WO1998011230A1 - Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant - Google Patents

Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant Download PDF

Info

Publication number
WO1998011230A1
WO1998011230A1 PCT/US1996/014791 US9614791W WO9811230A1 WO 1998011230 A1 WO1998011230 A1 WO 1998011230A1 US 9614791 W US9614791 W US 9614791W WO 9811230 A1 WO9811230 A1 WO 9811230A1
Authority
WO
WIPO (PCT)
Prior art keywords
ala
leu
nucleic acid
gly
val
Prior art date
Application number
PCT/US1996/014791
Other languages
English (en)
Inventor
Toshikazu Oki
Tohru Dairi
Original Assignee
Bristol-Myers Squibb Company
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 Bristol-Myers Squibb Company filed Critical Bristol-Myers Squibb Company
Priority to PCT/US1996/014791 priority Critical patent/WO1998011230A1/fr
Publication of WO1998011230A1 publication Critical patent/WO1998011230A1/fr

Links

Classifications

    • 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
    • C12P29/00—Preparation of compounds containing a naphthacene ring system, e.g. tetracycline
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin

Definitions

  • the present invention relates, inter alia, to purified nucleic acids encoding polyketide synthase genes for pradimicin biosynthesis, and purified polypeptides having polyketide synthase activity.
  • Polyketide metabolites are natural products made by microorganisms and plants from simple fatty acids.
  • Many polyketides are used as human and animal pharmaceuticals such as antibiotics, chemotherapeutics and growth promoting agents, as well as flavoring agents and pigments. Biosynthesis of polyketides is believed to occur by a series of condensations of carbon units in a manner similar to that of long chain fatty acids which are formed by fatty acid synthase.
  • the fatty acids are formed by a process in which a chain starter, usually a 2-carbon acetate residue, which is joined by condensation to a chain extender unit, such as malonate, to form an even-numbered chain.
  • a chain extender unit such as malonate
  • the resulting /?-keto group is then processed, by ?-ketoacyl reduction, dehydration and enoyl reduction.
  • the cycle then begins again with the condensation of a new extender unit.
  • a typical fatty acid synthase is a multivalent system involving eight functional units, acetyl, malonyl and palmit ⁇ l transferases, acyl carrier protein, ketoacyl synthase, ketoacyl reductase, dehydratase and enoyl reductase.
  • the organization of these units varies in different organisms. See, for example, EMBO J. 8:2717-2725 (1989).
  • the fatty acid synthesis process differs from polyketide synthesis since most polyketides contain structural complexities due to the use of different starter and extender units, such as acetate, propionate and butyrate.
  • the polyketide synthesis is further complicated by variations in the extent of processing of the ⁇ -carbon (jff-ketoreduction, dehydration, eno ⁇ lreduction) as well as the introduction of chiral carbons. See, for example, Science 252:675-679 (1991 ).
  • tetracenomycin C polyketide synthase genes (tmcf) from Streptomyces glaucescens, for example, have been sequenced, and the sequence data revealed three complete open reading frames.
  • An analysis of the sequence data resulted in a conclusion that polyketide synthesis in S.glaucescens involves a multienzyme complex consisting of at least five types of enzymes. These enzymes, which are homologous to counterparts involved in fatty acid synthesis, are presumably involved in the assembly of the tetracenomycin C decaketide.
  • the structure and function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber has also been studied.
  • This gene cluster has six open reading frames, thereby indicating that the granaticin-producing polyketide synthesis likely consists of at least six separate enzymes involved in carbon chain assembly.
  • Streptomyces polyketide synthase gene clusters involved in the biosynthesis of actinorhodin and the whi ⁇ . spore pigment have also been described. See J. Biol. Chem. 267: 19278-19290 (1992) and Gene 130: 107-1 16 (1993).
  • the molecular organization of the polyketide biosynthesis genes of Saccharopolyspora erythr ⁇ ea, which govern synthesis of the polyketide portion of the macrolide antibiotic erythromycin, is similarly complex.
  • the genes are organized in six repeated units that encode fatty acid synthase-like activities. Two repeated units are contained in a single open reading frame. It is believed that each repeated unit encodes a functional synthase unit and each synthase unit participates in one of six fatty acid synthase-like elongation steps required for the formation of the polyketide. See EMBO J. 8:2727-2736 (1989).
  • each synthase unit carries the elements required for the condensation process, for selecting the particular extender unit to be incorporated, and for the extent of processing that the 0-carbon will undergo.
  • ACP acyl carrier protein
  • Pradimicin A has a unique dihydro- benzo[a]naphthacenequinone aglycon substituted with D-alanine and two sugars, and is a potent antifungal antibiotic produced, for example, by Actinomadura hibisca and Actinomadura verrucosospora subsp. neohibisca. See, for example, J. Antibiot.
  • Pradimicin is an antibiotic useful for multiple purposes, particularly for use as a pharmaceutical.
  • pradimicin has been shown to have activity against system fungal infections caused by Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. Further, pradimicin is active in vitro against a wide variety of fungi and yeasts, some Gram-positive bacteria, and viruses. J. Org. Chem. 54:2536-2539 (1989). Purified polypeptides having polyketide synthase activity and purified nucleic acids encoding such polypeptides are therefore desirable, for example, to provide pharmaceutically useful products.
  • One preferred embodiment of the present invention is a substantially pure nucleic acid comprising a nucleic acid sharing at least about 75% nucleic acid identity with an open reading frame (ORF) of an Actinomadura polyketide synthase gene, and more preferably, at least about 80% identity, and most preferably, at least about 90% identity.
  • the nucleic acid comprises a nucleic acid selected from the group consisting of SEQ ID NO: 1-12.
  • a further preferred embodiment is a substantially pure nucleic acid comprising a nucleic acid encoding an Actinomadura polyketide synthase gene sharing at least about 75% amino acid identity, and more preferably, at least about 80% identity, and most preferably, at least about 90% identity with a polypeptide encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1 -12.
  • the substantially pure nucleic acid comprises a nucleic acid encoding a polypeptide differing from an Actinomadura polyketide synthase gene by no more than about 20 amino acid substitutions, and more preferably, no more than about 10 amino acid substitutions.
  • the substitutions cause a conservative substitution in the amino acid sequence of the encoded polyketide synthase.
  • the nucleic acids of the invention also include nucleic acid analogs.
  • the present invention provides a substantially pure nucleic acid comprising a nucleic acid encoding a polypeptide sharing at least about 75% amino acid identity with a polyketide synthase for biosynthesis of a benzo(a)naphthacenequinone.
  • the nucleic acid encodes a polypeptide sharing at least about 80%, and more preferably, at least about 90% amino acid identity with a polyketide synthase for biosynthesis of a benzo(a)naphthacenequinone.
  • the polyketide synthase is an Actinomadura polyketide synthase
  • the polyketide is preferably a dihydrobenzo(a)naphthacenequinone aglycon, and preferably pradimicin, such as Pradimicin A, B, C, D, E, FA-1 , FA-2, FL, FS, H, 1 1-O-L-xylosylpradimicin H, L, S, T1 , T2 or BMS181 184.
  • Yet another embodiment of the invention is a substantially pure nucleic acid comprising a nucleic acid that hybridizes, under stringent conditions, to a nucleic acid comprising a nucleic acid encoding a polypeptide sharing at least about 75% amino acid identity with an actinomadura polyketide synthase. More preferably, the nucleic acid hybridizes to a nucleic acid comprising a nucleic acid encoding a polypeptide sharing at least about 80% amino acid identity with an Actinomadura polyketide synthase, and even more preferably, encoding a polypeptide sharing at least about 90% amino acid identity with an Actinomadura polyketide synthase.
  • the nucleic acid hybridizes with a nucleic acid comprising a nucleic acid selected from the group consisting of SEQ ID NO:1-12.
  • a hybridizing nucleic acid can be used, for example, to screen for organisms that produce pradimicin.
  • the invention additionally includes vectors capable of reproducing in a eukaryotic or prokaryotic cell having a nucleic acid described above as well as transformed eukaryotic or prokaryotic cells having such nucleic acid.
  • another preferred embodiment is a transformed eukaryotic or prokaryotic cell comprising a nucleic acid encoding a polypeptide sharing at least about 70% amino acid identity with an Actinomadura polyketide synthase gene, and more preferably, at least about 80% identity, and most preferably, at least about 90% identity.
  • the nucleic acid sequence comprises a nucleic acid selected from the group consisting of SEQ ID NO: 1-12.
  • the transformed cell expresses one of the Actinomadura polyketide synthase genes described herein.
  • Yet another preferred embodiment is a vector capable of reproducing in a eukaryotic or prokaryotic cell comprising a nucleic acid encoding a polypeptide sharing at least about 70% nucleic acid identity with an Actinomadura polyketide synthase gene, and more preferably, at least about 80% identity, and most preferably, at least about 90% identity.
  • the nucleic acid comprises a nucleic acid selected from the group consisting of SEQ ID NO: 1-12.
  • the inventive vector expresses, intracellularly or extracellularly, one of the Actinomadura polyketide synthases described herein.
  • Another embodiment of the present invention provides a substantially pure polypeptide comprising an amino acid sequence sharing at least about 75% amino acid identity with an Actinomadura polyketide synthase, and more preferably, at least about 80% identity, and most preferably, at least about 90% identity.
  • the polypeptide shares at least about 75% amino acid identity with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13-15.
  • Yet another preferred embodiment is a method of preparing pradimicin or a pradimicin analog thereof, comprising transforming a eukaryotic or prokaryotic cell with an expression vector for expressing intracellularly or extracellularly a nucleic acid comprising a nucleic acid encoding a polypeptide sharing at least about 70% amino acid identity with an Actinomadura polyketide synthase, growing the transformed cell in culture, and isolating the pradimicin or analog thereof from the transformed cell or the culture medium.
