccaagttgta cgagttcacc agcaccggca gcggattcaa cagcccggtc 14820
    aaggtctggg acagcaacga cgaccccgtc aagagctgga actggaacgc cggcaagctc 14880
    accgtcggcg acttcaacgg tgacggcaag accgacatcg gcgtcctcta cgactccggc 14940
    aagaccgact ccggcaaccg caccggactg tggaccttca ccagcaacgg cactggattc 15000
    aacagcccga aacaggtgtg ggacagcaag agcgacccgg tgaaaagctg gaactgggcc 15060
    gcgagcaagc cggtcgcggg cgatttcaac ggtgacggca agaccgatat cggggtgctt 15120
    tacgactacg gcaaagatgg cgaccgcagc cgcaccggac tgtggacctt caccagcacg 15180
    ggcagcggat tcaacagccc caagcagacc tgggacagcg ggtcggaaag ctggagatgg 15240
    tcggcggcca aggtggtcgg cggcgacttc aacggtgacg gcaaggccga catcggggtg 15300
    ctgtacgacc tcggcaggaa cggcgaccgc aaccgcaccg aactgttcac gttcgcgggc 15360
    aacggcaccg gcctcaacac accggccaag gtgtgggaca gccaggacga cagcgcggtg 15420
    aagagctgga actgggccgc gagcaagccg gtcgcaggtg acttcaacgg cgacggaaag 15480
    acggatatcg gcgtcctcta cgactacggc cagaccgact ccggcaaccg caccgggctg 15540
    tggaccttca ccagcgacgg cagtggattc gccggcccca agctcacctg ggacagccgg 15600
    accgaccccg tcaagagctg gaactggaac atgagcaaga ccggctgagc cattcatgcc 15660
    gtacagaaga gaagaggaag gatgaaatac cgaccgggaa cactgctcac ttccataaca 15720
    gtcttgtgtg ccctgctcgt tccggtgcgt tcggcggctc aggcggccag gcccgagcag 15780
    ggacgttccg tggtggccgc ggccgccgta ctggagcaaa gtccgccgac gctgctcgcc 15840
    gagccggaaa tgcgcgtcgt ctcctggaac atctgcggtg aggcgggcgg ggtgcgcggg 15900
    gagggcggct actgccccta ccgcaacgat ccccaggcga aagtcgacca gatcgcgcag 15960
    gtggtcgcgg agcgcagtgc caatgtcgtc atgctccagg aagtgtgcgg cgaggcgccc 16020
    ggcagccata tggagcggct gcgcgcggcc ctgggcagcg gatggtcgat cgcgcacgcc 16080
    ccgggggccc gcccggacga cggaaccacg aactgccggg gcgggctcag cggcatattg 16140
    ggcgtgggga tcgcggtgaa ggggcgcgtc accgacacca ccgcgacgaa caccgtgccc 16200
    gggggcggcg gtgacaagca gaccctgccc atcctctgtg tacgtgtcga gggctggtcg 16260
    tccaggatct gcaccaccca catcctgtcc gaccctgccg atccgcgcag gccggggcag 16320
    atccagaacg tcaagaacga gatctggccg gaccgctatc agctggtgct cggcggcgac 16380
    ttcaacatgt tccccgactc cgccgggctc aagccgatct cggacgaatt cgacgagtgc 16440
    gaccgccgct cctacggcgc cggtgacatg gtcaacgagg tcacccatca ctcctgggag 16500
    aaaaagggcg gacacatatg gcgcaagcgt gaccacatct tcgcctcgtg gggagagtcc 16560
    gggagccagt tcacatcctg cgaggtcgac cggacccgga tggacaccac cgagaacgcg 16620
    cccgaaagcg gtccgcccaa cgggtattcg gaccatgcgc cgatcatcgg ctacctcaag 16680
    ccgccgcggc acctgagcac gtccggggac ttcgacggcg acggcaaggc cgacctcgcg 16740
    gtcctctacg ggcaggggaa gaccccggac ggccacaacc ggtccagcct gtggatctca 16800
    ggcggttccg gtaccggagc ggagaccgga ttcgccgcgc cgcgcgaggt ctgggacagc 16860
    ggtgccgaca gctggaactg gtccgcgagc gcgctgacct ccggggactt cgacggcgac 16920
    ggcaagaccg acatcggcgt cctctacaac tacggcaggg acggcgaccg caaccgcacc 16980
    gcgctgtgga ccttcaaggg gacatcgaac ggcttcgagg cgccccgcaa ggtgtgggac 17040
    agccacgacg acacggccgt tcccagctgg aactggtcca cgagcaagct cgtcgcgggc 17100
    gatttcaacg gcgacggcaa agcggacatc ggcgtcctgt acgactacgg caggaccgcc 17160
    tccggcaacc gcaccggact gtggaccttc accagcaccg gcaccggatt cggcaagccc 17220
    cacctggcgt gggacagctc caccgacccg gtgaagagct ggaactgggc cgcgagcaag 17280
    ccggtcgcag gtgacttcaa cggcgatggc aagaccgaca tcggcgtcct ctacgactac 17340
    ggcaaccaca ccgccctatg gaccttcacc agcaacggca ccggattcgc cggccccaag 17400
    caggcctggg acagcggacc ggagaactgg aactggtccg ccgccaagcc ggtcgccggg 17460
    gacttcgacg gcgacggcag gaccgacatc gcggtcctgt acgactacgg caggaccgcc 17520
    tccggcaacc gcaccggact gtggaccttc accggcaccg gcaccggatt cggcaagccc 17580
    cacctggcgt gggacagctc caccgacccg gtgaagagct ggaactgggc cgcgagcgag 17640
    ccggtcgctg gtgacttcaa cggggacggc agggccgacc tcgcggtgat gtacgactac 17700
    gggaacgcga ccaacggccg caaccgcacc gcgctgtggt ccttcaccag ccgcggcacg 17760
    gacttcgccg ccccgcgggc gaactgggac agcagcaacg ccgctgacca gctgaaatcg 17820
    ggcgagctga gggcggctcc gctcagcggg tcctagttct ccatgatcgg tccgtcgccc 17880
    tccagaccgg ccgctctccc ggtcagcgtc gcggccagtg cgtcagcgtc gcgaccgagt 17940
    ccgtaacagc gcatcccggc gatcgcgaag tacgcctggt cgagccagac gcgggccgcg 18000
    ccagtgctgc cgcgcggcga agtacggcga gctc 18034
    <210> SEQ ID NO 76
    <211> LENGTH: 53500
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 76
    gtccgggccg gcggcaccgg ccgagccact gctcgtacgg cagtcctgag ggctcacggc 60
    cacccgaaga acgcgcggta gcacggaacc tgctgctcca gcatatggat gccgtggtgc 120
    acacggcgcc cggcggtggc ggccgcgctc agcagcgccg tctcgtgcgg cttcatgacg 180
    acgtcgacca ccacggcatc cggtcgcacc ctcgcggggt cgaagggcag cgggtcctcg 240
    gaacgcatgc ccagaggcgt cgcgttgacg gcgaaatcgg ccgcctccag atcgccgggc 300
    cccagcgccc ggatcccgtc cggccggcgg gacccgagcc gcagcagcag cgcgtcgagc 360
    tgggcgcggt cggtgtcgtg cacggacacc cgcgcggcgt cggccatcag cagcgccgtg 420
    gcgatcgcgc tgcccgcccc tccggcgccg accagtgcca catgcctgtc gcgcaccgtg 480
    tgcccggccg cctgaagacc ctggacgaac ccgagcccgt cgaagttctc ggcgtaccag 540
    cggccgtcgg gttcgcgccg catcgcgttg gccgtcccga tgagggcggc cgccggcccg 600
    agcccgtccg cgagcccgca cagggccgcc ttgtgcggca cggtgaccag cagaccgtcc 660
    agattgccga tccgcttgag cccctcgacc acctcggcga gatcccgcgc ccggacgtgc 720
    accggcacca ccacggcgtc cagaccgctt tcgctcagca gggggttgag cagaccgggc 780
    gccttgacct gggcgacggg atcacccagc accgcgtaca gccgcgtggc gcccgagaca 840
    ccggccgccg gcccgaggaa ttccatcagc cgatcctctc tgtacccccg acggatgttg 900
    ccctacggtg ctggagatgc tccacagctt tgccgtgacc gccggtcggc acaaccctgc 960
    gtgcccctga cgcgccaggc cctccaggta gttgctcccg gcggatcccg acagctcccg 1020
    accggtcccg acggagggaa gaagccatca gatacctggg aatcgacgtc ggaggcacga 1080
    aggtcgccct gcgggtgacg ggggacaccg acggtgcggg cggcggcgac gtgacgttcc 1140
    gctggcccgc cgccggcgac gtcaccgcgg atctggacct gctcgccgcg cgggtccgcg 1200
    gtcttctggg acaccgcgag gaccccctcg ccggggtcgg cgtggccatg cccgcgatct 1260
    gcgacgcggc cgggacggtc cgcacgtggc cgggacggcc gagctgggcg ggcctgaacc 1320
    tgacggccgc cttcgggcag ttgctgcccg gcaccccggt cgcctgcgcc gacgacggtg 1380
    acctggccgc gctggcggag tcccgcgccg ccggctgccg gcatctgctg tacgtggggg 1440
    tcggcacggg catcggcggc ggcatcgtcc atgagggccg cgcctggccg ggccccggac 1500
    gcggctcgtg cgaggtcggc catgtcgtcg tcgaccgctc gggcccacgc tgcgactgcg 1560
    ggcgcgccgg ctgcgtccag gcggtcgcgt cgggaccggc gaccctccgg cgggccgccg 1620
    aacggcgcgg ccgggagacc ggcttcgacg aactggcctc cggggcgcgc ttgcacgccc 1680
    cgtgggcgga agcggccgtc gacgagagcg ccgcggccct ggccaccgcc gtgaccggca 1740
    tctgcgagct ggcccacccc gaactcgtcc tcgtcggcgg cgggttcgcg gcgggcgtgc 1800
    cgggatacgt ggcctcggtg gcggcgcacg tcgagcggct gacccgcccg ggaacggatc 1860
    ccgtgcgggt gcgcccggcg gtgctcggcg ggcggtcctc cctgcacggc gcactgctgc 1920
    tcgcgcggga ggcacacggg cggggaaacc ggccgccgga gagtgaccgt gtttcttccg 1980
    atgtttcttc cgatgtttct ttcgggggag tgacagacag ggccgttggc cggtccgact 2040
    gagcacaatc acaggtgatt tcgcccaggt tcaccacgcc tcgtgtgctc ggggtcggca 2100
    gaaggagtca gagtcatgct cgacaggcgg agcgtcattc gcgtcggcgc cggggtggcg 2160
    gcggccgccg ccgtggccgg tacggccgcc accggtgcgg cggccgtggg gctgccgggt 2220
    gtacggggac gcgcggcgtc gcgcggggtc gactgggcct ccttacgccg tcatctgtcg 2280
    ggcgagctcg tcctgccggc ggacaccgga tacgagcggg ccaggaagct ctacagcggc 2340
    cagttcgacg gcatccgccc gcaggccgtc gcctactgcc ggaccgagga ggacgtgcgg 2400
    acgaccctcg cgttcgccca ggaccacgcg ctgcccctca ccccgcgcag tggcgggcac 2460
    agcttcggcg gctactccac gaccgacgga atcgtcctgg acgtctccgg cttccacgcg 2520
    gtgagcctca cccggaacac cgtcgtcatg ggcgcgggca cccagcaggt ggacgccctc 2580
    accgccctgt cgccgcgcgg tgtcgccgtg gcgagcggca actgcgcggg cgtctgtccc 2640
    ggcggcttcg tccagggcgg cggactgggc tggcagagcc gcaagttcgg catggcgtgc 2700
    gaccggctcg tctccgcccg ggtcgtgctc gccgacggcc gcgccgtgac cgcctccgcc 2760
    accgaacacc ccgacctttt ctgggcgatg cgcggcggag gcggcggcaa cttcggcgtc 2820
    gtcaccggct tcgagctgcg ccccaccgac gtcccctccg tcgtcagcta caacctcacc 2880
    tggccgtggg agtcggcgcg gcgcgtcatc gaggcgtggc agcactggat catcgacggc 2940
    ccccgcgacc tcggtgccgc gatggccgtg cagtggcccg acgccgggac cggcacgccg 3000
    gtcgtggtcg tcaccggcgc ctggctgggc gcggccgacg cgctcacccc cgtgctggac 3060
    tccctggtgg cctccgtggg cagcgcgccc gccacccgct cggccaaggc gctctcccag 3120
    cacgacgcga tgatggcgca gtacggctgc gccgacctca cgcccgagca gtgccacacg 3180
    gtcggctact cgcccgaggc cgcgctgccc cggcagaact tctccatgga ccgcaaccgg 3240
    ctcttctccc gggccatcgg gcaaggaggc gtcgagcgga tcctggaggc gttcgccgcc 3300
    gacccgcgcg ccggacagtt ccgcttcctg agcttcttcg ccctcggcgg cgccgccaac 3360
    cgccccgacc gcaccaccac cgcctacgtt caccgcgaca ccgagttcta cctcggtttc 3420
    tcgatcgggc tgaacgaccc ggagtacacg gcggaggacg agaggctcgg ccgcgcctgg 3480
    gccgcgcgag gactgcgcac gctcgatccc cactccaacg gcgagagcta ccagaacttc 3540
    atcgacccgg agctcgacga ctggaagtcg gcctactacg ccgagaacta cgtgcgcctg 3600
    gccgccgtca aggcggccta cgacccgcac cggctcttct ccttcgcgca ggccgtctga 3660
    cctctcccga aagacccctg ccggcctgct cccctccgcg gctcctgtgg gcactggtgc 3720
    gcccgcgcac ttctgtgtga ttgagtgaag tccgggcgtg cagagctcag ttgccgtgga 3780
    gggggcgcca gttgcgagca tcagcggtgg agagggtgga gctgatccgc tggccggtgg 3840
    agtccgagcg gcgggagcgc tgccgcgacc ggggcgtcat gcggatcctg gtgctggagg 3900
    cgggggccga ggcacccttg tgcgtggacc ccaaggagga ctgggtccgc gctcccgtca 3960
    gcaccgacga cctgcgggcc cgcgtcgagg ccctgcgcct tcggggagcc gccgccgagt 4020
    cccggcccga ggtcgacccg aacggagtgc tgcgtttccg gtggcgctcc gccctgctct 4080
    cgcccaccga ggcccggctc gtcgcccggc tcgccgagtc ctatgccgag gtcgtcgccc 4140
    gcgacgacct gctccgcccg cccccgggcc gtaccgtgcc gagccgtaac gcgctcgacc 4200
    tccacatcat gcggatccga cggcgcctcg ccgcgctggg cctgagggtg cgcaccgtcc 4260
    gggggcgtgg ctacgtcctg gagagcgcgg aaggagtctg accgacgggc gtggccgcgc 4320
    accgcaccga ccgcccctac gagcgaggag cccgaagtgc agcagcctca tcacagccgc 4380
    gtcgacgtgg aactgggcga gaggtcctac cccgtccacg tcggaccggg ggtccgccac 4440
    ctcctgcccg gcatcgtcgc ctccctcggc gcgcaccgcg ccgccgtcgt gaccgcacgg 4500
    ccccccgacc tggtgcccga tcccggcgtg cccgcgctga tcgtgcgggc acgtgacggc 4560
    gagcggcaca agacgctcgc caccgtcgag gacctgtgcc gcaagttcac caccttcggc 4620
    atcacgcgcc acgacgtcgt cgtctcctgc ggaggaggct cgacgaccga caccgtcggc 4680
    ctggcggcgg cgctgcacca ccgtggggtg ccggtggtgc acctgccgac caccctcctg 4740
    gcccaggtgg acgcgagcgt cggcggcaag acggcggtca acctgcccga gggcaagaac 4800
    ctcgtcggcg cctactggca gcccaaggcc gtgctgtgcg acaccacgta tctccagacg 4860
    ctgcccgccg aggagtgggt caacggctac ggcgagatag cgcgctgcca cttcatcggt 4920
    gccggcgacc tccgcggcct cgccgtccac gaccaggtca ccgcgagcct gcggctgaag 4980
    gcgtccgtcg tcgcggccga cgagcgggac accggcctgc ggcacatcct caactacggc 5040
    catacgctgg gccacgcact ggagaccgcc accggcttcg ggctgcggca cggactcggc 5100
    gtggcgatcg ggacggtctt cgcgggccgg ctcgcggagg cgctgggccg catcggcgcc 5160
    gaccgcgcgc gggagcacac cgaggtcgtc cgccactacg gacttcccga cagcctcccg 5220
    ggaaacaccg acatcaccga gctcgtcgcg ctgatgaggc acgacaagaa ggccacgtcg 5280
    ggactgacct tcgtgctcga cgggccttcc ggcgtggagc tggtgtccgg gatcccggag 5340
    gacgtcgtcc tgcgtacgct cgcggcgatg ccgcgaggaa cggcctgacc gagtgttccg 5400
    tcttccgagg ggaagtgacc gtttcgtgtc ggcagagctg tcagaaccgc tgaagaaggc 5460
    cctggactcc ctggtgttcg gcgtcgtggc gacgaccgac cccgacggcc gcccgcacca 5520
    gtcggtggtg tgggtccggc gcgagggctc cgacgtgctg ttctcgatca cgcgcggcag 5580
    ccgcaaggag aggaacatcc tgcgcgaccc gcgtgtgagc gtgctgatca gcccggcgga 5640
    ctcgccgtac acctacgccg cgatccgggg caccgcgcac ttcgaggacg tgccggaccc 5700
    gggcgcgtac ctcgacacgt tctccataaa gtaccacggc gtgccctacc gggagtcgtt 5760
    ccccgagccg ccggaggtga gcaccattct cgccgtccgg ctcgttccga cgtcggtcta 5820
    cgagcagtgg tgagggcgta ggcgtcccga agccccggca gcgtcccgaa tgccgctgcc 5880
    ggggcttccc gtgggagccc tacgccggtt tccgcgcggt gaccaccgag tagccgacct 5940
    cctccaccga gcccatgcgg tcgatgccgt cgaccatgcg gtggaacgcc tcgtcgtcca 6000
    tgtgggagcc gagctcgtcc ctggccgcac gcatcttcgc cgccaccgcc tcgtaggagg 6060
    gccgcacctc gtccccgatg tcgaggaact ccaccacctc cagccccacc gaccgcatgc 6120
    agtcctcgta cgcctcgcgg gtgaggacgg ggccctgctg gaagttgtcg ttggcggtgt 6180
    cgacgatcgt cctggacgcg tcgctcaggg gccggcgcag cacgaagtcc gtcaccgtca 6240
    cccggccgcc cgggcgcagc acccgggcga tctcggtgaa gacgtcggcc cgttccaggg 6300
    cgtggcagat gctctcgatc gcgtaacagg cgtcgaacga gccgtcggga aaggggagcg 6360
    cgagcatgtc accgatgcgg aactcggtcg cctcgtcgcc ctccttctcg gcgagctgcc 6420
    gcgacagacc cacctggtag gggttgatgg agaccccggt ggcccgcacc ccgtgccggg 6480
    cggcgatgcg caaggtggcc ttgccgttgc ccgaccccac gtcgagcacc cgctccccgg 6540
    gggcgaggcg caggcgctcc gagacgtagt cggtcagccg gtcgcctgcc tcttccaccg 6600
    tcgtggggac gtcgggtccc gcccagtagc caccgtgcat gtagccgcct tccgcgtgca 6660
    ccatcaagtc ggtgacgcgg ttgtagagtt cgaccatccg gtcggaggcg gacgcggttt 6720
    ccgtcatgcc gctcactttc ccgggcgctg ggcgaccagc agcagatagc cgaactcctt 6780
    gacgccgacg aggtcgccgg ggtcgaactg gttcaccatc tcctcgccga actgcgtctc 6840
    cagcctctgc ttcgaggagt tgatgcgctc cgagagcagc ctgaaggtct tctccagggt 6900
    ctggtcgctg atgtcgagga actcctccag ccacaggccc gccccccgca gcaggggagg 6960
    gtacgcctcg gcgctgacca tggtcatcat gaagtcgtgg aggtagcgct ggacggcggc 7020
    ccgcccctcg ggggcgaggg gggcccgctc gaagaagtcg gtgagcacca gacggccccc 7080
    gggccgcagc acccggccga cctgggcgag cacctgggcg cggtcgggca tgtggatgat 7140
    cgattcgagg gcgatgacgg cgtcgaagct ctcgtcctcg aaggggaggt ccatcgcgtc 7200
    ggcccgctgg aagcgcgccc ggtcggcgag cccggcctcc tcggccagcg cgttggcccg 7260
    gacgacctgc tcatggctca ccgagatgcc cgtgacatgc gctccgctga gccgggcgat 7320
    gcgtacgccc ggggtcccca cgccgcagcc gaggtccagg acgcgggagc cggcgccgat 7380
    gcgcagccgc tcggccatca tgtcggtgag ccggtcggtg gcctcggcca gcggcacctg 7440
    gctgtcgggg gagtcccagt agccgaagtg caggttctcg ccgagggagg cggctcccag 7500
    cgcggtgaac cggtcgtaga gcgcgcccac ttcctcggag gcgggtgagg gcatggggag 7560
    ttcggacagc tcggagtgcg gcatggacga tccctctcgt gaaaggtcgg gggtgggtcg 7620
    ggcagtcggt gtcaggagag acggaggtcc tggtagacgg cgtcggcgag gcggacgatg 7680
    cgctcgcggt gccgttcgtg ctccaccttc ccgttgaggt ggagggcgac gacgagagcg 7740
    gcgtccgggt cggcgaagac cacggcggtc cacagcccgt cgtgcccgaa ggaccggggg 7800
    gaggcgtacg agccgaagct ggtgaaccgc ggatccagct gacggcattc gaggcggaac 7860
    cccatgcccc agtcggcgtt gccgtagcgg tcctggaggc cggtgcggtg ccgggccgtg 7920
    agggcggcga cggtgggcgg cgccaggacg cgcccgccgg gagcgtcccc gccgcgcagc 7980
    agcatctcga agagcctgcc catgtcccgc agcgggccac gggtgttgac ccccgggatg 8040
    cagcgtgtgg tggccgcctc cgtcgaccac cagtgggtgg gcagcgggcc gccctcgggg 8100
    ttgctcacat ggatcagcgg cagctcgccc ccgagcgcgg cgaactcctc gcgatccagg 8160
    tggacacggg tgccggacat gccgcacggc ccgaggatct cctcctggac gtacgcgcgg 8220
    tactccctgc cgtcgacgac cggaaggatg cgcgccagga cgaaccaggc ccaccactgg 8280
    ctgtagttga tgccgggcgt gccccccgga cgcggtgcca ccggcacctc gaaggcacgg 8340
    cgcacacgct cctcgtccgg gccggccacg atgccgtgca gcgggtcgtc gccggtgggc 8400
    agcgggcccg tatgcgtcag cagttccatc gaggtgatgg actccttgcc ccggttgccg 8460
    aactccggca gatagtgcgc gacgggcaga tacgggtcgt acgctcccgc ctcccacagc 8520
    cggcccaggg cgaccgacag cagtggcttg gcgcagcagt accacagggg cagcgaccgg 8580
    tgggtcatcg ccaccccggg gcgggccagc cccaacccgg cgtccgccag agggactccg 8640
    tcgcgggaga cgtagatctg cgcccccggg gtcgaggtgc cgacctcgcg ctccagctcc 8700
    cgcatggtgc cgggaagcgc cgcacgggcg cgccggacgg cctcggccgc gtcctcggtg 8760
    ccgggcggcg gggccgcttc ccgcgccgtg gtaccgggcg tgcccctgag cgcggccaca 8820
    tggcccgagg cgtcccgctc cagtgcggcg tcgatgaaga acagcaggct gaaattgccc 8880
    tcggcccgta cgtcgccccg ctcgaatacg ccggtccgat cccgcgcggg gccgagcagc 8940
    agggcgcggg cggcctccag gggaagccgg atccgcagtc ccggactctc accggccgcg 9000
    gccgcgaggc cgagccgccc ggcgacgagg tccacctgga cgtcgacggc cgggggttcg 9060
    ccgggcggtg ggtcggtgag ctccaggcgg agcgcgaccg tgccgcgttc gcccacgggc 9120
    gggcgttcga gcctcgcggc cccggcgagc caggccgtgg acaggaacgg cgcggtggac 9180
    agggtgttca gcacgggctc actcctccgc ttccttggcg gcccccggcg ccacctgggg 9240
    gacgacctcc tggaacagcc gctccaggct ggtggtggcc gggtagtcgc ggaaccacag 9300
    cacgaactcc gccacgcccg cggcgaccag gcgccgggcg cgctcggtga gctgttccgg 9360
    cgtcccgtac aggctgcgcc gcgccagcag gtcgggatgg tggctccaga acagcacctc 9420
    gtgcggggtg gagagccagc ggtcgcgttc caggacggag tcgaagatcc ggcactccgc 9480
    ggcccaggcg tgccggacgc cgtccggatc gagcccgagc tccgtgcgac ggcggcggaa 9540
    cgcggtgacg gccgcggcga cctgagcggg ctcaccggtc cactggacat gagcgcactc 9600
    ccgcacggtg gcgtcggcgg gccgcagcgc gccgctcccg gcgtccccgg ccggggtgcg 9660
    cagcgcgagg gggaggggct gctgccgtgg ggcgggcacg cagtgcgccg aagtgaggcg 9720
    gatgtgttcc ccggtgaagg tgacgggctg tccgccccac agcgcgcgca gggcctcgac 9780
    cgtctcgccg agagcccggt ggccggcggc ctcctcctcg tccgcctcca ggcccgtggg 9840
    cacctcgcgc cccgtcgagt ggtgctccgg caggtactcg cgggccggga agccgagggt 9900
    gagccggccg tcgcagacga cgtccagggt cgcggcccgc ttggcgatca gcgcggcgtt 9960
    gcggaacggc ggggccgagg agagcagtcc cagatccaga ccgggcaccg cgcccgcgag 10020
    ggccgccagc gccgtccagc cctcccagac cggctcgggc tcgcgccggg gcagggtgtc 10080
    ggtgcggtcc agcagccaga gggcgccacg gccgtgccgg tggacggtgc gggcggtctc 10140
    caggaggagc cgccagccct cgcggccgcc cgtaccggcc agttcgaggt tggtgccctg 10200
    cggggcgacg acgctccagc gcggggtcgc gggcgtcatg cgtggccgcc gctgcggacc 10260
    agcgcgagca ggctctgcgg cttgtccagc aggaagtcgg gccgctcggc gcgcagttgg 10320
    ccggccgcgc cctcgcccca ggtcgcgccc acggtggcgg tcccggcggc gcggccgctg 10380
    cggatgtcga tcaccgcgtc gccgaccatg accgcgtcct cgggcgccgc gtccagccgg 10440
    cgcagtgcct cccgcacgat gtcggggtgc ggcttgggcc tcggcacctc gtcgctgccc 10500
    accacctcgt ccagcagggg cagcagcccg accgcctcca gcacggcgcg cgcccgggag 10560
    ccggacttgc cggtggcgat cgcggtgccg acgccgtccg cgcgcagctc cgccagcaac 10620
    tgcggcacgt ccgggtacac ctcgacgcgg tccatcagcc ggtggctctc ccggacgaac 10680
    ggttcctcca tctcgccggg caggcccatc agccgcatga tgtccgggaa gtagcggccc 10740
    tggtgcgtgc ggtactcctc gaagggcggc tcgcccggcc ccaccacttc gcggtaggcc 10800
    acggcgaacg cctcgcgcat caccgcgaaa ctgtcgatga ggacgccgtc gaggtcgaag 10860
    accacggtgg tgaaccggca cgggagagcg gtggcgggcg cgtccgccgc ggcccgcgag 10920
    gcgggcgggg ggttcagggg catggcagga gctccgtggg tagggacgtc ggggcccaga 10980
    ccccggcgtg gctgacgact ttcgtgaagc ggcccgcgag ccggtggtag acggcccccg 11040
    gcgagctgtg cggcgggtgg gccgccgcgt ccagcccgcc ccaggcggcc atggccctca 11100
    gcagcagcgg atcggccggc acccagcggc cgcccaccag gaactcggcc cagcagtgcg 11160
    gtgtggagta cggcttggcc accagcagcc cgaaggagaa ccgcacgtcg agcccgcgcc 11220
    gccggccctc cgccaccagc caggccgcgg cgccgccgca gtcggccatg tgcgtgctcc 11280
    acaggaaccc cgggtcccag cggatcgctt cgggcagcag gaagaagccg accggctcca 11340
    gggtgcgcag caggtcgagg acggccgggg ggaagtccgg ccagtggccg cgcgaccgtg 11400
    tgtacagccg cgccagcgtg gtctcccgcg gcgaagccgc ctccaccggg cggcgggcgc 11460
    cgggcagcag cactccgtag cggcacgggc ccgcgtggcc gggcaccggg caggacgccg 11520
    tgacgtcgac gcgccagcgc gggctctccg cggccgaggc ggtccgcagg gaaccggccc 11580
    acgagcggat ggcccggcgc tgcaccgagg acaggccaag gtgcagggcc gcgttgccca 11640
    ggtcgtagcc gtcgaagagg cgtcccgcgc cgcttcccac gaagggcacg cccgcctcgc 11700
    acagcgcgga cagcagctcc ggtccgatcc ggtgcagccg gcgcgcgctc tgctcgtcga 11760
    cggtgaaacg gcggtgctcg tcgggcacga gcttcagacg ctccacgagc gcctggagct 11820
    cctgggtgga ggggggccgg gccggcaggt agacgctgat ctcctcaatc ctttcgccgg 11880
    ggcgggcagc cggtgcggga cggccggggc ggccgccggt ggggtgccgg gtcagtcgat 11940
    gtagcccaga gcctggaggc gggccgccac ctgctgctcg tcgctcgcgg tgtacgccgg 12000
    ctcctgcggg cgcggcgcgt agggggagaa ggcgcgcagt gccggggcca cctcgtgcgc 12060
    gaacagttcc atggactcga tgcggtgccg gtccgccagc tcgccgaagg tgaactcgcc 12120
    cagcagcgtg tcgaaatggc cctccaggga cgcggccctg atccggtcgg ccacggtcgc 12180
    cggcgagccg acgaagacga ggttgttctc cagcaggaag cgcaggtcgc cggagcccgc 12240
    caggatgtcc gtcgtgctga gcctgctggg tggggcgtgc tgatcgcggg aacgggacac 12300
    cgagccgtac cgcagcgtct tggtgaacgt ctcggtgatg tgcgcggccg cccgctcctc 12360
    cgcctcggcg tccgtccggg ccacgtggac cagcgtccag tagccgatgc gcgggtcgct 12420
    cgcgtggccg tggccgcgcc accagtcgag gtagtgccgg tagaccagcg ccatggaggc 12480
    gcgcggcacg atcatcgtgg aactggtgtg ggcaccggcc tcgctgagat agcgcaaggt 12540
    gttgcggttg gtggtgggca cccacatcgg cgggtgcggg cgctgcaccg gcgggaagga 12600
    cagggcgatg tcgcgcagcc ggtgcgcggg cccgtcgaag tcgaaggcgc cccgcgcggc 12660
    gaacgcggca cgcagcagct ccagggcctc ccgggtcagg aggtgacggt tgtcgaaatc 12720
    ggcgtcgaag ggcaggaagg ggtcacggct gacgcccgag gcgagcccca cctccagccg 12780
    cccgcccagg agctggtcca gggtcgcgac ctcctcgacc aggtgcagcg ggtggcgcag 12840
    cggcggcacc caccccatcg ggccgacccg gatgcgctcg gtgcgcagcg cggcgcccgt 12900
    gcagaagacc gcgggcgagg gcatccagct ctcgtgcggc gtgcagtggt gctccaccga 12960
    gaacgcgtag tcgaagccga gccggtcggc gtcctcgacc tcgcgccaca gctcttcgta 13020
    gcgctcgccc ggcgtgatac cgggacgtcc ccagacatgg gagaaataag cgaatttcat 13080
    tggcttcccc gggtgggcag gacacgtgga caactggacg tgctgggcgc cgcgtcgctc 13140
    tgcggccggt gcaggggcga gggagggtcg acgccggcgg ccgaatagat gtggtcgatg 13200
    caggaggcca ggatggtcac ttcggccatc gacccctcgc ccaggcccga caccgggccc 13260
    tgcccgtcgg ccgagccgcc gagcctgcgg gcgagttcgt ccacctgccg ccggtactcc 13320
    acgcccacgg gctcgtcggg cagcgcgacg gtctcctcca ccccctggcg caggaccacg 13380
    aggctcgact tctgtagccg gtgggggctg aagccgaagg tgccgcgcag cgtcgccacc 13440
    ccctcggtgc cctccacggt gatcgtggtg acgtccagcg cctggtggga ggcccagcgt 13500
    gtctccaggg agatgcccac gtcggtgtcg gtgacgagga agccgcgggc ggtgtcctcc 13560
    accgtctccc cggggcctgg ccgcgccgtg ccggaggagc gccggctcca gtcggccgtg 13620
    gcctcgcccc ggctcatcca gtccgcggac atcgtgctcg ccgcccggac cacgcgcggc 13680
    caccccagca ggtgcaggcc cacgtccagc aggtgccagc ccaggtcgag cagcgcgccg 13740
    cccccggcga gccggcggtc gacgaaccac ccggtgcgct gcgggatgcc ggtggcccgg 13800
    atccagctca gcccgacact gcgcacggtg cccagcgagg gcagcagctc cgccaggcgg 13860
    cagacatcgg tgcggtgccg ggcggcgctc caggcgtaga gggtgatgtc cccgatgctg 13920
    tcgccccgcg cctggtggtc cagggcgagc gcctgggcct cgaagagcgt gcggcacacc 13980
    ggcttctcga cgaacaccgg cacgtcccgc tccaggaggg ccttggccac ggggagatgg 14040
    aggtggttgg gcagggcgat gatggccgcg tccacgcttc tgggggcgag ctcttccggt 14100
    ctgctcagga cgcgggtccg cgcgccctcc ggcagggccg acctggccgc cacggggtcg 14160
    tcgtccacga ggaagtcgac ccggaacgcc gggtgttccg ccagcagcgg cagccacacc 14220
    ttgcgcgaga cccatcccgc gcccagcact gccatccgga ggggttcgcg agctgtcgtc 14280
    ggcggtgtca cgacgggtgc cttctccgtg aaagtcatca gaagcgggca ccaccgtcga 14340
    cgacgagcgt attgccgttg acgtacgagg ccgacggcga cagcaggaac gacaccgccg 14400
    ccgcgacctc ctccggctca ccgagccgcc ccgccccgat gaactgcagg accagatcgc 14460
    gctcggcctc cgagaagtcc atcacctcgg cgcggatcgc cccgggcgcg acgacgttga 14520
    cggtgatgcc gtgcttgccg acctcacccg ccaccgacgc cgcccacggc tggagcgccc 14580
    ccttggtggc ggcgtacgcg ctgtggccgg gcagcccgct ggtcccggcc cgggagccga 14640
    agagcacgat ccgcccgtac ctggcgcgca tcatcggctt caggcacgcc ttggcgagac 14700
    ccacggaacc ggccaggttg acccgcagca gcttctccag gctccgggcg tccgtggcca 14760
    tcgcgagccg gcgcgtacgc aagcccgcag cagccacaca gccgtccacc cgcccgaacc 14820
    gttcgacggc cgccgccacc agcgcgtcgg cgccctcggg ttcgctcagg tccgccgcca 14880
    cgggcacgag ggtgccgccc tggccctcca cctgctcccg cagcttgcgg atcgcctgct 14940
    cgccgctgtg gtagccggcg acgacggtgg cgccgagcgc ggcgagctcc agcgcgcacg 15000
    cgccgcctat ctgcccggac gcgcccgaga ccacgaccac gcggccgctc tgtcccaggc 15060
    gctccgtggc ccggtcggtg acggtgctca tgaaccggcc tccttggcga tgatcagatg 15120
    acagggggac gcgtccagcg gcacgtgccg tgcgacctcc gccccctctt gcagcagccc 15180
    ggcgatcagc tcgccggtgg ccggccacgc ggtcccgccg tgcgtgagcc agtccagagc 15240
    cagttcgctc cccggcccgg acgccggcag gaagacgtcg tcgaccagca gccgcccgcc 15300
    cggccgcagg gagccgagca gggcaccgag agcgctgccc ggccccgggc cgtgcacggc 15360
    gttggcgacc acgcagaagt cggcgtagcc gacgggcagt tccgtcccca cggtcaccct 15420
    gccctcctcg accgccgcgg cgacggccga ggagagcggc ccgctcagcc ggccgacggt 15480
    gaccagatgg ccgctcgccc cggggtccga ggcgagcagg cgttccagat agcggcccgg 15540
    gcccgcggtc acctccacca cccgggctcc cggcccgggc cgcaggagcc gcaggccgag 15600
    cgcggcccgg gcccgtgcgc cggggccgtc catcgcgccc tggtacaggg cgacgagcgg 15660
    accgaggctc tcggggggac gctcctcgaa gggacgccgc gccgtcccgc tccgggccac 15720
    cgcgacgagc tcctcgcgcg tgaccagccc acgggagagg tgctcctcca gcgccacgaa 15780
    cgcggccagc tccccggccc ggacccggtc cccgggctct tgcgccccgg tggtcagcac 15840
    ccccagagcg gtggccgtgc gcagcagcca ctccagggca tccgcgtcgc accccagctc 15900
    cccggcgagg agagccgtgc cggcaccctg ggcgagtgcc tccagggcgc ccaagtcgtg 15960
    cagcgcgaag agcacttcgg atgccttgta cgcccgggcg gcctccgccg cgctccgggt 16020
    gaggcggtag acggacgccg cccgcacctt gcccgcggcg ttgaccggca ggctctcccg 16080
    caggacgaac tcgtcgggca ccttgtgcgg ggccagctcc cggcgggcgt gctcgcgcag 16140
    cgcctcgggg gtgagccccg gccccgccgc cgagacctcc gcgacgatcc cgtcctcgcc 16200
    ccggtgccgc ccccgccggg cgcccacccg cacattcacc acgtccggat gaccgcgcag 16260
    cacctcctcg atctccagcg gggagaccca gcgcccgccc cggcggatcg cccggtcctc 16320
    gcgtcccagg atgcgcaggc ccccgggcac ggccacggcg agatcgccca tggcgtacgg 16380
    ccggccgtcg acccgtacgc tcagcagccc cggggtgccg gcgggcggca ccacgccctc 16440
    cgggccggtc agttcgcact ccacccccgg caggggagca ccggcgcaca agggctccag 16500
    ccccgccggt ccggcgagca cggcgcccgt ctccgtggaa ccgtagttgc gggcgagacc 16560
    ggtcccgaac gcctcggtga acgcgcggtc cagctgctcg tccaccggcc ccgcacccac 16620
    catggccagc cggagaccgg gagcggcggg cgcccgcccg gccgctgctc cccgcagccg 16680
    ccgggtcgcc agcagccggg ccacactggg caccagggcc accacggtcg caccaccgga 16740
    cagctccgcg gcgatgcggc cgagggcggt cggcggtacg gggcgcagcg cggcacccgt 16800
    cagcagtccg ccgaacagcc agcccagcgc gtacgcgtgg gacagcggca ccggcagcag 16860
    cagggtgtcc tctcccgtca gcccgacccc gtcgcggtag cggcggccct ccgcgagcag 16920
    gctctcctcg ctgcgggcga cgagcttgct cgcaccggtc gaccccgagg tcgggagcag 16980
    cacggcgggc ggggcgccgg agggttctcc gggcgagccg gtcaacgtca ggcggaggcc 17040
    gtcgccggtc ccggggacga ccagggacct gccgcccccg gcagcccgca gcagccgcgc 17100
    ggtctcgggg ccgggggtgt cgggttcgag cagcaggggc ctggcgccgg atgccagcag 17160
    ggagaggaag gcgacgaccc accgcgggct gttgggcgcg cgcagggcca ccgcctcgcc 17220
    ctccaccgcc tcggccttga gctgtgccgc ggccgtacgc acctgctcca gcagggagtt 17280
    cacatcggtc ccgggcagcg gtatccggcc ggacggcagc cgttccaccg cgcccaggag 17340
    ggtgtccgcc tcgtgaccgg tcgcctgttc agtcatggcc gccctggagg tagtcggcct 17400
    tcgcgtcgct gagcatctgc cggatgcggg gcggggagta ggtgggagcg gcgcacagct 17460
    cgtcgagggt gatctcgtcc tcgaagtaca tcgagagcgt gtcccagggc gtgaagcagc 17520
    cgtggcacgc cttgagccgg ggcttggggg cgagcagggc acggtagagg ccgctggtgc 17580
    ccaccgtgtc cagcatctcg ctccagtcgt cctccagggc gttgcccatg tcggagaacc 17640
    agatgttggg gcagggcgtg acgacgccgt cgctgaagct ggagacgacc agccgcggca 17700
    gatggcagcg gaaggtgcgg cggccctcgc ggtagaagct cgtcagccgg tcgaagtagg 17760
    gccgcggcgg gaggacccgc gcgaactcgt cgtagcggtc gacgagttcc tggatgtggc 17820
    cgaactgccc gggccgcacc ttgaagtcct ccgagtccgg gccccgcacc gggaagggga 17880
    agtagacggg aggccgggag aatcccgaca gccactcggc gaacgcgcag acctccgtga 17940
    cgctccggtc gttgagcact gaatagatct ccaccggcag ccccgagtcc aggatccggg 18000
    cgatggcggc gacgaccttc tcgtgcaggc tcccggacgg cacacgatgg ctgttgccgt 18060
    ggtggaggtg gctgtcgagg gagacctgga gcacgacgtt gccccacgag cggaaccgct 18120
    ccaggtgctc ctcgcgcacc aggacaccgt tggtctggat gaccagcacg tcgtatttac 18180
    gggcctcctg ctccaggaag tccatgatcc cccggaccag gaagatctcg cctccggtca 18240
    ccttgagcag cggcaggccg aagcggtccc ggatccggtc ggcgaccttg tccatgcgct 18300
    gccccagccc gctgtccttg gcgtagctgt cgcgccgcgg gggctcgaag atcagttgaa 18360
    gggagtggcc ctccttgagg ttgctctgtc cggtgaggca gtaggtgcag ctgaggttgc 18420
    aggagtcctc gttgatgacc aggtcgttgc cgatcagcgg caggcgtcgc ctggtgcccg 18480
    cgtcgggggt ggctgtcggc gggtgggtgc tacgggacat gagtggcctc tctcgtggtg 18540
    gggctgcgca cggcgtgggt cacggccccc tttccacggg tgccggccgg gcctcgcccc 18600
    ggtcgtcctg gccgacgggc acccagtggc ggtcggcgtg gccgggccgg ccgggcccgc 18660
    cgtgtgcgcg ggcgcgggcg aacagggcct cgcgcagggc ggccgcaccg ccgaagtggg 18720
    cgctcgcctg gctggagtag tgaccggcgg cggtcagcca gcggtccacc gcctcggacg 18780
    gcagccgttc cgtgcgcggc cgcaggttcg tgtggtcacg ttcgcgcagc cggtagggga 18840
    agtcctcgta gaagacggta cgggcggggg acaggggctc caccgcgccg cggaccaggc 18900
    ggtggtcgac gtgccggccc gccgccaggg gaacgtggac gctcgacgcc cccgcgcaca 18960
    gcggcagcag agccgcccgc acctcctcca gcagcgggag gtcggccggg tgccaggggc 19020
    cgaagagccg gcgcggggaa gcgtagagat aggcgcccga ggccgtacgc agtgccgcgt 19080
    cggtgaagcc cagcggcacg tggcgcacgc ccagttcggc acatgccgcc cggtcctcgg 19140
    cccggcgcac cgcgggatcg gcggcgctcc gccacgactc gggcttcccg gccgcgggcc 19200
    cggcgaagac cgtgacgacg gtcggccgcg ggccctcggc ggcccagcgc gccagccgcc 19260
    cgcccaggga ccacacggcg tcgtccgcgt gcggggagag caccacggga ccgtacggcg 19320
    cggtggccgg tgtgccgctc atcgcgccgc gtcccgtgcc ggtgcggcct cttcggccag 19380
    gacggcgcgc acatacgcct ccggcgtgcc gtcgggcccg agcagggcct ccaaggcgcg 19440
    taccgacggg tgcgggtggc cggcggagaa gtgcgtgagg ttgcggtggt gctgctccca 19500
    gcggccgggg cgggcgtggc tgaggtgcac accgagggcg tcgggggcga gggtcctgcg 19560
    cagcccggcg gcgtgcaggc ggaagccgaa ctccaggtcc tcgcaccccc aggtgagccc 19620
    gaactcctcg tcgaatccgc cggtatgctc ccatgcggcc ttgtcgaggg cggtgttcgc 19680
    gccgatgaag ccgagccagg gggcgacgtc cggcagggag ccgccggcca tggcctccac 19740
    ggcccgctcc agggcgttgg cgacgagccg ccggtgcggt tggcgccgct cggaggcggc 19800
    cggggcagcg ggttcgagtc cggcgcgggc gcggcggacc tcggtcgggg cggccttctc 19860
    gacagcggcg aggaaccgcg ccgcggtggg gagttcgcgc agccggccgt gggtgaaggc 19920
    gtccggttcc gcggccgcgg cgtgtgcggc gaggaagccg ggccccacca ggacgtcgtc 19980
    gtcgaggaag accagccggg gcgcgagggc cgcggccgcc ccggcgttcc gggcggcggc 20040
    ccgcccccgc agcggtcccc gcaccacgcg cagcgggaga aggccgctca tctcgcctgt 20100
    cacggcgatc agttgatcac cggcgtcgcc cccgtcgttg tcgtcgacga cgaccacttc 20160
    gaagggcggc gttcccgggg aggggccggc aaggcatgcg agggtcgcgc gcaggcgtgc 20220
    cgcgggcccc cggctgggga cgacgacgct cagccggggg gcggtggtgc cgttgggggc 20280
    ccgcatcggg tcagagcgcg ccgacgaggc cggagaagac ctccgccagc cggtccagcg 20340
    tggtcacgtc ggcgagcagg acgcgatggt gcagccagag gcagtcgctg ccgatctcct 20400
    ccgccacggg acagctcttg gccagctcct cggcgtccgc cggcgccggg ccgcgcgcga 20460
    aaccctcggt gcggtagacc ggcgggaagc cgacgaacgc gggcactccc cgctcgacca 20520
    gcgcgtccac cagcgcgagg cggcgccggg ccgagatgcc gggcagccgg accatggcca 20580
    tgtagtggga gtggaggtcg ccgcgctcgt cgcgcccctg cggcaccacg ccgtcgatgg 20640
    cggccagtgc cgtacgcagc tgggcccagc gctcctccct gatgcgcaac tgatccttca 20700
    agcgcttcag ttgagcacgc aggacgctcg cggagaactc gttcatgcgg tagttggagc 20760
    cctgcgtcag atggcggtag acgcggtccc cgggcgggcg gccgcagcag tgctggagga 20820
    acgcctcgtg gaaggactcg tcgtccggca ggagcagggc gccgccctcg ccggcggtca 20880
    tcagcttgcc gttctggaag ctgaaggcgg cgatcgagcc gagctccccg acccggcggc 20940
    cctgccactg cgcgccgtgg gcgtgcgcgg cgtcctggag gaccggcacg cccgtcgcga 21000
    cggagagctt ctccagggcg tccatgtcgg cgaactggcc cgccatgtgg accggcatga 21060
    tcgctttggt gcgtggcgtc accagcgccg ccgccgcgtc ggcatcgagg cagtaggtgt 21120
    cgggccgtac gtccgccggc accggcaccg cgcccatgcg ctgcacggcc agcgacgacg 21180
    agatgaaggt gaacgcgggg acgatgacct cgtcaccggg gccgatcccc atgaccccca 21240
    gggcgagttc cagggcgtgg gtgccgttcg tcgtggcgat cgcgtgcggg gcgccgtggt 21300
    ggtcggcgaa ctcccgctcg aagagatcga cctcctgccc cgcgtcgcgc caccacccct 21360
    tctggtccag ggcccgcagg agtccggcgc gctcctccgc gccgtgttgc ggccatgagg 21420
    gaaaggacag gacgtcatca ccggacgtag gtgtcattga gcagcctttc ggtcctgcgg 21480
    gtgcggcggc acggtcgact tcggggcgct gtacggcggg agggcgggtg tcgaggcctt 21540
    tgccttcggt ggccgtggct tcacggcccg gttccgttgt gttcgccccg cgtcgggaag 21600
    ggtggtgcgt gacccgacgg gaaacgccgt tcctcggggg cggcgggaaa tccggcccgc 21660
    ggtgtgaggg gtggccggag cgggcatgtg atccggcccc cggtcatagg ccgggatcgg 21720
    atgccagaca cgagcctcca tagggcagtt gccggagtca acacccttgc cgggaaggtc 21780
    tgccccgacc gccggtcggc ggtggattct cgtcagcagg gtggttgagc agtgaaactg 21840
    ccttattccc aagggaattg atccagttca ggggctgctc ggcgggcctg tcggcaacgt 21900
    tatccggcgt cgaagtggct caaaccgcac ggctggaggg agcgggaagc gtcgcgtatg 21960
    gtgggcgcga caccgtcctg gtatgtgctg tgtgcatggt tcattgagcc gaatcccact 22020
    ccggccctcg gatccgggcg ccatacgatc accgttgtcc ggtctgtgga cgcaccggtg 22080
    aggggctgtt acagtcctcg gatcatcgat gagcggcggc agtttctgcc tgcaatcgtg 22140
    atgagttctc agagctggag gcaatttcgt gccaccctct ccccgcgccc tcgtcatcgg 22200
    aatcgacgga ggcacattcg atacggtcga cccgctgatc gagtgcggtc tgctgcccca 22260
    tatggcgaag ttgctgcgcg agagcgccag tgccgccacg gactgcacct ggcccgccca 22320
    cacggcgccg gggtggagca cgttcgtctc cgccagcgat cccggcggtc acgggatcta 22380
    tcagttctac gacacccagg acccggccta cggggcccgc gtcacgcgct ccggcgacct 22440
    gggccggtcc tgcgcctggg actggctcgc cgcgcaggaa tattcgctgg gcctcatcaa 22500
    catcccgatg tcgcacccgc cggccgacct ccccggctat caggtcacct ggccgctgga 22560
    gcggacactc aagcactgcc gcccggattc cctgctgcgc gaactcgccg cggccaaggc 22620
    ccatttccag tcggacctcg cgaccatgtt ccggggcgac atggcctatc tggaggaggc 22680
    cgagcgcaat gtggcggcgc gggtccgctc cgtacggcat ctgatgagca cccggcccac 22740
    cgatgtcgtg atggtcgtgc tcaccgaggc cgaccgggtc ggccaccact actggcacta 22800
    cggcgacccc ggtcacccgg gccaccggcc cgccccggag ggcagcggct gggacgtcgc 22860
    catgccccgg atctaccagg ccatcgacca cgcggtgggc gagctcctgg agctcgtgga 22920
    cgaggacacc tccgtcgtgc tcgtctccga ccacggcctg ggcaccgggc gccacggcct 22980
    gtcggtgcac accctcctgg aggaggccgg gctgctggcc accgcaccgg gggaggagcc 23040
    gcaggacgcg gcggcgagct ggttcgcggg caacggccgg cacgtcgact tccgccgcac 23100
    cagcgtctac atgcccgtcc ccggcagcta cggcctcaac atcaacgtac gcggacgcca 23160
    gcagcgcggc accgtcgcac cccgcgaccg cgaacgcgtc atggacgagg tcacgggcct 23220
    gctctccggg ctgaccggcc ccgagggaca gcaggtcttc cgggccgtcc gcccgcgcga 23280
    agaggcgtac ccagggccgc acaccggccg ggcacccgac ctcctcctcg tcccgcggga 23340
    cgagaccgtc ctgcccgtcc ccgacctcgg cggtgacgtg tggcggccga gcgcgcagac 23400
    cggcctgcac cgctaccgcg gcctgtgggc gcaccgctcg ccccgcgtcc gccccggccg 23460
    cctgcccggc accgtcgcgc tcaccgacac cctgcccacc ctgctcaccg acctcggggc 23520
    cgcatggccc agcgacatcc acggccgccc cgtgaccgcc gtcctcgacg acggcgtacg 23580
    cgtcccgccc tccgaccccc gggtcgaggc caccggcacc ccggccacca cgatcccggc 23640
    cgccgcttcg gccgctgatg ccgccgagga cgcgtacacc agcgaccgct tgcgcgaaat 23700
    gggctacctg taagcaccgc cgggccgtac cggcgcttgt ccccaccgga gtcccgccgc 23760
    tcgcggcggc gtggaggaga gaggtatttc tgccatggag accctgacga ccgacaagat 23820
    caaggaccgg ctgcgcaagg tgctcgtcga ttccctcgaa ctgtccctgg acccctcggc 23880
    cgtacccgac gagggactcg tggagaagct gggcctggac tcgatcaaca ccatcgaatt 23940
    cctcatctgg gtcgagagcg aattcggcat agagatcgcc gacgaggacc tgtcgatcaa 24000
    gctcatcgac agtctcgacc tcctcgccgg ctatgtgtcc gagcgcgtga acggcgtcac 24060
    cgcacccgcc gaatgacggc cgtgcgcgcg ctcgcctccg ggcccactcc ccgcagcgga 24120
    aggacgtgag cacgatggac cggcacgccc tggtgatcgg gctcgacggc atgccgagga 24180
    ccctgctgac ccgcctggcc ggcgacggga ccatgccgca caccgcggcg ctgctcgccg 24240
    agggccactg cgcggaactg ctggcacccg taccggagat cagctccacc tcctgggcca 24300
    ccttcctcac cggcaccaac ccgggccggc acggcatcta cggcttcacc gacctcgccc 24360
    ccggcgacgg ctaccgcatc accttccccg gtgtgcggca gctgcgcgaa cccccgctgt 24420
    gggaactcgc cgcccgcgcc ggccgcagga ccgtgtgcct gaacgtgccg ggcacctacc 24480
    ccgcccccgc catcgacggc gtgctggtct ccggcttcgt cgcgcccgaa ctggagcgcg 24540
    ccgtcagccc gccacggctg ctgccgctgc tgcgcggcct cgactacgaa ctcgacgtcg 24600
    aggtcggcga cgtcgccgcc gacccggccg ccttcctcgg gcgggccgtc cgggccctgc 24660
    gcgcccgcac ccgggcgatg gaacacctgc tgcgccagga gacctgggac ctcgcggtcg 24720
    ccgtgctcac cgagaccgac cgcgtccacc acttcctgtg gcgcgcggtc gccgaccccg 24780
    ccgaccccct ccacggggac gtcctcgcct tctaccgcct cgtggacgac tgcgtcgcca 24840
    ccctggtgag caccctccca ccgggcggcg aactcttcct gatgagcgac cacggcttcg 24900
    gacccgccgc ctgtcaggtc tatctgaacg cgtggctcag ggagtccggc tggctggccg 24960
    ggctcgacgt ctgtccggac ctcaccgcgg tcgacgctcg cagcaccgcc ttcgcgctcg 25020
    accccgcccg catccacctc aaccgcaaga gccgcttccc cggcggcggc ctgaccgacg 25080
    cggaggcgga cgaggccgcc cacgagatcg cgcgcgagct gtccgccctg cgctgcgacg 25140
    gcacccgcct gggccccgac gtcgacggac ccctgctcgt ccgcgacctc taccgcgctc 25200
    aggagatcta ccacggcccg ctgttgggca acgcccccga cctggtggcc gtaccggccc 25260
    ccggggtgca gctgcgcggc ggctggggcg gcacgcacac cgtacgcaac gacatcctca 25320
    ccggcaccca cacccgcgac gacgcggtct tctaccggcg cggcgcgccc gcgcccgccc 25380
    ccggggcgga cgacggcccc ctcgacatga cggacgtcgc cccgaccgtc ctcgcctccc 25440
    tgggcatcca ccccggcggg ctcgacggcg cggccgtact cggcaccacg ggacccgcgt 25500
    ccggtcacgg ccgcacggac ccccctctcg acatcaggga gctctgatga agcacgacct 25560
    cggtctggca ccatcggcac ccaaaccggg aacactcgac ctgagcctgg acccacgcat 25620
    cacggacccc gcttccttcc gggtcagttt cctgatcctc ctcgacggcg acctcgtgat 25680
    gtcccccgaa cacctcggcg tcgcctacat ggccggtgtg ctgcgccata cgggcttcac 25740
    cgcggagatc cgggaggtgg agcacggcga cgaccaggcg gccgccaccg tcgaggcgct 25800
    caaggagtac cggcccgacc tcgtctgctt caccctgatg agcctgaacc tgggcagctg 25860
    tctgaccctg tgccggatgc tgcgggagga gctgccgggg acgacgatcg cctgcggcgg 25920
    cccagccggg accttcgcgg gcctggacgt cctgcggaac aacccctgga ccgacgtcgt 25980
    cgccgtgggg gagggcgagc ccaccatcct cgacctcgtc caacggctct acctcaagga 26040
    gccgttgtcc gcctgcaagg ggatctgcta ccgcgacgag gacggcacac cgcgccagaa 26100
    ccccgcccgc cccctgatcc acaacctgga ggacctcccc ttccccgccc gggaccagct 26160
    gcgccagcac ggcgacaagc tggagtacgt ccgggtcagc accagccggg gctgcgtcgc 26220
    caactgcgcc ttctgctccg ccccgcacct gaagaaccgc gtccaggcgg gcaaggcgtg 26280
    gcgcggccgc gggccggaac agatcgtgga cgaggtcgcc gagatcgtcg aacgccacca 26340
    gttccggacc ttcgacttcg tcgactccac cttcgaggac cccgacggcg gccgggtcgg 26400
    caagaaacgg gtcgccgcca tcgcgaacgg catcctggag cgcggcctcg acatctacta 26460
    caacgtctgc atgcgggccg agaactggca cgacaccccc gaggaccacg ccctgctcga 26520
    cctgctggtc gcctcgggcc tggagaaggt caacgtcggc atcgaggccg gcaccgccga 26580
    ggaactgctc ctctgggaga agcgcgccac cgtcgaggac aacgtcacca tcatcaggat 26640
    gctgcgggaa cacggcatct atctcgccat gggattcatt cccttccacc cctacgcgac 26700
    cctggagacc atcgtcacca acgcggcctt cctgcgcgac aattccggcc acaacctccg 26760
    gcgcatgacc gaacgcctgg agatctaccc cggaacggcc atcgtcagcc gcatgcgggc 26820
    cgacggactc ctcggcgaga gctatctcga agggctcgac ccctacggct acgcattcaa 26880
    ggatccccgc gtcggacggc tcgccaagca tttcgcccag ctctacaaca acgacgacta 26940
    ccaccggcac ggcgtcatca ccgagcagtc ctccgtcttc gccttcgaga cctacaacgt 27000
    cgtactccag accttcatct cccggctgca ccgccggttc accaccctgc cgggggtgga 27060
    cgaggtgatg gaggcattca aggcccgggt gcacgagatc cgccaggaga tgggccggca 27120
    caactacggc ttcttcatgt ccaatgtcga ggcggtcatg aacgacaccc tcgacccgga 27180
    gaagcagcgc cggcaggtgg tggacgtcga gcacttcttc cgcgaccgcc tcgatgtgtt 27240
    gcgcagcgag caattgcgcg tcggcaaggc cctcacccgg ctcggcgccc gggtgacgga 27300
    ggtcagctcg accattccca aggagcgccc cggcggactg ccgcgccagt acacgggaga 27360
    gggcagcggt gccacgtggt gagacgggaa ccgccgcggc gcgggtggcg gtctgcacgc 27420
    tgagcagcag ggaactggtc ggcccgctgg cccggttgcc cggtgtggcg gccgcgggca 27480
    cgctgatgac cgccaacctg ggcatcgagc aggtgatcaa ggccctgcgg tgcgaccgga 27540
    cggtccgcgg cctgctcgtg tgcggccgcg actcaccccg cttccgcgcc ggccagagcc 27600
    tgatcgccct cttccgccac ggcctgcgcc ccgaggacgg gcacatccgg ggagccaccg 27660
    gctatctccc cgtcctgagg tcggtgacgg cgcgggagac cgaggaggta cgcgcccgcg 27720
    tcgagctggt ggacgcccgt ggcgagcgcg acgtcgagac gctgcgcgcc gaggtcgcgg 27780
    cactcctcgc ccgcgtacgg cgcaccccgg ccctcccctc ccgcgagcac gacggcggcc 27840
    aacccagctt cgtggagccg gacttcggac ggctgcatcc tgtcggccgc cgccgctccc 27900
    tggacgcggg catcggcggg ttcgtgctca tcagcgtcga ccgtgagcac cggcggatcc 27960
    tgctgcgcca ctacacctcc gatgtgcggc cccggcacga gatgtggggc acccgcgggg 28020
    aggcgatgct gctcgggctg ctggaggccg gcgtcatcga ggaccccgcc cacgccggat 28080
    acctcggcgc cgaactggcc aaggccgaga cggcgctgcg gctcggcctg cactacgaac 28140
    aggacctgcc cctgcgcccg ccgggcaggc cgcccggccc tgtgcggcgc cggaccgcga 28200
    aggagcgaac gaccatggcg caagcacccg cgctggagga cttcctgcgt ctcgtgacga 28260
    ggacgctggg ggccgaggac gccgtcctgg acctgcacac gccgctcggc gagcaactgg 28320
    cggtggactc cgcccggctc atcgaactca ccgtcgtcct ggaggaggag ctcggcgcgg 28380
    acctccccga cgacgccgac ctcgccaggg ccacccccgc ggaactccac aaagcactcg 28440
    tgggctgagg aggagaccga catgcgcagc gtgctgttgc tcaacggacc caacctgggg 28500
    acgctcggca agcggcaacc ggagatctac ggaaccgaca ccctggccga gatcgaggcc 28560
    gccgtggccg aggaggtggg agcgcgcggc tgggaggtgg tctccgaaca gcgcaacggc 28620
    gagggggaac tggtcgatgt gctccagcgc cacgacgacg tggtgggcgc cgtggtcaac 28680
    cccggcgccc tgatgatcgc cggctggtca ctgcgcgacg cgctcgccga cttcgccccg 28740
    ccctgggtgg aggtgcacct gagcaacgtg tggggacgcg aggcattccg gcacacctcc 28800
    gtcacggccc cgctggcctc cggcgtcgtg atggggatgg gggcgctggg ctaccggctg 28860
    gcagcgcgcg ccctcacccg gctggtcccc gaggactgac ggtgacccgg cccggcccgt 28920
    acgcacctcc agatgggacc ggcccgcccg gcagggacgc cacctcggcg cccggcccgt 28980
    acgcacgctc aggcgggcca cacccgcagc tcctccttga tcacctgagc gccggcctgg 29040
    tcgcacgccc cgggcagcgg gcaggccgcc gggaggatcc gcacggtgaa cggcccctcg 29100
    gtcaggccgc gccaggcggg gaccacggcg cgaccgcggt ccacctccgc ggacaccaag 29160
    gccgtgaccg gacagggaaa ttgaccggag acctccccgc ccaccccttc gccgggcccg 29220
    cggctgccga gccacagcag cacatgcacc ggcgggcgcg cggcccgctc ggcggcccgc 29280
    agcgccgcgc ccagcccggc accggcacag accagcgccc acggcccgcc gtccccggcc 29340
    gcctccgcga tcgcctcgac gccgtgctcc accaccagtt cgggggcgag cgcctgccgc 29400
    cagccctccg cgtccggccc gtccgtcacg gcgaccagcc ggaccaccgg caccaccacg 29460
    cctccggcgt tcccctcagc cgtacgcgac atccccagac cctctcttcc gtaccgtccc 29520
    acccgccctc gctctcccgc ccggcgccgc tacggcaggc cgtcggtcat cccgagggag 29580
    aagtagttct cgtaccccag cagccggcgc agttcgggcg tcgcgcgggt ggcgacgtcc 29640
    tcccgcagac ccgagaagaa gttctgctgc tgccggacgt agccgcggca gtagtagttg 29700
    aggatgccgt gccgcggccg gtcggtggtg ttggcgcccg tctggtgcca caggcgcccg 29760
    tcgaagacca tcacgctccc ggccggcgcg cacacggcga ccgtctcggt gttcccctcg 29820
    ccccggtcgt agtccggctg ccggcccagc agatgggagc cgggcaccag gcgggtcgcg 29880
    ccgttgtcct cggtgaagtc gtccagcatc cacatgctgt tggccaccag cggatacggg 29940
    ggccacggcg ggcgggcgaa ggtctggtcc gcgtgcagat gcatccggga accgccgggg 30000
    cccgcgatat tggcgtgcgt gctggagagc aggaagccga agcccaggat ctcctccatc 30060
    aggagcatga cggtgggatc ctgcacgttc tgctcgaatt cctcgccctt gttcagcagg 30120
    ctgaagacgc gttggttgcc gccgtcgtag agaaaggccg agccgttctc acgctcctgc 30180
    tcggcgacct ccagcagccg ccctctgagc ttttcgaaga ccgcggccgg caaggggcac 30240
    tcgatcaggc agtatccggc ttcgaccaga tcccgggagg ctttctcgac gtcattcgtc 30300
    aaagtcgcat ccatatggcg aggctagcag ccgaaatctc ggccgcacca tagcgcgaaa 30360
    acgccggtcc atgatttttt cacgtgcggg aaggacggat tttccatggc acactcaccg 30420
    cggcggccgg acggccccct ccgcatcggg gtctggctgg ccccccagca cacctcggtg 30480
    gccgaactgc gcgccgcctg gcgcgcggcc gactccctgg gcgtggactc gctgtggctg 30540
    tgggaccact tcttcccgct caccggggac cccgacggca gccacttcga ggcctggacc 30600
    ctgctggcgg ccatggccgc cgacacccgc gccgcccgcc tgggcaccct ggtgtccaac 30660
    tacgcctacc gcaaccccga cctcctggcc gacatggccc gcacggtcga ccacatcggc 30720
    gacggccgcc tgatcctcgg catgggcgcc ggctgggtcg aacgcgacct gaaggagtac 30780
    ggctacccca cgcccggcgc gggggagcgg gtggacgggc tcatcgaggc ggtggagcgc 30840
    gtcgaccgca gactcggccg gctgcgcccc gggccgctcg gcgacctccc cctgctcatc 30900
    ggcggggacg ggcagcggcg cctgctgcgc ttcgccgccg aacgggccgc catctggaac 30960
    accatggcct ggcgcttcgc cgagggcaat cgcgtgctgg acgagtggtg cgcgcgggtc 31020
    ggccgcgacc cggcggagat cgagcgcagc gccttcgtca cccgcgacca gaccgacgag 31080
    gagctgcgct gcctggtggc gacgggcgtc cagcacctga tcttccaggt cgggcacccc 31140
    ttccgcttcg acggcgtgga gcgggccctg cgcttcgcgg gcggctggag caaggggtaa 31200
    ggccagggcc cggacgcgcc ccgcgtcgcc actagagcaa cgcgtccgcc agccggtcca 31260
    cttgggacag cgccgccgcc gtggggtgga ggacgacctc gtccaccccg ccgtcggcga 31320
    gcgccgagac cgccgcgcgg agctgccccg cggtgcgcgg ggtcttcgcc acgaactcct 31380
    ccgcctcctc gcccagcacc gcgaagtagt cccggacgaa ggccgccgac tcctgggcca 31440
    cgtcctcgcc cagggtgtag cgcgccagcg ccaccacatg cggcgccccg gcgcgtcccg 31500
    cctcgctcca ggcgcggcgc acccgttccg cgaccggcac gatccgctcc ggctccaggc 31560
    cgggcgccgt ccagccgtcg gcccagcgcg ccacgcggcg cacggccgcc gcgctgaccc 31620
    cgccgacgag gaccggcaca ccggggccct ccgcgcccgg ccgggcgccc cggccgagca 31680
    gctccagctg ctcctcgaac gccgcgcgcc ggtcgtcgaa ggcgcggccg gcggcctcga 31740
    agtcgtcctc gcgcacgccg ggcccgaccc ccagggtgaa ccgcccgccc gacagcgagt 31800
    ccagactcgc gaccgccttc gccagcacag gcgcggtgcg cagcgggccg atcaggacat 31860
    tggtgagcag cccgatccgg gaggtcgccc cggccgccgc cgccagcgcc agcagcggat 31920
    cgtggcccgg atacaccagg cgctcggtgg ccgcgagcga ggcgaatccc cgctcctcgg 31980
    cccgccgcgc ccaatcggtt atcaggcgcc cgtccgcgcc gggcacggtg ttcggcagag 32040
    caatgctgat cttcattggt ctccccgggg gttcgcagga tttccggtcg aatgtgacag 32100
    gggattccgg cacggccggc gtgattgcgg caggagttca ccagcggccc ggcgcggaga 32160
    aatgcggcgg catttccacg gccccctgtc ggaccgccgg accgccgtgt acgtttttcg 32220
    gaaagcaacg tcgtacggtg cgcacagcga gaggaatccg cgatgcccgc tgccggaaaa 32280
    gtcgccgtga taggactcga ctccgcgact ccgcagtaca tgttcgaccg gttcgccgag 32340
    gacatgccgg tgttcaccgc cctcaggcgc aagtccctgt ggggtccgat gcgcagcatc 32400
    gacccgccca tcaccatgcc cgcctggtcc tgcatgatgt ccggccgctc gcccggcgaa 32460
    ctcggcgtct acggattccg cgaccgcggc gcctacgact acgggccgtt gaagttcgcc 32520
    acctcccaca gcatccaagc cccccggatc tgggacgaga tgacggccgc cgggcgctcc 32580
    agcgtggtcc tgggcgtccc cggcacctat cctcccgccc ccatccgcgg ggccatggtc 32640
    tcctgcttcc tggctccctc cacacagtcg cgctacacct ccccgcccgg cctcgccgac 32700
    gagctggaga agctcaccgg cggctacgcc ctggacgtgg aggacttccg ctccaccgac 32760
    ctggaacgcg tatcccagcg cgtcttcgac atgagcgagc agcgcttcga ggtcgcgcgc 32820
    cacctggcga ccacccagga gtgggacttc ctctccttcg tggacatggg ccccgaccgc 32880
    ctccaccacg gcttctggaa atactgcgac cccgaccacc cgcgccacga gccgggcaac 32940
    gcctacgccg gtctcttccg cgactactac cgcgccctcg accggcacct cggccgcttc 33000
    ctggagagcc tgcccgagaa cacgaccgtc ctggtcgtct ccgaccacgg cgcccagccg 33060
    atggtgggcg ggctcttcgt caacgagtgg ctgcgcaagg agggttacct cgtcctgacc 33120
    gaggagcccg ccggacccac ccccgtcgcc caggccgccg tcgactggaa gcggaccacc 33180
    gcctgggccg aaggcggcta ctacggacgg atcttcctca acgtcgaggg ccgggagccg 33240
    cagggcacca tcccggccgc ggagtacgag agcacccgcg acctcatcgc ctccgccctg 33300
    gaagcgctgc ccgacgacca ggggcagccg atgggcaccc gcgccctgcg ccccggcgag 33360
    ctctacggag aggtcaacgg catcgccccc gacctcctgg tctacgtcgg caacctgcgc 33420
    tggcgggccc tggccaccct cggcatgggc aagggcctct acacgacgga gaacgacacc 33480
    ggccctgacc acgccaacca cggggacacc ggcatcttcg ccctcagcgc ccccggcatc 33540
    acccccggcc gcgcggacgg cctgtcgctg tacgacgtgg cccccaccct gcgggaactg 33600
    ctgggtctcg cgccgcaggg ctcccgcggc tccctcctcg gctgacatca cccgcccagc 33660
    agcgcgtagg gagtgggcgg cgccggcacc ccgcccgctc ccgcaccgcc accgtgcacc 33720
    acgtgcttgt ggcggtaggc gtccagctcg ttggtgagcc ggtcccagac ggcggagcgg 33780
    ggcccggctg tgccgggcag ctccaggtcg accagccggt agtcgttgat ccatacccgg 33840
    tccgcgcgca gccgctcggc caccgcgcgg gcccgcgcgg gatcggcgga ccacaccccg 33900
    gcgctgagcc ggtagcggga gccgttggcg atgcgcaccg cgtcgtcgtc ggacccggcc 33960
    cggacgaccg cgagcaccgg gccgaagatc tcctcctgcg cgacggcgtc gtccgcgccg 34020
    accgacgcca gcaccgtggg caggaaatac gccccggcgt ccagcccggg cggcagctcg 34080
    tccgccgcgg gcgcccggcc gccgcacacg agctccgcgc cctgggagag cccgagttcg 34140
    gtgaagcgcc tggccgtacg cgcctggttg cgcgagacca gcggccccag gtcggtggcc 34200
    gggtccagcg gatcaccgac gcgcagccgg cccacccgtt cgctcagcag ccgcaggaag 34260
    tcgtcgtgga cgtcggcgtg caccaccgcg cgggtaccgg ccatgcacac ctgcccgttg 34320
    tgcaggaacg ctccccacgt gacgccggtg accgcccggt ccagatcggc gtccgcgagc 34380
    acgatgttgg gggacttgcc ccccaggtcc agccgggcgc tcgtccccgc cgcggcggca 34440
    ccctcccgta cggcggcccc ggtctcgtcc gagccggtga acgccaccag gtcgacgccg 34500
    ggcgagcgca ccagctgctc cccggcgacc ccgcccggcc ccgtgaccac gttgaccacg 34560
    cccggcggca ggccgcactc gtggagcagc tccaccagtc gcagcgtgga gagcgaggcg 34620
    aacgaagccg gtttgatcac acaggtgttg cccgcggcga tggcgggcgc gatgcgccag 34680
    gccgccagca gcagcggcag attccacggc acgatcgcgg cgacgacccc caccggccgc 34740
    cacacgacgt aggaacccga accgggcgcc tccggctgcc gttcgggcac gtgctccgcc 34800
    caccacgcgc tccactcgaa ggctgccgcg gcccccggca catcggcccc gagagccttg 34860
    cgcagcgtcg agccgttgtc gcgggcctcc aactcggcca gcggctccgc ttcttcacgc 34920
    aagcgctgtg cggccttgcg cagcaggccc gcccgctcgc ccggcgccat ccgcggccac 34980
    gggccctcgt cgaaggcccg ccgggcggcg gacaccgccc ggcggacgtc ctccgcgccg 35040
    ccgctgggaa ggtcggccag gtggcgccgc gtggccggct cgaaggtgcg caggacggcg 35100
    ccgtcgtggg cctgcacggc ctgcccgtcg atgtacatcg ggaaccgctc gaccgctctg 35160
    tccacccggt ccatcgcctt caccttctcc ttctgctgac ccgtggggat gcgcccggcc 35220
    gggcccgccc gcggccgcgg ccgtaccgga acacccgccc cggagcggcc gcgcccgcgg 35280
    tcaggccggc aggggcggga tgttggggtt gaaccggaag acgttgcccg ggtcgtactg 35340
    cgacttcagg gcctggagcc gcgcgtagtc ctccggcgtg taggcgctgc gggtcgtctc 35400
    cctcgatgtg ttgtgacccg cgaggaagtt caggcacacc ccgggcgtcg tccacggccg 35460
    catgctgtcg acgaactcct gctgcgccgc gtccacggcc gccagggtgt ccgggtcgac 35520
    cagcgagccc acgtaggcgt tgaacaccgc ctccgggaag tggcccaccg cgctcgggtg 35580
    ccggggcggc cgggcgaggg cgccgcccag gtgccgcagc tccaccccga acagcgcgtc 35640
    cgtgcccggc cccgcgagcc tgaggatctc gtcgacggcg atctcgtcca gctgcccgaa 35700
    catgaccgtt ttgctgtgac tcgacaccgg ggccggcgga tcgttgtgga tgatcccggc 35760
    ccgcgtgtac gggagcgtgt ccaccgtatc catgacgacc gtgccggcgg cccggagctc 35820
    ggcgaaccgg cgctcaccct cctcggggtc tcccagccag gccagccgga tgtgggtgac 35880
    gaaccggccg cgcagcggtc cgggcacccc ctcggcatcg ggatacgcgg ccaggaacac 35940
    cgacgacgcc atgtcctcgg gcatccgggg cgcccactgg agataggtgt tcagcacggc 36000
    gcgcgtggag ccggcgtcga agaacagccc tccgccgtac acctgggtga cggggaacag 36060
    cccgacctcg acggaggtga cgatgccgag gttccccctg ctgccgcgca cgccccagaa 36120
    gagatcgggg tgttcctcgg cggagacctg gagaaaccgc ccgtcggccg tcaccaggtc 36180
    gagcgagacg acatggtcgc cggcgaaccc gtacttccgc gacagaagcc cgagcccgcc 36240
    gccgaggagg taggagaccg cgccgacgaa cggcgccgag ccgctcagcg gtgcaagacc 36300
    gtgcgccgcc gcctcgtgga tcacctgctc ccagcgcacg cccgcctcga tccgggcggt 36360
    ccgggcccgc gggtcgaccc tgacgccggt catccgccgg gtgctgatga ggacgtcggt 36420
    ggccgccgag gacttcccgt gaccggtggc ctggacggcg atcccaaggc cccgggccct 36480
    ggcgaagcgc acggcggcga tgacatccgc ggcaccggtg gcgacgacga cgagggcggg 36540
    gcggtgttcc acggacagtt cgaagccgga gcgctcctcg tcgtacccct cgtccccggg 36600
    caggaggacg gatccggcga cctgcgccgc gagctcttcg gcggccgcgg cgccgagggc 36660
    cgcggacgtg tccgtcacgg agtggctggc tggtttcacc gaggaacctt tctggctgga 36720
    gcttcgagaa gcgcgccgcg cgtgcgcggg cagggccgcg gggctcgccg gcccttggaa 36780
    cggagcgggc cccgtcagtt gcgcgggccg gggaccaccg gcagtgaccg gacgcccagg 36840
    ggcaggagtt cggagctgtg ctcgacctct tcgggaggca cggccagggc gatgcggggg 36900
    aaggccgcca gcaggcggcc gatgccgacg gtcagttcgg tcacggccag cccggtcgcg 36960
    gggcaggcgt gctgcccggc gccgaacgcg atgccgcggt cggccacccg ctcgatgtcg 37020
    agcacgtggg gatcggcgaa gacctcgggg tcgcgggagg ccgccgagac cagtggcagc 37080
    acggcgtccc cggcggcgac gcgcctgccg gagagcacca cgtcctcggt ggcgacccgc 37140
    agcaggccgt cgttgctgga ggggtagtag cgcagcaact cctgtacggc agagggcagt 37200
    acggaggggt cctcgcgcag ccgggccgcg aggccggggg aggagagcac gccgaagagg 37260
    tgccgggcca gcaggtcgcg gatggtgatg aagcccgaga tgatcaggcc gtggagcagc 37320
    aggcgccggt cgtcgtcggt gagctcctcc gcgtccagca gcgtgtcggt gacgctgtcg 37380
    cccggctcgg ccctgcgggc cgcgagcagt tcgtccagca cctggccgag cctgccgcgg 37440
    gcctccttca gcgcctgctc ggtggcaccg cgcggaagca gcagcagctc gacgtcggag 37500
    gtgacgtcct gccaccggtc cccgggcagc ccgaggaact cggctgtgac gcggccggcg 37560
    aagggcgcgg tgtacgccgc gacgaggtcc accgtgccgc tgccggcggg cagccggtcc 37620
    agggccgcct cggcggccgc ctcgatgcgg ggtgcgaacc gcgccgtgcg cgaggcaccg 37680
    aacgcggcca ccaccggacc gcgcagccgg ccgtgctccg gcgggtcgag gtccacgatg 37740
    cccgagccct gggaccggcc gaagcccgag cccggcagca tggcggcgcg atggcggctg 37800
    aagcggccgt cggtgagcac cgtgcgcacg tcctcgtgcc gggtcaccag ccagatgcgc 37860
    gagccgtccg ccaggcgcac ctcggcgacc gggtcatcgg tgagcagccg cgcgtactcg 37920
    ggcggcaccg tgccgccggg gccgggcggg aagggaaagg cgggcggggc ggctgaggtc 37980
    atgcgccccg gctcctctca ccggccggcg cgccggcgcg ggcgtggccc ggccaggtga 38040
    agtccttcgc caggacgcgg ttgtccagct ggtgttccac gacgatgttg ccgcagccgt 38100
    agtcgtcggc gatgacctcg acgtcctcgg cggacaccag gtgtccgggg tcggccagca 38160
    gggtcgccac gcccttggcg ggggccagga cctccagggc gaagccgcgg gccacgtgct 38220
    ggcggtgggt gtgcgccgcc gcgatcagga aggtgtcgtc ggggtcgcag atcagatgcc 38280
    acagcgcgcg ggcctggagg aagcggcgcg ggcggtcggt gaagcgccgg gaggagacca 38340
    ggaccggccc gtccgcgccc ctgcgggtgc cgagggcgac caggccccgg tccatatagg 38400
    ggacctgctc ggtgcggtag cccatcaggg gggccgggtc gaagggcgcg gtgtcgtcca 38460
    gacccgccca cgcgcgcacc cggtgcgcca cctcgtagcc gaggtcccag gggttgtcga 38520
    gtcgccccgg agcgaagaac ttctcgctca agtcggcgca gtccttgcgc agttccacca 38580
    aggcgggagg gagcggggta tccgcgggcg cggtgcccat cagggtgcgg acgcgcgcga 38640
    tccactggag ctggtcggct atccgctcag gccgcacccc gttgaagaag tcactggcga 38700
    gcggttccgc caactgctcg gcggccttga ggatgtccgc ctcgtacggc tcggcctcgg 38760
    cgtaggggtc caggcccagc cgtgccgcga tgcggcagaa ggcggcctcg tcctcgtcgg 38820
    tggcgcggac ggcggcccac tcctcctgga gcggggtgcc ggtgatgccc tggtccgtga 38880
    ggcgctcggt caccgcgtcg acgaacgagg ccagtgtggc ggtgaacgcg gcgctctcca 38940
    cacaggagtt gccccggctc gcgaagcggt tcccgggtcg tacgtcgggg cccatgtccg 39000
    gcatccatac gatccgggtc tcccggccct cgggcacgaa gagcatgtcc ggccagcgga 39060
    agccgtcgca ggcggcgcgc aggatgtgac ggcgcgaacg catccaccac gaaccgcggt 39120
    tgtcgcccac accgtggcgg taggcgaagc gcagctggga tatctgggtg ccgggccgcg 39180
    cgtcggccac cagcgaccac cagttgaagg cgatccactc ggccaggggg tagagcgagc 39240
    cggtcgtgtg ctcccggaag gtcccctgcc cgggctcctg gacgagtgtg acggtctcgg 39300
    cgcccacggc gatgcgcagc cgggcccagg tcgcttgcag ctcgcctccg ccgccggccg 39360
    gggcgtcgag ccaattccac tgcaattgga actcaggaag catggtccgc cagcccttcc 39420
    ggccattcgc tcgggtggag ttcgtatccg gtgtattcgc ccggcgcacg gcccgtcagc 39480
    cggaattcca cgacggagtc accggaccgg tgccagacat agcgcgggaa gccgtcctgc 39540
    cagggaccgc cgaggaatcc ggtgcggatc cctgaacgga gggtgcccag cggggcggcc 39600
    gacagggaac cgggcacggc gagcagatcg gccggggccg ggccgtattc ccggcgggcg 39660
    aatgtcaccc gcccgcagag ttcttcccgt tcgctgcggt cgggcagtgg cgcgatcgcg 39720
    cgacgtggct cgcggcggcg cccgggcgct cgtaggacca tgatgtccgc ctttcgggga 39780
    acgtgccggt gagctgggcc ggcggggccc ggacgcggcg tgcgtccggg ccccgcccag 39840
    ggtgttacgg gaggggcgcg aagaggtcca ccacgttgcc gtcggggtcc ttgacgatgg 39900
    cgtagcgctg accccacacg gcgttccacg gcttgaggtg gccctcgtag ccggcgtcga 39960
    cgagctcggc gtacttcttg tccacgctcg cggtgtcggg gaactcgaac gcgatggcga 40020
    agcggtggcc gccggtgggg gcctgccact cggggtcgta gctgcgcacc gtctccacgg 40080
    tgtcccaggc gagccggatg ccgccgtcga gcacggcctc cgtgtgcggc gcggagtcgg 40140
    cctcggcggg gatctcgacg cccagcttcc ggtagaactc cagcgacttg gccatgtcct 40200
    cgaccaccac ggcgaagagg gaaatccttg ctgacatgcg cgttcctttc ttgcactttt 40260
    aaattggtct ccggtgccgg gccgtctgaa ttctccgggg ccggccggac cacgaagtcc 40320
    gaatgtgctg gacgcgccgt acgctagtga ctgcgcgctg actttggcca atcggggtat 40380
    cccccgccgg agtcaacgcc gctgacagga caacgatttc aggacagcgg cacgccgtcc 40440
    cagtcgttcg gcacgtcacg cccgagcaga gcgcatacgg tcgcgggaat atcgacgggg 40500
    gcggcggtgc gggtctgccg cgccccaccg gtgaagccgg ggccgacggc cgaccagtag 40560
    gactcccggc ggtgccagcc gctctgccag tcgcggtgga cgtgcgaggc ccagtgcggg 40620
    tcgcccagcg gaagatagcg ccagtccgcc ggctccagga tgaggtcggg ggcgtgctgg 40680
    gtggcctcgc cgggatagac ctcctcccgg cggcgcaccg cgtcgaagaa cagcctgccg 40740
    gtacgggggt tgcgccgctc cagcagcgcg gccgcgacgt cggtgcggac cttctcgtag 40800
    tcgcgctccg ggaccaggcc gtgcttgtag cggtcgcgca ggttgatgtt caccccgtgc 40860
    gtgccctgca ccgcctcgaa ggccgcgctg ccggcccact cgacgcggcc gtcctcggcg 40920
    gtggccagga aacccgcctg ctccatctcg tcgttgatgg aacagtagtt gcgcagcggc 40980
    ccgaagccta tctccgagaa ggccacgaca ctggtgcggt cgtcggccgc ccgcagggcg 41040
    tcctggatga cctggtcgca ggtgcggtag gcggcgaaga cggcgctctc ccgctcgtgc 41100
    tcggggccgt gctccagctc ctgccagtag atgtgcgaac agcggtcgat gctcgtgagg 41160
    ttgacgatca cgacatcgga ctcctccagc agagccaatg ccgcgcgccc gcgctgcacg 41220
    tccgcctcca gcagggaagg cagcagctcg tcgcggtcct gcccggtcca gaagatcgac 41280
    acgtcgtgga ccggacggat gcccttcttc gccagggtgc gctggaggct gcgcgggtgg 41340
    caggcgtgga gggtggcata catcggatag gtgatcaggg aaccgtcgaa gggctccggg 41400
    ggatgggtgc cgaagaggcc tatcgaggcg aacctgacgc cctggaacac ctcgtgctgc 41460
    cacagcagtg ggtggcggcg gtgctcgggg gtgaggacct gcggcgcgta ctccgggtcg 41520
    tgacaggtcc agtaggagta gaagccgtgg tccgcggcgc gccggccggt caggacgctc 41580
    agcaggcccg gcggttcgta gggggtgccc tcggcgtgga gcggcccgga agccccctgc 41640
    gagcgcaggg cggcgaagcc gggcagcagc ccctgggcac accagcggtc gagcagctcg 41700
    ggtgccgctc cctcggtgat gacgacgacg acacgctggc ggattgtcac gtgcgactcc 41760
    ctcgggttgc gtggcagttg gcatgccgtc atccgggagg cgccggaaag gccgaggcgt 41820
    tccggcgccg gacaggcgtc gatcgtcgga tcaagctaac agcgggacga ggactctctc 41880
    cagacgacgg tacggaggaa attgagagag ggctgagaga gggctgagag agggcagagg 41940
    cgggggagtg gcgtggggtc acacggtgcg caggaggcgc agacgttccc gtaccgcctt 42000
    gggcagcccc gccaccacgc gatcgtacga atgctccacc atctcccgca gctcctccac 42060
    gggaaccgtg ccgttcagga caaccgtgtt ccagtggcgc ttgttgacgt ggtagccggg 42120
    caccaccgcc gcgtactgct cgcgcaggtg cagcgccaga tccggttcgc acttcagcgt 42180
    gacctgcggc gggcggtcct cggaggcgtc ctggagaatg gcgaagacct tcttctccac 42240
    cttgaagacc gcggctccgg ggccgaacgc ctcgtcgtcc accgcctccg gcagctccag 42300
    cgcgaagtcg gagagttcct ctggtgtcat cgccggtcct tcttcctgcg gcacggcagc 42360
    gagcggccga accgcgtggt catggggtcg gccaacagac tagaggcgca ggaggagttg 42420
    ccgtgcggca gggcgcggac gctgatccac gatggccgaa acactgcggg gagttccggt 42480
    cgcggcggga cggcgacctt gacgggcggt cctgccattg gcacagtttg gctggctcca 42540
    cacaggtttt cggtggaccg ttcgttcctc tcccggtgct gcccggtcgc ggtaccggtg 42600
    tccgcgcgat ccgtgtgccg cccgcgccgt cccgaaccgg cccgtgcgcc cactctcccg 42660
    gccctccgcc gccggtctcc gtaccgccgc cccgcccttg ccggggcggc gccgacgccc 42720
    gcaccccggc cttggccctg cccacggccg catccgcgca cccccctcac cccggcgccg 42780
    gccatgcccc cgtgccgcct gccccccttg atgccctgtg gaggaacccc cgtatgaccg 42840
    tggagcagac ccccgagaat cccgggaccg cggcccgcgc cgccgcggaa gagaccgtga 42900
    acgacatcct gcaaggggcg tggaaggccc gcgccatcca cgtggccgtc gaactcggcg 42960
    tcccggaact gctccaggag ggcccccgca ccgcgaccgc cctcgccgag gccaccggcg 43020
    cccacgagca gaccctgcgc agactgctcc gactgctcgc cacggtgggc gtcttcgacg 43080
    acctcggcca cgacgacctg ttcgcccaga acgccctctc cgccgtcctg ctgcccgacc 43140
    ccgcgagccc ggtcgccacc gacgcgcgct tccaggcggc cccctggcac tggcgggcct 43200
    gggaacagct cacgcacagc gtccgcaccg gtgaggcgtc ctttccttcg acgtggccaa 43260
    cggcacctcg ttctggcagc tcacccacga gggaccccaa ggcgcgcgaa ctgttcaacc 43320
    gcgccatggg gtcggtctcc ctcaccgagg ccggacaggt cgccgcggcc tacgacttct 43380
    ccggcgccgc gaccgccgtg gacatcggcg gcggccgcgg cagcctcatg gcggccgtcc 43440
    tcgacgcctt ccccggcctg cgcggaaccc tgctggagcg cccgcccgtc gccgaggagg 43500
    cccgtgagct cctcaccggc cgcggcctcg cggaccggtg cgagatcctg cccggcgact 43560
    tcttcgagac catccccgac ggcgccgacg tctacctcat caagcacgtg ctgcacgact 43620
    gggacgacga cgacgtcgta cgcatcctcc gccggatcgc caccgccatg aagccggact 43680
    cccggctcct ggtcatcgac aacctcatcg acgagcggcc cgccgcatcg acgctcttcg 43740
    tcgacctgct gctgctcgtc ctcgtcggcg gcgccgaacg ctcggagagc gaattcgccg 43800
    cgctgctgga gaagtcgggc ctgagggtgg agcgctcgct gccctgcggc gccggcccgg 43860
    tgcgcatcgt cgagatccgc agggcctgaa accgcccctc ctgaccgaag ccggccacag 43920
    ctgaaggagc aatgacacca tgacggtgct gggtctgggt ggatccggac atgactgggc 43980
    ctcctgtgcc accgacggcc gacggctggt ggcgatcgac gaggagcggc tggtccgcag 44040
    caagtacggc ctgggagcgg acctcctggc gggccacagc cggcgcgccg tcctcgacgc 44100
    cctcggcacg agtgccgagg ccgtggaaca cgtggtggcc tgcgagctcg taccacgccc 44160
    cttctaccac tcgttccgca ggcgcgtgac ggtcgtcaac caccatctcg cccacgccta 44220
    cagcgcgttc ggggcctccg ggatgacccg cgccgccgta ctggtctgcg acaactccgg 44280
    cagcctggtg acgggcctga agtccggccc agggccgcgc gaggcggaga cgatcagctg 44340
    ctacaccgcc gacgcctccg ggctgcgcct ggtcaaccgg gtcgccggga cacacgccgt 44400
    ggacgcctcc tccgagagcg cctactacca gcccggcgag accgacaatt ccctcggcca 44460
    cttctaccgc tcggccagcc tcgcactcgg cctcgcctac tccggtccca agacccgcta 44520
    ccccgtcagc gaggacggca agaccatggg cctcgcgccc tacggcgacg accgcttcgt 44580
    cgacgaggtc gcggagctgg tcaccctgct gcccgagggc ggcgtgcaga tctcggcgag 44640
    caaggtgaac cacctcttcg aacgcctcgt ggaatcgggt gagttcgagg accgggcggc 44700
    cttggcctac gccgcccagg agacgctgga acgcgccctg ctgcactgcg cccgcgacct 44760
    gcaccgccgc accggcctga cggacctgtg catcgccggc ggcgtcggcc tcaacagcgt 44820
    cgccaacggc cggatcctgc gcgagacccc cttcgagcgg gtcttcgtcg tcccggccgc 44880
    gggcgacaac gggatcagcc tcggctgcgc ctactacggc ctccacgagc tggaggggcg 44940
    cgcgccgtcg gagctccccg ccctcgacac cgcctacctc gggcccgact accccgccga 45000
    gcgcgtcgac gcggcgctgg ccggctcggg cttcaccgtg gagacccccg acgacctgcc 45060
    cggcagggtc gccggcctgc tcgccgaagg gaagatcatc ggctggttcg acggccgctc 45120
    cgaattcggc ccgcgcgcac tgggacaccg cagcatcctc gccgcaccct tccccgcctc 45180
    cgtgcgggac cacctcaacg acaacgtcaa acaccgcgag tggttccgcc cctacgcccc 45240
    catcgtccgc gaggaccggg cggcggacta cttcgacctc gtccagccct ccccgttcat 45300
    gctggtcgtc gcgcgcgtga cccggcagga cgccatcccc gccgccaccc acgtggacgg 45360
    caccgcccgg ctccagacgc tgaacgccgc acagaacccg aaggtctacg agctgctcgg 45420
    caggttcgag gcgctcaccg gctgcgccgt gctgctcaac acctccttca acgtcgccgg 45480
    ccagcccatc gtcgagaccc cggaggacgc cgtcgaggcg ttcgcgggca tgcgcctgga 45540
    ccacctcgtc gtgggggacc ggctggcgac caagccctga cagcacgccg aggcccgcga 45600
    ccggcaggga ggagagccaa gcggtggacg tccccgtgct cgtggtcgga ggaggaccga 45660
    cgggcttggc gatggcgctc ttcctcgcac gccacggcgt cggctgcctg ctggtcgaac 45720
    ggcggacgac cacctcgccc gtcccgcgcg ccacccacgt cagccgccgc tccatggaac 45780
    tcttccgcga ggcgggcctg gaggaggaga tccgccgggc cgggttcgag gtcgtgcgcg 45840
    aggacgaccc acggctgcgg acccggcccg aacgccacct gccccgggtg gtcctgcaag 45900
    ccgcctcgct cgccggcccc ggcccggtgg gggtcctgga gaccggtgac gaggaactgg 45960
    ccgtacccgg cccctgcgca cccttctggt gcggccagga ccggatggaa cccctgctcg 46020
    ccaaggccgc ggcgcgccac ggcgccgatg tgcgcttcgg ccacgaactg accggcctgt 46080
    ggccggggga ggacagcaca cgggcccgcg tccgggcagc gggaacggga cggacctaca 46140
    ccgtcgacgc ccgcttcgtc atcgccgccg acggggcgcg cggcgagatc gccgagcgcg 46200
    tgggcatcgc gcgggagggc ctgggcacgg tcgcccaccg ggtgagcatc ctcttccgcg 46260
    ccgacccggg gcgctgggcc cgcgaccggc ggttcttcat gtgcatgatc cagaacccgg 46320
    ggttcgacgg ggcggtgatg gagctcaaca ccccgggccg ctggtgcgcc gcggtggact 46380
    acgacccggc ccgcgccgaa cccgacggca cctactccgc acgcacctgc ctcgacctgg 46440
    tccgggccgc cgtcggtgac gaccggagcg acgcggcggt cgacaccgtc ttccactgga 46500
    aggcccggca ccgcatagcg gccgcctacc gcagtggggc ggtgttcctc atcggcgacg 46560
    ccgcccacct ccacccgccc tccggcggct acggatccaa cgtcggcttc caggacgcgc 46620
    acaacctcgc ctggaagatc gccgccgtgc tcggcggctg ggccggaccg cggctgctgg 46680
    acacctacga cgaagagcgc cgccccgtgg gaaaggcgac ggcggagcag tcgatgctcc 46740
    tcgacggcgt gccaccggaa ccactggggg gaagcgtcgt ccgctgcgat ccccgcaccc 46800
    tgatcatggg ataccgctac cactccgccg ccgtcctcgg ccccccgcac ggccccgcct 46860
    tccccgcggc cttcaccctg cgcggagacc cgggcacccg gctgccgcac gtatggctgc 46920
    gtacggacgc gggggaacgc gtctccacgc tcgacctgtg ccacgggcac ttcgtcctgc 46980
    tctccgccga cccggtctgg gcggcggccg cggcgcgctc ggcgaaggag acgggcgtac 47040
    cgctgcgggg ccaccacctg gcggccaccg gaagcgaact cgccgacccc tccggcgagt 47100
    tcccgcggag ctgcgggacc gggcccgcgg gggccgtgct cgtacggccg gacggcatgg 47160
    tcgcctggcg cacggcccgc gccgtgcccc cggacccgga cagcgcgcag gacctggtca 47220
    cggcagcggt gagacgtgtc ctcgcactgc cggagcgcgc ggcgccaccg gtgctcggtc 47280
    cgccgcggtt gtcacgcggt tcctatcggc gagtcgggag cgacgggtga agcctcattc 47340
    cttctgcacg tgctggccgg gcgccaccgt atggctgacg ggcccaccgg gcgcgggcaa 47400
    gacgacgatc gcccgcgcac tggcggagcg gctgcgcgaa cggggccggc gcgtggaggt 47460
    gctcgacggc gacgcgaccc gcgcgctcct gaccgcgggc tcctcgtggg aggaccgtgg 47520
    caccggcctc cagcgggtcg gcctgatggc cgaggtcctg gcgcgcaacg gcatcgtcgt 47580
    cctcgtcccg gtgaccgcgg cccgcgcgga cagccgcgaa gccgtacgca gacgccacga 47640
    gcggtccggc accgcgcacc tggaagtgcg ggtggtccgg gacgcagtgc ctccgagcgg 47700
    gctccccgcg ccgcccggcc cagatctgcg gatcgcggcg cacgagcaga gcgccgagga 47760
    gtcggcgcgg gcactgcacc ggctcctggc ggagagggag ctggcgtgaa ccccgggcgc 47820
    ggtggagcgt acgccgcggg gcgcgacggg acccgcggga cgcgacgccc tcacggtctg 47880
    tcgcacctgg atctgctgga gtcggagtcg gtccacatct tccgtgaggt ggcgggcgag 47940
    ttcgagcggc cggtgatcct cttctccggc ggcaaggact cgatcgtcat gctgcacctg 48000
    gcgctgaagt ccttcgctcc cgcacccgtg ccgttcgcgc tgctgcacgt ggacaccggc 48060
    cacaacttcc ccgaggtgat cgcctaccgg gaccgcgtcg tggcggcgct cggtctgcgg 48120
    ctggaagtgg cctccgtgca ggacttcatc gacaacggca ccttgcgcga acgcccggac 48180
    ggcacccgca atccgctgca gacggtgcca ctgctggacg cgatcgggcg ccaccgcttc 48240
    gacgccgtct tcggcggcgg ccgccgcgac gaggagaagg cccgcgcgaa ggagcgggtg 48300
    ttctccctgc gcgacgagtt cggcggctgg gacccgcgcc gccagcgccc cgaactgtgg 48360
    cggctctaca acggccgcca cgcacccggc gagcacgtcc gcgtcttccc cctctccaac 48420
    tggaccgagc tcgacgtgtg gcagtacgtc gcccgcgagg agatcgaact ccccaccatc 48480
    tactacgccc acgagcgcga ggtcttccgc cgcggcggca tgtggctggc accgggggag 48540
    tggggcggcc cacgcgaggg ggaagcggtg gagaagcgac gggtgcgcta ccgcacggtg 48600
    ggggacatgt cctgcaccgg cgcggtggac tcggcggcgg ccaccgtggc cgacgtcgtc 48660
    gccgagatcg ccacgtcccg cctcacggaa cggggcgcga cccgggccga cgacaagctg 48720
    tcggaagccg cgatggagga ccgcaagcgc gaggggtatt tctagcgcgg cggggccggt 48780
    gcggcccaca agcggaggac tagtccctaa gtatgaagtc ccctactccg tttgtctgtt 48840
    gagggcaggg gcgccgtctg aggatgatgc agtccatgtc acagttactt tccgggaagg 48900
    acggcgccca ggaggcgcca agtcgcggcg ggtccacgtg ggtggcggtc ctcgccgcgt 48960
    gcgtggggca gttcgtggtg gtcctcgacg tgtccgtcat caatgtcgcg ctgccgtcga 49020
    tccgttccgg cctcgacatc ggcgagacgg gcctgcagtg ggtggtcaac gcctacgtca 49080
    tcgccttcgc gggcttcctg ctgctcggcg gccgggcctc cgacctcttc ggccgcaagg 49140
    ccgtgttcgt cttcggcctc ggggtgttca ccgccgcgag cctgctcggc ggcctcgcgc 49200
    aggcgccgtg gatgctcatc gtcgcccgcg ccctgcaagg catcggggcg gccgtgctct 49260
    cacccgccac cctcgcgatc ctcaccacca cgttccccga gggtccggcg cgcatcaaag 49320
    ccgtcgcgat ctggacggcc gtgggcacgg gcggcggcgc ggccggcggc ctcatcggcg 49380
    gcctgctcac cgactacctc tcgtggcgct gggtgttgct gatcaacgtg ccgctgggcc 49440
    ttgtcgtgat cgtcgcgacc gtcgcctggc tggccgagag ccgcagcgac caggcacacc 49500
    gacgccggct ggacctcccg ggagcggtgc tggtgaccct gggcgtcggc agcctggcct 49560
    acggcatctc gcagagcgag ggccacggct ggggctcgcc gcggacgctc accttcctga 49620
    tcgtcggtgt cgtggcgctc ctcgccttcg tcgccgtgga gcagcgcacg cgcgagccgt 49680
    tgatgccgct cggtgtcttc cgggtgcgct cggtgtcggc ggccaacgcc atcaccatcg 49740
    tcagtggcat gggcttctac gcgatgtggt acttcctctc gctctacatg cagaacgtgc 49800
    tgaaatactc cgccgtacag accggcctgg ccctgcttcc ccacaccgcc accatcatcc 49860
    tctccgcgca gttcgcaccc cgcctgatgc ggtggatcaa ggggcgcacc ctcctcgtga 49920
    tcgcgggact gctgaccgcc gcgggcttca tctggcaggg gaacatggac gccgacggct 49980
    ccttcctggc gaccctgctc ggcccgggaa tcgtcttctc cttcggcgcg ggcctgatga 50040
    tgacgctcct cgcggtctcc gccacgacgg gcgtggagct ctccgaatcg ggcctggtgg 50100
    ccggcctcgc caacacctcg cgcaccatgg gcggcgcgct cggcctgtcg gtcctcgcgt 50160
    ccgtcgccgc ccgccgcacg gccgacgtgg ggcccggcgc ggagggcctg gcctccggct 50220
    acggtcgggc gttcgtcgtg tccggggcca tcatcctcgt gagcatgctg atgatcccct 50280
    tcctgcccaa gccccagccc cagaccccgg cggaatgacc tgtgagcacg gacatacgag 50340
    gaggcttcgt ggggcaggac agccggccgc ggtggctcac cgacgaggaa caacgcgtgt 50400
    ggcgcggcta tctgcgggcc accaggctgg tggaggacca cctggaccgc cgcctccagc 50460
    gggaagcgga catgccgcac ctctattacg gtcttctcgt ccagctctcc gaggccccgc 50520
    gccgggggat ccggatgacc gaccttgccc gcaacgcgaa gatcacccgc ccgcggctct 50580
    cgcacgcgat cacccgcctg gagaagctcg gctgggtgcg ccgggaatcg tgccacggcg 50640
    acaggcgcgg ccagaacgcc gtcctcacgg aagagggccg cgaggttctg gagaagtcgg 50700
    cgccgggcca tgtcgccgct gtgcgcgcgg ccgtcttcga cagcctcacc ccggaacagg 50760
    tcgggcaact gggccggatc tgccaggcga tagagaaggg gctggaccgg gaaggcgcgg 50820
    acctgccgtg gctgcgctga ggcgggaagc cgtcgcgagc gcgcggggcc gtcaggctct 50880
    gacggccccc gccgcccgcg tacgggatcg ggccgaccgc gccccggatt cacgcgagtc 50940
    cgggagcaga ccggacgaca cggatattct ggatgccgtg gaacgacacg acggggcacc 51000
    gggctggggc ttcacccata cccagtacag cgcggaccac ggtgaacgcg gcgccacccg 51060
    cagggccggg gccctgctct ccgcgcggcc cctgccgcag aaccagcaca tcatgggctg 51120
    gggcgcggag aatcccgaac cggcgcccgg acgctacgac ttcgaggtcc tcgacgagcg 51180
    cgtcgccctg atgcgcgcga cgggggccac gcccgtcctg accctgtgtg ccgcccccga 51240
    ctggatgaag ggcggccggc ccggccgcac cgactggtcg cgactggaga ccgcccccga 51300
    cccccggcac tacgcggact tcgcccggct cgcgggcgtg atcgcccaac gctacccgga 51360
    catcaggcac ttcctcgtgt ggaacgagct gaagggcttc tacgacgagg acaggcggcg 51420
    ctgggattat gagggataca cccggctgta caacctcgtc cacgccgagc tgaagcggcg 51480
    gaacccgcgc aatctggtgg gcggccccta tgcggtggtc gaccacgacc cgcccgccga 51540
    ggacgcggcg gaccgctcgc gcgaactgcg cggtccctgg ggcgagctgg accagcgctc 51600
    cgccgacgtc atccgctatt ggaacgccca caaggcgggc gcggacttcg tcgtcgtcga 51660
    cgggtccagc tacacccgcg agggccaccg ggcgattccg gacgagttcg ccgccaccga 51720
    gaagttcgcc gacgtcaccc gctgggtcag gagcgtgacc ggactcccgg tgtggtgggc 51780
    cgagtggtac gtcgagccgc ccgccgagga cgaccggccg ggcggccggg acggctgggg 51840
    cgaggggcac cgcaccgccg tgcaggccac cgcgatgatg cggctggcgg agagcggcgc 51900
    gtcggccgcc ttctactgga acccgcagcg gaccgggaag gcgtgccccg gctgcctgtg 51960
    gcggagcacc cacttgcgcg acgggggagg ggagttgccc atggcgggtc tcctgagccg 52020
    gttcgctcgc gaattccctc cgggcaccgc cttccggccg gtcgccgtca cctgcgggag 52080
    cggtgacagg gtcgaggccc tcgccgacga ggccgccgtg ctcgtcgtca acaccgagtg 52140
    ccggccggtg gccgccaggg tggacgggca ggcgctgtcc ctcgcgccgt acgaggtgcg 52200
    ctggctgacc cgcccgtaat ccagtggggc ggcgcacggg cgcggacagg gaattgcgga 52260
    acagggaagt tcacgaataa ggagaacgcg ggaaagcgct cgggcggagc gtgaaacccc 52320
    tgtcggcgct cacgatatcc acccagctga tttgcaggtg aaacgggcgg tcgcctcgac 52380
    ggtgccgccc gtttcctgtt gcccgaaagg gcaatcgggc atcagcagga gagattgccg 52440
    cccggcgcca cgccgaggat cttggtgaat cgctggtagt tcgcgacgcg gctctggacc 52500
    tgggcggggt tgtggccgtc gcactccagg gcgccgttga tgctgcggat ggtctgcccg 52560
    aagccgcggt ggttgaccat ggcctcgtgc ggggtcatgg tgccggggcc gcgctgggtg 52620
    ttccagtacc acaggccggt cttccaggag acggccgcgt ccttctgcac cagcgagggg 52680
    ttgtggagca ggtcgatgcc gagggcgtca cccgccgcct tgtagttgaa gttccagctg 52740
    atctggagcg ggccgcgacc gtagtaggcg gcctggcctg ccggacagcc gtagggccgg 52800
    ctccggtcgc agtagtgggg gtagttggcg gtgttctgct ccacgacata gaccagtccg 52860
    ccggtctcgt gggcgacgtt ggcgaggaag gcggcggcct cctgcttccg gacctcggcg 52920
    ctgccggtgc ccgcgaagcc cgggtacgcc ttgagcgcgg cgaccaggcc cttgtacgta 52980
    tagaacgcgt tccgcttcgg gaacatctgc ttgaactggg cctcgctcac ggggaatgcg 53040
    gcggcctgcg aggtgccgcc gtgcggtgcg gccgcgctcg ccgtggtggc gggggcgagg 53100
    acggatatgc cgaccagtgc cagagcggcc ggcagcaggg cggcgatgcg atttcttctc 53160
    atggcggctc ccgtggggga aagggtgagt gacgcccgcc gacggtgaat cgggcccgtt 53220
    gggcgccttc gcgtcatcgc gcagtgaata actcccgtga gtttggtgtc aatggcatgc 53280
    gccgtgtccg gccgaaccag gtgcactgag caatgagttc aggacaactg cggccgatag 53340
    ggcttgcggg agcaacgagg accatgacct catatgccgg aagccggaca cgtgccgaga 53400
    aatgccgctg tcctgtggct ccttgggtga cctgtgaaac ccggctggct catgaacgag 53460
    ccgattgaac gagccgattg aacaagccga tgaacaagga 53500
    <210> SEQ ID NO 77
    <400> SEQUENCE: 77
    000
    <210> SEQ ID NO 78
    <400> SEQUENCE: 78
    000
    <210> SEQ ID NO 79
    <211> LENGTH: 621
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 79
    gtgccccgga atctcctgga cgtcagggac gtccaccaca cctacggcac ccgcagggtg 60
    ctgcgcggca tcgacttgtc cctgcgcccc ggaacgctgg ccggcgtcgt gggcgagaac 120
    ggcgcgggga agtcgacgct cctgaagatc ctcgccggtg agctgcggcc gcagcgcggg 180
    caggtccact acggcggccg gttcggttac tgcccgcagc atctcgtcct gaaccaggct 240
    ctcaccgtcc gccagcacct ggagtatttc cgggtggcgt acggcctcgc caccctcagc 300
    catgccgaga gaatcatgga cgtgctccgg ctgtccgact acggggacga gcgggtcagc 360
    gtgctcagcg gcggcacgaa acagaagctc aacctcacac tggccctgat gcacgaccct 420
    gacctgctcc tcctggacga gccctaccag ggcttcgact gggacaccca ccagcggttc 480
    tggagcctgg ccgccggtct gcgcgaacgc gggcggtccg tgctggtcgt ctcccacctg 540
    gcctacgacg ccgaacggct cgacgaactc cggcacctcg acggcggact gctgcactca 600
    aggacgacgg caccggtatg a 621
    <210> SEQ ID NO 80
    <211> LENGTH: 1350
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 80
    atgaccggcg ccctcctctg tcccagggac atctccccga ctttcgcggc gaacgcggac 60
    ttcatccggc agcgcatcga ccgcacggcc gaacgcatca accatcacat cgaccgcctg 120
    tgcccgaacg cgtcggcaga tgcttgctcc acgtggctgc caccgggcca catcaccggg 180
    acgaccgggc ctggcacgcc gccggtcgtc gcccagcggc tgcaccgggc cctgacctct 240
    cccgtccgcc atctgaccga tgccggagga cagcgctggc ggccggtgct ggcctgggag 300
    gccatcggtc tgatgggagg tgacagcgaa tcctgcggcc tgctgatcgc ggcgagtgag 360
    ctgctccaca ccggatccct catcgtcgac gacgtccagg acgcctcacc gctgcgccgc 420
    ggacaaccgg ccgtgcacac catgttcggc atgccgactg cggtgaacgc gggtacggcc 480
    gcctatttcc tctgggagcg ggccgttcag ctcacctttc ccgacgacgc ctcgcggtgc 540
    ggggagttgc gggcactggg tctggccgcg ctgcgagcgg ctcacgccgg tcaggcactg 600
    gatctccaag gtcaccggga agagatggac caggccgtgg ccggcgacga ccggcacact 660
    gtgctggaac tggtccgtct gacacaccgg ttgaagtccg gggccccggt ctcggcggcc 720
    atggaggcag caggggtcgt cacgggtgcc gagccggaac tgcggagagc actgggggct 780
    ttcggttcag cggtgggcac cgcctaccag atcgccgacg acgtcgccga cctgagcggt 840
    gtcacacggg cgggggcacc gacgaagcag gccaccgagg acctgcggag cggcaaggtc 900
    accatgccac tggcccacgc cgtggtccgg ctgccgcaga gcccggctga accagctctg 960
    gcagcaggtc aaggacggct cgggcagcgc gacggcggtg gccgaggtgt gccgggacct 1020
    gaacgcctgc ggagcggtga gagcctgcct ggaggaagcc gaccagttgg tgagcgacgc 1080
    gtggaacaag ctcgagcccc tgctgccgtc ggcaggccac agccacttgt tgtacgagct 1140
    ggccctctcg gtcgtccggc gcgaccaggt cgcctgagga gaggaagccg tgccccggaa 1200
    tctcctggac gtcagggacg tccaccacac ctacggcacc cgcagggtgc tgcgcggcat 1260
    cgacttgtcc ctgcgccccg gaacgctggc cggcgtcgtg ggcgagaacg gcgcggggaa 1320
    gtcgacgctc ctgaagatcc tcgccggtga 1350
    <210> SEQ ID NO 81
    <211> LENGTH: 1425
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 81
    atgcgggcgg gaggacgcct gatgaacgga gattcgaagg cggactccgt tgtgccgatg 60
    gctcctgact gcagtcggtt gctcgggcat gcccgctccg tgcggttctt gatggatctg 120
    agggagatcg cggcccggct cggcaaggcg gggcctgtgg tcagggtgaa ggcgggcccg 180
    ttcgtcgggt acctgctcaa tgactcctcg ctgatccggc aggctgctcg cgacgaggac 240
    accttcatgt tctggggcag ggaaccccat gtgcgggtga tcgtaagaga gggactgctg 300
    agcaccgagg gaacggtcca ccgtgaccgg cgggcggtga tgaggcccgc gttcgccgtg 360
    ccccggccgg cggacctcgg ggcgtccgtg cggaccgaga cccgaagtct gctcgccacc 420
    cttcccgcgg accgtccggt cgacatgagc cacgagatga cccggctcac tttccatctc 480
    gccgtcaggt gcgtcttgcg cagcgaggtc tcgcccggaa cgctgaccgc gctggccgcg 540
    gcgcatgcca cgctgtcgca ggtcggggcc ttgcggtttc tcctgtcccc gtggccttgg 600
    gtacccgtgc caagacaacg cgcgctccgc cgtgccctcg cggtgctgga cgaggccacc 660
    cgggaagtcc tcgcgcgcca tcggccggct gaggacgggt gtgatgtggt ctccctgctg 720
    aagcaagcgt ggcgcgagcc ctcggaagcg gcggtgcagg acgtgcgcac gctgctgttc 780
    accggcgggg aggccaccgc ctcgacactc gcgtgggcct gctacgaact gggccgccac 840
    ccccaccatc aacaggcctt gcaggaggag gccgacgcgg cactcggcct ggggcactcg 900
    caggccggcg tcggaataaa ccggttgccc cggacagccg ccttcgtcaa ggaagtcatc 960
    cggcttcacg gcctcccggt ccttgtgcgt tgcacgcgcc gcgagacgtg gctgggcggc 1020
    caccggctgc ccgcgaaggc gacggtgttc ctccacctcg gggcgatgag ccgtgacccc 1080
    gcccacttcg aacggcctga tgtcttcgat ccgacgcgct ggatgccgga cgctcagccc 1140
    tcggtctccc ctgctgcctg gcttccctat gcgctcgggc cccgctactg ccctggtgcc 1200
    gcggtggccg acgtcatggt gcccgtcgcg ctcgccactc tggtcgccac gcggaccgta 1260
    cgcacggcgc gtccgggccg gacggtgcgc tccggcttcg aactcgccgc gatgccacgc 1320
    ggcctgacca tgatcgccgc actccgcgag ccgtgcccga gcagtccctc cgcggcatcg 1380
    ggccggcgac cccatgaggg agcctcgtcc atcgcccgcc cctga 1425
    <210> SEQ ID NO 82
    <211> LENGTH: 1821
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 82
    atgaagatct ctcgaatagg ccgcgcgtca tccatcgccg ccctggtgac aaccgcactc 60
    gctttcacgg cagttggcac cgtcgctccc acggccgtcg ccgactcccg cgcggccgcc 120
    gcttccggga cgcagaatga ccacccgagc tcggggcagg gcacctccac ctctgagctc 180
    cggcgcaagg gcctggtccc gtcgagtctc gtggccaagc ccatcacccg cagcgagacc 240
    ctcagacgcg ccgccagctg gttcggcaag ggtctccact acagcgggga caacacctat 300
    cagggctggc gcacggactg ctccggcttc gtctccatgg cctggggact gcccggcccg 360
    ggtgagacca ccgattcgtt cattcccggg ggcgtggccc acgaaatctc caaggacgaa 420
    ctgaagcccg gcgacgcgct caacaacaag gcgctcggca acgacggtca cgtcgtcctg 480
    ttcgagaagt gggccgattc ctcccagtcc tcctactggg gttatgagtt cagcagcagc 540
    ggtctgcacc accgtgtgat cccgtacgcc tacttctcca ggtccgagca gtaccgcccg 600
    atccgcttca acaccatcgt ggacgacgac acggccgcag ggcccgccga ggacaacgcc 660
    cgggtccagg gtgacttcga cggcgacggc cgcgacgacg tggcggtgct ctacgactac 720
    ggcaggaagg acgaccgcag tcgctcggcc ctgtggacgt tcaacagcaa cggcagcggt 780
    ttcaacagtc ccaagcaggt gtgggacagc gggacgtcgg agagctggaa ctgggcctcc 840
    agcaagttga cggtcggtga cttcaacggc gacggcaagg ccgacatcgg cgtcctctac 900
    aacatgggcg cgaccgagga cggccgcaac cgcaccaagc tgttcgtgtt caccagcacc 960
    ggcagcggat tcgccgcccc ggtcaaggtc tgggacagca acgacgaccc cgtgaagagc 1020
    tggaactgga acgccagcaa gctcaccgtc ggcgacttca acggcgacgg caaggccgac 1080
    atcggggtgc tgtacgacta cggcaaggac gacgaccaca accggacagg gctctggacg 1140
    ttcaccagca ccggcagcgg gttcaacagc ccgaagcagg tgtgggacag caacaacgac 1200
    cccgtgaaga gctggaactg ggaagccagc aagcccgtct ccggggactt caacggcgac 1260
    ggcaaggccg acatcggcgt cctctacgac tacggcaaga ccgactccgg cagccgcacc 1320
    ggactctgga cgttcaacgg caatggcaac gggttcaaca gcccgaagca ggtgtgggac 1380
    agcaacaacg accccgtgaa gagctggaac tgggaagccg gcaagcccgt ttccggcgac 1440
    ttcaacggcg acggcaagag cgacatcggc gtcctctacg acatgggtcg caccgaggac 1500
    ggccgcaacc gcaccaagct gttcaccttc accggcacgg cgaccggttt caacagcccg 1560
    gtcaaggtgt gggacagcaa cgacgacccc gtgaagagct ggaacgcgtc cgcgagcaag 1620
    cccgtcgcag gtgacttcaa cggcgacggc aaggcggaca tcggcgtcct ctacgactac 1680
    ggcaagaccg actccggcaa ccgcagcgga ctgtggacct tcaccagcaa cggcagcggc 1740
    agcgacagcc ccaagcttgg ctgggacagc agcgcggacc ccgtcaagag ctggaactgg 1800
    agcgcgagca agctcggctg a 1821
    <210> SEQ ID NO 83
    <211> LENGTH: 2466
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 83
    atgcgaacca tacgaatacg aagaacgaac ggcgtggcct tcgccgccgc tgccgccctg 60
    atggccctcg tcgcctccgg caccgccacg gtccaggccg cgccctcgca cgccggaccc 120
    tccggcacca ctccgatcac ctaccgtggc ctcaccctcg acataccctc cgggtggccg 180
    gtcgtggacc tggagaaaga cccgcacacg tgtgtgcggt tcgaccgcca cacggtgtac 240
    ttgggccacc ccggcaccga acagtcctgc ccctcccatc tggtcgcgga caagacggac 300
    gccctgatat tggagccgat caccggagcg ggcggccagg acgcctccca cgcgctgcgc 360
    atccctgccg gggccccgat gccgcacgag ctgccggtga cgtacgacca cgagacgaag 420
    gtcgccgtcg aaggcgccgg agtcatggtc acgtcctcct acggcacgtc cagtacaacg 480
    gtcgccgccg tcctcggctc ggcccgcacg gacgcgacag ccaagccgac ccccctgccc 540
    ggcaaggcgg gcaggggcct cgcggctcca ccggttgccg ccgtcgcggc cgacaaggga 600
    tacacagggc tgggcttcga gtcctgcacc gccccttcgt ccgccgcgat gaaggcatgg 660
    aaggcctcgt cgccctacgg ggccgtcggc atctacatcg gcggtcgcaa gcggggctgt 720
    gcgcaaccgc agctcaccgg cgactgggtg cgtcagcaga ccgccgacgg ctggcacctg 780
    ctgcctctct tcgtggacct ccaggccggc gacatctctc cggccaccgc ggacgcgcag 840
    ggccgcgagt ccgcggacgc cgccgtggcc aaggcggcgg acctgggcct gggccccggg 900
    acggtcatct acagcgacat ggagcactac gacagccgct cgtaccgggc ccgggtcatc 960
    gactacgtgt cggggtggac cagccgcctc cacgaacatg gctaccgctc cggtgtgtac 1020
    gcgggtgaaa cgagcggcat cccggacctc gcctcggtgg ccgacgacac caactacgca 1080
    tcacccgacg tgctgtggtc ggcgaactgg aacctcaagg ccgatgtgtc ggacgcgtcg 1140
    atgggacttc cgggccccgg ctactggccc aatgggcggc gcatccacca gtaccgcggc 1200
    caggtgaacg acacctacgg cggtgtcacc ctcgccatcg accgcgacta cgtcgatgtc 1260
    gccgcggact cggccctgcc cgcacccggc ggagaggacg gttcctcgcg cgtcaagggc 1320
    gacttcgacg gcgacggccg cgacgacgtg gccgtgctgt acgactacgg caaggagggc 1380
    ggcgtcagcc ggtccgcgct gtggacgttc gcggggaccg gcagcggctt cggcgccccg 1440
    aagaaggtgt gggacagcgg atcggacagc tggagttggt cggccgccaa gctgacggcc 1500
    ggcgatttca acggagacgg caaggccgac atcgcggtcc tgtacgacat gggtcgcact 1560
    gaggacggcc gcaaccgcac caagttgtac gagttcacca gcaccggcag cggattcaac 1620
    agcccggtca aggtctggga cagcaacgac gaccccgtca agagctggaa ctgggcctcc 1680
    agcaagctga ccgtcggcga cttcgacggc gacggcaagg ccgacatcgc ggttctgtac 1740
    gactacggca gggacggcga ccgcagccgt acgggcctgt ggaccttcac cagcaccggt 1800
    gccgccttca ccggccccaa gctggtgtgg gacagcaaca acgacccggt caagagctgg 1860
    aactggaacg ccagcaagcc caccgtcggc gacttcaacg gcgacggcaa ggccgacatc 1920
    ggcgtcctct acgacatggg tcgcaccgag gacggccgca accgcaccaa gctgttcacc 1980
    ttcaccggca cggcgaccgg tttcaacagc ccggtcaagg tgtgggacag caacgacgac 2040
    cccgtgaaga gctggaactg ggacgccgtc aaggtagtgg gaggcgactt caacggcgac 2100
    ggcaagagcg acatcggggt gttgtacgac tacggcaagg acggcgaccg cagccgcacc 2160
    ggactgtgga ccttcaccag caacggcagc gggttcaaca gcccgaagca ggtgtgggac 2220
    agcagcaacg acccggtgaa gagctggaac tgggccgcga gcaagccggt cgcaggggac 2280
    ttcaacggcg acggaaaaac ggatatcggc gtgctctacg actacggcag gaccgattcc 2340
    ggcaatcgca ccggactgtg gaccttcacc agcgacggca ccggattcgg tacacccctc 2400
    ctgggctggg acagcgtgac ggatgccgtg aagagctgga actggcgtgc cagcaaggtg 2460
    agttga 2466
    <210> SEQ ID NO 84
    <211> LENGTH: 1575
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 84
    gtggatccct tgacgcgcaa gacccgcacc ccccgcaaga agggcagacg cgcgagcgcg 60
    gcggcgatgt cggcctccgg catgctgctc gccttggtgg ccaccgccgc ccccgtcccc 120
    gcccaggcgg catcactcgc cacctgggaa aagatggccc agtgcgagag cagcggggac 180
    tggggataca accagccacc gtactacggc ggcctgcaat tcctggagag tacgtgggtg 240
    gcgtaccacg gaacggacta tgcgccatac ccctatcagg ccaccaagga acagcagatc 300
    cgggtcgcgc agcggctcct cgacaatgag ggcgcggctc cctggccgta ctgcggaaag 360
    aaggtggggc tggctgacga cgacgcacgc cccttccccg acgcgccgga cgacgacgcc 420
    tccgcccgga tcaacggtga cttcgacggc gacggatgcg acgacgtggc cgtgctctat 480
    gactacggca aggagggcgg cgtcagccgg tccgggctgt ggacgttctc cgggagcggt 540
    accggcctcg gcagcccgaa gaaggtgtgg gacagcggat cggccagctg gagttggtcg 600
    gccgccaaac tggccgtcgg cgatttcaac ggcgacggca aggccgacat cgcggtcctg 660
    tacgacatgg gccgcactga ggacggccgc aaccgcacca agttgtacga gttcaccagc 720
    accggcagcg gattcaacag cccggtcaag gtctgggaca gcaacgacga ccccgtcaag 780
    agctggaact ggaacgccgg caagctcacc gtcggcgact tcaacggtga cggcaagacc 840
    gacatcggcg tcctctacga ctccggcaag accgactccg gcaaccgcac cggactgtgg 900
    accttcacca gcaacggcac tggattcaac agcccgaaac aggtgtggga cagcaagagc 960
    gacccggtga aaagctggaa ctgggccgcg agcaagccgg tcgcgggcga tttcaacggt 1020
    gacggcaaga ccgatatcgg ggtgctttac gactacggca aagatggcga ccgcagccgc 1080
    accggactgt ggaccttcac cagcacgggc agcggattca acagccccaa gcagacctgg 1140
    gacagcgggt cggaaagctg gagatggtcg gcggccaagg tggtcggcgg cgacttcaac 1200
    ggtgacggca aggccgacat cggggtgctg tacgacctcg gcaggaacgg cgaccgcaac 1260
    cgcaccgaac tgttcacgtt cgcgggcaac ggcaccggcc tcaacacacc ggccaaggtg 1320
    tgggacagcc aggacgacag cgcggtgaag agctggaact gggccgcgag caagccggtc 1380
    gcaggtgact tcaacggcga cggaaagacg gatatcggcg tcctctacga ctacggccag 1440
    accgactccg gcaaccgcac cgggctgtgg accttcacca gcgacggcag tggattcgcc 1500
    ggccccaagc tcacctggga cagccggacc gaccccgtca agagctggaa ctggaacatg 1560
    agcaagaccg gctga 1575
    <210> SEQ ID NO 85
    <211> LENGTH: 2175
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 85
    atgaaatacc gaccgggaac actgctcact tccataacag tcttgtgtgc cctgctcgtt 60
    ccggtgcgtt cggcggctca ggcggccagg cccgagcagg gacgttccgt ggtggccgcg 120
    gccgccgtac tggagcaaag tccgccgacg ctgctcgccg agccggaaat gcgcgtcgtc 180
    tcctggaaca tctgcggtga ggcgggcggg gtgcgcgggg agggcggcta ctgcccctac 240
    cgcaacgatc cccaggcgaa agtcgaccag atcgcgcagg tggtcgcgga gcgcagtgcc 300
    aatgtcgtca tgctccagga agtgtgcggc gaggcgcccg gcagccatat ggagcggctg 360
    cgcgcggccc tgggcagcgg atggtcgatc gcgcacgccc cgggggcccg cccggacgac 420
    ggaaccacga actgccgggg cgggctcagc ggcatattgg gcgtggggat cgcggtgaag 480
    gggcgcgtca ccgacaccac cgcgacgaac accgtgcccg ggggcggcgg tgacaagcag 540
    accctgccca tcctctgtgt acgtgtcgag ggctggtcgt ccaggatctg caccacccac 600
    atcctgtccg accctgccga tccgcgcagg ccggggcaga tccagaacgt caagaacgag 660
    atctggccgg accgctatca gctggtgctc ggcggcgact tcaacatgtt ccccgactcc 720
    gccgggctca agccgatctc ggacgaattc gacgagtgcg accgccgctc ctacggcgcc 780
    ggtgacatgg tcaacgaggt cacccatcac tcctgggaga aaaagggcgg acacatatgg 840
    cgcaagcgtg accacatctt cgcctcgtgg ggagagtccg ggagccagtt cacatcctgc 900
    gaggtcgacc ggacccggat ggacaccacc gagaacgcgc ccgaaagcgg tccgcccaac 960
    gggtattcgg accatgcgcc gatcatcggc tacctcaagc cgccgcggca cctgagcacg 1020
    tccggggact tcgacggcga cggcaaggcc gacctcgcgg tcctctacgg gcaggggaag 1080
    accccggacg gccacaaccg gtccagcctg tggatctcag gcggttccgg taccggagcg 1140
    gagaccggat tcgccgcgcc gcgcgaggtc tgggacagcg gtgccgacag ctggaactgg 1200
    tccgcgagcg cgctgacctc cggggacttc gacggcgacg gcaagaccga catcggcgtc 1260
    ctctacaact acggcaggga cggcgaccgc aaccgcaccg cgctgtggac cttcaagggg 1320
    acatcgaacg gcttcgaggc gccccgcaag gtgtgggaca gccacgacga cacggccgtt 1380
    cccagctgga actggtccac gagcaagctc gtcgcgggcg atttcaacgg cgacggcaaa 1440
    gcggacatcg gcgtcctgta cgactacggc aggaccgcct ccggcaaccg caccggactg 1500
    tggaccttca ccagcaccgg caccggattc ggcaagcccc acctggcgtg ggacagctcc 1560
    accgacccgg tgaagagctg gaactgggcc gcgagcaagc cggtcgcagg tgacttcaac 1620
    ggcgatggca agaccgacat cggcgtcctc tacgactacg gcaaccacac cgccctatgg 1680
    accttcacca gcaacggcac cggattcgcc ggccccaagc aggcctggga cagcggaccg 1740
    gagaactgga actggtccgc cgccaagccg gtcgccgggg acttcgacgg cgacggcagg 1800
    accgacatcg cggtcctgta cgactacggc aggaccgcct ccggcaaccg caccggactg 1860
    tggaccttca ccggcaccgg caccggattc ggcaagcccc acctggcgtg ggacagctcc 1920
    accgacccgg tgaagagctg gaactgggcc gcgagcgagc cggtcgctgg tgacttcaac 1980
    ggggacggca gggccgacct cgcggtgatg tacgactacg ggaacgcgac caacggccgc 2040
    aaccgcaccg cgctgtggtc cttcaccagc cgcggcacgg acttcgccgc cccgcgggcg 2100
    aactgggaca gcagcaacgc cgctgaccag ctgaaatcgg gcgagctgag ggcggctccg 2160
    ctcagcgggt cctag 2175
    <210> SEQ ID NO 86
    <211> LENGTH: 27
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A primer
    <400> SEQUENCE: 86
    tcagaattcg gatccgaggg ccggagt 27
    <210> SEQ ID NO 87
    <211> LENGTH: 18
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 87
    acctactgcc tcgatgcc 18
    <210> SEQ ID NO 88
    <211> LENGTH: 15
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 88
    ctgatccttc aagcg 15
    <210> SEQ ID NO 89
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Amycolatopsis mediterranei
    <400> SEQUENCE: 89
    gcgtccgtgc tgcgcgcgca 20
    <210> SEQ ID NO 90
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Amycolatopsis mediterranei
    <400> SEQUENCE: 90
    tgcgcgcgca gcacggacgc 20
    <210> SEQ ID NO 91
    <211> LENGTH: 6
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 91
    gaaagg 6
    <210> SEQ ID NO 92
    <211> LENGTH: 80
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A conserved motif
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)...(80)
    <223> OTHER INFORMATION: Where present in this sequence, Xaa represents
    variable amino acid.
    <400> SEQUENCE: 92
    Gly Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa
    1 5 10 15
    Xaa Xaa Xaa Xaa Xaa Glu Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    20 25 30
    Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    35 40 45
    Xaa Xaa Xaa Lys Xaa Xaa Xaa Xaa Gly Glu Gly Gly Xaa Xaa Xaa Xaa
    50 55 60
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
    65 70 75 80
    <210> SEQ ID NO 93
    <211> LENGTH: 6
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 93
    ggaacg 6
    <210> SEQ ID NO 94
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A primer
    <400> SEQUENCE: 94
    gggaattcca tatgatgcag tccatgtcac 30
    <210> SEQ ID NO 95
    <211> LENGTH: 30
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A primer
    <400> SEQUENCE: 95
    gggaattcaa gctttcattc cgccggggtc 30
    <210> SEQ ID NO 96
    <211> LENGTH: 18331
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 96
    ggatcccgat cgtctcggac atgaccggcg accttctcgg cgcgcgggag gcccaggacc 60
    ccgcctactg ggtgtcccac atccgccgcg cggtgcgctt ccacgaccag atccgccgtc 120
    tgcagcgcta cggggccggg gccttcgtcg aggtcggccc ggacacggtg ctcagctcgg 180
    ccggccaggc gtgcctgacg gaccaggcgg gcaggagcgc gcccgtcctg gtgtccctcg 240
    cgcacgccga gcgcgcggag gtgcccgcgc tcctgaccgc tctggccacc ctgcacaccc 300
    gtggcgtggc cgtggactgg cgggcgtggt tcggcgacgg gccgcgcgcg gccggcctgc 360
    ccacatacgc gttccagaag cagcactact ggccgtcggg ccccaccggt tggcggtccg 420
    ggcccgcccc cgtacccctg ccccaggccg gaacggagga cgccgaaagg cccggtcgcg 480
    ccgcggagtg gcgggcgctg ccgcccggtg agcggtacga cgcgctgctg cggatggtgc 540
    gcggcgaagc cgccgccgtg atggggcacg ccgggccgga ggcggtggag ccggagcgcg 600
    gcttcctcga ccacggcttc gactcggtga tggccgtgaa gctgcgcgac cgtctcgtgg 660
    ccgggacggg gcgggagctg ccgacgaccc tgctgttcga ccaccccacg cccgcggccg 720
    tcgccgacta cctgctggcg gggacgggcg aggccgagac ggcgccgtcc gtgtccctgt 780
    cggaccagct cgaccgcctg gaggccgacc tcgcgcggct gccggccgac gaccggcagc 840
    gcgcccgcgt cgccgagcgg ctcaagggcc tgctcgcggt ccacgcgccg gaccggggcg 900
    ccgggagcga ggacgcgccg gaccaggacg cgctggacac ggcgaccgac gacgagatgt 960
    tcgagctgat cgagaaggaa ctccgccgtg gatgagacca acgagaccaa actccgcgag 1020
    tacctgcggc tggtcacggc cgatctgcgg cgaacccgca ggcagttgga ggaggccgag 1080
    gacgcggccc gcgagcccgt cgcgatcgtg ggcatggcgt gccgcttccc cggggacgtg 1140
    gcatcgccgg acgacctgtg gcagctggtc gccgagggcc gggacgccgt caccgagttc 1200
    cccgccgacc ggggctggga cgtcgacgcc gtctacgacc ccgagccggg caccccgggc 1260
    aggacgtacg cgcgccacgg cggcttcctc aaggacgccg ccggattcga cgccgccttc 1320
    ttcggcatca cgccgcgcga ggcgctcgcc atggacccgc agcagcgcat gatcatggag 1380
    gtctcctggg aggcgttcga gcaggcgggc ctcgacgcga ccaccctgcg gggcgaggac 1440
    gtcggcgtct tcgtcggctc caacagcaac gactacctga tcaacgtgct cgacgcgcgg 1500
    gacgtcgccg agggcttcat cgggaccggc aactccgcca gcatcctctc cggccgcgtc 1560
    gcctacacct tcggcttcga gggcccggcc gtgtccgtcg acaccgcctg ctcctcctcg 1620
    ctggtcgcgc tgcacctggc cgcgcagtcc ctgcggcagg gggagtgctc cctggcgctg 1680
    gcgggcggcg cgacggtgat ggccacgccg accgccttca tcgagttcag ccgccagcgg 1740
    ggcctggccc ccgacggccg ctgcaagtcc ttctcggcga ccgccgacgg caccacctgg 1800
    tccgagggcg cggccgtgct gctgctggcc cggctctcgg acgcccgccg cctgggctac 1860
    cccgtgcacg cggtcatccg gggcagcgcc gtcaaccagg acggcgcgag cgcgggcctg 1920
    accgcgccca acggaccggc gcaacagcgg gtgatccggc aggcactggc caacgcacgg 1980
    ctgacggccg acagcgtcga cgcggtcgag gcacacggca ccggcacccc gctgggcgac 2040
    ccgatcgagg cccaggccct cctcgccacc tacgggcggg cccgcggcga gggcaggccg 2100
    ctgtggctgg gctcgctgaa gtcgaacctg ggccacaccc agtccgcggc cggcgcgggc 2160
    ggcgtcatca agatggtgat ggccatgcgg cacgggacgc tgccccgcac gctgcacctc 2220
    acggagccca ccccgcgcgt cgactggtcc gccggtgacg tacggctgct gaccgaggcc 2280
    caggactggc cggacaccgg acagccgcgc cgtgcggccg tctcgtcctt cggcgtcagc 2340
    ggcaccaacg cccatgtgat cctggagggc ccgcccgccg aggaggcacc ggacgcgccg 2400
    ctgccggacg tctcctcgca gccgcggggc ccgctgccgt gggtcgtctc cggccgcagc 2460
    gaggcggccg tccgagcgca ggccgagcgc ctggcggccc acctgaccgc gcgcccgcac 2520
    ctggcaccgg ccgacgtggc caccgcgctg gccaccacgc gggcggcctt cgaccaccgg 2580
    gccgccgtcg tcggccggga ccgtgaggaa ctgctcgccg gcctcgcggc cctggccacc 2640
    ggaacccgcg cgcccggcct ggtcaccggc cggaccccgc cgtccggcgg caaggccgcc 2700
    ttcctcttca ccggacaggg cagccagcag cccggcatgg gccgcgaact ggcggctcac 2760
    agcaccgtgt tcgccgacgc cctggacgag gtctgcgccc agctcgaccg gcacctcgac 2820
    cggccgctgc gcgaggtgct gttcgccgcg gacggcacgc ccgaggccgc cctgctcgac 2880
    acgacggcct acacccagcc cgcgctgttc gccgtcgagg tcgcgctgct gcggctgctg 2940
    gaggactggg gcttgcggcc cggcatggtc gcgggccact cggtcggcga actgaccgcc 3000
    gcctacgccg ccggggtctg gtcgctcgcc gacgcctgcg ccctggtcgc cgcccgcggc 3060
    cggctgaccc aggcactgcc cgcgggcggc gccatggtcg ccgtgcaggc gaccgaggac 3120
    gaggtgcgcg cccaactcgc cgacggccgc cccggcgtgg acatcgccgc cgtcaacgga 3180
    ccggaagcgg tggtgctgtc cggcgacgag gccgccgtca cggacctggc gcgcgagtgg 3240
    gccgcccgcg gccgggagac caggaggctg cgggtcagcc acgccttcca ctccgcccac 3300
    ctggacgcca tgaccgaggc gttcgccgag gtcgcacgag gggtgtccta cagcgcgccg 3360
    tccctcccgg tggtctccac gctcaccggg gcccccgtca ccgacgagct ccgcaggccg 3420
    gaacactggg tgcggcacgt ccgggagacg gtgcgcttcc acgacgcggt ccgcgccctg 3480
    cgcgaccgcg gggccaccgc gttcctggag gtcgggcccg gcggcgtgct gacggccgcg 3540
    gcacgccgat gcctgcccga cgccgccccc gagacgttcg tccccgtgct gcggcgccgc 3600
    aggcccgaac ccgagtccgt gctgacggcc gtcgcgcagg cccacacgat cggcctctcg 3660
    ccggcgtggg accgcctgct gcccaaggcc cggacgcgcg tggacctgcc cacgtacgcc 3720
    ttccagcgcg gccactactg gctggcgggc atggccggag cgggcaccgc gcggccggtg 3780
    cggccggaag tgcaggagcc caccgccccc tccggtacgc cgccgctgtc gcgacggctg 3840
    gccgacgcgt cggaggagga gcgcggccac ctgctgctga cgctggtacg cgagcagtcg 3900
    gccaccgtga tgggcggcgt cgaccccgcg caggtcgaac ccgaccgccc cttcctggag 3960
    ctcggcttcg actccctgat gggcgtcgag ctgcgcaccg cgctcgccgc cgactgcgca 4020
    ctgcccctgc cgcccggcct gatcttcgac caccccacgc ccgccgccct ggccgccttc 4080
    ctcggcgagc agctcgcggc ggcggcctcc ggcaccccca cggcggcggc accctcgccg 4140
    tactccctgg aggcgctgta ccgcaacgcc aacaccctcg accggcccga ggacgcgctc 4200
    gccctcacca aggccgcctc ccggctgcgc ccggtcttcg ccagcgtggc cgaggcgggg 4260
    caggacccgg tcacggtgga gctggcacag gccaccggcc ttccgggcct gatctgctgc 4320
    ccggcacccg tgccgctgta cggggcacag cagtacagcc ggctcgcagc cgccttccgc 4380
    ggcacgcgcg gagtctcggc cctgctcgcc cccggcttct ccccgggcga actgctgccc 4440
    gccgacttcg aggtgatgca ggacttcctc gccgaggggg tccggcggca gaccgacggc 4500
    gcgcccttcg tcctcctggg ccactcctcc gggggctggt tcgcctacag cctggcggcc 4560
    cacctggcgc gcaccgggcc gcgcccggag gccgtcgtgc tgctggacac ctatcagctg 4620
    cacgacccgg cgctgcaccg catgcagcgc gaactcgccc agggcgtcct ggaccgcgag 4680
    gaggacttcg gggcgatgac ggacgtacgg ctgagtgcca tgggcaaata cttcgacttc 4740
    ttcaccgact gggtggccga ggacgccggt gtcccgacgc tgctgctgcg ggcctccgag 4800
    cctctgggcg aggtcgtcga gggccaggag tggcgctcca cctggccgtt cgacagcacg 4860
    gtcctcgaca cggaaggcga ccacttcgcc atggtcaacg accacgcgcc gcggacggcc 4920
    caggccgtga acggctggct gtcgggcctc accggcggaa ggggctgagc gccggtggag 4980
    acacgcaacg ccgaacggcc gtggatacgc agcttccacc ccgctcccca ggcccctgtg 5040
    cggctgctgt gcctgccgca cgccgggggc tccgcgagcg cctacttcgc gctgtcgagg 5100
    gaactggcgc cccgggtgga ggtgctcgcc gtgcagtacc ccgggcggca ggaccggcgc 5160
    gacgagccgc tgctggactc gatcgaggcc ctgcgcgacg gggtcgccga ggccctgacg 5220
    ccctggctgg accggccggt cgccctcttc ggccacagca tgggcgccgt ggtggcctac 5280
    gagctggcgc ggctgctgtg ccaggacgcg ggcgtgccgc tcacccacct cttcgtctcc 5340
    ggacgccggg gatccgaccg aagtctccgt ccttgccgcc gtgttccgga attcaccgtg 5400
    acaccgccgc gcggctcttc ttccgaagtc ctccagatcc ggcacgagtt tgtatccgaa 5460
    cggggttctg cgtgcgaaat actctcttcg aattgggtga catacccccg atcggcaccg 5520
    tacccgagca gatgtacgcc tcggtgatcc gacgggagcg ctacggacag ccccaccagg 5580
    cgttccgcag cgaggtcgtg gacgtgccga aggtggggcc cggtcaggcg ctggtcctcg 5640
    tgatggccgc gggcatcaac tacaacaacg tctgggcctc cctggggcag ccggtcgacg 5700
    tgatctccgc gcggcagaag cagggccaca gcgaggactt ccacatcggc gggtccgagg 5760
    gctccggcgt ggtgtgggcg gtgggggagg gcgtcaccca ggtcgcggtg ggcgacgaag 5820
    tgatcctctc cggctgccag tggacggaga cggccgccga catccggctc ggcgccgacc 5880
    ccatgacctc cggctcgcag tcggtgtggg gatacgaggg caactacggc tccttcgccc 5940
    agttcgccct cgtcgacgac tatcagtgcc accccaagcc gcccggcctg acctgggagg 6000
    aagccgcctg cttcctgctc accggggcca ccgcctaccg ccagctgtgc ggctggcagc 6060
    cgcacgacgt gcggccgggc gacccggtcc tcatctgggg cggggccggc gggctcggct 6120
    ccatggccat ccagatcacc cgggcgcggg gcggcatccc cgtcgccgtg gtctccgacg 6180
    aggagcgggc ccgctactgc cgggagctcg gcgcccaggg caccatcaac cgcctggact 6240
    tcgaccactg gggacggctg cccgacatcg gcgaccacga ggcgatgggc cgctggaccg 6300
    agggtgtacg ggccttcggc cggcgcttct gggaggtgct gggcgagcgc aggtccccgc 6360
    gcatcgtcct ggagcacagc ggccaggcca ccatccccac ctcgatgtac ctgtgcgaca 6420
    acgcgggcat ggtcgtcatc tgcggcggca ccaccggcta caacgccgac atcgacctgc 6480
    gcttcctgtg gatgcgtcag aagcgcttgc agggctcgca cttcgccaac ctgcggcagt 6540
    gccgcgacgt catccacatg gtcgcgaacg gccagctcga cccgtgcctg tcgtggaccg 6600
    gcggcttcga cgacatcggc aaggcacacc agctgatgca cgacaaccag cacccccagg 6660
    gcaaccaggc cgtcctggtc aacgcgccgc ggaccggcct gaccaccttc gcctgaacca 6720
    ccgccccggt gttccgacgt cttcccccca cacttaccga ccaaggagag atcaccatgg 6780
    acaagctcga catcctctgg agcgagcgcg agatccgtgc cgtgctgcag cgctactgcc 6840
    gcgggctcga ccgcctcgac gaggaactgg tcaagtccgc ctaccacgag gacgcgcacg 6900
    acgaccgcgg cgtcatccgc ggcaacgcac acgacttcgt caagcagatc gtcccgctcc 6960
    tgcgcgacgc ctacaccggc accctgcaca ccctgcacgg cagcacgatc gagatcgacg 7020
    gggatgccgc gggcgtggag tcctactgca ccgcctacca ctaccgcgag agcgacggca 7080
    tcaagcgggt ggagcagttc gccgggcgct acgtcgaccg cttcgagcgg cgcgacggcg 7140
    tctggaagat cgcccgccgg ctcgtgctga acgacttcag cctcgcccag gaggtgccgc 7200
    tcgaccccgc cgaggcccag gccggcttca acccctccca ccgcgacctc accgacgcca 7260
    gctaccaggt gctgccgctg cgcggcccgg acgcccccac cctctgagcc gtccggccgc 7320
    cccaactcgc cccacctcac caggagtcac caccgtgtcc gacaccgagc agcacgcgcc 7380
    cacgctgccg cggcagcgca cctgcccctt ctcgccgccg cccgagctcg aggagctgcg 7440
    gcgcaccgat cccatcagca ggatgcggtt cgccgacgac tccccgggat ggctgctgac 7500
    ccgccacgcc gacgtccgcg ccgcgctggc cgaccccggc gtcagctcgc accccggcaa 7560
    ggcaccccag ccctggcgca acctcgcccc cgagatgcgc gccgagcact acctgccggg 7620
    cttcctgatc ttcatggacc cgccggacca cacccgctac cgccgcctgc tcaccaagtg 7680
    gttcaccatg cgggccatcc gcaagctcga acccaggatc gagcagatcg tcaccgagac 7740
    cctcgacgcc atggaggccc agggcggcac cgtcgacctg gtgcagtcct tcgcgctgcc 7800
    gatcccgctg ctggtcatct gcgagctgat gggcatccgc tacgaggagc gcgaggagtt 7860
    catggacatg gtcctgcgac tccaggccct ggacgccacg cccgaggaac tcggggccct 7920
    cggcgccagg atgaacgagt tcatgatgaa gctcgccgcc gccaagcgcg cgaaccccgg 7980
    cgacgacctg ctcagccacc tcgcccacga ccccgacgcc gacccggcgc tcacggatct 8040
    ggagatcgcc ggcatcggcg tgctgatgct catcgcgggg cacgagacct cggccaacat 8100
    gctgggcgtc ggcacctaca ccctgctgga gaacgccgac cagtgggccc tgctccgtga 8160
    cgacatcagc ctgatcgacc gggccgtcga ggagctgctg cgccaccaga ccatcgtcca 8220
    gcagggcctg ccgcgcggcg tcacccggga catggagatc gccgggcacc aggtgaagac 8280
    cggggagtcc ctgctggcct cgctgcccgc cgccaaccgc gaccccgccg tcttccccga 8340
    ccccgaccgc ctcgacatca cgcgcgagca caacccgcac ctcgccttcg gccacggcat 8400
    ccacctctgc ctgggcatgg agctcgcccg ggtggagatg cgccaggcgt ggcgcggcct 8460
    cgtcacgcgc ttccccggcc tgcgcatggc cgccgcgccc gaggacatcc gctggcgcga 8520
    cgaccagatc gtctacggcg tgtacaacct cccggtgacc tgggacgagg ccaagtgacc 8580
    ggccccgagg ccgcggtgcg cgggtgcccc ttcggcgccg gcgaggcgcc cgcgtacccc 8640
    ttccacgccc ccgaccggct ggagcccgac ccgtactggg agccgctgcg ccgcgagcgg 8700
    ccgctgcaac gcgtcacgct gccgtacggc ggcgaggcgt ggctcgccac ccgctatcag 8760
    gacgtgcgcg cggtcttcgc cgaccgcagg ttctcccggc agctcgccgt cgcgcccggc 8820
    gctccgcgct tcctcccgca ccagccgccg ccggacgccg tcctgagcgt cgagggcccc 8880
    gaccacgcgc ggctgcgccg gctggtcggg aaggtcttca cgccgcgccg cgtggaggac 8940
    atgcgtccgc tcatccagcg caccgccgac ggactcctcg acgcgatgga ggagatgggg 9000
    ccgcccgcgg acctggtcga ggacttctcc ctgcccttcg ccgtgtccat gatctgcgag 9060
    ctgctcggcg tgccgcccga ggaccgcaag cggttctgcg tctggtcgga cgcgctgctg 9120
    acgaccaccg cgcacacccc cgcccaggtg cgcgactaca tgatgcagat gcacgactac 9180
    ctcggcgggc tcgtcgcgca gcgccgggtg cggcccaccg cggacctgat cggctccctc 9240
