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WO2000040596A1 - Gene cluster for production of the enediyne antitumor antibiotic c-1027 - Google Patents

Gene cluster for production of the enediyne antitumor antibiotic c-1027 Download PDF

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Publication number
WO2000040596A1
WO2000040596A1 PCT/US2000/000446 US0000446W WO0040596A1 WO 2000040596 A1 WO2000040596 A1 WO 2000040596A1 US 0000446 W US0000446 W US 0000446W WO 0040596 A1 WO0040596 A1 WO 0040596A1
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orf
nucleic acid
enediyne
open reading
gene cluster
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PCT/US2000/000446
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French (fr)
Inventor
Ben Shen
Wen Liu
Steven D. Christenson
Scott Standage
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The Regents Of The University Of California
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Priority claimed from US09/477,962 external-priority patent/US6927286B1/en
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO2000040596A1 publication Critical patent/WO2000040596A1/en

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    • 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
    • C12P19/58—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound through only acyclic carbon atoms to a non-saccharide heterocyclic ring, e.g. bleomycin, phleomycin
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/14—Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring
    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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

Definitions

  • This invention relates to the field of enediyne antibiotics.
  • this invention elucidates the gene cluster controlling the biosynthesis ofthe C-1027 enediyne.
  • the enediyne antibiotics are currently the focus of intense research activity in the fields of chemistry, biology, and medical sciences, because of their unique molecular architecture, biological activities, and modes of actions (Doyle and Borders (1995) Enediyne antibiotics as antitumor agents. Marcel-Dekker, New York, Thorson et al. (1999) Bioorg. Chem., 27: 172-188). Since the unveiling ofthe structure of neocarzinostatin chromophore (Edo et al. (1985) Tetrahedron Lett. 26: 331-340) in 1985, the enediyne family has grown steadily.
  • the calicheamicin/esperamicin type which includes the calicheamicins, the esperamicins, and namenamicin
  • the dynemicin type the chromoprotein type, consisting of an apoprotein and an unstable enediyne chromophore.
  • the latter group includes neocarzinostatin, kedarcidin, C-1027 (Fig.
  • the enediyne antibiotics are the most potent, highly active antitumor agents ever discovered. Some members are 1000 times more potent than adriamycin, one ofthe most effective, clinically used antitumor antibiotics (Zhen et al. (1989) J. Antibiot. 42: 1294-1298). All members of this family contain a unit consisting of two acetylenic groups conjugated to a double bond or incipient double bond within a nine or ten-membered ring; i.e., the enediyne core as exemplified by C-1027 in Fig. 1.
  • Elucidation of these aspects provides access to rational engineering of enediyne biosynthesis for novel drug leads and makes it possible to construct enediyne overproducing strains by de-regulating the biosynthetic machinery.
  • elucidation of an enediyne gene cluster contributes to the general field of combinatorial biosynthesis by expanding the repertoire of novel polyketide synthase (PKS) and deoxysugar biosynthesis genes as well as other genes uniquely associated with enediyne biosynthesis, leading to the making of novel enediynes via combinatorial biosynthesis.
  • PKS polyketide synthase
  • This invention provides nucleic acid sequences and characterization ofthe gene cluster responsible for the biosynthesis ofthe enediyne C-1027 (produced by Streptomyces globisporus).
  • structural and functional characterization is provided for the 50 open reading frames (ORFs) comprising this gene cluster.
  • this invention provides an isolated nucleic acid comprising a nucleic acid selected from the group consisting of a nucleic acid encoding any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA), a nucleic acid encoding a polypeptide encoded by any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA); and a nucleic acid amplified by polymerase chain reaction (PCR) using primer pairs that amplify any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA).
  • PCR polymerase chain reaction
  • preferred nucleic acids comprise a nucleic acid encoding at least two (more preferably at least three or more) open reading frames (ORFs) selected from the group consisting of ORF- 1 through ORF 42, excluding ORF 9 (cagA).
  • ORFs open reading frames
  • this invention provides an isolated nucleic acid comprising a nucleic acid that specifically hybridizes under stringent conditions to an open reading frame (ORF) ofthe C-1027 biosynthesis gene cluster, excluding ORF 9 (cagA), and can substitute for the ORF to which it specifically hybridizes to direct the synthesis of an enediyne.
  • this also includes nucleic acids that would stringently hybridizes indicated above, but for, the degeneracy ofthe nucleic acid code.
  • nucleic acids comprises a nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42.
  • Particularly preferred isolated nucleic acid comprises a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42.
  • the nucleic acid may comprises a nucleic acid that is a single nucleotide polymorphism (SNP) of a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 81, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42.
  • SNP single nucleotide
  • This invention also provides an isolated gene cluster comprising open reading frames encoding polypeptides sufficient to direct the assembly of a C-1027 enediyne or a C- 1027 enediyne analogue.
  • the gene cluster may be present in a cell, more preferably in a bacterial cell (e.g. Actinomycetes, Actinoplanetes, Actinomadura, Micromonospora, or Streptomycetes).
  • bacterial cell e.g. Actinomycetes, Actinoplanetes, Actinomadura, Micromonospora, or Streptomycetes.
  • Particular preferred bacterial cells include, but are not limited to
  • the gene cluster may contain one or more open reading frames is operatively linked to a heterologous promoter (e.g. a constitutive or an inducible promoter).
  • a heterologous promoter e.g. a constitutive or an inducible promoter
  • This invention also provides for an polypeptide encoded by any one or more ofthe nucleic acids described herein.
  • host cell(s) e.g. eukaryotic cells or bacterial cells as described herein
  • Preferred host cells are transformed with an exogenous nucleic acid comprising a gene cluster encoding polypeptides sufficient to direct the assembly of a C-1027 enediyne or a C- 1027 enediyne analogue.
  • heterologous nucleic acid may comprise only a portion ofthe gene cluster, but the cell will still be able to express an enediyne.
  • This invention also provides methods of chemically modifying a biological molecule.
  • the methods involve contacting a biological molecule that is a substrate for a polypeptide encoded by a C-1027 biosynthesis gene cluster open reading frame, with a polypeptide encoded by a C-1027 biosynthesis gene cluster open reading frame whereby the polypeptide chemically modifies the biological molecule.
  • the polypeptide is an enzyme selected from the group consisting of a hydroxylase, a homocysteine synthase, a dNDP-glucose dehydrogenase, a citrate carrier protein, a C-methyl transferase, an N-methyl transferase, an aminotransferase, a CagA apoprotein, an NDP- glucose synthase, an epimerase, an acyl transferase, a coenzyme F390 synthase, and epoxidase hydrolase, an anthranilate synthase, a glycosyl transferase, a monooxygenase, a type II condensation protein, an aminomutase, a type II adenylation protein, an O-methyl transferase, a P-450 hydroxylase, an oxidoreductase, and a proline oxidase.
  • a hydroxylase a homocysteine synthas
  • the method involves contacting the biological molecule with at least two (preferably at least three or more) different polypeptides encoded by C-1027 biosynthesis gene cluster open reading frames.
  • the contacting may be in a host cell (e.g. a eukaryotic cell or a bacterial cell) or the contacting can be ex vivo.
  • the biological molecule can be an endogenous metabolite produced by said host cell or an exogenous supplied metabolite.
  • the host cell is a bacterial cell or eukaryotic cell (e.g., a mammalian cell, a yeast cell, a plant cell, a fungal cell, an insect cell, etc.).
  • the host cell synthesizes sugars and glycosylates the biological molecule. In other preferred embodiments, the host cell synthesizes deoxysugars.
  • the method can further involve contacting the biological molecule with a polyketide synthase or a non-ribosomal polypeptide synthetase. The contacting can be in a cell (e.g., a bacterial cell) or ex vivo.
  • the method comprises contacting the biological molecule with at substantially all ofthe polypeptides encoded by C-1027 biosynthesis gene cluster open reading frames and said method produces an enediyne or enediyne analogue.
  • the biological molecule is a fatty acid and the biological molecule is contacted with a C-1027 orf polyeptide selected from the group consisting of an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase.
  • a C-1027 orf polyeptide selected from the group consisting of an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase.
  • the biological molecule is a fatty acid and said biological molecule is contacted with a plurality of C-1027 orf polypeptides comprising an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase.
  • the biological molecule is contacted with polypeptides encoded by ORF 17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38.
  • the biological molecule is contacted with polypeptides encoded by ORF 15, ORF 16, ORF 28, ORF3, ORF 14, and ORF 13, and, in certain embodiments, ORF 4 and ORF 3 as well.
  • the method may comprise contacting a sugar with one or more C-1027 open reading frame polypeptides selected from the group consisting of a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N-methyltransferase, and a glycosyl transferase.
  • Particularly preferred variant of this method comprise contacting a dNDP-glucose with a plurality of C- 1027 open reading frame polypeptides comprising a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N- methyltransferase, and a glycosyl transferase.
  • the method comprises contacting an amino acid with one or one or more C-1027 open reading frame polypeptides selected from the group consisting of a hydroxylase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein.
  • C-1027 open reading frame polypeptides selected from the group consisting of a hydroxylase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein.
  • the amino acid is a tyrosine.
  • This invention also provides a method of synthesizing a chromaprotein type enediyne core, said method comprising contacting a fatty acid with one or more C-1027 orf polypeptides selected from the group consisting of an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase.
  • the fatty acid may be contacted with a plurality of C-1027 orf polypeptides comprising an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase.
  • the fatty acid is contacted with polypeptides encoded by ORF 17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38.
  • this invention provides a method of synthesizing a deoxysugar. This method involves contacting a sugar with one or more C-
  • 1027 open reading frame polypeptides selected from the group consisting of a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C- methyltransferase, an N-methyltransferase, and a glycosyl transferase.
  • this method involves contacting a dNDP-glucose with a plurality of C-1027 open reading frame polypeptides comprising a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N- methyltransferase, and a glycosyl transferase.
  • the dNDP-glucose is contacted with polypeptides encoded by ORF 17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38.
  • This invention also provides methods of synthesizing a beta amino acid by contacting an amino acid with one or one or more C-1027 open reading frame polypeptides selected from the group consisting of a hydroxylase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein.
  • the method preferably comprises contacting an amino acid with a plurality of C- 1027 open reading frame polypeptides comprising a hydroxylase, a halogenase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein.
  • Particularly preferred embodiments comprise contacting the amino acid (e.g. tyrosine) with polypeptides encoded by ORF 4, ORF11, ORF24, ORF23, ORF25, and ORF26.
  • the gene cluster is present in a bacterium (e.g., Actinomycetes, Actinoplanetes, Actinomadura, Micromonospora, or Streptomycetes).
  • Particularly preferred bacteria include, but are not limited to Streptomyces globisporus, Streptomyces lividans, Streptomyces coelicolor, Micromonospora echinospora spp. calichenisis, Actinomadura verrucosopora, Micromonospora chersina, Streptomyces carzinostaticus, and Actinomycete L585-6.
  • the gene cluster is present in a eukaryotic cell (e.g. a mammalian cell, a yeast cell, a plant cell, a fungal cell, an insect cell, etc.).
  • the host cell can be one that synthesizes sugars and glycosylates the enediyne or enediyne analogue.
  • the host can be one that synthesizes deoxysugars.
  • This invention also provides a method of making a cell (e.g., a bacterial or eukaryotic cell) resistant to an enediyne or an enediyne metabolite.
  • This method involves expressing in the cell one or more isolated C-1027 open reading frame nucleic acids that encode a protein selected from the group consisting of a CagA apoprotein, a SgcB transmembrane efflux protein, a transmembrane transport protein, a Na+/H+ transporter, an ABC transport, a glycerol phosphate tranporter, and a UvrA-like protein.
  • the isolated C-1027 open reading frame nucleic acids are selected from the group consisting of ORF 9, ORF2, ORF 27, ORF 0, ORF 1 c-terminus, ORF 2, and ORF 1 N-terminus. Certain embodiments exclude cagA (ORF 9).
  • this invention specifically excludes one or more of open reading frames -7 through 42.
  • this invention excludes cagA (ORF 9), and/or sgcA (ORF 1), and/or sgcB (ORF 2).
  • C-1027 open reading frame and “C-1027 ORF” refer to an open reading frame in the C-1027 biosynthesis gene cluster as isolated from Streptomyces globisporus.
  • the term also embraces the same open reading frames as present in other enediyne-synthesizing organisms (e.g. other strains and/or species of Streptomyces, Actinomyces, and the like).
  • the term encompasses allelic variants and single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • the C-1027 ORF is used synonymously with the polypeptide encoded by the C-1027 ORF and may include conservative substitutions in that polypeptide. The particular usage will be clear from context.
  • isolated refers to material which is substantially or essentially free from components which normally accompany it as found in its native state.
  • nucleic acids and/or polypeptides the term can refer to nucleic acids or polypeptides that are no longer flanked by the sequences typically flanking them in nature.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide.
  • nucleic acid or “oligonucleotide” or grammatical equivalents herein refer to at least two nucleotides covalently linked together.
  • a nucleic acid ofthe present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49:1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; SRocl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.
  • nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev. pp 169- 176). Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. These modifications ofthe ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
  • heterologous as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally associated with a region of a recombinant construct, and/or are not normally associated with a particular cell.
  • a heterologous region of a nucleic acid construct is an identifiable segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature.
  • a heterologous region of a construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature.
  • heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene).
  • a host cell transformed with a construct which is not normally present in the host cell would be considered heterologous for purposes of this invention.
  • a "coding sequence” or a sequence which "encodes" a particular polypeptide is a nucleic acid sequence which is ultimately transcribed and or translated into that polypeptide in vitro and/or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries ofthe coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from procaryotic or eucaryotic mRNA, genomic DNA sequences from procaryotic or eucaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • Expression "control sequences” refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Not all of these control sequences need always be present in a recombinant vector so long as the desired gene is capable of being transcribed and translated.
  • “Recombination” refers to the reassortment of sections of DNA or RNA sequences between two DNA or RNA molecules. "Homologous recombination” occurs between two DNA molecules which hybridize by virtue of homologous or complementary nucleotide sequences present in each DNA molecule.
  • stringent conditions or “hybridization under stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences.
  • “Stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • Expression vectors are defined herein as nucleic acid sequences that are direct the transcription of cloned copies of genes/cDNAs and/or the translation of their mRNAs in an appropriate host. Such vectors can be used to express genes or cDNAs in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Expression vectors include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. Specifically designed vectors allow the shuttling of DNA between hosts, such as bacteria-yeast or bacteria-animal cells.
  • An appropriately constructed expression vector preferably contains: an origin of replication for autonomous replication in a host cell, a selectable marker, optionally one or more restriction enzyme sites, optionally one or more constitutive or inducible promoters.
  • an expression vector is a replicable DNA construct in which a DNA sequence encoding a one or more PKS and/or NRPS domains and/or modules is operably linked to suitable control sequences capable of effecting the expression ofthe products of these synthase and/or synthetases in a suitable host.
  • Control sequences include a transcriptional promoter, an optional operator sequence to control transcription and sequences which control the termination of transcription and translation, and so forth.
  • conservative substitution is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) ofthe molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity).
  • the following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • ORF- 1 through ORF 42 refers to the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 81, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 9, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42 as identified in Tables I and II. In certain embodiments ORF 9 (cagA) is excluded.
  • a “biological molecule that is a substrate for a polypeptide encoded by a enediyne (e.g., C-1027) biosynthesis gene” refers to a molecule that is chemically modified by one or more polypeptides encoded by open reading frame(s) ofthe C-1027 biosynthesis gene cluster.
  • the "substrate” may be a native molecule that typically participates in the biosynthesis of an enediyne, or can be any other molecule that can be similarly acted upon by the polypeptide.
  • a “polymorphism” is a variation in the DNA sequence of some members of a species.
  • a polymorphism is thus said to be "allelic,” in that, due to the existence ofthe polymorphism, some members of a species may have the unmutated sequence (i.e. the original "allele") whereas other members may have a mutated sequence (i.e. the variant or mutant "allele").
  • the polymorphism is said to be diallelic.
  • diallelic diploid organisms three genotypes are possible. They can be homozygous for one allele, homozygous for the other allele or heterozygous.
  • diallelic haploid organisms they can have one allele or the other, thus only two genotypes are possible.
  • the occurrence of alternative mutations can give rise to trialleleic, etc. polymo ⁇ hisms.
  • An allele may be referred to by the nucleotide(s) that comprise the mutation.
  • Single nucleotide polymo ⁇ hism or "SNPs are defined by their characteristic attributes.
  • a central attribute of such a polymo ⁇ hism is that it contains a polymo ⁇ hic site, "X,” most preferably occupied by a single nucleotide, which is the site of the polymo ⁇ hism's variation (Goelet and Knapp U.S. patent application Ser. No.
  • Abbreviations used herein include LB, Luria-Bertani; NGDH, dNDP-glucose 4,6-dehydratase ; nt, nucleotide; ORF, open reading frame; PCR, polymerase chain reaction; PEG, polyethyleneglycol; PKS, polyketide synthase; RBS, ribosomal binding site; Apr, apramycin; R, resistant; Th, thiostrepton; WT, wild-type; and TS, temperature sensitive
  • Figure 1 illustrates the structures of C-1027 chromophore and the benzenoid diradical intermediate proposed to initiate DNA cleavage.
  • Figure 2 illustrates a scheme using C-1027 open reading frame polypeptides for the synthesis of deoxysugars.
  • Figure 3 A illustrates a scheme using C-1027 open reading frame polypeptides for the synthesis of a ⁇ -amino acid.
  • Figure 3B illustrates a scheme using C-1027 open reading frame polypeptides for the synthesis of a benzoxazolinate.
  • Figure 4 illustrates the synthesis of the enediyne core and final assembly of the C-1027 enediyne.
  • Figures 5A, 5B, and 5C illustrate the organization ofthe C-1027 enediyne biosynthetic gene cluster.
  • Figure 5 A shows a restriction map ofthe 75-kb sgc gene cluster from S. globisporus as represented by three cosmid clones.
  • Figure 5B illustrates the genetic organization ofthe sgcA, sgcB, and cagA genes, showing that they are clustered in the sgc gene cluster.
  • Probe 1 the 0.55-kb dNDP-glucose 4,6-dehydratase gene fragment from pBS1002.
  • Probe 2 the 0.73-kb cagA fragment from pBS1003.
  • A Apal; B, R ⁇ mHI; E, EcoRI; K, Kp ⁇ l, S, S ⁇ cII; Sp, Sphl.
  • Figure 5C shows the genetic organization ofthe C-1027 biosynthesis gene cluster.
  • Figure 6 shows the DNA and deduced amino acid sequences ofthe 3.0-kb R ⁇ mHI fragment from pBS1007, showing the sgcA and sgcB genes. Possible RBSs are boxed. The presumed translational start and stop sites are in boldface. Restriction enzyme sites of interest are underlined. The amino acids, according to which the degenerated PCR primer were designed for amplifying the dNDP-glucose 4,6-dehydratase gene from S. globisporus, are underlined.
  • Figure 7 shows the amino acid sequence alignment of SgcA with three other dNDP-glucose 4,6-dehydratases.
  • Gdh TDP-glucose 4,6-dehydratase of S. erythraea (AAA68211); Mtm ⁇ , TDP-glucose 4,6-dehydratase in the mithramycin pathway of S. argillaceus (CAA71847); TylA2, TDP-glucose 4,6-dehydratase in the tylosin pathway of S. fradiae (S49054).
  • Given in parentheses are protein accession numbers. The ⁇ fold with the NAD + -binding motif of GxGxxG is boxed.
  • Figures 8 A and 8B show disruption of sgcA by single crossover homologous recombination.
  • Figure 8 A shows construction of sgcA disruption mutant and restriction maps ofthe wild-type S. globisporus C-1027 and S. globisporus SB 1001 mutant strains showing predicted fragment sizes upon R ⁇ mHI digestion.
  • Figures 8B and 8C show a Southern analysis of S. globisporus C-1027 (lane 1) and S.
  • globisporus SB 1001 (lanes 2, 3, and 4, three individual isolates) genomic DNA, digested with R ⁇ mHI, using ( Figure 8B) ⁇ OJ260 vector or ( Figure 8C) the 0.75-kb SacIVKpnl fragment of sgcA from pBS1012 as a probe, respectively.
  • B R ⁇ mHI; K, Kpnl; S, S ⁇ cII.
  • Figures 9 A, 9B, 9B, and 9D illustrate the determination of C-1027 production in various S. globisporus strains by assaying their antibacterial activity against M. luteus.
  • Figure 9A 1, S. globisporusC-1021; 2,3, and 4, S. globisporus SBIOOI (three individual isolates); 5, S. globisporus AF67; 6, S. globisporus AF40.
  • Figure 9B 1, S. globisporus C- 1027; 2, S. globisporus SB1001 ( ⁇ WHM3); 3 and 4, S. globisporus SBIOOI (pBS1015)(two individual isolates). Both S. globisporus SB1001 (pWHM3) and S.
  • globisporus SBIOOI (pBS 1015) were grown in the presence of 5 ⁇ g/mL thiostrepton.
  • Figure 9C 1 , S. globisporusC-1021; 2, S. globisporus SB1001 (pBS1015); 3. S. globisporus SB1001; 4. S. globisporus SB 1001 (pWHM3); 5. S. globisporus AF40; 6. S. globisporus AF44. All S. globisporus strains were grown in the absence of thiostrepton.
  • Figure 9D 1. S. globisporus (pKCl 139); 2. S. globisporus (pBS1018).
  • C-1027 is produced by Streptomyces globisporus C-1027 and consists of an apoprotein (encoded by the cagA gene) and a non-peptidic chromophore.
  • the C-1027 chromophore could be viewed as being derived biosynthetically from a benzoxazohnate, a deoxyamino hexose, a ⁇ -amino acid, and an enediyne core.
  • Adopting a strategy to clone the C-1027 biosynthesis gene cluster by mapping a putative dNDP-glucose 4,6-dehydratase (NGDH) gene to cagA we localized 75 kb contiguous DNA from S. globisporus encoding a complete C-1027 gene cluster.
  • Initial sequencing ofthe cloned gene cluster revealed two genes, sgcA and sgcB, that encode an NGDH enzyme and a transmembrane efflux protein, respectively, and confirmed that the cagA gene resides approximately 14 kb upstream ofthe sgcA,B locus.
  • the involvement ofthe cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the sgcA gene to generate C-1027-nonproducing mutants and by complementing the sgcA mutants in vivo to restore C-1027 production.
  • the C-1027 open reading frames encode polypeptides exhibiting a wide variety of enzymatic activities (e.g., epoxide hydrase, monooxygenase, oxidoreductase, P-450 hydroxylase, etc.).
  • the isolated C-1027 gene cluster can be used to synthesize C-1027 enediyne antibiotics and/or analogues thereof.
  • the C-1027 gene cluster can be modified and/or augmented to increase C-1027 and/or C-1027 analogue production.
  • various components ofthe C-1027 gene cluster can be used to synthesize and/or chemically modify a wide variety of metabolites.
  • ORF 6 C-methyltransferase
  • ORF 12 an epimerase
  • the ORFs can be combined in their native configuration or in modified configurations to synthesize a wide variety of biomolecules/metabolites.
  • C-1027 open reading frames can be used to synthesize an enediyne core, to synthesize a deoxy sugar, to synthesize a ⁇ -amino acid, to make a benzoxazolinate, etc (see, e.g., Figures 2, 3, and 4).
  • the native C-1027 gene cluster ORFs can be re-ordered, modified, and combined with other biosynthetic units (e.g. polyketide synthases (PKSs) or catalytic domains thereof and/or non-ribosomal polypeptide synthetases (NRPSs) or catalytic domains thereof) to produce a wide variety of molecules.
  • PKSs polyketide synthases
  • NRPSs non-ribosomal polypeptide synthetases
  • Large chemical libraries can be produced and then screened for a desired activity.
  • the C-1027 gene cluster also includes a number of drug resistance genes (see, e.g., Table 2) that confer resistance to C-1027 and/or metabolites involved in C-1027 biosynthesis thereby permitting the cell to complete the enediyne biosynthesis.
  • These resistance genes can be used to confer enediyne resistance on a cell lacking such resistance or to augment the enediyne resistance of a cell that does tolerate enediynes.
  • Such cells can be used to produce high levels of enediynes and/or enediyne metabolites, and/or enediyne analogues.
  • ORF 9 CagA apoprotein Drug sequestering
  • ORF 2 SgcB transmembrane efflux protein Drug exporting
  • the C-1027 gene cluster nucleic acids can be isolated, optionally modified, and inserted into a host cell to create and/or modify a metabolic (biosynthetic) pathway and thereby enable that host cell to synthesize and/or modify various metabolites.
  • the C-1027 gene cluster nucleic acids can be expressed in the host cell and the encoded C- 1027 polypeptide(s) recovered for use as chemical reagents, e.g. in the ex vivo synthesis and/or chemical modification of various metabolites.
  • Either application typically entails insertion of one or more nucleic acids encoding one or more isolated and/or modified C-1027 enediyne open reading frames in a suitable host cell.
  • the nucleic acid(s) are typically in an expression vector, a construct containing control elements suitable to direct expression ofthe C-1027 polypeptides.
  • the expressed C-1027 polypeptides in the host cell then act as components of a metabolic/biosynthetic pathway (in which case the synthetic product ofthe pathway is typically recovered) or the C-1027 polypeptides themselves are recovered.
  • cloning and expression of C- 1027 nucleic acids can be accomplished using routine and well known methods.
  • A) C-1027 nucleic acids A) C-1027 nucleic acids.
  • the nucleic acids comprising the C-1027 gene cluster are identified in Tables I and are listed in the sequence listing provided herein.
  • Table 1 identifies genes and functions of open reading frames (ORFs) in the C-1027 enediyne biosynthesis gene cluster and identifies primers suitable for the amplification/isolation of any one or more of the C-1027 open reading frames.
  • ORFs open reading frames
  • primers suitable for the amplification/isolation of any one or more of the C-1027 open reading frames can be determined according to standard methods well known to those of skill in the art (e.g. using Vector NTI SuiteTM, InforMax, Gaithersberg, MD, USA).
  • amplifications will utilize the DNA or RNA of an organism containing the requisite genes (e.g. Streptomyces globisporus) as a template.
  • Typical amplification conditions include the following PCR temperature program: initial denaturing at 94°C for 5 min, 24-36 cycles of 45 sec at 94°C, 1 min at 60°C, 2 min at 72°C, followed by additional 7 min at 72°C.
  • PCR temperature program initial denaturing at 94°C for 5 min, 24-36 cycles of 45 sec at 94°C, 1 min at 60°C, 2 min at 72°C, followed by additional 7 min at 72°C.
  • optimization of such a protocol e.g. to improve yield, etc. is routine (see, e.g., U.S. Patent No. 4,683,202; Innis (1990) PCR Protocols A Guide to Methods and Applications Academic Press Inc. San Diego, CA, etc).
  • primer may be designed to introduce restriction sites and so facilitate cloning of the amplified sequence
  • this invention provides nucleic acids for the recombinant expression of an enediyne (e.g. a C-1027 enediyne or an analogue thereof).
  • nucleic acids include isolated gene cluster(s) comprising open reading frames encoding polypeptides sufficient to direct the assembly ofthe enediyne.
  • the C-1027 open reading frames may be unchanged, but the control elements (e.g. promoters, enhancers, etc.) may be modified.
  • the nucleic acids may encode selected components (e.g.
  • C-1027 or modified C-1027 open reading frames may optionally contain other heterologous biosynthetic elements including, but not limited to polyketide synthase (PKS) and/or non-ribosomal polypeptide synthetase (NRPS) modules or enzymatic domains.
  • PKS polyketide synthase
  • NRPS non-ribosomal polypeptide synthetase
  • variations may be introduced by design, for example to modify a known molecule in a specific way, e.g. by replacing a single substituent ofthe enediyne with another, thereby creating a derivative enediyne molecule of predicted structure.
  • variations can be made randomly, for example by making a library of molecular variants of a known enediyne by systematically or haphazardly replacing one or open reading frames in the biosynthetic pathway. Production of alternative/modified enediyne, and hybrid enediyne PKSs and/or NRPSs and hybrid systems is described below. Using the information provided herein other approaches to cloning the desired sequences will be apparent to those of skill in the art.
  • the enediyne, and/or optionally PKS and/or NRPS modules or enzymatic domains of interest can be obtained from an organism that expresses such, using recombinant methods, such as by screening cDNA or genomic libraries, derived from cells expressing the gene, or by deriving the gene from a vector known to include the same.
  • the gene can then be isolated and combined with other desired biosynthetic elements using standard techniques. If the gene in question is already present in a suitable expression vector, it can be combined in situ, with, e.g., other PKS subunits, as desired.
  • the gene of interest can also be produced synthetically, rather than cloned.
  • the nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. In general, one will select preferred codons for the intended host in which the sequence will be expressed.
  • the complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence (see, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223: 1299; Jay et al. (1984) J. Biol. Chem. 259:6311). In addition, it is noted that custom gene synthesis is commercially available (see, e.g. Operon Technologies, Alameda, CA).
  • the choice of expression vector depends on the sequence(s) that are to be expressed. Any fransducible cloning vector can be used as a cloning vector for the nucleic acid constructs of this invention. However, where large clusters are to be expressed, it phagemids, cosmids, Pis, YACs, BACs, PACs, HACs or similar cloning vectors be used for cloning the nucleotide sequences into the host cell. Phagemids, cosmids, and BACs, for example, are advantageous vectors due to the ability to insert and stably propagate therein larger fragments of DNA than in M13 phage and lambda phage, respectively.
  • Phagemids which will find use in this method generally include hybrids between plasmids and filamentous phage cloning vehicles.
  • Cosmids which will find use in this method generally include lambda phage-based vectors into which cos sites have been inserted.
  • Recipient pool cloning vectors can be any suitable plasmid.
  • the cloning vectors into which pools of mutants are inserted may be identical or may be constructed to harbor and express different genetic markers (see, e.g., Sambrook et al., supra). The utility of employing such vectors having different marker genes may be exploited to facilitate a determination of successful transduction.
  • vectors are used to introduce C- 1027 biosynthesis genes or gene clusters into host (e.g. Streptomyces) cells.
  • host e.g. Streptomyces
  • Numerous vectors for use in particular host cells are well known to those of skill in the art. For example described in Malpartida and Hopwook, (1984) Nature, 309:462-464; Kao et al., (1994), Science, 265: 509-512; and Hopwood et al, (1987) Methods Enzymol, 153:116-166 all describe vectors for use in various Streptomyces hosts.
  • Streptomyces vectors are used that include sequences that allow their introduction and maintenance in E. coli.
  • Such Streptomyces! E. coli shuttle vectors have been described (see, for example, Vara et al, (1989) J. Bacteriol, 171:5872-5881; Guilfoile & Hutchinson (1991) Proc. Natl. Acad. Sci. USA, 88: 8553-8557.)
  • the wildtype and/or modified C-1027 enediyne open reading frame(s) of this invention can be inserted into one or more expression vectors, using methods known to those of skill in the art.
  • Expression vectors will include control sequences operably linked to the desired open reading frame.
  • Suitable expression systems for use with the present invention include systems that function in eucaryotic and/or prokaryotic host cells. However, as explained above, prokaryotic systems are prefe ⁇ ed, and in particular, systems compatible with Streptomyces spp. are of particular interest. Control elements for use in such systems include promoters, optionally containing operator sequences, and ribosome binding sites. Particularly useful promoters include control sequences derived from enediyne, and/or PKS, and/or NRPS gene clusters. Other promoters (e.g. ermE* as illustrated in Example 1) are also suitable.
  • bacterial promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, will also find use in the present constructs. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the beta -lactamase (bid) promoter system, bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Patent 4,551,433), which do not occur in nature also function in bacterial host cells.
  • promoters In Streptomyces, numerous promoters have been described including constitutive promoters, such as ErmE and TcmG (Shen and Hutchinson, (1994) J. Biol Chem. 269: 30726-30733), as well as controllable promoters such as actl and actlll (P leper et al, (1995) Nature, vol. 378: 263-266; Pieper et al, (1995) J. Am. Chem. Soc, 117: 11373-11374; and Wiesmann et al, (1995) Chem. & Biol. 2: 583-589).
  • constitutive promoters such as ErmE and TcmG (Shen and Hutchinson, (1994) J. Biol Chem. 269: 30726-30733)
  • controllable promoters such as actl and actlll (P leper et al, (1995) Nature, vol. 378: 263-266; Pieper et al, (1995) J
  • regulatory sequences may also be desirable which allow for regulation of expression ofthe enediyne open reading frame(s) relative to the growth ofthe host cell.
  • Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • Selectable markers can also be included in the recombinant expression vectors.
  • markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid.
  • the various enediyne cluster open reading frames, and/or PKS, and/or NRPS clusters or subunits of interest can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter.
  • the various open reading frames can include flanking restriction sites to allow for the easy deletion and insertion of other open reading frames so that hybrid synthetic pathways can be generated. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site- directed mutagenesis and PCR.
  • nucleic acid sequences of well over lOOkb have been introduced into cells, including prokaryotic cells, using vector-based methods (see, for example, Osoegawa et al, (1998) Genomics, 52: 1-8; Woon et al, (1998) Genomics, 50: 306-316; Huang et al, (1996) Nucl. Acids Res., 24: 4202-4209).
  • vector-based methods see, for example, Osoegawa et al, (1998) Genomics, 52: 1-8; Woon et al, (1998) Genomics, 50: 306-316; Huang et al, (1996) Nucl. Acids Res., 24: 4202-4209).
  • the cloning and expression of C-1027 enediyne is illustrated in Example 1.
  • the vectors described above can be used to express various protein components ofthe enediyne, and/or enediyne shunt metabolites, and/or other modified metabolites for subsequent isolation and/or to provide a biological synthesis of one or more desired biomolecules (e.g. C-1027 and/or a C-1027 analogue, etc.).
  • desired biomolecules e.g. C-1027 and/or a C-1027 analogue, etc.
  • the proteins are expressed in any prokaryotic or eukaryotic cell suitable for protein expression. In one prefe ⁇ ed embodiment, the proteins are expressed in E. coli.
  • Host cells for the recombinant production ofthe subject enediynes, enediyne metabolites, shunt metabolites, etc. can be derived from any organism with the capability of harboring a recombinant enediyne gene cluster and/or subset thereof.
  • the host cells of the present invention can be derived from either prokaryotic or eucaryotic organisms.
  • Prefe ⁇ ed host cells are those of species or strains (e.g. bacterial strains) that naturally express enediynes. Such host cells include, but are not limited to Actinomycetes,
  • Streptomyces lividans Streptomyces coelicolor, Micromonospora echinospora spp. calichenisis, Actinomadura verrucosopora, Micromonospora chersina, Streptomyces carzinostaticus, and Actinomycete L585-6.
  • Other suitable host cells include, but are not limited to S. verticillis S. ambofaciens, S. avermitilis, S. azureus, S. cinnamonensis, S. coelicolor, S. curacoi, S. erythraeus, S.fradiae, S. galilaeus, S.
  • a eukaryotic host cell is prefe ⁇ ed (e.g. where certain glycosylation patterns are desired).
  • Suitable eukaryotic host cells are well known to those of skill in the art.
  • Such eukaryotic cells include, but are not limited to yeast cells, insect cells, plant cells, fungal cells, and various mammalian cells (e.g. COS, CHO HeLa cells lines and various myeloma cell lines).
  • the expression product e.g., enediyne, enediyne analogue, enediyne biosynthetic pathway polypeptide, etc.
  • the proteins can be expressed with a convenient tag to facilitate isolation (e.g. a His 6 ) tag.
  • Other standard protein purification techniques are suitable and well known to those of skill in the art (see, e.g., (Quadri et ⁇ /. (1998) Biochemistry 37: 1585-1595; Nakano et ⁇ /. (1992) Mol. Gen. Genet. 232: 313-321, etc.).
  • components e.g. enediyne biosynthetic cluster orfs
  • various biomolecules e.g. enediynes, enediyne analogues, shunt metabolites, etc.
  • the desired product and/or shunt metabolite(s) are isolated according to standard methods well know to those of skill in the art (see, e.g., Ca ⁇ eras and Khosla (1998) Biochemistry 37: 2084-2088, Deutscher (1990) Methods in Enzymology Volume 182: Guide to Protein Purification, M. Deutscher, ed. etc.).
  • C-1027 open reading frames in directed biosynthesis.
  • Elements (e.g. open reading frames) ofthe C-1027 biosynthetic gene cluster and/or variants thereof can be used in a wide variety of "directed" biosynthetic processes (i.e. where the process is designed to modify and/or synthesize one or more particular preselected metabolite(s)).
  • the entire C-1027 gene cluster can be used to synthesize a C-1027 enediyne and/or a C-1027 enediyne analogue.
  • Individual C-1027 cluster open reading frames can be used to perform chemically modifications on particular substrates and/or to synthesize various metabolites.
  • ORF 6 C-methyltransferase can be used to methylate a carbon
  • ORF 7 N-methyltransferase
  • ORF 12 an amino transferase
  • ORF 8 an amino transferase
  • combinations of C-1027 open reading frames can be used to direct the synthesis of various metabolites (e.g. ⁇ -amino acids, deoxysugars, benzoxazolinates, and the like). These examples, are merely illustrative.
  • the biosyntheses can be performed in vivo, e.g. by providing a host cell comprising the desired C-1027 gene cluster open reading frames and/or in vivo, e.g., by providing the polypeptides encoded by the C-1027 gene cluster ORFs and the appropriate substrates and/or cofactors.
  • this invention provides for the synthesis of C-1027 enediynes and/or C-1027 analogues or derivatives.
  • this is accomplished by providing a cell comprising a C-1027 gene cluster and culturing the cell under conditions whereby the desired enediyne or enediyne analogue is synthesized.
  • the cell can be a cell that does not normally synthesize an enediyne and the entire gene cluster can be transfected into the cell.
  • a cell that typically synthesizes enediynes can be utilized and all or part ofthe C-1027 gene cluster can be introduced into the cell.
  • Enediyne derivatives/analogues can be produced by varying the order of, or kind of, gene cluster subunits present in the cell, and/or by changing the host cell (e.g. to a eukaryotic cell that glycosylates the biosynthetic product), and/or by providing altered metabolites (e.g. adding exogenous aglycones to a host that carries a gene cassette ofthe deoxysugar biosynthesis and glycosylation genes for the production of glycosylated metabolites), etc.
  • the host cell need not be transfected with an entire C-
  • C-1027 gene cluster Rather, various components of a C-1027 gene cluster can be altered within a cell already harboring a C-1027 cluster. By varying or adding various biosynthetic open reading frames, C-1027 enediyne variants can be produced.
  • enediyne and/or other metabolite e.g. shunt metabolite
  • an organism that naturally synthesizes an enediyne e.g. S. globisporus
  • an organism that is modified to synthesize an enediyne e.g. S. globisporus
  • control sequences that alter the expression of various open reading frames can be introduced that alter the amount and/or timing of enediyne production.
  • control sequences that alter the expression of various open reading frames can be introduced that alter the amount and/or timing of enediyne production.
  • ermE* constitutive promoter
  • ORF 2 transmembrane eflux protein
  • ORF 9 CagA apoprotein
  • ORF 10 TDP-glucose synthase
  • ORF 11 Hydroxylase/halogenase
  • Example 1 depicts an assay of antibacterial activity against M. luteus as described by Hu et al. (1988) J. Antibiot. 41: 1575- 1579).
  • Other suitable assays for enediyne activity will be known to those of skill in the art.
  • C-1027 open reading frames to synthesize an enediyne core.
  • the C-1027 open reading frames described herein, or variants thereof, can be used to synthesize an enediyene core, e.g., from a fatty acid precursor.
  • One such synthetic pathway is illustrated in Figure 4.
  • This reaction scheme utilizes ORF 17 (epoxide hydrase), ORF 20 (monooxygenase), ORF 21 (iron-sulfur flavoprotein), ORF 29 (P-450 hydroxylase, ORF 30 (oxidoreductase), ORF 32 (oxidoreductase), ORF 35 (proline oxidase), and ORF 38 (P-450 hydroxylase) to synthesize anenediyne core.
  • ORF 17 epoxide hydrase
  • ORF 20 monooxygenase
  • ORF 21 iron-sulfur flavoprotein
  • ORF 29 P-450 hydroxylase
  • ORF 30 oxidoreductase
  • ORF 32 oxidoreductase
  • ORF 35 proline oxidase
  • ORF 38 P-450 hydroxylase
  • the C-1027 gene cluster includes an NAGDH enzyme which can be exploited to synthesize a variety of deoxy sugars.
  • FIG. 2 One illustrative synthetic pathway is shown in Figure 2.
  • This biosynthetic scheme utilizes ORF 10 (dNDP-glucose synthase), ORF 1 (dNDP-glucose dehydratase), ORF 12 (epimerase), ORF 8 (aminotransferase), ORF 6 (C-methyltransferase), ORF 7 (N- methyltransferase) and ORF 19 (glycosyl transferase).
  • ORF 10 dNDP-glucose synthase
  • ORF 1 dNDP-glucose dehydratase
  • ORF 12 epimerase
  • ORF 8 aminotransferase
  • ORF 6 C-methyltransferase
  • ORF 7 N- methyltransferase
  • ORF 19 glycose
  • C-1027 biosynthetic polypeptides can be used in the biosynthesis of ⁇ -amino acids.
  • One illustrative synthetic pathway is shown in Figure 3 A. This biosynthetic scheme utilizes ORF 4 (hydroxylase), ORF 11 (hydroxylase/halogenase), ORF 24 (aminomutase), ORF 23 (type II NRPS condensation enzyme), ORF 25 (type II NRPS adenylation enzyme), and ORF 26 (type II peptidyl carrier protein).
  • this synthetic pathway is not considered limiting, but merely illustrative. Using this as a model, one of ordinary skill in the art can design numerous other synthetic schemes to produce other beta amino acids.
  • the C-1027 biosynthetic gene cluster open reading frames can be utilized, by themselves or in combination with other biosynthetic subunits (e.g. NRPS and/or PKS modules and/or enzymatic domains of other PKS and/or NRPS systems) to produce a wide variety of compounds including, but not limited to various enediyne or enediyne derivatives, various polyketides, polypeptides, polyketide/polypeptide hybrids, various thiazoles, various sugars, various methylated polypeptides/polyketides, and the like.
  • biosynthetic subunits e.g. NRPS and/or PKS modules and/or enzymatic domains of other PKS and/or NRPS systems
  • such compounds can be produced, in vivo or in vitro, by catalytic biosynthesis, e.g., using large, enediyne cluster units and/or modular PKSs, NRPSs, and hybrid PKS/NRPS systems.
  • catalytic biosynthesis e.g., using large, enediyne cluster units and/or modular PKSs, NRPSs, and hybrid PKS/NRPS systems.
  • large combinatorial libraries of cells harboring various megasynthetases can be produced by the random or directed modification of particular pathways and then selected for the production of a molecule or molecules of interest. It will be appreciated that, in certain embodiments, such libraries of megasynthetases/modified pathways, can be used to generate large, complex combinatorial libraries of compounds which themselves can be screened for a desired activity.
  • Such combinatorial libraries can be created by the deliberate modification variation of selected biosynthetic pathways and/or by random/haphazard modification of such pathways.
  • novel polyketides, polypeptides, and combinations thereof are created by modifying the entediyne gene cluster ORFs and/or known PKSs, and/or NRPSs so as to introduce variations into metabolites synthesized by the enzymes.
  • Such variations may be introduced by design, for example to modify a known molecule in a specific way, e.g. by replacing a single monomeric unit within a polymer with another, thereby creating a derivative molecule of predicted structure.
  • Such variations can also be made by adding one or more modules or enzymatic domains to a known PKS or NRPS or enediyne cluster, or by removing one or more module from a known PKS or NRPS.
  • Such mutations can be made to the native sequences using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a NRPS and/or PKS subunit using restriction endonuclease digestion, (see, e.g., Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82: 448; Geisselsoder et al. (1987) BioTechniques 5: 786).
  • the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA co ⁇ esponding to the RNA sequence), at a temperature below the melting temperature ofthe mismatched duplex.
  • the primer can be made specific by keeping primer length and base composition within relatively na ⁇ ow limits and by keeping the mutant base centrally located (Zoller and Smith (1983) Meth, Enzymol 100: 468).
  • Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation ofthe primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe.
  • the technique is also applicable for generating multiple point mutations (see, e.g., Dalbie-McFarland et al. (1982) Proc. Natl Acad. Sci USA 79:6409). PCR mutagenesis will also find use for effecting the desired mutations.
  • variations can be made randomly, for example by making a library of molecular variants (e.g. of a known enediyne) by randomly mutating one or more elements ofthe subject gene cluster or by randomly replacing one or more open reading frames in a gene cluster with one or more of alternative open reading frames.
  • a library of molecular variants e.g. of a known enediyne
  • the various open reading frames can be combined into a single multi-modular enzyme, thereby dramatically increasing the number of possible combinations obtained using these methods.
  • These combinations can be made using standard recombinant or nucleic acid amplification methods, for example by shuffling nucleic acid sequences encoding various modules or enzymatic domains to create novel a ⁇ angements ofthe sequences, analogous to DNA shuffling methods described in Crameri et al. (1998) Nature 391: 288-291, and in U.S. Patents 5,605,793 and in 5,837,458.
  • novel combinations can be made in vitro, for example by combinatorial synthetic methods. Novel molecules or molecule libraries, can be screened for any specific activity using standard methods.
  • Random mutagenesis ofthe nucleotide sequences obtained as described above can be accomplished by several different techniques known in the art, such as by altering sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, by i ⁇ adiation with X-rays or ultraviolet light, by incorporating inco ⁇ ect nucleotides during in vitro DNA synthesis, by e ⁇ or-prone PCR mutagenesis, by preparing synthetic mutants or by damaging plasmid DNA in vitro with chemicals.
  • Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like.
  • plasmid DNA or DNA fragments are treated with chemicals, transformed into E. coli and propagated as a pool or library of mutant plasmids.
  • nucleic acids encoding novel combinations of gene cluster ORFs are introduced into a cell.
  • nucleic acids encoding one or more enediyne synthetic cluster ORFS and or PKS and/or NRPS domains are introduced into a cell so as to replace one or more domains of an endogenous gene cluster within a cell. Endogenous gene replacement can be accomplished using standard methods, such as homologous recombination.
  • Nucleic acids encoding an entire enediyne, enediyne ORF, PKS, NRPS, or combination thereof can also be introduced into a cell so as to enable the cell to produce the novel enzyme, and, consequently, synthesize the novel polymer.
  • such nucleic acids are introduced into the cell optionally along with a number of additional genes, together called a 'gene cluster,' that influence the expression ofthe genes, survival ofthe expressing cells, etc.
  • such cells do not have any other enediyne and/or PKS- and/or NRPS- encoding genes or gene clusters, thereby allowing the straightforward isolation ofthe molecule(s) synthesized by the genes introduced into the cell.
  • the recombinant vector(s) can include genes from a single enediyne and/or PKS and/or NRPS gene cluster, or may comprise hybrid replacement PKS gene clusters with, e.g., a gene for one cluster replaced by the co ⁇ esponding gene from another gene cluster.
  • PKS and/or NRPS gene cluster e.g., a gene for one cluster replaced by the co ⁇ esponding gene from another gene cluster.
  • ACPs are readily interchangeable among different synthases without an effect on product structure.
  • a given KR can recognize and reduce polyketide chains of different chain lengths. Accordingly, these genes are freely interchangeable in the constructs described herein.
  • the replacement clusters ofthe present invention can be derived from any combination of PKS and/or NRPS gene sets that ultimately function to produce an identifiable polyketide.
  • hybrid replacement clusters include, but are not limited to, clusters with genes derived from two or more ofthe act gene cluster, the whiE gene cluster, frenolicin (fre ), granaticin (gra), tetracenomycin (tcm), 6-methylsalicylic acid (6-msas), oxytetracycline (otc), tetracycline (tet), erythromycin (ery), griseusin (gris), nanaomycin, medermycin, daunorubicin, tylosin, carbomycin, spiramycin, avermectin, monensin, nonactin, curamycin, rifamycin and candicidin synthase gene clusters, among others.
  • frenolicin frenolicin
  • gra granaticin
  • tcm tetracenomycin
  • 6-methylsalicylic acid 6-methylsalicylic acid
  • otc oxytetracycline
  • hybrid gene clusters have been constructed, having components derived from the act.fren, tcm, gris and gra gene clusters (see, e.g., U.S. Patent 5,712,146).
  • Other hybrid gene clusters, as described above, can easily be produced and screened using the disclosure herein, for the production of identifiable polyketides, polypeptides or polyketide/polypeptide hybrids.
  • Host cells can be transformed with one or more vectors, collectively encoding a functional PKS/NRPS set, or a cocktail comprising a random assortment of enediyne ORFs and/or PKS and/or NRPS genes, modules, active sites, or portions thereof.
  • the vector(s) can include native or hybrid combinations of enediyne ORFs, and/or PKS and/or NRPS subunits or cocktail components, or mutants thereof.
  • the gene cluster need not co ⁇ espond to the complete native gene cluster but need only encode the necessary enediyne ORFs and/or PKS and/or NRPS components to catalyze the production ofthe desired product(s). IV. Variation of starter and/or extender units, and/or host cells.
  • variations in the products produced by the gene cluster(s) can be obtained by varying the the host cell, the starter units and/or the extender units.
  • different fatty acids can be utilized in the enediyne synthetic pathway resulting in different enediyne variants.
  • different intermediate metabolites can be provided (e.g. endogenously produced by the host cell, or produced by an introduced herterologous construct, and/or supplied from an exogenous source (e.g. the culture media)).
  • varying the host cell can vary the resulting product(s).
  • a gene cassette carrying the enediyne biosynthesis genes can be introduced into a deoxysugar-synthesizing host for the production of glycosylated enediyne metabolites.
  • the antibiotic C-1027 and metabolites present in C-1027 biosynthesis are highly potent cytotoxins. Accordingly the biosynthesis of C-1027 is facilitated by the presence of one or more antibiotic (e.g. enediyne) resistance genes.
  • antibiotic e.g. enediyne
  • CagA and SgcB function cooperatively to provide resistance. It is believed that the C-1027 chromophore is first sequestered by binding to the preaproprotein CagA (ORF 9) to form a complex, which is then transported out ofthe cell by the efflux pump SgcB (ORF 2) and processed by removing the leader peptide to yield the chromoprotein.
  • genes that appear to mediate resistance in the C-1027 biosynthesis gene cluster include a transmembrane transport protein (ORF 27), a Na ' VH * transporter (ORF 0), an ABC transporter (ORF -1, C-terminus), a glycerol phosphate transporter (ORF -2), and a UvrA-like protein (ORF -1, N-terminus) (see, e.g., Table 2).
  • ORFs and/or the polypeptides encoded by these ORFs can be utilized alone, or in combination with one or more other C- 1027 ORFs to confer resistance to enediyne or enediyne metabolites on a cell. This is useful in a wide variety of contexts. For example, to increase production of enediynes. For example, it is believed that C-1027 resistance could be a limiting factor at the onset of C-1027 production. Provision of an extra copy ofthe plasmid-born sgcB, and overexpression of sgcB under the control ofthe constitutive ermE* promoter resulted in increase of C-1027 production (see example 1).
  • enediyne In a therapeutic context, it is sometimes desirable to confer resistance on certain vulnerable cells.
  • transfection of vulnerable, but healthy cells e.g. liver cells remote from the tumor site, stem cells, etc.
  • vector(s) expressing the resistance gene(s) permits administration ofthe enediyne at a higher dosage with fewer adverse effects to the organism.
  • MDR1 multi-drug resistance gene
  • vectors are provided containing one or more resistance genes of this invention under control of a constitutive and/or inducible promoter thereby providing a "ready-made" expression system suitable for the expression of an enediyne or enediyne metabolite at high concentration.
  • the resistance genes are expected to confer resistance to compounds other than enediynes.
  • the resistance genes are expected to confer resistance to essentially any cytotoxic compound that can act as a substrate for the resistance gene(s) of this invention.
  • kits for practice ofthe methods described herein.
  • the kits comprise one or more containers containing nucleic acids encoding one or more ofthe C-1027 biosynthesis gene cluster open reading frames.
  • Certain kits may comprise vectors encoding the sgc gene cluster orfs and/or cells containing such vectors.
  • the kits may optionally include any reagents and/or apparatus to facilitate practice ofthe methods described herein.
  • Such reagents include, but are not limited to buffers, labels, labeled antibodies, bioreactors, cells, etc.
  • kits may include instructional materials containing directions (i.e., protocols) for the practice ofthe methods of this invention.
  • directions i.e., protocols
  • Prefe ⁇ ed instructional materials provide protocols utilizing the kit contents for creating or modifying C-1027 gene cluster and/or for synthesizing or modifying a molecule using one or more sgc gene cluster ORFs.
  • the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • Example 1 Genes for production of the enediyne antitumor antibiotic C-1027 in Streptomyces globisporus are clustered with the ca ⁇ A gene that encodes the C-1027 apoprotein
  • C-1027 consists of a non-peptidic chromophore and an apoprotein, CagA [also called C-1027 AG (Otani et al. (1991) Agri. Biol. Chem. 55: 407- 417)].
  • the C-1027 chromophore is extremely unstable in the protein-free state, the structure of which was initially deduced from an inactive but more stable degradation product (Minami et al. (1993) Tetrahedron Lett.
  • the CagA apoprotein of C-1027 has been isolated, its amino acid sequence has been determined, and the co ⁇ esponding cagA gene has been cloned and sequenced (Otani et al. (1991) Agri. Biol. Chem. 55: 407-417; Sakata et al. (1992) Biosci. Biotech. Biochem. 56: 1592-1595). Since genes encoding secondary metabolite production in actinomycetes have invariably been found to be clustered in one region ofthe microbial chromosome (Hopwood (1997) Chem. Rev.
  • mapping the cagA gene with either a putative PKS gene, a deoxysugar biosynthesis gene, or both to the same region ofthe S. globisporus chromosome should be viewed as strong evidence supporting the proposition that the cloned genes constitute the C-1027 biosynthesis gene cluster.
  • sgcA Streptomyces globisporus C-1027
  • sgcB that encode a dNDP-glucose 4,6-dehydratase (NGDH) and a transmembrane efflux protein, respectively.
  • the sgcA,B locus is indeed clustered with the cagA gene, leading to the localization of a 75-kb gene cluster from S. globisporus.
  • the involvement ofthe cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the sgcA gene to generate C-1027-nonproducing mutants and by complementing the sgcA mutants in vivo to restore C-1027 production.
  • Escherichia coli DH5 ⁇ was used as a general host for routine subcloning
  • E. coli XL 1-Blue MR (Stratagene, La Jolla, CA) was used as the transduction host for cosmid library construction.
  • E. coli SI 7-1 was used as the donor host for E. coli-S. globisporus conjugation (Mazodier et al. (1989) J. Bacteriol. 171: 3583-3585).
  • Micrococcus luteus ATCC9431 was used as the testing organism to assay the antibacterial activity of C-1027 (Hu et al. (1988) J. Antibiot. 41: 1575-1579).
  • the pG ⁇ M-3zf, -5zf, and -7zf and pGEM-T vectors were from Promega (Madison, WI). S. globisporus strains and other plasmids in this study are listed in Table 3
  • T pBS 1002 0.55-kb PCR product amplified from S. globisporus with NGDH gene primers in pGEM-T pBS 1003 0.73-kb PCR product amplified from pBS 1005 with cagA primers in pGEM-T pBS 1004 pOJ446 S. globisporus genomic library cosmid pBS 1005 pOJ446 S. globisporus genomic library cosmid pBS 1006 pOJ446 S.
  • globisporus genomic library cosmid pBS 1007 3.0-kb Bam ⁇ l fragment from pBS 1005 in pGEM-3zf, sgcA, sgcB pBS 1008 4.0-kb Bat ⁇ l fragment from pBS 1005 in pGEM-3zf, cagA ⁇ BS1009 1.0-kb Kpnl truncated fragment of sgcA from pBS1007 in pGEM-3zf pBSlOlO 0.75-kb SaclVSphl internal fragment of sgcA from pBS1009 in pGEM-5zf pBSlOl 1 0.75-kb SacVSphl internal fragment of sgcA from pBSlOlO in pGEM-3zf pBS1012 0.75-kb £coRI/Hi III internal fragment of sgcA from pBSlOlO in pOJ260 pBS1013 0.75-kb EcoKU
  • Ampicillin, apramycin, nalidixic acid, and thiostrepton were from Sigma (St. Louis, MO). Unless specified otherwise, restriction enzymes and other molecular biology reagents were from standard commercial sources.
  • E. coli strains carrying plasmids were grown in Luria-Bertani (LB) medium and were selected with appropriate antibiotics.
  • S. globisporus strains were grown on ISP-4 (Difco Laboratories, Detroit, MI) or R2YE at 28°C for sporulation and in TSB (Hopwood et al (1985) Genetic manipulation of Streptomyces: a laboratory manual John Innes
  • S. globisporus strains were grown either on R2YE or ISP-4 agar medium at 28°C or in liquid medium by a two-stage fermentation.
  • the seed inoculum was prepared by inoculating 50 mL medium (consisting of 2% glycerol, 2% dextrin, 1% fish meal, 0.5% peptone, 0.2% (NH ) 2 SO 4 , and 0.2% CaCO 3 , pH
  • Plasmid preparation and DNA extraction were carried out by using commercial kits (Qiagen, Santa Clarita, CA).
  • Total S. globisporus DNA was isolated according to literature protocols (Hopwood et al. (1985) Genetic manipulation of Streptomyces: a laboratory manual John Innes Foundation, Norwich, UK; Rao et al. (1987) Methods Enzymol. 153: 166-198). Restriction endonuclease digestion and ligation followed standard methods (Sambrook et al. (1989) Molecular cloning, a laboratory manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). For Southern analysis, digoxigenin labeling of DNA probes, hybridization, and detection were performed according to the protocols provided by the manufacturer (Boehringer Mannheim Biochemicals, Indianapolis, IN).
  • Automated DNA sequencing was carried out on an ABI Prism 377 DNA Sequencer using the ABI Prism dye terminator cycle sequencing ready reaction kit and Ampli-T ⁇ DNA polymerase FS (Perkin-Elmer/ABI, Foster City, CA). Sequencing service was provided by either the DBS Automated DNA Sequencing Facility, UC Davis, or Davis Sequencing Inc. (Davis, CA). Data were analyzed by ABI Prism Sequencing 2.1.1 software and the Genetics Computer Group program (Madison, WI).
  • PCR Polymerase chain reaction
  • PCR was carried out on a Gene Amp PCR System 2400 (Perkin-Elmer/ABI) with Taq polymerase and buffer from Promega.
  • a typical PCR mixture consisted of 5 ng of S. globisporus genomic or plasmid DNA as template, 25 pmoles of each primers, 25 ⁇ M dNTP, 5% DMSO, 2 units of Taq polymerase, 1 x buffer, with or without 20% glycerol in a final volume of 50 ⁇ L.
  • the PCR temperature program was as follows: initial denaturing at 94°C for 5 min, 24-36 cycles of 45 sec at 94°C, 1 min at 60°C, 2 min at 72°C, followed by additional 7 min at 72°C.
  • cagA For cagA, the following pair of primers, flanking its coding region, were used— 5'-AG GTG GAG GCG CTC ACC GAG-3' (forward, SEQ ID NO:l 1 l)/5'-G GGC GTC AGG CCG TAA GAA G-3' (reverse, SEQ ID NO: 112) (Sakata et al. (1992) Biosci. Biotechnol Biochem., 56: 159201595). A distinctive product with the predicted size of 0.73 kb was amplified from pBS1005 and cloned into pGEM-T to yield pBS1003.
  • S. globisporus genomic DNA was partially digested with Mbol to yield a smear around 60 kb, as monitored by electrophoresis on a 0.3% agarose gel.
  • This sample was dephosphorylated upon treatment with shrimp alkaline phosphatase and ligated into the E. coli-Streptomyces shuttle vector pOJ446 (Bierman et al. (1992) Gene 116: 43-69) that was prepared by digestion with Hp ⁇ l, shrimp alkaline phosphatase treatment, and additional digestion with R ⁇ m ⁇ I.
  • the resulting ligation mixture was packaged with the Gigapack II XL two-component packaging extract (Stratagene). The package mixture was transduced into E.
  • colonies from five LB plates containing apramycin were transfe ⁇ ed to nylon transfer membranes (Micro Separations, Inc., Westborough, MA) and screened by colony hybridization with the PCR-amplified 0.55-kb NGDH fragment from pBS 1002 as a probe.
  • the positive cosmid clones were re-screened by PCR with primers for NGDH and confirmed by Southern hybridization (Sambrook et al, supra.).
  • An internal fragment of sgcA was moved sequentially as a 0.75-kb SaclVSphl fragment from pBS1009 into the same sites of pGEM-5zf to yield pBSlOlO and as a 0.75-kb SacVSphl fragment from pBSlOlO into the same sites of pGEM-3zf to yield pBS 1011.
  • E. coli S17-l( ⁇ BS1012) or E. coli SI 7-1 was grown to an OD 600 of 0.3 to 0.4. Cells from a 20-mL culture were pelleted by centrifugation, washed in LB, and resuspended in 2 mL of LB as the E. coli donors.
  • globisporus spores (10 3 to 10 9 ) were washed, resuspended in TSB, and incubated at 50°C for 10 min to activate germination. After additional incubation at 37°C for 2 to 5 hrs, the spores were pelleted and resuspended in LB as the S. globisporus recipients. The donors (100 ⁇ L) and recipients (100 ⁇ L) were mixed and spread equally onto two modified ISP-4 or AS-1 plates supplemented freshly with 10 mM MgCl 2 (see Media and culture conditions). The plates were incubated at 28°C for 16 to 22 hrs. After removal of most ofthe E.
  • coli SI 7-1 donors by washing the surface with sterile water, the plates were overlaid with 3 mL of soft LB (0.7% agar) containing nalidixic acid (50 ⁇ g/mL, final concentration) and apramycin (100 ⁇ g/mL, final concentration) and incubated at 28°C until exconjugants appeared (in approximately 5 days).
  • pBS1013 bears a temperature-sensitive Streptomyces replication origin (Bierman et al. (1992) Gene 116: 43-69; Muth et al. (1989) Mol. Gen. Genet. 219: 341-348) that is unable to replicate at temperatures above 34°C (Table 3), while the S. globisporus wild-type strain grows normally up to 37°C.
  • spores of S. globisporus (pBS1013), from either the transformants or the exconjugants, were spread onto R2YE plates containing apramycin (100 ⁇ g/mL).
  • the plates were incubated directly at 37°C, and mutants, resulting from single crossover homologous recombination between pBS 1013 and the S. globisporus chromosome, were readily obtained in 7 to 10 days. Alternatively, the plates were first incubated at 28°C for 2 days until pinpoint-size colonies became visible and then shifted to 37°C to continue incubation. Mutants resulting from single crossover homologous recombination grew out ofthe original pinpoint-size colonies as easily distinguishable sectors in 7 to 10 days.
  • pBS1007 was digested with EcoRI, and made blunt-ended by treatment with the Klenow fragment of DNA polymerase I. Upon additional digestion with Sphl, the resulting 2.0-kb blunt-ended Sphl fragment containing the intact sgcA gene was cloned into the Small Sphl sites of pWHM79 (Shen et al. (1996) Proc. Natl. Acad. Sci., USA, 93: 6600- 6604) to yield pBS1014.
  • pBS1007 was digested with Kpnl, removing most ofthe sgcA gene, and the 5.2-kb Kpnl fragment was recovered and self-ligated to yield pBS1016.
  • the ermE* promoter was subcloned from pWHM79 (Shen et al (1996) Proc. Natl. Acad. Sci., USA, 93: 6600-6604) as a 0.45-kb EcoRI/S ⁇ cI fragment and cloned into the same sites of pBS1016 to yield pBS1017.
  • C-1027 production The production of C- 1027 was detected by assaying its antibacterial activity against M. luteus (Hu et al. (1988) J. Antibiot. 41: 1575-1579). From liquid culture, fermentation supernant (180 ⁇ L) was added to stainless steel cylinders placed on LB plates pre-seeded with overnight M. luteus culture (0.01% vol/vol). From solid culture, a small square block (0.5 x 0.5 x 0.5 cm 3 ) of agar from either R2Y ⁇ or ISP-4 medium was directly placed on M. /wtews-seeded LB plates. The plates were incubated at 37°C for 24 hrs, and C- 1027 production was estimated by measuring the size of inhibition zones.
  • ORFs Two complete open reading frames (ORFs) (sgcA and sgcB) were identified within the 3.0-kb R ⁇ mHI fragment of pBS1007, the 3,035-nt sequence of which is shown in Figure 6.
  • the sgcA gene most likely begins with an ATG at nt 101, preceded by a probable ribosome biding site (RBS), GGAGG, and ends with a TGA stop codon at nt 1099.
  • RBS probable ribosome biding site
  • GGAGG probable ribosome biding site
  • Sgc-4 should therefore encode a 332-amino acid protein with a molecular weight of 36,341 and an isoelectric point of 6.01.
  • a Gapped-BLAST search showed that the deduced sgcA gene product is highly homologous to various putative and known NGDH enzymes from antibiotic-producing actinomycetes, including Gdh from the erythromycin biosynthesis gene cluster in Saccharopolyspora erythraea (64% identity and 70% similarity) (Linton et al (1995) Gene 153: 33-40), MtmE from the mithramycin biosynthesis gene cluster in Streptomyces argillaceus (64% identity and 68% similarity) (Lombo et al. (1997) J. Bacteriol.
  • sgcA encodes the NGDH enzyme required for the biosynthesis of the 4,6-dideoxy-4-dimethylamino-5- methylrhamnose moiety ofthe C-1027 chromophore.
  • the sgcB gene is located 43 nt downstream of sgcA. It should begin with a GTG at nt 1143, preceded by a probable RBS, AGGAG, and end with a TGA at nt 2708 (Fig. 6).
  • sgcB should therefore encode a 521 -amino acid protein with a molecular weight of 52,952 and an isoelectric point of 4.64.
  • Database comparison ofthe deduced sgcB product revealed that SgcB is closely related to a family of membrane efflux pumps, such as LfrA from Mycobacterium smegmatis (43% identity and 50% similarity, protein accession number AAC43550) (Takiff et al
  • LfrA employs the transmembrane proton gradient in an antiporter mode to drive the efflux of intracellular antibiotics, resulting in fluoroquinolone resistance in M. smegmatis (Takiff et al. (1996) Proc. Natl Acad. Sci. USA 93: 362-366).
  • SgcB conferring resistance by exporting C-1027 from S. globisporus.
  • the cagA gene is clustered with the sgcA and sgcB locus.
  • PCR primers were designed according to the flanking regions of cagA (Sakata et al (1992) Biosci. Biotech. Biochem. 56: 1592-1595).
  • a single product with the predicted size of 0.73 kb was indeed amplified from several ofthe overlapping cosmids (which cover the 75-kb sgc cluster), including pBS1004 and pBS1005, the identity of which as cagA was confirmed by sequencing.
  • Restriction enzyme mapping and Southern hybridization analysis localized cagA to a single 4.0-kb R ⁇ mHI fragment that is approximately 14 kb upstream ofthe sgcA.B locus (Fig.
  • cagA encodes a 142-amino acid protein that is processed by cleavage of a 32-amino acid lead peptide to yield the mature CagA apoprotein (Sakata et al. (1992) Biosci. Biotech. Biochem. 56: 1592-1595).
  • sgcA was insertionally disrupted by a single crossover homologous recombination event to generate C- 1027-nonproducing mutant strains (Fig. 8A).
  • Two plasmids were used — pBS1012 (a pOJ260 derivative) and pBS1013 (a pKCl 139 derivative), each of which contain a 0.75-kb internal fragment from sgcA (Table 3). After introduction of pBS1012 into S. globisporus either by PEG-mediated protoplast transformation or E. coli-S.
  • pBS1012 is derived from the Streptomyces non-replicating plasmid of pOJ260, these isolates must have resulted from integration of pBS1012 into the S. globisporus chromosome by homologous recombination. Plasmid pBS1013 was similarly introduced into S. globisporus. However, since pBS1013 is derived from pKCl 139 that carries the temperature-sensitive Streptomyces replication origin from pSG5 and can replicate normally at 28°C (Muth et al. (1989) Mol. Gen. Genet.
  • S. globisporus SBIOOI and S. globisporus SB1002 are C-1027-nonproducing mutants.
  • the ability ofthe wild-type sgcA gene to complement the disrupted sgcA gene was tested in the S. globisporus SB1001 strain.
  • the effect oisgcB on C-1027 production was tested in the wild- type S. globisporus strain.
  • pBS 1018 and the pKC 1139 vector as a control were each introduced by conjugation into S. globisporus.
  • Culture supernatants from each exconjugant were harvested on days 3, 4, and 5, and assayed for C-1027 production by determining the antibacterial activity against M. luteus. While no apparent difference for C-1027 production was observed between the S. globisporus and S.
  • enediyne cores are structurally distinct from either the reduced or aromatic polyketides, the biosynthesis of which is well characterized by type I or type II PKS, respectively, it could be imagined that an enediyne PKS catalyzes the biosynthesis of a polyunsaturated linear heptaketide intermediate that is subsequently cyclized into the enediyne core structure (Hu et al. (1994) Mol. Microbiol. 14: 163-172; Spaink et al.
  • NGDH NGDH
  • S. globisporus S. globisporus by PCR and used it as a probe to screen an S. globisporus genomic library, resulting in the isolation ofthe 75-kb sgc gene cluster.
  • DNA sequence analysis of a 3.0-kb R ⁇ mHI fragment ofthe sgc cluster confirmed the presence ofthe NGDH protein, encoded by sgcA, along with sgcB that encodes a transmembrane efflux protein (Fig. 6).
  • the cagA gene indeed resides approximately 14 kb upstream of sgcA (Fig. 5);
  • DNA sequence analysis of a 4.0-kb R ⁇ mHI fragment confirmed the identity of cagA along with two additional ORFs (data not shown).
  • globisporus SB 1002 were characterized as C-1027-nonproducing on the basis ofthe antibacterial assay alone (Fig. 9 A), this phenotype was identical to that ofthe controls ofthe AF40, AF44, and AF67 mutants (Fig. 9A and 9C).
  • the latter strains were isolated previously upon randomly mutagenizing the wild-type S. globisporus strain with acriflavine and confirmed to be C-1027-nonproducing by both the antibacterial bioassay and an antitumor spermatogonial assay (Mao, et al. (1997) Chinese J. Biotechnol. 13: 195-199), providing strong support to the cu ⁇ ent study. Gene disruption and complementation in S.
  • globisporus were made possible by the recently developed genetic system that allowed us to introduce plasmid DNA into S. globisporus via either PEG-mediated protoplast transformation (Hopwood et al. (1985) Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation, Norwich, UK) or E. coli-S. globisporus conjugation (Bierman et al. (1992) Gene 116: 43-69; Matsushima and Baltz (1996) Microbiology 142: 261-267; Matsushima et al (1994) Gene 146: 39-45) for analyzing the sgc biosynthesis gene cluster in vivo.
  • PEG-mediated protoplast transformation Hopwood et al. (1985) Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation, Norwich, UK
  • E. coli-S. globisporus conjugation Bierman et al. (1992) Gene 116: 43-69; Matsushima and Balt
  • SgcB was probed by examining C-1027 production, following expression ofthe gene in the wild-type S. globisporus strain. Database comparison ofthe deduced amino acid sequence clearly suggested SgcB as a transmembrane efflux protein, conferring resistance by exporting C-1027 out ofthe cell. Hence, in addition to CagA, SgcB could be viewed as the second resistance element identified for C-1027 biosynthesis. Multiple resistance genes have been identified in numerous antibiotic biosynthesis gene clusters (Hopwood (1997) Chem. Rev. 97: 2465-2497).

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Abstract

This invention provides nucleic acid sequences and characterization of the gene cluster responsible for the biosynthesis of the enediyne C-1027 (produced by Streptomyces globisporus). Methods are provided for the biosynthesis of enediynes, enediyne analogs and other biological molecules.

Description

GENE CLUSTER FOR PRODUCTION OF THE ENEDIYNE ANTITUMOR ANTIBIOTIC C-1027
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 U.S.C. §119 of provisional application USSN 60/115,434, filed on January 6, 1999, which is herein incorporated by reference in its entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This work was supported in part by a grant from the Cancer Research Coordinating Committee, University of California, the National Institutes of Health grant CA78747, and the Searle Scholars Program/The Chicago Community Trust. The Government ofthe United States of America may have certain rights in this invention.
FIELD OF THE INVENTION
This invention relates to the field of enediyne antibiotics. In particular this invention elucidates the gene cluster controlling the biosynthesis ofthe C-1027 enediyne.
BACKGROUND OF THE INVENTION
The enediyne antibiotics are currently the focus of intense research activity in the fields of chemistry, biology, and medical sciences, because of their unique molecular architecture, biological activities, and modes of actions (Doyle and Borders (1995) Enediyne antibiotics as antitumor agents. Marcel-Dekker, New York, Thorson et al. (1999) Bioorg. Chem., 27: 172-188). Since the unveiling ofthe structure of neocarzinostatin chromophore (Edo et al. (1985) Tetrahedron Lett. 26: 331-340) in 1985, the enediyne family has grown steadily. Thus far, there have been three basic groups within the enediyne antibiotic family: (a) the calicheamicin/esperamicin type, which includes the calicheamicins, the esperamicins, and namenamicin, (b) the dynemicin type, and (c) the chromoprotein type, consisting of an apoprotein and an unstable enediyne chromophore. The latter group includes neocarzinostatin, kedarcidin, C-1027 (Fig. 1), and maduropeptin, whose enediyne chromophore structures have been established, as well as several others whose enediyne chromophore structures are yet to be determined due to their instability (Thorson et al. (1999) Bioorg. Chem., 27: 172-188). N1999A2, in contrast to the other chromoproteins, exists as an enediyne chromophore alone despite the fact that its structure is very similar to the other chromoprotein chromophore (Ando et α/.(1998) Tetra. Letts., 39: 6495-6480). As a family, the enediyne antibiotics are the most potent, highly active antitumor agents ever discovered. Some members are 1000 times more potent than adriamycin, one ofthe most effective, clinically used antitumor antibiotics (Zhen et al. (1989) J. Antibiot. 42: 1294-1298). All members of this family contain a unit consisting of two acetylenic groups conjugated to a double bond or incipient double bond within a nine or ten-membered ring; i.e., the enediyne core as exemplified by C-1027 in Fig. 1. As the consequence of this structural feature, these compounds share a common mechanism of action: the enediyne core undergoes an electronic rearrangement to form a transient benzenoid diradical, which is positioned in the minor groove of DNA so as to damage DNA by abstracting hydrogen atoms from deoxyriboses on both strands (Fig. 1). Reaction ofthe resulting deoxyribose carbon-centered radicals with molecular oxygen initiates a process that results in both single-strand and double-strand DNA cleavages (Doyle and Borders (1995) Enediyne antibiotics as antitumor agents. Marcel-Dekker, New York; Ikemoton et al. (1995) Proc. Natl. Acad. Sci. USA 92:10506-10510; Myers et al. (1997) J. Am. Chem. Soc. 119: 2965-2972; Stassinopoulos et al. (1996) Science 272: 1943-1946; Thorson et al. (1999) Bioorg. Chem., 21: 172-188; Xu et al. (1997) J. Am. Chem. Soc. 119: 1133-1134). This novel mechanism of DNA damage has important implications for their application as potent cancer chemotherapeutic agents (Doyle and Borders (1995) supra.; Sievers et al. (1999) Blood 93: 3678-3684).
As an alternative to making structural analogs of microbial metabolites by chemical synthesis, manipulations of genes governing secondary metabolism offer a promising alternative allowing preparation of these compounds biosynthetically (Cane et al. (1998) Science 282: 63-68; Hutchinson and Fujii. (1995) Ann. Rev. Microbiol. 49: 201-38; Katz and Donadio (1993) Ann. Rev. Microbiol. 47: 875-912). The success ofthe latter approach depends critically on the availability of novel genetic systems and on genes encoding novel enzyme activities. The enediynes offer a distinct opportunity to study the biosynthesis of their unique molecular scaffolds and the mechanism of self-resistance to extremely cytotoxic natural products. Elucidation of these aspects provides access to rational engineering of enediyne biosynthesis for novel drug leads and makes it possible to construct enediyne overproducing strains by de-regulating the biosynthetic machinery. In addition, elucidation of an enediyne gene cluster contributes to the general field of combinatorial biosynthesis by expanding the repertoire of novel polyketide synthase (PKS) and deoxysugar biosynthesis genes as well as other genes uniquely associated with enediyne biosynthesis, leading to the making of novel enediynes via combinatorial biosynthesis.
SUMMARY OF THE INVENTION
This invention provides nucleic acid sequences and characterization ofthe gene cluster responsible for the biosynthesis ofthe enediyne C-1027 (produced by Streptomyces globisporus). In particular structural and functional characterization is provided for the 50 open reading frames (ORFs) comprising this gene cluster. Thus, in one embodiment, this invention provides an isolated nucleic acid comprising a nucleic acid selected from the group consisting of a nucleic acid encoding any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA), a nucleic acid encoding a polypeptide encoded by any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA); and a nucleic acid amplified by polymerase chain reaction (PCR) using primer pairs that amplify any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA). In one embodiment, preferred nucleic acids comprise a nucleic acid encoding at least two (more preferably at least three or more) open reading frames (ORFs) selected from the group consisting of ORF- 1 through ORF 42, excluding ORF 9 (cagA). In another embodiment this invention provides an isolated nucleic acid comprising a nucleic acid that specifically hybridizes under stringent conditions to an open reading frame (ORF) ofthe C-1027 biosynthesis gene cluster, excluding ORF 9 (cagA), and can substitute for the ORF to which it specifically hybridizes to direct the synthesis of an enediyne. In certain embodiments this also includes nucleic acids that would stringently hybridizes indicated above, but for, the degeneracy ofthe nucleic acid code. In other words, if silent mutations could be made in the subject sequence so that it hybridizes to he indicated sequence(s) under stringent conditions, it would be included in certain embodiments. Particularly preferred nucleic acids comprises a nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42. Particularly preferred isolated nucleic acid comprises a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42. The nucleic acid may comprises a nucleic acid that is a single nucleotide polymorphism (SNP) of a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 81, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42.
This invention also provides an isolated gene cluster comprising open reading frames encoding polypeptides sufficient to direct the assembly of a C-1027 enediyne or a C- 1027 enediyne analogue. The gene cluster may be present in a cell, more preferably in a bacterial cell (e.g. Actinomycetes, Actinoplanetes, Actinomadura, Micromonospora, or Streptomycetes). Particular preferred bacterial cells include, but are not limited to
Streptomyces globisporus, Streptomyces lividans, Streptomyces coelicolor, Micromonospora echinospora spp. calichenisis, Actinomadura verrucosopora, Micromonospora chersina, Streptomyces carzinostaticus, and Actinomycete L585-6. The gene cluster may contain one or more open reading frames is operatively linked to a heterologous promoter (e.g. a constitutive or an inducible promoter).
This invention also provides for an polypeptide encoded by any one or more ofthe nucleic acids described herein.
Also provided are host cell(s) (e.g. eukaryotic cells or bacterial cells as described herein) transformed with one or more ofthe expression vectors described herein. Preferred host cells are transformed with an exogenous nucleic acid comprising a gene cluster encoding polypeptides sufficient to direct the assembly of a C-1027 enediyne or a C- 1027 enediyne analogue. In certain embodiments, heterologous nucleic acid may comprise only a portion ofthe gene cluster, but the cell will still be able to express an enediyne. This invention also provides methods of chemically modifying a biological molecule. The methods involve contacting a biological molecule that is a substrate for a polypeptide encoded by a C-1027 biosynthesis gene cluster open reading frame, with a polypeptide encoded by a C-1027 biosynthesis gene cluster open reading frame whereby the polypeptide chemically modifies the biological molecule. In one preferred embodiment, the polypeptide is an enzyme selected from the group consisting of a hydroxylase, a homocysteine synthase, a dNDP-glucose dehydrogenase, a citrate carrier protein, a C-methyl transferase, an N-methyl transferase, an aminotransferase, a CagA apoprotein, an NDP- glucose synthase, an epimerase, an acyl transferase, a coenzyme F390 synthase, and epoxidase hydrolase, an anthranilate synthase, a glycosyl transferase, a monooxygenase, a type II condensation protein, an aminomutase, a type II adenylation protein, an O-methyl transferase, a P-450 hydroxylase, an oxidoreductase, and a proline oxidase. In a preferred embodiment the method involves contacting the biological molecule with at least two (preferably at least three or more) different polypeptides encoded by C-1027 biosynthesis gene cluster open reading frames. The contacting may be in a host cell (e.g. a eukaryotic cell or a bacterial cell) or the contacting can be ex vivo. The biological molecule can be an endogenous metabolite produced by said host cell or an exogenous supplied metabolite. In preferred embodiments, the host cell is a bacterial cell or eukaryotic cell (e.g., a mammalian cell, a yeast cell, a plant cell, a fungal cell, an insect cell, etc.). In certain preferred embodiments, the host cell synthesizes sugars and glycosylates the biological molecule. In other preferred embodiments, the host cell synthesizes deoxysugars. The method can further involve contacting the biological molecule with a polyketide synthase or a non-ribosomal polypeptide synthetase. The contacting can be in a cell (e.g., a bacterial cell) or ex vivo. In one preferred embodiment the method comprises contacting the biological molecule with at substantially all ofthe polypeptides encoded by C-1027 biosynthesis gene cluster open reading frames and said method produces an enediyne or enediyne analogue. In another preferred embodiment, the biological molecule is a fatty acid and the biological molecule is contacted with a C-1027 orf polyeptide selected from the group consisting of an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase. In certain embodiments, the biological molecule is a fatty acid and said biological molecule is contacted with a plurality of C-1027 orf polypeptides comprising an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase. In one especially preferred embodiment ,the biological molecule is contacted with polypeptides encoded by ORF 17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38. In another especially preferred embodiment, the biological molecule is contacted with polypeptides encoded by ORF 15, ORF 16, ORF 28, ORF3, ORF 14, and ORF 13, and, in certain embodiments, ORF 4 and ORF 3 as well.
In certain embodiments, the method may comprise contacting a sugar with one or more C-1027 open reading frame polypeptides selected from the group consisting of a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N-methyltransferase, and a glycosyl transferase. Particularly preferred variant of this method comprise contacting a dNDP-glucose with a plurality of C- 1027 open reading frame polypeptides comprising a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N- methyltransferase, and a glycosyl transferase.
In certain other embodiments, the method comprises contacting an amino acid with one or one or more C-1027 open reading frame polypeptides selected from the group consisting of a hydroxylase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein. These methods may involve contacting an amino acid with a plurality of C-1027 open reading frame polypeptides comprising a hydroxylase, a halogenase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein. In particularly preferred embodiments, the amino acid is a tyrosine.
This invention also provides a method of synthesizing a chromaprotein type enediyne core, said method comprising contacting a fatty acid with one or more C-1027 orf polypeptides selected from the group consisting of an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase. In preferred embodiments, the fatty acid may be contacted with a plurality of C-1027 orf polypeptides comprising an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase. In particularly preferred embodiments, the fatty acid is contacted with polypeptides encoded by ORF 17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38.
In still yet another embodiment, this invention provides a method of synthesizing a deoxysugar. This method involves contacting a sugar with one or more C-
1027 open reading frame polypeptides selected from the group consisting of a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C- methyltransferase, an N-methyltransferase, and a glycosyl transferase. In preferred embodiments, this method involves contacting a dNDP-glucose with a plurality of C-1027 open reading frame polypeptides comprising a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N- methyltransferase, and a glycosyl transferase. In particularly preferred embodiments, the dNDP-glucose is contacted with polypeptides encoded by ORF 17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38.
This invention also provides methods of synthesizing a beta amino acid by contacting an amino acid with one or one or more C-1027 open reading frame polypeptides selected from the group consisting of a hydroxylase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein. The method preferably comprises contacting an amino acid with a plurality of C- 1027 open reading frame polypeptides comprising a hydroxylase, a halogenase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein. Particularly preferred embodiments comprise contacting the amino acid (e.g. tyrosine) with polypeptides encoded by ORF 4, ORF11, ORF24, ORF23, ORF25, and ORF26.
Also provided are methods of synthesizing an enediyne or an enediyne analogue. These methods involve culturing a cell (e.g. a eukaryotic cell or a bacterium) comprising a recombinantly modified C-1027 gene cluster under conditions whereby said cell expresses said enediyne or enediyne analogue; and recovering the enediyne or enediyne analogue. In preferred embodiments, the gene cluster is present in a bacterium (e.g., Actinomycetes, Actinoplanetes, Actinomadura, Micromonospora, or Streptomycetes). Particularly preferred bacteria include, but are not limited to Streptomyces globisporus, Streptomyces lividans, Streptomyces coelicolor, Micromonospora echinospora spp. calichenisis, Actinomadura verrucosopora, Micromonospora chersina, Streptomyces carzinostaticus, and Actinomycete L585-6. In another preferred embodiment, the gene cluster is present in a eukaryotic cell (e.g. a mammalian cell, a yeast cell, a plant cell, a fungal cell, an insect cell, etc.). The host cell can be one that synthesizes sugars and glycosylates the enediyne or enediyne analogue. The host can be one that synthesizes deoxysugars. This invention also provides a method of making a cell (e.g., a bacterial or eukaryotic cell) resistant to an enediyne or an enediyne metabolite. This method involves expressing in the cell one or more isolated C-1027 open reading frame nucleic acids that encode a protein selected from the group consisting of a CagA apoprotein, a SgcB transmembrane efflux protein, a transmembrane transport protein, a Na+/H+ transporter, an ABC transport, a glycerol phosphate tranporter, and a UvrA-like protein. In preferred embodiments, the isolated C-1027 open reading frame nucleic acids are selected from the group consisting of ORF 9, ORF2, ORF 27, ORF 0, ORF 1 c-terminus, ORF 2, and ORF 1 N-terminus. Certain embodiments exclude cagA (ORF 9). In one embodiment, this invention specifically excludes one or more of open reading frames -7 through 42. In particular, in one embodiment this invention excludes cagA (ORF 9), and/or sgcA (ORF 1), and/or sgcB (ORF 2).
DEFINITIONS
The terms "C-1027 open reading frame", and "C-1027 ORF" refer to an open reading frame in the C-1027 biosynthesis gene cluster as isolated from Streptomyces globisporus. The term also embraces the same open reading frames as present in other enediyne-synthesizing organisms (e.g. other strains and/or species of Streptomyces, Actinomyces, and the like). The term encompasses allelic variants and single nucleotide polymorphisms (SNPs). In certain instances the C-1027 ORF is used synonymously with the polypeptide encoded by the C-1027 ORF and may include conservative substitutions in that polypeptide. The particular usage will be clear from context.
The terms "isolated" "purified" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. With respect to nucleic acids and/or polypeptides the term can refer to nucleic acids or polypeptides that are no longer flanked by the sequences typically flanking them in nature.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide. The terms "nucleic acid" or "oligonucleotide" or grammatical equivalents herein refer to at least two nucleotides covalently linked together. A nucleic acid ofthe present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49:1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica Scripta 26: 141 9), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111 :2321, 0- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31 : 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996) Nαtwre 380: 207). Other analog nucleic acids include those with positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Patent Νos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate
Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996) and non-ribose backbones, including those described in U.S. Patent Νos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev. pp 169- 176). Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. These modifications ofthe ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.
The term "heterologous" as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally associated with a region of a recombinant construct, and/or are not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct is an identifiable segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Similarly, a host cell transformed with a construct which is not normally present in the host cell would be considered heterologous for purposes of this invention.
A "coding sequence" or a sequence which "encodes" a particular polypeptide (e.g. a PKS, an NRPS, etc.), is a nucleic acid sequence which is ultimately transcribed and or translated into that polypeptide in vitro and/or in vivo when placed under the control of appropriate regulatory sequences. In certain embodiments, the boundaries ofthe coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from procaryotic or eucaryotic mRNA, genomic DNA sequences from procaryotic or eucaryotic DNA, and even synthetic DNA sequences. In preferred embodiments, a transcription termination sequence will usually be located 3' to the coding sequence. Expression "control sequences" refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Not all of these control sequences need always be present in a recombinant vector so long as the desired gene is capable of being transcribed and translated.
"Recombination" refers to the reassortment of sections of DNA or RNA sequences between two DNA or RNA molecules. "Homologous recombination" occurs between two DNA molecules which hybridize by virtue of homologous or complementary nucleotide sequences present in each DNA molecule. The terms "stringent conditions" or "hybridization under stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. "Stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays, Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.
An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
Expression vectors are defined herein as nucleic acid sequences that are direct the transcription of cloned copies of genes/cDNAs and/or the translation of their mRNAs in an appropriate host. Such vectors can be used to express genes or cDNAs in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Expression vectors include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. Specifically designed vectors allow the shuttling of DNA between hosts, such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector preferably contains: an origin of replication for autonomous replication in a host cell, a selectable marker, optionally one or more restriction enzyme sites, optionally one or more constitutive or inducible promoters. In preferred embodiments, an expression vector is a replicable DNA construct in which a DNA sequence encoding a one or more PKS and/or NRPS domains and/or modules is operably linked to suitable control sequences capable of effecting the expression ofthe products of these synthase and/or synthetases in a suitable host. Control sequences include a transcriptional promoter, an optional operator sequence to control transcription and sequences which control the termination of transcription and translation, and so forth.
The term "conservative substitution" is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) ofthe molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). The "group consisting of ORF- 1 through ORF 42" refers to the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 81, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 9, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42 as identified in Tables I and II. In certain embodiments ORF 9 (cagA) is excluded.
A "biological molecule that is a substrate for a polypeptide encoded by a enediyne (e.g., C-1027) biosynthesis gene" refers to a molecule that is chemically modified by one or more polypeptides encoded by open reading frame(s) ofthe C-1027 biosynthesis gene cluster. The "substrate" may be a native molecule that typically participates in the biosynthesis of an enediyne, or can be any other molecule that can be similarly acted upon by the polypeptide. A "polymorphism" is a variation in the DNA sequence of some members of a species. A polymorphism is thus said to be "allelic," in that, due to the existence ofthe polymorphism, some members of a species may have the unmutated sequence (i.e. the original "allele") whereas other members may have a mutated sequence (i.e. the variant or mutant "allele"). In the simplest case, only one mutated sequence may exist, and the polymorphism is said to be diallelic. In the case of diallelic diploid organisms, three genotypes are possible. They can be homozygous for one allele, homozygous for the other allele or heterozygous. In the case of diallelic haploid organisms, they can have one allele or the other, thus only two genotypes are possible. The occurrence of alternative mutations can give rise to trialleleic, etc. polymoφhisms. An allele may be referred to by the nucleotide(s) that comprise the mutation.
"Single nucleotide polymoφhism" or "SNPs are defined by their characteristic attributes. A central attribute of such a polymoφhism is that it contains a polymoφhic site, "X," most preferably occupied by a single nucleotide, which is the site of the polymoφhism's variation (Goelet and Knapp U.S. patent application Ser. No.
08/145,145). Methods of identifying SNPs are well known to those of skill in the art (see, e.g., U.S. Patent 5,952,174).
Abbreviations used herein include LB, Luria-Bertani; NGDH, dNDP-glucose 4,6-dehydratase ; nt, nucleotide; ORF, open reading frame; PCR, polymerase chain reaction; PEG, polyethyleneglycol; PKS, polyketide synthase; RBS, ribosomal binding site; Apr, apramycin; R, resistant; Th, thiostrepton; WT, wild-type; and TS, temperature sensitive
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the structures of C-1027 chromophore and the benzenoid diradical intermediate proposed to initiate DNA cleavage. Figure 2 illustrates a scheme using C-1027 open reading frame polypeptides for the synthesis of deoxysugars.
Figure 3 A illustrates a scheme using C-1027 open reading frame polypeptides for the synthesis of a β-amino acid.
Figure 3B illustrates a scheme using C-1027 open reading frame polypeptides for the synthesis of a benzoxazolinate. Figure 4 illustrates the synthesis of the enediyne core and final assembly of the C-1027 enediyne.
Figures 5A, 5B, and 5C illustrate the organization ofthe C-1027 enediyne biosynthetic gene cluster. Figure 5 A shows a restriction map ofthe 75-kb sgc gene cluster from S. globisporus as represented by three cosmid clones. Figure 5B illustrates the genetic organization ofthe sgcA, sgcB, and cagA genes, showing that they are clustered in the sgc gene cluster. Probe 1, the 0.55-kb dNDP-glucose 4,6-dehydratase gene fragment from pBS1002. Probe 2, the 0.73-kb cagA fragment from pBS1003. A, Apal; B, RαmHI; E, EcoRI; K, Kpήl, S, SαcII; Sp, Sphl. Figure 5C shows the genetic organization ofthe C-1027 biosynthesis gene cluster.
Figure 6 shows the DNA and deduced amino acid sequences ofthe 3.0-kb RαmHI fragment from pBS1007, showing the sgcA and sgcB genes. Possible RBSs are boxed. The presumed translational start and stop sites are in boldface. Restriction enzyme sites of interest are underlined. The amino acids, according to which the degenerated PCR primer were designed for amplifying the dNDP-glucose 4,6-dehydratase gene from S. globisporus, are underlined.
Figure 7 shows the amino acid sequence alignment of SgcA with three other dNDP-glucose 4,6-dehydratases. Gdh, TDP-glucose 4,6-dehydratase of S. erythraea (AAA68211); MtmΕ, TDP-glucose 4,6-dehydratase in the mithramycin pathway of S. argillaceus (CAA71847); TylA2, TDP-glucose 4,6-dehydratase in the tylosin pathway of S. fradiae (S49054). Given in parentheses are protein accession numbers. The αβα fold with the NAD+-binding motif of GxGxxG is boxed.
Figures 8 A and 8B show disruption of sgcA by single crossover homologous recombination. Figure 8 A shows construction of sgcA disruption mutant and restriction maps ofthe wild-type S. globisporus C-1027 and S. globisporus SB 1001 mutant strains showing predicted fragment sizes upon RαmHI digestion. Figures 8B and 8C show a Southern analysis of S. globisporus C-1027 (lane 1) and S. globisporus SB 1001 (lanes 2, 3, and 4, three individual isolates) genomic DNA, digested with RαmHI, using (Figure 8B) ρOJ260 vector or (Figure 8C) the 0.75-kb SacIVKpnl fragment of sgcA from pBS1012 as a probe, respectively. B, RαmHI; K, Kpnl; S, SαcII.
Figures 9 A, 9B, 9B, and 9D illustrate the determination of C-1027 production in various S. globisporus strains by assaying their antibacterial activity against M. luteus. Figure 9A:1, S. globisporusC-1021; 2,3, and 4, S. globisporus SBIOOI (three individual isolates); 5, S. globisporus AF67; 6, S. globisporus AF40. Figure 9B: 1, S. globisporus C- 1027; 2, S. globisporus SB1001 (ρWHM3); 3 and 4, S. globisporus SBIOOI (pBS1015)(two individual isolates). Both S. globisporus SB1001 (pWHM3) and S. globisporus SBIOOI (pBS 1015) were grown in the presence of 5 μg/mL thiostrepton. Figure 9C: 1 , S. globisporusC-1021; 2, S. globisporus SB1001 (pBS1015); 3. S. globisporus SB1001; 4. S. globisporus SB 1001 (pWHM3); 5. S. globisporus AF40; 6. S. globisporus AF44. All S. globisporus strains were grown in the absence of thiostrepton. Figure 9D: 1. S. globisporus (pKCl 139); 2. S. globisporus (pBS1018).
DETAILED DESCRIPTION
This invention provides a complete gene cluster regulating the biosynthesis of C-1027, the most potent member ofthe enediyne antitumor antibiotic family. C-1027 is produced by Streptomyces globisporus C-1027 and consists of an apoprotein (encoded by the cagA gene) and a non-peptidic chromophore. The C-1027 chromophore could be viewed as being derived biosynthetically from a benzoxazohnate, a deoxyamino hexose, a β-amino acid, and an enediyne core. Adopting a strategy to clone the C-1027 biosynthesis gene cluster by mapping a putative dNDP-glucose 4,6-dehydratase (NGDH) gene to cagA, we localized 75 kb contiguous DNA from S. globisporus encoding a complete C-1027 gene cluster. Initial sequencing ofthe cloned gene cluster revealed two genes, sgcA and sgcB, that encode an NGDH enzyme and a transmembrane efflux protein, respectively, and confirmed that the cagA gene resides approximately 14 kb upstream ofthe sgcA,B locus. The involvement ofthe cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the sgcA gene to generate C-1027-nonproducing mutants and by complementing the sgcA mutants in vivo to restore C-1027 production.
Subsequent DNA sequence analysis provided the complete enediyne C-1027 gene cluster sequence (SEQ ID NOs: 1 and 2) revealing 50 open reading frames which are summarized in Tables I and II. These results represent the first cloning of a gene cluster for enediyne anti-tumor antibiotic biosynthesis. Table I. Summary ofthe C-1027 gene cluster open reading frames. Table 1. C-1027 gene cluster open reading frames (-7 to 26), primers for ORF amplification, and proposed functions
orf# Size Relative Primers Function Seq position ID
No. orf- 648 658-11 F d: ATG GGC ATG ACG GGT very weak 3
(-7) bp Rev: CTA GAG GAT CCC GGG homology to 4 putative hydroxylase orf- 549 1478- Fwd: ATG CCG CGG ATT CCC Viral 5
(-6) bp 930 Rev: TCA GCT GTC GAT GTC infectivity 6 potentiator protein orf- 1065 2713- Fwd: ATG ACC ATC GCC ACT N-truncated 7
(-5) bp 1649 Rev: TCA GAG GCC GAG CAC Methionine 8 synthase (likely psuedogene) orf- 387 3238- Fwd: ATG AGC TCG CTA CTG Viral 9
(-4) bp 2851 Rev: CTA GGA GCC GGT CGC transcription 10 factor orf- 1530 4971- Fwd: ATG AGC AGC AGC GCC Viral Homolog 11
(-3) bp 3442 Rev: TCA TTC GTC GGC TGC possibly primase 12 orf- 3027 5982- Fwd: GTG AGG GCT CTG CCG Glycerol- 13
(-2) bp 7478 Rev: TCA GAC GGC GGA GGG Phosphate ABC 14 Transporter (SnoX drug resistance) orf- 2328 9900- Fwd: GTG AGC GTC ACC GAC UvrA-like drug 15
(-1) bp 7573 Rev: TCA ACC CGC CCT GCG resistance pump 16 orf- 1368 11349- Fwd: ATG AGG ATG CTG GTG Na+/H+ efflux 17
0 bp 9982 Rev: GTG GCT GTG CTC GCA pump 18 orf- 999 28590- Fwd: ATG AGG ATG CTG GTG d TP-glucose 19
1 bp 29588 Rev: TCA GCC GAC GGC GTC dehydratase 20 orf- 1566 29632- Fwd: GTG ACA GCA GTC AAG Transmembrane 21
2 bp 31197 Rev: TCA TGT GGC CGG TTG efflux protein 22 orf- 1311 31280- Fwd: GTG GAG TAC TGG AAC Coenzyme F390 23
3 bp 32590 Rev: TCA GGC CTG AGG GGC synthase 24 phenylacetyl-CoA ligase orf- 1584 32809- Fwd: GTG CCC CAC GGT GCA phenol 25
4 bp 34392 Rev: CTA CAG CCC TCC GAG hydroxylase 26 chloropheno1-4- monooxygenase orf- bp 35274- Fwd: ATG TCT TCA ACC CGT citrate 27
5 34458 Rev: TCA GCC GCG CAG GAA transport 28 protein orf- 1272 17924- Fwd: ATG CTG GAG AAA TGC C-methyl 29
6 16653 Rev: TCA GAC GAG CTC CTT transferase 30 hydroxylase orf- 735 16653- Fwd: ATG GAG TAC GGC CCC N- 31 bp 15919 Rev: TCA TGC CGT GCG CAC methyltransferas 32 e orf- 1233 15922- Fwd: ATG AGC GGC GGC CCG Aminotransferase 33 8 bp 14690 Rev: TCA CCT CGC CGG ACG 34 orf- 432 14643- Fwd: ATG TCG TTA CGT CAC CagA 35 9 bp 14212 Rev: TCA GCC GAA GGT CAG 36 orf- 1068 13012- Fwd ATG AAG GCA CTT GTA dNTP-glucose 37
10 bp 14079 Rev TCA GGC CGC GAT CTC synthase 38 orf- 1485 12835- Fwd GTG GAC GTG TCA GCG Hydroxy1ase, 39
11 bp 11351 Rev TCA GGA CCG CGC ACC Halogenase 40 orf- 579 25564- Fwd: ATG AAG CCG ATC GGG dNTP-4-keto-6- 41
12 bp 24986 Rev: TCAGGA CGA CTT GTT deoxyglucose 42 3, 5-epimerase orf- 1137 24702- Fwd: ATG CCT TCC CCC TTC 3-0- 43 13 bp 23566 Rev: TCA GGT GCG CTC GGC acyltransferase 44 orf- 1455 22878- Fwd: GTG AGA GAC GGC CGG Coenzyme F-390 45 14 bp 21424 Rev: TCA CGT GGT GAT GGC Synthase 46 Phenylacetyl CoA Ligase orf- 1482 21407- Fwd: ATG ACC GAC CAG TGC Anthranilate 47 15 bp 19926 Rev: TCA CAG CAA CTC CTC Synthase I 48 orf- 663 19929- Fwd: GTG AGC TTG TGG TCT Anthranilate 49
16 bp 19267 Rev: TCA GGC CGG TTC GGC Synthase" II 50 orf- 1161 19191- Fwd: GTG CGT CCC TTC CGT epoxide 51
17 bp 18031 Rev: TCA GCG GAG CGG ACG hydrolase 52 orf- 423 35938- Fwd: ATG CCA GCA CCG ACT Unknown 53
18 bp 35516 Rev: TCA GTC GTT GCC GCG 54 orf- 1380 27214- Fwd: ATG CGG GTG ATG ATC glycosyl 55
19 bp 28593 Rev: TCA TCG GTC CGC CTC transferase 56 orf- 1356 25815- Fwd: ATG ACC AAG CAC GCC squalene 57
20 bp 27170 Rev: TCA TAC GGC GGC GCC monooxygenase 58 orf- 672 23546- Fwd: GTG AGC GCA CAA CTC hypothetical Fe- 59
21 bp 22875 Rev: TCA CGG CTG TGC CTG S flavoprotein 60 orf- 816 35274- Fwd: ATG TCT TCA ACC CGT haloacetate 61
22 bp 34458 Rev: TCA GCC GCG CAG GAA dehalogenase 62 hydrolase orf- 1380 37559- Fwd: ATG ACG ACG TCC GAC peptide 63
23 bp 38938 Rev. TCA GGA GGT GAA GGG synthetase 64 orf- 1620 40986- Fwd: ATG GCA TTG ACT CAA Histidine 65
24 bp 39367 Rev: TCA GCG CAG CTG GAT Ammonia lyase 66 orf- 1560 42611 Fwd: ATG ACG CGG CCG GTG Type II 67
25 bp 41052 Rev: TCA GCG GGT GAG CCG adenylation 68 protein orf- 282 38983 Fwd: GTG TCC ACC GTT TCC Type II peptidyl 69
26 bp 39264 Rev: TCA CTG CGT TCC GGA carrier protein 70 Table II. C-1027 gene cluster open reading frames (27 to 42), primers for ORF amplification, and proposed functions
ORF Relative Primers Function SEQ
Position ID
NO. orf- 43945- -46023 Fwd GTG TGC CCG GTG ACA GAC Antibiotic 71
27 Rev TCA GCC CAC GGG CTG GGA Transporter 72 orf- 46167- -47171 Fwd GTG TTG GGC GAT GAG GAC 0- 73
28 Rev TCA GAC CGC GGA CAT CTG methyltransfer 74 ase orf-- 47227- -48485 Fwd ATG GCC GGC CTG GTC ATG p450 75
29 Rev TCA GGA CCC GAG GGT CAC hydroxylase 76 orf- 48610- -49714 Fwd GTG GAC CAG ACG TCT ACG Oxidoreductase 77
30 Rev TCA TGC AGG TGC AGC GTG 78 orf- 50350- -51390 Fwd ATG AGG CCG CTC GTT CGG Unknown 79
31 Rev TCA TCC CGG CCC GGC GGC Protein 80 orf- 51420- -52341 Fwd ATG AGA ACG CGG CGA CGC Oxidoreductase 81
32 Rev TCA CGG CCG GAG GCG TAC 82 orf- 53241- -54074 Fwd GTG TAT CAG CCG GAC TGT Unknown 83
33 Rev CTA CTC ATT CCA GTT GTG Protein 84 orf- 54230- -55379 Fwd ATG TCT ACG GGC TAT CTC Unknown 85
34 Rev TCA GCC GCC GGT GGC GCC Protein 86 orf- 56027- -56881 Fwd ATG TTC TCC CCC GCC GCC Oxidase/ 87
35 Rev TCA GTA CGC CTG GTG GGC Dehydrogenase 88 orf- 56928- -57730 Fwd ATG AAT TCG CTC GAC GAC Unknown 89
36 Rev : TCA GCT CCC GGT CGC CGC Protein 90 orf- 57834 -58304 Fwd ATG ACC GCG ACG AAT CCT Regulatory 91
37 Rev CTA GGC GGC GCG TCC CGC 92 orf- 58440 -60091 Fwd : ATG AGC ACC ACG GCC GAG Oxidoreductase 93
38 Rev : TCA GCC GCG CGC CGA CGG 94 orf- 60092 -60622 Fwd : ATG ACC CTG GAG GCC TAC Regulatory 95
39 Rev : TCA TGC GGG GCT CCC GGT 96 orf- 60940 -62020 Fwd : GTG AAA AGT GAC TCT GCC Regulatory 97
40 Rev : TCA ACG GCG AGT TGG CTG 98 orf- 62045 -62899 Fwd : GTG ACC ACG AAC ACC ATC Regulatory 99
41 Rev : TCA CCC GCG ATC TCG ATC 100 orf- 62788 -63164 Fwd : (partial ORF) p450 101
42 Rev : TCA CCT CGC CGT ACT CAC hydroxylase 102
Surprisingly, sequence analysis failed to reveal any gene that resembles a polyketide synthase. The C-1027 open reading frames, however, encode polypeptides exhibiting a wide variety of enzymatic activities (e.g., epoxide hydrase, monooxygenase, oxidoreductase, P-450 hydroxylase, etc.). The isolated C-1027 gene cluster can be used to synthesize C-1027 enediyne antibiotics and/or analogues thereof. The C-1027 gene cluster can be modified and/or augmented to increase C-1027 and/or C-1027 analogue production. Alternatively, various components ofthe C-1027 gene cluster can be used to synthesize and/or chemically modify a wide variety of metabolites. Thus, for example, ORF 6 (C-methyltransferase) can be used to methylate a carbon, while ORF 12, an epimerase, can be used to change the conformation of a sugar. The ORFs can be combined in their native configuration or in modified configurations to synthesize a wide variety of biomolecules/metabolites. Thus, for example, various combinations of C-1027 open reading frames can be used to synthesize an enediyne core, to synthesize a deoxy sugar, to synthesize a β-amino acid, to make a benzoxazolinate, etc (see, e.g., Figures 2, 3, and 4).
The native C-1027 gene cluster ORFs can be re-ordered, modified, and combined with other biosynthetic units (e.g. polyketide synthases (PKSs) or catalytic domains thereof and/or non-ribosomal polypeptide synthetases (NRPSs) or catalytic domains thereof) to produce a wide variety of molecules. Large chemical libraries can be produced and then screened for a desired activity.
The C-1027 gene cluster also includes a number of drug resistance genes (see, e.g., Table 2) that confer resistance to C-1027 and/or metabolites involved in C-1027 biosynthesis thereby permitting the cell to complete the enediyne biosynthesis. These resistance genes can be used to confer enediyne resistance on a cell lacking such resistance or to augment the enediyne resistance of a cell that does tolerate enediynes. Such cells can be used to produce high levels of enediynes and/or enediyne metabolites, and/or enediyne analogues.
Table III. C-1027 cluster drug resistance genes.
ORF Protein Mechanism
ORF 9: CagA apoprotein Drug sequestering
ORF 2: SgcB transmembrane efflux protein Drug exporting
ORF 27 Transmembrane transport protein Drug exporting
ORF 0 Na'VH* transporter Drug exporting
ORF -1 ABC transport (C-terminus) Drug exporting
ORF -2 Glycerol phosphate transporter Drug exporting
ORF -1 UvrA-like protein (N-terminus) DNA repairing I. Isolation, preparation, and expression of C-1027 nucleic acids.
The C-1027 gene cluster nucleic acids can be isolated, optionally modified, and inserted into a host cell to create and/or modify a metabolic (biosynthetic) pathway and thereby enable that host cell to synthesize and/or modify various metabolites. Alternatively the C-1027 gene cluster nucleic acids can be expressed in the host cell and the encoded C- 1027 polypeptide(s) recovered for use as chemical reagents, e.g. in the ex vivo synthesis and/or chemical modification of various metabolites. Either application typically entails insertion of one or more nucleic acids encoding one or more isolated and/or modified C-1027 enediyne open reading frames in a suitable host cell. The nucleic acid(s) are typically in an expression vector, a construct containing control elements suitable to direct expression ofthe C-1027 polypeptides. The expressed C-1027 polypeptides in the host cell then act as components of a metabolic/biosynthetic pathway (in which case the synthetic product ofthe pathway is typically recovered) or the C-1027 polypeptides themselves are recovered. Using the sequence information provided herein, cloning and expression of C- 1027 nucleic acids can be accomplished using routine and well known methods.
A) C-1027 nucleic acids.
The nucleic acids comprising the C-1027 gene cluster are identified in Tables I and are listed in the sequence listing provided herein. In particular, Table 1 identifies genes and functions of open reading frames (ORFs) in the C-1027 enediyne biosynthesis gene cluster and identifies primers suitable for the amplification/isolation of any one or more of the C-1027 open reading frames. Of course, using the sequence information provided herein, other primers suitable for amplification/isolation of one or more C-1027 open reading frames can be determined according to standard methods well known to those of skill in the art (e.g. using Vector NTI Suite™, InforMax, Gaithersberg, MD, USA).
Typically such amplifications will utilize the DNA or RNA of an organism containing the requisite genes (e.g. Streptomyces globisporus) as a template. Typical amplification conditions include the following PCR temperature program: initial denaturing at 94°C for 5 min, 24-36 cycles of 45 sec at 94°C, 1 min at 60°C, 2 min at 72°C, followed by additional 7 min at 72°C. One of skill will appreciate that optimization of such a protocol, e.g. to improve yield, etc. is routine (see, e.g., U.S. Patent No. 4,683,202; Innis (1990) PCR Protocols A Guide to Methods and Applications Academic Press Inc. San Diego, CA, etc). In addition, primer may be designed to introduce restriction sites and so facilitate cloning of the amplified sequence into a vector.
In one embodiment, this invention provides nucleic acids for the recombinant expression of an enediyne (e.g. a C-1027 enediyne or an analogue thereof). Such nucleic acids include isolated gene cluster(s) comprising open reading frames encoding polypeptides sufficient to direct the assembly ofthe enediyne. In other embodiments of this invention, the C-1027 open reading frames may be unchanged, but the control elements (e.g. promoters, enhancers, etc.) may be modified. In still other embodiments, the nucleic acids may encode selected components (e.g. one or more C-1027 or modified C-1027 open reading frames) and/or may optionally contain other heterologous biosynthetic elements including, but not limited to polyketide synthase (PKS) and/or non-ribosomal polypeptide synthetase (NRPS) modules or enzymatic domains.
Such variations may be introduced by design, for example to modify a known molecule in a specific way, e.g. by replacing a single substituent ofthe enediyne with another, thereby creating a derivative enediyne molecule of predicted structure. Alternatively, variations can be made randomly, for example by making a library of molecular variants of a known enediyne by systematically or haphazardly replacing one or open reading frames in the biosynthetic pathway. Production of alternative/modified enediyne, and hybrid enediyne PKSs and/or NRPSs and hybrid systems is described below. Using the information provided herein other approaches to cloning the desired sequences will be apparent to those of skill in the art. For example, the enediyne, and/or optionally PKS and/or NRPS modules or enzymatic domains of interest can be obtained from an organism that expresses such, using recombinant methods, such as by screening cDNA or genomic libraries, derived from cells expressing the gene, or by deriving the gene from a vector known to include the same. The gene can then be isolated and combined with other desired biosynthetic elements using standard techniques. If the gene in question is already present in a suitable expression vector, it can be combined in situ, with, e.g., other PKS subunits, as desired. The gene of interest can also be produced synthetically, rather than cloned. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. In general, one will select preferred codons for the intended host in which the sequence will be expressed. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence (see, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223: 1299; Jay et al. (1984) J. Biol. Chem. 259:6311). In addition, it is noted that custom gene synthesis is commercially available (see, e.g. Operon Technologies, Alameda, CA). Examples of such techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel (1989) Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Ausubel (19 1994) Current Protocols in Molecular Biology, Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., U.S. Patent 5,017,478; and European Patent No. 0,246,864.
B) Expression of f C-1027 open reading frames.
The choice of expression vector depends on the sequence(s) that are to be expressed. Any fransducible cloning vector can be used as a cloning vector for the nucleic acid constructs of this invention. However, where large clusters are to be expressed, it phagemids, cosmids, Pis, YACs, BACs, PACs, HACs or similar cloning vectors be used for cloning the nucleotide sequences into the host cell. Phagemids, cosmids, and BACs, for example, are advantageous vectors due to the ability to insert and stably propagate therein larger fragments of DNA than in M13 phage and lambda phage, respectively. Phagemids which will find use in this method generally include hybrids between plasmids and filamentous phage cloning vehicles. Cosmids which will find use in this method generally include lambda phage-based vectors into which cos sites have been inserted. Recipient pool cloning vectors can be any suitable plasmid. The cloning vectors into which pools of mutants are inserted may be identical or may be constructed to harbor and express different genetic markers (see, e.g., Sambrook et al., supra). The utility of employing such vectors having different marker genes may be exploited to facilitate a determination of successful transduction.
In preferred embodiments of this invention, vectors are used to introduce C- 1027 biosynthesis genes or gene clusters into host (e.g. Streptomyces) cells. Numerous vectors for use in particular host cells are well known to those of skill in the art. For example described in Malpartida and Hopwook, (1984) Nature, 309:462-464; Kao et al., (1994), Science, 265: 509-512; and Hopwood et al, (1987) Methods Enzymol, 153:116-166 all describe vectors for use in various Streptomyces hosts.
In one prefeπed embodiment, Streptomyces vectors are used that include sequences that allow their introduction and maintenance in E. coli. Such Streptomyces! E. coli shuttle vectors have been described (see, for example, Vara et al, (1989) J. Bacteriol, 171:5872-5881; Guilfoile & Hutchinson (1991) Proc. Natl. Acad. Sci. USA, 88: 8553-8557.) The wildtype and/or modified C-1027 enediyne open reading frame(s) of this invention, can be inserted into one or more expression vectors, using methods known to those of skill in the art. Expression vectors will include control sequences operably linked to the desired open reading frame. Suitable expression systems for use with the present invention include systems that function in eucaryotic and/or prokaryotic host cells. However, as explained above, prokaryotic systems are prefeπed, and in particular, systems compatible with Streptomyces spp. are of particular interest. Control elements for use in such systems include promoters, optionally containing operator sequences, and ribosome binding sites. Particularly useful promoters include control sequences derived from enediyne, and/or PKS, and/or NRPS gene clusters. Other promoters (e.g. ermE* as illustrated in Example 1) are also suitable. Other bacterial promoters, such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, will also find use in the present constructs. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the beta -lactamase (bid) promoter system, bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Patent 4,551,433), which do not occur in nature also function in bacterial host cells. In Streptomyces, numerous promoters have been described including constitutive promoters, such as ErmE and TcmG (Shen and Hutchinson, (1994) J. Biol Chem. 269: 30726-30733), as well as controllable promoters such as actl and actlll (P leper et al, (1995) Nature, vol. 378: 263-266; Pieper et al, (1995) J. Am. Chem. Soc, 117: 11373-11374; and Wiesmann et al, (1995) Chem. & Biol. 2: 583-589).
Other regulatory sequences may also be desirable which allow for regulation of expression ofthe enediyne open reading frame(s) relative to the growth ofthe host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences. Selectable markers can also be included in the recombinant expression vectors. A variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid.
The various enediyne cluster open reading frames, and/or PKS, and/or NRPS clusters or subunits of interest can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter. The various open reading frames can include flanking restriction sites to allow for the easy deletion and insertion of other open reading frames so that hybrid synthetic pathways can be generated. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site- directed mutagenesis and PCR. Methods of cloning and expressing large nucleic acids such as gene clusters, including PKS- or NRPS-encoding gene clusters, in cells including Streptomyces are well known to those of skill in the art (see, e.g., Stutzman-Engwall and Hutchinson (1989) Proc. Natl. Acad. Sci. USA, 86: 3135-3139; Motamedi and Hutchinson (1987) Proc. Natl. Acad. Sci. USA, 84: 4445-4449; Grim et al. (1994) Gene, 151: 1-10; Kao et al (1994) Science, 265: 509-512; and Hopwood et al (1987) Meth. Enzymol, 153: 116-166). In some examples, nucleic acid sequences of well over lOOkb have been introduced into cells, including prokaryotic cells, using vector-based methods (see, for example, Osoegawa et al, (1998) Genomics, 52: 1-8; Woon et al, (1998) Genomics, 50: 306-316; Huang et al, (1996) Nucl. Acids Res., 24: 4202-4209). In addition, the cloning and expression of C-1027 enediyne is illustrated in Example 1.
O Host cells.
The vectors described above can be used to express various protein components ofthe enediyne, and/or enediyne shunt metabolites, and/or other modified metabolites for subsequent isolation and/or to provide a biological synthesis of one or more desired biomolecules (e.g. C-1027 and/or a C-1027 analogue, etc.). Where one or more proteins ofthe enediyne biosynthetic gene cluster are expressed (e.g. overexpressed) for subsequent isolation and/or characterization, the proteins are expressed in any prokaryotic or eukaryotic cell suitable for protein expression. In one prefeπed embodiment, the proteins are expressed in E. coli.
Host cells for the recombinant production ofthe subject enediynes, enediyne metabolites, shunt metabolites, etc. can be derived from any organism with the capability of harboring a recombinant enediyne gene cluster and/or subset thereof. Thus, the host cells of the present invention can be derived from either prokaryotic or eucaryotic organisms.
Prefeπed host cells are those of species or strains (e.g. bacterial strains) that naturally express enediynes. Such host cells include, but are not limited to Actinomycetes,
Actinoplanetes, and Streptomycetes, Actinomadura, Micromonospra, and the like. Particularly prefeπed host cells include, but are not limited to Streptomyces globisporus,
Streptomyces lividans, Streptomyces coelicolor, Micromonospora echinospora spp. calichenisis, Actinomadura verrucosopora, Micromonospora chersina, Streptomyces carzinostaticus, and Actinomycete L585-6. Other suitable host cells include, but are not limited to S. verticillis S. ambofaciens, S. avermitilis, S. azureus, S. cinnamonensis, S. coelicolor, S. curacoi, S. erythraeus, S.fradiae, S. galilaeus, S. glaucescens, S. hygroscopicus, S. lividans, S. parvulus, S. peucetius, S. rimosus, S. roseofulvus, S. thermotolerans, and S. violaceoruber (see, e.g., Hopwood and Sherman (1990) Ann. Rev.
Genet. 24: 37-66; O'Hagan (1991) The Polyketide Metabolites, Ellis Horwood Limited, etc.). In certain embodiments, a eukaryotic host cell is prefeπed (e.g. where certain glycosylation patterns are desired). Suitable eukaryotic host cells are well known to those of skill in the art. Such eukaryotic cells include, but are not limited to yeast cells, insect cells, plant cells, fungal cells, and various mammalian cells (e.g. COS, CHO HeLa cells lines and various myeloma cell lines).
D) Recovery of the expression product.
Recovery ofthe expression product (e.g., enediyne, enediyne analogue, enediyne biosynthetic pathway polypeptide, etc.) is accomplished according to standard methods well known to those of skill in the art. Thus, for example where enediyne biosynthetic gene cluster proteins are to be expressed and isolated, the proteins can be expressed with a convenient tag to facilitate isolation (e.g. a His6) tag. Other standard protein purification techniques are suitable and well known to those of skill in the art (see, e.g., (Quadri et α/. (1998) Biochemistry 37: 1585-1595; Nakano et α/. (1992) Mol. Gen. Genet. 232: 313-321, etc.).
Similarly where components (e.g. enediyne biosynthetic cluster orfs) are used to synthesize and/or modify various biomolecules (e.g. enediynes, enediyne analogues, shunt metabolites, etc.) the desired product and/or shunt metabolite(s) are isolated according to standard methods well know to those of skill in the art (see, e.g., Caπeras and Khosla (1998) Biochemistry 37: 2084-2088, Deutscher (1990) Methods in Enzymology Volume 182: Guide to Protein Purification, M. Deutscher, ed. etc.).
II. Use of C-1027 open reading frames in directed biosynthesis. Elements (e.g. open reading frames) ofthe C-1027 biosynthetic gene cluster and/or variants thereof can be used in a wide variety of "directed" biosynthetic processes (i.e. where the process is designed to modify and/or synthesize one or more particular preselected metabolite(s)). Essentially the entire C-1027 gene cluster can be used to synthesize a C-1027 enediyne and/or a C-1027 enediyne analogue. Individual C-1027 cluster open reading frames can be used to perform chemically modifications on particular substrates and/or to synthesize various metabolites. Thus, for example, ORF 6 (C-methyltransferase can be used to methylate a carbon), while ORF 7 (N-methyltransferase) can be used to methylate a nitrogen. ORF 12, and epimerase, can be used to change the conformation of a sugar, and ORF 8 (an amino transferase) can be used to aminate a suitable substrate. Similarly, combinations of C-1027 open reading frames can be used to direct the synthesis of various metabolites (e.g. β-amino acids, deoxysugars, benzoxazolinates, and the like). These examples, are merely illustrative. One of skill in the art, utilizing the information provided here, can perform literally countless chemical modifications and/or syntheses using either "native" enediyne biosynthesis metabolites as the substrate molecule, or other molecules capable of acting as substrates for the particular enzymes in question. Other substrates can be identified by routine screening. Methods of screening enzymes for specific activity against particular substrates are well known to those of skill in the art.
The biosyntheses can be performed in vivo, e.g. by providing a host cell comprising the desired C-1027 gene cluster open reading frames and/or in vivo, e.g., by providing the polypeptides encoded by the C-1027 gene cluster ORFs and the appropriate substrates and/or cofactors. A) Synthesis of enediynes and enediyne analogues.
In one embodiment, this invention provides for the synthesis of C-1027 enediynes and/or C-1027 analogues or derivatives. In a prefeπed embodiment, this is accomplished by providing a cell comprising a C-1027 gene cluster and culturing the cell under conditions whereby the desired enediyne or enediyne analogue is synthesized. The cell can be a cell that does not normally synthesize an enediyne and the entire gene cluster can be transfected into the cell. Alternatively, a cell that typically synthesizes enediynes can be utilized and all or part ofthe C-1027 gene cluster can be introduced into the cell.
Enediyne derivatives/analogues can be produced by varying the order of, or kind of, gene cluster subunits present in the cell, and/or by changing the host cell (e.g. to a eukaryotic cell that glycosylates the biosynthetic product), and/or by providing altered metabolites (e.g. adding exogenous aglycones to a host that carries a gene cassette ofthe deoxysugar biosynthesis and glycosylation genes for the production of glycosylated metabolites), etc. In certain embodiments, the host cell need not be transfected with an entire C-
1027 gene cluster. Rather, various components of a C-1027 gene cluster can be altered within a cell already harboring a C-1027 cluster. By varying or adding various biosynthetic open reading frames, C-1027 enediyne variants can be produced.
The use of standard techniques of molecular biology (gene disruption, gene replacement, gene supplement) can be used to modulate and or otherwise alter enediyne and/or other metabolite (e.g. shunt metabolite) production in an organism that naturally synthesizes an enediyne (e.g. S. globisporus) or an organism that is modified to synthesize an enediyne.
In addition, or alternatively, control sequences that alter the expression of various open reading frames can be introduced that alter the amount and/or timing of enediyne production. Thus, for example, by placing particular C-1027 open reading frames under control of a constitutive promoter (ermE*) C-1027 production was increased by as much as 4-fold (see, e.g. Table 3 and Example 1).
Table 3. Alteration of C-1027 production by engineering the C-1027 biosynthesis gene cluster.
Strain Yield (%) WT 100
WT/pKC1139 100
WT/ermE*/ORF 2 >150
WT/ORF 9 >100
WT/ermE*/ORF 9 <10
WT/ORF 10, 11 >100
WT/errnE*/ORF 10, 11 >100
WT/ ORF 9, 10, 11 >400
ORF 2: transmembrane eflux protein; ORF 9: CagA apoprotein; ORF 10: TDP-glucose synthase; ORF 11; Hydroxylase/halogenase
Where enediyne analogues are synthesized, it will often prove desirable to assay them for biological activity. Such assays are well know to those of skill in the art. One such assay is illustrated in Example 1. Briefly, this example depicts an assay of antibacterial activity against M. luteus as described by Hu et al. (1988) J. Antibiot. 41: 1575- 1579). Other suitable assays for enediyne activity will be known to those of skill in the art.
B) Use of C-1027 open reading frames to synthesize an enediyne core. The C-1027 open reading frames described herein, or variants thereof, can be used to synthesize an enediyene core, e.g., from a fatty acid precursor. One such synthetic pathway is illustrated in Figure 4. This reaction scheme utilizes ORF 17 (epoxide hydrase), ORF 20 (monooxygenase), ORF 21 (iron-sulfur flavoprotein), ORF 29 (P-450 hydroxylase, ORF 30 (oxidoreductase), ORF 32 (oxidoreductase), ORF 35 (proline oxidase), and ORF 38 (P-450 hydroxylase) to synthesize anenediyne core.
This synthetic pathway, is not considered limiting, but merely illustrative. Using this as a model, one of ordinary skill in the art can design numerous other synthetic schemes to produce enediyne cores and/or core variants.
O Use of C-1027 open reading frames to synthesize deoxy sugars. The biosynthesis of various deoxy sugars (e.g., deoxyhexoses) typically share a common key intermediate ~4-keto-6-deoxyglucose nucleoside diphosphate or its analogs, whose formation from glucose nucleoside diphosphate is catalyzed by the NGDH enzyme, an NAD+-dependent oxidoreductase (Liu and Thorson (1994) Ann. Rev. Microbiol. 48: 223-
256; Piepersberg (1997) pp. 81-163. In Biotechnology of antibiotics, 2nd ed. W. R. Strohl (ed). Marcel Dekker, New York.). Similarly, the C-1027 gene cluster includes an NAGDH enzyme which can be exploited to synthesize a variety of deoxy sugars.
One illustrative synthetic pathway is shown in Figure 2. This biosynthetic scheme utilizes ORF 10 (dNDP-glucose synthase), ORF 1 (dNDP-glucose dehydratase), ORF 12 (epimerase), ORF 8 (aminotransferase), ORF 6 (C-methyltransferase), ORF 7 (N- methyltransferase) and ORF 19 (glycosyl transferase).
This synthetic pathway, is not considered limiting, but merely illustrative. Using this as a model, one of ordinary skill in the art can design numerous other synthetic schemes to produce various deoxy sugars.
D) Use of C-1027 open reading frames to synthesize β-amino acids.
In still another embodiment, C-1027 biosynthetic polypeptides can be used in the biosynthesis of β-amino acids. One illustrative synthetic pathway is shown in Figure 3 A. This biosynthetic scheme utilizes ORF 4 (hydroxylase), ORF 11 (hydroxylase/halogenase), ORF 24 (aminomutase), ORF 23 (type II NRPS condensation enzyme), ORF 25 (type II NRPS adenylation enzyme), and ORF 26 (type II peptidyl carrier protein).
Again, this synthetic pathway, is not considered limiting, but merely illustrative. Using this as a model, one of ordinary skill in the art can design numerous other synthetic schemes to produce other beta amino acids.
E) Use of C-1027 open reading frames to synthesize benzoxazolinates. The C-1027 open reading frames can also be used to synthesize a benzoxazohnate. One illustrative synthetic pathway is shown in Figure 3B. This biosynthetic scheme utilizes ORF 15 (anthranilate synthase I, ORF 16 (anthranilate synthase II), ORF 4 (phenol hydroxylase/chlorophenol-4-monooxygenase), ORF 11 (Hydroxylase/Halogenase), ORF 28 (O-methyltransferase), ORF 3 (coenzyme F390 synthetase, ORF 14 (coenzyme F390 synthetase), and ORF 13 (O-acyltransferase). Again, this synthetic pathway, is not considered limiting, but merely illustrative. Using this as a model, one of ordinary skill in the art can design numerous other synthetic schemes to produce other beta amino acids. III. Generation of chemical diversity.
In addition to the directed modification and/or biosynthesis of various metabolites as described above, the C-1027 biosynthetic gene cluster open reading frames can be utilized, by themselves or in combination with other biosynthetic subunits (e.g. NRPS and/or PKS modules and/or enzymatic domains of other PKS and/or NRPS systems) to produce a wide variety of compounds including, but not limited to various enediyne or enediyne derivatives, various polyketides, polypeptides, polyketide/polypeptide hybrids, various thiazoles, various sugars, various methylated polypeptides/polyketides, and the like. As with the directed production of various metabolites described above, such compounds can be produced, in vivo or in vitro, by catalytic biosynthesis, e.g., using large, enediyne cluster units and/or modular PKSs, NRPSs, and hybrid PKS/NRPS systems. In a prefeπed embodiment large combinatorial libraries of cells harboring various megasynthetases can be produced by the random or directed modification of particular pathways and then selected for the production of a molecule or molecules of interest. It will be appreciated that, in certain embodiments, such libraries of megasynthetases/modified pathways, can be used to generate large, complex combinatorial libraries of compounds which themselves can be screened for a desired activity.
Such combinatorial libraries can be created by the deliberate modification variation of selected biosynthetic pathways and/or by random/haphazard modification of such pathways.
A) Directed engineering of novel synthetic pathways.
In numerous embodiments of this invention, novel polyketides, polypeptides, and combinations thereof are created by modifying the entediyne gene cluster ORFs and/or known PKSs, and/or NRPSs so as to introduce variations into metabolites synthesized by the enzymes. Such variations may be introduced by design, for example to modify a known molecule in a specific way, e.g. by replacing a single monomeric unit within a polymer with another, thereby creating a derivative molecule of predicted structure. Such variations can also be made by adding one or more modules or enzymatic domains to a known PKS or NRPS or enediyne cluster, or by removing one or more module from a known PKS or NRPS.
Using any of these methods, it is possible to introduce PKS domains, NRPS domains, and entediyne domains into a megasynthetase. Mutations can be made to the native enediyne, and/or NRPS, and/or PKS subunit sequences and such mutants used in place ofthe native sequence, so long as the mutants are able to function with other subunits (domains) in the synthetic pathway. Such mutations can be made to the native sequences using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a NRPS and/or PKS subunit using restriction endonuclease digestion, (see, e.g., Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82: 448; Geisselsoder et al. (1987) BioTechniques 5: 786). Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA coπesponding to the RNA sequence), at a temperature below the melting temperature ofthe mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively naπow limits and by keeping the mutant base centrally located (Zoller and Smith (1983) Meth, Enzymol 100: 468). Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation ofthe primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations (see, e.g., Dalbie-McFarland et al. (1982) Proc. Natl Acad. Sci USA 79:6409). PCR mutagenesis will also find use for effecting the desired mutations.
B) Random modification of enediyne pathways. In another embodiment, variations can be made randomly, for example by making a library of molecular variants (e.g. of a known enediyne) by randomly mutating one or more elements ofthe subject gene cluster or by randomly replacing one or more open reading frames in a gene cluster with one or more of alternative open reading frames.
The various open reading frames can be combined into a single multi-modular enzyme, thereby dramatically increasing the number of possible combinations obtained using these methods. These combinations can be made using standard recombinant or nucleic acid amplification methods, for example by shuffling nucleic acid sequences encoding various modules or enzymatic domains to create novel aπangements ofthe sequences, analogous to DNA shuffling methods described in Crameri et al. (1998) Nature 391: 288-291, and in U.S. Patents 5,605,793 and in 5,837,458. In addition, novel combinations can be made in vitro, for example by combinatorial synthetic methods. Novel molecules or molecule libraries, can be screened for any specific activity using standard methods. Random mutagenesis ofthe nucleotide sequences obtained as described above can be accomplished by several different techniques known in the art, such as by altering sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, by iπadiation with X-rays or ultraviolet light, by incorporating incoπect nucleotides during in vitro DNA synthesis, by eπor-prone PCR mutagenesis, by preparing synthetic mutants or by damaging plasmid DNA in vitro with chemicals. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like.
Generally, plasmid DNA or DNA fragments are treated with chemicals, transformed into E. coli and propagated as a pool or library of mutant plasmids.
Large populations of random enzyme variants can be constructed in vivo using "recombination-enhanced mutagenesis." This method employs two or more pools of, for example, 106 mutants each ofthe wild-type encoding nucleotide sequence that are generated using any convenient mutagenesis technique, described more fully above, and then inserted into cloning vectors.
O Incorporation and/or modification of non-C-1027 cluster elements.
In either the directed or random approaches, nucleic acids encoding novel combinations of gene cluster ORFs are introduced into a cell. In one embodiment, nucleic acids encoding one or more enediyne synthetic cluster ORFS and or PKS and/or NRPS domains are introduced into a cell so as to replace one or more domains of an endogenous gene cluster within a cell. Endogenous gene replacement can be accomplished using standard methods, such as homologous recombination. Nucleic acids encoding an entire enediyne, enediyne ORF, PKS, NRPS, or combination thereof can also be introduced into a cell so as to enable the cell to produce the novel enzyme, and, consequently, synthesize the novel polymer. In a prefeπed embodiment, such nucleic acids are introduced into the cell optionally along with a number of additional genes, together called a 'gene cluster,' that influence the expression ofthe genes, survival ofthe expressing cells, etc. In a particularly prefeπed embodiment, such cells do not have any other enediyne and/or PKS- and/or NRPS- encoding genes or gene clusters, thereby allowing the straightforward isolation ofthe molecule(s) synthesized by the genes introduced into the cell. Furthermore, the recombinant vector(s) can include genes from a single enediyne and/or PKS and/or NRPS gene cluster, or may comprise hybrid replacement PKS gene clusters with, e.g., a gene for one cluster replaced by the coπesponding gene from another gene cluster. For example, it has been found that ACPs are readily interchangeable among different synthases without an effect on product structure. Furthermore, a given KR can recognize and reduce polyketide chains of different chain lengths. Accordingly, these genes are freely interchangeable in the constructs described herein. Thus, the replacement clusters ofthe present invention can be derived from any combination of PKS and/or NRPS gene sets that ultimately function to produce an identifiable polyketide. Examples of hybrid replacement clusters include, but are not limited to, clusters with genes derived from two or more ofthe act gene cluster, the whiE gene cluster, frenolicin (fre ), granaticin (gra), tetracenomycin (tcm), 6-methylsalicylic acid (6-msas), oxytetracycline (otc), tetracycline (tet), erythromycin (ery), griseusin (gris), nanaomycin, medermycin, daunorubicin, tylosin, carbomycin, spiramycin, avermectin, monensin, nonactin, curamycin, rifamycin and candicidin synthase gene clusters, among others. (For a discussion of various PKSs, see, e.g., Hopwood and Sherman (1990) Ann. Rev. Genet. 24: 37-66; OΗagan (1991) The Polyketide Metabolites, Ellis Horwood Limited.
A number of hybrid gene clusters have been constructed, having components derived from the act.fren, tcm, gris and gra gene clusters (see, e.g., U.S. Patent 5,712,146). Other hybrid gene clusters, as described above, can easily be produced and screened using the disclosure herein, for the production of identifiable polyketides, polypeptides or polyketide/polypeptide hybrids.
Host cells (e.g. Streptomyces) can be transformed with one or more vectors, collectively encoding a functional PKS/NRPS set, or a cocktail comprising a random assortment of enediyne ORFs and/or PKS and/or NRPS genes, modules, active sites, or portions thereof. The vector(s) can include native or hybrid combinations of enediyne ORFs, and/or PKS and/or NRPS subunits or cocktail components, or mutants thereof. As explained above, the gene cluster need not coπespond to the complete native gene cluster but need only encode the necessary enediyne ORFs and/or PKS and/or NRPS components to catalyze the production ofthe desired product(s). IV. Variation of starter and/or extender units, and/or host cells.
In addition to varying the nucleic acids comprising the subject gene cluster, variations in the products produced by the gene cluster(s) can be obtained by varying the the host cell, the starter units and/or the extender units. Thus, for example different fatty acids can be utilized in the enediyne synthetic pathway resulting in different enediyne variants. Similarly different intermediate metabolites can be provided (e.g. endogenously produced by the host cell, or produced by an introduced herterologous construct, and/or supplied from an exogenous source (e.g. the culture media)). Similarly, varying the host cell can vary the resulting product(s). For example, a gene cassette carrying the enediyne biosynthesis genes can be introduced into a deoxysugar-synthesizing host for the production of glycosylated enediyne metabolites.
V. Use of C-1027 resistance genes.
The antibiotic C-1027 and metabolites present in C-1027 biosynthesis are highly potent cytotoxins. Accordingly the biosynthesis of C-1027 is facilitated by the presence of one or more antibiotic (e.g. enediyne) resistance genes. Without being bound to a particular theory, it is believed that CagA and SgcB function cooperatively to provide resistance. It is believed that the C-1027 chromophore is first sequestered by binding to the preaproprotein CagA (ORF 9) to form a complex, which is then transported out ofthe cell by the efflux pump SgcB (ORF 2) and processed by removing the leader peptide to yield the chromoprotein. Other genes that appear to mediate resistance in the C-1027 biosynthesis gene cluster include a transmembrane transport protein (ORF 27), a Na'VH* transporter (ORF 0), an ABC transporter (ORF -1, C-terminus), a glycerol phosphate transporter (ORF -2), and a UvrA-like protein (ORF -1, N-terminus) (see, e.g., Table 2).
These ORFs and/or the polypeptides encoded by these ORFs can be utilized alone, or in combination with one or more other C- 1027 ORFs to confer resistance to enediyne or enediyne metabolites on a cell. This is useful in a wide variety of contexts. For example, to increase production of enediynes. For example, it is believed that C-1027 resistance could be a limiting factor at the onset of C-1027 production. Provision of an extra copy ofthe plasmid-born sgcB, and overexpression of sgcB under the control ofthe constitutive ermE* promoter resulted in increase of C-1027 production (see example 1).
In a therapeutic context, it is sometimes desirable to confer resistance on certain vulnerable cells. Thus, for example, where an enediyne is used as a chemotherapeutic, transfection of vulnerable, but healthy cells (e.g. liver cells remote from the tumor site, stem cells, etc.) with vector(s) expressing the resistance gene(s) permits administration ofthe enediyne at a higher dosage with fewer adverse effects to the organism. Such approaches have been taken using the multi-drug resistance gene (MDR1) expressing p-glycoprotein.
In another embodiment vectors are provided containing one or more resistance genes of this invention under control of a constitutive and/or inducible promoter thereby providing a "ready-made" expression system suitable for the expression of an enediyne or enediyne metabolite at high concentration. It is also noted that the resistance genes are expected to confer resistance to compounds other than enediynes. The resistance genes are expected to confer resistance to essentially any cytotoxic compound that can act as a substrate for the resistance gene(s) of this invention.
VI. Kits. In still another embodiment, this invention provides kits for practice ofthe methods described herein. In one prefeπed embodiment, the kits comprise one or more containers containing nucleic acids encoding one or more ofthe C-1027 biosynthesis gene cluster open reading frames. Certain kits may comprise vectors encoding the sgc gene cluster orfs and/or cells containing such vectors. The kits may optionally include any reagents and/or apparatus to facilitate practice ofthe methods described herein. Such reagents include, but are not limited to buffers, labels, labeled antibodies, bioreactors, cells, etc.
In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice ofthe methods of this invention. Prefeπed instructional materials provide protocols utilizing the kit contents for creating or modifying C-1027 gene cluster and/or for synthesizing or modifying a molecule using one or more sgc gene cluster ORFs. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1 Genes for production of the enediyne antitumor antibiotic C-1027 in Streptomyces globisporus are clustered with the caεA gene that encodes the C-1027 apoprotein
We have been studying the biosynthesis of C-1027 in Streptomyces globisporus C-1027 as a model for the enediyne family of antitumor antibiotics (Thorson et al. (1999) Bioorg. Chem., 27: 172-188). C-1027 consists of a non-peptidic chromophore and an apoprotein, CagA [also called C-1027 AG (Otani et al. (1991) Agri. Biol. Chem. 55: 407- 417)]. The C-1027 chromophore is extremely unstable in the protein-free state, the structure of which was initially deduced from an inactive but more stable degradation product (Minami et al. (1993) Tetrahedron Lett. 34: 2633-2636) and subsequently confirmed by spectroscopic analysis ofthe natural product (Yoshida et al. (1993) Tetrahedron Lett. 34: 2637-2640) (Fig. 1). While the absolute stereochemistry ofthe deoxysugar moiety was established by total synthesis (Iida et al. (1993) Tetrahedron Lett. 34: 4079-4082), the 8S, 9S, 13S and 17R configuration ofthe C-1027 chromophore were based only on computer modeling (Okuno et al. (1994) J. Med. Chem. 37: 2266-2273). Although no biosynthetic study has been carried out specifically on C-1027, the polyketide origin ofthe enediyne cores has been implicated by feeding experiments with 13C-labeled acetate for the neocarzinostatin chromophore A (Hensens et al. (1989) J. Am. Chem. Soc. Ill: 3295-3299), dynemicin (Tokiwa et al (1992) J. Am. Chem. Soc. 114: 4107-4110), and esperamicin (Lam et al. (1993) J. Am. Chem. Soc. 115: 12340-12345); and deoxysugar biosynthesis has been well characterized in actinomycetes (Liu and Thorson (1994) Ann. Rev. Microbiol 48: 223- 256; Piepersberg ( 1997) pp. 81 - 163. In Biotechnology of antibiotics, 2nd ed. W. R. Strohl (ed). Marcel Dekker, New York). Given the structural similarity of C-1027 to the other enediyne cores and to deoxysugars found in other secondary metabolites, we decided to clone either a PKS or a deoxysugar biosynthesis gene as the first step of identifying the C- 1027 gene cluster from S. globisporus. Furthermore, the CagA apoprotein of C-1027 has been isolated, its amino acid sequence has been determined, and the coπesponding cagA gene has been cloned and sequenced (Otani et al. (1991) Agri. Biol. Chem. 55: 407-417; Sakata et al. (1992) Biosci. Biotech. Biochem. 56: 1592-1595). Since genes encoding secondary metabolite production in actinomycetes have invariably been found to be clustered in one region ofthe microbial chromosome (Hopwood (1997) Chem. Rev. 97: 2465-2497), we further reasoned that mapping the cagA gene with either a putative PKS gene, a deoxysugar biosynthesis gene, or both to the same region ofthe S. globisporus chromosome should be viewed as strong evidence supporting the proposition that the cloned genes constitute the C-1027 biosynthesis gene cluster.
We report here the cloning and sequencing of two genes, sgcA (Streptomyces globisporus C-1027) and sgcB, that encode a dNDP-glucose 4,6-dehydratase (NGDH) and a transmembrane efflux protein, respectively. The sgcA,B locus is indeed clustered with the cagA gene, leading to the localization of a 75-kb gene cluster from S. globisporus. The involvement ofthe cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the sgcA gene to generate C-1027-nonproducing mutants and by complementing the sgcA mutants in vivo to restore C-1027 production. Our results, together with similar effort in the Thorson laboratory on the calicheamicin gene cluster (Thorson et al. (1999) Bioorg. Chem., 27: 172-188), represent the first cloning of a gene cluster for enediyne antitumor antibiotic biosynthesis.
Materials and methods.
Bacterial strains and plasmids. Escherichia coli DH5α was used as a general host for routine subcloning
(Sambrook et al (1989) Molecular cloning, a laboratory manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). E. coli XL 1-Blue MR (Stratagene, La Jolla, CA) was used as the transduction host for cosmid library construction. E. coli SI 7-1 was used as the donor host for E. coli-S. globisporus conjugation (Mazodier et al. (1989) J. Bacteriol. 171: 3583-3585). Micrococcus luteus ATCC9431 was used as the testing organism to assay the antibacterial activity of C-1027 (Hu et al. (1988) J. Antibiot. 41: 1575-1579). The pGΕM-3zf, -5zf, and -7zf and pGEM-T vectors were from Promega (Madison, WI). S. globisporus strains and other plasmids in this study are listed in Table 3
Table 3. Strains and plasmids.
Strain or Relevant Characteristics plasmid
S. globisporus
C-1027 Wild-type (Hu et al. (1988) J. Antibiot. 41 : 1575-1579)
AF40 Mutant resulted from acriflavine treatment of S. globisporus C-1027, C-
1027-nonproducing (Mao et al. (1997) Chinese ! Biotechnol. 13: 195-
199) AF44 Mutant resulted from acriflavine treatment of S. globisporus C-1027, C-
1027-nonproducing (Mao et al, supra) AF67 Mutant resulted from acriflavine treatment of S. globisporus C-1027, C-
1027-nonproducing (Mao et al, supra) SB 1001 sgcA -disrupted mutant resulted from integration of pB S 1012 into S. globisporus C-1027 Apr11, C-1027-nonproducing SB 1002 sgcA-disuxpted mutant resulted from integration ofpBS1013 into S. globisporus C-1027 Apr11, C-1027-nonproducing
Plasmids: pOJ446 E. coli-Streptomyces shuttle cosmid, Apr11 (Bierman et al. (1992) Gene, 116: 43- pOJ260 E. coli vector, non-replicating in Streptomyces, Apr11 (Bierman et al. supra) pKCl 139 E. coli-Streptomyces shuttle vector, rep78, Apr11 (Bierman et al. supra) pWHM3 E. coli-Streptomyces shuttle vector, ThR (Vara et al. ( 1989) J. Bacteriol. 171:
5872-5881) pWHM79 ermE* promoter in pGEM-3zf (Shen and Hutchinson ( 1996) Proc. Natl. Acad.
Sci. USA 93: 6600-6604) pBSlOOl 0.75-kb PCR product amplified from S. globisporus with type I PKS primers in pGEM-
T pBS 1002 0.55-kb PCR product amplified from S. globisporus with NGDH gene primers in pGEM-T pBS 1003 0.73-kb PCR product amplified from pBS 1005 with cagA primers in pGEM-T pBS 1004 pOJ446 S. globisporus genomic library cosmid pBS 1005 pOJ446 S. globisporus genomic library cosmid pBS 1006 pOJ446 S. globisporus genomic library cosmid pBS 1007 3.0-kb BamΑl fragment from pBS 1005 in pGEM-3zf, sgcA, sgcB pBS 1008 4.0-kb Bat Αl fragment from pBS 1005 in pGEM-3zf, cagA ρBS1009 1.0-kb Kpnl truncated fragment of sgcA from pBS1007 in pGEM-3zf pBSlOlO 0.75-kb SaclVSphl internal fragment of sgcA from pBS1009 in pGEM-5zf pBSlOl 1 0.75-kb SacVSphl internal fragment of sgcA from pBSlOlO in pGEM-3zf pBS1012 0.75-kb £coRI/Hi III internal fragment of sgcA from pBSlOlO in pOJ260 pBS1013 0.75-kb EcoKUHindlll internal fragment of sgcA from pBSlOlO in pKCl 139 pBS1014 2.0-kb EcotU Sphl fragment from pBS1007 in the SmaVSphl sites of pWΗM79, ermE*, sgcA pBS1015 2.5-kb EcoTO/Hinalll fragment from pBS1014 in pWHM3, ermE*, sgcA pBS1016 Self-ligation ofthe 5.2-kb Kpnl fragment from pBS1007 pBS1017 0.45-kb EcoKUSacl fragment from pWHM79 in EcoKUSacl sites of pBS1016, ermE*, sgcB pBS 1018 2.5-kb EcoRI/Hindlll fragment from pBS 1017 in pKC 1139, ermE*, sgcB
Biochemicals and chemicals.
Ampicillin, apramycin, nalidixic acid, and thiostrepton were from Sigma (St. Louis, MO). Unless specified otherwise, restriction enzymes and other molecular biology reagents were from standard commercial sources.
Media and culture conditions.
E. coli strains carrying plasmids were grown in Luria-Bertani (LB) medium and were selected with appropriate antibiotics. S. globisporus strains were grown on ISP-4 (Difco Laboratories, Detroit, MI) or R2YE at 28°C for sporulation and in TSB (Hopwood et al (1985) Genetic manipulation of Streptomyces: a laboratory manual John Innes
Foundation, Norwich, UK) supplemented with 5 mM MgCl2 and 0.5% glycine at 28°C, 250 rpm for isolation of genomic DNA. For transformation, S. globisporus strains were grown in YEME (Hopwood et al, supra.) for preparation of protoplasts and on R2YE for protoplast regeneration. For conjugation, both the E. coli SI 7-1 donors and the S. globisporus recipients (upon germination in TSB) were prepared in LB, and donors/recipients were grown on either ISP-4 medium with 0.05% yeast extract and 0.1% tryptone or AS-1 medium (Baltz (1980) Dev. Ind. Microbiol. 21: 43-54; Bierman et al. (1992) Gene 116: 43-69) at 30°C for isolation of exconjugants.
For C-1027 production, S. globisporus strains were grown either on R2YE or ISP-4 agar medium at 28°C or in liquid medium by a two-stage fermentation. For liquid culture, the seed inoculum was prepared by inoculating 50 mL medium (consisting of 2% glycerol, 2% dextrin, 1% fish meal, 0.5% peptone, 0.2% (NH )2SO4, and 0.2% CaCO3, pH
7.0) with an aliquot of spore suspension, incubating at 28°C, 250 rpm for two days. To a fresh 50 mL ofthe same medium was then added the seed culture (5%), and incubation continued at 28°C, 250 rpm for three to six days (Hu et al. (1988) J. Antibiot. 41 : 1575- 1579). The fermentation supernatants were harvested by centrifugation (Eppendorf 5415C, 4°C, 10 min, 14,000 rpm) on day 3, 4 and 5, and assayed for their antibacterial activity against M. luteus (Hu et al. (1988) J. Antibiot., 41 : 1575-1579).
DNA isolation and manipulation.
Plasmid preparation and DNA extraction were carried out by using commercial kits (Qiagen, Santa Clarita, CA). Total S. globisporus DNA was isolated according to literature protocols (Hopwood et al. (1985) Genetic manipulation of Streptomyces: a laboratory manual John Innes Foundation, Norwich, UK; Rao et al. (1987) Methods Enzymol. 153: 166-198). Restriction endonuclease digestion and ligation followed standard methods (Sambrook et al. (1989) Molecular cloning, a laboratory manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). For Southern analysis, digoxigenin labeling of DNA probes, hybridization, and detection were performed according to the protocols provided by the manufacturer (Boehringer Mannheim Biochemicals, Indianapolis, IN).
DNA sequencing.
Automated DNA sequencing was carried out on an ABI Prism 377 DNA Sequencer using the ABI Prism dye terminator cycle sequencing ready reaction kit and Ampli-Tαα DNA polymerase FS (Perkin-Elmer/ABI, Foster City, CA). Sequencing service was provided by either the DBS Automated DNA Sequencing Facility, UC Davis, or Davis Sequencing Inc. (Davis, CA). Data were analyzed by ABI Prism Sequencing 2.1.1 software and the Genetics Computer Group program (Madison, WI).
Polymerase chain reaction (PCR). Primers were synthesized at the Protein Structure Laboratory, UC Davis.
PCR was carried out on a Gene Amp PCR System 2400 (Perkin-Elmer/ABI) with Taq polymerase and buffer from Promega. A typical PCR mixture consisted of 5 ng of S. globisporus genomic or plasmid DNA as template, 25 pmoles of each primers, 25 μM dNTP, 5% DMSO, 2 units of Taq polymerase, 1 x buffer, with or without 20% glycerol in a final volume of 50 μL. The PCR temperature program was as follows: initial denaturing at 94°C for 5 min, 24-36 cycles of 45 sec at 94°C, 1 min at 60°C, 2 min at 72°C, followed by additional 7 min at 72°C.
For type II PKS, the following two pairs of degenerate primers were used — 5'-AGC TCC ATC AAG TCS ATG RTC GG-3' (forward, SEQ ID NO:103) / 5'-CC GGT GTT SAC SGC GTA GAA CCA GGC G-3 ' (reverse, SEQ ID NO: 104) and 5 '-GAC ACV GCN TGY TCB TCV-3' (forward, SEQ ID NO: 105)/5'-RTG SGC RTT VGT NCC RCT-3' (SEQ ID NO: 106) (B, C+G+T; N, A+C+G+T; R, A+G; S, C+G; V, A+C+G; Y, C+T) (reverse) (Seow et al. (1997) J. Bacteriol, 179: 7360-7368). No product was amplified under all conditions tested. For type I PKS, the following pair of degenerate primers were used— 5'-GCS TCC CGS GAC CTG GGC TTC GAC TC-3' (forward, SEQ ID NO:107) / 5'-AG SGA SGA SGA GCA GGC GGT STC SAC-3' (S, G+Q (reverse, SEQ ID NO: 108) (Kakavas et al (1997) J. Bacteriol, 179: 7515-7522). A distinctive product with the predicted size of 0.75 kb was amplified in the presence of 20% glycerol and cloned into pGEM-T according to the protocol provided by the manufacturer (Promega) to yield pBSlOOl.
For NGDH, the following pair of degenerate primers were used — 5'-CS GGS GSS GCS GGS TTC ATC GG-3' (forward, SEQ ID NO: 109) / 5'-GG GWR CTG GYR SGG SCC GTA GTT G-3' (R, A+G; S, C+G; W, A+T; Y, C+T) (reverse, SEQ ID NO:l 10) (Decker, et al. (1996) FEMS Lett., 141: 195-201). A distinctive product with the predicted size of 0.55 kb was amplified and cloned into pGEM-T to yield pBS 1002.
For cagA, the following pair of primers, flanking its coding region, were used— 5'-AG GTG GAG GCG CTC ACC GAG-3' (forward, SEQ ID NO:l 1 l)/5'-G GGC GTC AGG CCG TAA GAA G-3' (reverse, SEQ ID NO: 112) (Sakata et al. (1992) Biosci. Biotechnol Biochem., 56: 159201595). A distinctive product with the predicted size of 0.73 kb was amplified from pBS1005 and cloned into pGEM-T to yield pBS1003.
Genomic library construction and screening.
S. globisporus genomic DNA was partially digested with Mbol to yield a smear around 60 kb, as monitored by electrophoresis on a 0.3% agarose gel. This sample was dephosphorylated upon treatment with shrimp alkaline phosphatase and ligated into the E. coli-Streptomyces shuttle vector pOJ446 (Bierman et al. (1992) Gene 116: 43-69) that was prepared by digestion with Hpαl, shrimp alkaline phosphatase treatment, and additional digestion with RαmΗI. The resulting ligation mixture was packaged with the Gigapack II XL two-component packaging extract (Stratagene). The package mixture was transduced into E. coli XL 1-Blue MR. The transduced cells were spread onto LB plates containing apramycin (100 μg/mL) and incubated at 37°C overnight. The titer ofthe primary library was approximately 6,000 colony-forming units per μg of DNA. Restriction enzyme analysis of twelve randomly selected cosmids confirmed that the average size of inserts was about 35 to 45 kb (Rao et al. (1987) Meth. Enzymol, 153: 166-198).
To screen the genomic library, colonies from five LB plates containing apramycin (100 μg/mL, with approximately 2,000 colonies per plate) were transfeπed to nylon transfer membranes (Micro Separations, Inc., Westborough, MA) and screened by colony hybridization with the PCR-amplified 0.55-kb NGDH fragment from pBS 1002 as a probe. The positive cosmid clones were re-screened by PCR with primers for NGDH and confirmed by Southern hybridization (Sambrook et al, supra.). Further restriction enzyme mapping and chromosomal walking of these overlapping cosmids led to the genetic localization ofthe 75-kb sgc gene cluster, as represented by pBS1004, pBS1005, and pBS 1006 (Fig. 5 A). A 3.0-kb RαmHI fragment from pBS 1005 that hybridized to the NGDH probe was cloned into the same sites of pGΕM-3zf to yield pBS1007. Similarly, a 4.0-kb RαmHI fragment from pBS1005 that hybridizes to the PCR-amplified 0.73-kb cagA probe from pBS1003 was cloned into the same sites of pGEM-3zf to yield pBS1008 (Fig. 5B).
Generation of s cA mutants by insert-directed homologous recombination in S. globisporus.
A 1.0-kb Kpήl fragment from pBS1007, containing the C-terminal truncated sgcA, was subcloned into pGEM-3zf to yield pBS1009. An internal fragment of sgcA was moved sequentially as a 0.75-kb SaclVSphl fragment from pBS1009 into the same sites of pGEM-5zf to yield pBSlOlO and as a 0.75-kb SacVSphl fragment from pBSlOlO into the same sites of pGEM-3zf to yield pBS 1011. The latter plasmid was digested with EcoRI and Hindlll, and the resulting 0.75-kb EcoRI/Hi/iilll fragment was cloned into the same sites of pOJ260 and pKCl 139 (Bierman et al. (1992) Gene, 116: 43-69 to yield pBS1012 and pBS1013, respectively.
Introduction of pBS1012 and pBS1013 into S. globisporus was carried out by either polyethyleneglycol (PΕG)-mediated protoplast transformation (Hopwood et al. ( 1985) Genetic manipulation of Streptomyces: a laboratory manual John Innes Foundation, Norwich, UK) or E. coli-S. globisporus conjugation (Bierman et al. (1992) Gene 116: 43-69; Matsushima and Baltz (1996) Microbiology 142: 261-267; Matsushima et al. (1994) Gene 146: 39-45), methods for both of which were developed recently in our laboratory. In brief, for transformation, pBS1012 and pBS1013 were propagated in E. coli ΕT12567 (MacNeil et al. (1992) Gene 111: 61-68), and the resulting double strand plasmid DNA was denatured by alkaline treatment (Ho and Chater (1997) J. Bacteriol. 179: 122-127). The latter DNA (5 μL) and 200 μL of 25% PEG 1000 in P buffer (Hopwood et al. supra) were sequentially added to 50 μL of S. globisporus protoplasts (109) in P buffer. The resulting suspension was mixed immediately and spread on R2YE plates. After incubation at 28°C for 16 to 20 hrs, the plates were overlaid with soft R2YE (0.7% agar) containing apramycin (100 μg/mL, final concentration); incubation continued until colonies appeared (in 5 to 7 days). For conjugation, E. coli S17-l(ρBS1012) or E. coli SI 7-1 (pBS1013) was grown to an OD600 of 0.3 to 0.4. Cells from a 20-mL culture were pelleted by centrifugation, washed in LB, and resuspended in 2 mL of LB as the E. coli donors. S. globisporus spores (103 to 109) were washed, resuspended in TSB, and incubated at 50°C for 10 min to activate germination. After additional incubation at 37°C for 2 to 5 hrs, the spores were pelleted and resuspended in LB as the S. globisporus recipients. The donors (100 μL) and recipients (100 μL) were mixed and spread equally onto two modified ISP-4 or AS-1 plates supplemented freshly with 10 mM MgCl2 (see Media and culture conditions). The plates were incubated at 28°C for 16 to 22 hrs. After removal of most ofthe E. coli SI 7-1 donors by washing the surface with sterile water, the plates were overlaid with 3 mL of soft LB (0.7% agar) containing nalidixic acid (50 μg/mL, final concentration) and apramycin (100 μg/mL, final concentration) and incubated at 28°C until exconjugants appeared (in approximately 5 days).
Unlike pBS1012, which is a Streptomyces non-replicating plasmid, pBS1013 bears a temperature-sensitive Streptomyces replication origin (Bierman et al. (1992) Gene 116: 43-69; Muth et al. (1989) Mol. Gen. Genet. 219: 341-348) that is unable to replicate at temperatures above 34°C (Table 3), while the S. globisporus wild-type strain grows normally up to 37°C. Thus, spores of S. globisporus (pBS1013), from either the transformants or the exconjugants, were spread onto R2YE plates containing apramycin (100 μg/mL). The plates were incubated directly at 37°C, and mutants, resulting from single crossover homologous recombination between pBS 1013 and the S. globisporus chromosome, were readily obtained in 7 to 10 days. Alternatively, the plates were first incubated at 28°C for 2 days until pinpoint-size colonies became visible and then shifted to 37°C to continue incubation. Mutants resulting from single crossover homologous recombination grew out ofthe original pinpoint-size colonies as easily distinguishable sectors in 7 to 10 days.
Construction of the sscA and sεcB expression plasmids. pBS1007 was digested with EcoRI, and made blunt-ended by treatment with the Klenow fragment of DNA polymerase I. Upon additional digestion with Sphl, the resulting 2.0-kb blunt-ended Sphl fragment containing the intact sgcA gene was cloned into the Small Sphl sites of pWHM79 (Shen et al. (1996) Proc. Natl. Acad. Sci., USA, 93: 6600- 6604) to yield pBS1014. The latter was digested with EcoRI and Hindlll, and the resulting 2.5-kb EcoRA/Hindlϊl fragment was cloned into the same sites of pWHM3 (Vara et al (1989) J. Bacteriol 111: 5872-5881) to yield pBS1015, in which the expression of sgcA is under the control ofthe ermE* promoter (Bibb et al. (1994) Mol. Microbiol 14: 533-545).
Alternatively, pBS1007 was digested with Kpnl, removing most ofthe sgcA gene, and the 5.2-kb Kpnl fragment was recovered and self-ligated to yield pBS1016. The ermE* promoter was subcloned from pWHM79 (Shen et al (1996) Proc. Natl. Acad. Sci., USA, 93: 6600-6604) as a 0.45-kb EcoRI/SαcI fragment and cloned into the same sites of pBS1016 to yield pBS1017. The latter was digested with EcoRI and Hindlll, and the resulting 2.5-kb EcoRI/Hindlll fragment was cloned into the same sites of pKCl 139 to yield pBS1018, in which the expression ϊsgcB is under the control ofthe ermE* promoter.
Determination of C-1027 production. The production of C- 1027 was detected by assaying its antibacterial activity against M. luteus (Hu et al. (1988) J. Antibiot. 41: 1575-1579). From liquid culture, fermentation supernant (180 μL) was added to stainless steel cylinders placed on LB plates pre-seeded with overnight M. luteus culture (0.01% vol/vol). From solid culture, a small square block (0.5 x 0.5 x 0.5 cm3) of agar from either R2YΕ or ISP-4 medium was directly placed on M. /wtews-seeded LB plates. The plates were incubated at 37°C for 24 hrs, and C- 1027 production was estimated by measuring the size of inhibition zones.
Nucleotide sequence accession number.
The nucleotide sequence reported here has been deposited in the GenBank database with the accession number AF201913. Results.
No polyketide synthase gene was amplified by PCR from S. elobisporus.
On the assumption that the C-1027 enediyne core is of polyketide origin, the PCR approach was adopted to screen S. globisporus for any putative PKS genes, although it is far from certain α priori if the biosynthesis of the enediyne core invokes a PKS and, if so, whether the enediyne PKS will exhibit a type I or type II structural organization. PCR methods for cloning either type I or type II PKS genes have been developed, and these methods have proven to be very effective in cloning PKS genes from various polyketide- producing actinomycetes (Kakavas et al. (1997) J. Bacteriol. 179: 7515-7522; Seow et al. (1997) J. Bacteriol. 179: 7360-7368). While no distinctive product was amplified under all conditions examined with both pairs of primers designed for type II PKS, a single product with the expected size of 0.75 kb was readily amplified by PCR from S. globisporus with primers designed for type I PKS, which was subsequently cloned (pBSlOOl). Intriguingly, sequence analysis of six randomly selected pBSlOOl clones yielded an identical product — indicative of a specific PCR amplification — the deduced amino acid sequence of which, however, showed no homology to known PKSs (data not shown), excluding the possibility of using PKS as a probe to identify the sgc biosynthesis gene cluster.
Cloning of a putative NGDH gene by PCR from S. globisporus.
The biosynthesis of various deoxyhexoses share a common key intermediate — 4-keto-6-deoxyglucose nucleoside diphosphate or its analogs — whose formation from glucose nucleoside diphosphate is catalyzed by the NGDH enzyme, an NAD+-dependent oxidoreductase (Liu and Thorson (1994) Ann. Rev. Microbiol. 48: 223- 256; Piepersberg (1997) pp. 81-163. In Biotechnology of antibiotics, 2nd ed. W. R. Strohl (ed). Marcel Dekker, New York). The PCR method was adopted to clone the putative NGDH gene from S. globisporus with primers designed according to the homologous regions of various NGDH enzymes from actinomycetes (Decker et al. (1996) FEMSLett. 141: 195-201), resulting in the amplification of a single product with the expected size of 0.55 kb (pBS1002). Sequence analysis of pBS1002 confirmed its identity as a part of a putative NGDH gene. To clone the complete NGDH gene, an S. globisporus genomic library, constructed in the E. coli-Streptomyces shuttle vector pOJ446 (Bierman et al. (1992) Gene 116: 43-69; Rao et al. (1987) Methods Enzymol. 153: 166-198), was analyzed by Southern hybridization with the PCR-amplified 0.55-kb fragment from pBS1002 as a probe. Ofthe 10,000 colonies screened, 36 positive colonies were identified, 9 of which were confirmed by PCR to harbor the DGDH gene. Restriction enzyme mapping showed that all of them contained a single 3.0-kb RαmHI fragment hybridizing to the NGDH probe. Additional chromosomal walking from this locus eventually led to the localization ofthe 75-kb sgc gene cluster, covered by 18 overlapping cosmids as represented by pBS1004, pBS1005, and pBS1006 (Fig. 5A). The 3.0-kb RαmHI fragment was subcloned (pBS1007) (Fig. 5B), and its nucleotide (nt) sequence was determined.
Analysis of the DNA sequences of the sgcA and sgcB genes.
Two complete open reading frames (ORFs) (sgcA and sgcB) were identified within the 3.0-kb RαmHI fragment of pBS1007, the 3,035-nt sequence of which is shown in Figure 6. The sgcA gene most likely begins with an ATG at nt 101, preceded by a probable ribosome biding site (RBS), GGAGG, and ends with a TGA stop codon at nt 1099. Sgc-4 should therefore encode a 332-amino acid protein with a molecular weight of 36,341 and an isoelectric point of 6.01. A Gapped-BLAST search showed that the deduced sgcA gene product is highly homologous to various putative and known NGDH enzymes from antibiotic-producing actinomycetes, including Gdh from the erythromycin biosynthesis gene cluster in Saccharopolyspora erythraea (64% identity and 70% similarity) (Linton et al (1995) Gene 153: 33-40), MtmE from the mithramycin biosynthesis gene cluster in Streptomyces argillaceus (64% identity and 68% similarity) (Lombo et al. (1997) J. Bacteriol. 179: 3354-3357), and TylA2 from the tylosin biosynthesis gene cluster in Streptomyces fradiae (62% identity and 68% similarity) (Merson-Davies and Cundliffe (1994) Mol Microbiol 13: 349-355) (Fig. 7). A conserved sequence of 14 amino acid residues close to the N-termini can be easily identified in these proteins, which has been described as a βαβ fold with an NAD+-binding motif, GxGxxG, (Fig. 7, boxed), consistent with their biochemical role in deoxyhexose biosynthesis (Liu and Thorson (1994) Ann. Rev. Microbiol. 48: 223-256; Piepersberg (1997) pp. 81-163. In Biotechnology of antibiotics, 2nd ed. W. R. Strohl (ed). Marcel Dekker, New York). The function of Gdh and MtmE as TDP- glucose 4,6-dehydratases, requiring NAD+ as a cofactor, has been confirmed by an enzyme assay following expression ofthe gdh (Linton et al (1995) Gene 153: 33-40) and mtmE gene (Lombo et al. (1997) J. Bacteriol 179: 3354-3357) in E. coli, respectively, and by purification ofthe Gdh protein from Sacc. erythraea (Vara et al. (1989) J. Bacteriol. 171: 5872-5881). From these data, it is reasonable to suggest that sgcA encodes the NGDH enzyme required for the biosynthesis of the 4,6-dideoxy-4-dimethylamino-5- methylrhamnose moiety ofthe C-1027 chromophore. Transcribed in the same direction as sgc A, the sgcB gene is located 43 nt downstream of sgcA. It should begin with a GTG at nt 1143, preceded by a probable RBS, AGGAG, and end with a TGA at nt 2708 (Fig. 6). Coπespondingly, sgcB should therefore encode a 521 -amino acid protein with a molecular weight of 52,952 and an isoelectric point of 4.64. Database comparison ofthe deduced sgcB product revealed that SgcB is closely related to a family of membrane efflux pumps, such as LfrA from Mycobacterium smegmatis (43% identity and 50% similarity, protein accession number AAC43550) (Takiff et al
(1996) Proc. Natl. Acad. Sci. USA 93: 362-366), OrfA from Streptomyces cinnamomeus (42% identity and 47% similarity, protein accession number AAB71209) (Sommer et al.
(1997) Appl. Environ. Microbiol. 63: 3553-3560), and RifP from the rifamycin biosynthesis gene cluster in Amycolatopsis mediterranei (35% identity and 44% similarity, protein accession number AAC01725) Augus et al. (1998) Chem. Biol. 5: 69-79). These proteins are membrane-localized transporters involved in the transport of antibiotics (conferring resistance), sugars, and other substances. While direct evidence is lacking for RifP conferring rifamycin resistance in A. mediterranei by transporting it out ofthe cells (August et al (1998) Chem. Biol, 5: 68-79), it has been proven that LfrA employs the transmembrane proton gradient in an antiporter mode to drive the efflux of intracellular antibiotics, resulting in fluoroquinolone resistance in M. smegmatis (Takiff et al. (1996) Proc. Natl Acad. Sci. USA 93: 362-366). On the basis ofthe high degree of amino acid sequence conservation, an equivalent role could be proposed for SgcB, conferring resistance by exporting C-1027 from S. globisporus.
The cagA gene is clustered with the sgcA and sgcB locus.
To determine if cagA is clustered with the sgcA and sgcB locus, PCR primers were designed according to the flanking regions of cagA (Sakata et al (1992) Biosci. Biotech. Biochem. 56: 1592-1595). A single product with the predicted size of 0.73 kb was indeed amplified from several ofthe overlapping cosmids (which cover the 75-kb sgc cluster), including pBS1004 and pBS1005, the identity of which as cagA was confirmed by sequencing. Restriction enzyme mapping and Southern hybridization analysis localized cagA to a single 4.0-kb RαmHI fragment that is approximately 14 kb upstream ofthe sgcA.B locus (Fig. 5B). The 4.0-kb RαmHI fragment was subcloned (pBS1008), and its nt sequence was determined, revealing the cagA gene along with two additional ORFs (data not shown) (Fig. 5). As reported earlier, cagA encodes a 142-amino acid protein that is processed by cleavage of a 32-amino acid lead peptide to yield the mature CagA apoprotein (Sakata et al. (1992) Biosci. Biotech. Biochem. 56: 1592-1595).
Disruption of the sgcA gene in S. globisporus.
To examine if the cloned sgc cluster encodes C-1027 biosynthesis, sgcA was insertionally disrupted by a single crossover homologous recombination event to generate C- 1027-nonproducing mutant strains (Fig. 8A). Two plasmids were used — pBS1012 (a pOJ260 derivative) and pBS1013 (a pKCl 139 derivative), each of which contain a 0.75-kb internal fragment from sgcA (Table 3). After introduction of pBS1012 into S. globisporus either by PEG-mediated protoplast transformation or E. coli-S. globisporus conjugation, transformants or exconjugants that were resistant to apramycin were isolated in all cases. Since pBS1012 is derived from the Streptomyces non-replicating plasmid of pOJ260, these isolates must have resulted from integration of pBS1012 into the S. globisporus chromosome by homologous recombination. Plasmid pBS1013 was similarly introduced into S. globisporus. However, since pBS1013 is derived from pKCl 139 that carries the temperature-sensitive Streptomyces replication origin from pSG5 and can replicate normally at 28°C (Muth et al. (1989) Mol. Gen. Genet. 219: 341-348), these isolates were subjected to incubation at the non-permissive temperature of 37°C to eliminate free plasmids from the host cells. As expected, normal growth stopped except for the recombinants that continue to grow at 37°C, indicative of integration of pBS1013 into S. globisporus by homologous recombination. The apramycin-resistant S. globisporus SB 1001 and S. globisporus SB 1002 strains were chosen as representatives of mutant strains with disrupted sgcA gene resulted from integration of pBS1012 and pBS1013, respectively.
To confirm that targeted sgcA disruption has occuπed by a single crossover homologous recombination event, Southern analysis ofthe DNA from the mutant strains was performed as exemplified for S. globisporus SBIOOI with either pOJ260 or the 0.75-kb SaclllKpnl internal fragment of sgcA from pBSlOlO as a probe. As shown in Fig. 8B, a distinctive band ofthe predicted size of 6.3 kb was detected with the pOJ260 vector as a probe in all mutant strains (lanes 2, 3, and 4); this band was absent from the wild-type strain (lane 1). Complementarity, when using the 0.75-kb SaclllKpnl internal fragment of sgcA as a probe (Fig. 8C), the 3.0-kb band in the wild-type strain (lane 1) was split into two fragments with the size of 6.3 kb and 1.0 kb in the mutant strains (lanes 2, 3, and 4), as would be expected for disruption of sgc A by a single crossover homologous recombination event.
S. globisporus SBIOOI and S. globisporus SB1002 are C-1027-nonproducing mutants.
No apparent difference in growth characteristics and morphologies between the wild-type S. globisporus and mutant S. globisporus SB 1001 and S. globisporus SB 1002 strains was observed. While C-1027 production in the wild-type S. globisporus strain could be detected on day 3, peaked on day 5, and continued for a few more days, as judged by assaying the antibacterial activity ofthe culture supernant against M. luteus (Hu et al. (1988) J. Antibiot. 41: 1575-1579), C-1027 production is completely abolished in the sgcA mutant strains S. globisporus SB 1001 and S. globisporus SB 1002 (Fig. 9 A). The latter phenotype was identical to that ofthe AF40, AF44, and AF67 mutants, C-1027-nonproducing S. globisporus strains that have been characterized previously (Fig. 9A and 9C) (Mao, et al. (1997) Chinese J. Biotechnol 13: 195-199).
In vivo complementation of S. globisporus SBIOOI.
The ability ofthe wild-type sgcA gene to complement the disrupted sgcA gene was tested in the S. globisporus SB1001 strain. The construction of pBS1015, in which the expression of sgcA is under the control ofthe constitutive ermE* promoter, was described in Materials and Methods. Both the pBS1015 construct and the pWHM3 vector as a control were introduced by transformation into the S. globisporus SBIOOI mutant strains. Culture supernants from each transformant were bioassayed against M. luteus for C-1027 production. pBS1015 restored C-1027 production to S. globisporus SB1001 to the wild-type level; no C- 1027 production was detected in the control in which pWHM3 was introduced into S. globisporus BS1001 (Fig. 9B and 9C). A significant reduction of C-1027 production was observed when S. globisporus SB1001(pBS1015) was cultured under identical conditions but without thiostrepton (Fig. 9B vs. 6C), indicative that pBS1015 may be unstable in S. globisporus SB1001 in the absence of antibiotic selection pressure. Expression oisgcB in S. globisporus.
The effect oisgcB on C-1027 production was tested in the wild- type S. globisporus strain. The construction of pBS1018, in which the expression of sgcB is under the control ofthe constitutive ermE* promoter, was described in Materials and Methods. pBS 1018 and the pKC 1139 vector as a control were each introduced by conjugation into S. globisporus. Culture supernatants from each exconjugant were harvested on days 3, 4, and 5, and assayed for C-1027 production by determining the antibacterial activity against M. luteus. While no apparent difference for C-1027 production was observed between the S. globisporus and S. globisporus (pKCl 139) strains, a significant increase in C-1027 production (150±25%) was evident in the early stage of S. globisporus (pBS1018) fermentation (Fig. 9D, day 3). However, such effect on C-1027 production leveled off as the fermentation proceeded and became insignificant when the culture reached the late stationary phase of fermentation (Fig. 9D, day 4 and 5).
Discussion. Our inability to clone the putative enediyne PKS gene by PCR, with degenerate primers designed according to the highly conserved amino acid sequences of either type I or type II PKSs, or by DNA hybridization, with homologous type I or type II PKS as probes (data not shown), was unexpected, since feeding experiments by incorporation of [1-13C]- and [l,2-13C]acetate into the enediyne cores of esperamicin (Lam et al. (1993) J. Am. Chem. Soc. 115: 12340-12345), dynemicin (Tokiwa et al. (1992) J. Am. Chem. Soc. 114: 4107-4110), and neocarzinostatin (Hensens et al. (1989) J. Am. Chem. Soc. Il l: 3295-3299) supported their polyketide origin. Although the enediyne cores are structurally distinct from either the reduced or aromatic polyketides, the biosynthesis of which is well characterized by type I or type II PKS, respectively, it could be imagined that an enediyne PKS catalyzes the biosynthesis of a polyunsaturated linear heptaketide intermediate that is subsequently cyclized into the enediyne core structure (Hu et al. (1994) Mol. Microbiol. 14: 163-172; Spaink et al. (1991) Nature 354: 125-130; Thorson et al. (1999) Bioorg. Chem., 21: 172-188). Alternatively, Hensens and co-workers proposed a fatty acid origin for the enediyne core that was also consistent with the isotope labeling results. These authors suggested oleate as a precursor that is shortened by loss of carbons from both ends and is desaturated via the oleate-crepenynate pathway to furnish the enediyne core (Hensens et al (1989) J. Am. Chem. Soc. I l l: 3295-3299). The latter pathway resembles polyacetylene biosynthesis in higher plants and fungi and requires an acetylene forming enzyme — a plant gene encoding such an enzyme was identified recently (Lee et al. (1998) Science 280: 915-918). Our DNA sequence analysis of approximately 60 kb ofthe sgc gene cluster, fails to reveal any gene that resembles PKS. Although little is known about the resistance mechanism for the enediyne antibiotics in general, the apoproteins ofthe chromoprotein type of enediynes could be viewed as resistance elements that confer self-resistance to the producing organisms by drug sequestration (Thorson et al. (1999) Bioorg. Chem., 27: 172-188). Such a resistance mechanism is in fact well established in antibiotic-producing actinomycetes, for example, BlmA, the bleomycin-binding protein from Streptomyces verticillus (Shen et al. (1999)
Bioorg. Chem. 27: 155-171). Given the fact that antibiotic production genes have invariably been found to be clustered in one region ofthe microbial chromosome, consisting of structural, resistance, and regulatory genes, we adopted a strategy to clone the sgc gene cluster by mapping a putative C-1027 structural gene to the previously cloned cagA gene, considered as a resistance gene that encodes the C-1027 apoprotein.
We chose NGDH as the putative C-1027 structural gene on the basis ofthe 4,6-dideoxy-4-dimethylamino-5-methyhhamnose moiety ofthe C-1027 chromophore. It has been well established that all deoxyhexoses could be derived from the common intermediate of 4-keto-6-deoxyglucose nucleoside diphosphate, the biosynthesis of which from glucose nucleoside diphosphate is catalyzed by an NGDH enzyme. We cloned the NGDH gene from S. globisporus by PCR and used it as a probe to screen an S. globisporus genomic library, resulting in the isolation ofthe 75-kb sgc gene cluster. DNA sequence analysis of a 3.0-kb RαmHI fragment ofthe sgc cluster confirmed the presence ofthe NGDH protein, encoded by sgcA, along with sgcB that encodes a transmembrane efflux protein (Fig. 6). The cagA gene indeed resides approximately 14 kb upstream of sgcA (Fig. 5); DNA sequence analysis of a 4.0-kb RαmHI fragment confirmed the identity of cagA along with two additional ORFs (data not shown). These results underline once again the effectiveness of cloning natural product biosynthesis gene clusters by exploiting the clustering phenomenon between resistance and structural genes. The involvement ofthe cloned gene cluster in C-1027 biosynthesis was demonstrated by disrupting the sgcA gene to generate S. globisporus mutants, the ability of which to produce C-1027 was completely abolished (Fig. 9 A), and by complementing the sgcA mutants in vivo upon expression of sgcA in trans to restore C-1027 production (Fig. 9B and 6C). These data unambiguously establish that sgc A is essential for C-1027 production, and thus support the conclusion that the cloned gene cluster encodes C-1027 biosynthesis. It should be pointed out that, although the sgcA mutants S. globisporus SB 1001 and S. globisporus SB 1002 were characterized as C-1027-nonproducing on the basis ofthe antibacterial assay alone (Fig. 9 A), this phenotype was identical to that ofthe controls ofthe AF40, AF44, and AF67 mutants (Fig. 9A and 9C). The latter strains were isolated previously upon randomly mutagenizing the wild-type S. globisporus strain with acriflavine and confirmed to be C-1027-nonproducing by both the antibacterial bioassay and an antitumor spermatogonial assay (Mao, et al. (1997) Chinese J. Biotechnol. 13: 195-199), providing strong support to the cuπent study. Gene disruption and complementation in S. globisporus were made possible by the recently developed genetic system that allowed us to introduce plasmid DNA into S. globisporus via either PEG-mediated protoplast transformation (Hopwood et al. (1985) Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation, Norwich, UK) or E. coli-S. globisporus conjugation (Bierman et al. (1992) Gene 116: 43-69; Matsushima and Baltz (1996) Microbiology 142: 261-267; Matsushima et al (1994) Gene 146: 39-45) for analyzing the sgc biosynthesis gene cluster in vivo. Given the difficulties encountered with calicheamicin biosynthesis in Micromonospora echinospora, into which all attempts to introduce plasmid DNA have failed (Thorson et al. (1999) Bioorg. Chem., 27: 172-188), the latter results underscore the importance of selecting C-1027 as a model system for enediyne biosynthesis so that many of the genetic tools developed in Streptomyces species can now be directly applied to the study of enediyne biosynthesis.
Finally, the function of sgcB was probed by examining C-1027 production, following expression ofthe gene in the wild-type S. globisporus strain. Database comparison ofthe deduced amino acid sequence clearly suggested SgcB as a transmembrane efflux protein, conferring resistance by exporting C-1027 out ofthe cell. Hence, in addition to CagA, SgcB could be viewed as the second resistance element identified for C-1027 biosynthesis. Multiple resistance genes have been identified in numerous antibiotic biosynthesis gene clusters (Hopwood (1997) Chem. Rev. 97: 2465-2497). It could be imagined that CagA and SgcB function cooperatively to provide resistance — the C-1027 chromophore is first sequestered by binding to the preaproprotein CagA to form a complex, which is then transported out of he cell by the efflux pump SgcB and processed by removing the leader peptide to yield the chromoprotein, although we do not have any experimental data to substantiate this speculation. Since it is known that yields for antibiotic production could be profoundly altered by the introduction of extra copies of regulatory, resistance, or structural genes into wild-type organisms (Hutchinson (1994) Bio/Technology 12: 375-380), we tested the effect of overexpressing sgcB in S. globisporus on C-1027 production. While no apparent adverse effect on C-1027 production was observed upon introduction ofthe pKCl 139 vector into S. globisporus (data not shown), a significant increase in C-1027 production (150±25%) was observed in the early stage of S. globisporus (pBS1017) fermentation (Fig. 9D, day 3), supporting the predicted function for SgcB in C-1027 biosynthesis. We propose that C-1027 resistance could be a limiting factor at the onset of C- 1027 production, which is circumvented by the extra copy ofthe plasmid-born sgcB, and overexpression of sgcB under the control ofthe constitutive ermE* promoter results in increase of C-1027 production. However, as the S. globisporus (pBS1017) fermentation proceeds to its stationary phase, C-1027 resistance is no longer a limiting factor for overall C-1027 production, and the effect of extra copy of SgcB on C-1027 production consequently became insignificant (Fig. 9D, day 5).
In conclusion, genetic analysis of enediyne biosynthesis has heretofore met with little success in spite of considerable effort (Thorson et al. (1999) Bioorg. Chem., 27: 172-188). The localization ofthe sgc gene cluster and characterization ofthe sgcA and sgcB genes have now provided an excellent basis for genetic and biochemical investigations and/or modification of C-1027 biosynthesis, and gene disruption and overexpression in S. globisporus clearly demonstrated the potential to construct enediyne-overproducing strains and to produce novel enediynes that may have enhanced potency as novel anticancer drugs using combinatorial biosynthesis and targeted mutagenesis. We envisage that the results from C-1027 biosynthesis should facilitate the cloning and characterization of biosynthesis gene clusters of other enediyne antibiotics in Streptomyces as well as in other actinomycetes, and could have a great impact on the overall field of combinatorial biosynthesis. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope ofthe appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMSWhat is claimed is:
1. An isolated nucleic acid comprising a nucleic acid selected from the group consisting of a nucleic acid encoding any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA); a nucleic acid encoding a polypeptide encoded by any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA); and a nucleic acid amplified by polymerase chain reaction (PCR) using primer pairs that amplify any of C- 1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA).
2. The isolated nucleic acid of claim 1 , wherein said nucleic comprises a nucleic acid encoding at least two open reading frames (ORFs) selected from the group consisting of ORF-1 through ORF 42, excluding ORF 9 (cagA).
3. The isolated nucleic acid of claim 1, wherein said nucleic comprises a nucleic acid encoding at least three open reading frames (ORFs) selected from the group consisting of ORF-1 through ORF 42, excluding ORF 9 (cagA).
4. An isolated nucleic acid comprising a nucleic acid that specifically hybridizes under stringent conditions to an open reading frame (ORF) ofthe C-1027 biosynthesis gene cluster, excluding ORF 9 (cagA), and can substitute for the ORF to which it specifically hybridizes to direct the synthesis of an enediyne.
5. The isolated nucleic acid of claim 4, wherein said isolated nucleic acid comprises a nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF - 2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, and ORF 14.
6. The isolated nucleic acid of claim 4, wherein said isolated nucleic acid comprises a nucleic acid that specifically hybridizes under stringent conditions to a nucleic acid selected from the group consisting of ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42.
7. The isolated nucleic acid of claim 5, wherein said isolated nucleic acid comprises a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, and ORF 14.
8. The isolated nucleic acid of claim 6, wherein said isolated nucleic acid comprises a nucleic acid selected from the group consisting of ORF 15, ORF 16, ORF 17,
ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42.
9. The isolated nucleic acid of claim 4, wherein said nucleic acid comprises a nucleic acid that is a single nucleotide polymorphism (SNP) of a nucleic acid selected from the group consisting of ORF -7, ORF -6, ORF -5, ORF -4, ORF -3, ORF -2, ORF -1, ORF 0, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 81, ORF 1, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, ORF 7, ORF 8, ORF 10, ORF 11, ORF 12, ORF 13, ORF 14, ORF 15, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 23, ORF 24, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 34, ORF 35, ORF 36, ORF 37, ORF 38, ORF 39, ORF 40, ORF 41, and ORF 42.
10. An isolated gene cluster comprising open reading frames encoding polypeptides sufficient to direct the assembly of a C-1027 enediyne or a C-1027 enediyne analogue.
11. The gene cluster of claim 10, wherein said gene cluster is present in a bacterium.
12. The gene cluster of claim 11, wherein said gene cluster is present in a bacterium selected from the group consisting of Actinomycetes, Actinoplanetes, Actinomadura, Micromonospora, and Streptomycetes.
13. The gene cluster of claim 11, wherein said gene cluster is present in a bacterium selected from the group consisting Streptomyces globisporus, Streptomyces lividans, Streptomyces coelicolor, Micromonospora echinospora spp. calichenisis, Actinomadura verrucosopora, Micromonospora chersina, Streptomyces carzinostaticus, and Actinomycete L585-6.
14. The gene cluster of claim 13, wherein one or more open reading frames is operatively linked to a heterologous promoter.
15. An isolated polypeptide comprising a catalytic domain encoded by a nucleic acid of a C-1027 gene cluster wherein said nucleic acid comprises a nucleic acid selected from the group consisting of a nucleic acid encoding any of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA); and a nucleic acid amplified by polymerase chain reaction (PCR) using any one ofthe primer pairs identified in Tables I and II that specifically amplify one or more of (ORFs) -7 through 42, excluding ORF 9 (cagA).
16. The polypeptide of claim 15 , wherein said polypeptide is encoded by at least two open reading frames selected from the group consisting of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA).
17. The polypeptide of claim 15, wherein said polypeptide is encoded by at least three open reading frames selected from the group consisting of C-1027 open reading frames (ORFs) -7 through 42, excluding ORF 9 (cagA).
18. An expression vector comprising a nucleic acid of any one of claims 1 through 9.
19. A host cell transformed with an expression vector of claim 18.
20. The host cell of claim 19, wherein said cell is transformed with an exogenous nucleic acid comprising a gene cluster encoding polypeptides sufficient to direct the assembly of a C-1027 enediyne or a C-1027 enediyne analogue.
21. The host cell of claim 19, wherein said host cell is a bacterium.
22. The host cell of claim 21 , wherein said bacterium is selected from the group consisting of Actinomycetes, Actinoplanetes, Actinomadura, Micromonospora, and Streptomycetes.
23. The host cell of claim 21 , wherein said bacterium is selected from the group consisting of Streptomyces globisporus, Streptomyces lividans, Streptomyces coelicolor, Micromonospora echinospora spp. calichenisis, Actinomadura verrucosopora, Micromonospora chersina, Streptomyces carzinostaticus, and Actinomycete L585-6.
24. A method of chemically modifying a biological molecule, said method comprising contacting a biological molecule that is a substrate for a polypeptide encoded by a C-1027 biosynthesis gene cluster open reading frame, with a polypeptide encoded by a C- 1027 biosynthesis gene cluster open reading frame whereby said polypeptide chemically modifies said biological molecule.
25. The method of claim 24, wherein said polypeptide is an enzyme selected from the group consisting of a hydroxylase, a homocysteine synthase, a dNDP- glucose dehydrogenase, a citrate carrier protein, a C-methyl transferase, an N-methyl transferase, an aminotransferase, a CagA apoprotein, an NDP-glucose synthase, an epimerase, an acyl transferase, a coenzyme F390 synthase, and epoxidase hydrolase, an anthranilate synthase, a glycosyl transferase, a monooxygenase, a type II condensation protein, an aminomutase, a type II adenylation protein, an O-methyl transferase, a P-450 hydroxylase, an oxidoreductase, and a proline oxidase.
26. The method of claim 24, wherein said method comprising contacting said biological molecule with at least two different polypeptides encoded by C-1027 biosynthesis gene cluster open reading frames.
27. The method of claim 24, wherein said method comprising contacting said biological molecule with at least three different polypeptides encoded by C-1027 biosynthesis gene cluster open reading frames.
28. The method of claim 24, wherein said contacting is in a host cell.
29. The method of claim 28, wherein said host cell is a bacterium.
30. The method of claim 24, wherein said contacting ex vivo.
31. The method of claim 28, wherein said biological molecule is an endogenous metabolite produced by said host cell.
32. The method of claim 28, wherein said biological molecule is an exogenous supplied metabolite.
33. The method of claim 28, wherein said host cell is a eukaryotic cell.
34. The method of claim 33, wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, a yeast cell, a plant cell, a fungal cell, and an insect cell.
35. The method of claim 28, wherein said host cell synthesizes sugars and glycosylates the biological molecule.
36. The method of claim 35, wherein said host cell synthesizes deoxysugars.
37. The method of claim 24, wherein said method further comprises contacting said biological molecule with a polyketide synthase or a non-ribosomal polypeptide synthetase.
38. The method of claim of claim 24, wherein said contacting is in a bacterial cell.
39. The method of claim of claim 24, wherein said contacting is ex vivo.
40. The method of claim 24, wherein said method comprises contacting said biological molecule with at substantially all ofthe polypeptides encoded by C-1027 biosynthesis gene cluster open reading frames and said method produces an enediyne or enediyne analogue.
41. The method of claim 24, wherein said biological molecule is a fatty acid and said biological molecule is contacted with a C-1027 orf polyeptide selected from the group consisting of an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p- 450 hydroxylase, an oxidoreductase, and a proline oxidase.
42. The method of claim 41 , wherein said biological molecule is a fatty acid and said biological molecule is contacted with a plurality of C-1027 orf polypeptides comprising an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase.
43. The method of claim 42, wherein said biological molecule is contacted with polypeptides encoded by ORF17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38.
44. The method of claim 41 , wherein said biological molecule is contacted with polypeptides encoded by ORF 15, ORF 16, ORF 28, ORF3, ORF 14, and ORF 13.
45. The method of claim 44 wherein said biological molecule is also contacted with polypeptides encoded by ORF 4 and ORF 3.
46. The method of claim 24, wherein said method comprises contacting a sugar with one or more C-1027 open reading frame polypeptides selected from the group consisting of a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N-methyltransferase, and a glycosyl transferase.
47. The method of claim 46, wherein said method comprises contacting a dNDP-glucose with a plurality of C-1027 open reading frame polypeptides comprising a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N-methyltransferase, and a glycosyl transferase.
48. The method of claim 24, wherein said method comprises contacting an amino acid with one or one or more C-1027 open reading frame polypeptides selected from the group consisting of a hydroxylase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein.
49. The method of claim 48, wherein said method comprises contacting an amino acid with a plurality of C-1027 open reading frame polypeptides comprising a hydroxylase, a halogenase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein.
50. The method of claim 48, wherein said amino acid is a tyrosine.
51. A method of synthesizing a chromaprotein type enediyne core, said method comprising contacting a fatty acid with one or more C-1027 orf polypeptides selected from the group consisting of an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase.
52. The method of claim 51 , wherein said fatty acid is contacted with a plurality of C-1027 orf polypeptides comprising an epoxide hydrase, a monooxygenase, an iron-sulfer flavoprotein, a p-450 hydroxylase, an oxidoreductase, and a proline oxidase.
53. The method of claim 52, wherein said fatty acid is contacted with polypeptides encoded by ORF17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38.
54. A method of synthesizing a deoxysugar, said method comprising contacting a sugar with one or more C-1027 open reading frame polypeptides selected from the group consisting of a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N-methyltransferase, and a glycosyl transferase.
55. The method of claim 54, wherein said method comprises contacting a dNDP-glucose with a plurality of C- 1027 open reading frame polypeptides comprising a dNDP-glucose synthase, a dNDP glucose dehydratase, an epimerase, an aminotransferase, a C-methyltransferase, an N-methyltransferase, and a glycosyl transferase.
56. The method of claim 55, wherein said dNDP-glucose is contacted with polypeptides encoded by ORF17, ORF20, ORF21, ORF29, ORF30, ORF32, ORF35, and ORF38.
57. A method of synthesizing a beta amino acid, said method comprising contacting an amino acid with one or one or more C-1027 open reading frame polypeptides selected from the group consisting of a hydroxylase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein.
58. The method of claim 57, wherein said method comprises contacting an amino acid with a plurality of C-1027 open reading frame polypeptides comprising a hydroxylase, a halogenase, an aminomutase, a type II NRPS condensation enzyme, a type II NRPS adenylation enzyme, and a type II peptidyl carrier protein.
59. The method of claim wherein said amino acid is contacted with polypeptides encoded by ORF 4, ORFl 1, ORF24, ORF23, ORF25, and ORF26.
60. The method of claim 57, wherein said amino acid is a tyrosine.
61. A method of synthesizing an enediyne or an enediyne analogue said method comprising: culturing a cell comprising a recombinantly modified C-1027 gene cluster under conditions whereby said cell expresses said enediyne or enediyne analogue; and recovering said enediyne or enediyne analogue.
62. The method of claim 61 , wherein said gene cluster is present in a bacterium.
63. The gene cluster of claim 62, wherein said gene cluster is present in a bacterium selected from the group consisting of Actinomycetes, Actinoplanetes, Actinomadura, Micromonospora, and Streptomycetes.
64. The gene cluster of claim 62, wherein said gene cluster is present in a bacterium selected from the group consisting Streptomyces globisporus, Streptomyces lividans, Streptomyces coelicolor, Micromonospora echinospora spp. calichenisis, Actinomadura verrucosopora, Micromonospora chersina, Streptomyces carzinostaticus, and Actinomycete L585-6.
65. The method of claim 61 , wherein said gene cluster is present in a eukaryotic cell.
66. The method of claim 65, wherein said eukaryotic cell is selected from the group consisting of a mammalian cell, a yeast cell, a plant cell, a fungal cell, and an insect cell.
67. The method of claim 61, wherein said host cell synthesizes sugars and glycosylates said enediyne or enediyne analogue.
68. The method of claim 67, wherein said host cell synthesizes deoxy sugars.
69. A method of making a cell resistant to an enediyne or an enediyne metabolite, said method comprising expressing in said cell one or more isolated C-1027 open reading frame nucleic acids that encode a protein selected from the group consisting of a CagA apoprotein, a SgcB transmembrane efflux protein, a transmembrane transport protein, a Na+/H+ transporter, an ABC transport, a glycerol phosphate tranporter, and a UvrA-like protein.
70. The method of claim 69, wherein said isolated C- 1027 open reading frame nucleic acids are selected from the group consisting of ORF 9, ORF2, ORF 27, ORF 0,
ORF 1 c-terminus, ORF 2, and ORF 1 N-terminus.
71. The method of claim 69, wherein said cell is a bacterial cell.
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LIU ET AL.: "Genes for production of the enediyne antitumor antibiotic C1027 in Streptomyces globisporus are clustered with the cagA gene that encodes the C1027 apoprotein", ANTIMICROB. AGENTS CHEMOTHER.,, vol. 44, no. 2, February 2000 (2000-02-01), pages 382 - 392, XP002926776 *
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WO2002079477A2 (en) 2001-03-30 2002-10-10 Dow Agrosciences Llc Biosynthetic genes for butenyl-spinosyn insecticide production
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