  • the polypeptide shares at least about 80% amino acid identity with an Actinomadura polyketide synthase, and more preferably, the polypeptide shares at least about 90% amino acid identity with an Actinomadura polyketide synthase.
  • the expression vector comprises a nucleic acid encoding all polyketide synthase genes necessary for synthesis of pradimicin, such as SEQ ID NO:1.
  • Figure 1 shows the chemical structure of two types of pradimicin, pradimicin A and pradimicin S.
  • Figure 2 shows conserved amino acid sequences in ⁇ - ketosynthases and acyl transferases for granaticin, tetracenomycin and actinorhodin. These conserved sequences were used to create two probes for cloning the polyketide synthase genes in Actinomadura.
  • Figure 3 shows a restriction map of Actinomadura polyketide synthase genes, ORFs 1-1 1.
  • Figure 4 provides an alignment of the Actinomadura ORF1 gene product ("A") (SEQ ID NO: 13) with a Streptomyces polyketide synthase gene product for tetracenomycin biosynthesis (“B”).
  • A Actinomadura ORF1 gene product
  • B Streptomyces polyketide synthase gene product for tetracenomycin biosynthesis
  • Figure 5 provides an alignment of the Actinomadura ORF2 gene product ("A") (SEQ ID NO: 14) with a Streptomyces polyketide synthase gene product for actinorhodin biosynthesis ("B").
  • A Actinomadura ORF2 gene product
  • B Streptomyces polyketide synthase gene product for actinorhodin biosynthesis
  • the present invention provides, inter alia, nucleic acids and corresponding amino acid sequences of Actinomadura polyketide synthase genes.
  • the polyketide synthases are responsible for the biosynthesis of pradimicin, such as zwitterionic pradimicins A, B and C, which are produced, for example, by Actinomadura hibisca, and pradimicin S, which is produced, for example, by Actinomadura spinosa.
  • pradimicin such as zwitterionic pradimicins A, B and C, which are produced, for example, by Actinomadura hibisca
  • pradimicin S which is produced, for example, by Actinomadura spinosa.
  • Figure 1 which provides the chemical structures of pradimicins A and S. See also J. Antibiot. 43:755-762 (1990).
  • Pradimicin is useful, for example, as an antibiotic, including use as an anti-fungal and an antiviral
  • pradimicin has been shown to have activity against system fungal infections caused by Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. Further, pradimicin is active in vitro against a wide variety of fungi and yeasts, some Gram-positive bacteria, and viruses. J. Org. Chem. 54:2536-2539 (1989). For instance, pradimicin is believed to be active against HIV. See, for example, J. Antibiot. 41 : 1708 (1988) and Virology 176:467 (1990). Techniques used in the prior art were not applicable for cloning pradimicin A biosynthetic genes from Actinomadura hibisca.
  • antibiotic biosynthetic genes including self-defense genes in actinomycetes are clustered in a genomic region.
  • the close linkage between antibiotic biosynthetic genes and self-defense genes has provided a useful tool for cloning of antibiotic biosynthetic genes, since transformants carrying antibiotic resistance determinants can be selected.
  • this technique could not be applied to the cloning of the pradimicin A biosynthetic gene cluster because pradimicin A had not been shown to have significant antibacterial activity.
  • polyketide synthase genes for pradimicin A biosynthesis were cloned from Actinomadura hibisca using oligonucleotide probes based on the conserved amino acid sequences of other polyketide synthase genes, followed by cloning of the flanking region of pradimicin A polyketide synthase genes.
  • certain amino acid sequences of ⁇ -keto synthase, acyl transferase and acyl carrier protein of polyketide synthases are strongly conserved in Streptomyces strains producing polyketide antibiotics. See Annu. Rev. Microbiol. 47:875-912 (1993) and J. Biol. Chem. 267: 19278-19290 (1992). Based on these sequences, two oligonucleotide probes were synthesized, as shown in Figure 2. See also Example 1 , which provides experimental details of the cloning of the pradimicin A polyketide synthase genes.
  • ORF1 spans from position 72 (beginning with GTG) to position 1347 (ending with TGA); ORF2 spans from 1346 (GTG) to 2567 (TGA); ORF3 spans from 2594 (ATG) to 2855 (TGA); ORF4 spans from 2854 (ATG) to 3313 (TGA); ORF5 spans from 3312 (GTG) to 3771 (TGA); ORF6 spans from 3794 (ATG) to 4817 (TGA); ORF7 spans from 4857 (ATG) to 5595 (TGA); ORF8 spans from 5594 (GTG) to 5933 (TGA); ORF9 spans from 5932 (GTG) to 6241 (TAA); ORF10 spans, in reverse direction, from 7534 (ATG) to 6301 (TAG) and ORF1 1 spans from 7668 (ATG) to 8010 (TGA).
  • ORF1 , ORF2 and ORF3 have particularly strong similarities (50% - 70% amino acid identity) with polyketide synthases for actinorhodin biosynthesis. See, for example, Figure 4, which provides an alignment of the ORF1 gene product with a Streptomyces polyketide synthase gene product for tetracenomycin biosynthesis, and Figure 5, which provides an alignment of the ORF2 gene product with a Streptomyces polyketide synthase gene product for actinorhodin biosynthesis. See also Table 1 below.
  • ORF6 37,004 tcm protein of S. glaucescens (47%/330) ⁇ l
  • ORF1 1 1 15 13,036 Hypothetical protein 7 of S. coelicolor (51 % 107) 6 c ⁇ rG protein of S. cyaneus (45%/106) 7 ' tcm ⁇ protein of S. glaucescens (35%/105) 191