    gtgaccgcgc gcgacgagga ggacaagctc accgagggcg agctggtgcg gctggccgag 9300
    gccatcctca tcgccggcta cgagacctcg gcgagccaga tccccaactt cctctacgtc 9360
    ctcttccgcc acccgcagct gctggagcgg atcaggaacg accacgacct catccccgac 9420
    gccgtcgagg aactgctgcg cttcgtgccc atcggcaccg tggacggctt tccccgtacg 9480
    gccaccgagg acgtcgagct cgggggagtc ctggtcaggg ccggggagac ggtcgtgccg 9540
    tcgatgggcg ccgccaaccg cgaccccgag ctgttcacgg accccgacga gctggacctc 9600
    gcgcggcggc cgaatccgca cctgggcttc ggcgcgggac cgcaccactg cctgggcgcc 9660
    caactggccc gggtggagct ccagatcacg ctcacgacgc tgttccgcag atacccccgc 9720
    ctgcggctgg ccgtgccgga ggagagcctc tcgtggaagg aggggctgat ggtccgcggc 9780
    atgcacacca tgccggtcac ctggtgagga caccggcgtc ctcctgacct tcccggcgtt 9840
    ctcacgccgt cccggcagcc ttccttccga cacgagcgca cagagggtga agcgaccgca 9900
    atgagcacca tcgacgaatg ggaacacagc acgaaggagg cgggcatgga ccccgcggcc 9960
    ctcagacgcc tgaccgatgt ggtgcgggcg aggggcggcg cggcgcagct gtgcgtcatg 10020
    cggcggggca ccgtggtcct ggaccgctcg ttcggctgct cctccgactc cctcttcctc 10080
    gtctacgcgg ccaccaagcc cgtcgccgcc ctcgccgtgc acgcgctcgc cgagcggggc 10140
    ctgatcgggc tggaccggcc ggtggccgaa tactggccgc agttcgcccg gcacggcaag 10200
    ggtgacgtga ccgtccgtca tgtcctccag caccgggccg gggtgccggt cggccggggc 10260
    atcgtgcgca cgatgcgcac cgccggcgac tgggagcgct ccgtgcgcga ccttgagcag 10320
    tcccggccca agtggcccgg cggcgaggtc gccgcctacc acttcatgag tttcggattc 10380
    attctcggcg aactggtgca gcgcgtcacc gggcggtcgt tccgagattt cgtgacttcc 10440
    gagctcttcg ccccacttgg gctgaatgat ttgcacatgg gattgcccgg cagtgcctgg 10500
    ccccggcatg tgcccgcgcg ggccgcccac ccctccgaat ggcccaatca gtggatgagc 10560
    aaccgccgcg gctaccgcca ggccgtcatt ccgtccgccg gtctttccgg aaccgccgca 10620
    caaatggccc gcttttacca gatgcttatg gagggcggct cgctcgacgg catccgcgtg 10680
    ctgcggcccg aaactgtgga ggaagccaga aaaccgtcca atgacggcgg aatcgacgct 10740
    tccctcaagc gtccggtccg ctggtcccac ggattcatgc tcggtggtcc gggcccggac 10800
    ccgcgggggc tgtccaatgt gctgggccgc acgagcgacc cgagcgcctt cgggcacgcg 10860
    ggcaccacgt ccagcgtcgt gtgggccgac cccacgcgcg agctggtcct cgcctacctc 10920
    tccaacatcc agcccgagtt cggagcgggt atcgagcggc tccgcgaggt cagtgacctc 10980
    gcgctcggtg cctgcgaggc aggctgaccc gagccgtgcc gccacggccc ggcgcccgcc 11040
    cgatccgatc gggtccggtg ggggccggcc gggtccgggc ggggacgcac ttcccccggc 11100
    gtccccgccc gggccccggt gcgaaccggg cgcaaaggcg gccgatcgcc cggcgcggcc 11160
    ggatgccccc gaacggtgtg aaacgttctt atcagcctct gaccagcacc gagtgatcta 11220
    ctgcacagcc cgaggccgcg attccggcag tatcttgatc ttgacggggc accaatgcga 11280
    gcgggctatt cgccgcggtt ttccctgacg tcggatgcag atgacaccgg aggagggcca 11340
    gtgctgaatc tgcccaaagg aatggagcgc gcgcatccgc attctccgcc acaggtggga 11400
    atactcggac ccttggaagt ccgctcggcc ggaggtgccg gaacgggagc cgcggtaagc 11460
    ggtattcgcg tacgcacatt gcttgccgcg ttgactgccc gcctggggca ggcgatgtcg 11520
    accgagcgca tcctcaaaga ggtctgggcc gacaacccgc ccgcgaccga tcgcaaggcg 11580
    gtggccgtcg ccgtcctgcg gctgcggcgg gtcctcggcg acaacgaagg acggtggctg 11640
    ctcacccgcc cctccggtta cgtcctggac atccccccgg accacctcga cgccgtacgc 11700
    gcggagaccc tggtgcggga aggccgggcc gccctggccg ccggcgaccc acgcgtcgcg 11760
    gcccgccacc tcacgcgcgc cctcgaccag tggcggggcg agccctacgc ggacgccaac 11820
    gccatctcga ccgtgtccca gcgcatcacg gagctggaga acctcaggtc cgaggccgtc 11880
    caggcgcaca tcgacgccag gctcgaactg ggtcaccacc aggaactggt cggcgaactc 11940
    cgctcgctga ccgccgcgaa ccccctgcac gagccgcact ggctgcagct gatgctcgcc 12000
    ctctaccgct ccggcaagca ggccgaggct ctcgccgcct atatgcagct gcggcaggcg 12060
    ctggccgaga acctgggcat cgacccgggt cgtcagctcc aggaactgca cctgcggatc 12120
    ctgcgcgccg acgcgggcct gctgacgggg tccgggccgg cggcaccggc cgagccactg 12180
    ctcgtacggc agtcctgagg gctcacggcc acccgaagaa cgcgcggtag cacggaacct 12240
    gctgctccag catatggatg ccgtggtgca cacggcgccc ggcggtggcg gccgcgctca 12300
    gcagcgccgt ctcgtgcggc ttcatgacga cgtcgaccac cacggcatcc ggtcgcaccc 12360
    tcgcggggtc gaagggcagc gggtcctcgg aacgcatgcc cagaggcgtc gcgttgacgg 12420
    cgaaatcggc cgcctccaga tcgccgggcc ccagcgcccg gatcccgtcc ggccggcggg 12480
    acccgagccg cagcagcagc gcgtcgagct gggcgcggtc ggtgtcgtgc acggacaccc 12540
    gcgcggcgtc ggccatcagc agcgccgtgg cgatcgcgct gcccgcccct ccggcgccga 12600
    ccagtgccac atgcctgtcg cgcaccgtgt gcccggccgc ctgaagaccc tggacgaacc 12660
    cgagcccgtc gaagttctcg gcgtaccagc ggccgtcggg ttcgcgccgc atcgcgttgg 12720
    ccgtcccgat gagggcggcc gccggcccga gcccgtccgc gagcccgcac agggccgcct 12780
    tgtgcggcac ggtgaccagc agaccgtcca gattgccgat ccgcttgagc ccctcgacca 12840
    cctcggcgag atcccgcgcc cggacgtgca ccggcaccac cacggcgtcc agaccgcttt 12900
    cgctcagcag ggggttgagc agaccgggcg ccttgacctg ggcgacggga tcacccagca 12960
    ccgcgtacag ccgcgtggcg cccgagacac cggccgccgg cccgaggaat tccatcagcc 13020
    gatcctctct gtacccccga cggatgttgc cctacggtgc tggagatgct ccacagcttt 13080
    gccgtgaccg ccggtcggca caaccctgcg tgcccctgac gcgccaggcc ctccaggtag 13140
    ttgctcccgg cggatcccga cagctcccga ccggtcccga cggagggaag aagccatcag 13200
    atacctggga atcgacgtcg gaggcacgaa ggtcgccctg cgggtgacgg gggacaccga 13260
    cggtgcgggc ggcggcgacg tgacgttccg ctggcccgcc gccggcgacg tcaccgcgga 13320
    tctggacctg ctcgccgcgc gggtccgcgg tcttctggga caccgcgagg accccctcgc 13380
    cggggtcggc gtggccatgc ccgcgatctg cgacgcggcc gggacggtcc gcacgtggcc 13440
    gggacggccg agctgggcgg gcctgaacct gacggccgcc ttcgggcagt tgctgcccgg 13500
    caccccggtc gcctgcgccg acgacggtga cctggccgcg ctggcggagt cccgcgccgc 13560
    cggctgccgg catctgctgt acgtgggggt cggcacgggc atcggcggcg gcatcgtcca 13620
    tgagggccgc gcctggccgg gccccggacg cggctcgtgc gaggtcggcc atgtcgtcgt 13680
    cgaccgctcg ggcccacgct gcgactgcgg gcgcgccggc tgcgtccagg cggtcgcgtc 13740
    gggaccggcg accctccggc gggccgccga acggcgcggc cgggagaccg gcttcgacga 13800
    actggcctcc ggggcgcgct tgcacgcccc gtgggcggaa gcggccgtcg acgagagcgc 13860
    cgcggccctg gccaccgccg tgaccggcat ctgcgagctg gcccaccccg aactcgtcct 13920
    cgtcggcggc gggttcgcgg cgggcgtgcc gggatacgtg gcctcggtgg cggcgcacgt 13980
    cgagcggctg acccgcccgg gaacggatcc cgtgcgggtg cgcccggcgg tgctcggcgg 14040
    gcggtcctcc ctgcacggcg cactgctgct cgcgcgggag gcacacgggc ggggaaaccg 14100
    gccgccggag agtgaccgtg tttcttccga tgtttcttcc gatgtttctt tcgggggagt 14160
    gacagacagg gccgttggcc ggtccgactg agcacaatca caggtgattt cgcccaggtt 14220
    caccacgcct cgtgtgctcg gggtcggcag aaggagtcag agtcatgctc gacaggcgga 14280
    gcgtcattcg cgtcggcgcc ggggtggcgg cggccgccgc cgtggccggt acggccgcca 14340
    ccggtgcggc ggccgtgggg ctgccgggtg tacggggacg cgcggcgtcg cgcggggtcg 14400
    actgggcctc cttacgccgt catctgtcgg gcgagctcgt cctgccggcg gacaccggat 14460
    acgagcgggc caggaagctc tacagcggcc agttcgacgg catccgcccg caggccgtcg 14520
    cctactgccg gaccgaggag gacgtgcgga cgaccctcgc gttcgcccag gaccacgcgc 14580
    tgcccctcac cccgcgcagt ggcgggcaca gcttcggcgg ctactccacg accgacggaa 14640
    tcgtcctgga cgtctccggc ttccacgcgg tgagcctcac ccggaacacc gtcgtcatgg 14700
    gcgcgggcac ccagcaggtg gacgccctca ccgccctgtc gccgcgcggt gtcgccgtgg 14760
    cgagcggcaa ctgcgcgggc gtctgtcccg gcggcttcgt ccagggcggc ggactgggct 14820
    ggcagagccg caagttcggc atggcgtgcg accggctcgt ctccgcccgg gtcgtgctcg 14880
    ccgacggccg cgccgtgacc gcctccgcca ccgaacaccc cgaccttttc tgggcgatgc 14940
    gcggcggagg cggcggcaac ttcggcgtcg tcaccggctt cgagctgcgc cccaccgacg 15000
    tcccctccgt cgtcagctac aacctcacct ggccgtggga gtcggcgcgg cgcgtcatcg 15060
    aggcgtggca gcactggatc atcgacggcc cccgcgacct cggtgccgcg atggccgtgc 15120
    agtggcccga cgccgggacc ggcacgccgg tcgtggtcgt caccggcgcc tggctgggcg 15180
    cggccgacgc gctcaccccc gtgctggact ccctggtggc ctccgtgggc agcgcgcccg 15240
    ccacccgctc ggccaaggcg ctctcccagc acgacgcgat gatggcgcag tacggctgcg 15300
    ccgacctcac gcccgagcag tgccacacgg tcggctactc gcccgaggcc gcgctgcccc 15360
    ggcagaactt ctccatggac cgcaaccggc tcttctcccg ggccatcggg caaggaggcg 15420
    tcgagcggat cctggaggcg ttcgccgccg acccgcgcgc cggacagttc cgcttcctga 15480
    gcttcttcgc cctcggcggc gccgccaacc gccccgaccg caccaccacc gcctacgttc 15540
    accgcgacac cgagttctac ctcggtttct cgatcgggct gaacgacccg gagtacacgg 15600
    cggaggacga gaggctcggc cgcgcctggg ccgcgcgagg actgcgcacg ctcgatcccc 15660
    actccaacgg cgagagctac cagaacttca tcgacccgga gctcgacgac tggaagtcgg 15720
    cctactacgc cgagaactac gtgcgcctgg ccgccgtcaa ggcggcctac gacccgcacc 15780
    ggctcttctc cttcgcgcag gccgtctgac ctctcccgaa agacccctgc cggcctgctc 15840
    ccctccgcgg ctcctgtggg cactggtgcg cccgcgcact tctgtgtgat tgagtgaagt 15900
    ccgggcgtgc agagctcagt tgccgtggag ggggcgccag ttgcgagcat cagcggtgga 15960
    gagggtggag ctgatccgct ggccggtgga gtccgagcgg cgggagcgct gccgcgaccg 16020
    gggcgtcatg cggatcctgg tgctggaggc gggggccgag gcacccttgt gcgtggaccc 16080
    caaggaggac tgggtccgcg ctcccgtcag caccgacgac ctgcgggccc gcgtcgaggc 16140
    cctgcgcctt cggggagccg ccgccgagtc ccggcccgag gtcgacccga acggagtgct 16200
    gcgtttccgg tggcgctccg ccctgctctc gcccaccgag gcccggctcg tcgcccggct 16260
    cgccgagtcc tatgccgagg tcgtcgcccg cgacgacctg ctccgcccgc ccccgggccg 16320
    taccgtgccg agccgtaacg cgctcgacct ccacatcatg cggatccgac ggcgcctcgc 16380
    cgcgctgggc ctgagggtgc gcaccgtccg ggggcgtggc tacgtcctgg agagcgcgga 16440
    aggagtctga ccgacgggcg tggccgcgca ccgcaccgac cgcccctacg agcgaggagc 16500
    ccgaagtgca gcagcctcat cacagccgcg tcgacgtgga actgggcgag aggtcctacc 16560
    ccgtccacgt cggaccgggg gtccgccacc tcctgcccgg catcgtcgcc tccctcggcg 16620
    cgcaccgcgc cgccgtcgtg accgcacggc cccccgacct ggtgcccgat cccggcgtgc 16680
    ccgcgctgat cgtgcgggca cgtgacggcg agcggcacaa gacgctcgcc accgtcgagg 16740
    acctgtgccg caagttcacc accttcggca tcacgcgcca cgacgtcgtc gtctcctgcg 16800
    gaggaggctc gacgaccgac accgtcggcc tggcggcggc gctgcaccac cgtggggtgc 16860
    cggtggtgca cctgccgacc accctcctgg cccaggtgga cgcgagcgtc ggcggcaaga 16920
    cggcggtcaa cctgcccgag ggcaagaacc tcgtcggcgc ctactggcag cccaaggccg 16980
    tgctgtgcga caccacgtat ctccagacgc tgcccgccga ggagtgggtc aacggctacg 17040
    gcgagatagc gcgctgccac ttcatcggtg ccggcgacct ccgcggcctc gccgtccacg 17100
    accaggtcac cgcgagcctg cggctgaagg cgtccgtcgt cgcggccgac gagcgggaca 17160
    ccggcctgcg gcacatcctc aactacggcc atacgctggg ccacgcactg gagaccgcca 17220
    ccggcttcgg gctgcggcac ggactcggcg tggcgatcgg gacggtcttc gcgggccggc 17280
    tcgcggaggc gctgggccgc atcggcgccg accgcgcgcg ggagcacacc gaggtcgtcc 17340
    gccactacgg acttcccgac agcctcccgg gaaacaccga catcaccgag ctcgtcgcgc 17400
    tgatgaggca cgacaagaag gccacgtcgg gactgacctt cgtgctcgac gggccttccg 17460
    gcgtggagct ggtgtccggg atcccggagg acgtcgtcct gcgtacgctc gcggcgatgc 17520
    cgcgaggaac ggcctgaccg agtgttccgt cttccgaggg gaagtgaccg tttcgtgtcg 17580
    gcagagctgt cagaaccgct gaagaaggcc ctggactccc tggtgttcgg cgtcgtggcg 17640
    acgaccgacc ccgacggccg cccgcaccag tcggtggtgt gggtccggcg cgagggctcc 17700
    gacgtgctgt tctcgatcac gcgcggcagc cgcaaggaga ggaacatcct gcgcgacccg 17760
    cgtgtgagcg tgctgatcag cccggcggac tcgccgtaca cctacgccgc gatccggggc 17820
    accgcgcact tcgaggacgt gccggacccg ggcgcgtacc tcgacacgtt ctccataaag 17880
    taccacggcg tgccctaccg ggagtcgttc cccgagccgc cggaggtgag caccattctc 17940
    gccgtccggc tcgttccgac gtcggtctac gagcagtggt gagggcgtag gcgtcccgaa 18000
    gccccggcag cgtcccgaat gccgctgccg gggcttcccg tgggagccct acgccggttt 18060
    ccgcgcggtg accaccgagt agccgacctc ctccaccgag cccatgcggt cgatgccgtc 18120
    gaccatgcgg tggaacgcct cgtcgtccat gtgggagccg agctcgtccc tggccgcacg 18180
    catcttcgcc gccaccgcct cgtaggaggg ccgcacctcg tccccgatgt cgaggaactc 18240
    caccacctcc agccccaccg accgcatgca gtcctcgtac gcctcgcggg tgaggacggg 18300
    gccctgctgg aagttgtcgt tggcggtgtc g 18331
    <210> SEQ ID NO 97
    <211> LENGTH: 1167
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 97
    atgacaccta cgtccggtga tgacgtcctg tcctttccct catggccgca acacggcgcg 60
    gaggagcgcg ccggactcct gcgggccctg gaccagaagg ggtggtggcg cgacgcgggg 120
    caggaggtcg atctcttcga gcgggagttc gccgaccacc acggcgcccc gcacgcgatc 180
    gccacgacga acggcaccca cgccctggaa ctcgccctgg gggtcatggg gatcggcccc 240
    ggtgacgagg tcatcgtccc cgcgttcacc ttcatctcgt cgtcgctggc cgtgcagcgc 300
    atgggcgcgg tgccggtgcc ggcggacgta cggcccgaca cctactgcct cgatgccgac 360
    gcggcggcgg cgctggtgac gccacgcacc aaagcgatca tgccggtcca catggcgggc 420
    cagttcgccg acatggacgc cctggagaag ctctccgtcg cgacgggcgt gccggtcctc 480
    caggacgccg cgcacgccca cggcgcgcag tggcagggcc gccgggtcgg ggagctcggc 540
    tcgatcgccg ccttcagctt ccagaacggc aagctgatga ccgccggcga gggcggcgcc 600
    ctgctcctgc cggacgacga gtccttccac gaggcgttcc tccagcactg ctgcggccgc 660
    ccgcccgggg accgcgtcta ccgccatctg acgcagggct ccaactaccg catgaacgag 720
    ttctccgcga gcgtcctgcg tgctcaactg aagcgcttga aggatcagtt gcgcatcagg 780
    gaggagcgct gggcccagct gcgtacggca ctggccgcca tcgacggcgt ggtgccgcag 840
    gggcgcgacg agcgcggcga cctccactcc cactacatgg ccatggtccg gctgcccggc 900
    atctcggccc ggcgccgcct cgcgctggtg gacgcgctgg tcgagcgggg agtgcccgcg 960
    ttcgtcggct tcccgccggt ctaccgcacc gagggtttcg cgcgcggccc ggcgccggcg 1020
    gacgccgagg agctggccaa gagctgtccc gtggcggagg agatcggcag cgactgcctc 1080
    tggctgcacc atcgcgtcct gctcgccgac gtgaccacgc tggaccggct ggcggaggtc 1140
    ttctccggcc tcgtcggcgc gctctga 1167
    <210> SEQ ID NO 98
    <211> LENGTH: 819
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 98
    gtggtcgtcg tcgacgacaa cgacgggggc gacgccggtg atcaactgat cgccgtgaca 60
    ggcgagatga gcggccttct cccgctgcgc gtggtgcggg gaccgctgcg ggggcgggcc 120
    gccgcccgga acgccggggc ggccgcggcc ctcgcgcccc ggctggtctt cctcgacgac 180
    gacgtcctgg tggggcccgg cttcctcgcc gcacacgccg cggccgcgga accggacgcc 240
    ttcacccacg gccggctgcg cgaactcccc accgcggcgc ggttcctcgc cgctgtcgag 300
    aaggccgccc cgaccgaggt ccgccgcgcc cgcgccggac tcgaacccgc tgccccggcc 360
    gcctccgagc ggcgccaacc gcaccggcgg ctcgtcgcca acgccctgga gcgggccgtg 420
    gaggccatgg ccggcggctc cctgccggac gtcgccccct ggctcggctt catcggcgcg 480
    aacaccgccc tcgacaaggc cgcatgggag cataccggcg gattcgacga ggagttcggg 540
    ctcacctggg ggtgcgagga cctggagttc ggcttccgcc tgcacgccgc cgggctgcgc 600
    aggaccctcg cccccgacgc cctcggtgtg cacctcagcc acgcccgccc cggccgctgg 660
    gagcagcacc accgcaacct cacgcacttc tccgccggcc acccgcaccc gtcggtacgc 720
    gccttggagg ccctgctcgg gcccgacggc acgccggagg cgtatgtgcg cgccgtcctg 780
    gccgaagagg ccgcaccggc acgggacgcg gcgcgatga 819
    <210> SEQ ID NO 99
    <211> LENGTH: 783
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 99
    atgagcggca caccggccac cgcgccgtac ggtcccgtgg tgctctcccc gcacgcggac 60
    gacgccgtgt ggtccctggg cgggcggctg gcgcgctggg ccgccgaggg cccgcggccg 120
    accgtcgtca cggtcttcgc cgggcccgcg gccgggaagc ccgagtcgtg gcggagcgcc 180
    gccgatcccg cggtgcgccg ggccgaggac cgggcggcat gtgccgaact gggcgtgcgc 240
    cacgtgccgc tgggcttcac cgacgcggca ctgcgtacgg cctcgggcgc ctatctctac 300
    gcttccccgc gccggctctt cggcccctgg cacccggccg acctcccgct gctggaggag 360
    gtgcgggcgg ctctgctgcc gctgtgcgcg ggggcgtcga gcgtccacgt tcccctggcg 420
    gcgggccggc acgtcgacca ccgcctggtc cgcggcgcgg tggagcccct gtcccccgcc 480
    cgtaccgtct tctacgagga cttcccctac cggctgcgcg aacgtgacca cacgaacctg 540
    cggccgcgca cggaacggct gccgtccgag gcggtggacc gctggctgac cgccgccggt 600
    cactactcca gccaggcgag cgcccacttc ggcggtgcgg ccgccctgcg cgaggccctg 660
    ttcgcccgcg cccgcgcaca cggcgggccc ggccggcccg gccacgccga ccgccactgg 720
    gtgcccgtcg gccaggacga ccggggcgag gcccggccgg cacccgtgga aagggggccg 780
    tga 783
    <210> SEQ ID NO 100
    <211> LENGTH: 383
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 100
    Met Ser Arg Ser Thr His Pro Pro Thr Ala Thr Pro Asp Ala Gly Thr
    1 5 10 15
    Arg Arg Arg Leu Pro Leu Ile Gly Asn Asp Leu Val Ile Asn Glu Asp
    20 25 30
    Ser Cys Asn Leu Ser Cys Thr Tyr Cys Leu Thr Gly Gln Ser Asn Leu
    35 40 45
    Lys Glu Gly His Ser Leu Gln Leu Ile Phe Glu Pro Pro Arg Arg Asp
    50 55 60
    Ser Tyr Ala Lys Asp Ser Gly Leu Gly Gln Arg Met Asp Lys Val Ala
    65 70 75 80
    Asp Arg Ile Arg Asp Arg Phe Gly Leu Pro Leu Leu Lys Val Thr Gly
    85 90 95
    Gly Glu Ile Phe Leu Val Arg Gly Ile Met Asp Phe Leu Glu Gln Glu
    100 105 110
    Ala Arg Lys Tyr Asp Val Leu Val Ile Gln Thr Asn Gly Val Leu Val
    115 120 125
    Arg Glu Glu His Leu Glu Arg Phe Arg Ser Trp Gly Asn Val Val Leu
    130 135 140
    Gln Val Ser Leu Asp Ser His Leu His His Gly Asn Ser His Arg Val
    145 150 155 160
    Pro Ser Gly Ser Leu His Glu Lys Val Val Ala Ala Ile Ala Arg Ile
    165 170 175
    Leu Asp Ser Gly Leu Pro Val Glu Ile Tyr Ser Val Leu Asn Asp Arg
    180 185 190
    Ser Val Thr Glu Val Cys Ala Phe Ala Glu Trp Leu Ser Gly Phe Ser
    195 200 205
    Arg Pro Pro Val Tyr Phe Pro Phe Pro Val Arg Gly Pro Asp Ser Glu
    210 215 220
    Asp Phe Lys Val Arg Pro Gly Gln Phe Gly His Ile Gln Glu Leu Val
    225 230 235 240
    Asp Arg Tyr Asp Glu Phe Ala Arg Val Leu Pro Pro Arg Pro Tyr Phe
    245 250 255
    Asp Arg Leu Thr Ser Phe Tyr Arg Glu Gly Arg Arg Thr Phe Arg Cys
    260 265 270
    His Leu Pro Arg Leu Val Val Ser Ser Phe Ser Asp Gly Val Val Thr
    275 280 285
    Pro Cys Pro Asn Ile Trp Phe Ser Asp Met Gly Asn Ala Leu Glu Asp
    290 295 300
    Asp Trp Ser Glu Met Leu Asp Thr Val Gly Thr Ser Gly Leu Tyr Arg
    305 310 315 320
    Ala Leu Leu Ala Pro Lys Pro Arg Leu Lys Ala Cys His Gly Cys Phe
    325 330 335
    Thr Pro Trp Asp Thr Leu Ser Met Tyr Phe Glu Asp Glu Ile Thr Leu
    340 345 350
    Asp Glu Leu Cys Ala Ala Pro Thr Tyr Ser Pro Pro Arg Ile Arg Gln
    355 360 365
    Met Leu Ser Asp Ala Lys Ala Asp Tyr Leu Gln Gly Gly His Asp
    370 375 380
    <210> SEQ ID NO 101
    <211> LENGTH: 707
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 101
    Met Ala Leu Arg Ala Pro Asn Ser Pro Arg Trp Val Val Ala Phe Leu
    1 5 10 15
    Ser Leu Leu Ala Ser Gly Ala Arg Pro Leu Leu Leu Glu Pro Asp Thr
    20 25 30
    Pro Gly Pro Glu Thr Ala Arg Leu Leu Arg Ala Ala Gly Gly Gly Arg
    35 40 45
    Ser Leu Val Val Pro Gly Thr Gly Asp Gly Leu Arg Leu Thr Leu Thr
    50 55 60
    Gly Ser Pro Gly Glu Pro Ser Gly Ala Pro Pro Ala Val Leu Leu Pro
    65 70 75 80
    Thr Ser Gly Ser Thr Gly Ala Ser Lys Leu Val Ala Arg Ser Glu Glu
    85 90 95
    Ser Leu Leu Ala Glu Gly Arg Arg Tyr Arg Asp Gly Val Gly Leu Thr
    100 105 110
    Gly Glu Asp Thr Leu Leu Leu Pro Val Pro Leu Ser His Ala Tyr Ala
    115 120 125
    Leu Gly Trp Leu Phe Gly Gly Leu Leu Thr Gly Ala Ala Leu Arg Pro
    130 135 140
    Val Pro Pro Thr Ala Leu Gly Arg Ile Ala Ala Glu Leu Ser Gly Gly
    145 150 155 160
    Ala Thr Val Val Ala Leu Val Pro Ser Val Ala Arg Leu Leu Ala Thr
    165 170 175
    Arg Arg Leu Arg Gly Ala Ala Ala Gly Arg Ala Pro Ala Ala Pro Gly
    180 185 190
    Leu Arg Leu Ala Met Val Gly Ala Gly Pro Val Asp Glu Gln Leu Asp
    195 200 205
    Arg Ala Phe Thr Glu Ala Phe Gly Thr Gly Leu Ala Arg Asn Tyr Gly
    210 215 220
    Ser Thr Glu Thr Gly Ala Val Leu Ala Gly Pro Ala Gly Leu Glu Pro
    225 230 235 240
    Leu Cys Ala Gly Ala Pro Leu Pro Gly Val Glu Cys Glu Leu Thr Gly
    245 250 255
    Pro Glu Gly Val Val Pro Pro Ala Gly Thr Pro Gly Leu Leu Ser Val
    260 265 270
    Arg Val Asp Gly Arg Pro Tyr Ala Met Gly Asp Leu Ala Val Ala Val
    275 280 285
    Pro Gly Gly Leu Arg Ile Leu Gly Arg Glu Asp Arg Ala Ile Arg Arg
    290 295 300
    Gly Gly Arg Trp Val Ser Pro Leu Glu Ile Glu Glu Val Leu Arg Gly
    305 310 315 320
    His Pro Asp Val Val Asn Val Arg Val Gly Ala Arg Arg Gly Arg His
    325 330 335
    Arg Gly Glu Asp Gly Ile Val Ala Glu Val Ser Ala Ala Gly Pro Gly
    340 345 350
    Leu Thr Pro Glu Ala Leu Arg Glu His Ala Arg Arg Glu Leu Ala Pro
    355 360 365
    His Lys Val Pro Asp Glu Phe Val Leu Arg Glu Ser Leu Pro Val Asn
    370 375 380
    Ala Ala Gly Lys Val Arg Ala Ala Ser Val Tyr Arg Leu Thr Arg Ser
    385 390 395 400
    Ala Ala Glu Ala Ala Arg Ala Tyr Lys Ala Ser Glu Val Leu Phe Ala
    405 410 415
    Leu His Asp Leu Gly Ala Leu Glu Ala Leu Ala Gln Gly Ala Gly Thr
    420 425 430
    Ala Leu Leu Ala Gly Glu Leu Gly Cys Asp Ala Asp Ala Leu Glu Trp
    435 440 445
    Leu Leu Arg Thr Ala Thr Ala Leu Gly Val Leu Thr Thr Gly Ala Gln
    450 455 460
    Glu Pro Gly Asp Arg Val Arg Ala Gly Glu Leu Ala Ala Phe Val Ala
    465 470 475 480
    Leu Glu Glu His Leu Ser Arg Gly Leu Val Thr Arg Glu Glu Leu Val
    485 490 495
    Ala Val Ala Arg Ser Gly Thr Ala Arg Arg Pro Phe Glu Glu Arg Pro
    500 505 510
    Pro Glu Ser Leu Gly Pro Leu Val Ala Leu Tyr Gln Gly Ala Met Asp
    515 520 525
    Gly Pro Gly Ala Arg Ala Arg Ala Ala Leu Gly Leu Arg Leu Leu Arg
    530 535 540
    Pro Gly Pro Gly Ala Arg Val Val Glu Val Thr Ala Gly Pro Gly Arg
    545 550 555 560
    Tyr Leu Glu Arg Leu Leu Ala Ser Asp Pro Gly Ala Ser Gly His Leu
    565 570 575
    Val Thr Val Gly Arg Leu Ser Gly Pro Leu Ser Ser Ala Val Ala Ala
    580 585 590
    Ala Val Glu Glu Gly Arg Val Thr Val Gly Thr Glu Leu Pro Val Gly
    595 600 605
    Tyr Ala Asp Phe Cys Val Val Ala Asn Ala Val His Gly Pro Gly Pro
    610 615 620
    Gly Ser Ala Leu Gly Ala Leu Leu Gly Ser Leu Arg Pro Gly Gly Arg
    625 630 635 640
    Leu Leu Val Asp Asp Val Phe Leu Pro Ala Ser Gly Pro Gly Ser Glu
    645 650 655
    Leu Ala Leu Asp Trp Leu Thr His Gly Gly Thr Ala Trp Pro Ala Thr
    660 665 670
    Gly Glu Leu Ile Ala Gly Leu Leu Gln Glu Gly Ala Glu Val Ala Arg
    675 680 685
    His Val Pro Leu Asp Ala Ser Pro Cys His Leu Ile Ile Ala Lys Glu
    690 695 700
    Ala Gly Ser
    705
    <210> SEQ ID NO 102
    <211> LENGTH: 257
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 102
    Met Ser Thr Val Thr Asp Arg Ala Thr Glu Arg Leu Gly Gln Ser Gly
    1 5 10 15
    Arg Val Val Val Val Ser Gly Ala Ser Gly Gln Ile Gly Gly Ala Cys
    20 25 30
    Ala Leu Glu Leu Ala Ala Leu Gly Ala Thr Val Val Ala Gly Tyr His
    35 40 45
    Ser Gly Glu Gln Ala Ile Arg Lys Leu Arg Glu Gln Val Glu Gly Gln
    50 55 60
    Gly Gly Thr Leu Val Pro Val Ala Ala Asp Leu Ser Glu Pro Glu Gly
    65 70 75 80
    Ala Asp Ala Leu Val Ala Ala Ala Val Glu Arg Phe Gly Arg Val Asp
    85 90 95
    Gly Cys Val Ala Ala Ala Gly Leu Arg Thr Arg Arg Leu Ala Met Ala
    100 105 110
    Thr Asp Ala Arg Ser Leu Glu Lys Leu Leu Arg Val Asn Leu Ala Gly
    115 120 125
    Ser Val Gly Leu Ala Lys Ala Cys Leu Lys Pro Met Met Arg Ala Arg
    130 135 140
    Tyr Gly Arg Ile Val Leu Phe Gly Ser Arg Ala Gly Thr Ser Gly Leu
    145 150 155 160
    Pro Gly His Ser Ala Tyr Ala Ala Thr Lys Gly Ala Leu Gln Pro Trp
    165 170 175
    Ala Ala Ser Val Ala Gly Glu Val Gly Lys His Gly Ile Thr Val Asn
    180 185 190
    Val Val Ala Pro Gly Ala Ile Arg Ala Glu Val Met Asp Phe Ser Glu
    195 200 205
    Ala Glu Arg Asp Leu Val Leu Gln Phe Ile Gly Ala Gly Arg Leu Gly
    210 215 220
    Glu Pro Glu Glu Val Ala Ala Ala Val Ser Phe Leu Leu Ser Pro Ser
    225 230 235 240
    Ala Ser Tyr Val Asn Gly Asn Thr Leu Val Val Asp Gly Gly Ala Arg
    245 250 255
    Phe
    <210> SEQ ID NO 103
    <211> LENGTH: 404
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 103
    Met Thr Pro Pro Thr Thr Ala Arg Glu Pro Leu Arg Met Ala Val Leu
    1 5 10 15
    Gly Ala Gly Trp Val Ser Arg Lys Val Trp Leu Pro Leu Leu Ala Glu
    20 25 30
    His Pro Ala Phe Arg Val Asp Phe Leu Val Asp Asp Asp Pro Val Ala
    35 40 45
    Ala Arg Ser Ala Leu Pro Glu Gly Ala Arg Thr Arg Val Leu Ser Arg
    50 55 60
    Pro Glu Glu Leu Ala Pro Arg Ser Val Asp Ala Ala Ile Ile Ala Leu
    65 70 75 80
    Pro Asn His Leu His Leu Pro Val Ala Lys Ala Leu Leu Glu Arg Asp
    85 90 95
    Val Pro Val Phe Val Glu Lys Pro Val Cys Arg Thr Leu Phe Glu Ala
    100 105 110
    Gln Ala Leu Ala Leu Asp His Gln Ala Arg Gly Asp Ser Ile Gly Asp
    115 120 125
    Ile Thr Leu Tyr Ala Trp Ser Ala Ala Arg His Arg Thr Asp Val Cys
    130 135 140
    Arg Leu Ala Glu Leu Leu Pro Ser Leu Gly Thr Val Arg Ser Val Gly
    145 150 155 160
    Leu Ser Trp Ile Arg Ala Thr Gly Ile Pro Gln Arg Thr Gly Trp Phe
    165 170 175
    Val Asp Arg Arg Leu Ala Gly Gly Gly Ala Leu Leu Asp Leu Gly Trp
    180 185 190
    His Leu Leu Asp Val Gly Leu His Leu Leu Gly Trp Pro Arg Val Val
    195 200 205
    Arg Ala Ala Ser Thr Met Ser Ala Asp Trp Met Ser Arg Gly Glu Ala
    210 215 220
    Thr Ala Asp Trp Ser Arg Arg Ser Ser Gly Thr Ala Arg Pro Gly Pro
    225 230 235 240
    Gly Glu Thr Val Glu Asp Thr Ala Arg Gly Phe Leu Val Thr Asp Thr
    245 250 255
    Asp Val Gly Ile Ser Leu Glu Thr Arg Trp Ala Ser His Gln Ala Leu
    260 265 270
    Asp Val Thr Thr Ile Thr Val Glu Gly Thr Glu Gly Val Ala Thr Leu
    275 280 285
    Arg Gly Thr Phe Gly Phe Ser Pro His Arg Leu Gln Lys Ser Ser Leu
    290 295 300
    Val Val Leu Arg Gln Gly Val Glu Glu Thr Val Ala Leu Pro Asp Glu
    305 310 315 320
    Pro Val Gly Val Glu Tyr Arg Arg Gln Val Asp Glu Leu Ala Arg Arg
    325 330 335
    Leu Gly Gly Ser Ala Asp Gly Gln Gly Pro Val Ser Gly Leu Gly Glu
    340 345 350
    Gly Ser Met Ala Glu Val Thr Ile Leu Ala Ser Cys Ile Asp His Ile
    355 360 365
    Tyr Ser Ala Ala Gly Val Asp Pro Pro Ser Pro Leu His Arg Pro Gln
    370 375 380
    Ser Asp Ala Ala Pro Ser Thr Ser Ser Cys Pro Arg Val Leu Pro Thr
    385 390 395 400
    Arg Gly Ser Gln
    <210> SEQ ID NO 104
    <211> LENGTH: 382
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 104
    Met Lys Phe Ala Tyr Phe Ser His Val Trp Gly Arg Pro Gly Ile Thr
    1 5 10 15
    Pro Gly Glu Arg Tyr Glu Glu Leu Trp Arg Glu Val Glu Asp Ala Asp
    20 25 30
    Arg Leu Gly Phe Asp Tyr Ala Phe Ser Val Glu His His Cys Thr Pro
    35 40 45
    His Glu Ser Trp Met Pro Ser Pro Ala Val Phe Cys Thr Gly Ala Ala
    50 55 60
    Leu Arg Thr Glu Arg Ile Arg Val Gly Pro Met Gly Trp Val Pro Pro
    65 70 75 80
    Leu Arg His Pro Leu His Leu Val Glu Glu Val Ala Thr Leu Asp Gln
    85 90 95
    Leu Leu Gly Gly Arg Leu Glu Val Gly Leu Ala Ser Gly Val Ser Arg
    100 105 110
    Asp Pro Phe Leu Pro Phe Asp Ala Asp Phe Asp Asn Arg His Leu Leu
    115 120 125
    Thr Arg Glu Ala Leu Glu Leu Leu Arg Ala Ala Phe Ala Ala Arg Gly
    130 135 140
    Ala Phe Asp Phe Asp Gly Pro Ala His Arg Leu Arg Asp Ile Ala Leu
    145 150 155 160
    Ser Phe Pro Pro Val Gln Arg Pro His Pro Pro Met Trp Val Pro Thr
    165 170 175
    Thr Asn Arg Asn Thr Leu Arg Tyr Leu Ser Glu Ala Gly Ala His Thr
    180 185 190
    Ser Ser Thr Met Ile Val Pro Arg Ala Ser Met Ala Leu Val Tyr Arg
    195 200 205
    His Tyr Leu Asp Trp Trp Arg Gly His Gly His Ala Ser Asp Pro Arg
    210 215 220
    Ile Gly Tyr Trp Thr Leu Val His Val Ala Arg Thr Asp Ala Glu Ala
    225 230 235 240
    Glu Glu Arg Ala Ala Ala His Ile Thr Glu Thr Phe Thr Lys Thr Leu
    245 250 255
    Arg Tyr Gly Ser Val Ser Arg Ser Arg Asp Gln His Ala Pro Pro Ser
    260 265 270
    Arg Leu Ser Thr Thr Asp Ile Leu Ala Gly Ser Gly Asp Leu Arg Phe
    275 280 285
    Leu Leu Glu Asn Asn Leu Val Phe Val Gly Ser Pro Ala Thr Val Ala
    290 295 300
    Asp Arg Ile Arg Ala Ala Ser Leu Glu Gly His Phe Asp Thr Leu Leu
    305 310 315 320
    Gly Glu Phe Thr Phe Gly Glu Leu Ala Asp Arg His Arg Ile Glu Ser
    325 330 335
    Met Glu Leu Phe Ala His Glu Val Ala Pro Ala Leu Arg Ala Phe Ser
    340 345 350
    Pro Tyr Ala Pro Arg Pro Gln Glu Pro Ala Tyr Thr Ala Ser Asp Glu
    355 360 365
    Gln Gln Val Ala Ala Arg Leu Gln Ala Leu Gly Tyr Ile Asp
    370 375 380
    <210> SEQ ID NO 105
    <211> LENGTH: 290
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 105
    Met Glu Arg Leu Lys Leu Val Pro Asp Glu His Arg Arg Phe Thr Val
    1 5 10 15
    Asp Glu Gln Ser Ala Arg Arg Leu His Arg Ile Gly Pro Glu Leu Leu
    20 25 30
    Ser Ala Leu Cys Glu Ala Gly Val Pro Phe Val Gly Ser Gly Ala Gly
    35 40 45
    Arg Leu Phe Asp Gly Tyr Asp Leu Gly Asn Ala Ala Leu His Leu Gly
    50 55 60
    Leu Ser Ser Val Gln Arg Arg Ala Ile Arg Ser Trp Ala Gly Ser Leu
    65 70 75 80
    Arg Thr Ala Ser Ala Ala Glu Ser Pro Arg Trp Arg Val Asp Val Thr
    85 90 95
    Ala Ser Cys Pro Val Pro Gly His Ala Gly Pro Cys Arg Tyr Gly Val
    100 105 110
    Leu Leu Pro Gly Ala Arg Arg Pro Val Glu Ala Ala Ser Pro Arg Glu
    115 120 125
    Thr Thr Leu Ala Arg Leu Tyr Thr Arg Ser Arg Gly His Trp Pro Asp
    130 135 140
    Phe Pro Pro Ala Val Leu Asp Leu Leu Arg Thr Leu Glu Pro Val Gly
    145 150 155 160
    Phe Phe Leu Leu Pro Glu Ala Ile Arg Trp Asp Pro Gly Phe Leu Trp
    165 170 175
    Ser Thr His Met Ala Asp Cys Gly Gly Ala Ala Ala Trp Leu Val Ala
    180 185 190
    Glu Gly Arg Arg Arg Gly Leu Asp Val Arg Phe Ser Phe Gly Leu Leu
    195 200 205
    Val Ala Lys Pro Tyr Ser Thr Pro His Cys Trp Ala Glu Phe Leu Val
    210 215 220
    Gly Gly Arg Trp Val Pro Ala Asp Pro Leu Leu Leu Arg Ala Met Ala
    225 230 235 240
    Ala Trp Gly Gly Leu Asp Ala Ala Ala His Pro Pro His Ser Ser Pro
    245 250 255
    Gly Ala Val Tyr His Arg Leu Ala Gly Arg Phe Thr Lys Val Val Ser
    260 265 270
    His Ala Gly Val Trp Ala Pro Thr Ser Leu Pro Thr Glu Leu Leu Pro
    275 280 285
    Cys Pro
    290
    <210> SEQ ID NO 106
    <211> LENGTH: 235
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 106
    Met Pro Leu Asn Pro Pro Pro Ala Ser Arg Ala Ala Ala Asp Ala Pro
    1 5 10 15
    Ala Thr Ala Leu Pro Cys Arg Phe Thr Thr Val Val Phe Asp Leu Asp
    20 25 30
    Gly Val Leu Ile Asp Ser Phe Ala Val Met Arg Glu Ala Phe Ala Val
    35 40 45
    Ala Tyr Arg Glu Val Val Gly Pro Gly Glu Pro Pro Phe Glu Glu Tyr
    50 55 60
    Arg Thr His Gln Gly Arg Tyr Phe Pro Asp Ile Met Arg Leu Met Gly
    65 70 75 80
    Leu Pro Gly Glu Met Glu Glu Pro Phe Val Arg Glu Ser His Arg Leu
    85 90 95
    Met Asp Arg Val Glu Val Tyr Pro Asp Val Pro Gln Leu Leu Ala Glu
    100 105 110
    Leu Arg Ala Asp Gly Val Gly Thr Ala Ile Ala Thr Gly Lys Ser Gly
    115 120 125
    Ser Arg Ala Arg Ala Val Leu Glu Ala Val Gly Leu Leu Pro Leu Leu
    130 135 140
    Asp Glu Val Val Gly Ser Asp Glu Val Pro Arg Pro Lys Pro His Pro
    145 150 155 160
    Asp Ile Val Arg Glu Ala Leu Arg Arg Leu Asp Ala Ala Pro Glu Asp
    165 170 175
    Ala Val Met Val Gly Asp Ala Val Ile Asp Ile Arg Ser Gly Arg Ala
    180 185 190
    Ala Gly Thr Ala Thr Val Gly Ala Thr Trp Gly Glu Gly Ala Ala Gly
    195 200 205
    Gln Leu Arg Ala Glu Arg Pro Asp Phe Leu Leu Asp Lys Pro Gln Ser
    210 215 220
    Leu Leu Ala Leu Val Arg Ser Gly Gly His Ala
    225 230 235
    <210> SEQ ID NO 107
    <211> LENGTH: 346
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 107
    Met Thr Pro Ala Thr Pro Arg Trp Ser Val Val Ala Pro Gln Gly Thr
    1 5 10 15
    Asn Leu Glu Leu Ala Gly Thr Gly Gly Arg Glu Gly Trp Arg Leu Leu
    20 25 30
    Leu Glu Thr Ala Arg Thr Val His Arg His Gly Arg Gly Ala Leu Trp
    35 40 45
    Leu Leu Asp Arg Thr Asp Thr Leu Pro Arg Arg Glu Pro Glu Pro Val
    50 55 60
    Trp Glu Gly Trp Thr Ala Leu Ala Ala Leu Ala Gly Ala Val Pro Gly
    65 70 75 80
    Leu Asp Leu Gly Leu Leu Ser Ser Ala Pro Pro Phe Arg Asn Ala Ala
    85 90 95
    Leu Ile Ala Lys Arg Ala Ala Thr Leu Asp Val Val Cys Asp Gly Arg
    100 105 110
    Leu Thr Leu Gly Phe Pro Ala Arg Glu Tyr Leu Pro Glu His His Ser
    115 120 125
    Thr Gly Arg Glu Val Pro Thr Gly Leu Glu Ala Asp Glu Glu Glu Ala
    130 135 140
    Ala Gly His Arg Ala Leu Gly Glu Thr Val Glu Ala Leu Arg Ala Leu
    145 150 155 160
    Trp Gly Gly Gln Pro Val Thr Phe Thr Gly Glu His Ile Arg Leu Thr
    165 170 175
    Ser Ala His Cys Val Pro Ala Pro Arg Gln Gln Pro Leu Pro Leu Ala
    180 185 190
    Leu Arg Thr Pro Ala Gly Asp Ala Gly Ser Gly Ala Leu Arg Pro Ala
    195 200 205
    Asp Ala Thr Val Arg Glu Cys Ala His Val Gln Trp Thr Gly Glu Pro
    210 215 220
    Ala Gln Val Ala Ala Ala Val Thr Ala Phe Arg Arg Arg Arg Thr Glu
    225 230 235 240
    Leu Gly Leu Asp Pro Asp Gly Val Arg His Ala Trp Ala Ala Glu Cys
    245 250 255
    Arg Ile Phe Asp Ser Val Leu Glu Arg Asp Arg Trp Leu Ser Thr Pro
    260 265 270
    His Glu Val Leu Phe Trp Ser His His Pro Asp Leu Leu Ala Arg Arg
    275 280 285
    Ser Leu Tyr Gly Thr Pro Glu Gln Leu Thr Glu Arg Ala Arg Arg Leu
    290 295 300
    Val Ala Ala Gly Val Ala Glu Phe Val Leu Trp Phe Arg Asp Tyr Pro
    305 310 315 320
    Ala Thr Thr Ser Leu Glu Arg Leu Phe Gln Glu Val Val Pro Gln Val
    325 330 335
    Ala Pro Gly Ala Ala Lys Glu Ala Glu Glu
    340 345
    <210> SEQ ID NO 108
    <211> LENGTH: 520
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 108
    Met Leu Asn Thr Leu Ser Thr Ala Pro Phe Leu Ser Thr Ala Trp Leu
    1 5 10 15
    Ala Gly Ala Ala Arg Leu Glu Arg Pro Pro Val Gly Glu Arg Gly Thr
    20 25 30
    Val Ala Leu Arg Leu Glu Leu Thr Asp Pro Pro Pro Gly Glu Pro Pro
    35 40 45
    Ala Val Asp Val Gln Val Asp Leu Val Ala Gly Arg Leu Gly Leu Ala
    50 55 60
    Ala Ala Ala Gly Glu Ser Pro Gly Leu Arg Ile Arg Leu Pro Leu Glu
    65 70 75 80
    Ala Ala Arg Ala Leu Leu Leu Gly Pro Ala Arg Asp Arg Thr Gly Val
    85 90 95
    Phe Glu Arg Gly Asp Val Arg Ala Glu Gly Asn Phe Ser Leu Leu Phe
    100 105 110
    Phe Ile Asp Ala Ala Leu Glu Arg Asp Ala Ser Gly His Val Ala Ala
    115 120 125
    Leu Arg Gly Thr Pro Gly Thr Thr Ala Arg Glu Ala Ala Pro Pro Pro
    130 135 140
    Gly Thr Glu Asp Ala Ala Glu Ala Val Arg Arg Ala Arg Ala Ala Leu
    145 150 155 160
    Pro Gly Thr Met Arg Glu Leu Glu Arg Glu Val Gly Thr Ser Thr Pro
    165 170 175
    Gly Ala Gln Ile Tyr Val Ser Arg Asp Gly Val Pro Leu Ala Asp Ala
    180 185 190
    Gly Leu Gly Leu Ala Arg Pro Gly Val Ala Met Thr His Arg Ser Leu
    195 200 205
    Pro Leu Trp Tyr Cys Cys Ala Lys Pro Leu Leu Ser Val Ala Leu Gly
    210 215 220
    Arg Leu Trp Glu Ala Gly Ala Tyr Asp Pro Tyr Leu Pro Val Ala His
    225 230 235 240
    Tyr Leu Pro Glu Phe Gly Asn Arg Gly Lys Glu Ser Ile Thr Ser Met
    245 250 255
    Glu Leu Leu Thr His Thr Gly Pro Leu Pro Thr Gly Asp Asp Pro Leu
    260 265 270
    His Gly Ile Val Ala Gly Pro Asp Glu Glu Arg Val Arg Arg Ala Phe
    275 280 285
    Glu Val Pro Val Ala Pro Arg Pro Gly Gly Thr Pro Gly Ile Asn Tyr
    290 295 300
    Ser Gln Trp Trp Ala Trp Phe Val Leu Ala Arg Ile Leu Pro Val Val
    305 310 315 320
    Asp Gly Arg Glu Tyr Arg Ala Tyr Val Gln Glu Glu Ile Leu Gly Pro
    325 330 335
    Cys Gly Met Ser Gly Thr Arg Val His Leu Asp Arg Glu Glu Phe Ala
    340 345 350
    Ala Leu Gly Gly Glu Leu Pro Leu Ile His Val Ser Asn Pro Glu Gly
    355 360 365
    Gly Pro Leu Pro Thr His Trp Trp Ser Thr Glu Ala Ala Thr Thr Arg
    370 375 380
    Cys Ile Pro Gly Val Asn Thr Arg Gly Pro Leu Arg Asp Met Gly Arg
    385 390 395 400
    Leu Phe Glu Met Leu Leu Arg Gly Gly Asp Ala Pro Gly Gly Arg Val
    405 410 415
    Leu Ala Pro Pro Thr Val Ala Ala Leu Thr Ala Arg His Arg Thr Gly
    420 425 430
    Leu Gln Asp Arg Tyr Gly Asn Ala Asp Trp Gly Met Gly Phe Arg Leu
    435 440 445
    Glu Cys Arg Gln Leu Asp Pro Arg Phe Thr Ser Phe Gly Ser Tyr Ala
    450 455 460
    Ser Pro Arg Ser Phe Gly His Asp Gly Leu Trp Thr Ala Val Val Phe
    465 470 475 480
    Ala Asp Pro Asp Ala Ala Leu Val Val Ala Leu His Leu Asn Gly Lys
    485 490 495
    Val Glu His Glu Arg His Arg Glu Arg Ile Val Arg Leu Ala Asp Ala
    500 505 510
    Val Tyr Gln Asp Leu Arg Leu Ser
    515 520
    <210> SEQ ID NO 109
    <211> LENGTH: 283
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 109
    Met Pro His Ser Glu Leu Ser Glu Leu Pro Met Pro Ser Pro Ala Ser
    1 5 10 15
    Glu Glu Val Gly Ala Leu Tyr Asp Arg Phe Thr Ala Leu Gly Ala Ala
    20 25 30
    Ser Leu Gly Glu Asn Leu His Phe Gly Tyr Trp Asp Ser Pro Asp Ser
    35 40 45
    Gln Val Pro Leu Ala Glu Ala Thr Asp Arg Leu Thr Asp Met Met Ala
    50 55 60
    Glu Arg Leu Arg Ile Gly Ala Gly Ser Arg Val Leu Asp Leu Gly Cys
    65 70 75 80
    Gly Val Gly Thr Pro Gly Val Arg Ile Ala Arg Leu Ser Gly Ala His
    85 90 95
    Val Thr Gly Ile Ser Val Ser His Glu Gln Val Val Arg Ala Asn Ala
    100 105 110
    Leu Ala Glu Glu Ala Gly Leu Ala Asp Arg Ala Arg Phe Gln Arg Ala
    115 120 125
    Asp Ala Met Asp Leu Pro Phe Glu Asp Glu Ser Phe Asp Ala Val Ile
    130 135 140
    Ala Leu Glu Ser Ile Ile His Met Pro Asp Arg Ala Gln Val Leu Ala
    145 150 155 160
    Gln Val Gly Arg Val Leu Arg Pro Gly Gly Arg Leu Val Leu Thr Asp
    165 170 175
    Phe Phe Glu Arg Ala Pro Leu Ala Pro Glu Gly Arg Ala Ala Val Gln
    180 185 190
    Arg Tyr Leu His Asp Phe Met Met Thr Met Val Ser Ala Glu Ala Tyr
    195 200 205
    Pro Pro Leu Leu Arg Gly Ala Gly Leu Trp Leu Glu Glu Phe Leu Asp
    210 215 220
    Ile Ser Asp Gln Thr Leu Glu Lys Thr Phe Arg Leu Leu Ser Glu Arg
    225 230 235 240
    Ile Asn Ser Ser Lys Gln Arg Leu Glu Thr Gln Phe Gly Glu Glu Met
    245 250 255
    Val Asn Gln Phe Asp Pro Gly Asp Leu Val Gly Val Lys Glu Phe Gly
    260 265 270
    Tyr Leu Leu Leu Val Ala Gln Arg Pro Gly Lys
    275 280
    <210> SEQ ID NO 110
    <211> LENGTH: 275
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 110
    Met Thr Glu Thr Ala Ser Ala Ser Asp Arg Met Val Glu Leu Tyr Asn
    1 5 10 15
    Arg Val Thr Asp Leu Met Val His Ala Glu Gly Gly Tyr Met His Gly
    20 25 30
    Gly Tyr Trp Ala Gly Pro Asp Val Pro Thr Thr Val Glu Glu Ala Gly
    35 40 45
    Asp Arg Leu Thr Asp Tyr Val Ser Glu Arg Leu Arg Leu Ala Pro Gly
    50 55 60
    Glu Arg Val Leu Asp Val Gly Ser Gly Asn Gly Lys Ala Thr Leu Arg
    65 70 75 80
    Ile Ala Ala Arg His Gly Val Arg Ala Thr Gly Val Ser Ile Asn Pro
    85 90 95
    Tyr Gln Val Gly Leu Ser Arg Gln Leu Ala Glu Lys Glu Gly Asp Glu
    100 105 110
    Ala Thr Glu Phe Arg Ile Gly Asp Met Leu Ala Leu Pro Phe Pro Asp
    115 120 125
    Gly Ser Phe Asp Ala Cys Tyr Ala Ile Glu Ser Ile Cys His Ala Leu
    130 135 140
    Glu Arg Ala Asp Val Phe Thr Glu Ile Ala Arg Val Leu Arg Pro Gly
    145 150 155 160
    Gly Arg Val Thr Val Thr Asp Phe Val Leu Arg Arg Pro Leu Ser Asp
    165 170 175
    Ala Ser Arg Thr Ile Val Asp Thr Ala Asn Asp Asn Phe Gln Gln Gly
    180 185 190
    Pro Val Leu Thr Arg Glu Ala Tyr Glu Asp Cys Met Arg Ser Val Gly
    195 200 205
    Leu Glu Val Val Glu Phe Leu Asp Ile Gly Asp Glu Val Arg Pro Ser
    210 215 220
    Tyr Glu Ala Val Ala Ala Lys Met Arg Ala Ala Arg Asp Glu Leu Gly
    225 230 235 240
    Ser His Met Asp Asp Glu Ala Phe His Arg Met Val Asp Gly Ile Asp
    245 250 255
    Arg Met Gly Ser Val Glu Glu Val Gly Tyr Ser Val Val Thr Ala Arg
    260 265 270
    Lys Pro Ala
    275
    <210> SEQ ID NO 111
    <211> LENGTH: 146
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 111
    Met Phe Arg Leu Pro Arg Gly Ser Asp Arg Phe Val Ser Ala Glu Leu
    1 5 10 15
    Ser Glu Pro Leu Lys Lys Ala Leu Asp Ser Leu Val Phe Gly Val Val
    20 25 30
    Ala Thr Thr Asp Pro Asp Gly Arg Pro His Gln Ser Val Val Trp Val
    35 40 45
    Arg Arg Glu Gly Ser Asp Val Leu Phe Ser Ile Thr Arg Gly Ser Arg
    50 55 60
    Lys Glu Arg Asn Ile Leu Arg Asp Pro Arg Val Ser Val Leu Ile Ser
    65 70 75 80
    Pro Ala Asp Ser Pro Tyr Thr Tyr Ala Ala Ile Arg Gly Thr Ala His
    85 90 95
    Phe Glu Asp Val Pro Asp Pro Gly Ala Tyr Leu Asp Thr Phe Ser Ile
    100 105 110
    Lys Tyr His Gly Val Pro Tyr Arg Glu Ser Phe Pro Glu Pro Pro Glu
    115 120 125
    Val Ser Thr Ile Leu Ala Val Arg Leu Val Pro Thr Ser Val Tyr Glu
    130 135 140
    Gln Trp
    145
    <210> SEQ ID NO 112
    <211> LENGTH: 343
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 112
    Met Gln Gln Pro His His Ser Arg Val Asp Val Glu Leu Gly Glu Arg
    1 5 10 15
    Ser Tyr Pro Val His Val Gly Pro Gly Val Arg His Leu Leu Pro Gly
    20 25 30
    Ile Val Ala Ser Leu Gly Ala His Arg Ala Ala Val Val Thr Ala Arg
    35 40 45
    Pro Pro Asp Leu Val Pro Asp Pro Gly Val Pro Ala Leu Ile Val Arg
    50 55 60
    Ala Arg Asp Gly Glu Arg His Lys Thr Leu Ala Thr Val Glu Asp Leu
    65 70 75 80
    Cys Arg Lys Phe Thr Thr Phe Gly Ile Thr Arg His Asp Val Val Val
    85 90 95
    Ser Cys Gly Gly Gly Ser Thr Thr Asp Thr Val Gly Leu Ala Ala Ala
    100 105 110
    Leu His His Arg Gly Val Pro Val Val His Leu Pro Thr Thr Leu Leu
    115 120 125
    Ala Gln Val Asp Ala Ser Val Gly Gly Lys Thr Ala Val Asn Leu Pro
    130 135 140
    Glu Gly Lys Asn Leu Val Gly Ala Tyr Trp Gln Pro Lys Ala Val Leu
    145 150 155 160
    Cys Asp Thr Thr Tyr Leu Gln Thr Leu Pro Ala Glu Glu Trp Val Asn
    165 170 175
    Gly Tyr Gly Glu Ile Ala Arg Cys His Phe Ile Gly Ala Gly Asp Leu
    180 185 190
    Arg Gly Leu Ala Val His Asp Gln Val Thr Ala Ser Leu Arg Leu Lys
    195 200 205
    Ala Ser Val Val Ala Ala Asp Glu Arg Asp Thr Gly Leu Arg His Ile
    210 215 220
    Leu Asn Tyr Gly His Thr Leu Gly His Ala Leu Glu Thr Ala Thr Gly
    225 230 235 240
    Phe Gly Leu Arg His Gly Leu Gly Val Ala Ile Gly Thr Val Phe Ala
    245 250 255
    Gly Arg Leu Ala Glu Ala Leu Gly Arg Ile Gly Ala Asp Arg Ala Arg
    260 265 270
    Glu His Thr Glu Val Val Arg His Tyr Gly Leu Pro Asp Ser Leu Pro
    275 280 285
    Gly Asn Thr Asp Ile Thr Glu Leu Val Ala Leu Met Arg His Asp Lys
    290 295 300
    Lys Ala Thr Ser Gly Leu Thr Phe Val Leu Asp Gly Pro Ser Gly Val
    305 310 315 320
    Glu Leu Val Ser Gly Ile Pro Glu Asp Val Val Leu Arg Thr Leu Ala
    325 330 335
    Ala Met Pro Arg Gly Thr Ala
    340
    <210> SEQ ID NO 113
    <211> LENGTH: 164
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 113
    Met Glu Arg Val Glu Leu Ile Arg Trp Pro Val Glu Ser Glu Arg Arg
    1 5 10 15
    Glu Arg Cys Arg Asp Arg Gly Val Met Arg Ile Leu Val Leu Glu Ala
    20 25 30
    Gly Ala Glu Ala Pro Leu Cys Val Asp Pro Lys Glu Asp Trp Val Arg
    35 40 45
    Ala Pro Val Ser Thr Asp Asp Leu Arg Ala Arg Val Glu Ala Leu Arg
    50 55 60
    Leu Arg Gly Ala Ala Ala Glu Ser Arg Pro Glu Val Asp Pro Asn Gly
    65 70 75 80
    Val Leu Arg Phe Arg Trp Arg Ser Ala Leu Leu Ser Pro Thr Glu Ala
    85 90 95
    Arg Leu Val Ala Arg Leu Ala Glu Ser Tyr Ala Glu Val Val Ala Arg
    100 105 110
    Asp Asp Leu Leu Arg Pro Pro Pro Gly Arg Thr Val Pro Ser Arg Asn
    115 120 125
    Ala Leu Asp Leu His Ile Met Arg Ile Arg Arg Arg Leu Ala Ala Leu
    130 135 140
    Gly Leu Arg Val Arg Thr Val Arg Gly Arg Gly Tyr Val Leu Glu Ser
    145 150 155 160
    Ala Glu Gly Val
    <210> SEQ ID NO 114
    <211> LENGTH: 514
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 114
    Met Leu Asp Arg Arg Ser Val Ile Arg Val Gly Ala Gly Val Ala Ala
    1 5 10 15
    Ala Ala Ala Val Ala Gly Thr Ala Ala Thr Gly Ala Ala Ala Val Gly
    20 25 30
    Leu Pro Gly Val Arg Gly Arg Ala Ala Ser Arg Gly Val Asp Trp Ala
    35 40 45
    Ser Leu Arg Arg His Leu Ser Gly Glu Leu Val Leu Pro Ala Asp Thr
    50 55 60
    Gly Tyr Glu Arg Ala Arg Lys Leu Tyr Ser Gly Gln Phe Asp Gly Ile
    65 70 75 80
    Arg Pro Gln Ala Val Ala Tyr Cys Arg Thr Glu Glu Asp Val Arg Thr
    85 90 95
    Thr Leu Ala Phe Ala Gln Asp His Ala Leu Pro Leu Thr Pro Arg Ser
    100 105 110
    Gly Gly His Ser Phe Gly Gly Tyr Ser Thr Thr Asp Gly Ile Val Leu
    115 120 125
    Asp Val Ser Gly Phe His Ala Val Ser Leu Thr Arg Asn Thr Val Val
    130 135 140
    Met Gly Ala Gly Thr Gln Gln Val Asp Ala Leu Thr Ala Leu Ser Pro
    145 150 155 160
    Arg Gly Val Ala Val Ala Ser Gly Asn Cys Ala Gly Val Cys Pro Gly
    165 170 175
    Gly Phe Val Gln Gly Gly Gly Leu Gly Trp Gln Ser Arg Lys Phe Gly
    180 185 190
    Met Ala Cys Asp Arg Leu Val Ser Ala Arg Val Val Leu Ala Asp Gly
    195 200 205
    Arg Ala Val Thr Ala Ser Ala Thr Glu His Pro Asp Leu Phe Trp Ala
    210 215 220
    Met Arg Gly Gly Gly Gly Gly Asn Phe Gly Val Val Thr Gly Phe Glu
    225 230 235 240
    Leu Arg Pro Thr Asp Val Pro Ser Val Val Ser Tyr Asn Leu Thr Trp
    245 250 255
    Pro Trp Glu Ser Ala Arg Arg Val Ile Glu Ala Trp Gln His Trp Ile
    260 265 270
    Ile Asp Gly Pro Arg Asp Leu Gly Ala Ala Met Ala Val Gln Trp Pro
    275 280 285
    Asp Ala Gly Thr Gly Thr Pro Val Val Val Val Thr Gly Ala Trp Leu
    290 295 300
    Gly Ala Ala Asp Ala Leu Thr Pro Val Leu Asp Ser Leu Val Ala Ser
    305 310 315 320
    Val Gly Ser Ala Pro Ala Thr Arg Ser Ala Lys Ala Leu Ser Gln His
    325 330 335
    Asp Ala Met Met Ala Gln Tyr Gly Cys Ala Asp Leu Thr Pro Glu Gln
    340 345 350
    Cys His Thr Val Gly Tyr Ser Pro Glu Ala Ala Leu Pro Arg Gln Asn
    355 360 365
    Phe Ser Met Asp Arg Asn Arg Leu Phe Ser Arg Ala Ile Gly Gln Gly
    370 375 380
    Gly Val Glu Arg Ile Leu Glu Ala Phe Ala Ala Asp Pro Arg Ala Gly
    385 390 395 400
    Gln Phe Arg Phe Leu Ser Phe Phe Ala Leu Gly Gly Ala Ala Asn Arg
    405 410 415
    Pro Asp Arg Thr Thr Thr Ala Tyr Val His Arg Asp Thr Glu Phe Tyr
    420 425 430
    Leu Gly Phe Ser Ile Gly Leu Asn Asp Pro Glu Tyr Thr Ala Glu Asp
    435 440 445
    Glu Arg Leu Gly Arg Ala Trp Ala Ala Arg Gly Leu Arg Thr Leu Asp
    450 455 460
    Pro His Ser Asn Gly Glu Ser Tyr Gln Asn Phe Ile Asp Pro Glu Leu
    465 470 475 480
    Asp Asp Trp Lys Ser Ala Tyr Tyr Ala Glu Asn Tyr Val Arg Leu Ala
    485 490 495
    Ala Val Lys Ala Ala Tyr Asp Pro His Arg Leu Phe Ser Phe Ala Gln
    500 505 510
    Ala Val
    <210> SEQ ID NO 115
    <211> LENGTH: 315
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 115
    Met Thr Gly Asp Thr Asp Gly Ala Gly Gly Gly Asp Val Thr Phe Arg
    1 5 10 15
    Trp Pro Ala Ala Gly Asp Val Thr Ala Asp Leu Asp Leu Leu Ala Ala
    20 25 30
    Arg Val Arg Gly Leu Leu Gly His Arg Glu Asp Pro Leu Ala Gly Val
    35 40 45
    Gly Val Ala Met Pro Ala Ile Cys Asp Ala Ala Gly Thr Val Arg Thr
    50 55 60
    Trp Pro Gly Arg Pro Ser Trp Ala Gly Leu Asn Leu Thr Ala Ala Phe
    65 70 75 80
    Gly Gln Leu Leu Pro Gly Thr Pro Val Ala Cys Ala Asp Asp Gly Asp
    85 90 95
    Leu Ala Ala Leu Ala Glu Ser Arg Ala Ala Gly Cys Arg His Leu Leu
    100 105 110
    Tyr Val Gly Val Gly Thr Gly Ile Gly Gly Gly Ile Val His Glu Gly
    115 120 125
    Arg Ala Trp Pro Gly Pro Gly Arg Gly Ser Cys Glu Val Gly His Val
    130 135 140
    Val Val Asp Arg Ser Gly Pro Arg Cys Asp Cys Gly Arg Ala Gly Cys
    145 150 155 160
    Val Gln Ala Val Ala Ser Gly Pro Ala Thr Leu Arg Arg Ala Ala Glu
    165 170 175
    Arg Arg Gly Arg Glu Thr Gly Phe Asp Glu Leu Ala Ser Gly Ala Arg
    180 185 190
    Leu His Ala Pro Trp Ala Glu Ala Ala Val Asp Glu Ser Ala Ala Ala
    195 200 205
    Leu Ala Thr Ala Val Thr Gly Ile Cys Glu Leu Ala His Pro Glu Leu
    210 215 220
    Val Leu Val Gly Gly Gly Phe Ala Ala Gly Val Pro Gly Tyr Val Ala
    225 230 235 240
    Ser Val Ala Ala His Val Glu Arg Leu Thr Arg Pro Gly Thr Asp Pro
    245 250 255
    Val Arg Val Arg Pro Ala Val Leu Gly Gly Arg Ser Ser Leu His Gly
    260 265 270
    Ala Leu Leu Leu Ala Arg Glu Ala His Gly Arg Gly Asn Arg Pro Pro
    275 280 285
    Glu Ser Asp Arg Val Ser Ser Asp Val Ser Ser Asp Val Ser Phe Gly
    290 295 300
    Gly Val Thr Asp Arg Ala Val Gly Arg Ser Asp
    305 310 315
    <210> SEQ ID NO 116
    <211> LENGTH: 514
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 116
    Met Pro Pro Ser Pro Arg Ala Leu Val Ile Gly Ile Asp Gly Gly Thr
    1 5 10 15
    Phe Asp Thr Val Asp Pro Leu Ile Glu Cys Gly Leu Leu Pro His Met
    20 25 30
    Ala Lys Leu Leu Arg Glu Ser Ala Ser Ala Ala Thr Asp Cys Thr Trp
    35 40 45
    Pro Ala His Thr Ala Pro Gly Trp Ser Thr Phe Val Ser Ala Ser Asp
    50 55 60
    Pro Gly Gly His Gly Ile Tyr Gln Phe Tyr Asp Thr Gln Asp Pro Ala
    65 70 75 80
    Tyr Gly Ala Arg Val Thr Arg Ser Gly Asp Leu Gly Arg Ser Cys Ala
    85 90 95
    Trp Asp Trp Leu Ala Ala Gln Glu Tyr Ser Leu Gly Leu Ile Asn Ile
    100 105 110
    Pro Met Ser His Pro Pro Ala Asp Leu Pro Gly Tyr Gln Val Thr Trp
    115 120 125
    Pro Leu Glu Arg Thr Leu Lys His Cys Arg Pro Asp Ser Leu Leu Arg
    130 135 140
    Glu Leu Ala Ala Ala Lys Ala His Phe Gln Ser Asp Leu Ala Thr Met
    145 150 155 160
    Phe Arg Gly Asp Met Ala Tyr Leu Glu Glu Ala Glu Arg Asn Val Ala
    165 170 175
    Ala Arg Val Arg Ser Val Arg His Leu Met Ser Thr Arg Pro Thr Asp
    180 185 190
    Val Val Met Val Val Leu Thr Glu Ala Asp Arg Val Gly His His Tyr
    195 200 205
    Trp His Tyr Gly Asp Pro Gly His Pro Gly His Arg Pro Ala Pro Glu
    210 215 220
    Gly Ser Gly Trp Asp Val Ala Met Pro Arg Ile Tyr Gln Ala Ile Asp
    225 230 235 240
    His Ala Val Gly Glu Leu Leu Glu Leu Val Asp Glu Asp Thr Ser Val
    245 250 255
    Val Leu Val Ser Asp His Gly Leu Gly Thr Gly Arg His Gly Leu Ser
    260 265 270
    Val His Thr Leu Leu Glu Glu Ala Gly Leu Leu Ala Thr Ala Pro Gly
    275 280 285
    Glu Glu Pro Gln Asp Ala Ala Ala Ser Trp Phe Ala Gly Asn Gly Arg
    290 295 300
    His Val Asp Phe Arg Arg Thr Ser Val Tyr Met Pro Val Pro Gly Ser
    305 310 315 320
    Tyr Gly Leu Asn Ile Asn Val Arg Gly Arg Gln Gln Arg Gly Thr Val
    325 330 335
    Ala Pro Arg Asp Arg Glu Arg Val Met Asp Glu Val Thr Gly Leu Leu
    340 345 350
    Ser Gly Leu Thr Gly Pro Glu Gly Gln Gln Val Phe Arg Ala Val Arg
    355 360 365
    Pro Arg Glu Glu Ala Tyr Pro Gly Pro His Thr Gly Arg Ala Pro Asp
    370 375 380
    Leu Leu Leu Val Pro Arg Asp Glu Thr Val Leu Pro Val Pro Asp Leu
    385 390 395 400
    Gly Gly Asp Val Trp Arg Pro Ser Ala Gln Thr Gly Leu His Arg Tyr
    405 410 415
    Arg Gly Leu Trp Ala His Arg Ser Pro Arg Val Arg Pro Gly Arg Leu
    420 425 430
    Pro Gly Thr Val Ala Leu Thr Asp Thr Leu Pro Thr Leu Leu Thr Asp
    435 440 445
    Leu Gly Ala Ala Trp Pro Ser Asp Ile His Gly Arg Pro Val Thr Ala
    450 455 460
    Val Leu Asp Asp Gly Val Arg Val Pro Pro Ser Asp Pro Arg Val Glu
    465 470 475 480
    Ala Thr Gly Thr Pro Ala Thr Thr Ile Pro Ala Ala Ala Ser Ala Ala
    485 490 495
    Asp Ala Ala Glu Asp Ala Tyr Thr Ser Asp Arg Leu Arg Glu Met Gly
    500 505 510
    Tyr Leu
    <210> SEQ ID NO 117
    <211> LENGTH: 93
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 117
    Met Glu Thr Leu Thr Thr Asp Lys Ile Lys Asp Arg Leu Arg Lys Val
    1 5 10 15
    Leu Val Asp Ser Leu Glu Leu Ser Leu Asp Pro Ser Ala Val Pro Asp
    20 25 30
    Glu Gly Leu Val Glu Lys Leu Gly Leu Asp Ser Ile Asn Thr Ile Glu
    35 40 45
    Phe Leu Ile Trp Val Glu Ser Glu Phe Gly Ile Glu Ile Ala Asp Glu
    50 55 60
    Asp Leu Ser Ile Lys Leu Ile Asp Ser Leu Asp Leu Leu Ala Gly Tyr
    65 70 75 80
    Val Ser Glu Arg Val Asn Gly Val Thr Ala Pro Ala Glu
    85 90
    <210> SEQ ID NO 118
    <211> LENGTH: 470
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 118
    Met Asp Arg His Ala Leu Val Ile Gly Leu Asp Gly Met Pro Arg Thr
    1 5 10 15
    Leu Leu Thr Arg Leu Ala Gly Asp Gly Thr Met Pro His Thr Ala Ala
    20 25 30
    Leu Leu Ala Glu Gly His Cys Ala Glu Leu Leu Ala Pro Val Pro Glu
    35 40 45
    Ile Ser Ser Thr Ser Trp Ala Thr Phe Leu Thr Gly Thr Asn Pro Gly
    50 55 60
    Arg His Gly Ile Tyr Gly Phe Thr Asp Leu Ala Pro Gly Asp Gly Tyr
    65 70 75 80
    Arg Ile Thr Phe Pro Gly Val Arg Gln Leu Arg Glu Pro Pro Leu Trp
    85 90 95
    Glu Leu Ala Ala Arg Ala Gly Arg Arg Thr Val Cys Leu Asn Val Pro
    100 105 110
    Gly Thr Tyr Pro Ala Pro Ala Ile Asp Gly Val Leu Val Ser Gly Phe
    115 120 125
    Val Ala Pro Glu Leu Glu Arg Ala Val Ser Pro Pro Arg Leu Leu Pro
    130 135 140
    Leu Leu Arg Gly Leu Asp Tyr Glu Leu Asp Val Glu Val Gly Asp Val
    145 150 155 160
    Ala Ala Asp Pro Ala Ala Phe Leu Gly Arg Ala Val Arg Ala Leu Arg
    165 170 175
    Ala Arg Thr Arg Ala Met Glu His Leu Leu Arg Gln Glu Thr Trp Asp
    180 185 190
    Leu Ala Val Ala Val Leu Thr Glu Thr Asp Arg Val His His Phe Leu
    195 200 205
    Trp Arg Ala Val Ala Asp Pro Ala Asp Pro Leu His Gly Asp Val Leu
    210 215 220
    Ala Phe Tyr Arg Leu Val Asp Asp Cys Val Ala Thr Leu Val Ser Thr
    225 230 235 240
    Leu Pro Pro Gly Gly Glu Leu Phe Leu Met Ser Asp His Gly Phe Gly
    245 250 255
    Pro Ala Ala Cys Gln Val Tyr Leu Asn Ala Trp Leu Arg Glu Ser Gly
    260 265 270
    Trp Leu Ala Gly Leu Asp Val Cys Pro Asp Leu Thr Ala Val Asp Ala
    275 280 285
    Arg Ser Thr Ala Phe Ala Leu Asp Pro Ala Arg Ile His Leu Asn Arg
    290 295 300
    Lys Ser Arg Phe Pro Gly Gly Gly Leu Thr Asp Ala Glu Ala Asp Glu
    305 310 315 320
    Ala Ala His Glu Ile Ala Arg Glu Leu Ser Ala Leu Arg Cys Asp Gly
    325 330 335
    Thr Arg Leu Gly Pro Asp Val Asp Gly Pro Leu Leu Val Arg Asp Leu
    340 345 350
    Tyr Arg Ala Gln Glu Ile Tyr His Gly Pro Leu Leu Gly Asn Ala Pro
    355 360 365
    Asp Leu Val Ala Val Pro Ala Pro Gly Val Gln Leu Arg Gly Gly Trp
    370 375 380
    Gly Gly Thr His Thr Val Arg Asn Asp Ile Leu Thr Gly Thr His Thr
    385 390 395 400
    Arg Asp Asp Ala Val Phe Tyr Arg Arg Gly Ala Pro Ala Pro Ala Pro
    405 410 415
    Gly Ala Asp Asp Gly Pro Leu Asp Met Thr Asp Val Ala Pro Thr Val
    420 425 430
    Leu Ala Ser Leu Gly Ile His Pro Gly Gly Leu Asp Gly Ala Ala Val
    435 440 445
    Leu Gly Thr Thr Gly Pro Ala Ser Gly His Gly Arg Thr Asp Pro Pro
    450 455 460
    Leu Asp Ile Arg Glu Leu
    465 470
    <210> SEQ ID NO 119
    <211> LENGTH: 611
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 119
    Met Lys His Asp Leu Gly Leu Ala Pro Ser Ala Pro Lys Pro Gly Thr
    1 5 10 15
    Leu Asp Leu Ser Leu Asp Pro Arg Ile Thr Asp Pro Ala Ser Phe Arg
    20 25 30
    Val Ser Phe Leu Ile Leu Leu Asp Gly Asp Leu Val Met Ser Pro Glu
    35 40 45
    His Leu Gly Val Ala Tyr Met Ala Gly Val Leu Arg His Thr Gly Phe
    50 55 60
    Thr Ala Glu Ile Arg Glu Val Glu His Gly Asp Asp Gln Ala Ala Ala
    65 70 75 80
    Thr Val Glu Ala Leu Lys Glu Tyr Arg Pro Asp Leu Val Cys Phe Thr
    85 90 95
    Leu Met Ser Leu Asn Leu Gly Ser Cys Leu Thr Leu Cys Arg Met Leu
    100 105 110
    Arg Glu Glu Leu Pro Gly Thr Thr Ile Ala Cys Gly Gly Pro Ala Gly
    115 120 125
    Thr Phe Ala Gly Leu Asp Val Leu Arg Asn Asn Pro Trp Thr Asp Val
    130 135 140
    Val Ala Val Gly Glu Gly Glu Pro Thr Ile Leu Asp Leu Val Gln Arg
    145 150 155 160
    Leu Tyr Leu Lys Glu Pro Leu Ser Ala Cys Lys Gly Ile Cys Tyr Arg
    165 170 175
    Asp Glu Asp Gly Thr Pro Arg Gln Asn Pro Ala Arg Pro Leu Ile His
    180 185 190
    Asn Leu Glu Asp Leu Pro Phe Pro Ala Arg Asp Gln Leu Arg Gln His
    195 200 205
    Gly Asp Lys Leu Glu Tyr Val Arg Val Ser Thr Ser Arg Gly Cys Val
    210 215 220
    Ala Asn Cys Ala Phe Cys Ser Ala Pro His Leu Lys Asn Arg Val Gln
    225 230 235 240
    Ala Gly Lys Ala Trp Arg Gly Arg Gly Pro Glu Gln Ile Val Asp Glu
    245 250 255
    Val Ala Glu Ile Val Glu Arg His Gln Phe Arg Thr Phe Asp Phe Val
    260 265 270
    Asp Ser Thr Phe Glu Asp Pro Asp Gly Gly Arg Val Gly Lys Lys Arg
    275 280 285
    Val Ala Ala Ile Ala Asn Gly Ile Leu Glu Arg Gly Leu Asp Ile Tyr
    290 295 300
    Tyr Asn Val Cys Met Arg Ala Glu Asn Trp His Asp Thr Pro Glu Asp
    305 310 315 320
    His Ala Leu Leu Asp Leu Leu Val Ala Ser Gly Leu Glu Lys Val Asn
    325 330 335
    Val Gly Ile Glu Ala Gly Thr Ala Glu Glu Leu Leu Leu Trp Glu Lys
    340 345 350
    Arg Ala Thr Val Glu Asp Asn Val Thr Ile Ile Arg Met Leu Arg Glu
    355 360 365
    His Gly Ile Tyr Leu Ala Met Gly Phe Ile Pro Phe His Pro Tyr Ala
    370 375 380
    Thr Leu Glu Thr Ile Val Thr Asn Ala Ala Phe Leu Arg Asp Asn Ser
    385 390 395 400
    Gly His Asn Leu Arg Arg Met Thr Glu Arg Leu Glu Ile Tyr Pro Gly
    405 410 415
    Thr Ala Ile Val Ser Arg Met Arg Ala Asp Gly Leu Leu Gly Glu Ser
    420 425 430
    Tyr Leu Glu Gly Leu Asp Pro Tyr Gly Tyr Ala Phe Lys Asp Pro Arg
    435 440 445
    Val Gly Arg Leu Ala Lys His Phe Ala Gln Leu Tyr Asn Asn Asp Asp
    450 455 460
    Tyr His Arg His Gly Val Ile Thr Glu Gln Ser Ser Val Phe Ala Phe
    465 470 475 480
    Glu Thr Tyr Asn Val Val Leu Gln Thr Phe Ile Ser Arg Leu His Arg
    485 490 495
    Arg Phe Thr Thr Leu Pro Gly Val Asp Glu Val Met Glu Ala Phe Lys
    500 505 510
    Ala Arg Val His Glu Ile Arg Gln Glu Met Gly Arg His Asn Tyr Gly
    515 520 525
    Phe Phe Met Ser Asn Val Glu Ala Val Met Asn Asp Thr Leu Asp Pro
    530 535 540
    Glu Lys Gln Arg Arg Gln Val Val Asp Val Glu His Phe Phe Arg Asp
    545 550 555 560
    Arg Leu Asp Val Leu Arg Ser Glu Gln Leu Arg Val Gly Lys Ala Leu
    565 570 575
    Thr Arg Leu Gly Ala Arg Val Thr Glu Val Ser Ser Thr Ile Pro Lys
    580 585 590
    Glu Arg Pro Gly Gly Leu Pro Arg Gln Tyr Thr Gly Glu Gly Ser Gly
    595 600 605
    Ala Thr Trp
    610
    <210> SEQ ID NO 120
    <211> LENGTH: 359
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 120
    Met Pro Arg Gly Glu Thr Gly Thr Ala Ala Ala Arg Val Ala Val Cys
    1 5 10 15
    Thr Leu Ser Ser Arg Glu Leu Val Gly Pro Leu Ala Arg Leu Pro Gly
    20 25 30
    Val Ala Ala Ala Gly Thr Leu Met Thr Ala Asn Leu Gly Ile Glu Gln
    35 40 45
    Val Ile Lys Ala Leu Arg Cys Asp Arg Thr Val Arg Gly Leu Leu Val
    50 55 60
    Cys Gly Arg Asp Ser Pro Arg Phe Arg Ala Gly Gln Ser Leu Ile Ala
    65 70 75 80
    Leu Phe Arg His Gly Leu Arg Pro Glu Asp Gly His Ile Arg Gly Ala
    85 90 95
    Thr Gly Tyr Leu Pro Val Leu Arg Ser Val Thr Ala Arg Glu Thr Glu
    100 105 110
    Glu Val Arg Ala Arg Val Glu Leu Val Asp Ala Arg Gly Glu Arg Asp
    115 120 125
    Val Glu Thr Leu Arg Ala Glu Val Ala Ala Leu Leu Ala Arg Val Arg
    130 135 140
    Arg Thr Pro Ala Leu Pro Ser Arg Glu His Asp Gly Gly Gln Pro Ser
    145 150 155 160
    Phe Val Glu Pro Asp Phe Gly Arg Leu His Pro Val Gly Arg Arg Arg
    165 170 175
    Ser Leu Asp Ala Gly Ile Gly Gly Phe Val Leu Ile Ser Val Asp Arg
    180 185 190
    Glu His Arg Arg Ile Leu Leu Arg His Tyr Thr Ser Asp Val Arg Pro
    195 200 205
    Arg His Glu Met Trp Gly Thr Arg Gly Glu Ala Met Leu Leu Gly Leu
    210 215 220
    Leu Glu Ala Gly Val Ile Glu Asp Pro Ala His Ala Gly Tyr Leu Gly
    225 230 235 240
    Ala Glu Leu Ala Lys Ala Glu Thr Ala Leu Arg Leu Gly Leu His Tyr
    245 250 255
    Glu Gln Asp Leu Pro Leu Arg Pro Pro Gly Arg Pro Pro Gly Pro Val
    260 265 270
    Arg Arg Arg Thr Ala Lys Glu Arg Thr Thr Met Ala Gln Ala Pro Ala
    275 280 285
    Leu Glu Asp Phe Leu Arg Leu Val Thr Arg Thr Leu Gly Ala Glu Asp
    290 295 300
    Ala Val Leu Asp Leu His Thr Pro Leu Gly Glu Gln Leu Ala Val Asp
    305 310 315 320
    Ser Ala Arg Leu Ile Glu Leu Thr Val Val Leu Glu Glu Glu Leu Gly
    325 330 335
    Ala Asp Leu Pro Asp Asp Ala Asp Leu Ala Arg Ala Thr Pro Ala Glu
    340 345 350
    Leu His Lys Ala Leu Val Gly
    355
    <210> SEQ ID NO 121
    <211> LENGTH: 145
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 121
    Met Arg Ser Val Leu Leu Leu Asn Gly Pro Asn Leu Gly Thr Leu Gly
    1 5 10 15
    Lys Arg Gln Pro Glu Ile Tyr Gly Thr Asp Thr Leu Ala Glu Ile Glu
    20 25 30
    Ala Ala Val Ala Glu Glu Val Gly Ala Arg Gly Trp Glu Val Val Ser
    35 40 45
    Glu Gln Arg Asn Gly Glu Gly Glu Leu Val Asp Val Leu Gln Arg His
    50 55 60
    Asp Asp Val Val Gly Ala Val Val Asn Pro Gly Ala Leu Met Ile Ala
    65 70 75 80
    Gly Trp Ser Leu Arg Asp Ala Leu Ala Asp Phe Ala Pro Pro Trp Val
    85 90 95
    Glu Val His Leu Ser Asn Val Trp Gly Arg Glu Ala Phe Arg His Thr
    100 105 110
    Ser Val Thr Ala Pro Leu Ala Ser Gly Val Val Met Gly Met Gly Ala
    115 120 125
    Leu Gly Tyr Arg Leu Ala Ala Arg Ala Leu Thr Arg Leu Val Pro Glu
    130 135 140
    Asp
    145
    <210> SEQ ID NO 122
    <211> LENGTH: 177
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 122
    Met Gly Arg Tyr Gly Arg Glu Gly Leu Gly Met Ser Arg Thr Ala Glu
    1 5 10 15
    Gly Asn Ala Gly Gly Val Val Val Pro Val Val Arg Leu Val Ala Val
    20 25 30
    Thr Asp Gly Pro Asp Ala Glu Gly Trp Arg Gln Ala Leu Ala Pro Glu
    35 40 45
    Leu Val Val Glu His Gly Val Glu Ala Ile Ala Glu Ala Ala Gly Asp
    50 55 60
    Gly Gly Pro Trp Ala Leu Val Cys Ala Gly Ala Gly Leu Gly Ala Ala
    65 70 75 80
    Leu Arg Ala Ala Glu Arg Ala Ala Arg Pro Pro Val His Val Leu Leu
    85 90 95
    Trp Leu Gly Ser Arg Gly Pro Gly Glu Gly Val Gly Gly Glu Val Ser
    100 105 110
    Gly Gln Phe Pro Cys Pro Val Thr Ala Leu Val Ser Ala Glu Val Asp
    115 120 125
    Arg Gly Arg Ala Val Val Pro Ala Trp Arg Gly Leu Thr Glu Gly Pro
    130 135 140
    Phe Thr Val Arg Ile Leu Pro Ala Ala Cys Pro Leu Pro Gly Ala Cys
    145 150 155 160
    Asp Gln Ala Gly Ala Gln Val Ile Lys Glu Glu Leu Arg Val Trp Pro
    165 170 175
    Ala
    <210> SEQ ID NO 123
    <211> LENGTH: 254
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 123
    Met Asp Ala Thr Leu Thr Asn Asp Val Glu Lys Ala Ser Arg Asp Leu
    1 5 10 15
    Val Glu Ala Gly Tyr Cys Leu Ile Glu Cys Pro Leu Pro Ala Ala Val
    20 25 30
    Phe Glu Lys Leu Arg Gly Arg Leu Leu Glu Val Ala Glu Gln Glu Arg
    35 40 45
    Glu Asn Gly Ser Ala Phe Leu Tyr Asp Gly Gly Asn Gln Arg Val Phe
    50 55 60
    Ser Leu Leu Asn Lys Gly Glu Glu Phe Glu Gln Asn Val Gln Asp Pro
    65 70 75 80
    Thr Val Met Leu Leu Met Glu Glu Ile Leu Gly Phe Gly Phe Leu Leu
    85 90 95
    Ser Ser Thr His Ala Asn Ile Ala Gly Pro Gly Gly Ser Arg Met His
    100 105 110
    Leu His Ala Asp Gln Thr Phe Ala Arg Pro Pro Trp Pro Pro Tyr Pro
    115 120 125
    Leu Val Ala Asn Ser Met Trp Met Leu Asp Asp Phe Thr Glu Asp Asn
    130 135 140
    Gly Ala Thr Arg Leu Val Pro Gly Ser His Leu Leu Gly Arg Gln Pro
    145 150 155 160
    Asp Tyr Asp Arg Gly Glu Gly Asn Thr Glu Thr Val Ala Val Cys Ala
    165 170 175
    Pro Ala Gly Ser Val Met Val Phe Asp Gly Arg Leu Trp His Gln Thr
    180 185 190
    Gly Ala Asn Thr Thr Asp Arg Pro Arg His Gly Ile Leu Asn Tyr Tyr
    195 200 205
    Cys Arg Gly Tyr Val Arg Gln Gln Gln Asn Phe Phe Ser Gly Leu Arg
    210 215 220
    Glu Asp Val Ala Thr Arg Ala Thr Pro Glu Leu Arg Arg Leu Leu Gly
    225 230 235 240
    Tyr Glu Asn Tyr Phe Ser Leu Gly Met Thr Asp Gly Leu Pro
    245 250
    <210> SEQ ID NO 124
    <211> LENGTH: 264
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 124
    Met Ala His Ser Pro Arg Arg Pro Asp Gly Pro Leu Arg Ile Gly Val
    1 5 10 15
    Trp Leu Ala Pro Gln His Thr Ser Val Ala Glu Leu Arg Ala Ala Trp
    20 25 30
    Arg Ala Ala Asp Ser Leu Gly Val Asp Ser Leu Trp Leu Trp Asp His
    35 40 45
    Phe Phe Pro Leu Thr Gly Asp Pro Asp Gly Ser His Phe Glu Ala Trp
    50 55 60
    Thr Leu Leu Ala Ala Met Ala Ala Asp Thr Arg Ala Ala Arg Leu Gly
    65 70 75 80
    Thr Leu Val Ser Asn Tyr Ala Tyr Arg Asn Pro Asp Leu Leu Ala Asp
    85 90 95
    Met Ala Arg Thr Val Asp His Ile Gly Asp Gly Arg Leu Ile Leu Gly
    100 105 110
    Met Gly Ala Gly Trp Val Glu Arg Asp Leu Lys Glu Tyr Gly Tyr Pro
    115 120 125
    Thr Pro Gly Ala Gly Glu Arg Val Asp Gly Leu Ile Glu Ala Val Glu
    130 135 140
    Arg Val Asp Arg Arg Leu Gly Arg Leu Arg Pro Gly Pro Leu Gly Asp
    145 150 155 160
    Leu Pro Leu Leu Ile Gly Gly Asp Gly Gln Arg Arg Leu Leu Arg Phe
    165 170 175
    Ala Ala Glu Arg Ala Ala Ile Trp Asn Thr Met Ala Trp Arg Phe Ala
    180 185 190
    Glu Gly Asn Arg Val Leu Asp Glu Trp Cys Ala Arg Val Gly Arg Asp
    195 200 205
    Pro Ala Glu Ile Glu Arg Ser Ala Phe Val Thr Arg Asp Gln Thr Asp
    210 215 220
    Glu Glu Leu Arg Cys Leu Val Ala Thr Gly Val Gln His Leu Ile Phe
    225 230 235 240
    Gln Val Gly His Pro Phe Arg Phe Asp Gly Val Glu Arg Ala Leu Arg
    245 250 255
    Phe Ala Gly Gly Trp Ser Lys Gly
    260
    <210> SEQ ID NO 125
    <211> LENGTH: 274
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 125
    Met Lys Ile Ser Ile Ala Leu Pro Asn Thr Val Pro Gly Ala Asp Gly
    1 5 10 15
    Arg Leu Ile Thr Asp Trp Ala Arg Arg Ala Glu Glu Arg Gly Phe Ala
    20 25 30
    Ser Leu Ala Ala Thr Glu Arg Leu Val Tyr Pro Gly His Asp Pro Leu
    35 40 45
    Leu Ala Leu Ala Ala Ala Ala Gly Ala Thr Ser Arg Ile Gly Leu Leu
    50 55 60
    Thr Asn Val Leu Ile Gly Pro Leu Arg Thr Ala Pro Val Leu Ala Lys
    65 70 75 80
    Ala Val Ala Ser Leu Asp Ser Leu Ser Gly Gly Arg Phe Thr Leu Gly
    85 90 95
    Val Gly Pro Gly Val Arg Glu Asp Asp Phe Glu Ala Ala Gly Arg Ala
    100 105 110
    Phe Asp Asp Arg Arg Ala Ala Phe Glu Glu Gln Leu Glu Leu Leu Gly
    115 120 125
    Arg Gly Ala Arg Pro Gly Ala Glu Gly Pro Gly Val Pro Val Leu Val
    130 135 140
    Gly Gly Val Ser Ala Ala Ala Val Arg Arg Val Ala Arg Trp Ala Asp
    145 150 155 160
    Gly Trp Thr Ala Pro Gly Leu Glu Pro Glu Arg Ile Val Pro Val Ala
    165 170 175
    Glu Arg Val Arg Arg Ala Trp Ser Glu Ala Gly Arg Ala Gly Ala Pro
    180 185 190
    His Val Val Ala Leu Ala Arg Tyr Thr Leu Gly Glu Asp Val Ala Gln
    195 200 205
    Glu Ser Ala Ala Phe Val Arg Asp Tyr Phe Ala Val Leu Gly Glu Glu
    210 215 220
    Ala Glu Glu Phe Val Ala Lys Thr Pro Arg Thr Ala Gly Gln Leu Arg
    225 230 235 240
    Ala Ala Val Ser Ala Leu Ala Asp Gly Gly Val Asp Glu Val Val Leu
    245 250 255
    His Pro Thr Ala Ala Ala Leu Ser Gln Val Asp Arg Leu Ala Asp Ala
    260 265 270
    Leu Leu
    <210> SEQ ID NO 126
    <211> LENGTH: 460
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 126
    Met Pro Ala Ala Gly Lys Val Ala Val Ile Gly Leu Asp Ser Ala Thr
    1 5 10 15
    Pro Gln Tyr Met Phe Asp Arg Phe Ala Glu Asp Met Pro Val Phe Thr
    20 25 30
    Ala Leu Arg Arg Lys Ser Leu Trp Gly Pro Met Arg Ser Ile Asp Pro
    35 40 45
    Pro Ile Thr Met Pro Ala Trp Ser Cys Met Met Ser Gly Arg Ser Pro
    50 55 60
    Gly Glu Leu Gly Val Tyr Gly Phe Arg Asp Arg Gly Ala Tyr Asp Tyr
    65 70 75 80
    Gly Pro Leu Lys Phe Ala Thr Ser His Ser Ile Gln Ala Pro Arg Ile
    85 90 95
    Trp Asp Glu Met Thr Ala Ala Gly Arg Ser Ser Val Val Leu Gly Val
    100 105 110
    Pro Gly Thr Tyr Pro Pro Ala Pro Ile Arg Gly Ala Met Val Ser Cys
    115 120 125
    Phe Leu Ala Pro Ser Thr Gln Ser Arg Tyr Thr Ser Pro Pro Gly Leu
    130 135 140
    Ala Asp Glu Leu Glu Lys Leu Thr Gly Gly Tyr Ala Leu Asp Val Glu
    145 150 155 160
    Asp Phe Arg Ser Thr Asp Leu Glu Arg Val Ser Gln Arg Val Phe Asp
    165 170 175
    Met Ser Glu Gln Arg Phe Glu Val Ala Arg His Leu Ala Thr Thr Gln
    180 185 190
    Glu Trp Asp Phe Leu Ser Phe Val Asp Met Gly Pro Asp Arg Leu His
    195 200 205
    His Gly Phe Trp Lys Tyr Cys Asp Pro Asp His Pro Arg His Glu Pro
    210 215 220
    Gly Asn Ala Tyr Ala Gly Leu Phe Arg Asp Tyr Tyr Arg Ala Leu Asp
    225 230 235 240
    Arg His Leu Gly Arg Phe Leu Glu Ser Leu Pro Glu Asn Thr Thr Val
    245 250 255
    Leu Val Val Ser Asp His Gly Ala Gln Pro Met Val Gly Gly Leu Phe
    260 265 270
    Val Asn Glu Trp Leu Arg Lys Glu Gly Tyr Leu Val Leu Thr Glu Glu
    275 280 285
    Pro Ala Gly Pro Thr Pro Val Ala Gln Ala Ala Val Asp Trp Lys Arg
    290 295 300
    Thr Thr Ala Trp Ala Glu Gly Gly Tyr Tyr Gly Arg Ile Phe Leu Asn
    305 310 315 320
    Val Glu Gly Arg Glu Pro Gln Gly Thr Ile Pro Ala Ala Glu Tyr Glu
    325 330 335
    Ser Thr Arg Asp Leu Ile Ala Ser Ala Leu Glu Ala Leu Pro Asp Asp
    340 345 350
    Gln Gly Gln Pro Met Gly Thr Arg Ala Leu Arg Pro Gly Glu Leu Tyr
    355 360 365
    Gly Glu Val Asn Gly Ile Ala Pro Asp Leu Leu Val Tyr Val Gly Asn
    370 375 380
    Leu Arg Trp Arg Ala Leu Ala Thr Leu Gly Met Gly Lys Gly Leu Tyr
    385 390 395 400
    Thr Thr Glu Asn Asp Thr Gly Pro Asp His Ala Asn His Gly Asp Thr
    405 410 415
    Gly Ile Phe Ala Leu Ser Ala Pro Gly Ile Thr Pro Gly Arg Ala Asp
    420 425 430
    Gly Leu Ser Leu Tyr Asp Val Ala Pro Thr Leu Arg Glu Leu Leu Gly
    435 440 445
    Leu Ala Pro Gln Gly Ser Arg Gly Ser Leu Leu Gly
    450 455 460
    <210> SEQ ID NO 127
    <211> LENGTH: 511
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 127
    Met Lys Ala Met Asp Arg Val Asp Arg Ala Val Glu Arg Phe Pro Met
    1 5 10 15
    Tyr Ile Asp Gly Gln Ala Val Gln Ala His Asp Gly Ala Val Leu Arg
    20 25 30
    Thr Phe Glu Pro Ala Thr Arg Arg His Leu Ala Asp Leu Pro Ser Gly
    35 40 45
    Gly Ala Glu Asp Val Arg Arg Ala Val Ser Ala Ala Arg Arg Ala Phe
    50 55 60
    Asp Glu Gly Pro Trp Pro Arg Met Ala Pro Gly Glu Arg Ala Gly Leu
    65 70 75 80
    Leu Arg Lys Ala Ala Gln Arg Leu Arg Glu Glu Ala Glu Pro Leu Ala
    85 90 95
    Glu Leu Glu Ala Arg Asp Asn Gly Ser Thr Leu Arg Lys Ala Leu Gly
    100 105 110
    Ala Asp Val Pro Gly Ala Ala Ala Ala Phe Glu Trp Ser Ala Trp Trp
    115 120 125
    Ala Glu His Val Pro Glu Arg Gln Pro Glu Ala Pro Gly Ser Gly Ser
    130 135 140
    Tyr Val Val Trp Arg Pro Val Gly Val Val Ala Ala Ile Val Pro Trp
    145 150 155 160
    Asn Leu Pro Leu Leu Leu Ala Ala Trp Arg Ile Ala Pro Ala Ile Ala
    165 170 175
    Ala Gly Asn Thr Cys Val Ile Lys Pro Ala Ser Phe Ala Ser Leu Ser
    180 185 190
    Thr Leu Arg Leu Val Glu Leu Leu His Glu Cys Gly Leu Pro Pro Gly
    195 200 205
    Val Val Asn Val Val Thr Gly Pro Gly Gly Val Ala Gly Glu Gln Leu
    210 215 220
    Val Arg Ser Pro Gly Val Asp Leu Val Ala Phe Thr Gly Ser Asp Glu
    225 230 235 240
    Thr Gly Ala Ala Val Arg Glu Gly Ala Ala Ala Ala Gly Thr Ser Ala
    245 250 255
    Arg Leu Asp Leu Gly Gly Lys Ser Pro Asn Ile Val Leu Ala Asp Ala
    260 265 270
    Asp Leu Asp Arg Ala Val Thr Gly Val Thr Trp Gly Ala Phe Leu His
    275 280 285
    Asn Gly Gln Val Cys Met Ala Gly Thr Arg Ala Val Val His Ala Asp
    290 295 300
    Val His Asp Asp Phe Leu Arg Leu Leu Ser Glu Arg Val Gly Arg Leu
    305 310 315 320
    Arg Val Gly Asp Pro Leu Asp Pro Ala Thr Asp Leu Gly Pro Leu Val
    325 330 335
    Ser Arg Asn Gln Ala Arg Thr Ala Arg Arg Phe Thr Glu Leu Gly Leu
    340 345 350
    Ser Gln Gly Ala Glu Leu Val Cys Gly Gly Arg Ala Pro Ala Ala Asp
    355 360 365
    Glu Leu Pro Pro Gly Leu Asp Ala Gly Ala Tyr Phe Leu Pro Thr Val
    370 375 380
    Leu Ala Ser Val Gly Ala Asp Asp Ala Val Ala Gln Glu Glu Ile Phe
    385 390 395 400
    Gly Pro Val Leu Ala Val Val Arg Ala Gly Ser Asp Asp Asp Ala Val
    405 410 415
    Arg Ile Ala Asn Gly Ser Arg Tyr Arg Leu Ser Ala Gly Val Trp Ser
    420 425 430
    Ala Asp Pro Ala Arg Ala Arg Ala Val Ala Glu Arg Leu Arg Ala Asp
    435 440 445
    Arg Val Trp Ile Asn Asp Tyr Arg Leu Val Asp Leu Glu Leu Pro Gly
    450 455 460
    Thr Ala Gly Pro Arg Ser Ala Val Trp Asp Arg Leu Thr Asn Glu Leu
    465 470 475 480
    Asp Ala Tyr Arg His Lys His Val Val His Gly Gly Gly Ala Gly Ala
    485 490 495
    Gly Gly Val Pro Ala Pro Pro Thr Pro Tyr Ala Leu Leu Gly Gly
    500 505 510
    <210> SEQ ID NO 128
    <211> LENGTH: 472
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 128
    Met Lys Pro Ala Ser His Ser Val Thr Asp Thr Ser Ala Ala Leu Gly
    1 5 10 15
    Ala Ala Ala Ala Glu Glu Leu Ala Ala Gln Val Ala Gly Ser Val Leu
    20 25 30
    Leu Pro Gly Asp Glu Gly Tyr Asp Glu Glu Arg Ser Gly Phe Glu Leu
    35 40 45
    Ser Val Glu His Arg Pro Ala Leu Val Val Val Ala Thr Gly Ala Ala
    50 55 60
    Asp Val Ile Ala Ala Val Arg Phe Ala Arg Ala Arg Gly Leu Gly Ile
    65 70 75 80
    Ala Val Gln Ala Thr Gly His Gly Lys Ser Ser Ala Ala Thr Asp Val
    85 90 95
    Leu Ile Ser Thr Arg Arg Met Thr Gly Val Arg Val Asp Pro Arg Ala
    100 105 110
    Arg Thr Ala Arg Ile Glu Ala Gly Val Arg Trp Glu Gln Val Ile His
    115 120 125
    Glu Ala Ala Ala His Gly Leu Ala Pro Leu Ser Gly Ser Ala Pro Phe
    130 135 140
    Val Gly Ala Val Ser Tyr Leu Leu Gly Gly Gly Leu Gly Leu Leu Ser
    145 150 155 160
    Arg Lys Tyr Gly Phe Ala Gly Asp His Val Val Ser Leu Asp Leu Val
    165 170 175
    Thr Ala Asp Gly Arg Phe Leu Gln Val Ser Ala Glu Glu His Pro Asp
    180 185 190
    Leu Phe Trp Gly Val Arg Gly Ser Arg Gly Asn Leu Gly Ile Val Thr
    195 200 205
    Ser Val Glu Val Gly Leu Phe Pro Val Thr Gln Val Tyr Gly Gly Gly
    210 215 220
    Leu Phe Phe Asp Ala Gly Ser Thr Arg Ala Val Leu Asn Thr Tyr Leu
    225 230 235 240
    Gln Trp Ala Pro Arg Met Pro Glu Asp Met Ala Ser Ser Val Phe Leu
    245 250 255
    Ala Ala Tyr Pro Asp Ala Glu Gly Val Pro Gly Pro Leu Arg Gly Arg
    260 265 270
    Phe Val Thr His Ile Arg Leu Ala Trp Leu Gly Asp Pro Glu Glu Gly
    275 280 285
    Glu Arg Arg Phe Ala Glu Leu Arg Ala Ala Gly Thr Val Val Met Asp
    290 295 300
    Thr Val Asp Thr Leu Pro Tyr Thr Arg Ala Gly Ile Ile His Asn Asp
    305 310 315 320
    Pro Pro Ala Pro Val Ser Ser His Ser Lys Thr Val Met Phe Gly Gln
    325 330 335
    Leu Asp Glu Ile Ala Val Asp Glu Ile Leu Arg Leu Ala Gly Pro Gly
    340 345 350
    Thr Asp Ala Leu Phe Gly Val Glu Leu Arg His Leu Gly Gly Ala Leu
    355 360 365
    Ala Arg Pro Pro Arg His Pro Ser Ala Val Gly His Phe Pro Glu Ala
    370 375 380
    Val Phe Asn Ala Tyr Val Gly Ser Leu Val Asp Pro Asp Thr Leu Ala
    385 390 395 400
    Ala Val Asp Ala Ala Gln Gln Glu Phe Val Asp Ser Met Arg Pro Trp
    405 410 415
    Thr Thr Pro Gly Val Cys Leu Asn Phe Leu Ala Gly His Asn Thr Ser
    420 425 430
    Arg Glu Thr Thr Arg Ser Ala Tyr Thr Pro Glu Asp Tyr Ala Arg Leu
    435 440 445
    Gln Ala Leu Lys Ser Gln Tyr Asp Pro Gly Asn Val Phe Arg Phe Asn
    450 455 460
    Pro Asn Ile Pro Pro Leu Pro Ala
    465 470
    <210> SEQ ID NO 129
    <211> LENGTH: 395
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 129
    Met Thr Ser Ala Ala Pro Pro Ala Phe Pro Phe Pro Pro Gly Pro Gly
    1 5 10 15
    Gly Thr Val Pro Pro Glu Tyr Ala Arg Leu Leu Thr Asp Asp Pro Val
    20 25 30
    Ala Glu Val Arg Leu Ala Asp Gly Ser Arg Ile Trp Leu Val Thr Arg
    35 40 45
    His Glu Asp Val Arg Thr Val Leu Thr Asp Gly Arg Phe Ser Arg His
    50 55 60
    Arg Ala Ala Met Leu Pro Gly Ser Gly Phe Gly Arg Ser Gln Gly Ser
    65 70 75 80
    Gly Ile Val Asp Leu Asp Pro Pro Glu His Gly Arg Leu Arg Gly Pro
    85 90 95
    Val Val Ala Ala Phe Gly Ala Ser Arg Thr Ala Arg Phe Ala Pro Arg
    100 105 110
    Ile Glu Ala Ala Ala Glu Ala Ala Leu Asp Arg Leu Pro Ala Gly Ser
    115 120 125
    Gly Thr Val Asp Leu Val Ala Ala Tyr Thr Ala Pro Phe Ala Gly Arg
    130 135 140
    Val Thr Ala Glu Phe Leu Gly Leu Pro Gly Asp Arg Trp Gln Asp Val
    145 150 155 160
    Thr Ser Asp Val Glu Leu Leu Leu Leu Pro Arg Gly Ala Thr Glu Gln
    165 170 175
    Ala Leu Lys Glu Ala Arg Gly Arg Leu Gly Gln Val Leu Asp Glu Leu
    180 185 190
    Leu Ala Ala Arg Arg Ala Glu Pro Gly Asp Ser Val Thr Asp Thr Leu
    195 200 205
    Leu Asp Ala Glu Glu Leu Thr Asp Asp Asp Arg Arg Leu Leu Leu His
    210 215 220
    Gly Leu Ile Ile Ser Gly Phe Ile Thr Ile Arg Asp Leu Leu Ala Arg
    225 230 235 240
    His Leu Phe Gly Val Leu Ser Ser Pro Gly Leu Ala Ala Arg Leu Arg
    245 250 255
    Glu Asp Pro Ser Val Leu Pro Ser Ala Val Gln Glu Leu Leu Arg Tyr
    260 265 270
    Tyr Pro Ser Ser Asn Asp Gly Leu Leu Arg Val Ala Thr Glu Asp Val
    275 280 285
    Val Leu Ser Gly Arg Arg Val Ala Ala Gly Asp Ala Val Leu Pro Leu
    290 295 300
    Val Ser Ala Ala Ser Arg Asp Pro Glu Val Phe Ala Asp Pro His Val
    305 310 315 320
    Leu Asp Ile Glu Arg Val Ala Asp Arg Gly Ile Ala Phe Gly Ala Gly
    325 330 335
    Gln His Ala Cys Pro Ala Thr Gly Leu Ala Val Thr Glu Leu Thr Val
    340 345 350
    Gly Ile Gly Arg Leu Leu Ala Ala Phe Pro Arg Ile Ala Leu Ala Val
    355 360 365
    Pro Pro Glu Glu Val Glu His Ser Ser Glu Leu Leu Pro Leu Gly Val
    370 375 380
    Arg Ser Leu Pro Val Val Pro Gly Pro Arg Asn
    385 390 395
    <210> SEQ ID NO 130
    <211> LENGTH: 474
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 130
    Met Leu Pro Glu Phe Gln Leu Gln Trp Asn Trp Leu Asp Ala Pro Ala
    1 5 10 15
    Gly Gly Gly Gly Glu Leu Gln Ala Thr Trp Ala Arg Leu Arg Ile Ala
    20 25 30
    Val Gly Ala Glu Thr Val Thr Leu Val Gln Glu Pro Gly Gln Gly Thr
    35 40 45
    Phe Arg Glu His Thr Thr Gly Ser Leu Tyr Pro Leu Ala Glu Trp Ile
    50 55 60
    Ala Phe Asn Trp Trp Ser Leu Val Ala Asp Ala Arg Pro Gly Thr Gln
    65 70 75 80
    Ile Ser Gln Leu Arg Phe Ala Tyr Arg His Gly Val Gly Asp Asn Arg
    85 90 95
    Gly Ser Trp Trp Met Arg Ser Arg Arg His Ile Leu Arg Ala Ala Cys
    100 105 110
    Asp Gly Phe Arg Trp Pro Asp Met Leu Phe Val Pro Glu Gly Arg Glu
    115 120 125
    Thr Arg Ile Val Trp Met Pro Asp Met Gly Pro Asp Val Arg Pro Gly
    130 135 140
    Asn Arg Phe Ala Ser Arg Gly Asn Ser Cys Val Glu Ser Ala Ala Phe
    145 150 155 160
    Thr Ala Thr Leu Ala Ser Phe Val Asp Ala Val Thr Glu Arg Leu Thr
    165 170 175
    Asp Gln Gly Ile Thr Gly Thr Pro Leu Gln Glu Glu Trp Ala Ala Val
    180 185 190
    Arg Ala Thr Asp Glu Asp Glu Ala Ala Phe Cys Arg Ile Ala Ala Arg
    195 200 205
    Leu Gly Leu Asp Pro Tyr Ala Glu Ala Glu Pro Tyr Glu Ala Asp Ile
    210 215 220
    Leu Lys Ala Ala Glu Gln Leu Ala Glu Pro Leu Ala Ser Asp Phe Phe
    225 230 235 240
    Asn Gly Val Arg Pro Glu Arg Ile Ala Asp Gln Leu Gln Trp Ile Ala
    245 250 255
    Arg Val Arg Thr Leu Met Gly Thr Ala Pro Ala Asp Thr Pro Leu Pro
    260 265 270
    Pro Ala Leu Val Glu Leu Arg Lys Asp Cys Ala Asp Leu Ser Glu Lys
    275 280 285
    Phe Phe Ala Pro Gly Arg Leu Asp Asn Pro Trp Asp Leu Gly Tyr Glu
    290 295 300
    Val Ala His Arg Val Arg Ala Trp Ala Gly Leu Asp Asp Thr Ala Pro
    305 310 315 320
    Phe Asp Pro Ala Pro Leu Met Gly Tyr Arg Thr Glu Gln Val Pro Tyr
    325 330 335
    Met Asp Arg Gly Leu Val Ala Leu Gly Thr Arg Arg Gly Ala Asp Gly
    340 345 350
    Pro Val Leu Val Ser Ser Arg Arg Phe Thr Asp Arg Pro Arg Arg Phe
    355 360 365
    Leu Gln Ala Arg Ala Leu Trp His Leu Ile Cys Asp Pro Asp Asp Thr
    370 375 380
    Phe Leu Ile Ala Ala Ala His Thr His Arg Gln His Val Ala Arg Gly
    385 390 395 400
    Phe Ala Leu Glu Val Leu Ala Pro Ala Lys Gly Val Ala Thr Leu Leu
    405 410 415
    Ala Asp Pro Gly His Leu Val Ser Ala Glu Asp Val Glu Val Ile Ala
    420 425 430
    Asp Asp Tyr Gly Cys Gly Asn Ile Val Val Glu His Gln Leu Asp Asn
    435 440 445
    Arg Val Leu Ala Lys Asp Phe Thr Trp Pro Gly His Ala Arg Ala Gly
    450 455 460
    Ala Pro Ala Gly Glu Arg Ser Arg Gly Ala
    465 470
    <210> SEQ ID NO 131
    <211> LENGTH: 443
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 131
    Met Thr Ile Arg Gln Arg Val Val Val Val Ile Thr Glu Gly Ala Ala
    1 5 10 15
    Pro Glu Leu Leu Asp Arg Trp Cys Ala Gln Gly Leu Leu Pro Gly Phe
    20 25 30
    Ala Ala Leu Arg Ser Gln Gly Ala Ser Gly Pro Leu His Ala Glu Gly
    35 40 45
    Thr Pro Tyr Glu Pro Pro Gly Leu Leu Ser Val Leu Thr Gly Arg Arg
    50 55 60
    Ala Ala Asp His Gly Phe Tyr Ser Tyr Trp Thr Cys His Asp Pro Glu
    65 70 75 80
    Tyr Ala Pro Gln Val Leu Thr Pro Glu His Arg Arg His Pro Leu Leu
    85 90 95
    Trp Gln His Glu Val Phe Gln Gly Val Arg Phe Ala Ser Ile Gly Leu
    100 105 110
    Phe Gly Thr His Pro Pro Glu Pro Phe Asp Gly Ser Leu Ile Thr Tyr
    115 120 125
    Pro Met Tyr Ala Thr Leu His Ala Cys His Pro Arg Ser Leu Gln Arg
    130 135 140
    Thr Leu Ala Lys Lys Gly Ile Arg Pro Val His Asp Val Ser Ile Phe
    145 150 155 160
    Trp Thr Gly Gln Asp Arg Asp Glu Leu Leu Pro Ser Leu Leu Glu Ala
    165 170 175
    Asp Val Gln Arg Gly Arg Ala Ala Leu Ala Leu Leu Glu Glu Ser Asp
    180 185 190
    Val Val Ile Val Asn Leu Thr Ser Ile Asp Arg Cys Ser His Ile Tyr
    195 200 205
    Trp Gln Glu Leu Glu His Gly Pro Glu His Glu Arg Glu Ser Ala Val
    210 215 220
    Phe Ala Ala Tyr Arg Thr Cys Asp Gln Val Ile Gln Asp Ala Leu Arg
    225 230 235 240
    Ala Ala Asp Asp Arg Thr Ser Val Val Ala Phe Ser Glu Ile Gly Phe
    245 250 255
    Gly Pro Leu Arg Asn Tyr Cys Ser Ile Asn Asp Glu Met Glu Gln Ala
    260 265 270
    Gly Phe Leu Ala Thr Ala Glu Asp Gly Arg Val Glu Trp Ala Gly Ser
    275 280 285
    Ala Ala Phe Glu Ala Val Gln Gly Thr His Gly Val Asn Ile Asn Leu
    290 295 300
    Arg Asp Arg Tyr Lys His Gly Leu Val Pro Glu Arg Asp Tyr Glu Lys
    305 310 315 320
    Val Arg Thr Asp Val Ala Ala Ala Leu Leu Glu Arg Arg Asn Pro Arg
    325 330 335
    Thr Gly Arg Leu Phe Phe Asp Ala Val Arg Arg Arg Glu Glu Val Tyr
    340 345 350
    Pro Gly Glu Ala Thr Gln His Ala Pro Asp Leu Ile Leu Glu Pro Ala
    355 360 365
    Asp Trp Arg Tyr Leu Pro Leu Gly Asp Pro His Trp Ala Ser His Val
    370 375 380
    His Arg Asp Trp Gln Ser Gly Trp His Arg Arg Glu Ser Tyr Trp Ser
    385 390 395 400
    Ala Val Gly Pro Gly Phe Thr Gly Gly Ala Arg Gln Thr Arg Thr Ala
    405 410 415
    Ala Pro Val Asp Ile Pro Ala Thr Val Cys Ala Leu Leu Gly Arg Asp
    420 425 430
    Val Pro Asn Asp Trp Asp Gly Val Pro Leu Ser
    435 440
    <210> SEQ ID NO 132
    <211> LENGTH: 123
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 132
    Met Thr Pro Glu Glu Leu Ser Asp Phe Ala Leu Glu Leu Pro Glu Ala
    1 5 10 15
    Val Asp Asp Glu Ala Phe Gly Pro Gly Ala Ala Val Phe Lys Val Glu
    20 25 30
    Lys Lys Val Phe Ala Ile Leu Gln Asp Ala Ser Glu Asp Arg Pro Pro
    35 40 45
    Gln Val Thr Leu Lys Cys Glu Pro Asp Leu Ala Leu His Leu Arg Glu
    50 55 60
    Gln Tyr Ala Ala Val Val Pro Gly Tyr His Val Asn Lys Arg His Trp
    65 70 75 80
    Asn Thr Val Val Leu Asn Gly Thr Val Pro Val Glu Glu Leu Arg Glu
    85 90 95
    Met Val Glu His Ser Tyr Asp Arg Val Val Ala Gly Leu Pro Lys Ala
    100 105 110
    Val Arg Glu Arg Leu Arg Leu Leu Arg Thr Val
    115 120
    <210> SEQ ID NO 133
    <211> LENGTH: 351
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 133
    Met Thr Val Glu Gln Thr Pro Glu Asn Pro Gly Thr Ala Ala Arg Ala
    1 5 10 15
    Ala Ala Glu Glu Thr Val Asn Asp Ile Leu Gln Gly Ala Trp Lys Ala
    20 25 30
    Arg Ala Ile His Val Ala Val Glu Leu Gly Val Pro Glu Leu Leu Gln
    35 40 45
    Glu Gly Pro Arg Thr Ala Thr Ala Leu Ala Glu Ala Thr Gly Ala His
    50 55 60
    Glu Gln Thr Leu Arg Arg Leu Leu Arg Leu Leu Ala Thr Val Gly Val
    65 70 75 80
    Phe Asp Asp Leu Gly His Asp Asp Leu Phe Ala Gln Asn Ala Leu Ser
    85 90 95
    Ala Val Leu Leu Pro Asp Pro Ala Ser Pro Val Ala Thr Asp Ala Arg
    100 105 110
    Phe Gln Ala Ala Pro Trp His Trp Arg Ala Trp Glu Gln Leu Thr His
    115 120 125
    Ser Val Arg Thr Gly Glu Ala Ser Phe Pro Ser Thr Trp Pro Thr Ala
    130 135 140
    Pro Arg Ser Gly Ser Ser Pro Thr Arg Asp Pro Lys Ala Arg Glu Leu
    145 150 155 160
    Phe Asn Arg Ala Met Gly Ser Val Ser Leu Thr Glu Ala Gly Gln Val
    165 170 175
    Ala Ala Ala Tyr Asp Phe Ser Gly Ala Ala Thr Ala Val Asp Ile Gly
    180 185 190
    Gly Gly Arg Gly Ser Leu Met Ala Ala Val Leu Asp Ala Phe Pro Gly
    195 200 205
    Leu Arg Gly Thr Leu Leu Glu Arg Pro Pro Val Ala Glu Glu Ala Arg
    210 215 220
    Glu Leu Leu Thr Gly Arg Gly Leu Ala Asp Arg Cys Glu Ile Leu Pro
    225 230 235 240
    Gly Asp Phe Phe Glu Thr Ile Pro Asp Gly Ala Asp Val Tyr Leu Ile
    245 250 255
    Lys His Val Leu His Asp Trp Asp Asp Asp Asp Val Val Arg Ile Leu
    260 265 270
    Arg Arg Ile Ala Thr Ala Met Lys Pro Asp Ser Arg Leu Leu Val Ile
    275 280 285
    Asp Asn Leu Ile Asp Glu Arg Pro Ala Ala Ser Thr Leu Phe Val Asp
    290 295 300
    Leu Leu Leu Leu Val Leu Val Gly Gly Ala Glu Arg Ser Glu Ser Glu
    305 310 315 320
    Phe Ala Ala Leu Leu Glu Lys Ser Gly Leu Arg Val Glu Arg Ser Leu
    325 330 335
    Pro Cys Gly Ala Gly Pro Val Arg Ile Val Glu Ile Arg Arg Ala
    340 345 350
    <210> SEQ ID NO 134
    <211> LENGTH: 546
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 134
    Met Thr Val Leu Gly Leu Gly Gly Ser Gly His Asp Trp Ala Ser Cys
    1 5 10 15
    Ala Thr Asp Gly Arg Arg Leu Val Ala Ile Asp Glu Glu Arg Leu Val
    20 25 30
    Arg Ser Lys Tyr Gly Leu Gly Ala Asp Leu Leu Ala Gly His Ser Arg
    35 40 45
    Arg Ala Val Leu Asp Ala Leu Gly Thr Ser Ala Glu Ala Val Glu His
    50 55 60
    Val Val Ala Cys Glu Leu Val Pro Arg Pro Phe Tyr His Ser Phe Arg
    65 70 75 80
    Arg Arg Val Thr Val Val Asn His His Leu Ala His Ala Tyr Ser Ala
    85 90 95
    Phe Gly Ala Ser Gly Met Thr Arg Ala Ala Val Leu Val Cys Asp Asn
    100 105 110
    Ser Gly Ser Leu Val Thr Gly Leu Lys Ser Gly Pro Gly Pro Arg Glu
    115 120 125
    Ala Glu Thr Ile Ser Cys Tyr Thr Ala Asp Ala Ser Gly Leu Arg Leu
    130 135 140
    Val Asn Arg Val Ala Gly Thr His Ala Val Asp Ala Ser Ser Glu Ser
    145 150 155 160
    Ala Tyr Tyr Gln Pro Gly Glu Thr Asp Asn Ser Leu Gly His Phe Tyr
    165 170 175
    Arg Ser Ala Ser Leu Ala Leu Gly Leu Ala Tyr Ser Gly Pro Lys Thr
    180 185 190
    Arg Tyr Pro Val Ser Glu Asp Gly Lys Thr Met Gly Leu Ala Pro Tyr
    195 200 205
    Gly Asp Asp Arg Phe Val Asp Glu Val Ala Glu Leu Val Thr Leu Leu
    210 215 220
    Pro Glu Gly Gly Val Gln Ile Ser Ala Ser Lys Val Asn His Leu Phe
    225 230 235 240
    Glu Arg Leu Val Glu Ser Gly Glu Phe Glu Asp Arg Ala Ala Leu Ala
    245 250 255
    Tyr Ala Ala Gln Glu Thr Leu Glu Arg Ala Leu Leu His Cys Ala Arg
    260 265 270
    Asp Leu His Arg Arg Thr Gly Leu Thr Asp Leu Cys Ile Ala Gly Gly
    275 280 285
    Val Gly Leu Asn Ser Val Ala Asn Gly Arg Ile Leu Arg Glu Thr Pro
    290 295 300
    Phe Glu Arg Val Phe Val Val Pro Ala Ala Gly Asp Asn Gly Ile Ser
    305 310 315 320
    Leu Gly Cys Ala Tyr Tyr Gly Leu His Glu Leu Glu Gly Arg Ala Pro
    325 330 335
    Ser Glu Leu Pro Ala Leu Asp Thr Ala Tyr Leu Gly Pro Asp Tyr Pro
    340 345 350
    Ala Glu Arg Val Asp Ala Ala Leu Ala Gly Ser Gly Phe Thr Val Glu
    355 360 365
    Thr Pro Asp Asp Leu Pro Gly Arg Val Ala Gly Leu Leu Ala Glu Gly
    370 375 380
    Lys Ile Ile Gly Trp Phe Asp Gly Arg Ser Glu Phe Gly Pro Arg Ala
    385 390 395 400
    Leu Gly His Arg Ser Ile Leu Ala Ala Pro Phe Pro Ala Ser Val Arg
    405 410 415
    Asp His Leu Asn Asp Asn Val Lys His Arg Glu Trp Phe Arg Pro Tyr
    420 425 430
    Ala Pro Ile Val Arg Glu Asp Arg Ala Ala Asp Tyr Phe Asp Leu Val
    435 440 445
    Gln Pro Ser Pro Phe Met Leu Val Val Ala Arg Val Thr Arg Gln Asp
    450 455 460
    Ala Ile Pro Ala Ala Thr His Val Asp Gly Thr Ala Arg Leu Gln Thr
    465 470 475 480
    Leu Asn Ala Ala Gln Asn Pro Lys Val Tyr Glu Leu Leu Gly Arg Phe
    485 490 495
    Glu Ala Leu Thr Gly Cys Ala Val Leu Leu Asn Thr Ser Phe Asn Val
    500 505 510
    Ala Gly Gln Pro Ile Val Glu Thr Pro Glu Asp Ala Val Glu Ala Phe
    515 520 525
    Ala Gly Met Arg Leu Asp His Leu Val Val Gly Asp Arg Leu Ala Thr
    530 535 540
    Lys Pro
    545
    <210> SEQ ID NO 135
    <211> LENGTH: 568
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 135
    Met Asp Val Pro Val Leu Val Val Gly Gly Gly Pro Thr Gly Leu Ala
    1 5 10 15
    Met Ala Leu Phe Leu Ala Arg His Gly Val Gly Cys Leu Leu Val Glu
    20 25 30
    Arg Arg Thr Thr Thr Ser Pro Val Pro Arg Ala Thr His Val Ser Arg
    35 40 45
    Arg Ser Met Glu Leu Phe Arg Glu Ala Gly Leu Glu Glu Glu Ile Arg
    50 55 60
    Arg Ala Gly Phe Glu Val Val Arg Glu Asp Asp Pro Arg Leu Arg Thr
    65 70 75 80
    Arg Pro Glu Arg His Leu Pro Arg Val Val Leu Gln Ala Ala Ser Leu
    85 90 95
    Ala Gly Pro Gly Pro Val Gly Val Leu Glu Thr Gly Asp Glu Glu Leu
    100 105 110
    Ala Val Pro Gly Pro Cys Ala Pro Phe Trp Cys Gly Gln Asp Arg Met
    115 120 125
    Glu Pro Leu Leu Ala Lys Ala Ala Ala Arg His Gly Ala Asp Val Arg
    130 135 140
    Phe Gly His Glu Leu Thr Gly Leu Trp Pro Gly Glu Asp Ser Thr Arg
    145 150 155 160
    Ala Arg Val Arg Ala Ala Gly Thr Gly Arg Thr Tyr Thr Val Asp Ala
    165 170 175
    Arg Phe Val Ile Ala Ala Asp Gly Ala Arg Gly Glu Ile Ala Glu Arg
    180 185 190
    Val Gly Ile Ala Arg Glu Gly Leu Gly Thr Val Ala His Arg Val Ser
    195 200 205
    Ile Leu Phe Arg Ala Asp Pro Gly Arg Trp Ala Arg Asp Arg Arg Phe
    210 215 220
    Phe Met Cys Met Ile Gln Asn Pro Gly Phe Asp Gly Ala Val Met Glu
    225 230 235 240
    Leu Asn Thr Pro Gly Arg Trp Cys Ala Ala Val Asp Tyr Asp Pro Ala
    245 250 255
    Arg Ala Glu Pro Asp Gly Thr Tyr Ser Ala Arg Thr Cys Leu Asp Leu
    260 265 270
    Val Arg Ala Ala Val Gly Asp Asp Arg Ser Asp Ala Ala Val Asp Thr
    275 280 285
    Val Phe His Trp Lys Ala Arg His Arg Ile Ala Ala Ala Tyr Arg Ser
    290 295 300
    Gly Ala Val Phe Leu Ile Gly Asp Ala Ala His Leu His Pro Pro Ser
    305 310 315 320
    Gly Gly Tyr Gly Ser Asn Val Gly Phe Gln Asp Ala His Asn Leu Ala
    325 330 335
    Trp Lys Ile Ala Ala Val Leu Gly Gly Trp Ala Gly Pro Arg Leu Leu
    340 345 350
    Asp Thr Tyr Asp Glu Glu Arg Arg Pro Val Gly Lys Ala Thr Ala Glu
    355 360 365
    Gln Ser Met Leu Leu Asp Gly Val Pro Pro Glu Pro Leu Gly Gly Ser
    370 375 380
    Val Val Arg Cys Asp Pro Arg Thr Leu Ile Met Gly Tyr Arg Tyr His
    385 390 395 400
    Ser Ala Ala Val Leu Gly Pro Pro His Gly Pro Ala Phe Pro Ala Ala
    405 410 415
    Phe Thr Leu Arg Gly Asp Pro Gly Thr Arg Leu Pro His Val Trp Leu
    420 425 430
    Arg Thr Asp Ala Gly Glu Arg Val Ser Thr Leu Asp Leu Cys His Gly
    435 440 445
    His Phe Val Leu Leu Ser Ala Asp Pro Val Trp Ala Ala Ala Ala Ala
    450 455 460
    Arg Ser Ala Lys Glu Thr Gly Val Pro Leu Arg Gly His His Leu Ala
    465 470 475 480
    Ala Thr Gly Ser Glu Leu Ala Asp Pro Ser Gly Glu Phe Pro Arg Ser
    485 490 495
    Cys Gly Thr Gly Pro Ala Gly Ala Val Leu Val Arg Pro Asp Gly Met
    500 505 510
    Val Ala Trp Arg Thr Ala Arg Ala Val Pro Pro Asp Pro Asp Ser Ala
    515 520 525
    Gln Asp Leu Val Thr Ala Ala Val Arg Arg Val Leu Ala Leu Pro Glu
    530 535 540
    Arg Ala Ala Pro Pro Val Leu Gly Pro Pro Arg Leu Ser Arg Gly Ser
    545 550 555 560
    Tyr Arg Arg Val Gly Ser Asp Gly
    565
    <210> SEQ ID NO 136
    <211> LENGTH: 160
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 136
    Met Lys Pro His Ser Phe Cys Thr Cys Trp Pro Gly Ala Thr Val Trp
    1 5 10 15
    Leu Thr Gly Pro Pro Gly Ala Gly Lys Thr Thr Ile Ala Arg Ala Leu
    20 25 30
    Ala Glu Arg Leu Arg Glu Arg Gly Arg Arg Val Glu Val Leu Asp Gly
    35 40 45
    Asp Ala Thr Arg Ala Leu Leu Thr Ala Gly Ser Ser Trp Glu Asp Arg
    50 55 60
    Gly Thr Gly Leu Gln Arg Val Gly Leu Met Ala Glu Val Leu Ala Arg
    65 70 75 80
    Asn Gly Ile Val Val Leu Val Pro Val Thr Ala Ala Arg Ala Asp Ser
    85 90 95
    Arg Glu Ala Val Arg Arg Arg His Glu Arg Ser Gly Thr Ala His Leu
    100 105 110
    Glu Val Arg Val Val Arg Asp Ala Val Pro Pro Ser Gly Leu Pro Ala
    115 120 125
    Pro Pro Gly Pro Asp Leu Arg Ile Ala Ala His Glu Gln Ser Ala Glu
    130 135 140
    Glu Ser Ala Arg Ala Leu His Arg Leu Leu Ala Glu Arg Glu Leu Ala
    145 150 155 160
    <210> SEQ ID NO 137
    <211> LENGTH: 319
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 137
    Met Asn Pro Gly Arg Gly Gly Ala Tyr Ala Ala Gly Arg Asp Gly Thr
    1 5 10 15
    Arg Gly Thr Arg Arg Pro His Gly Leu Ser His Leu Asp Leu Leu Glu
    20 25 30
    Ser Glu Ser Val His Ile Phe Arg Glu Val Ala Gly Glu Phe Glu Arg
    35 40 45
    Pro Val Ile Leu Phe Ser Gly Gly Lys Asp Ser Ile Val Met Leu His
    50 55 60
    Leu Ala Leu Lys Ser Phe Ala Pro Ala Pro Val Pro Phe Ala Leu Leu
    65 70 75 80
    His Val Asp Thr Gly His Asn Phe Pro Glu Val Ile Ala Tyr Arg Asp
    85 90 95
    Arg Val Val Ala Ala Leu Gly Leu Arg Leu Glu Val Ala Ser Val Gln
    100 105 110
    Asp Phe Ile Asp Asn Gly Thr Leu Arg Glu Arg Pro Asp Gly Thr Arg
    115 120 125
    Asn Pro Leu Gln Thr Val Pro Leu Leu Asp Ala Ile Gly Arg His Arg
    130 135 140
    Phe Asp Ala Val Phe Gly Gly Gly Arg Arg Asp Glu Glu Lys Ala Arg
    145 150 155 160
    Ala Lys Glu Arg Val Phe Ser Leu Arg Asp Glu Phe Gly Gly Trp Asp
    165 170 175
    Pro Arg Arg Gln Arg Pro Glu Leu Trp Arg Leu Tyr Asn Gly Arg His
    180 185 190
    Ala Pro Gly Glu His Val Arg Val Phe Pro Leu Ser Asn Trp Thr Glu
    195 200 205
    Leu Asp Val Trp Gln Tyr Val Ala Arg Glu Glu Ile Glu Leu Pro Thr
    210 215 220
    Ile Tyr Tyr Ala His Glu Arg Glu Val Phe Arg Arg Gly Gly Met Trp
    225 230 235 240
    Leu Ala Pro Gly Glu Trp Gly Gly Pro Arg Glu Gly Glu Ala Val Glu
    245 250 255
    Lys Arg Arg Val Arg Tyr Arg Thr Val Gly Asp Met Ser Cys Thr Gly
    260 265 270
    Ala Val Asp Ser Ala Ala Ala Thr Val Ala Asp Val Val Ala Glu Ile
    275 280 285
    Ala Thr Ser Arg Leu Thr Glu Arg Gly Ala Thr Arg Ala Asp Asp Lys
    290 295 300
    Leu Ser Glu Ala Ala Met Glu Asp Arg Lys Arg Glu Gly Tyr Phe
    305 310 315
    <210> SEQ ID NO 138
    <211> LENGTH: 163
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 138
    Met Gly Gln Asp Ser Arg Pro Arg Trp Leu Thr Asp Glu Glu Gln Arg
    1 5 10 15
    Val Trp Arg Gly Tyr Leu Arg Ala Thr Arg Leu Val Glu Asp His Leu
    20 25 30
    Asp Arg Arg Leu Gln Arg Glu Ala Asp Met Pro His Leu Tyr Tyr Gly
    35 40 45
    Leu Leu Val Gln Leu Ser Glu Ala Pro Arg Arg Gly Ile Arg Met Thr
    50 55 60
    Asp Leu Ala Arg Asn Ala Lys Ile Thr Arg Pro Arg Leu Ser His Ala
    65 70 75 80
    Ile Thr Arg Leu Glu Lys Leu Gly Trp Val Arg Arg Glu Ser Cys His
    85 90 95
    Gly Asp Arg Arg Gly Gln Asn Ala Val Leu Thr Glu Glu Gly Arg Glu
    100 105 110
    Val Leu Glu Lys Ser Ala Pro Gly His Val Ala Ala Val Arg Ala Ala
    115 120 125
    Val Phe Asp Ser Leu Thr Pro Glu Gln Val Gly Gln Leu Gly Arg Ile
    130 135 140
    Cys Gln Ala Ile Glu Lys Gly Leu Asp Arg Glu Gly Ala Asp Leu Pro
    145 150 155 160
    Trp Leu Arg
    <210> SEQ ID NO 139
    <211> LENGTH: 413
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 139
    Met Glu Arg His Asp Gly Ala Pro Gly Trp Gly Phe Thr His Thr Gln
    1 5 10 15
    Tyr Ser Ala Asp His Gly Glu Arg Gly Ala Thr Arg Arg Ala Gly Ala
    20 25 30
    Leu Leu Ser Ala Arg Pro Leu Pro Gln Asn Gln His Ile Met Gly Trp
    35 40 45
    Gly Ala Glu Asn Pro Glu Pro Ala Pro Gly Arg Tyr Asp Phe Glu Val
    50 55 60
    Leu Asp Glu Arg Val Ala Leu Met Arg Ala Thr Gly Ala Thr Pro Val
    65 70 75 80
    Leu Thr Leu Cys Ala Ala Pro Asp Trp Met Lys Gly Gly Arg Pro Gly
    85 90 95
    Arg Thr Asp Trp Ser Arg Leu Glu Thr Ala Pro Asp Pro Arg His Tyr
    100 105 110
    Ala Asp Phe Ala Arg Leu Ala Gly Val Ile Ala Gln Arg Tyr Pro Asp
    115 120 125
    Ile Arg His Phe Leu Val Trp Asn Glu Leu Lys Gly Phe Tyr Asp Glu
    130 135 140
    Asp Arg Arg Arg Trp Asp Tyr Glu Gly Tyr Thr Arg Leu Tyr Asn Leu
    145 150 155 160
    Val His Ala Glu Leu Lys Arg Arg Asn Pro Arg Asn Leu Val Gly Gly
    165 170 175
    Pro Tyr Ala Val Val Asp His Asp Pro Pro Ala Glu Asp Ala Ala Asp
    180 185 190
    Arg Ser Arg Glu Leu Arg Gly Pro Trp Gly Glu Leu Asp Gln Arg Ser
    195 200 205
    Ala Asp Val Ile Arg Tyr Trp Asn Ala His Lys Ala Gly Ala Asp Phe
    210 215 220
    Val Val Val Asp Gly Ser Ser Tyr Thr Arg Glu Gly His Arg Ala Ile
    225 230 235 240
    Pro Asp Glu Phe Ala Ala Thr Glu Lys Phe Ala Asp Val Thr Arg Trp
    245 250 255
    Val Arg Ser Val Thr Gly Leu Pro Val Trp Trp Ala Glu Trp Tyr Val
    260 265 270
    Glu Pro Pro Ala Glu Asp Asp Arg Pro Gly Gly Arg Asp Gly Trp Gly
    275 280 285
    Glu Gly His Arg Thr Ala Val Gln Ala Thr Ala Met Met Arg Leu Ala
    290 295 300
    Glu Ser Gly Ala Ser Ala Ala Phe Tyr Trp Asn Pro Gln Arg Thr Gly
    305 310 315 320
    Lys Ala Cys Pro Gly Cys Leu Trp Arg Ser Thr His Leu Arg Asp Gly
    325 330 335
    Gly Gly Glu Leu Pro Met Ala Gly Leu Leu Ser Arg Phe Ala Arg Glu
    340 345 350
    Phe Pro Pro Gly Thr Ala Phe Arg Pro Val Ala Val Thr Cys Gly Ser
    355 360 365
    Gly Asp Arg Val Glu Ala Leu Ala Asp Glu Ala Ala Val Leu Val Val
    370 375 380
    Asn Thr Glu Cys Arg Pro Val Ala Ala Arg Val Asp Gly Gln Ala Leu
    385 390 395 400
    Ser Leu Ala Pro Tyr Glu Val Arg Trp Leu Thr Arg Pro
    405 410
    <210> SEQ ID NO 140
    <211> LENGTH: 270
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 140
    Met Glu Phe Leu Gly Pro Ala Ala Gly Val Ser Gly Ala Thr Arg Leu
    1 5 10 15
    Tyr Ala Val Leu Gly Asp Pro Val Ala Gln Val Lys Ala Pro Gly Leu
    20 25 30
    Leu Asn Pro Leu Leu Ser Glu Ser Gly Leu Asp Ala Val Val Val Pro
    35 40 45
    Val His Val Arg Ala Arg Asp Leu Ala Glu Val Val Glu Gly Leu Lys
    50 55 60
    Arg Ile Gly Asn Leu Asp Gly Leu Leu Val Thr Val Pro His Lys Ala
    65 70 75 80
    Ala Leu Cys Gly Leu Ala Asp Gly Leu Gly Pro Ala Ala Ala Leu Ile
    85 90 95
    Gly Thr Ala Asn Ala Met Arg Arg Glu Pro Asp Gly Arg Trp Tyr Ala
    100 105 110
    Glu Asn Phe Asp Gly Leu Gly Phe Val Gln Gly Leu Gln Ala Ala Gly
    115 120 125
    His Thr Val Arg Asp Arg His Val Ala Leu Val Gly Ala Gly Gly Ala
    130 135 140
    Gly Ser Ala Ile Ala Thr Ala Leu Leu Met Ala Asp Ala Ala Arg Val
    145 150 155 160
    Ser Val His Asp Thr Asp Arg Ala Gln Leu Asp Ala Leu Leu Leu Arg
    165 170 175
    Leu Gly Ser Arg Arg Pro Asp Gly Ile Arg Ala Leu Gly Pro Gly Asp
    180 185 190
    Leu Glu Ala Ala Asp Phe Ala Val Asn Ala Thr Pro Leu Gly Met Arg
    195 200 205
    Ser Glu Asp Pro Leu Pro Phe Asp Pro Ala Arg Val Arg Pro Asp Ala
    210 215 220
    Val Val Val Asp Val Val Met Lys Pro His Glu Thr Ala Leu Leu Ser
    225 230 235 240
    Ala Ala Ala Thr Ala Gly Arg Arg Val His His Gly Ile His Met Leu
    245 250 255
    Glu Gln Gln Val Pro Cys Tyr Arg Ala Phe Phe Gly Trp Pro
    260 265 270
    <210> SEQ ID NO 141
    <211> LENGTH: 271
    <212> TYPE: PRT
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 141
    Met Thr Arg Arg Arg Pro Thr Gly Pro Ile His Arg Arg Arg Ala Ser
    1 5 10 15
    Leu Thr Leu Ser Pro Thr Gly Ala Ala Met Arg Arg Asn Arg Ile Ala
    20 25 30
    Ala Leu Leu Pro Ala Ala Leu Ala Leu Val Gly Ile Ser Val Leu Ala
    35 40 45
    Pro Ala Thr Thr Ala Ser Ala Ala Ala Pro His Gly Gly Thr Ser Gln
    50 55 60
    Ala Ala Ala Phe Pro Val Ser Glu Ala Gln Phe Lys Gln Met Phe Pro
    65 70 75 80
    Lys Arg Asn Ala Phe Tyr Thr Tyr Lys Gly Leu Val Ala Ala Leu Lys
    85 90 95
    Ala Tyr Pro Gly Phe Ala Gly Thr Gly Ser Ala Glu Val Arg Lys Gln
    100 105 110
    Glu Ala Ala Ala Phe Leu Ala Asn Val Ala His Glu Thr Gly Gly Leu
    115 120 125
    Val Tyr Val Val Glu Gln Asn Thr Ala Asn Tyr Pro His Tyr Cys Asp
    130 135 140
    Arg Ser Arg Pro Tyr Gly Cys Pro Ala Gly Gln Ala Ala Tyr Tyr Gly
    145 150 155 160
    Arg Gly Pro Leu Gln Ile Ser Trp Asn Phe Asn Tyr Lys Ala Ala Gly
    165 170 175
    Asp Ala Leu Gly Ile Asp Leu Leu His Asn Pro Ser Leu Val Gln Lys
    180 185 190
    Asp Ala Ala Val Ser Trp Lys Thr Gly Leu Trp Tyr Trp Asn Thr Gln
    195 200 205
    Arg Gly Pro Gly Thr Met Thr Pro His Glu Ala Met Val Asn His Arg
    210 215 220
    Gly Phe Gly Gln Thr Ile Arg Ser Ile Asn Gly Ala Leu Glu Cys Asp
    225 230 235 240
    Gly His Asn Pro Ala Gln Val Gln Ser Arg Val Ala Asn Tyr Gln Arg
    245 250 255
    Phe Thr Lys Ile Leu Gly Val Ala Pro Gly Gly Asn Leu Ser Cys
    260 265 270
    <210> SEQ ID NO 142
    <211> LENGTH: 391
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A consensus sequence
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)...(391)
    <223> OTHER INFORMATION: Where present in this sequence, Xaa represents
    an amino acid that varied between the sequences used
    to generate this consensus sequence.
    <400> SEQUENCE: 142
    Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Pro Xaa Trp Pro
    1 5 10 15
    Gln Xaa Asp Asp Ala Glu Arg Xaa Gly Leu Xaa Arg Ala Leu Xaa Gln
    20 25 30
    Gly Gln Trp Trp Arg Xaa Gly Gly Xaa Glu Val Xaa Xaa Phe Glu Arg
    35 40 45
    Glu Phe Ala Xaa Xaa His Gly Ala Xaa His Ala Leu Ala Val Thr Asn
    50 55 60
    Gly Thr His Ala Leu Glu Leu Ala Leu Xaa Val Met Gly Val Gly Pro
    65 70 75 80
    Gly Thr Glu Val Ile Val Pro Ala Phe Thr Phe Ile Ser Ser Ser Gln
    85 90 95
    Ala Xaa Gln Arg Leu Gly Ala Val Xaa Val Pro Val Asp Val Asp Pro
    100 105 110
    Xaa Thr Tyr Cys Leu Asp Xaa Xaa Ala Ala Ala Xaa Ala Val Thr Pro
    115 120 125
    Arg Thr Xaa Ala Ile Met Pro Val His Met Ala Gly Gln Xaa Ala Asp
    130 135 140
    Met Asp Ala Leu Xaa Lys Xaa Ser Ala Xaa Thr Gly Val Pro Xaa Xaa
    145 150 155 160
    Gln Asp Ala Ala His Ala His Gly Ala Xaa Trp Xaa Gly Xaa Arg Val
    165 170 175
    Gly Glu Leu Gly Ser Ile Ala Xaa Phe Ser Phe Gln Asn Gly Lys Leu
    180 185 190
    Met Thr Ala Gly Glu Gly Gly Ala Val Leu Phe Pro Asp Xaa Glu Xaa
    195 200 205
    Xaa Xaa Xaa Glu Xaa Ala Phe Leu Xaa His Ser Cys Gly Arg Pro Xaa
    210 215 220
    Xaa Asp Arg Xaa Tyr Phe His Xaa Thr Xaa Gly Ser Asn Xaa Arg Xaa
    225 230 235 240
    Asn Glu Phe Ser Ala Ser Val Leu Arg Ala Gln Leu Xaa Arg Leu Asp
    245 250 255
    Xaa Gln Ile Xaa Xaa Arg Xaa Glu Arg Trp Xaa Xaa Leu Ser Xaa Leu
    260 265 270
    Leu Ala Xaa Ile Asp Gly Val Val Pro Gln Xaa Xaa Asp Xaa Arg Xaa
    275 280 285
    Asp Arg Asn Xaa His Tyr Met Ala Met Phe Arg Xaa Pro Gly Xaa Thr
    290 295 300
    Glu Glu Arg Arg Xaa Ala Xaa Val Asp Xaa Leu Val Glu Arg Gly Xaa
    305 310 315 320
    Pro Ala Phe Xaa Ala Phe Arg Xaa Val Tyr Arg Thr Xaa Ala Phe Trp
    325 330 335
    Glu Xaa Gly Ala Pro Asp Xaa Xaa Xaa Xaa Glu Leu Ala Xaa Arg Cys
    340 345 350
    Pro Xaa Xaa Xaa Xaa Ile Xaa Xaa Asp Cys Xaa Trp Leu His His Arg
    355 360 365
    Val Leu Leu Xaa Xaa Glu Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Val Xaa
    370 375 380
    Ala Asp Xaa Val Xaa Xaa Xaa
    385 390
    <210> SEQ ID NO 143
    <211> LENGTH: 393
    <212> TYPE: DNA
    <213> ORGANISM: Streptomyces lavendulae
    <400> SEQUENCE: 143
    atgtcagcaa ggatttccct cttcgccgtg gtggtcgagg acatggccaa gtcgctggag 60
    ttctaccgga agctgggcgt cgagatcccc gccgaggccg actccgcgcc gcacacggag 120
    gccgtgctcg acggcggcat ccggctcgcc tgggacaccg tggagacggt gcgcagctac 180
    gaccccgagt ggcaggcccc caccggcggc caccgcttcg ccatcgcgtt cgagttcccc 240
    gacaccgcga gcgtggacaa gaagtacgcc gagctcgtcg acgccggcta cgagggccac 300
    ctcaagccgt ggaacgccgt gtggggtcag cgctacgcca tcgtcaagga ccccgacggc 360
    aacgtggtgg acctcttcgc gcccctcccg taa 393
    <210> SEQ ID NO 144
    <211> LENGTH: 16
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A motif
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)...(16)
    <223> OTHER INFORMATION: Where present in this sequence, Xaa represents
    an amino acid that varied in this motif.
    <400> SEQUENCE: 144
    Val Xaa Gly Xaa Leu Xaa Asp Xaa Xaa Gly Arg Lys Xaa Xaa Xaa Leu
    1 5 10 15
    <210> SEQ ID NO 145
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: A motif
    <220> FEATURE:
    <221> NAME/KEY: SITE
    <222> LOCATION: (1)...(11)
    <223> OTHER INFORMATION: Where present in this sequence, Xaa represents
    an amino acid that varied in this conserved motif.
    <400> SEQUENCE: 145
    Leu Asp Xaa Thr Val Xaa Asn Val Ala Leu Pro
    1 5 10

Claims (58)

What is claimed is:
1. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising a mitomycin biosynthetic gene cluster, a variant or a fragment thereof.
2. The isolated and purified nucleic acid molecule of claim 1 which encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.
3. The isolated and purified nucleic acid molecule of claim 1 which encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
4. The isolated and purified nucleic acid molecule of claim 1 which is from Streptomyces spp.
5. An expression cassette comprising the nucleic acid molecule of claim 1, 2 or 3 operably linked to a promoter functional in a host cell.
6. A recombinant bacterial host cell in which at least a portion of a nucleic acid molecule comprising a mitomycin biosynthetic gene cluster is disrupted to as to result in a recombinant host cell that produces altered levels of mitomycin relative to a corresponding nonrecombinant bacterial host cell.
7. The recombinant host cell of claim 6 in which mitomycin levels are increased.
8. The recombinant host cell of claim 6 in which mitomycin levels are decreased.
9. The host cell of claim 6 wherein the nucleic acid molecule which is disrupted encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.
10. The host cell of claim 6 wherein the nucleic acid molecule which is disrupted encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
11. A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid molecule comprising a mitomycin biosynthetic gene cluster operably linked to a promoter functional in the host cell.
12. The recombinant host cell of claim 11 in which mitomycin levels are increased.
13. The recombinant host cell of claim 11 in which mitomycin levels are decreased.
14. The host cell of claim 11 wherein the genome is augmented with a nucleic acid molecule that encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any combination thereof.
15. The host cell of claim 11 wherein the genome is augmented with a nucleic acid molecule that encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
16. A recombinant host cell comprising a mitomycin biosynthetic gene cluster, the genome of which is augmented by a recombinant nucleic acid molecule, wherein the recombinant nucleic acid does not comprise a mitomycin biosynthetic gene, and wherein the recombinant host cell produces a biologically active agent that is not produced by the corresponding non-recombinant host cell.
17. A product produced by the recombinant host cell of claim 6 or 11 which is not produced by the corresponding non-recombinant host cell.
18. The product of claim 17 which comprises a biologically active agent.
19. The product of claim 18 which is a mitomycin.
20. The product of claim 18 is not a mitomycin.
21. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence which encodes polyketide biosynthetic enzymes or a fragment thereof, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74.
22. An isolated and purified polypeptide comprising MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
23. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising sugar metabolism genes or a fragment thereof, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.
24. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence which encodes an aminoDAHP synthase from Streptomyces strains that produce mitomycin.
25. A recombinant host cell in which at least a portion of a nucleic acid sequence which encodes polyketide biosynthetic enzymes is disrupted so as to result in a recombinant host cell that produces altered polyketide levels or polyketides of altered composition relative to a corresponding nonrecombinant cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74
26. A recombinant host cell in which at least a portion of a nucleic acid sequence which encodes sugar metabolism enzymes is disrupted so as to result in a recombinant host cell that produces altered sugar levels or molecules with altered sugar composition relative to a corresponding nonrecombinant cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.
27. A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid sequence which encodes polyketide biosynthetic enzymes operably linked to a promoter functional in the host cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to SEQ ID NO:74.
28. A recombinant host cell, the genome of which is augmented with at least a portion of a nucleic acid sequence which encodes sugar metabolism enzymes operably linked to a promoter functional in the host cell, wherein the nucleic acid sequence hybridizes under hybridizing conditions to a DNA comprising SEQ ID NO:75.
29. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence that hybridizes under hybridizing conditions to a nucleic acid segment comprising SEQ ID NO:96, or a fragment thereof.
30. The isolated and purified nucleic acid molecule of claim 29 which is plant nucleic acid.
31. The isolated and purified nucleic acid molecule of claim 29 which is prokaryotic nucleic acid.
32. A method to introduce exogenous DNA into a refractory Streptoinyces strain, comprising:
a) contacting a bacterial donor cell comprising a conjugative plasmid with a Streptomyces cell so as to yield a transformed Streptomyces cell comprising at least a portion of the plasmid; and
b) identifying the transformed Streptomyces cell.
33. The method of claim 32 wherein the Streptomyces strain produces a mitomycin.
34. A method to identify a nucleic acid molecule that is related to at least a portion of a nucleic acid molecule comprising a mitomycin gene cluster, comprising:
a) contacting a sample comprising nucleic acid with an amount of a probe comprising at least a portion of a nucleic acid molecule comprising a mitomycin gene so as to form a complex;
b) detecting the presence or absence of the complex.
35. A method to identify a nucleic acid molecule that is related to at least a portion of a nucleic acid molecule comprising a mitomycin gene cluster comprising:
a) contacting a sample comprising nucleic acid with at least one oligonucleotide under conditions effective to amplify the nucleic acid so as to yield an amplification product, wherein the oligonucleotide specifically hybridizes to nucleic acid comprising a mitomycin gene cluster; and
b) detecting or determining the presence or absence of the product.
36. The method of claim 34 or 35 wherein the sample is obtained from a plant.
37. The method of claim 34 or 35 wherein the sample is obtained from a microorganism.
38. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence comprising a gene product selected from MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, MmcW, MmcX, MmcY, or any combination thereof.
39. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding at least one gene necessary for mitomycin biosynthesis.
40. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding at least one gene for mitomycin transport.
41. An isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide that regulates mitomycin biosynthesis or resistance.
42. A method for preparing a compound or a pharmaceutically acceptable salt thereof from a recombinant host cell comprising culturing the host cell of claim 6, 11 or 16 in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under aerobic fermentation conditions so as to yield an increase in the compound relative to the level of the compound produced by the corresponding non-recombinant host cell.
43. A method for preparing a mitomycin or a pharmaceutically acceptable salt thereof from a recombinant host cell comprising culturing the host cell of claim 6, 11 or 16 in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under aerobic fermentation conditions so as to yield an increase in the mitomycin relative to the level of the mitomycin produced by the corresponding non-recombinant host cell.
44. A product produced by the recombinant host cell of claim 16 which is a mitomycin.
45. A product produced by the recombinant host cell of claim 16 which is not a mitomycin.
46. An isolated and purified nucleic acid molecule comprising at least a fragment of a nucleic acid sequence comprising a mitomycin biosynthetic gene cluster (mit/mmc), which fragment encodes an enzyme that during the biosyntheis of mitomycin modifies mitosane, and which fragment has at least 80% nucleic acid sequence identity with at least one of SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62, 68, or comprising the complement of the fragment.
47. The isolated and purified nucleic acid molecule of claim 46 which encodes MitK having SEQ ID NO: 107, MitH having SEQ ID NO: 104, or MitF having SEQ ID NO: 102.
48. The isolated and purified nucleic acid molecule of claim 46 which encodes MmcE having SEQ ID NO: 120, MmcI having SEQ ID NO: 124, MmcJ having SEQ ID NO: 125, MmcL having SEQ ID NO: 127, MmcN having SEQ ID NO: 129, or MmcT having SEQ ID NO: 135.
49. The isolated and purified nucleic acid molecule of claim 46 which is from a naturally-occurring Streptomyces spp.
50. An expression cassette comprising the nucleic acid molecule of claim 46 operably linked to a promoter functional in a host cell.
51. A recombinant host cell comprising a recombinant nucleic acid molecule comprising at least a fragment of a mitomycin biosynthetic gene cluster (mit/mmc) operably linked to a promoter functional in the host cell, which fragment encodes an enzyme that during the biosynthesis of mitomycin modifies mitosane, and which fragment has at least 80% nucleic acid sequence identity with at least one of SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62, 68, or comprising the complement of the fragment.
52. The recombinant host cell of claim 51 in which the levels of the enzyme are increased.
53. The recombinant host cell of claim 51 in which the levels of the enzyme are decreased.
54. The recombinant host cell of claim 51 wherein the fragment encodes MitK having SEQ ID NO: 107, MitH having SEQ ID NO: 104, or MitF having SEQ ID NO: 102.
55. The recombinant host cell of claim 51 wherein the fragment encodes MmcE having SEQ ID NO: 120, MmcI having SEQ ID NO: 124, MmcJ having SEQ ID NO: 125, MmcL having SEQ ID NO: 127, MmcN having SEQ ID NO: 129, or MmcT having SEQ ID NO: 135.
56. The isolated and purified nucleic acid molecule of claim 46 wherein the enzyme is a hydroxylase, a reductase, a dehydrogenase, methyltransferase, or converts a carboxyl group to a methyl group.
57. The isolated and purified nucleic acid molecule of claim 46 wherein the fragment comprises SEQ ID NO:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62 or 68.
58. A method to prepare an enzyme that catalyzes a step in mitomycin biosynthesis: expressing a recombinant DNA molecule in a host cell so as to yield an enzyme that catalyzes a step in mitomycin biosynthesis, wherein the recombinant DNA molecule comprises a promoter operably linked to a DNA sequence which encodes an enzyme that modifies mitosane, and wherein the DNA sequence has at least 80% nucleic acid sequence identity with at least one of SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62, or 68.
US10/267,255 1999-03-12 2002-10-09 Mitomycin biosynthetic gene cluster Abandoned US20030124689A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/267,255 US20030124689A1 (en) 1999-03-12 2002-10-09 Mitomycin biosynthetic gene cluster

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US09/266,965 US6495348B1 (en) 1993-10-07 1999-03-12 Mitomycin biosynthetic gene cluster
US10/267,255 US20030124689A1 (en) 1999-03-12 2002-10-09 Mitomycin biosynthetic gene cluster

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09/266,965 Division US6495348B1 (en) 1993-10-07 1999-03-12 Mitomycin biosynthetic gene cluster

Publications (1)

Publication Number Publication Date
US20030124689A1 true US20030124689A1 (en) 2003-07-03

Family

ID=23016731

Family Applications (2)

Application Number Title Priority Date Filing Date
US09/266,965 Expired - Fee Related US6495348B1 (en) 1993-10-07 1999-03-12 Mitomycin biosynthetic gene cluster
US10/267,255 Abandoned US20030124689A1 (en) 1999-03-12 2002-10-09 Mitomycin biosynthetic gene cluster

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US09/266,965 Expired - Fee Related US6495348B1 (en) 1993-10-07 1999-03-12 Mitomycin biosynthetic gene cluster

Country Status (6)

Country Link
US (2) US6495348B1 (en)
EP (1) EP1165800A2 (en)
JP (1) JP2002537833A (en)
AU (1) AU3876300A (en)
CA (1) CA2365904A1 (en)
WO (1) WO2000053737A2 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090075283A1 (en) * 2007-07-27 2009-03-19 Wyeth Vectors and Methods for Cloning Gene Clusters or Portions Thereof
WO2016182819A3 (en) * 2015-05-05 2016-12-22 William Marsh Rice University Identifying ligands from bacterial sensors
WO2017031399A1 (en) * 2015-08-20 2017-02-23 Genomatica, Inc. Compositions and multiplexed systems for coupled cell-free transcription-translation and protein synthesis and methods for using them
CN106916836A (en) * 2015-12-24 2017-07-04 武汉臻智生物科技有限公司 The biological synthesis gene cluster of compound and its application

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6831165B1 (en) * 1999-06-25 2004-12-14 Basf Aktiengesellschaft Corynebacterium glutamicum genes encoding proteins involved in homeostasis and adaptation
US7257562B2 (en) 2000-10-13 2007-08-14 Thallion Pharmaceuticals Inc. High throughput method for discovery of gene clusters
WO2002059322A2 (en) * 2000-10-17 2002-08-01 Cubist Pharmaceuticlas, Inc. Compositions and methods relating to the daptomycin biosynthetic gene cluster
US7189549B2 (en) * 2002-06-14 2007-03-13 Kosan Biosciences, Inc. Recombinant polynucleotides encoding pro-geldanamycin producing polyketide synthase and accessory proteins, and uses thereof
US7326782B2 (en) * 2003-08-21 2008-02-05 Wisconsin Alumni Research Foundation Metabolic engineering of viomycin biosynthesis
CN100465277C (en) * 2005-07-01 2009-03-04 中国科学院上海有机化学研究所 Biosynthetic gene cluster of chlorthricin and its application
EP1918375B1 (en) * 2005-08-02 2015-03-11 Kaneka Corporation D-amino acid oxidase, and method for production of l-amino acid, 2-oxo acid or cyclic imine
KR100860502B1 (en) * 2006-09-29 2008-09-26 한국생명공학연구원 Derivatives Prepared from Biosynthetic Gene Mutations of Zeldanamycin
JP5524053B2 (en) 2008-06-04 2014-06-18 エーザイ・アール・アンド・ディー・マネジメント株式会社 DNA encoding a polypeptide involved in the biosynthesis of herboxidiene
RU2420568C2 (en) * 2009-05-20 2011-06-10 Государственное учреждение научно-исследовательский институт по изысканию новых антибиотиков им. Г.Ф. Гаузе Российской академии медицинских наук Strain and biosynthesis method of producing antibiotic mitomycin
US8292863B2 (en) 2009-10-21 2012-10-23 Donoho Christopher D Disposable diaper with pouches
CN114045252B (en) * 2021-11-18 2023-08-22 陕西麦可罗生物科技有限公司 Method for improving titer of production of antibiotics in Streptomyces lilacinus Hainan variety

Citations (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3214440A (en) * 1964-11-02 1965-10-26 American Cyanamid Co Reaction products of mitomycins and porfiromycin
US3219530A (en) * 1961-05-03 1965-11-23 American Cyanamid Co Porfiromycin antibiotic and production thereof
US3272696A (en) * 1964-04-27 1966-09-13 Upjohn Co Porfiromycin insecticide
US3306821A (en) * 1962-09-04 1967-02-28 Upjohn Co Porfiromycin derivatives and method of making same
US3332944A (en) * 1964-11-02 1967-07-25 American Cyanamid Co Antibiotic derivatives of mitomycins a, b, c and porfiromycin
US4395558A (en) * 1979-08-24 1983-07-26 Kyowa Hakko Kogyo Kabushiki Kaisha 9-Epi-mitomycin B and D compounds
US4885251A (en) * 1985-04-22 1989-12-05 Eli Lilly And Company Recombinant DNA expression vectors and DNA compounds which encode isopenicillin N synthetase
US4892819A (en) * 1985-11-25 1990-01-09 Eli Lilly And Company Recombinant DNA expression vectors and DNA compounds that encode isopenicillin N synthetase from penicillium chrysogenum
US4935340A (en) * 1985-06-07 1990-06-19 Eli Lilly And Company Method of isolating antibiotic biosynthetic genes
US4975278A (en) * 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
US5023253A (en) * 1987-12-21 1991-06-11 University Patents, Inc. 6-substituted mitomycin analogs
US5032512A (en) * 1987-01-15 1991-07-16 Rijksuniversiteit Te Groningen Process for producing compounds containing a terminal hydroxyl or epoxy group and microorganisms suitable therefor
US5140013A (en) * 1989-11-28 1992-08-18 Universite Laval Maleic anhydride derivatives used as conjugation agents of anti-tumor agents on desired carriers
US5229279A (en) * 1987-06-29 1993-07-20 Massachusetts Institute Of Technology Method for producing novel polyester biopolymers
US5245023A (en) * 1987-06-29 1993-09-14 Massachusetts Institute Of Technology Method for producing novel polyester biopolymers
US5250430A (en) * 1987-06-29 1993-10-05 Massachusetts Institute Of Technology Polyhydroxyalkanoate polymerase
US5252673A (en) * 1991-03-18 1993-10-12 Agency Of Industrial Science & Technology Macromolecular mitomycin C derivative and method for production thereof
US5256685A (en) * 1991-03-26 1993-10-26 Kyowa Hakko Kogyo Co., Ltd. Mitomycin derivatives
US5256688A (en) * 1988-07-18 1993-10-26 E. R. Squibb & Sons, Inc. Method for inhibiting myocardial cell necrosis and preserving heart function during myocardial ischemia and/or reperfusion
US5352798A (en) * 1990-03-08 1994-10-04 Bristol-Myers Squibb Company Process for preparing dithio-ethylanino 9A-methoxymitosanes
US5374739A (en) * 1987-06-12 1994-12-20 Bristol-Myers Squibb Company Mitomycin analogs
US5462862A (en) * 1988-08-11 1995-10-31 Gist-Brocades N.V. Method and compositions for enhancing production of secondary metabolites using clustered biosynthetic genes
US5514544A (en) * 1991-07-26 1996-05-07 Eli Lilly And Company Activator gene for macrolide biosynthesis
US5545553A (en) * 1994-09-26 1996-08-13 The Rockefeller University Glycosyltransferases for biosynthesis of oligosaccharides, and genes encoding them
US5554638A (en) * 1993-05-24 1996-09-10 Duke University Methods for improving therapeutic effectiveness of agents for the treatment of solid tumors and other disorders
US5589385A (en) * 1990-07-26 1996-12-31 American Cyanamid Company Cloning of the biosynthetic pathway for chlortetracycline and tetracycline formation and cosmids useful therein
US5629427A (en) * 1993-08-26 1997-05-13 Peterson; Dwight M. 2,7-diaminomitosene analogues
US5672497A (en) * 1986-03-21 1997-09-30 Eli Lilly And Company Method for increasing the antibiotic-producing ability of antibiotic-producing microorganisms
US5716849A (en) * 1994-06-08 1998-02-10 Novartis Finance Corporation Genes for the biosynthesis of soraphen
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
US5744350A (en) * 1993-11-02 1998-04-28 Merck & Co., Inc. DNA encoding triol polyketide synthase
US5747469A (en) * 1991-03-06 1998-05-05 Board Of Regents, The University Of Texas System Methods and compositions comprising DNA damaging agents and p53
US5763239A (en) * 1996-06-18 1998-06-09 Diversa Corporation Production and use of normalized DNA libraries
US5824513A (en) * 1991-01-17 1998-10-20 Abbott Laboratories Recombinant DNA method for producing erythromycin analogs
US5830750A (en) * 1993-09-20 1998-11-03 The John Innes Institute Recombinant production of novel polyketides
US5962290A (en) * 1993-09-20 1999-10-05 The Leland Stanford Junior University Recombinant production of novel polyketides
US6033883A (en) * 1996-12-18 2000-03-07 Kosan Biosciences, Inc. Production of polyketides in bacteria and yeast
US6090601A (en) * 1998-01-23 2000-07-18 Kosan Bioscience Sorangium polyketide synthase
US6117659A (en) * 1997-04-30 2000-09-12 Kosan Biosciences, Inc. Recombinant narbonolide polyketide synthase
US6210935B1 (en) * 1995-08-30 2001-04-03 Novartis Ag Staurosporin biosynthesis gene clusters

Family Cites Families (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61205484A (en) 1985-03-09 1986-09-11 Sanraku Inc Novel plasmid
US5024948A (en) 1985-10-17 1991-06-18 American Cyanamid Company Genetic system for micromonospora
IL81892A (en) 1986-03-21 1995-10-31 Lilly Co Eli Recombinant DNA molecule comprising a sequence encoding macrocin o-methyltransferase
US5665564A (en) 1988-05-27 1997-09-09 Pharmacia & Upjohn S.P.A. Isolation and characterisation of genes resistant to anthracycline antibiotics
US5108918A (en) 1988-08-11 1992-04-28 Gist-Brocades Method for identifying and using biosynthetic genes for enhanced production of secondary metabolites
EP0361905A3 (en) 1988-09-29 1991-07-10 Eli Lilly And Company Carbomycin biosynthetic genes, designated carl and carm, for use in streptomyces and other organisms
ES2117630T3 (en) 1990-07-26 1998-08-16 American Cyanamid Co BIFUNCTIONAL COSMIDE USEFUL TO CLONE ACTINOMYTICAL DNA.
EP0468220A3 (en) 1990-07-26 1992-09-23 American Cyanamid Company Cloning of the biosynthetic pathway genes for chlortetracycline production from streptomyces aureofaciens & their expression in steptomyces lividans
WO1993013663A1 (en) 1992-01-17 1993-07-22 Abbott Laboratories Method of directing biosynthesis of specific polyketides
US5107370A (en) 1991-01-17 1992-04-21 Eastman Kodak Company Astronomical data collection method employing a novel optical reticle projector
AU2028792A (en) 1991-05-17 1992-12-30 Uab Research Foundation Sequence specific dna binding drugs
FR2696189B1 (en) 1992-09-25 1994-11-10 Rhone Poulenc Rorer Sa Polypeptides involved in the biosynthesis of streptogramins, nucleotide sequences coding for these polypeptides and their use.
JPH06261765A (en) 1993-03-12 1994-09-20 Asahi Chem Ind Co Ltd Dna comprising biosynthetic gene of macrolide antibiotic substance
US6066721A (en) 1995-07-06 2000-05-23 Stanford University Method to produce novel polyketides
WO1995009926A1 (en) 1993-10-07 1995-04-13 Regents Of The University Of Minnesota Genes encoding resistance to dna alkylating agents
FR2722210B1 (en) 1994-07-08 1996-08-14 Rhone Poulenc Rorer Sa NOVEL STREPTOGRAMINS AND PROCESS FOR THE PREPARATION OF STREPTOGRAMINS BY MUTASYNTHESIS
AU3517895A (en) 1994-09-28 1996-04-19 Novo Nordisk A/S Process for the production of secondary metabolites
NZ313383A (en) 1995-07-06 1999-08-30 Univ Leland Stanford Junior Cell-free synthesis of polyketides using pks (polyketide synthase)
US5958672A (en) 1995-07-18 1999-09-28 Diversa Corporation Protein activity screening of clones having DNA from uncultivated microorganisms
US5998194A (en) 1995-12-21 1999-12-07 Abbott Laboratories Polyketide-associated sugar biosynthesis genes
CA2197524A1 (en) 1996-02-22 1997-08-22 Bradley Stuart Dehoff Polyketide synthase genes
CA2197160C (en) 1996-02-22 2007-05-01 Stanley Gene Burgett Platenolide synthase gene
US5958745A (en) 1996-03-13 1999-09-28 Monsanto Company Methods of optimizing substrate pools and biosynthesis of poly-β-hydroxybutyrate-co-poly-β-hydroxyvalerate in bacteria and plants
JPH09268190A (en) 1996-04-02 1997-10-14 Sagami Chem Res Center Mitomycin c derivative and nonreceptor type tyrosine kinase inhibitor
WO1997039135A1 (en) 1996-04-17 1997-10-23 Board Of Regents, The University Of Texas System Enhanced expression of transgenes
EP0806480A3 (en) 1996-05-07 1999-11-24 F. Hoffmann-La Roche Ag Frenolicin gene cluster
GB9710962D0 (en) 1997-05-28 1997-07-23 Univ Cambridge Tech Polyketides and their synthesis
US5932613A (en) 1996-07-03 1999-08-03 Millennium Pharmaceuticals, Inc. Anticancer agents
EA001744B1 (en) 1996-07-05 2001-08-27 Байотика Текнолоджи Лимитед (Gb) Novel erythromycins and process for their preparation
US5811272A (en) 1996-07-26 1998-09-22 Massachusetts Institute Of Technology Method for controlling molecular weight of polyhydroxyalkanoates
WO1998007868A1 (en) 1996-08-20 1998-02-26 Novartis Ag Rifamycin biosynthesis gene cluster
CN1273475C (en) 1996-09-04 2006-09-06 艾博特公司 6-0-substituted ketolides with antibacterial activity
WO1998011230A1 (en) 1996-09-13 1998-03-19 Bristol-Myers Squibb Company Polyketide synthases for pradimicin biosynthesis and dna sequences encoding same
WO1998036078A1 (en) 1997-02-13 1998-08-20 James Madison University Methods of making polyhydroxyalkanoates comprising 4-hydroxybutyrate monomer units
JP2001524829A (en) 1997-04-30 2001-12-04 コーサン バイオサイエンシーズ，インコーポレイテッド Combinatorial polyketide library generated using modular PKS gene cluster as scaffold
US6297007B1 (en) 1997-05-22 2001-10-02 Terragen Diversity Inc. Method for isolation of biosynthesis genes for bioactive molecules

Patent Citations (45)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3219530A (en) * 1961-05-03 1965-11-23 American Cyanamid Co Porfiromycin antibiotic and production thereof
US3306821A (en) * 1962-09-04 1967-02-28 Upjohn Co Porfiromycin derivatives and method of making same
US3272696A (en) * 1964-04-27 1966-09-13 Upjohn Co Porfiromycin insecticide
US3214440A (en) * 1964-11-02 1965-10-26 American Cyanamid Co Reaction products of mitomycins and porfiromycin
US3332944A (en) * 1964-11-02 1967-07-25 American Cyanamid Co Antibiotic derivatives of mitomycins a, b, c and porfiromycin
US4395558A (en) * 1979-08-24 1983-07-26 Kyowa Hakko Kogyo Kabushiki Kaisha 9-Epi-mitomycin B and D compounds
US4885251A (en) * 1985-04-22 1989-12-05 Eli Lilly And Company Recombinant DNA expression vectors and DNA compounds which encode isopenicillin N synthetase
US4935340A (en) * 1985-06-07 1990-06-19 Eli Lilly And Company Method of isolating antibiotic biosynthetic genes
US4892819A (en) * 1985-11-25 1990-01-09 Eli Lilly And Company Recombinant DNA expression vectors and DNA compounds that encode isopenicillin N synthetase from penicillium chrysogenum
US5672497A (en) * 1986-03-21 1997-09-30 Eli Lilly And Company Method for increasing the antibiotic-producing ability of antibiotic-producing microorganisms
US5032512A (en) * 1987-01-15 1991-07-16 Rijksuniversiteit Te Groningen Process for producing compounds containing a terminal hydroxyl or epoxy group and microorganisms suitable therefor
US5374739A (en) * 1987-06-12 1994-12-20 Bristol-Myers Squibb Company Mitomycin analogs
US5229279A (en) * 1987-06-29 1993-07-20 Massachusetts Institute Of Technology Method for producing novel polyester biopolymers
US5245023A (en) * 1987-06-29 1993-09-14 Massachusetts Institute Of Technology Method for producing novel polyester biopolymers
US5250430A (en) * 1987-06-29 1993-10-05 Massachusetts Institute Of Technology Polyhydroxyalkanoate polymerase
US5023253A (en) * 1987-12-21 1991-06-11 University Patents, Inc. 6-substituted mitomycin analogs
US4975278A (en) * 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
US5256688A (en) * 1988-07-18 1993-10-26 E. R. Squibb & Sons, Inc. Method for inhibiting myocardial cell necrosis and preserving heart function during myocardial ischemia and/or reperfusion
US5462862A (en) * 1988-08-11 1995-10-31 Gist-Brocades N.V. Method and compositions for enhancing production of secondary metabolites using clustered biosynthetic genes
US5140013A (en) * 1989-11-28 1992-08-18 Universite Laval Maleic anhydride derivatives used as conjugation agents of anti-tumor agents on desired carriers
US5352798A (en) * 1990-03-08 1994-10-04 Bristol-Myers Squibb Company Process for preparing dithio-ethylanino 9A-methoxymitosanes
US5866410A (en) * 1990-07-26 1999-02-02 American Cyanamid Company Cloning of the biosynthetic pathway for chlortetracycline and tetracycline formation and cosmids useful therein
US5589385A (en) * 1990-07-26 1996-12-31 American Cyanamid Company Cloning of the biosynthetic pathway for chlortetracycline and tetracycline formation and cosmids useful therein
US5824513A (en) * 1991-01-17 1998-10-20 Abbott Laboratories Recombinant DNA method for producing erythromycin analogs
US5747469A (en) * 1991-03-06 1998-05-05 Board Of Regents, The University Of Texas System Methods and compositions comprising DNA damaging agents and p53
US5252673A (en) * 1991-03-18 1993-10-12 Agency Of Industrial Science & Technology Macromolecular mitomycin C derivative and method for production thereof
US5256685A (en) * 1991-03-26 1993-10-26 Kyowa Hakko Kogyo Co., Ltd. Mitomycin derivatives
US5334611A (en) * 1991-03-26 1994-08-02 Kyowa Hakko Kogyo Co., Ltd. 6-ethyl mitomycin derivatives
US5514544A (en) * 1991-07-26 1996-05-07 Eli Lilly And Company Activator gene for macrolide biosynthesis
US5554638A (en) * 1993-05-24 1996-09-10 Duke University Methods for improving therapeutic effectiveness of agents for the treatment of solid tumors and other disorders
US5788958A (en) * 1993-05-24 1998-08-04 Duke University Methods for improving therapeutic effectiveness of agents for the treatment of solid tumors and other disorders
US5629427A (en) * 1993-08-26 1997-05-13 Peterson; Dwight M. 2,7-diaminomitosene analogues
US6022731A (en) * 1993-09-20 2000-02-08 The Leland Stanford Junior University Recombinant production of novel polyketides
US5830750A (en) * 1993-09-20 1998-11-03 The John Innes Institute Recombinant production of novel polyketides
US5962290A (en) * 1993-09-20 1999-10-05 The Leland Stanford Junior University Recombinant production of novel polyketides
US6077696A (en) * 1993-09-20 2000-06-20 The Johns Innes Institute Recombinant production of novel polyketides
US5744350A (en) * 1993-11-02 1998-04-28 Merck & Co., Inc. DNA encoding triol polyketide synthase
US5716849A (en) * 1994-06-08 1998-02-10 Novartis Finance Corporation Genes for the biosynthesis of soraphen
US5545553A (en) * 1994-09-26 1996-08-13 The Rockefeller University Glycosyltransferases for biosynthesis of oligosaccharides, and genes encoding them
US6210935B1 (en) * 1995-08-30 2001-04-03 Novartis Ag Staurosporin biosynthesis gene clusters
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
US5763239A (en) * 1996-06-18 1998-06-09 Diversa Corporation Production and use of normalized DNA libraries
US6033883A (en) * 1996-12-18 2000-03-07 Kosan Biosciences, Inc. Production of polyketides in bacteria and yeast
US6117659A (en) * 1997-04-30 2000-09-12 Kosan Biosciences, Inc. Recombinant narbonolide polyketide synthase
US6090601A (en) * 1998-01-23 2000-07-18 Kosan Bioscience Sorangium polyketide synthase

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090075283A1 (en) * 2007-07-27 2009-03-19 Wyeth Vectors and Methods for Cloning Gene Clusters or Portions Thereof
WO2016182819A3 (en) * 2015-05-05 2016-12-22 William Marsh Rice University Identifying ligands from bacterial sensors
WO2017031399A1 (en) * 2015-08-20 2017-02-23 Genomatica, Inc. Compositions and multiplexed systems for coupled cell-free transcription-translation and protein synthesis and methods for using them
CN106916836A (en) * 2015-12-24 2017-07-04 武汉臻智生物科技有限公司 The biological synthesis gene cluster of compound and its application

Also Published As

Publication number Publication date
WO2000053737A2 (en) 2000-09-14
JP2002537833A (en) 2002-11-12
AU3876300A (en) 2000-09-28
CA2365904A1 (en) 2000-09-14
EP1165800A2 (en) 2002-01-02
WO2000053737A3 (en) 2000-12-21
US6495348B1 (en) 2002-12-17

Similar Documents

Publication Publication Date Title
US6495348B1 (en) Mitomycin biosynthetic gene cluster
DK2271666T3 (en) NRPS-PKS GROUP AND ITS MANIPULATION AND APPLICABILITY
KR20100039443A (en) Compositions and methods relating to the daptomycin biosynthetic gene cluster
KR20190099396A (en) Compositions and Methods for the Production of Compounds
CN107868789B (en) Colimycin biosynthesis gene cluster
CA2399689A1 (en) Gene cluster encoding a nystatin polyketide synthase and its manipulation and utility
KR20040099138A (en) Cloning genes from Streptomyces cyaneogriseus subsp. noncyanogenus for biosynthesis of antibiotics and methods of use
EP0929681A1 (en) Rifamycin biosynthesis gene cluster
CN111607603B (en) Hangtaimycin biosynthesis gene cluster and application thereof
CA2394616C (en) Gene cluster for ramoplanin biosynthesis
CN101691575B (en) Biosynthetic gene cluster of sanglifehrin
US20030175888A1 (en) Discrete acyltransferases associated with type I polyketide synthases and methods of use
WO2002059322A9 (en) Compositions and methods relating to the daptomycin biosynthetic gene cluster
KR100882692B1 (en) Biosynthetic Genes for Butenyl-Spinosine Pesticide Production
KR101189475B1 (en) Genes and proteins for biosynthesis of tricyclocompounds
KR20100034055A (en) Vectors and methods for cloning gene clusters or portions thereof
US20030171562A1 (en) Genes and proteins for the biosynthesis of polyketides
US20030134398A1 (en) Mitomycin biosynthetic gene cluster
US20030064491A1 (en) Genes and proteins involved in the biosynthesis of enediyne ring structures
CN101175767A (en) Actinomadura chromoprotein, apoprotein, and gene cluster
US20030113874A1 (en) Genes and proteins for the biosynthesis of rosaramicin
KR102017788B1 (en) Recombinant Microorganisms Producing Milbemycin D and Method of Preparing Milbemycin D Using the Same
US20030157654A1 (en) Biosynthesis of enediyne compounds by manipulation of C-1027 gene pathway
CA2450691C (en) Genes and proteins involved in the biosynthesis of lipopeptides
US20040161828A1 (en) Gene cluster for production of the enediyne antitumor antibiotic C-1027

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION