[go: up one dir, main page]

WO2015092575A1 - Groupe de gènes pour la biosynthèse de la pamamycine - Google Patents

Groupe de gènes pour la biosynthèse de la pamamycine Download PDF

Info

Publication number
WO2015092575A1
WO2015092575A1 PCT/IB2014/066284 IB2014066284W WO2015092575A1 WO 2015092575 A1 WO2015092575 A1 WO 2015092575A1 IB 2014066284 W IB2014066284 W IB 2014066284W WO 2015092575 A1 WO2015092575 A1 WO 2015092575A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
polypeptide
polynucleotide
activity
pamamycin
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.)
Ceased
Application number
PCT/IB2014/066284
Other languages
English (en)
Inventor
Lutz Petzke
Andrea Herold
Christian Fleck
Katrin TREIER-MARXEN
Peter ÖDMAN
Joachim Dickhaut
Melanie WEINGARTEN
Andriy LUZHETSKY
Yuriy REBETS
Elke BRÖTZ
Niko MANDERSCHEID
Maksym MYRONOVSKYI
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.)
BASF China Co Ltd
BASF SE
Original Assignee
BASF China Co Ltd
BASF SE
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 BASF China Co Ltd, BASF SE filed Critical BASF China Co Ltd
Publication of WO2015092575A1 publication Critical patent/WO2015092575A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin

Definitions

  • the invention pertains to the field of production of natural products and, in particular, in the field of production of pamamycin. It provides polynucleotides and polypeptides involved in the biosynthesis of pamamycin and pamamycin precursors as well as vectors and recombinant microorganisms comprising such polynucleotides and polypeptides. Also provided are methods for the production of pamamycins, in particular pamamycin 607 and pamamycin 621 , and their precursors.
  • Pamamycins are a group of macrolide compounds and have been described as early as 1979 (McCann and Pogell, JOURNAL OF ANTIBIOTICS (1979) Vol.: 32(7) pages: 673 to 678). They are produced by several Streptomyces species and have raised scientific interest, because they stimulate the formation of aerial mycelium in Streptomycete species which produce pamamycins, as well as in other streptomycetes which are not known to produce pamamycins.
  • BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY (201 1 ) Vol. :75(9) pages: 1722 to 1726). Further investigations showed, that the different side chains located on the ring structure of pamamycins, as well as their dimethylamino group have different effects on the capacity of pamamycins to induce aerial mycelia, to inhibit growth of Streptomyces albonigeror to work as antibiotic on Bacillus subtilis, see Natsume, M. et al. JOURNAL OF ANTIBIOTICS (1995) Vol.:48(10); pages:1 159 to 1 164 and Natsume, M. et al.
  • C-13- and N-15-labeled precursor units provided further information on the metabolic pathway leading to the production of pamamycins.
  • N-15-labeling suggests that the nitrogen atom of the dimethylamino group present in many pamamycins was derived from the alpha-amino group of an amino acid which has been introduced into the pamaycin structure by transamination, followed by N-methylation.
  • Feeding experiments with C-13- labeled acetate or propionate indicates that the carbon skeleton of pamamycins is derived from six acetate, four propionate and three succinate units, see Hashimoto, M. et al.
  • Organisms which produce pamamycins, as well as fermentation methods for the production of pamamycins and methods to purify pamamycins have been disclosed for example in US4283391 , JP62135476A, DE4134168, DE4316836, Natsume, M. et al.
  • pamamycin Another fragment of pamamycin produced via hydrolysation is tetrahydorfuranyl-propionic acid, which can be used to transport organic cations into cells and to enhance the availability of cationic therapeutics. Further information can be found in DE4316836. However, the available methods to produce pamamycins via use of their natural producers are limited by the natural production level of these organism. Thus, there is still a need to develop further methods for the production of pamamycins.
  • the technical problem underlying the present invention can be seen as the provision of additional means and methods for the production of pamamycins, in particular pamamycin 607 and pamamycin 621 .
  • the technical problem is solved by the embodiments characterized in the claims and herein below.
  • the invention provides for polypeptides and polynucleotides as well as gene clusters, expression cassettes and vectors comprising one or several of those polynucleotides for the production of pamamycins, which can be used to construct, identify and improve
  • microorganisms having the capacity to produce one or several pamamycins, in particular to produce pamamycin 607 and/or pamamycin 621.
  • the provided polypeptides have different activities and can be used in different combinations to enhance, or down regulate the capacity of microorganisms to produce one or several pamamycins.
  • the invention encompasses recombinant microorganism comprising: a) at least one polypeptide having pamC activity, and b) at least one polypeptide having pamG activity, and c) at least one polypeptide having pamF activity, and d) at least one polypeptide having pamA activity, and e) at least one polypeptide having pamB activity, and f) at least one polypeptide having pamD activity, and g) at least one polypeptide having pamE activity, and h) at least one polypeptide having pamO activity, and i) at least one polypeptide having pamK activity, and j) at least one polypeptide having pamJ activity, and k) at least one polypeptide having pamM activity, and I) at least one polypeptide having pamN activity, and m) at least one polypeptide having pamL activity, and n) at least one polypeptide having pamX activity, and o) at least one polypeptide having pamM activity
  • the information on gene clusters being involved in pamamycin production capacity of microorganisms allow to identify new microorganisms suitable to be used for the production of pamamycins and to identify new cluster variants comprised in such microorganisms, as well as to design new variants of these clusters, having new gene orders, gene orientation, gene expression, and/or gene numbers for the encoded
  • the invention comprises also recombinant microorganisms comprising i) at least one polynucleotide having at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, ii) at least one polynucleotide having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 and comprising at least one expression cassette for a) at least one polypeptide having pamC activity, and b) at least one polypeptide having pamG activity, and c) at least one polypeptide having pamF activity, and d) at least one polypeptide having pamA activity
  • the recombinant microorganisms may also comprise at least one expression cassette for q) a polypeptide having pamR1 activity, or r) a polypeptide having pamR2 activity, or s) a polypeptide having pamW activity, or t) a polypeptide having pamH activity, or u) a polypeptide q), r) s) and t) and at least one expression cassette for a polypeptide of q), r) s) and t), which encodes a different polypeptide than the first expression cassette.
  • recombinant microorganism as described above comprising i) at least one polynucleotide having at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 , ii) at least one polynucleotide having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 and comprising at least one expression cassette for a) at least one polynucleotide having at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to S
  • polypeptide having pamC activity and b) at least one polypeptide having pamG activity, and c) at least one polypeptide having pamF activity, and d) at least one polypeptide having pamA activity, and e) at least one polypeptide having pamB activity, and f) at least one polypeptide having pamD activity, and g) at least one polypeptide having pamE activity, and h) at least one polypeptide having pamO activity, and i) at least one polypeptide having pamK activity, and j) at least one polypeptide having pamJ activity, and k) at least one polypeptide having pamM activity, and I) at least one polypeptide having pamN activity, and m) at least one polypeptide having pamL activity, and n) at least one polypeptide having pamX activity, and o) at least one polypeptide having pamY activity, and p) at least one polypeptide having pamS activity, and q) at least one
  • the invention provides also the possibility to enhance the expression of polypeptides for the production of pamamycins via the overexpression of a polypeptide having pamR1 activity.
  • the invention comprises recombinant microorganisms comprising i) at least one polynucleotide having at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, ii) at least one polynucleotide having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2 and comprising at least one expression cassette for a) at least one polypeptide having pamC activity, and b) at least one polypeptide having
  • the recombinant microorganisms as described above and variants therof will ususally have the capacity to produce pamamycins, in particular pamamycin 607 or pamamycin 621 or both. Accordingly, the recombinant microorganisms can be used in methods to produce at least one pamamycin, so that the invention provides for method for the production of pamamycin comprising the steps of: i) cultivating a recombinant microorganism as described above under conditions which allow for the production of pamamycin by said recombinant microorganism; ii) obtaining produced pamamycin, preferably obtaining pamamycin 607 and/or pamamycin 621.
  • Pamamycins produced by the recombinant microorganisms of the invention can be further modified, e.g. by hydrolyzing in order to produce compounds of Formular IV or Formula V, or both.
  • the invention comprises methods for the production of compounds of the Formula IV or Formula V comprising the steps of: i) cultivating a recombinant microorganism as described above under conditions which allow for the production of pamamycin by said recombinant microorganism; ii) obtaining produced pamamycin, preferably obtaining pamamycin 607 and/or pamamycin 621 , iii) hydrolysing pamamycin, preferably hydrolysing pamamycin 607 and/or pamamycin 621 and iv) obtaining compounds of Formula IV or obtaining compounds of Formula V or obtaining compounds of Formula IV and Formula V.
  • the produced pamamycins may be obtained from the culture medium (culture broth), or from the produced biomass, or from both.
  • the produced pamamycins or compounds of Formula iV or Formula V may be obtained in the form of their agriculturally or pharmacologically acceptable salts.
  • the gene clusters provided by the invention comprise promoter and terminator sequences as well as transcription factors (pamR1 and pamR2), which regulate the activity of the provided promoter sequences.
  • the promoters and transcription factors can be used to construct systems for coordinated gene expression in recombinant microorganisms comprising 1 ) at least one expression cassette comprising a promoter being able to provide for gene expression in said microorganism and being operably linkted to q1 ) a polynucleotide encoding a polypeptide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 16, or q2) a polynucleotide encoding a polypeptide which is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
  • polynucleotides of SEQ ID NO: 143, 144 or 145 per primer or iv) a polynucleotide of at least one of i), ii) or iii) and being obtainable from a pamamycin producing organism, or v) a polynucleotide of at least one of i), ii), iii) or iv) and comprising promoter activity in
  • Streptomyces alboniger These systems can be used to coordinate the expression of any polypeptide encoding region operatively linked to at least one of the respective promoters.
  • the polypeptide encoding regions encode one or several of the polypeptides involved in pamamycin production as described above.
  • the recombinant microorganisms comprising such a system for coordinated gene expression are preferably bacteria, yeasts or filamentous fungi, even more preferred they belong to the genus
  • Streptomyces in particular to the species Streptomyces albus or Streptomyces alboniger.
  • the information about polypeptides, polynucleotides and gene clusters involved in the production of pamamycin can be used to identify microorganisms which are potentially capable to produce at least one pamamycin, in particular at least one of pamamycin 607 and 621.
  • the invention comprises methods to identify pamaycin producing microorganisms or microorganisms potentially capable to produce pamamycin comprising the steps of a) providing a microorganism, b) isolating DNA and/or cDNA from such organism, c) identifying polynucleotide sequences encoding polypeptides having at least one of pamC activity, pam G activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity, pamS activity, by I) hybridizing the isolated DNA or RNA under medium or high stringent conditions with polynucleotide fragments of SEQ ID NO: 1 or 2 or both, or II) performing PCR reactions with PCR-primer suitable to amplify fragments of SEQ ID NO: 1 or 2 or both III) sequencing the isolated DNA and/or
  • the invention does also provide for methods to enhance the production of pamamycin, preferably the production of pamamycin 607 or pamamycin 621 or both, in a microorganism comprising i) at least one polynucleotide having at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 and/or 2, preferably 2, ii) at least one polynucleotide having at least 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 and/or 2, preferably 2, and comprising at least one expression cassette for each one of a) to p) as described above; iii) two or more fragment
  • polynucleotide of at least two of q 1 ), q2), q3), q4), q5) and q6).
  • the invention comprises also the use of one or several of them in any method, in particular in a method involving the production of at least one pamamycin, the identification of microorganisms potentially being able to produce at least one pamamycin, methods to improve the capacity of a microorganism to produce at least one pamamycin.
  • Figure 1 shows schematic drawings of different pamamycin gene clusters.
  • the drawing of Cluster 1 represents a polynucleotide comprising polypeptide encoding regions for polypeptides having pamC, pamG, pamF, pamA, pamB, pamD, pamE, pamO, pamK, pamJ, pamM, pamN, pamL, pamX, pamY and pamS activity, respectively.
  • the drawing of Cluster 2 represents a cluster, which comprises polypeptide encoding regions for all of the activites mentioned for Cluster 1 , but comprises in addition thereto a polypeptide encoding region encoding a polypeptide having pamH activity.
  • Cluster 3 represents a cluster comprising polypeptide encoding regions for all activities mentioned for Cluster 1 , but comprises also a polypeptide encoding region encoding a polypeptide having pamR1 activity.
  • the drawing of Cluster 4 represents a cluster comprising polypeptide encoding regions for all activities mentioned for Cluster 1 , but comprises also a polypeptide encoding region encoding a polypeptide having pamH activity and a polypeptide encoding region encoding a polypeptide having pamR1 activity.
  • Cluster 5 represents a cluster comprising polypeptide encoding regions for all activities mentioned for Cluster 4, but comprises also a polypeptide encoding region encoding a polypeptide having pamR2 activity and a polypeptide encoding region encoding a polypeptide having pamW activity.
  • the boxed arrows represent the order and orientation of the polypeptide encoding regions, wherein the colour or pattern of the boxed arrows represent the general class or family of the encoded polypeptides based on sequence homology, e.g. the encoded polypeptides having pamG, pamF, pamA, pam D, pam E, pamK and pamJ activity are represendted by black boxed arrows.
  • the legend of boxed arrows provided under the representations of the clusters shows that these polypeptides belong to the homology class or family of ketosynthases " KS" .
  • the polypeptide having pamB activity, represented by a white boxed arrow with black borders belongs to the homology class of acetyltransferases " AT” .
  • KR/DH represent ketoreductases/dehydrogenases
  • ACP represent acyl carrier proteins
  • hydrolase represent hydrolases
  • AmT represents aminotransferases
  • MT represents methyltransferases
  • transport represent polypeptides having transporter functions
  • enoyl-CoA hydratases represents enoyl-CoA hydratases
  • CoA-ligase represents CoA-ligases
  • regulation represents polypeptides having regulatory functions.
  • the schematic drawings of the clusters are intended to represent polynucleotides comprising promoter and terminator sequences operatively connected to the polypeptide encoding regions, which are suited to enable expression of the respective polypeptide in a microorganism, preferably of the genus Streptomyces.
  • the schematic drawings of the clusters are intended to represent also polynucleotides comprising at least one of the different polypeptide encoding regions in a different order and/or orientation.
  • a preferred example of Cluster 1 is disclosed as SEQ ID NO: 1
  • a preferred example of Cluster 5 is disclosed as SEQ ID NO: 2.
  • Preferred examples of Cluster 2 and 4 are respective fragments of SEQ ID NO: 2, while a preferred version of Cluster 3 is a fragment of SEQ ID NO: 2, wherein expression of the polypeptide having pamH activity is prevented, e.g. by deletion or mutation of parts or all of the polypeptide encoding region or its promoter, or by substitution of parts or all of the pamD encoding region or expression cassette with a pamR1 encoding region or expression cassette.
  • Examples of Cluster 3 preferably express the polypeptide having pamR1 activity under control of a constitutive promoter.
  • Clusters 1 , 2, 3, 4 or 5 are preferably used for expression of the endocded polypeptides in microorganisms of the genus Streptomyces, such as, but not excluding others, Streptomyces alboniger and Streptomyces albus.
  • Figure 2 schows a schematic drawing of the metabolic pathway leading to pamamycins.
  • the first step involves the activity of pamA, followed by the activity of pamB, pamD and the activity of pamE.
  • the pathway splits in order to produce the compound S-chain via the activities of pamO, pamM, pamN, pamS and to procude the compound K-chain via the activities of pamF, pamG, pamO, pamM, pamN and pamS.
  • the produced compounds S-chain and L-chain are combined with Coenzyme A (CoASH) via the activitiy of pamL.
  • CoASH Coenzyme A
  • the final pamamycins are then produced by the combination of one compound S- chain-SCoA with one compound L-chain-SCoA, mediated by the activity of pamJ and pamK.
  • the residues described as R1 , R2, R3, R4 and R5 of all formulas are the same residues as listed in the definition of Formula (I) given below.
  • Figure 3 represents an amino acid sequence alignment of the polypeptide (SEQ ID NO: 4) encoded by gene pamC and variants thereof (SEQ ID NO: 5, 6, 7, 8, and 9).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment. Such sequence variants preferably have no change in the amino acids marked with a black arrow, being L29, D32, S33, E44 and F47. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 4a and Figure 4b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 1 1) encoded by gene pamG and variants thereof (SEQ ID NO: 12, 13, 14, 15 and 16).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being P19, A64, C1 16, L139, D163, A167, H258, G287 and N288. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Amino acid P19 may be substituted by another amino acid in preferred variants, if P20 is not changed.
  • Figures 5a to Figure 5c represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 18) encoded by gene pamF and variants thereof (SEQ ID NO: 19, 20, 21 , 22 and 23). The amino acids are represented according to the standard single letter amino acid code. Further sequence variants can be deduced from this alignment. Such sequence variants preferably have no change in the amino acids marked with a black arrow, being G16, A186, G255, G261 , G366 and H267. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 6a to Figure 6c represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 25) encoded by gene pamA and variants thereof (SEQ ID NO: 26, 27, 28, 29 and 30).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in one or all the amino acids marked with a black arrow, being G21 , C191 , G258, G264, H333, G371 and H272, more preferred, they also have no change in at least one of the amino acids A190, V192, G193, G334, T335 and Y370, e.g. having no change in the amino acids G21 , A190, G258, G264, G371 and H272. More preferred, the variants will only have
  • Figures 7a to Figure 7c represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 32) encoded by gene pamB and variants thereof (SEQ ID NO: 33, 34, 35, 36 and 37).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being T2, G3, P6, R22, A23, P59, L89, D218, G221 , N222, P260, P266, V270, G407, G409, E434, G436, G491 , G506, G508, G509. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • One amino acid of G506, G508, G509 may be substituted in the preferred variants, in case the other two are kept identical.
  • Figures 8a and Figure 8b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 39) encoded by gene pamD and variants thereof (SEQ ID NO: 40, 41 , 42, 43 and 44).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being P21 , P101 , C121 , H246, G275 and G308. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 9a and Figure 9b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 46) encoded by gene pamE and variants thereof (SEQ ID NO: 47, 48, 49, 50 and 51 ).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment. Such sequence variants preferably have no change in the amino acids marked with a black arrow, being P25, P103, C136, H270, G299 and G333. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 10a and Figure 10b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 53) encoded by gene pamO and variants thereof (SEQ ID NO: 54, 55, 56, 57 and 58).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being G1 1 , G17, D61 , Y164, K168, P194 and at least two of G256, G257 and G258. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 1 1a and Figure 1 1 b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 60) encoded by gene pamK and variants thereof (SEQ ID NO: 61 , 62, 63, 64 and 65).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being F33, C159, G314, G346 and G348.
  • G346 and G348 may be substituted, in case G347 is kept. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background..
  • Figures 12a to Figure 12c represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 67) encoded by gene pamJ and variants thereof (SEQ ID NO: 68, 69, 70, 71 and 72).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment. Such sequence variants preferably have no change in the amino acids marked with a black arrow, being G14, G246, G252, G256 and H257. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 13a to Figure 13c represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 74) encoded by gene pamM and variants thereof (SEQ ID NO: 75, 76, 77, 78 and 79).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being G1 1 , V139, G241 , G247, D291 , A319, Y384, K388, P414, G415, T419, G465, G473.
  • G473 may be substituted, in case G472 is kept identical.
  • the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 14a and Figure 14b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 81 ) encoded by gene pamN and variants thereof (SEQ ID NO: 82, 83, 84, 85 and 86). The amino acids are represented according to the standard single letter amino acid code. Further sequence variants can be deduced from this alignment. Such sequence variants preferably have no change in the amino acids marked with a black arrow, being G16, G22, D66, Y158, K162, P188 and G189. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 15a to Figure 15c represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 88) encoded by gene pamL and variants thereof (SEQ ID NO: 89, 90, 91 , 92 and 93).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being L82, V93, T214, S215, G216, T218, G219, K222, E352, G370, G394, Y410, T438, G439, D430, R445, G454, E463, P520, P530, G535 and K536.
  • T218 may be substituted, in case T217 is kept. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 16a to Figure 16c represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 95) encoded by gene pamX and variants thereof (SEQ ID NO: 96, 97, 98, 99 and 100).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being G41 , D46, G57, G1 13, H147, D241 , K271 , G276 and T308.
  • G41 may be substituted, in case G40 is kept. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 17a and Figure 17b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 102) encoded by gene pamY and variants thereof (SEQ ID NO: 103, 104, 105, 106 and 107).
  • the amino acids are
  • sequence variants can be deduced from this alignment. Such sequence variants preferably have no change in the amino acids marked with a black arrow, being G67, G69, D86, G154 and G244. G154 and/or G244 may be substituted, in case the neighboring G153 and G243 are kept. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 18a and Figure 18b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 109) encoded by gene pamS and variants thereof (SEQ ID NO: 1 10, 1 1 1 , 1 12, 1 13 and 1 14).
  • the amino acids are
  • sequence variants can be deduced from this alignment. Such sequence variants preferably have no change in the amino acids marked with a black arrow, being N41 , F76, G79, D81 , G1 16, G120, G122, D130, G148, G187 and K221. G120 or G122 may be substituted, in case G121 is kept. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 19a and Figure 19b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 1 16) encoded by gene pamR1 and variants thereof (SEQ ID NO: 1 17, 1 18, 1 19, 120 and 121 ).
  • the amino acids are represented according to the standard single letter amino acid code.
  • Further sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being K105, E153 and K190. E153 may be substituted, in case E154 is kept. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 20a and Figure 20b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 123) encoded by gene pamH and variants thereof (SEQ ID NO: 124, 125, 126, 127 and 128).
  • the amino acids are
  • Figures 21a and Figure 21 b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ I D NO: 130) encoded by gene pamR2 and variants thereof (SEQ ID NO: 131 , 132, 133, 134 and 135).
  • the amino acids are represented according to the standard single letter amino acid code.
  • sequence variants can be deduced from this alignment.
  • Such sequence variants preferably have no change in the amino acids marked with a black arrow, being E3, W19, A41 , G49, A59, Y70, L79, G169, P207, F226, L230 and G236.
  • A41 may be substituted, in case A40 is kept.
  • G49 may be substituted, in case G48 is kept.
  • L79 may be substituted, in case L80 is kept.
  • the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 22a to Figure 22c represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ I D NO: 137) encoded by gene pamW and variants thereof (SEQ ID NO: 138, 139, 140, 141 and 142).
  • the amino acids are
  • sequence variants can be deduced from this alignment. Such sequence variants preferably have no change in the amino acids marked with a black arrow, being D92, G95 and G131. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background.
  • Figures 23a to Figure 23c represent consecutive parts of an amino acid sequence alignment of polypeptides (SEQ ID NO: 349, 356, 363, 370 and 377, see also Table 8g) which show pamB activity and which are comprised by different
  • sequence variants are described by SEQ ID NO: 349, 356, 363, 370, 377). Further sequence variants can be deduced from the alignment in Figure 23, as well as from alignments of SEQ ID NO: 349 to 354, 356 to 361 , 363 to 368, 370 to 375 and 377 to 382). Such sequence variants preferably have no change in the amino acids marked with a black arrow in Figure 23, being G34, P46, L77, T146, P162, D188, G191 , P230, P226, V240, G369, G, 371 , E396, G471 and R586. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background in Figure 23.
  • Figures 24a to Figure 24b represent consecutive parts of an amino acid sequence alignment of the polypeptide (SEQ ID NO: 384, 391 , 398, 405 and 412, see also Table 1 1g) which show pamO activity and which are comprised by different polypeptide (SEQ ID NO: 384, 391 , 398, 405 and 412, see also Table 1 1g) which show pamO activity and which are comprised by different polypeptide (SEQ ID NO: 384, 391 , 398, 405 and 412, see also Table 1 1g) which show pamO activity and which are comprised by different
  • sequence variants are described by SEQ ID NO: 384, 391 , 398, 405 and 412). Further sequence variants can be deduced from the alignment in Figure 24, as well as from alignments of SEQ ID NOs: 284 to 389, 391 to 396, 398 to 403, 405 to 410 and 412 to 417). Such sequence variants preferably have no change in the amino acids marked with a black arrow in Figure 24, being G22, G28, D72, Y177, K181 , and P207. More preferred, the variants will only have conservative amino acid substitutions in the amino acids depicted as white letters on a black background in Figure 24.
  • Figure 25a depicts results of Example 4.
  • the production of pamamycin 607 of the natural pamamycin producing organism Streptomyces alboniger DSM 40043 and of a recombinant strain of Streptomyces albus J 1074 is confirmed by testing the SGG culture medium
  • the recombinant strain of Streptomyces albus J 1074 comprises the R2 cosmid with the pamamycin cluster depicted in Figure 1 as Cluster 5. It is shown that the recombinant strain of Streptomyces albus J 1074 (“ S. albus + R22" ) comprises less pamamycin in the produced biomass, but more pamamycin in the culture medium than the wildtype strain of Streptomyces albus J 1074.
  • Figure 25b depicts results of Example 5.
  • Three different strains of pamamycin producing microorganisms are analysed for their production of pamamycin 607 in two different media, and separately analysed for pamamycin 607 production in the culture supernatant (" PPM KF” and (“ SGG KF) or in the produced biomass (" PPM Mycel” and " SGG Mycel” ) of the respective media PPM and SGG described in Example 1.
  • the first strain is Streptomyces albus J 1074 comprising the R2 cosmid (“ S. albus + R2" )
  • the second strain is the wildtype of Streptomyces alboniger DSM 40043 (" S.
  • Figure 26 depicts results of Example 8.
  • the first strain is the wildtype of Streptomyces alboniger DSM 40043 (" S. alboniger” )
  • the second strain is a recombinant Streptomyces alboniger DSM 40043 comprising the R2 cosmid (" S. alboniger + R2"
  • the third strain is a recombinant Streptomyces alboniger DSM 40043 (" pTOSR1 P21 " ) overexpressing the pamR1 polypeptide from the
  • polypeptide encoding region (SEQ ID NO: 129) obtained from Streptomyces alboniger DSM 40043 under transcriptional control of the heterologous p21 promoter (SEQ ID NO: 193), while the fourth strain is a recombinant Streptomyces alboniger DSM 40043 overexpressing the pamR1 polypeptide from the polypeptide encoding region (SEQ ID NO: 129) obtained of Streptomyces alboniger DSM 40043 (" pTOSRI BX" ) under
  • pamamycin 593 means a compound of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 as defined in Table 1.
  • pamamycin 607 means a compound of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 as defined in Table 1.
  • pamamycin 621 means compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 621 A, 621 B, 621 C, or 621 D as defined in Table 1.
  • pamamycin 621 refers to a mixture of at least two of 621 A, 621 B, 621 C and 621 D.
  • 621 C" and “ pamamycin 621 D” mean compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 621 A, 621 B, 621 C, or 621 D as defined in Table 1.
  • pamamycin 635" means compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 635A, 635B, 635C, 635D, 635E, or 635F as defined in Table 1.
  • pamamycin 635 refers to a mixture of at least two of 635A, 635B, 635C, 635D, 635E, and 635F.
  • pamamycin 635A means compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 621 A, 621 B, 621C, 621 D, 621 E or 621 F as defined in Table 1.
  • pamamycin 649 means a mixture of compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 649A or 649B as defined in Table 1.
  • pamamycin 649A and pamamycin 649B mean compounds of Formula (I), having the respective residues R1 , R2, R3, R4 and R5 for 649A, or 6249B as defined in Table 1.
  • S-chain means compounds of Formula (II) AND Enantiomer
  • R4 and R5 can be H, CH 3 or CH2CH5 in combinations as shown for R4 and R5 in Table 1.
  • SCoA representing bound Coenzyme A, can also be OH, preferably it is OH.
  • L-chain means a compound of Formula (III),
  • R1 , R2 and R3 can be H, CH3 or CH2CH5 in combinations as shown for R1 , R2 and R3 in Table 1.
  • SCoA, representing bound Coenzyme A can also be OH, preferably it is OH.
  • K-chain means a compound of Formula (IV),
  • R1 is hydrogen, a carboxyl, hydroxyl, ester, amide or hydroxamate group, a halogenide or a salt with anorganic or organic catione, and
  • R2 is hydrogen, a hydroxyl, ester or ether group or a halogenide
  • the term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values-set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower), preferably 15 percent, more preferably 10 percent and most preferably 5 percent.
  • genomic DNA is referring to the heritable genetic information of a host organism.
  • Said genomic DNA comprises the entire genetic material of a cell or an organism, including the DNA of the nucleus (chromosomal DNA), extrachromosomal DNA, and organellar DNA (e.g. of mitochondria).
  • chromosomal DNA chromosomal DNA
  • extrachromosomal DNA e.g. of mitochondria
  • organellar DNA e.g. of mitochondria
  • chromosomal DNA or "chromosomal DNA sequence” is to be understood as the genomic DNA of the cellular nucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes or chromatids, they might be
  • PCR polymerase chain reaction
  • FISH fluorescence in situ hybridization
  • NGS next generation sequencing
  • promoter refers to a polynucleotide which directs the transcription of a structural gene to produce mRNA. Typically, a promoter is located in the 5' region of a gene/operon, proximal to the start codon of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter.
  • enhancer refers to a polynucleotide.
  • An enhancer can increase the efficiency with which a particular gene is transcribed into mRNA irrespective of the distance or orientation of the enhancer relative to the start site of transcription.
  • an enhancer is located close to a promoter, a 5' -untranslated sequence or in an intron.
  • a polynucleotide is "heterologous to" an organism or a second polynucleotide if it originates from a foreign species, or, if from the same species, is modified from its original form.
  • a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e. g. a genetically engineered coding sequence or an allele from a different ecotype or variety).
  • Transgene refers to a polynucleotide manipulated by man or a copy or complement of a polynucleotide manipulated by man.
  • a transgenic expression cassette comprising a promoter operably linked to a second polynucleotide may include a promoter that is heterologous to the second polynucleotide as the result of manipulation by man (e.g., by methods described in Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1 -3, John Wiley & Sons, Inc.
  • a recombinant expression cassette may comprise polynucleotides combined in such a way that the polynucleotides are extremely unlikely to be found in nature.
  • restriction sites or plasmid vector sequences manipulated by man may flank or separate the promoter from the second polynucleotide.
  • polynucleotides can be manipulated in many ways and are not limited to the examples above.
  • the term "recombinant” is used to specify an organism or cell, e.g. a microorganism, it is used to express that the organism or cell comprises at least one "transgene”,
  • transgenic or “recombinant” polynucleotide which is usually specified later on.
  • the term “recombinant” comprises also " man-made mutations” or “ man-made mutants” Both are not isolated from nature, but are the result of artificial mutagenesis and/or selection pressure created by interference from man. Usually such mutations are produced by exposing the respective organisms to mutagenic chemicals or mutagenic irradiation .
  • Man-made mutants are microorganisms comprising man-made mutations.
  • a polynucleotide "exogenous to" an individual organism is a polynucleotide which is introduced into the organism by any means other than by a sexual cross.
  • operable linkage or " operably linked” are generally understood as meaning an arrangement in which a genetic control sequence, e.g. a promoter, enhancer or terminator, is capable of exerting its function with regard to a polynucletide being operably linked to it, for example a polynucleotide encoding a polypeptide.
  • Function in this context, may mean for example control of the expression, i.e. transcription and/or translation, of the nucleic acid sequence.
  • Control in this context, encompasses for example initiating, increasing, governing or suppressing the expression, i.e. transcription and, if appropriate, translation.
  • Controlling in turn, may be, for example, growth stage- and / or cell type- specific. It may also be inducible, for example by certain chemicals, stress, pathogens and the like.
  • operable linkage is understood as meaning for example the sequential arrangement of a promoter, of the nucleic acid sequence to be expressed and, if
  • regulatory elements such as, for example, a terminator, in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is expressed.
  • An operably linkage does not necessarily require a direct linkage in the chemical sense.
  • Genetic control sequences such as, for example, enhancer sequences are also capable of exerting their function on the target sequence from positions located at a distance to the polynucleotide, which is operably linked. Preferred arrangements are those in which the nucleic acid sequence to be expressed is positioned after a sequence acting as promoter so that the two sequences are linked covalently to one another.
  • the distance between the promoter and the amino acid sequence encoding polynucleotide in an expression cassette is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
  • an expression cassette may also be constructed in such a way that the nucleic acid sequence to be expressed is brought under the control of an endogenous genetic control element, for example an endogenous promoter, for example by means of homologous recombination or else by random insertion.
  • Such constructs are likewise understood as being expression cassettes for the purposes of the invention.
  • expression cassette means those construct in which the nucleic acid sequence encoding an amino acid sequence to be expressed is linked operably to at least one genetic control element which enables or regulates its expression (i.e. transcription and / or translation).
  • the expression may be, for example, stable or transient, constitutive or inducible.
  • express refers to expression of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or reaction defined and described in this application) at a level that the resulting enzyme activity of this protein encoded for, or the pathway or reaction that it refers to allows metabolic flux through this pathway or reaction in the organism in which this gene/pathway is expressed.
  • the expression can be done by genetic alteration of the microorganism that is used as a starting organism.
  • a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product at an increased level relative to that produced by the starting microorganism or in a comparable microorganism which has not been altered.
  • Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g. by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins).
  • modifying proteins e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like
  • a microorganism can be physically or environmentally altered to express a gene product at an increased or lower level relative to level of expression of the gene product unaltered microorganism.
  • a microorganism can be treated with, or cultured in the presence of an agent known, or suspected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased.
  • a microorganism can be cultured at a temperature selected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased.
  • a gene that is altered or modified encodes an enzyme in a biosynthetic pathway, or a transport protein, such that the level or activity of the biosynthetic enzyme in the
  • microorganism is altered or modified, or that the transport specificity or efficiency is altered or modified.
  • at least one gene that encodes an enzyme in a biosynthetic pathway i.e. a polypeptide bringing about a specific activity in the biosynthetic pathway, is altered or modified such that the level or activity of the enzyme is enhanced or increased relative to the level in presence of the unaltered or wild type gene.
  • Deregulation also includes altering the coding region of one or more genes to yield, for example, an enzyme that is feedback resistant or has a higher or lower specific activity. Also, deregulation further encompasses genetic alteration of genes encoding transcriptional factors (e.g., activators, repressors) which regulate expression of genes coding for enzymes or transport proteins.
  • transcriptional factors e.g., activators, repressors
  • the terms “ deregulate,” “ deregulated” and “ deregulation” can further be specified in regard to the kind of deregulation present.
  • the term " up-regulated” is used.
  • the term " down -regulated” is used.
  • the recombinant microorganism having an " up-regulated” activity comprises additional expression cassettes for the expression of polypeptides having the respective up- regulated activity and/or may have an modified or exchanged promoter region for the respective endogenous expression cassette, in order to produce more of the polypeptide.
  • the activities may be also up-regulated according to other technologies known to a person skilled in the art, for example but not excluding others, modifiying the mRNA sequence in order to enhance its stability or promote its translation, by using a codon optimized mRNA sequence, or by down-regulating an repressor of the respective activity or expression of the polypeptide providing the respective activity.
  • deregulated includes expression of a gene product at a level lower or higher than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated.
  • the microorganism can be genetically manipulated (e.g., genetically engineered) to express a level of gene product at a lesser or higher level than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated.
  • Genetic manipulation can include, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by removing strong promoters, inducible promoters or multiple promoters), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, decreasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene routine in the art (including but not limited to use of antisense nucleic acid molecules, or other methods to knock-out or block expression of the target protein).
  • modifying proteins e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like
  • deregulated gene activity also means that a gene activity is introduced into a microorganism where the respective gene activity, has not been observed before, e.g. by introducing a recombinant gene, e.g. a heterologous gene, in one or more copies into the microorganism preferably by means of genetic engineering.
  • a recombinant gene e.g. a heterologous gene
  • deregulated pathway or reaction refers to a biosynthetic pathway or reaction in which at least one gene that encodes an enzyme in a biosynthetic pathway or reaction is altered or modified such that the level or activity of at least one biosynthetic enzyme is altered or modified.
  • deregulated pathway includes a biosynthetic pathway in which more than one gene has been altered or modified, thereby altering level and/or activity of the corresponding gene products/enzymes.
  • the ability to " deregulate" a pathway arises from the particular phenomenon of microorganisms in which more than one enzyme (e.g., two or three biosynthetic enzymes) are encoded by genes occurring adjacent to one another on a contiguous piece of genetic material termed a " cluster" or " gene cluster”
  • more than one enzyme e.g., two or three biosynthetic enzymes
  • cluster e.g., two or three biosynthetic enzymes
  • the DNA sequence encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into either microorganism.
  • expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.
  • overexpression refers to expression of a gene product, in particular to enhancing the expression of a gene product at a level greater than that present prior to a genetic alteration of the starting microorganism.
  • a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product at an increased level relative to that produced by the starting microorganism.
  • Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins).
  • Another way to overexpress a gene product is to enhance the stability of the gene product to increase its life time.
  • domain refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
  • motif or " consensus sequence” or “ signature” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain). Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res.
  • GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
  • the BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).
  • Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package
  • BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) when starting from a protein sequence.
  • the BLAST results may optionally be filtered.
  • the full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived.
  • the results of the first and second BLASTS are then compared.
  • a paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is derived, a BLAST back then ideally results in the query sequence amongst the highest hits; an orthologue is identified if a high-ranking hit in the first BLAST is not from the same species as from which the query sequence is derived, and preferably results upon BLAST back in the query sequence being among the highest hits.
  • High-ranking hits are those having a low E-value.
  • Computation of the E-value is well known in the art.
  • comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.
  • sequence identity between two nucleic acid sequences is understood as meaning the percent identity of the nucleic acid sequence over in each case the entire sequence length which is calculated by alignment with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:
  • Gap Weight 12 Length Weight: 4
  • sequence identity between two amino acid sequences is understood as meaning the percent identity of the nucleic acid sequence over in each case the entire sequence length which is calculated by alignment with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:
  • Gap Weight 8 Length Weight: 2
  • hybridisation is a process wherein substantially homologous complementary nucleotide sequences anneal to each other.
  • the hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution.
  • the hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin.
  • the hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips).
  • the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
  • stringency refers to the conditions under which a hybridisation takes place.
  • the stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20°C below T m , and high stringency conditions are when the temperature is 10°C below T m . High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.
  • the Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe.
  • the T m is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures.
  • the maximum rate of hybridisation is obtained from about 16°C up to 32°C below T m .
  • the presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored).
  • Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisation will be lowered.
  • Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes.
  • the Tm decreases about 1 °C per % base mismatch.
  • the T m may be calculated using the following equations, depending on the types of hybrids:
  • T m 81.5°C + 16.6xlogi 0 [Na + ] a + 0.41x%[G/C b ] - 500x[L c ]- 1 - 0.61x% formamide
  • T m 79.8°C+ 18.5 (logi 0 [Na + ] a ) + 0.58 (%G/C b ) + 1 1.8 (%G/C b ) 2 - 820/L c
  • c L length of duplex in base pairs.
  • Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase.
  • a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68°C to 42°C) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%).
  • annealing temperature for example from 68°C to 42°C
  • formamide concentration for example from 50% to 0%
  • hybridisation typically also depends on the function of post-hybridisation washes.
  • samples are washed with dilute salt solutions.
  • Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency.
  • a positive hybridisation gives a signal that is at least twice of that of the background.
  • suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions. For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65°C in 1x SSC or at 42°C in 1x SSC and 50% formamide, followed by washing at 65°C in 0.3x SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass
  • hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% formamide, followed by washing at 50°C in 2x SSC.
  • the length of the hybrid is the anticipated length for the hybridising nucleic acid.
  • the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein.
  • 1 xSSC is 0.15M NaCI and 15mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5x Denhardt's reagent, 0.5-1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
  • Homologues of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • a deletion refers to removal of one or more amino acids from a protein.
  • An insertion refers to one or more amino acid residues being introduced into a
  • Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues.
  • N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S- transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag* 100 epitope, c-myc epitope, FLAG ® -epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
  • a transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • glutathione S- transferase-tag glutathione S- transferase-tag
  • protein A maltose-binding protein
  • dihydrofolate reductase dihydrofolate reductase
  • Tag* 100 epitop
  • a substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a -helical structures or ⁇ -sheet structures).
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues.
  • the amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below).
  • an " endogenous" gene not only refers to the gene in question as found in an organism in its natural form (i.e., without there being any human intervention), but also refers to that same gene (or a substantially homologous nucleic acid/gene) in an isolated form subsequently (re)introduced into a microorganism (a transgene).
  • a transgenic microorganism containing such a transgene may encounter a substantial reduction of the transgene expression and/or substantial reduction of expression of the endogenous gene.
  • the isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis.
  • orthologues and " paralogues” encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.
  • splice variant encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the biological activity of the protein is substantially retained; this may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex (2005) BMC Bioinformatics 6: 25).
  • vector preferably, encompasses phage, plasmid, fosmid, cosmid, viral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below.
  • the vector encompassing the polynucleotide of the present invention preferably, further comprises selectable markers for propagation and/or selection in a recombinant microorganism. The vector may be incorporated into a recombinant microorganism by various techniques well known in the art.
  • the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous recombination or
  • Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” , conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a recombinant microorganism, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofection, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment.
  • foreign nucleic acid for example DNA
  • a recombinant microorganism including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofection, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment.
  • the present invention provides nucleic acid sequences which
  • pamamycin synthesis encompasses all steps of the biosynthesis of pamamycin.
  • a polypeptide which is involved in the synthesis of pamamycin may either convert a substrate into pamamycin or may produce any of the precursors which occur in pamamycin biosynthesis.
  • the polypeptide encoded by the polynucleotide of the present invention shall be capable of increasing the amount of pamamycin or a precursor thereof upon expression in an organism, preferably a recombinant microorganism as specified elsewhere herein. Precursors of pamamycin are depicted in Figure 2.
  • Such an increase is, preferably, statistically significant when compared to a control organism which lacks expression of the polynucleotide of the present invention.
  • the control organism is of the same species and even more preferred belongs to the same strain that was used to construct the recombinant microorganism. Whether an increase is significant can be determined by statistical tests well known in the art including, e.g., Student ' s t-test. More preferably, the increase is an increase of the amount of pamamycin of at least 5%, at least 10%, at least 15%, at least 20% or at least 30% compared to said control.
  • Suitable assays to identify and measure the amount of pamamycin are known by the person skilled in the art and are described for example in Hashimoto et al (2004) Biosynthetic Origin of the Carbon Skeleton and Nitrogen Atom of Pamamycin-607, a Nitrogen-Containing Polyketide, Biosci. Biotechnol. Biochem. 69; Hashimoto et al. (201 1 ) Effect of Pamamycin-607 on Secondary Metabolite Production by Streptomyces spp., Biosci. Biotechnol. Biochem. 75; Hashimoto et al. (2003) Relationship between Response to and Production of the Aerial Mycelium- inducing Substances Pamamycin-607 and A-factor, Biosci. Biotechnol. Biochem. 67;
  • One nucleic acid sequence provided by the invention is a gene cluster described by SEQ ID NO: 1 , which comprises expression cassette for polypeptides being involved in pamamycin synthesis.
  • a schematic representation of the gene cluster is provided by Figure 1 .
  • Table 3 Listing of genes and encoded polypeptides of the polnucleotidesequence described by SEQ ID NO: 2:
  • Table 3 provides a listing of the polypeptide (protein) encoding sequences of SEQ ID NO: 2, the respective ORF names, the number of the nucleotides in SEQ ID NO: 2, which are starting and endpoints of the polypeptide encoding sequences, the likely function of the encoded polypeptides and the respective SEQ ID NOs: of the polynucleotide and amino acid sequences in the sequence listings.
  • the starting points and end points given in Table 3 do not necessarily represent the 5-prime and 3-prime ends of the polypeptide encoding regions.
  • the orientation of the transcribed region is depicted in the sketch of Cluster 5 in Figure 1.
  • the provided recombinant polynucleotides can either be isolated from their natural genomic environment and modified after their isolation or produced artificially from pure sequence information.
  • a natural source of polynucleotides of the invention are pamamycin producing organisms and related species.
  • One way to isolate polynucleotides from pamamycin producing organisms is to perform a PCR reaction on genomic DNA of such organisms using primers comprising at least 15, preferably at least 20, consecutive polynucleotides of the 5-prime and 3-prime ends of the polypeptide encoding sequences disclosed in SEQ ID NO: 1 and/or 2.
  • Pamamycin producing organisms can, for example but not excluding others, be found in the group consisting of the genus Streptomyces preferably of the species Streptomyces alboniger, Streptomyces aurantiacus, or Streptomyces kitasatoensis, in particular in the strains Streptomyces alboniger ATCC 12461 Streptomyces alboniger DSM 40043, Streptomyces aurantiacus ATCC 19822 and Streptomyces kitasatoensis JCM5001 ,.
  • Pamamycin producing organisms can also be used to isolate or identify variants of SEQ ID NO: 1 and/or 2, by using PCR and/or other hybridisation techniques and/or by using bioinformatic searches of available genomic sequence information.
  • Variants of SEQ ID NO: 1 and/or 2 have preferably polypeptide encoding regions for most, preferably all of the polypeptides of the group of polypeptides having pamC, pamG, pamF, pamA, pamB, pamD, pamE, pamO, pamK, pamJ, pamM, pamN, pamL, pamX, pamY and/or pamS activity.
  • Streptomyces alboniger ATCC 12461 also deposited as DSM 40043 and IFO 12738 and isolated in 1952 from forest soil in Wisconsin (USA)
  • Streptomyces aurantiacus ATCC 19822 also deposited as DSM 40412
  • Streptomyces aurantiacus I MET 43917 Streptomyces aurantiacus J A 4570 and
  • Streptomyces kitasatoensis JCM5001 These strains are deposited under the respective deposit numbers and disclosed in DE4134168, DE4316836, US4283391 , JP62135476A, in Hashimoto et al. in Biosci. Biotechnol. Biochm. 2003, Vol 67(4), pages 803 to 808 and/or HAERTL et al. THE JOURNAL OF ANTIBIOTICS (1998) VOL. 51 , NO. 1 1 , pp. 1040-1046.
  • polynucleotides and polypeptides provided by the present invention are suitbable to identify and construct recombinant microorganisms having the ability to produce pamamycin or to produce recombinant versions of pamamycin producing microorganisms which have an enhanced or modified production of pamamycin.
  • microorganisms comprising:
  • Microorganisms comprising at least one expression cassette for the polypeptide activities a) to p) can be identified by testing microorganisms for the production of pamamycin and/or for the presence of polypeptide encoding regions for at least 3 activities selected from pamC activity, pamG activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity and pamS activity.
  • Microorganism which are selected to be tested for the production of pamamycin and/or to be used as sources to obtain sequence variants of SEQ ID NO: 1 or 2 or at least one of the polpypetide encoding regions, promoters or terminators comprised therein, have in order of preference
  • Microorganisms can be tested for the presence of expression cassettes for each one of the polypeptide activities of a) to p), by several ways known to the person skilled in the art. For example, but not excluding others, by testing the genome of a microorganism for
  • Another way to test for expression cassettes for each one of the polypeptide activities of a) to p) is to perform sequence comparisons of sequenced DNA of the microorganism, in order to identify DNA sequences having at least 70%, 80% or 90% sequence identity to at least one of the polypeptide encoding regions for pamC activity, pam G activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity and pamS activity comprised by SEQ ID NO: 1.
  • a further way to test for expression cassettes for each one of the polypeptide activities of a) to p) in a given microorganisms is to perform sequence comparisons of sequenced RNA of the microorganism in question, in order to identify microorganisms comprising mRNAs being at least 70%, 80% or 90% identical to at least one of the polypeptide encoding regions for pamC activity, pamG activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity and pamS activity comprised by SEQ ID NO: 1.
  • the recombinant microorgaisms of the invention are preferably microorganisms which have been intentionally modified via molecular biology techniques, like transformation or gene knock-out or gene replacement.
  • this term recombinant microorganism does also apply to non-naturally occurring microorganisms.
  • Non-naturally occurring microorganisms are microorganisms which have been created by single or repeated mutagenesis and selection performed on microorganism isolated from nature.
  • Non-naturally occurring microorganisms comprise at least one up-regulated activity of a polypeptide having pamC activity, pamG activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity, pamS activity, pamR1 activity, pamW activity, and/or at least one down-regulated pamR2 activity or pamH activity, in comparison to the unmodified version of such microorganism.
  • Recombinant microorganisms are preferably produced via transformation of a polypeptide having pamC activity, pamG activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity, pamS activity
  • microorganism with polynucletide constructs comprising at least one expression cassette encoding a polypeptide having pamC activity, pamG activity, pamF activity, pamA activity, pamB activity, pamD activity, pamE activity, pamO activity, pamK activity, pamJ activity, pamM activity, pamN activity, pamL activity, pamX activity, pamY activity, pamS activity, pamR1 activity, pamW activity, pamR2 activity or pamD activity.
  • Microorganisms can also be transformed with polynucleotide constructs which are able to modify expression of at least one expression cassette encoding such an activity and which is already present in such microorganism.
  • Such polynucleotide construes may replace the endogenous promoter with a heterologous promoter sequence providing for constitutive or inducible expression in such microorganism, or which provide for a stronger or weaker expression in such microorganism in comparison to the endogenous promoter.
  • the expression of at least one expression cassette can also be modified by providing the microorganism with an expression cassette for a transcriptional regulator which enhances or decreases expression from such expression cassette, or to provide the organism with an expression cassette encoding a polypeptide having one of the above described activities, but providing such activity with a higher activity per expressed polypeptide molecule or having a higher stability of such polypeptide molecules in order to prolong enzymatic activity per expressed polypeptide.
  • Polypeptides having pamC activity are polypeptides having pamC activity:
  • a polypeptide having pamC activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 4, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 3, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 4. Preferably it is the same promoter.
  • polypeptides having pamC activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 3.
  • polynucleotides encoding polypeptides having pamC activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 3 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 3, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 3.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 4 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 4.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 4 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 5, 6, 7, 8 and 9. Further variants can be deduced by the information provided by Figure 3 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 4 shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 4, 5, 6, 7, 8 and 9.
  • the parameters used for the pairwise sequence alignments are:
  • polypeptides having pamC activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamC activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 209, and/or are expressed from a polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 208, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 208 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 208, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 208.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 209 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 209.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 209 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 210, 21 1 , 212, 213 and 214.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 209, 210, 21 1 , 212, 213 and 214 to the information provided by Figure 3 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 4a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs 4, 209, 210, 21 1 , 212, 213 and 214.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamG activity are polypeptides having pamG activity:
  • a polypeptide having pamG activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 1 1 , or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 10, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complemented strain for the capability to produce pamamycin 607.
  • a successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ I D NO: 1 1. Preferably it is the same promoter.
  • polypeptides having pamC activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ I D NO: 1 1 , and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamG activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 10 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 10, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 10.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 1 1 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,
  • SEQ ID NO: 1 1 98%, 99%, or 100% identical SEQ ID NO: 1 1.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 1 1 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples are polypeptides of SEQ ID NO: 12, 13, 14, 15 and 16. Further variants can be deduced by the information provided by Figure 4 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 5 shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 1 1 , 12, 13, 14, 15 and 16. The parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamG activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamG activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 216, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 215, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 215 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 215, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 215.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 209 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 216.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 216 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples are polypeptides of SEQ ID NO: 217, 218, 219, 220 and 221.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 216, 217, 218, 219, 220 and 221 to the information provided by Figure 4 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 5a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 1 1 , 216, 217, 218, 219, 220 and 221 .
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • NO: 1 1 NO: 216 NO: 217 NO: 218 NO: 219 NO: 220 NO: 221
  • SEQ ID NO: 1 100% 90% 86% 85% 83% 81 % 81 %
  • Polypeptides having pamF activity are polypeptides having pamF activity:
  • a polypeptide having pamF activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 18, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 17, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 18. Preferably it is the same promoter.
  • polypeptides having pamC activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamF activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 17 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 17, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 17.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 18 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 18.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ I D NO: 18 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples of such sequence variants, but not excluding others, are polypeptides of SEQ ID NO: 19, 20, 21 , 22 and 23. Further variants can be deduced by the information provided by Figure 5 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 6 shows the % sequence identity of pairwise sequence alignments of SEQ I D NO: 18, 19, 20, 21 , 22 and 23.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamF activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamF activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 223, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 222, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 222 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 222, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 222.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 223 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 223.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 223 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 224, 225, 226, 227 and 228. Further variants can be deduced aligning the sequences of SEQ ID NO: 223, 224, 225, 226, 227 and 228 to the information provided by Figure 5 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 6a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 18, 223, 224, 225, 226, 227 and 228.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • a polypeptide having pamA activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 25, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 24, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complemented strain for the capability to produce pamamycin 607.
  • a successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 25. Preferably it is the same promoter.
  • polypeptides having pamA activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamA activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 24 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 24, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 24.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 25 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 25.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 25 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples are polypeptides of SEQ ID NO: 26, 27, 28, 29 and 30. Further variants can be deduced by the information provided by Figure 6 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 7 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 25, 26, 27, 28, 29 and 30. The parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamA activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamA activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 230, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 229, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 229 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 229, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 229.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 230 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 230.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 230 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 231 , 232, 233, 234 and 235.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 230, 231 , 232, 233, 234 and 235 to the information provided by Figure 6 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 7a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 25, 230, 231 , 232, 233, 234 and 235.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamB activity are polypeptides having pamB activity:
  • a polypeptide having pamB activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 32, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 31 , in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 32. Preferably it is the same promoter.
  • polypeptides having pamB activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31 , or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamB activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 31 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 31 , using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 31.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 32 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 32.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 32 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants are polypeptides of SEQ ID NO: 33, 34, 35, 36 and 37. Further variants can be deduced by the information provided by Figure 7 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 8 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 32, 33, 34, 35, 36 and 37.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamB activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamB activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 237, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 236, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 236.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 236 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 236, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 236.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 237 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 237.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 237 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ I D NO: 238, 239, 240, 241 and 242.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 237, 238, 239, 240, 241 and 242 to the information provided by Figure 7 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 8a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 32, 237, 238, 239, 240, 241 and 242.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamB activity which are derived from other metabolic pathways
  • polypeptides having pamB activity are polypeptides being obtainable from Streptomyces fulvissimus and their variants.
  • polypeptides having pamB activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 349, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 348, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 348.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 348 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 348, using genomic DNA or cDNA of Streptomyces fulvissimus as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 348.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 349 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 349.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 349 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 350, 351 , 352, 353 and 354. Further variants can be deduced aligning the sequences of SEQ ID NO: 349, 350, 351 , 352, 353 and 354 to the information provided by Figure 23 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 8b shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 349, 350, 351 , 352, 353 and 354.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamB activity are polypeptides being obtainable from Streptomyces griseus JA 4570 and their variants.
  • polypeptides having pamB activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 356, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 355, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 355.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 355 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 355, using genomic DNA or cDNA of Streptomyces griseus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 355.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 356 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 356.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 356 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples are polypeptides of SEQ ID NO: 357, 358, 359, 360 and 361. Further variants can be deduced aligning the Sequences of SEQ ID NO: 356, 357, 358, 359, 360 and 361 to the information provided by Figure 23 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 8c shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 356, 357, 358, 359, 360 and 361 .
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamB activity are polypeptides being obtainable from Streptomyces sp. SolWspMP and their variants.
  • polypeptides having pamB activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 363, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 362, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 362.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 362 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 362, using genomic DNA or cDNA of Streptomyces sp. SolWspMP as PCR template. Preferred primers are capable to produce a full length fragment of SEQ ID NO: 362.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 363 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 363.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 363 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 364, 365, 366, 367 and 368. Further variants can be deduced aligning the sequences of SEQ ID NO: 363, 364, 365, 366, 367 and 368 to the information provided by Figure 23 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 8d shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 363, 364, 365, 366, 367 and 368.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamB activity are polypeptides being obtainable from Streptomyces sp. W007 and their variants.
  • polypeptides having pamB activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 370, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 369, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 369.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 369 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 369, using genomic DNA or cDNA of Streptomyces sp. W007 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 369.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 370 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 370.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 370 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 371 , 372, 373, 374 and 375.
  • Further variants can be deduced aligning the Sequences of SEQ ID NO: 370, 371 , 372, 373, 374 and 375 to the information provided by Figure 23 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 8e shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 370, 371 , 372, 373, 374 and 375.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamB activity are polypeptides being obtainable from Streptomyces californicus and their variants.
  • polypeptides having pamB activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 377, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 376, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 376 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 376, using genomic DNA or cDNA of Streptomyces californicus as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 376.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 377 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 377.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 377 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 378, 379, 380, 381 and 382.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 377, 378, 379, 380, 381 and 382 to the information provided by Figure 23 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 8f shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 377, 378, 379, 380, 381 and 382.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Table 8g shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs 32, 237, 349, 356, 363, 370 and 377.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamD activity are polypeptides having pamD activity:
  • a polypeptide having pamD activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 39, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 38, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 39. Preferably it is the same promoter.
  • polypeptides having pamD activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamD activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 38 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 38, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 38.
  • Further variants can be deduced by the information provided by Figure 8 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 39 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 39.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 39 with other amino acids, preferably using conservative amino acid substitutions.
  • polypeptides of SEQ ID NO: 40, 41 , 42, 43 and 44 are polypeptides of SEQ ID NO: 40, 41 , 42, 43 and 44.
  • Table 9 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 39, 40, 41 , 42, 43 and 44.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamD activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamD activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 244, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 243, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 243 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 243, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 243.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 244 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 244.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 244 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ I D NO: 245, 246, 247, 248 and 249. Further variants can be deduced aligning the sequences of SEQ ID NO: 244, 245, 246, 247, 248 and 249 to the information provided by Figure 8 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 9a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 39, 244, 245, 246, 247, 248 and 249.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • a polypeptide having pamE activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 46, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 45, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 46. Preferably it is the same promoter.
  • polypeptides having pamE activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamE activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 45 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 45, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 45.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 46 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 46.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 46 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples of such sequence variants, but not excluding others, are polypeptides of SEQ ID NO: 47, 48, 49, 50 and 51. Further variants can be deduced by the information provided by Figure 9 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 10 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 46, 47, 48, 49, 50 and 51.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamE activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • the invention provides for polypeptides having pamE activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 251 , and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 250, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 250 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 250, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 250.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 251 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 251.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 251 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 252, 253, 254, 255 and 256. Further variants can be deduced aligning the sequences of SEQ ID NO: 251 , 252, 253, 254, 255 and 256 to the information provided by Figure 9 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 10a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 46, 251 , 252, 253, 254, 255 and 256.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • SEQ ID NO: 46 100% 94% 89% 89% 88% 84% 84% SEQ ID NO: 251 100% 95% 95% 93% 90% 90% SEQ ID NO: 252 100% 90% 90% 88% 85% SEQ ID NO: 253 100% 90% 90% 90% SEQ ID NO: 254 100% 85% 84%
  • Polypeptides having pamO activity are polypeptides having pamO activity:
  • a polypeptide having pamO activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 53, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 52, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 53. Preferably it is the same promoter.
  • polypeptides having pamO activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamO activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 52 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 52, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 52.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 53 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 53.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 53 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 54, 55, 56, 57 and 58. Further variants can be deduced by the information provided by Figure 10 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 1 1 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 53, 54, 55, 56, 57 and 58.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamO activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamO activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 258, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 257, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 257 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 257, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 257.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 258 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 258.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 258 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 259, 260, 261 , 262 and 263. Further variants can be deduced aligning the sequences of SEQ ID NO: 258, 259, 260, 261 , 262 and 263 to the information provided by Figure 10 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 1 1a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs 53, 258, 259, 260, 261 , 262 and 263.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamO activity which are derived from other metabolic pathways
  • polypeptides having pamO activity are polypeptides being obtainable from Streptomyces fulvissimus and their variants.
  • polypeptides having pamOB activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 384, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 383, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 383 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 383, using genomic DNA or cDNA of Streptomyces fulvissimus as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 383.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 384 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 384.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 384 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 385, 386, 387, 388 and 389.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 384, 385, 386, 387, 388 and 389 to the information provided by Figure 24 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 1 1 b shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 384, 385, 386, 387, 388 and 389.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamO activity are polypeptides being obtainable from Streptomyces griseus and their variants.
  • polypeptides having pamO activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 391 , and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 390, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 390 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 390, using genomic DNA or cDNA of Streptomyces griseus as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 390.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 391 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 391.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 391 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 392, 393, 394, 395 and 396. Further variants can be deduced aligning the sequences of SEQ ID NO: 391 , 392, 393, 394, 395 and 396 to the information provided by Figure 24 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 1 1c shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 391 , 392, 393, 394, 395 and 396.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamO activity are polypeptides being obtainable from Streptomyces sp. SolWspMP and their variants.
  • polypeptides having pamO activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 398, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 397, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 397 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 397, using genomic DNA or cDNA of Streptomyces sp. SolWspMP as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 397.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 398 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 398.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 398 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 399, 400, 401 , 402 and 403. Further variants can be deduced aligning the sequences of SEQ ID NO: 398, 399, 400, 401 , 402 and 403 to the information provided by Figure 24 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 1 1 d shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 398, 399, 400, 401 , 402 and 403.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamO activity are polypeptides being obtainable from Streptomyces sp. W007 and their variants.
  • polypeptides having pamO activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 405, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 404, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 404 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 404, using genomic DNA or cDNA of Streptomyces sp. W007 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 404.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 405 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 405.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 405 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 406, 407, 408, 409 and 410. Further variants can be deduced aligning the sequences of SEQ ID NO: 405, 406, 407, 408, 409 and 410 to the information provided by Figure 24 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 1 1e shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 405, 406, 407, 408, 409 and 410.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamO activity are chimeric polypeptides and polynucleotides comprising polypeptide and polynucleotide sequences obtainable from Streptomyces californicus. Also comprised are polypeptides having pamO activity and the respective encoding polynucleotides being obtainable from Streptomyces californicus.
  • polypeptides having pamO activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 412, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41 1 , or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 41 1 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 41 1 , using genomic DNA or cDNA of Streptomyces californicus as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 41 1.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 412 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 412.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 412 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 413, 414, 415, 416 and 417.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 412, 413, 414, 415, 416 and 417 to the information provided by Figure 24 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 1 1f shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 412, 413, 414, 415, 416 and 417.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Table 1 1g shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs 53, 258, 384, 391 , 398, 405 and 412.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamK activity are polypeptides having pamK activity:
  • a polypeptide having pamK activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 60, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 59, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complemented strain for the capability to produce pamamycin 607.
  • a successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 60. Preferably it is the same promoter.
  • polypeptides having pamK activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 59, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamK activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 59 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 59, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 59.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 60 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 60.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 60 with other amino acids, preferably using conservative amino acid substitutions. Examples of such sequence variants, but not excluding others, are polypeptides of SEQ ID NO: 61 , 62, 63, 64 and 65. Further variants can be deduced by the information provided by Figure 1 1 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 12 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 60, 61 , 62, 63, 64 and 65.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamK activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamK activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 265, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 264, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 264 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 264, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 264.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 265 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 265.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 265 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 266, 267, 268, 269 and 270. Further variants can be deduced aligning the sequences of SEQ ID NO: 265, 266, 267, 268, 269 and 270 to the information provided by Figure 1 1 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 12a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 60, 265, 266, 267, 268, 269 and 270.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • SEQ ID NO: 60 100% 95% 90% 90% 88% 85% 85% SEQ ID NO: 265 100% 95% 95% 93% 90% 90% SEQ ID NO: 266 100% 90% 89% 88% 86% SEQ ID NO: 267 100% 91 % 88% 89% SEQ ID NO: 268 100% 87% 85% SEQ ID NO: 269 100% 84% SEQ ID NO: 270 100%
  • Polypeptides having pamJ activity are polypeptides having pamJ activity:
  • a polypeptide having pamJ activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 67, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 66, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 67. Preferably it is the same promoter.
  • polypeptides having pamJ activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 67, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 66, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • polynucleotides encoding polypeptides having pamJ activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 66 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 66, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 66.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 67 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 67.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 67 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 68, 69, 70, 71 and 72. Further variants can be deduced by the information provided by Figure 12 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 13 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 67, 68, 69, 70, 71 and 72.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamJ activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • the invention provides for polypeptides having pamJ activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 272, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 271 , or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 271 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 271 , using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 271.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 272 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 272.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 272 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 273, 274, 275, 276 and 277. Further variants can be deduced aligning the sequences of SEQ ID NO: 272, 273, 274, 275, 276 and 277 to the information provided by Figure 12 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 13a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 67, 272, 273, 274, 275, 276 and 277.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamM activity are polypeptides having pamM activity:
  • a polypeptide having pamM activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 74, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 73, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 74. Preferably it is the same promoter.
  • polypeptides having pamM activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamM activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 73 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 73, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ I D NO: 73.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 74 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 74.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 74 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 75, 76, 77, 78 and 79. Further variants can be deduced by the information provided by Figure 13 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions. Table 14 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 74, 75, 76, 77, 78 and 79.
  • polypeptides having pamM activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • the invention provides for polypeptides having pamM activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 279, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 278, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 278 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 278, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 278.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 279 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ I D NO: 279.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 279 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 280, 281 , 282, 283 and 284.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 279, 280, 281 , 282, 283 and 284 to the information provided by Figure 13 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 14a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 74, 279, 280, 281 , 282, 283 and 284.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamN activity are polypeptides having pamN activity:
  • a polypeptide having pamN activity is a polypeptide being able to complement the activity of a polypeptide of SEQ I D NO: 81 , or a polypeptide being expressed from a polynucleotide sequence of SEQ I D NO: 80, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 81 . Preferably it is the same promoter.
  • polypeptides having pamN activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ I D NO: 81 , and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 80, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions, to a complement of SEQ ID NO: 80.
  • polynucleotides encoding polypeptides having pamN activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 80 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 80, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 80.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 81 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 81.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 81 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples are polypeptides of SEQ ID NO: 82, 83, 84, 85 and 86. Further variants can be deduced by the information provided by Figure 14 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 15 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 81 , 82, 83, 84, 85 and 86. The parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamN activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • the invention provides for polypeptides having pamN activity, which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 286, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 285, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 285 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 285, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 285.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 286 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 286.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 286 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples are polypeptides of SEQ ID NO: 287, 288, 289, 290 and 291 .
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 286, 287, 288, 289, 290 and 291 to the information provided by Figure 14 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 15a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 81 , 286, 287, 288, 289, 290 and 291 .
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamL activity are polypeptides having pamL activity:
  • a polypeptide having pamL activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 88, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 87, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 88. Preferably it is the same promoter.
  • polypeptides having pamL activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 88, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 87, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamL activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 87 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 87, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 87.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 88 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 88.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ I D NO: 88 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 89, 90, 91 , 92 and 93. Further variants can be deduced by the information provided by Figure 15 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 16 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 88, 89, 90, 91 , 92 and 93.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamL activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamL activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 293, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 292, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 292 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 292, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 292.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 293 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 293.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 293 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 294, 295, 296, 297 and 298. Further variants can be deduced aligning the sequences of SEQ ID NO: 293, 294, 295, 296, 297 and 298 to the information provided by Figure 15 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 15a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 88, 293, 294, 295, 296, 297 and 298.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • a polypeptide having pamX activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 95, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 94, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ I D NO: 95. Preferably it is the same promoter.
  • polypeptides having pamX activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ I D NO: 95, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 94, or are expressed from a polynucleotide which hybridises under medium stringency
  • hybridisation conditions preferably high stringency hybridisation conditions
  • polynucleotides encoding polypeptides having pamX activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 94 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 94, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 94.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 95 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 95.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 95 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 96, 97, 98, 99 and 100. Further variants can be deduced by the information provided by Figure 16 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 17 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 95, 96, 97, 98, 99 and 100.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite, #GAPMETHOD: NOGAPS, #GAPOPEN:10, GAPEXTEND: 0,5, MATRIX: EBLOSUM62
  • polypeptides having pamX activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamX activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 300, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 299, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 299 per primer, wherein the primers are capable to produce a PCR fragment of SEQ I D NO: 299, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 299.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 300 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 300.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 300 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 301 , 302, 303, 304 and 305.
  • Further variants can be deduced aligning the sequences of SEQ ID NO: 300, 301 , 302, 303, 304 and 305 to the information provided by Figure 16 and its description provided above.
  • Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 17a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 95, 300, 301 , 302, 303, 304 and 305.
  • the parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • a polypeptide having pamY activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 102, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 101 , in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complemented strain for the capability to produce pamamycin 607.
  • a successful complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 102. Preferably it is the same promoter.
  • polypeptides having pamY activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 102, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 101 , or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • polynucleotides encoding polypeptides having pamY activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 101 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 101 , using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 101.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 102 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 102.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ I D NO: 102 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples are polypeptides of SEQ ID NO: 103, 104, 105, 106 and 107. Further variants can be deduced by the information provided by Figure 17 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 18 shows the % sequence identity of pairwise sequence alignments of SEQ ID NO: 102, 103, 104, 105, 106 and 107. The parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • polypeptides having pamY activity are polypeptides being obtainable from Streptomyces aurantiacus JA 4570 and their variants.
  • polypeptides having pamY activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 307, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 306, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 306 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 306, using genomic DNA or cDNA of Streptomyces aurantiacus JA 4570 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 306.
  • the invention does also include sequence variants of polypeptides of SEQ ID NO: 307 which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical SEQ ID NO: 307.
  • sequence variants can be obtained by substitution of one or more amino acids of SEQ ID NO: 307 with other amino acids, preferably using conservative amino acid substitutions.
  • sequence variants examples include polypeptides of SEQ ID NO: 308, 309, 310, 31 1 and 312. Further variants can be deduced aligning the sequences of SEQ ID NO: 307, 308, 309, 310, 31 1 and 312 to the information provided by Figure 17 and its description provided above. Preferred variants comprise not more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions or deletions.
  • Table 18a shows the % sequence identity of pairwise sequence alignments of SEQ ID NOs: 102, 307, 308, 309, 310, 31 1 and 312. The parameters used for the pairwise sequence alignments are: Needleman-Wunsch algorithm of the EMBOSS Software Suite,
  • Polypeptides having pamS activity are polypeptides having pamS activity:
  • a polypeptide having pamS activity is a polypeptide being able to complement the activity of a polypeptide of SEQ ID NO: 109, or a polypeptide being expressed from a polynucleotide sequence of SEQ ID NO: 108, in a Streptomyces alboniger strain available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the DMS Nr. 40043. Complementation of the activity of such polypeptide can be tested by deleting the polynucleotide sequence encoding such polypeptide from the genome of Streptomyces alboniger DMS Nr. 40043 and transforming the resulting strain with an expression cassette capapble to express the polypeptide to be tested in such strain and testing the
  • complementation is achieved, if the complemented strain produces pamamycin 607 on a higher level than the strain comprising the deletion under growth conditions under which the unmodified Streptomyces alboniger DMS Nr. 40043 produces pamamycin 607, wherein the complemented strain uses a promoter to express the polypeptide to be tested, which has a similar or higher expression level under such growth conditions than the promoter of Streptomyces alboniger DMS Nr. 40043 driving expression of the polypeptide of SEQ ID NO: 109. Preferably it is the same promoter.
  • polypeptides having pamS activity which are at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 109, and/or
  • polynucleotide which is at least 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 108, or are expressed from a polynucleotide which hybridises under medium stringency hybridisation conditions, preferably high stringency hybridisation conditions, to a
  • polynucleotides encoding polypeptides having pamS activity can be obtained from an organism producing at least one of the pamamycins of Formula (I), preferably producing pamamycin 607.
  • polynucleotides are obtained from organisms of the genus Streptomyces or obtained via sequence database searches comprising sequence information of organisms of the genus Streptomyces.
  • a preferred method to obtain such polynucleotides is to perform PCR on genomic DNA or cDNA of pamamycin producing organisms using primer pairs of at least 15 consecutive polynucleotides of SEQ ID NO: 108 per primer, wherein the primers are capable to produce a PCR fragment of SEQ ID NO: 108, using genomic DNA or cDNA of Streptomyces alboniger DMS Nr. 40043 as PCR template.
  • Preferred primers are capable to produce a full length fragment of SEQ ID NO: 108.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des polynucléotides et des polypeptides impliqués dans la biosynthèse de la pamamycine et de précurseurs de la pamamycine ainsi que des vecteurs et micro-organismes recombinants comprenant lesdits polynucléotides et polypeptides. L'invention concerne également des procédés de production de la pamamycines, en particulier la pamamycine 607 et la pamamycine 621 et leurs précurseurs.
PCT/IB2014/066284 2013-12-20 2014-11-24 Groupe de gènes pour la biosynthèse de la pamamycine Ceased WO2015092575A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP13198688.7 2013-12-20
EP13198688 2013-12-20
EP14169265.7 2014-05-21
EP14169265 2014-05-21
EP14183171.9 2014-09-02
EP14183171 2014-09-02

Publications (1)

Publication Number Publication Date
WO2015092575A1 true WO2015092575A1 (fr) 2015-06-25

Family

ID=53402192

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2014/066284 Ceased WO2015092575A1 (fr) 2013-12-20 2014-11-24 Groupe de gènes pour la biosynthèse de la pamamycine

Country Status (1)

Country Link
WO (1) WO2015092575A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017037629A1 (fr) * 2015-09-04 2017-03-09 Basf Se Micro-organismes améliorés pour la biosynthèse de pamamycines
US10428361B2 (en) 2015-03-26 2019-10-01 Basf Se Biocatalytic production of l-fucose
US10440953B2 (en) 2015-08-07 2019-10-15 Basf Se Control of pests in maize by ginkgolides and bilobalide

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4283391A (en) * 1978-11-30 1981-08-11 St. Louis, University Pamamycin

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4283391A (en) * 1978-11-30 1981-08-11 St. Louis, University Pamamycin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 12 July 2014 (2014-07-12), "acyl carrier protein [Streptomyces sp. NRRL S-920]", accession no. P_030785007.1 *
DATABASE GENBANK 26 June 2013 (2013-06-26), "acyl carrier protein [Streptomyces aurantiacus]", accession no. P_016640658.1 *
MAKOTO HASHIMOTO ET AL.: "The Journal of Antibiotics", NITROGEN INCORPORATION IN THE BIOSYNTHETIC PATHWAY OF THE NITROGEN-CONTAINING POLYKETIDE, PAMAMYCIN IN STREPTOMYCES ALBONIGER, no. 58, 31 December 2005 (2005-12-31), pages 722 - 739 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10428361B2 (en) 2015-03-26 2019-10-01 Basf Se Biocatalytic production of l-fucose
US10440953B2 (en) 2015-08-07 2019-10-15 Basf Se Control of pests in maize by ginkgolides and bilobalide
WO2017037629A1 (fr) * 2015-09-04 2017-03-09 Basf Se Micro-organismes améliorés pour la biosynthèse de pamamycines

Similar Documents

Publication Publication Date Title
Ochi From microbial differentiation to ribosome engineering
Sakai et al. Construction of a citrinin gene cluster expression system in heterologous Aspergillus oryzae
Hesketh et al. The role of absC, a novel regulatory gene for secondary metabolism, in zinc‐dependent antibiotic production in Streptomyces coelicolor A3 (2)
Li et al. Stepwise increase of thaxtomins production in Streptomyces albidoflavus J1074 through combinatorial metabolic engineering
CN106687577A (zh) 用于产生香草醛的微生物和方法
CN111117942B (zh) 一种产林可霉素的基因工程菌及其构建方法和应用
WO2015092575A1 (fr) Groupe de gènes pour la biosynthèse de la pamamycine
Yu et al. RS24090, a TetR family transcriptional repressor, negatively affects the rimocidin biosynthesis in Streptomyces rimosus M527
CN102286398B (zh) 一种生产高活性和高纯度利福霉素sv的方法
US9631212B2 (en) Gene cluster for biosynthesis of cornexistin and hydroxycornexistin
US20210230573A1 (en) Microorganisms and the production of fine chemicals
CN109136253B (zh) 一种通过糖多孢红霉菌sace_5754基因途径提高红霉素产量的方法
JP2020089297A (ja) ポリリジン生産菌及びそれを用いるポリリジン製造方法
Jensen et al. Streptomyces clavuligerus has a second copy of the proclavaminate amidinohydrolase gene
US9796992B2 (en) Gene cluster for biosynthesis of cornexistin and hydroxycornexistin
Xie et al. Enhancement of himastatin bioproduction via inactivation of atypical repressors in Streptomyces hygroscopicus
Yu et al. High frequency of homologous gene disruption by single-stranded DNA in the taxol-producing fungus Pestalotiopsis microspora
Jnawali et al. Role of σ-factor (orf21) in clavulanic acid production in Streptomyces clavuligerus NRRL3585
Gomez‐Escribano et al. An rplKΔ29‐PALG‐32 mutation leads to reduced expression of the regulatory genes ccaR and claR and very low transcription of the ceaS2 gene for clavulanic acid biosynthesis in Streptomyces clavuligerus
Oh et al. Identification of the duplicated genes for S‐adenosyl‐l‐methionine synthetase (metK1‐sp and metK2‐sp) in Streptomyces peucetius var. caesius ATCC 27952
Zhang et al. Functional characterization of the first two actinomycete 4-amino-4-deoxychorismate lyase genes
WO2015177674A1 (fr) Groupe de gènes pour la biosynthèse de cornexistine et d'hydroconexistine
CN113667670A (zh) 一种适用于链霉菌的强启动子及其应用
CN101124242B (zh) 与糖异生作用有关的新颖的基因
WO2017037629A1 (fr) Micro-organismes améliorés pour la biosynthèse de pamamycines

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14870923

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14870923

Country of ref document: EP

Kind code of ref document: A1