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WO1992017496A1 - Isolement et sequence du gene acetyle coa=deacetyl-cephalosporin acetyltransferase - Google Patents

Isolement et sequence du gene acetyle coa=deacetyl-cephalosporin acetyltransferase Download PDF

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WO1992017496A1
WO1992017496A1 PCT/US1992/002087 US9202087W WO9217496A1 WO 1992017496 A1 WO1992017496 A1 WO 1992017496A1 US 9202087 W US9202087 W US 9202087W WO 9217496 A1 WO9217496 A1 WO 9217496A1
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cephalosporin
gene
seq
acetyltransferase
synthesis
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Lorilee Mathison
Charles L. Soliday
John A. Rambosek
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Panlabs Inc USA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/06—Cephalosporin C; Derivatives thereof

Definitions

  • the invention relates generally to genetic engineering involving recombinant DNA technology, and particularly to DNA sequences for the final gene in cephalosporin antibiotic synthesis that encodes Acetyl CoA.Deacetylcephalosporin acetyltransferase useful in recombinant and cell-free synthesis of intermediates in cephalosporin synthesis, and in increased synthesis of Cephalosput in * antibiotics.
  • Beta lactam antibiotics including the penicillins and cephalosporins have had incalculable social impact in freeing civilization from the scourge of bacterial diseases.
  • the emergence of antibiotic resistance in bacteria has spurred efforts to create, through chemical means and genetic engineering, new classes of antibiotics.
  • Cephalosporins are particularly useful because they affect both gram negative and gram positive bacteria, are more resistant to beta-lactamases than penicillins, and do not elicit allergic responses in those allergic to penicillins.
  • Intermediates in the synthesis of Cephalosporin C are very useful reagents in chemical synthesis of novel antibiotics, which, while retaining the penicillinase resistant beta-lactam ring characteristics of Cephalosporin C, have increased antibiotic potency.
  • CoArDeacetylcephalosporin acetyltransferase and epimerase have proven to be elusive, (in part because the enzymes they encode are unstable). It is the acetyltransferase gene which is a subject of the present disclosure. Throughout the specification, the notation "(#)" is used to refer to the documents in the appended Citation section.
  • Cephalosporin producing organisms are shown in Table I below. It has only recently become possible to study filamentous fungi with recombinant genetic engineering techniques, and of the cephalosporin producing organisms listed in
  • C. acremonium Molecular biology in C. acremonium is at an early stage compared with the extensive studies conducted with Saccharomyces cerevisiae. Several laboratories have independently developed transformation systems in filamentous fungi, and quite recently stable integrative transformation of C. acremonium was demonstrated, although at a low frequency (1-3). This has offered (for the first time) the possible opportunity to specifically and predictably alter antibiotic production in C. acremonium.
  • approaches commonly employed in other genetic systems are not easily amenable for routine use with C. acremonium: namely, screening for complementation of mutant phenotypes, gene disruption techniques, or gene replacement techniques.
  • Biosynthesis of Cephalosporin C involves the sequential action of at least 5 genes, and their 6 enzymatic activities, as depicted in FIGURE 1. (Unlike bacteria, in C. acremonium the cef E and cef F genes are fused (cef E/F), giving a total of 5 instead of six genes.)
  • the 6 enzymatic activities oxidatively condense L-alpha-aminoadipic acid, L-cysteine and L-valine precursors into Cephalosporin C through a series of intermediates. All of the intermediates with the exception of the first (delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine;
  • LLD-ACV or simply ACV contain a beta lactam ring.
  • the final step in the synthesis of Cephalosporin C involves the addition of an acetyl group from acetyl CoA to the cephem ring.
  • the reaction is catalyzed by acetyl CoA: Deacetyl cephalosporin acetyltransferase (also commonly known as 'Cephalosporin C synthetase', 'DAC acetyltransferase', 'acetyl CoA: cephalosporin acetyl ⁇ transferase' or 'cephalosporin acetyltransferase').
  • acetyl CoA Deacetyl cephalosporin acetyltransferase (also commonly known as 'Cephalosporin C synthetase', 'DAC acetyltransferase', 'acety
  • Cephalosporin C synthetase (cefG) has not previously been cloned.
  • cephalosporin pathway genes encoding 4 enzymatic activities
  • pcbAB plasmid, cephalo- sporin biosynthesis AB
  • ACV synthetase (4) encoding ACV synthetase (4)
  • pcbC encoding isopenicillin N synthetase
  • IPNS isopenicillin N synthetase
  • cef E/F gene encoding both deacetoxycephalosporin C synthetase (DAOCS, also commonly known as 'expandase') and deacetylcephalosporin C synthetase (DACS, also commonly known as 'hydroxylase'), respectively (6,7).
  • DOCS deacetoxycephalosporin C synthetase
  • DAS deacetylcephalosporin C synthetase
  • Nucleotide sequence ho ologies are reported between cephalosporin genes coding for the same enzyme in different genus and species of filamentous fungi (8- 12). In the case of IPNS, homology of about 74% exists at the nucleotide sequence level between the genes in C. acremonium and Penicillium chrysogenum (8). In comparing DACS/DAOCS encoded by cef E/F in C. acremonium with the bacterial cef E gene product of S. clavuligerus, it is reported that there is 57% identity at the amino acid level and 65% identity in the gene sequence (11).
  • Rate limiting steps in synthetic pathways are important points where improving efficiency may significantly decrease cost.
  • Two studies have recently attempted to identify points in Cephalosporin C synthesis where cellular enzyme activity may be rate limiting.
  • gene dosage of pcbC (the IPNS gene) was reported increased (ie. in transformants) and the effect on cephalosporin synthesis studied (2)).
  • the findings reported that IPNS enzyme may not be rate- limiting in this production strain.
  • quantitative measurements were made of the levels of the intermediates isopenicillin N, penicillin N, and
  • DNA sequences of the entire cefG gene are provided for encoding acetyl CoArcephalosporin acetyltransferase polypeptide, which is the last enzyme in Cephalosporin C synthesis in C. acremonium.
  • DNA sequences are useful in replicative cloning and shuttle vectors, and in expression vectors for transforming host cells.
  • the invention provides methods for constructing mutant DNA sequences related to the cefG gene by deletion and conservative substitution, and which encode polypeptides having modified enzyme activity.
  • Other aspects provide assays including nucleic acid probes and immunoassays for measuring cefG gene and its translation products. Methods are provided using these assays to identify and select novel variants and strains of filamentous fungi having relatives of the cefG gene.
  • Other aspects of the invention relate to novel methods and transformants with multiple copies of the cefG gene that synthesize remarkably greater amounts of
  • Cephalosporin C than non-transformed Cephalosporin C producing strains.
  • FIGURE 1 is a schematic drawing which shows the biosynthetic pathway, enzymes, and genes reportedly involved in synthesis of cephalosporin antibiotics, as described in the Background section of the specification.
  • FIGURE 2 is a flow chart which shows schematically the steps involved in cloning and sequencing the gene encoding acetylCoA:deacetylcephalosporin acetyltransferase (cefG), as described in Examples I-VIII of the Detailed Description of the Invention.
  • FIGURE 3 shows by Northern blot hybridization that transformation of the mutant M40 strain (incapable of synthesizing acetyltransferase) with the cefG gene resulted in expression of acetyltransferase mRNA, as described in Example XI.
  • FIGURE 4 shows by antibiotic bioassay that cefG transformed mutant M40 strains express Cephalosporin C which is fully active in neutralizing the bacterial growth of A. faecalis, as described in Example XIII.
  • FIGURE 5 shows by high pressure liquid chromatography that the cefG transformant strain M40-T 3 produces Cephalosporin C antibiotic, as described in Example XIV.
  • FIGURE 6 confirms by high pressure liquid chromatography that the Cephalosporin C produced by the cefG transformant strain M40-T, is indistinguishable from purified Cephalosporin C standard, as described in Example XIV.
  • FIGURE 7 shows by high pressure liquid chromatography that a second transformant M40-T g strain also produces cephalosporin antibiotic, as described in Example XIV.
  • FIGURE 8 shows by high pressure liquid chromatography that the mutant M40 strain, from which the cefG transformants were derived, is incapable of synthesizing Cephalosporin C.
  • FIGURE 9 shows the genomic organization of the acetylCoA:Deacetyl- cephalosporin Acetyltransferase gene (cefG) as deduced following nucleotide sequencing of the entire gene.
  • FIGURE 10 shows similarities in the amino acid sequences in the N-terminal open reading frame between the amino acid sequence of Acetyl
  • Acetyltransferase of Ascobollus immersus (MET2 & ASCIM).
  • FIGURE 11A shows the nucleotide sequence of bases 1-720 of cefG cDNA and FIGURE 11B shows the remaining nucleotide sequence (bases 721 -1183) of cefG cDNA.
  • FIGURE 12A shows the nucleotide sequence of bases 1-720 of cefG genomic
  • FIGURE 12B shows the nucleotide sequence of bases 721-1575
  • FIGURE 12C shows base 1575-1807 of cefG genomic DNA.
  • the present invention provides an isolated cefG DNA sequence which encodes acetyl CoA:cephalosporin acetyltransferase, the last enzyme in the synthesis of Cephalosporin C. Since it has proven difficult to obtain an amino acid sequence for this relatively unstable enzyme, two different interrelated cloning approaches were employed successfully to identify and isolate the cefG gene from
  • the protein coded for by this open reading frame show significant amino acid sequence similarity to the homoserine acetyltransferase from Ascobollus immersus (FIGURE 10; METZ & ASCIM is Ascobollus immersus amino acid sequence and AACACTDNA is the deduced amino acid sequence; colons indicate identical amino acids; FIGURE 10; SEQ ID No. 5).
  • the sequence similarity with an acetyltransferase, and the linkage with cef E/F indicated to us that this open reading frame may code for the Cephalosporin C acetyl ⁇ transferase.
  • Example XI M40 mutants lacking Cephalosporin C acetyltransferase were transformed.
  • DNA from the cefG gene was used to make restriction fragment probes that were used to screen a C. acremonium cDNA library (Examples V-VIII).
  • the present invention provides recombinant vectors containing the cefG gene capable of transforming a host cell to produce a recombinant host cell.
  • suitable host cells include E. coli, Streptomycetes, Cephalosporium, or Saccharomyces, while for production of Cephalosporin C (or intermediates as detailed for example in FIGURE 1), suitable host cells include, e.g., Cephalosporium or Streptomyces cells.
  • C. acremonium is a well-known microorganism, and several suitable host cell strains from the American Type Culture Collection under accession numbers such as ATCC 20339 ⁇ Cephalosporium sp. strain F12), ATCC 14553 (C. acremonium), and ATCC 36255 (Acremonium strictum, of which M-0198 is a nonproducing mutant.
  • Other strains of host cells known to those skilled in the art are of course also useful including many species of Streptomyces, for example, S. lipmanii (A16884, MM4550, MM13902), S. clavuligerus, S. lactamdurans, S. griseus, S. hygroscopicus, S. madayamensis (WS-3442-D), S. chartreusis (SF1623),
  • S. heteromorphus and S. panayensis C2081X
  • S. cinnamonensis S. fimbriatus, S. halstedii, S. rochei, and S. viridochromogenes
  • S. cattleya S. olivaceus, S. flavovirens, S. flavus, S. fulvoviridis, S. argenteolus and S. sioyaensis (MM4550 and MM13902).
  • Genetic transformation of Cephalosporium or Streptomyces host cells with plasmid vectors containing the nucleic acids of the present invention e.g., SEQ ID No.
  • FIGURE 11-12 is conveniently accomplished using for example the P-.T221 expression plasmid system described by Chapman et al., U.S. Patent No. 4,762,786 (incorporated herein by reference).
  • This vector and others which contain the DNA sequence of the invention operably linked (e.g., in the correct reading frame) to suitable control sequences, e.g., Ceph ⁇ osporium-functional autonomous replication sequences, transcriptional and translational activating sequences, promoters and enhancers and integration sequences, are useful for producing transformed host cells which express the polypeptide gene products of the invention (e.g., SEQ ID No. 5; FIGURE 10).
  • suitable control sequences e.g., Ceph ⁇ osporium-functional autonomous replication sequences, transcriptional and translational activating sequences, promoters and enhancers and integration sequences.
  • Useful shuttle vectors for transferring genetic material between E. coli, Cephalosporium, and many related Streptomyces species including "related Nocardia are for example pETS702 and pPLS221 and other plasmid vectors described in Chapman et al op.cit. These vectors and the plasmid and cosmid cloning vectors operative in Escherichia coli (e.g., pBR322, ⁇ gtlO, ⁇ gtll, etc.; ref. 23) are all referred to herein as replicative cloning vectors. Methods for preparing these replicative cloning vectors are well known to those skilled in the art (23).
  • Embodiments of the invention are also useful for preparing vectors containing nucleotide sequences that are related to the nucleotide sequence of the cefG gene (i.e., SEQ ID No. 4; FIGURE 12) or its m RNA (i.e., SEQ ID No. 3; FIGURE 11), e.g., by deletion or nucleotide substitution.
  • the invention provides that recombinant host cells transformed with vectors containing these related DNA sequences produce enzymes with modified enzyme activity, e.g., decreased, increased, showing altered response to regulatory control or improved commercial properties.
  • Recombinant host cells expressing decreased enzyme activity are useful for preparing intermediates while those expressing increased activity are useful for preparing Cephalosporin C, as well as the acetyl CoArcephalosporin acetyltransferase enzyme.
  • Recombinant host cells expressing enzymes with altered response to regulatory control, e.g., less sensitive to end-product feedback inhibition of enzyme activity, are useful in cell-free synthesis of antibiotics.
  • Recombinant host cells expressing enzyme with improved commercial properties include enzymes, e.g., with greater stability and improved performance in cell- free synthesis of antibiotics.
  • Cephalosporin biosynthesis For example, mutations created in the cefG gene
  • nucleotide sequences of the disclosure thus provides one skilled in the art the necessary road map to introduce specific mutations (e.g., deletions, insertions, substitutions).
  • the resultant mutagenized DNA sequences can then be used in replicative cloning vectors, shuttle vectors, or expression vectors (above).
  • Host cells transformed with the vectors are useful as a source of enzyme having modified activity (above).
  • Embodiments of the present invention thus anticipate production of mutations in the cefG gene, and in the related cefG genes in other strains of filamentous fungi (and bacteria) as well as selection methods for distinguishing among these mutants for desirable traits such as (but not limited to) increased production of intermediates in Cephalosporin C biosynthesis which may be useful for subsequent production of other chemically modified cephalosporin antibiotic derivatives.
  • the transformed host cells of the invention are a useful source for preparing cellular extracts for synthesis of antibiotics.
  • one illustrative method of preparing a cellular extract for synthesis of antibiotics is to lyse a protoplast pellet made from whole cells obtained from 40-70 hr. mycelia and treated, e.g., with Cytophagolytic enzyme - ⁇ preparation and Zymolyase-5000.
  • the extract After treatment with the enzymes the extract is clarified by eentrifuging, chilled (but not frozen) at sub-zero temperatures, and re-centrifuged.
  • the enzyme is used in substantially pure form, e.g., by chromatographie separation from the cell-free extract. Synthesis of Cephalosporin C and its intermediates may be accomplished using this cell-free extract for example as described by Demain et al., U.S. Patent No. 4,307,192 and A. Scheidegger et al., J. Antibiotics 3 ⁇ :522-531, 1984.
  • Semi-purified enzymes such as this can be prepared from numerous microorganisms, e.g., Cephalosporium acremonium (such as strains ATCC 48272 and ATCC 36225), Streptomyces clavuligerus and Streptomyces lipmanii.
  • Cephalosporium acremonium such as strains ATCC 48272 and ATCC 36225
  • Streptomyces clavuligerus Streptomyces lipmanii.
  • the cephalosporin products and intermediates obtained from these enzymatic processes are useful intermediates in synthesis of known cephalosporin semi-synthetic antibiotics.
  • the oral antibiotic cephalexin can be prepared by deacylating the 3-carboxyphenylacetyl group and the adipoyl group of the product is then deacylated to the corresponding 3-substituted 7-6-amino-3 cepham nucleus. The latter is then reacylated to provide the desired 7- ⁇ -acylamino cephal
  • the invention provides methods for modifying Cephalo ⁇ sporin C synthesis (e.g., increasing or decreasing synthesis) in Cephalosporin C producing organisms by transforming host cells of these organisms with vectors containing the cefG gene.
  • Synthesis of Cephalosporin C is increased when extra copies of the cefG gene are introduced into host cells to form transformed host cells (e.g., Example XVI).
  • Synthesis of Cephalosporin C may be decreased by introducing a mutant cefG gene into host cells and selecting transformed host cells with either decreased synthesis of Cephalosporin C; or, decreased levels of cefG mRNA; or, decreased cephalosporin acetyltransferase enzyme activity.
  • upstream regulatory regions i.e., upstream from the ATG start codon in SEQ. ID. No. 4 and FIGURE 12A
  • upstream regulatory regions are particularly attractive targets for site- directed mutagenesis to create the mutagenized cefG genes useful in this aspect-,— although other regions of the nucleotide sequence are also useful
  • the invention provides nucleic acid probes (labeled probes) for identifying the presence or the amount of the nucleotide sequence of the cefG gene and related nucleotide sequences (e.g., by deletion and/or nucleotide substitution) in cell extracts of organisms.
  • Detection of the cefG gene may be accomplished, for example, using labeled probes prepared from genomic fragments, DNA fragments, cDNA fragments (e.g., SEQ ID No. 3; FIGURE 11), synthetic and natural nucleic acids and oligonucleotides that hybridize to the cefG gene of Cephalosporium acremonium (as provided in SEQ ID No. 4; FIGURE 12) under stringent conditions.
  • These probes are useful in identifying the cefG gene in cellular extracts of other organisms, e.g., using suitable enzyme-, fluorescent- and
  • P-radiolabeling methods and stringent hybridization conditions For example, it is possible to identify related genes synthesizing Cephalosporin C synthetase in other strains of filamentous fungi (or bacteria) by using labeled oligonucleotide probes under stringent conditions for identification of positive clones in genomic and cDNA libraries. It is also possible to examine the restriction fragments of these clones using Northern and Southern blotting, respectively; and, it is routine for those skilled in the art to determine the nucleotide sequence of the related gene and to determine the relationships between nucleic acid sequences based on deletion and substitution, such as conservative substitution of a purine base for a purine base or a pyrimidine for a pyrimidine.
  • the labeled nucleic acid probes of the invention are useful in direct screening for new species and strains of filamentous fungi (and bacteria) having modified enzyme activity (as detailed above).
  • using the nucleic acid probes of the invention for Northern and Southern blotting one can rapidly screen cellular extracts containing DNA's or mRNA's prepared from hundreds of natural strains, mutagen treated cultures, or transformed recombinant host cells looking for increased or decreased expression of the cefG mRNA (e.g., by methods similar to those used in Examples II- VIII, below). In this way the invention provides screening and selection methods for identifying and isolating cephalosporin producing strains with novel properties.
  • polypeptides of the enzyme encoded by the cefG gene Peptide portions of the acetyltransferase enzyme (i.e., SEQ ID NO: 1]
  • FIGURE 10 are useful for constructing synthetic peptides and polypeptides identical to, homologous with, or related to certain portions of the Cephalosporin
  • useful homologous synthetic peptides may be at least 40% identical to a sequence of at least five amino acids; related peptides may be related by substitution of one amino acid for another amino acid of like physical properties (ie. a polar amino acid for a polar amino acid; a negatively charged for a negatively charged; a polar for a polar; a hydrophobic for a hydrophobic; etc.).
  • These amino acid substitutions can be engineered by changing a nucleotide base (or series of bases) or a deletion of nucleotide(s) in a nucleotide sequence of the cefG gene.
  • the synthetic polypeptides are useful for evoking specific binding partners, such as but not limited to monoclonal and polyclonal antibodies. They are also useful for regulating the activity of other enzymes in the Cephalosporin C biosynthesis pathway, such as through feedback inhibition of enzyme synthesis and/or specific negative and positive feedback regulatory interactions as they occur between the different enzymes in the Cephalosporin C biosynthetic pathway, e.g., mechanisms involved in coordinate control of cephalosporin biosynthesis.
  • Specific binding partners as exemplified by antibody reagents developed to synthetic peptides (above) are useful for qualitative and quantitative immunoassays such as those outlined below.
  • the antibody reagents are also useful for preparing substantially pure preparations of Cephalosporin C acetyltransferase (and portions thereof), as for example (but not limited to) by immunoaffinity chromatography.
  • the antibody reagents are also useful for blocking the activity of the Cephalosporin C acetyltransferase, such as (but not limited to) during cell- free synthesis of intermediates in Cephalosporin C synthesis using cellular extracts.
  • An aspect of the invention provides immunoassays for quantitatively and qualitatively assessing the amount and type acetyl CoArcephalosporin acetyltransferase produced by filamentous fungi.
  • Immunoassays for expression of acetyltransferase enzyme such as using monoclonal and polyclonal antibody reagents produced against synthetic peptides, are useful for selecting among natural, mutant, and recombinant cells to find those which express altered levels (e.g., intracellular or extracellular) or types (i.e., mutant) or amounts of
  • Cephalosporin C acetyltransferase The assays are also useful for identifying the sources, methods, and procedures for obtaining fragments and portions of Cephalosporin C acetyltransferase, as well as for assessing recombinant production of the Cephalosporin C acetyltransferase polypeptide or for assessing amounts of the enzyme in cellular extracts as that may be useful in cell-free synthesis of cephalosporins.
  • immunoassay formats examples include radioimmunoassay (RIA), enzyme- linked immunoassay (ELISA), fluorescence immunoassay (FIA), time resolved immunofluorescence assay, Western immunoblot assays, immunoelectrophoresis " " assays, and- adial immunodiffusion assays.
  • RIA radioimmunoassay
  • ELISA enzyme- linked immunoassay
  • FIA fluorescence immunoassay
  • time resolved immunofluorescence assay Western immunoblot assays
  • immunoelectrophoresis " assays immunoelectrophoresis " assays
  • - adial immunodiffusion assays examples include radioimmunoassay (RIA), enzyme- linked immunoassay (ELISA), fluorescence immunoassay (FIA), time resolved immunofluorescence assay, Western immunoblot assays, immunoelectrophoresis " " assays, and- adial immunodiffusion assays.
  • the antibody reagents are also useful for purifying acetyl CoArcephalosporin acetyltransferase, e.g., by affinity chromatography such as from cellular extracts or filamentous fungi, bacteria, or transformed recombinant host cells.
  • affinity chromatography such as from cellular extracts or filamentous fungi, bacteria, or transformed recombinant host cells.
  • isolated DNA sequence means a substantially pure molecule of deoxyribonucleic acid having a defining sequence of nucleotides.
  • Oligonucleotide sequence means a molecule having a sequence of nucleotide bases in an indicated manner.
  • “Complementary” nucleotide sequence means an oligonucleotide sequence that hybridizes with DNA or RNA because it contains nucleotide bases capable of base-pairing with the bases in the DNA or RNA (e.g., a G pairing with a C or an A with a T or a U).
  • “Encode” refers to the genetic code by which triplets of nucleotides within a nucleotide sequence directs the selection of a particular amino acid for inclusion in a chain of amino acids.
  • amino acid sequence is a proscribed sequence of amino acids in a chain.
  • Polypeptide means a chain of more than 30 amino acids having an amino acid sequence.
  • Protein means a chain of 5 to 29 amino acids.
  • Protein means a polypeptide chain which has an activity, eg. enzymatic, or antigenic.
  • Labeled probe means an oligonucleotide having a label which permits its detection or quantitation (e.g., an enzyme-label, fluorescent-label, or radioactive label such as P; Example III).
  • Cyclon C synthetase is used synonymously with DAC acetyltransferase, as they are commonly used in the art, to refer to the, acetyl CoArcephalosporin acetyltransferase.
  • “Deletion” means an oligonucleotide sequence which differs from the native oligonucleotide sequence by the absence of at least one nucleotide base.
  • “Homologous amino acid substitution” means that the replacement of one amino acid with another which has similar physical properties, e.g., an acidic amino acid by another acidic amino acid, or a basic with a basic, or polar with a polar, or a hydrophobic with a hydrophobic.
  • “Conservative nucleotide substitution” means the substitution of an nucleotide phosphate base with a corresponding complementary base, e.g. an A for a T; a T for an A; a G for C; or, a C for G.
  • Hybridizing under stringent conditions means binding (i.e., greater than 30% double-stranded) of one oligonucleotide sequence to another under conditions such as those included within the specification (e.g., Example VII), or alternatively, at page 387-389 of Maniatis et al. (23).
  • Replicative cloning vector means an oligonucleotide sequence capable of increasing in copy number when introduced into a cell.
  • “Expression vector” means an oligonucleotide sequence which contains at least regulatory elements, such as (but not limited to) a promoter, and a nucleotide sequence encoding Cephalosporin C acetyltransferase in such a manner that the introduction of the oligonucleotide sequence into a cell results in the expression of Cephalosporin C acetyltransferase.
  • “Expression” means transcription of an oligonucleotide sequence encoding
  • Cephalosporin C acetyltransferase into complementary mRNA or translation of the mRNA to obtain production of the encoded Cephalosporin C acetyltransferase.
  • To transform means to introduce into a cell nucleic acids, such as an isolated DNA sequence or oligonucleotide sequence, for the purpose of altering the phenotype of the cell, such as (but not limited; to) cases in which gene expression may be down-regulated, or alternatively, where expression of a gene may be turned on which was not previously present in the cell.
  • nucleic acids such as an isolated DNA sequence or oligonucleotide sequence
  • Cephalosporin acremonium strains used in these procedures include a
  • Cephalosporin C producing strain Panlabs production strain PF14-1
  • M40 mutant " ⁇ strain which does not produce Cephalosporin C. Analysis of M40 fermentations shows that the mutant strain produces the immediate
  • Cephalosporin C precursor deacetyicephalosporin. Strains were maintained on slants containing complete medium composed of sucrose, 20g; agar, 20g; peptone,
  • yeast extract 4g; NaN0 3 , 3g; KH 2 P0 4 , 0.5g; K 2 HP0 4 , 0.5g; KCl, 0.5g; MgSO 4 .7H 2 0, 0.5g; FeS0 4 .7H 2 0, O.Olg; in 1 liter of distilled water, pH 6.6. After ten days of growth at 28°C and 65% relative humidity, 6 ml of sterilized water was added to the slants, and the culture growth was scraped from the agar surface. The resulting suspension was transferred to a sterile screw-top tube containing glass beads.
  • a two-stage fermentation of the strains in shake flasks was used for the production of cephalosporins or for the production of mycelia as a source of DNA and RNA.
  • the seed stage was initiated by adding the inoculum to 15 ml/250 ml flask of medium with the following composition: glucose, 5g; sucrose, 40g; corn starch, 30g; beet molasses, 50g; soybean meal, 65g; CaSO 4 .2H 2 Q, 15.8g; ammonium acetate, 8g; CaCO 3 (pptd), 5g; (NH 4 ) 2 S0 4 , 7.5g; MgSO 4 .7H 2 O, 3.5g; KH 2 PO 4 , lg; soybean oil, 0.15 ml/flask in 1 liter of distilled water at pH 6.2.
  • Incubation was at 25°C and 65% relative humidity on a rotary shaker with a 70 mm diameter amplitude at 220 rpm. After 96 hours of incubation, the production stage was initiated by transferring 2 ml of vegetative seed to a fresh 15 ml/250 ml flask of the above-described media. Incubation was continued under the same conditions.
  • the strains were grown in 100 ml/500 ml flask of complete media composed of: glycerol, 20g; peptone, 4g; yeast extract, 4g; KH PO 4 , 0.5g; K 2 HP0 4 , 0.5g; KCl, 0.5g; MgS0 4 .7H 2 0, lg; NaN0 3 , 3g; FeS0 4 .7H 2 0, O.Olg in 1 liter of distilled water. Incubation was at 30°C on a rotary shaker at 200 rpm.
  • complete media composed of: glycerol, 20g; peptone, 4g; yeast extract, 4g; KH PO 4 , 0.5g; K 2 HP0 4 , 0.5g; KCl, 0.5g; MgS0 4 .7H 2 0, lg; NaN0 3 , 3g; FeS0 4 .7H 2 0, O.Olg in 1 liter of distilled water. Incubation was
  • C. acremonium PF14-1 genomic DNA obtained by the procedure of Example II was partially digested with 100 ⁇ g Sau3AI for 5, 10, and 20 minutes (23) to achieve an average fragment size of 10 to 20 kb. Centrifugation of the digest on a 5 to 20% NaCl density gradient provided a further enriched 10 to 20 kb fraction which was ligated into the BamHI site of the lambda vector 2001.
  • the ligation was mixed with a lambda DNA packaging system (Gigapack from Stratagene, La Jolla, CA) and the resulting plaque forming units (PFU) plated on agarose in NZCYM media (10 g NZ-amine, 5 g NaCl, 5 g yeast extract, 2 g MgSO 4 -7H 2 O, pH7.5) in five 157 mm petri dishes (approximately 10,000
  • oligonucleotide probe was constructed to complement the coding sequence of a DNA region at the 3'-end of the published sequence of the cef E/F gene (Samson et al., 1987 (12)). The probe was synthesized by cyanoethyl phosphoramidite chemistry (Pharmacia Gene Assembler instrumentation) and it is shown in SEQ ID No. 1. Those clones that hybridized with the SEQ ID No. 1 probe were isolated and their DNA prepared for restriction enzyme mapping.
  • a 7.2 Kb fragment thus shown to contain the cef E/F gene was gel purified from a BamHI digest of the DNA from a selected lambda clone and ligated into the BamHI site of a pUC18 vector. Restriction mapping of the resulting subclone, 104.80.1, and Southern blot analysis with the 3' and 5' cef E/F oligonucleotides probes (SEQ ID No. 1 and No. 2) revealed the position and orientation of the gene within the subclone. This clone provided a source of DNA for construction of fungal transformation vectors and subclones for DNA sequencing.
  • Example IV Subcloning of the cefG (acetyltransferase) gene into M13 Vectors for DNA Sequencing
  • a 5.0kb genomic fragment was isolated from a BamHI/Sstl digest of the 104.80.1 plasmid by separation in and elution from a 0.7% low melting point agarose gel.
  • the DNA fragment was ligated to M13mpl8 and M13mpl9 DNAs which had been previously digested with BamHI and Sstl.
  • the ligations were done in a final volume of 10 ⁇ l for one hour at room temperature. Dilutions of the ligation mixture were used to transfect competent MV1190 cells (Bio-Rad) using electroporation (Gene Pulser, Bio-Rad, Richmond, CA). Preparation of the competent MV1190 eells and electroporation conditions were both according to the manufacturer's recommendations.
  • the transfection mixture was plated on yeast tryptone (YT) plates in 3 ml of YT soft agar (5.0 g yeast extract, 8.0 g tryptone, 2.5 g NaCl per liter, pH 7.2) containing 40 ⁇ g/ml X-gal, 0.1 M IPTG, and 100 ⁇ l of an overnight culture of ⁇ MV1190 grown in YT broth. Following overnight incubation of the YT plates at 37°C, recombinant M13 phages were identified by the colorless plaque formation technique involving insertional inactivation of the plasmid vector ⁇ -galactosidase gene activity.
  • the colorless plaques were picked from the YT plates as agar plugs using a pasteur pipet and transferred into 1.5 ml of YT broth containing 10 ⁇ l of an MV1190 overnight culture. The cultures were grown six hours at 37°C on a rotary shaker and the cells pelleted by centrifuged at
  • Single-stranded template DNA was prepared by inoculating 1 ml of TY broth with 5 ⁇ l of M13mpl8 and M13mpl9 vector stock and 10 ⁇ l of a MV1190 overnight culture, incubating six hours at 37 °C on a rotary shaker, eentrifuging at 12000 xg for five minutes at 4°C and decanting the supernatant to fresh tubes. Two- hundred ⁇ l of 20% polyethylene glycol/(PEG, MW3350), 2.5M NaCl were added to the supernatant and the mixture was incubated for 15 minutes at room temperature. The tubes were spun at 12000xg for five minutes at 4°C and the supernatant was drawn off and discarded.
  • the pellet was resuspended in 100 ⁇ ls of TE buffer after which 50 ⁇ l of a Tris-saturated phenohchloroform (1:1, v:v) mix was added.
  • the tubes were vortexed 15 seconds, left and room temperature for five minutes, vortexed another 15 seconds, and then centrifuged at 12000xg for five minutes at 4°C.
  • the top 80 ul of the aqueous layer was removed and 3 ⁇ l of 3M sodium acetate and 200 ⁇ l of ethanol were added, and the mixture was then placed at -70°C for ten minutes.
  • the single-stranded DNA - was pelleted by centrifugation, washed with 70% ethanol, dried and resuspended in 18 ⁇ l of TE buffer.
  • RNA was pelleted at 12000xg for 20 minutes at 4°C, the pellet dissolved in 0.3 ml TE buffer plus 0.03 ml of 3M sodium acetate, and 2.5 volumes of ethanol were added to reprecipitate the RNA.
  • RNA concentration was determined spectrophotometrically using absorbance at 260 nm.
  • mRNA was isolated from total RNA using an mRNA Purification Kit per the manufacturer's instructions (Pharmacia LKB Biotechnology, Piscataway, New Jersey). The kit uses prepacked oligo(dT)-cellulose columns for affinity purification of polyadenylated RNA.
  • mRNA Purification Kit per the manufacturer's instructions (Pharmacia LKB Biotechnology, Piscataway, New Jersey). The kit uses prepacked oligo(dT)-cellulose columns for affinity purification of polyadenylated RNA.
  • cDNA Synthesis of cDNA was accomplished with a cDNA Synthesis Kit, per the manufacturer's instructions (Pharmacia LKB Biotechnology, Piscataway, New Jersey) and poly(A+) RNA isolated from C. acremonium PF14-1 as described above.
  • the cDNA synthesis kit created cDNAs with cohesive EcoRI ends which were ligated to EcoRI cut lambda gtlO arms (Bethesda Research Labs, Gaithersburg, MD). One ⁇ g of EcoRI cut lambda gtlO arms and the total cDNA synthesis product were ligated overnight at 15°C in a total volume of 10 ⁇ l.
  • cDNA in four ⁇ l of the ligation mixture was packaged using an in vitro packaging extract purchased as "Gigapack Gold-tm" (Stratagene, La Jolla, CA), and the resultant mixture was plated on NZCYM plates (above) using B. coli strains C600 (permissive) and C600 hflA150 (non-permissive), obtained (Bethesda Research Labs, Gaithersburg, MD), to determine the titer and frequency of recombinants. The permissive an non-permjssiye E. coli strains were grown overnight in NZCYM broth plus 100 ⁇ l of 20% maltose.
  • plaque forming units were screened by standard methods (Maniatis et al. 27) using E. coli strain C600hflA150 (above;
  • Example VI A 1.2kb Hindlll fragment (which contains 430bp of the 5' end of the C. acremonium cefG gene) was isolated from a Hindlll digest of the pUC18 vector subclone 104.80.1 (Example III, above) by separation in and elution from a 0.7% o o low melting point agarose gel, and was labeled with P for use as a probe to screen the cDNA library. Labeling of the isolated fragment was accomplished by o random-primer extension reaction with ( P) dCTP and an Oligolabeling Kit, per the manufacturer's instructions (Pharmacia, Piscataway, New Jersey).
  • Hybridization reactions were performed in the presence of 50% formamide, 5X SSC (0.15M NaCl, 0.015M sodium citrate pH7), 0.1% SDS, 5X Denhardt's (5g ficoll, 5g polyvinylpyrolidone, and 5g BSA for 500 ml of 50X stock) and 100 ⁇ g/ml calf thymus DNA, at 37°C overnight.
  • Hybridization assays were performed at least in duplicate. Three plaques termed, Clones # 1, #2, and #3 were identified having overlapping nucleotide sequence, a composite of which is provided in SEQ ID No. 3; FIGURE 11. Clones 1-3 cDNA hybridized strongly on duplicate filters with o the isolated P-labeled probe and these were rescreened under identical hybridization conditions and continued to show strong hybridization to the isolated o
  • Example VIII Subcloning of the acetyltransferase cDNA clones into M13 Vectors The phage in the supernatant of the three positive plaques (identified in Example VII) was transferred into 1 ml of SM (0.1M NaCl, 0.1M MgSO 4 , 0.05M
  • NCZYM broth was then added and the cultures were grown overnight at 37°C on a rotary shaker.
  • Cell debris from the resulting lysed cultures were removed by centrifugation at 150 Oxg for ten minutes at 4°C, and the supernatant was transferred to a new tube.
  • RNase A and DNase I were added to a final concentration of 1 ⁇ g/ml each and the mixture was placed at 37°C for 30 minutes.
  • An equal volume of SM containing 20% PEG and 2M NaCl was added to precipitate the phage and the mixture was left on ice for one hour.
  • the PEG- precipitated phage were pelleted by centrifugation at 12000xg for 20 minutes at 4°C, resuspended in 500 ⁇ l SM, and centrifuged again at 12000xg for 2 minutes at 4°C.
  • the supernatant, now containing the phage was transferred to a new tube and 5 ⁇ l of 10% SDS and 5 ⁇ l of 0.5M EDTA were added to remove the protein coat from the phage DNA. After the tube was incubated for 15 minutes at 68°C, the solution was extracted with an equal volume of Tris saturated phenol:chloroform.
  • the aqueous layer was transferred to a new tube; an equal volume of isopropanol was added; and, the tube was placed at -70°C for 20 minutes.
  • the phage DNA was pelleted, washed with 70% ethanol, dried under vacuum, and resuspended in TE buffer.
  • the DNA was digested with ECQRI (New England BioLabs, Beverly, MA) for 2 hours at 37°C and the desired 1.2 kb fragment was purified by electrophoresis on and elution from a 0.7% low melting point agarose gel.
  • the isolated fragment was ligated in an EcoRI cut M13mpl8 vector (Bethesda
  • the agar plugs were each transferred to 1.5 ml of TY broth containing 10 ⁇ l of an M VI 190 overnight culture.
  • the resulting cultures were grown for six hours at 37°C on a rotary shaker, and the resulting phage lysates were transferred to fresh tubes after first removing the residual cell debris by centrifugation. Two 5 such lysates were chosen for DNA sequence analysis.
  • the 7.2 kb isolated BamHI fragment was purified from a BamHI digest of the
  • pJL21 transformation vector was constructed by the addition to a pUC18 E. coli plasmid backbone to a 4.3 kb EcoRI fragment containing a beta-tubulin gene isolated from a benomyl resistant A. niger
  • pJL21 also contains a 550 bp lambda cos fragment which enables the vector to be used for cosmid formation when appropriate size inserts are included.
  • a C. acremonium transformation vector was constructed with a phleomycin resistant gene as a dominate selectable marker. This was accomplished first by isolating a 660 bp fragment, containing the phleomycin resistance gene (a).
  • IPNS-promoter fragment isolated in this manner was ligated into the" TM"
  • Example X Isolation of Plasmid DNA E. coli cultures containing the pCLS5 plasmid were grown in 500ml LB broth
  • the plasmid DNA was pelleted at 4000xg for 20 minutes at 4°C and then washed with 70% ethanol and dried briefly. The pellet was resuspended in 9 ml TE buffer, then 10 grams of CsCl and 0.387 ml of a lOmg/ml ethidium bromide solution were added. This solution was centrifuged at 313,100xg for 24 hours. The resulting plasmid band in the ethidium gradient was visualized with ultraviolet light, isolated, and then the ethidium bromide was removed using water saturated butanol for extraction. The
  • Protoplasts from strains PF14-1 and M40 were created by growing the cells for 48 hours at 30°C in CCM broth. Transformation of the C. acremonium protoplasts was carried out as described by Queener et al., 1985, with modifications described by Skatrud et al., 1987. Transformation vectors are described in Example IX, above. Northern Blotting
  • RNA molecular weight markers (0.16-1.77 kb ladder; and, 0.24-9.5 kb ladder, supplied by Bethesda Research Labs) were also applied to the gel for size comparisons.
  • the gel was blotted onto nitrocellulose (Schleicher and Schuell, Keene, NH), baked for one hour at 80°C in a vacuum oven, prehybridized in 50% formamide, 5X SSC, 0.1% SDS, 5X Denhardt's, and
  • FIGURE 3 show hybridization of the isolated cloned acetyltransferase DNA (prepared in Example IV, above) with mRNA (prepared in Example V, above) from the PF14-1 strain (expressing acetyltransferase), but not with m-RNA prepared from the M40 acetyltransferase mutant strain.
  • FIGURE 3 shows a Northern blot of total RNA extracted from Cephalosporin C producing cultures of PF14-1 (production strain), M40 (acetyl transferase mutant), and two transformants of M40.
  • the acetyltransferase gene was " also transferred into Aspergillus niger, i.e., which does not contain acetyltransferase activity.
  • the resultant transformant also expressed cefG mRNA (Example XII, below).
  • Example XII Transformation of Aspergillus niger
  • Spheroplasts were produced by first growing the conidia from A. niger strain NRRL 3 (Northern Regional Research Laboratory, U.S. Dept. of Agriculture, Peoria, IL) in Clutterbuck's complete medium (50 ml of 20X Clutterbuck's salts (120g Na2NO3, 10.4 g KCL, 10.4 g MgSO4-7H20, 30.4 g KH2P04, 2.0 ml Vogel's
  • PPC 50%PEG (MW3500), 20mM KPO4 pH6.3, 50mM CaC12
  • CM molten
  • CM molten
  • CM molten
  • agar containing 1 ⁇ g/ml benomyl
  • the transformation mixture was then distributed between 4 petri dishes and the dishes were incubated for 4 hours at 30°C or until onset of germ tube growth.
  • the plates were then overlayed with 1% peptone in 1% agar containing 1 ⁇ g/ml benomyl. The amount of the overlay was equal to the amount of the regeneration agar.
  • the plates were incubated at 35°C for 3-10 days and observed for generation sf transformant colonies.
  • FIGURE 4 shows a halo of inhibited growth of the indicator bacteria around the disk indicates the presence of Cephalosporin C in the filtrate.
  • FIGURE 4 shows a
  • Cephalosporin C Plate Assay where culture filtrates and a Cephalosporin C standard were spotted on filter disks and then overlayed with Alcaligenes faecalis in the presence and absence of penicillinase. An estimation of the concentration of cephalosporin was determined by comparing the ring diameter to a standard curve of the ring diameters resulting from known concentrations of a
  • Cephalosporin C standards (0.5 ⁇ g/ml to 10 ⁇ g/ml). Culture filtrates of the M40 (blocked) mutant strain had no effect on A. faecalis growth (FIGURE 4, M40) indicating that they did not produce cephalosporin, whereas filtrates of the Cephalosporin C producing strain (PF14-1 or M40 strains transformed with the M13 vector pCLS5), in this case M40-T g and M40-T 5 (FIGURE 3), show a halo around the disc representing growth inhibition of the indicator A. faecalis strain.
  • HPLC High performance liquid chromatography
  • Cephalosporin C in culture filtrates of the C. acremonium strains and from the acetyltransferase assay of A. niger transformants. The analysis was done on
  • the mobile phase (at a 1 ml/min. flow rate) consisted of a 15 minute, 5 to 25% linear gradient of methanol/0.02 M KH 2 P0 4 , pH 4.0 with 2 mM tetrabutylammonium bromide added to both the 5% and 25% solvents as an ion
  • FIGURE 5 shows cephalosporin synthesized by the M40-T 3 transformed strain
  • FIGURE 6 shows that the M40-T 3 product is indistinguishable from a cephalosporin standard (CPC STD, FIGURE 6) when mixed and co- separated on HPLC
  • FIGURE 7 shows the cephalosporin produced by the M40-Tg strain
  • FIGURE 8 shows that the control mutant M40 parent does not synthesize cephalosporin.
  • FIGURE 5 shows an HPLC tracing showing restoration of Cephalosporin C production in the supernatant culture fluid from an M40 (cefG mutant) transformed with the cefG gene. The transformant was designated T3.
  • FIGURE 6 shows an HPLC tracing of the supernatant culture fluid from transformant T3 spiked with Cephalosporin C.
  • FIGURE 7 shows an HPLC tracing demonstrating restoration of Cephalosporin C production in supernatant culture fluid from an M40 (cefG mutant) transformed with the cefG gene. The transformant was designated T5.
  • FIGURE 8 shows an HPLC tracing demonstrating the absence of Cephalosporin C production in supernatant culture fluid from a control M40 (cefG mutant).
  • Cell-free extracts of each strain were made from cultures incubated for 4 days at 28°C by extracting lOOmg of lyophilized vegetative growth with 500 ⁇ l of 0.01 M potassium phosphate buffer, pH 7.0, containing 1 m M 2-mercaptoethanol.
  • the enzyme assay was performed by adding 20 ⁇ l of the cell-free extract to the reaction mixture containing 30 ⁇ l of 0.5 M potassium phosphate buffer (pH 7.5, 10 ⁇ l of 22.8 mM deacetylcephalosporin C, 10 ⁇ l of 7.8 acetyl CoA, 10 ul of acetyl-l- 14 C-CoA (20 ⁇ Ci/mmole), and 20 ⁇ l of 20 mM MgS0 4 . After 20 minutes incubation at 30°C the reaction was stopped by the addition of 60 ⁇ l ethanol. After centrifugation, an aliquot was co-injected with Cephalosporin C standard into the HPLC and analyzed by the previous conditions described in Example XIV, above.
  • 0.5 M potassium phosphate buffer pH 7.5, 10 ⁇ l of 22.8 mM deacetylcephalosporin C, 10 ⁇ l of 7.8 acetyl CoA, 10 ul of acetyl-l-
  • ATCC #11550 was transformed with a vector pCLS5 which contained the Cephalosporium acetyltransferase gene and the resulting transformants were analyzed for copy number and Cephalosporin C production.
  • the copy number was determined by comparing intensities of hybridizing bands on a Southern blot which was probed with a cefG genomic DNA probe. The intensities of the bands were measured by a densitometer using a single copy control, i.e., the endogenous acetyltransferase gene.
  • the results are presented in Table II.
  • the T3 M40 transformant (M40-T3) showed 3-4 extra copies of the acetyltransferase gene and M40-T5 showed only 1 extra copy.
  • ATATCGCTCT TCACACTGGA ATCTGGCGTC ATCCTTCGCG ATGTACCCGT GGCATACAAA 120 TATAGCGAGA AGTGTGACCT TAGACCGCAG TAGGAAGCGC TACATGGGCA CCGTATGTTT
  • TTCGACACCC ACGACATCAG CAGAGGCCGG GCAGGATCAA TCCCGGAGGC TCTGGCAATG 960 AAGCTGTGGG TGCTGTAGTC GTCTCCGGCC CGTCCTAGTT AGGGCCTCCG AGACCGTTAC
  • GGTCATGACT TCTTTGTAAT GGAAGCGGAC AAGGTTTAAT GATGCCGTCA GAGGATTCCT 1140 CCAGTACTGA AGAAACATTA CCTTCGCCTG TTCCAAATTA CTACGGCAGT CTCCTAAGGA
  • GTCGGCCCAA GTGGCCCGTC TAAAGCCGGA CCCCTTTCCC CCGAGTCTCT CCCCGATCCC 120

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Abstract

Cette invention concerne une sequence d'ADN comprenant une séquence de nucléotide codant un polypeptide dans une cellule hôte présentant une activité de Céphalosporine C synthétase (acétyl-transférase DAC) qui est la dernière enzyme dans la synthèse des céphalosporines. Des modes de réalisation de l'invention concernent des procédés et des compositions permettant de détecter le gène ou son produit, des mutants ou des hybrides dudit gène, des vecteurs d'expression et de clonage réplicatif et des procédés de préparation du polypeptide acétyltransférase DAC recombiné utile pour la synthèse acellulaire d'antibiotiques constitués de céphalosporine naturelle ou synthétique.
PCT/US1992/002087 1991-03-18 1992-03-13 Isolement et sequence du gene acetyle coa=deacetyl-cephalosporin acetyltransferase Ceased WO1992017496A1 (fr)

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 182, No. 3, issued 14 February 1992, A. MATSUDA et al., "Molecular Cloning of Acetyl Coenzyme A: Deacetylcephalosporin C O-Acetyltransferase cDNA from Acremonium Chrysogenium: Sequence and Expression of Catalytic Activity in Yeast", pages 995-1001. *
BIOTECHNOLOGY, Volume 5, No. 11, issued November 1987, S.M. SAMSON et al., "Cloning and Expression of the Fungal Expandase/Hydroxylase Gene Involved in Cephalosporin Biosynthesis", pages 1207-1214. *
BIOTECHNOLOGY, Volume 7, No. 5, issued May 1989, P.L. SKATRUD et al., "Use of Recombinant DNA to Improve Production of Cephalosporin C by Cephalosporium Acremonium", pages 477-485. *
CHEMICAL ABSTRACTS, Volume 104, No. 2, issued January 1986, A. SCHEIDEGGER et al., "Investigation of Acetyl-CoA: Deacetyl-cephalosporin C O-Acetyltransferase of Cephalosporium Acremonium", see page 238, column 1, abstract No. 16857m; & JOURNAL OF BIOTECHNOLOGY, No. 3, pages 109-117. *
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CN101970462A (zh) * 2008-03-14 2011-02-09 安斯泰来制药株式会社 环状化合物及其盐
EP2261238A4 (fr) * 2008-03-14 2012-04-25 Astellas Pharma Inc Composé cyclique et son sel
US8241872B2 (en) 2008-03-14 2012-08-14 Astellas Pharma Inc. Microorganism producing cyclic compound
AU2009224302B2 (en) * 2008-03-14 2013-07-04 Astellas Pharma Inc. Cyclic compound and salt thereof
US8598115B2 (en) 2008-03-14 2013-12-03 Astellas Pharma Inc. Cyclic compound and salt thereof
AU2009224302C1 (en) * 2008-03-14 2014-01-30 Astellas Pharma Inc. Cyclic compound and salt thereof

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