  • the present invention provides, inter alia, nucleic acids encoding Actinomadura polyketide synthase genes and polypeptides and analogs thereof, including nucleic acids that bind to an Actinomadura polyketide synthase gene.
  • the nucleic acids can be used, for example, to screen for organisms that produce pradimicin or that have homologous polyketide synthase gene sequences. Further, the nucleic acids can be used, for instance, to synthesize polyketide synthases, which can in turn be used, for example, to produce pradimicin.
  • the Actinomadura species include but are not limited to Actinomadura hibisca, Actinomadura verrucosospora, and particularly subsp.
  • the present invention provides, inter alia, nucleic acids.
  • the nucleic acid embodiments of the invention are preferably deoxyribonucleic acids (DNAs), both single- and double-stranded, and most preferably double-stranded deoxyribonucleic acids. However, they can also be ribonucleic acids (RNAs), as well as hybrid RNA:DNA double-stranded molecules.
  • DNAs deoxyribonucleic acids
  • RNAs ribonucleic acids
  • Nucleic acids encoding an Actinomadura polyketide synthase gene include all Actinomadura polyketide synthase gene-encoding nucleic acids, whether native or synthetic, RNA, DNA, or cDNA, that encode an Actinomadura polyketide synthase gene, or the complementary strand thereof, including but not limited to nucleic acid found in an
  • Actinomadura polyketide synthase gene-expressing organism For recombinant expression purposes, codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic polyketide synthase- encoding nucleic acid.
  • the present invention provides a substantially pure nucleic acid comprising a nucleic acid encoding a polypeptide sharing at least about 75% amino acid identity with a polyketide synthase for biosynthesis of a benzo( ⁇ )naphthacenequinone.
  • the nucleic acid encodes a polypeptide sharing at least about 80%, and more preferably, at least about 90% amino acid identity with a polyketide synthase for biosynthesis of a benzo( ⁇ )naphthacenequinone.
  • the polyketide synthase is an Actinomadura polyketide synthase
  • the polyketide is preferably a dih ⁇ drobenzo(a)naphthacenequinone aglycon, and preferably pradimicin, such as Pradimicin A, B, C, D, E, FA-1 , FA-2, FL, FS, H, 1 1-O-L-xylosylpradimicin H, L, S, T1 , T2 or BMS181 184.
  • pradimicin such as Pradimicin A, B, C, D, E, FA-1 , FA-2, FL, FS, H, 1 1-O-L-xylosylpradimicin H, L, S, T1 , T2 or BMS181 184.
  • nucleic acids encoding an Actinomadura polyketide synthase gene includes nucleic acids encoding polypeptides that are homologous to or share a percentage amino acid identity with Actinomadura polyketide synthases. Numerous methods for determining percent homology are known in the art. One preferred method is to use version 6.0 of the GAP computer program for making sequence comparisons. The program is available from the University of Wisconsin Genetics Computer Group and utilizes the alignment method of Needleman and Wunsch, J. Mol.
  • determining percent identity is also known in the art, such as use of the FASTA computer program, which is also available from the University of Wisconsin.
  • the program used to determine percent identity is the DNASIS program, which is available from Hitachi Corp. (Tokyo, Japan).
  • nucleic acids of the invention include, for example, the nucleic acids of SEQ ID NO: 1
  • the invention is also directed to a nucleic acid encoding a segment of an Actinomadura polyketide synthase gene.
  • the encoded polypeptide will be effective to perform its function, such as an enzymatic function, that is performed by the full-size polyketide synthase.
  • one approach is to take an Actinomadura polyketide synthase gene cDNA and create deletional mutants lacking segments at either the 5' or the 3' end by, for instance, partial digestion with S1 nuclease, Bal 31 or Mung Bean nuclease (the latter approach described in literature available from Stratagene, San Diego, CA, in connection with a commercial deletion cloning kit).
  • the deletion mutants are constructed by subcloning restriction fragments of an Actinomadura polyketide synthase gene cDNA. The deletional constructs are cloned into expression vectors and tested for their polyketide synthase activity.
  • mutant genes can be altered by mutagenesis methods such as that described by Adelman et al., DNA, 2: 183 (1983) or through the use of synthetic nucleic acid strands. The products of mutant genes can be tested for polyketide synthase activity.
  • the nucleic acid sequences can be further mutated, for example, to incorporate useful restriction sites. See Maniatis et ai. Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Press, 1989). Such restriction sites can be used to create "cassettes," or regions of nucleic acid sequence that are facilely substituted using restriction enzymes and ligation reactions.
  • the cassettes can be used to substitute synthetic sequences encoding mutated Actinomadura polyketide synthase amino acid sequences. Actinomadura polyketide synthase gene-encoding sequences can be, for instance, substantially or fully synthetic. See, for example, Goeddel et al., Proc. Natl. Acad. Sci.
  • codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic Actinomadura polyketide synthase gene-encoding nucleic acid. Since the nucleic acid code is degenerate, numerous nucleic acid sequences can be used to create the same amino acid sequence.
  • the invention also relates to a mutated or deleted version of an Actinomadura polyketide synthase nucleic acid that encodes a polypeptide that preferably retains polyketide synthase activity. Conservative mutations are preferred. Such conservative mutations include mutations that switch one amino acid for another within one of the following groups:
  • Aromatic residues Phe, Tyr and Trp.
  • the types of substitutions selected may be based on the analysis of the frequencies of amino acid substitutions between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, (Springer- Verlag, 1978), pp. 14-16, on the analyses of structure-forming potentials developed by Chou and Fasman, Biochemistry 13: 21 1 (1974) or other such methods reviewed by Schulz et al, Principles in Protein Structure, (Springer-Verlag, 1978), pp. 108- 130, and on the analysis of hydrophobicity patterns in proteins developed by Kyte and Doolittle, J. Mol. Biol. 157: 105-132 (1982).
  • the present invention includes analogs of Actinomadura polyketide synthases that preferably retain polyketide synthase activity.
  • the analogs will share at least about 75% amino acid identity, more preferably, at least about 80% identity, even more preferably, at least about 85% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity to an Actinomadura polyketide synthase, such as the polypeptide of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO:15.
  • the polypeptides of the invention are made as follows, using a gene fusion.
  • fusion to maltose-binding protein MBP
  • MBP maltose-binding protein
  • the hybrid protein can be purified, for example, using affinity chromatography using the binding protein's substrate. See, for example, Gene 67: 21 -30 (1988).
  • a cross-linked amylose affinity chromatography column can be used to purify the protein.
  • the cDNA specific for a given polyketide synthase or analog thereof can also be linked using standard means to a cDNA for glutathione S-transf erase ("GST"), found on a commercial vector, for example.
  • GST glutathione S-transf erase
  • the fusion protein expressed by such a vector construct includes the polyketide synthase or analog and GST, and can be treated for purification.
  • the linkers are designed to lack structure, for instance using the rules for secondary structure-forming potential developed by Chou and Fasman, Biochemistry 13, 21 1 , 1974.
  • the linker is also designed to incorporate protease target amino acids, such as trypsin, arginine and lysine residues.
  • protease target amino acids such as trypsin, arginine and lysine residues.
  • standard synthetic approaches for making oligonucleotides are employed together with standard subcloning methodologies.
  • Other fusion partners other than GST or MBP can also be used.
  • Actinomadura polyketide synthases can be directly synthesized from nucleic acid (by the cellular machinery) without use of fusion partners.
  • nucleic acids having the sequence of any of SEQ ID NO: 1-12 are subcloned into an appropriate expression vector having an appropriate promoter and expressed in an appropriate organism.
  • Antibodies against Actinomadura polyketide synthases can be employed to facilitate purification.
  • a polypeptide or nucleic acid is "isolated” in accordance with the invention in that the molecular cloning of the nucleic acid of interest, for example, involves taking an Actinomadura polyketide synthase gene nucleic acid from a cell, and isolating it from other nucleic acids. This isolated nucleic acid may then be inserted into a host cell, which may be yeast or bacteria, for example.
  • a polypeptide or nucleic acid is "substantially pure” in accordance with the invention if it is predominantly free of other polypeptides or nucleic acids, respectively.
  • a macromolecule such as a nucleic acid or a polypeptide, is predominantly free of other polypeptides or nucleic acids if it constitutes at least about 50% by weight of the given macromolecule in a composition.
  • the polypeptide or nucleic acid of the present invention constitutes at least about 60% by weight of the total polypeptides or nucleic acids, respectively, that are present in a given composition thereof, more preferably about 80%, still more preferably about 90%, yet more preferably about 95%, and most preferably about 100%.
  • Such compositions are referred to herein as being polypeptides or nucleic acids that are 60% pure, 80% pure, 90% pure, 95% pure, or 100% pure, any of which are substantially pure.
  • the present invention provides methods for identifying polypeptides that are homologous to an Actinomadura polyketide synthase using an Actinomadura polyketide synthase cDNA, for example.
  • probes for Actinomadura polyketide synthase expression can be used, for example, to detect the presence of an Actinomadura polyketide synthase.
  • probes include antibodies directed against an Actinomadura polyketide synthase or fragments thereof, nucleic acid probes that hybridize, under stringent conditions, to an Actinomadura polyketide synthase mRNA, and oligonucleotides that specifically prime a PCR amplification of an Actinomadura polyketide synthase mRNA.
  • nucleic acid molecules that bind to an Actinomadura polyketide-encoding nucleic acid under high stringency conditions are identified functionally, or by using the hybridization rules reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Press, 1989). Many deletional or mutational analogs of nucleic acid sequences for an Actinomadura polyketide synthase are effective hybridization probes for Actinomadura polyketide synthase-encoding nucleic acid. Accordingly, the present invention relates to nucleic acids that hybridize with such Actinomadura polyketide synthase-encoding nucleic acids under stringent conditions. Preferably, the nucleic acid of the present invention hybridizes, under stringent conditions, with at least a segment of any of the nucleic acids described as SEQ ID NO: 1-12.
  • “Stringent conditions” refers to conditions that allow for the hybridization of substantially related nucleic acids, where relatedness is a function of the sequence of nucleotides in the respective nucleic acids. For instance, for a nucleic acid of 100 nucleotides, such conditions will generally allow hybridization thereto of a second nucleic acid having at least about 85% homology, and more preferably having at least about 90% homology. Such hybridization conditions are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Press, 1989).
  • PCR polymerase chain reaction
  • PCR methods of amplifying nucleic acids utilize at least two primers. One of these primers is capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction.
  • the other is capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence is initially hypothetical, but is synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer.
  • Conditions for conducting such amplifications are well known. See, for example, PCR Protocols (Cold Spring Harbor Press, 1991 ).
  • Antibodies against Actinomadura polyketide synthases can also be used to identify polypeptides that are homologous to Actinomadura polyketide synthases.
  • Antigens for eliciting the production of antibodies against an Actinomadura polyketide synthase can be produced recombinantly by expressing all of or a part of the nucleic acid of an Actinomadura polyketide synthase in a bacteria or a yeast or other eukaryotic cell line.
  • the recombinant protein is expressed as a fusion protein, with the non-Actinomadura polyketide synthase portion of the protein serving either to facilitate purification or to enhance the immunogenicity of the fusion protein.
  • the non-Actinomadura polyketide synthase portion comprises a protein for which there is a readily-available binding partner that is utilized for affinity purification of the fusion protein.
  • the antigen includes an "antigenic determinant," i.e., a minimum portion of amino acids sufficient to bind specifically with an ant ⁇ -Actinomadura polyketide synthase antibody.
  • Antisera to an Actinomadura polyketide synthase can be made, for example, by creating an Actinomadura polyketide synthase antigen by linking a portion of the cDNA for Actinomadura polyketide synthase to a cDNA for glutathione s-transferase ("GST") found on a commercial vector.
  • GST glutathione s-transferase
  • the resulting vector expresses a fusion protein containing an antigenic segment of an Actinomadura polyketide synthase and GST that is readily purified from the expressing bacteria using a glutathione affinity column.
  • the purified antigenic fusion protein is used to immunize rabbits.
  • the present invention also provides polyketides, including purified pradimicin and pradimicin analogs, and methods for synthesizing polyketides.
  • a vector containing a nucleic acid comprising SEQ ID NO:1 can be expressed in an organism, preferably Streptomyces, thereby resulting in pradimicin A synthesis.
  • all of the polyketide synthase genes required for polyketide synthesis are present in a single vector, and the genes are preferably in the same configuration as the cDNA.
  • Preferred Streptomyces organisms for polyketide synthesis include, for example, Streptomyces lividans, Streptomyces coelicor and Streptomyces griseus.
  • Preferred vectors for expression include, for example, plasmids plJ61 , plJ702 and plJ922, which are described in Hopwood et. al., Gene Manipulation of Streptomyces, A Laboratory Manual (The John Innes Foundation, Norwich, UK 1985).
  • the vector includes a promoter that functions well at idiophase, which is a stage of secondary metabolite production, such as the promoter of the mel gene, which is present in vector plJ702.
  • Preferred methods for preparing a polyketide such as pradimicin or an analog thereof comprise transforming a eukaryotic or prokaryotic cell with an expression vector for expressing intracellularly or extracellularly a nucleic acid comprising a nucleic acid encoding a polypeptide sharing at least about 70% amino acid identity with an Actinomadura polyketide synthase, growing the transformed cell in culture, and isolating the pradimicin or analog thereof from the transformed cell or the culture medium.
  • the polypeptide shares at least about 80% amino acid identity with an Actinomadura polyketide synthase, and more preferably, the polypeptide shares at least about 90% amino acid identity with an Actinomadura polyketide synthase.
  • the expression vector comprises a nucleic acid encoding all polyketide synthase genes necessary for synthesis of pradimicin, such as SEQ ID NO: 1.
  • SEQ ID NO: 1 The production of pradimicin A, for example, can be detected by the presence of a red pigment. Purification of pradimicin from Actinomadura, for example, is described in J. Antibiot. 41 : 1701 -1704 (1988).
  • the present invention is further exemplified by the following non- limiting example.
  • Escherichia coli XL1-Blue and pSE101 (Biosci. Biotech. Biochem. 59: 1835-1841 (1995)), a shuttle cosmid vector replicable in both Streptomyces lividans and E. coli, were used for preparation of an Actinomadura hibisca genomic library.
  • E coli XL1-Blue and plasmids pUC1 18 and pUC1 19 were used for sequencing analysis.
  • Plasmid and genomic DNA isolations were done by the method of Hopwood et. al., Gene Manipulation of Streptomyces, A Laboratory Manual (The John Innes Foundation, Norwich, UK 1985). Plasmids -from E. coli were prepared with the Qiagen Plasmid Kit (Qiagen Inc., Chatsworth, CA). All restriction enzymes, T4 ligase and calf intestinal alkaline phosphatase were obtained from Takara (Kyoto, Japan). The procedure for library preparation is described, for example, in Mol. Gen. Genet. 236:39-48 (1992).
  • the hybridization conditions employed for reactions with the oligonucleotide probe, 32 P-labeled with T4 kinase were as follows: a Nylon membrane with immobilized DNA was prehybridized at 40 °C for 4 hours in 6X SSC buffer, which contains 5X Denhardt's solution (Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1982)), 0.5% SDS and 100 /g/ml of heat denatured salmon sperm DNA. For overnight hybridization, the same buffer and temperature conditions were used. The genomic DNA blotted filter and plasmid DNA blotted filter were washed twice with 6X SSC buffer at 40 °C for 30 minutes and with 0.6X SSC buffer at 60 °C for 1 hour, respectively.
  • the other probe was synthesized based on the amino acid sequences of the Streptomyces acyl transferase around the serine residue which is believed to be a catalytic domain. See Figure 2, probe 2 (SEQ ID NO:17).
  • Genomic DNA from Actinomadura hibisca P157-2 (ATCC 53557) that was digested with several restriction enzymes was subjected to Southern blot analysis with probes 1 and 2, which were separately labeled with 32 P and then mixed. Weak but specific signals could be detected.
  • a library was prepared from the strain P157-2 and screened by the colony hybridization with probes 1 and 2 under the same conditions as that for genomic Southern analysis.
  • the 8.2-kb Sacl fragment prepared from pPRMI was cloned into the Sac ⁇ sites of pUC1 18 and pUC1 19 (pUC1 18 and pUC1 19 are available, for example, from Takara Syuzo, Kyoto, Japan).
  • pUC1 18 and pUC1 19 are available, for example, from Takara Syuzo, Kyoto, Japan.
  • helper phage M13 KO7 which is also available, for example, from Takara Syuzo.
  • Sequencing was done by the dideoxy chain termination method of Sanger et al., using an automatic DNA sequencer ALF (Pharmacia, Sweden), it was also done with [a- 35 S]-dCTP as the radioactive label.
  • Nucleotide sequence of the DNA fragment hybridized to the probe As one approach to examine whether the DNA fragment hybridized to the probes carries the PKS gene for biosynthesis of PRM A, the nucleotide sequence of the 8.2-kb Sacl fragment containing hybridized region was determined. Computer analysis of the DNA sequence, using Frame Analysis (See Gene 30:157-166 (1984)), revealed eleven ORFs (ORF1-1 1 ), which are oriented in the same direction except for ORF 10. To understand the functions of each the ORFs deduced by DNA sequencing, databases, including DNASIS, were searched using their translated products. The results are summarized in Table 1 , infra.
  • ORF1 , ORF2 and ORF3 gene products show strong similarities (44-73% amino acid identity) with ORF 1 , 2 and 3 gene products of gra (EMBO J. 8:2717-2725 (1989)), tcm (EMBO J. 8:2727- 2736 (1989)) and act (J. Biol. Chem. 267:19278-19290(1992)), which are known to encode condensing enzyme, acyltransferase and acyl carrier protein for granaticin, tetracenomycin and actinorhodin biosynthesis, respectively.
  • the proteins encoded by ORF4 and ORF6 have similarities with the N and C-terminal half of the TcmN protein (J. Bacteriol.
  • the ORF7 gene product is homologous to the fabG product of E coli (J. Biol. Chem. 267:5751-5754 (1992)) (3-ketoacyl-ACP reductase, 38% amino acid identity) and granaticin-producing polyketide synthase chains 5 and 6 (EMBO J. 8:2717-2725 (1989)) (30% and 35% amino acid identity, respectively).
  • ORF8 and ORF9 gene products have some similarity to hypothetical protein 1 participating in spore color formation in Streptomyces coelicolor (Mol. Microbiol. 4: 1679-1691 (1990)) (23 and 24% amino acid identity, respectively) in a limited region.
  • the ORF10 gene product has a significant similarity to a variety of monooxygenases, including cytochrome P450 (28-40% amino acid identity).
  • the ORF1 1 gene product shows similarity with the hypothetical protein 1 participating in spore color formation in Streptomyces coelicolor (Mol. Microbiol.
  • ADDRESSEE Dechert Price & Rhoads
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • AAGCGCCGGT CCGAGGTGAC GAACCGGACG CTGGCGTAGC GCGTCACGAC CCACGCGTGG 7380 TCGCCGGTCG GCAGCACCAC CTTGGCGACC GGGTCGGACG CGCGCAGGCG CGCGTGCTCG 7440 CACGGCGGCT GGAAGGGGTC GTCCGGCCGG AACGGGAAGG CCGGCGTGAC GTCGGGGCGG 7500 GGGTCGACGG TCGGGGCATC CTTCGAGGAG GGCATACGCC AGGCTTGCAA GGACGCCTCG 7560 AAGCGGGCTC .AACGCGGGCT CGCTCCACCG TCCTTCGAGC GGCCCCCGAG CTGCGGTGAC 7620 CACACTCTGC GGCTACCGGC TCACAGCCCC GACCGAGGGA TGGTTCCCAT GGACAGGTTC 7680 CTGATCGTCG CCCGCATGTC CCCCTCGTCG GAGAAGGAGG TGGCGCGCCT GTTCGCCGAG 7740 TC
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CACGCCCTGT AA 312 (2) INFORMATION FOR SEQ ID NO: 11:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CTGTTCGCCG AGTCCGAACG AGGGCACCGA GCTGCCGGAG GTGGCCGGGA CGGTCAGCCG 120
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "probe”
  • ANTI-SENSE NO
  • MOLECULE TYPE other nucleic acid

Landscapes

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

Abstract

La présente invention concerne, entre autre, des acides nucléiques et des séquences d'aminoacides correspondantes de plusieurs gènes de la synthase polykétide Actinomadura qui sont utiles, par exemple, dans la préparation de la pradimicine et des analogues de celle-ci.
PCT/US1996/014791 1996-09-13 1996-09-13 Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant WO1998011230A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US1996/014791 WO1998011230A1 (fr) 1996-09-13 1996-09-13 Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1996/014791 WO1998011230A1 (fr) 1996-09-13 1996-09-13 Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant

Publications (1)

Publication Number Publication Date
WO1998011230A1 true WO1998011230A1 (fr) 1998-03-19

Family

ID=22255792

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/014791 WO1998011230A1 (fr) 1996-09-13 1996-09-13 Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant

Country Status (1)

Country Link
WO (1) WO1998011230A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077222A1 (fr) * 1999-06-14 2000-12-21 Dsm N.V. Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee
US6265202B1 (en) 1998-06-26 2001-07-24 Regents Of The University Of Minnesota DNA encoding methymycin and pikromycin
US6495348B1 (en) 1993-10-07 2002-12-17 Regents Of The University Of Minnesota Mitomycin biosynthetic gene cluster
KR100834257B1 (ko) * 2007-01-25 2008-05-30 고려대학교 산학협력단 프라디마이신의 생합성효소인 ｏ―메틸트랜스퍼라제의유전자가 파괴된 액티노마두라 히비스카 변이균주 및 그생성물인 디메틸프라디마이신
CN114686452A (zh) * 2020-12-31 2022-07-01 中国科学院深圳先进技术研究院 一种人工蛋白骨架及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
C. LE GOUILL ET AL.: "Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase, ketoacyl reductase, acyl carrier protein, and biotin carboxyl carrier protein", MOL. GEN. GENET., vol. 240, 1993, pages 146 - 150, XP000654921 *
K. SAITOH ET AL.: "Pradimicin S, a new pradimicin analog. III. Application of the Frit-FAB LC/MS technique to the elucidation of the pradimicin S biosynthetic pathway", J. ANTIBIOTICS, vol. 48, 1995, pages 162 - 168, XP000654920 *
K. YLIHONKO ET AL.: "A gene cluster involved in nogalamycin biosynthesis from Streptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway", MOL. GEN. GENET., vol. 251, 1996, pages 113 - 120, XP000652375 *
M.A. FERNANDEZ-MORENO ET AL.: "Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin", J. BIOL. CHEM., vol. 267, 1992, pages 19278 - 19290, XP000652285 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6495348B1 (en) 1993-10-07 2002-12-17 Regents Of The University Of Minnesota Mitomycin biosynthetic gene cluster
US6265202B1 (en) 1998-06-26 2001-07-24 Regents Of The University Of Minnesota DNA encoding methymycin and pikromycin
WO2000077222A1 (fr) * 1999-06-14 2000-12-21 Dsm N.V. Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee
KR100834257B1 (ko) * 2007-01-25 2008-05-30 고려대학교 산학협력단 프라디마이신의 생합성효소인 ｏ―메틸트랜스퍼라제의유전자가 파괴된 액티노마두라 히비스카 변이균주 및 그생성물인 디메틸프라디마이신
CN114686452A (zh) * 2020-12-31 2022-07-01 中国科学院深圳先进技术研究院 一种人工蛋白骨架及其应用

Similar Documents

Publication Publication Date Title
AU758421B2 (en) Recombinant oleandolide polyketide synthase
EP2271666B1 (fr) Groupe de gènes nrps-pks, sa manipulation et son utilité
JP2002505881A (ja) スピノシン殺虫剤生産のための生合成遺伝子
CN107868789B (zh) 可利霉素生物合成基因簇
Suwa et al. Identification of two polyketide synthase gene clusters on the linear plasmid pSLA2-L in Streptomyces rochei
WO1999005283A2 (fr) Genes de biosynthese et de transfert des 6-desoxyhexoses chez saccharopolyspora erythraea et chez streptomyces antibioticus et leur utilisation
US6825013B2 (en) Isolation of biosynthesis genes for pseudo-oligosaccharides from Streptomyces glaucescens GLA.O, and their use
US20050003409A1 (en) Cloning genes from streptomyces cyaneogriseus subsp. noncyanogenus for biosynthesis of antibiotics and methods of use
WO1998011230A1 (fr) Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant
WO2002059322A9 (fr) Compositions et methodes liees a la famille de genes biosynthetiques de la daptomycine
US20030175888A1 (en) Discrete acyltransferases associated with type I polyketide synthases and methods of use
US7595187B2 (en) Elaiophylin biosynthetic gene cluster
KR100882692B1 (ko) 부테닐-스피노신 살충제 생산을 위한 생합성 유전자
US20040219645A1 (en) Polyketides and their synthesis
JP2004535175A (ja) ポリケチド生合成用遺伝子及びタンパク質
CN107164394B (zh) 一种非典型角环素类化合物nenestatin A的生物合成基因簇及其应用
JPH1094395A (ja) フレノリシン遺伝子クラスター
US20030143666A1 (en) Genetic locus for everninomicin biosynthesis
EP0792285B1 (fr) Procedes de production des anthracyclines et de leurs intermediaires
JP2004049100A (ja) ミデカマイシンの生合成遺伝子
CA2441275A1 (fr) Batterie de genes pour la biosynthese de la rabelomycine, et leur utilisation pour obtenir des composes en vue du criblage de medicaments
KR100549690B1 (ko) 에프알-008 폴리케타이드 합성에 관여하는 유전자
WO2000077222A1 (fr) Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee
CA2450691C (fr) Genes et proteines participant a la biosynthese des lipopeptides
CN101278050A (zh) 参与噻可拉林生物合成的基因和噻可拉林的异源产生

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: CA

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998513600

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase