WO2004018703A2 - Synthase par recombinaison de la chalcomycine et des polycetides et genes les modifiant - Google Patents

Synthase par recombinaison de la chalcomycine et des polycetides et genes les modifiant Download PDF

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WO2004018703A2
WO2004018703A2 PCT/US2003/026569 US0326569W WO2004018703A2 WO 2004018703 A2 WO2004018703 A2 WO 2004018703A2 US 0326569 W US0326569 W US 0326569W WO 2004018703 A2 WO2004018703 A2 WO 2004018703A2
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pks
chalcomycin
domain
recombinant
polyketide
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PCT/US2003/026569
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WO2004018703A3 (fr
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Leonard Katz
Ralph C. Reid
Zhihao Hu
Andreas Schirmer
Shannon L. Ward
Christopher Reeves
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Kosan Biosciences, Inc.
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Priority to AU2003260062A priority Critical patent/AU2003260062A1/en
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Publication of WO2004018703A3 publication Critical patent/WO2004018703A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the invention relates to recombinant polynucleotides that encode polypeptides or domains of the chalcomycin polyketide synthase gene cluster. Accordingly, the present invention is directed to the production of chalcomycin PKS enzymes, to polynucleotides that encode such enzymes, and to host cells that contain such polynucleotides. Further enhancements in the biological activities of chalcomycin and other polyketides, through production of derivatives thereof, is also made possible according to the practice of the invention by providing P450 hydroxylases that provide attachment points on the polyketide molecule for further modifications. Thus the present invention relates to the fields of molecular biology, chemistry, recombinant DNA technology, medicine, animal health, and agriculture.
  • Polyketides represent a large family of diverse compounds synthesized from 2 carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms including fungi and mycelial bacteria, in particular the actinomycetes. An appreciation for the wide variety of polyketide structures and for their biological activities, may be gained upon review of the extensive art, for example, published PCT Patent Publication WO 95/08548 and United States Patent Nos. 5,672,491 and 6,303,342
  • Polyketides are synthesized in nature by polyketide synthases ("PKS").
  • PKS polyketide synthases
  • the Type I or modular PKS comprise a set of separate catalytic active sites; each active site is termed a "domain”, and a set thereof is termed a "module”.
  • One module exists for each cycle of carbon chain elongation and modification.
  • Figure 9 of aforementioned WO95/08548 depicts a typical Type I PKS, in this case 6-deoxyerythronolide B synthase (“DEBS”), which is involved in the production of erythromycin.
  • DEBS 6-deoxyerythronolide B synthase
  • Six separate modules, each catalyzing a round of condensation and modification of a 2-carbon unit, are present in DEBS.
  • DEBS-1 The number and type of catalytic domains that are present in each module varies based on the needed chemistry, and the total of 6 modules is provided on 3 separate polypeptides (designated DEBS-1, DEBS-2, and DEBS-3, with 2 modules per each polypeptide).
  • Each of the DEBS polypeptides is encoded by a separate open reading frame (gene), see Caffrey et al, FEBS Letters, 304, pp. 205, 1992.
  • DEBS provides a representative example of a Type I PKS.
  • modules 1 and 2 reside on DEBS-1, modules 3 and 4 on DEBS-2, and modules 5 and 6 on DEBS-3, wherein module 1 is defined as the first module to act on the growing polyketide backbone, and module 6 the last.
  • the minimal PKS module is typified by module 3 of DEBS which contains a ketosynthase (“KS”) domain, an acyltransferase (“AT”) domain, and an acyl carrier protein (“ACP”) domain. These three enzyme activities are sufficient to activate a 2-carbon extender unit and attach it to the growing polyketide molecule. Additional domains that may be included in a module relate to reactions other than the actual condensation, and include domains for a ketoreductase activity (“KR”), a dehydratase activity (“DH”), and an enoylreductase activity (“ER”).
  • KR ketoreductase activity
  • DH dehydratase activity
  • ER enoylreductase activity
  • the first module thereof also contains an additional set of the AT and ACP activities because that module catalyzes initial condensation, and so begins with a "loading domain" (sometimes referred to as a loading module) that contains an AT and ACP, that bind the starter unit.
  • the "finishing" of the 6- deoxyerythronolide molecule is regulated by a thioesterase activity ("TE") in module 6 that catalyzes cyclization of the macrolide ring during release of the product of the PKS.
  • TE thioesterase activity
  • PKS genes can be engineered in a variety of ways to achieve biosynthesis of polyketides.
  • PKS genes can be inserted into a heterologous host to make a polyketide in a host that does not make it naturally.
  • Polyketides can also be made by hybrid or otherwise altered PKSs or polyketide biosynthetic gene clusters.
  • polyketides can be overproduced by increasing the pools of available starting polyketide biosynthetic precursors and by other means. See U.S. Pat. Nos. 5,672,491; 5,962,290; 6,080,555; 6,391,594; and 6,221,641 and PCT Patent Publications 00/47724, 01/27306, and 01/31035.
  • Chalcomycin is a 16-membered macrolide antibiotic produced by some strains of Streptomyces bikiniensis and possesses a broad spectrum of antimicrobial activity. Certain naturally occurring derivatives of chalcomycin produced by other Streptomyces organisms also have antimicrobial activity. For instance, the 8-deoxy chalcomycin derivative produced by Streptomycin hirsutus has antimicrobial activity against gram-positive bacteria. Chalcomycin has two modifying sugar molecules, D-mycinose and D-chalcose, the former being subject to post-glycosylation modification by O-methylation at two positions. For additional information regarding chalcomycin, see Woo, P.W.K.
  • Chalcomycin is synthesized by a Type I or modular PKS and modification enzymes.
  • Post-PKS modification reactions include P450 oxidation at three sites to add hydroxyl groups and glycosylation at the C5 hydroxyl to add D-chalcose, and at the C20 hydroxyl to add allose, which is then methylated at two positions to yield D-mycinose.
  • the present invention provides recombinant nucleic acids encoding polyketide synthases and polyketide modification enzymes.
  • the recombinant nucleic acids of the invention are useful in the production of polyketides, including but not limited to chalcomycin and chalcomycin analogs and derivatives in recombinant host cells.
  • the biosynthesis of chalcomycin is performed by a modular PKS and polyketide modification enzymes.
  • the chalcomycin synthase is made up of several proteins, each having one or more modules. The modules have domains with specific synthetic functions.
  • the present invention also provides domains and modules of the chalcomycin PKS and corresponding nucleic acid sequences encoding them and/or parts thereof. Such compounds are useful in the production of hybrid PKS enzymes and the recombinant genes that encode them.
  • the present invention also provides modifying genes of chalcomycin biosynthetic gene cluster in recombinant form, including but not limited to isolated form and incorporated into a vector or the chromosomal DNA of a host cell.
  • the present invention also provides recombinant P450 hydroxylases that provide hydroxyl attachment points useful for further chemical modification.
  • the P450 hydroxylases of the present invention include ChmHI, ChmPI and ChmPII hydroxylases.
  • the present invention also provides recombinant host cells that contain the nucleic acids of the invention.
  • the host cell provided by the invention is a Streptomyces host cell that produces a chalcomycin modification enzyme and/or a domain, module, or protein of the chalcomycin PKS. Methods for the genetic manipulation of Streptomyces are described in Kieser et al, "Practical Streptomyces Genetics," The John Innes Foundation, Norwich (2000), which is incorporated herein by reference in its entirety.
  • a recombinant PKS wherein at least 10, 15, 20, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules of chalcomycin polyketide synthase. Preferably at least an entire domain of a module of chalcomycin synthase is included.
  • Representative chalcomycin PKS domains useful in this aspect of the invention include, for example, KR, DH, ER, AT, ACP and KS domains.
  • the PKS is assembled from polypeptides encoded by DNA molecules that comprise coding sequences for PKS domains, wherein at least one encoded domain corresponds to a domain of chalcomycin PKS.
  • the coding sequences are operably linked to control sequences so that expression therefrom in host cells is effective.
  • chalcomycin PKS coding sequences or modules and/or domains can be made to encode PKS to biosynthesize compounds having antibiotic or other useful bioactivity other than chalcomycin.
  • Figure 1 illustrates the structure of the chalcomycin PKS biosynthetic gene cluster, and cosmids pKOS146.185.1 and pKOS146.185.10, which contain insert DNA encompassing the chalcomycin PKS gene cluster and associated modification enzyme genes.
  • ACP acyl carrier protein
  • chm chalcomycin gene
  • Orf open reading frame.
  • Figure 2 shows proposed pathways for post-PKS modification of the chalcomycin-spiramycin hybrid PKS macrolide product.
  • the invention provides recombinant materials for the production of polyketides.
  • the present invention provides recombinant nucleic acids encoding polyketide synthases that contain all or a portion of the chalcomycin PKS.
  • the biosynthesis of chalcomycin is performed by a modular PKS and modification enzymes.
  • the chalcomycin synthase is made up of five proteins, each having one or more modules, each module comprising domains with specific synthetic functions.
  • the present invention also provides the domains and modules of the chalcomycin PKS and corresponding nucleic acid sequences encoding them in recombinant form.
  • Modifying genes of the chalcomycin biosynthetic gene cluster are also provided, including but not limited to the genes for the ChmHI, ChmPI and ChmPII P450 hydroxylases that provide hydroxyl attachment points useful for further chemical modification.
  • Methods and host cells for using these genes to produce or modify a polyketide in recombinant host cells are also provided.
  • the nucleotide sequences encoding chalcomycin PKS and modifying proteins of the present invention were isolated from Streptomyces bikiniensis NRRL 2737 (obtained from the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, Illinois USA).
  • the chalcomycin PKS gene cluster and modifying genes are contained in cosmids pKOS 146.185.1 and pKOS146.185.10.
  • the cloning and characterization of the chalcomycin PKS gene cluster is described in Example 1, infra.
  • pKOS146-185.1 was deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA, 20110-2209, on 19 February 2003, with accession number PTA-4961.
  • pKOS146-185.10 was deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA, 20110-2209, on 19 February 2003, with accession number PTA-4962.
  • chalcomycin modifying enzymes can also be used to modify other polyketides and produce derivatives thereof with enhanced solubility and/or bioactivity, for instance as antibiotics, and/or sites for further enzymatic or chemical modification.
  • the nucleotide sequences of the chalcomycin biosynthetic gene cluster encoding chalcomycin PKS and modifying enzymes, and domains and/or modules of the PKS can be used, for example, to make antibiotics or other useful compounds in addition to chalcomycin, and in host cells in addition to Streptomyces bikiniensis.
  • nucleic acids there is a need for recombinant nucleic acids, host cells, and methods of expressing those nucleic acids in host cells resulting in production of chalcomycin and or its analogs or derivatives, and modifying enzymes, such as the cytochrome P450 hydroxylases that specifically attach hydroxyl groups on the resulting aglycone (which can then be used as attachment points for further modifications).
  • modifying enzymes such as the cytochrome P450 hydroxylases that specifically attach hydroxyl groups on the resulting aglycone (which can then be used as attachment points for further modifications).
  • the modifying P450's from the chalcomycin PKS cluster of the present invention can be used to make compounds in a host that does not naturally produce such compounds.
  • purified and isolated DNA molecules are provided that comprise one or more coding sequences for one or more domains or modules of chalcomycin synthase.
  • encoded domains include chalcomycin synthase KR, DH, ER, AT, ACP, and KS domains.
  • the invention provides DNA molecules in which the complete set of chalcomycin PKS-encoding sequences are operably linked to expression control sequences that are effective in suitable host cells to produce chalcomycin and/or its analogs or derivatives.
  • the invention provides polypeptides comprising a portion of the coding sequences for the proteins of the chalcomycin synthase.
  • Table 2 in Example 1 provides a description of genes in the chalcomycin PKS gene (i.e., SEQ ID NO:l and subsequences encoding modules, domains and ORFs, e.g., as indicated), as well as encoded proteins (including SEQ ID. NOS: 2-43) or domains. It will be apparent from Table 2, and Figures 1 and 2, which DNA strand comprises the coding sequence for a protein (i.e., the strand having the sequence of SEQ ID NO: 1 , or its complement.
  • the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, alternatively at least one module, alternatively at least one domain, involved in the biosynthesis of a chalcomycin.
  • the invention provides the present invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, alternatively at least one module, alternatively at least one domain, involved in the biosynthesis of a chalcomycin.
  • the invention also provides polypeptides comprising PKS interpolypeptide linker sequences, and polynucleotides encoding such linker sequences. Also provided by the invention are polypeptides comprising intrapolypeptide linker sequences, and polynucleotides encoding such linkers.
  • the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to at least one subsequence of SEQ ID NO:l or its complement.
  • the subsequence comprises a sequence encoding a chalcomycin PKS domain or module.
  • the invention provides a recombinant DNA molecule that encodes a polypeptide, module or domain derived from a chalcomycin polyketide synthase (PKS) gene cluster.
  • a polypeptide, module or domain is derived from a chalcomycin polyketide synthase (PKS) gene cluster when it is encoded by a DNA with substantial sequence identity to the corresponding coding region of the S.
  • the DNA encoding sequence of the polypeptide, module or domain hybridizes under stringent conditions to a nucleic acid having the sequence of SEQ ID NO:l (or its complement).
  • a polypeptide, module or domain is biologically active, i.e., has at least one enzymatic activity chracteristic of the polypeptide, module or domain encoded exactly by corresponding sequence of SEQ ID NO:l or its complement.
  • the biological activity of a polypeptide of the invention can be measured by methods well known to the art.
  • the invention provides the present invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes an open reading frame, module or domain having an amino acid sequence identical or substantially similar to an ORF, module or domain encoded by SEQ ID NO:l or its complement.
  • a polypeptide, module or domain having a sequence substantially similar to a reference sequence has substantially the same activity as the reference protein, module or domain (e.g., when integrated into an appropriate PKS framework using methods known in the art).
  • one or more activities of a substantially similar polypeptide, module or domain are modified or inactivated as described below.
  • the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain encoded by SEQ ID NO:l, e.g., a polypeptide, module or domain involved in the biosynthesis of a chalcomycin, wherein said nucleotide sequence comprises at least 20, 25, 30, 35, 40, 45, or 50 contiguous base pairs identical to a sequence of SEQ ID NO:l or its complement.
  • the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain encoded by SEQ ID NO:l, e.g., a polypeptide, module or domain involved in the biosynthesis of a chalcomycin, wherein said polypeptide, module or domain comprises at least 10, 15, 20, 30, or 40 contiguous residues of a corresponding polypeptide, module or domain encoded by SEQ ID NO:l or its complement.
  • the invention provides a recombinant DNA molecule, comprising a sequence of at least about 200, optionally at least about 500, basepairs with a sequence identical or substantially identical to a protein encoding region of SEQ ID NO: 1.
  • the DNA molecule encodes a polypeptide, module or domain derived from a chalcomycin polyketide synthase (PKS) gene cluster.
  • PKS chalcomycin polyketide synthase
  • SEQ ID NO: 1 was determined using the inserts of pKOS 146.185.1 and pKOS146-185.10. Accordingly, the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to a ORF encoding sequence of the insert of pKOS 146.185.1 or pKOS146-185.10.
  • Those of skill will recognize that, due to the degeneracy of the genetic code, a large number of DNA sequences encode the amino acid sequences of the domains, modules, and proteins of the chalcomycin PKS, the enzymes involved in chalcomycin modification and other polypeptides encoded by the genes of the chalcomycin biosynthetic gene cluster.
  • the present invention contemplates all such DNAs. For example, it may be advantageous to optimize sequence to account for the codon preference of a host organism.
  • the invention also contemplates naturally occurring genes encoding the chalcomycin PKS and tailoring enzymes that are polymorphic or other variants.
  • polypeptide, modules and domains of the invention may comprise one or more conservative amino acid substitutions relative to the polypeptides encoded by SEQ ID NO: 1, such as, for example, conservative substitutions include aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids.
  • conservative substitutions include aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids.
  • the terms "substantial identity,” “substantial sequence identity,” or “substantial similarity” in the context of nucleic acids refers to a measure of sequence similarity between two polynucleotides.
  • Substantial sequence identity can be determined by hybridization under stringent conditions, by direct comparison, or other means.
  • two polynucleotides can be identified as having substantial sequence identity if they are capable of specifically hybridizing to each other under stringent hybridization conditions.
  • Other degrees of sequence identity e.g., less than "substantial” can be characterized by hybridization under different conditions of stringency.
  • “Stringent hybridization conditions” refers to conditions in a range from about 5°C to about 20°C or 25°C below the melting temperature (Tm) of the target sequence and a probe with exact or nearly exact complementarity to the target.
  • Tm melting temperature
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands.
  • stringent hybridization conditions for probes greater than 50 nucleotides are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 50°C, preferably at least about 60°C.
  • stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed.
  • Exemplary conditions include hybridization at 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 pH 7.0, 1 mM EDTA at 50°C (or alternatively 65°C); wash with 2xSSC, 1% SDS, at 50°C (or alternatively 0.1 - 0.2 xSSC, 1% SDS, at 50°C or 65°C).
  • Other exemplary conditions for hybridization include (1) high stringency: O.l SSPE, 0.1% SDS, 65°C; (2) medium stringency: 0.2 ⁇ SSPE, 0.1% SDS, 50° C; and (3) low stringency: l.OxSSPE, 0.1% SDS, 50° C. Equivalent stringencies may be achieved using alternative buffers, salts and temperatures.
  • substantial sequence identity can be described as a percentage identity between two nucleotide or amino acid sequences.
  • Two nucleic acid sequences are considered substantially identical when they are at least about 70% identical, at least about 75% identical, or at least about 80% identical, or at least about 85% identical, or at least about 90% identical, or at least about 95% or 98% identical.
  • Two amino acid sequences are considered substantially identical when they are at least about 60%, sequence identical, more often at least about 70%, at least about 80%, or at least about 90% sequence identity to the reference sequence.
  • Percentage sequence (nucleotide or amino acid) identity is typically calculated using art known means to determine the optimal alignment between two sequences and comparing the two sequences.
  • Optimal alignment of sequences may be conducted using the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol. Biol 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by the BLAST algorithm of Altschul (1990) J Mol. Biol. 215: 403-410; and Shpaer (1996) Genomics 38:179-191, or by the Needleham et al. (1970) J Mol. Biol.
  • the term "recombinant” has its usual meaning in the art and refers to a polynucleotide synthesized or otherwise manipulated in vitro, or to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems.
  • a "recombinant" polynucleotide is defined either by its method of production or its structure.
  • a recombinant polynucleotide can be a polynucleotide made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature.
  • products made by transforming cells with any non-naturally occurring vector is encompassed, as are polynucleotides comprising sequence derived using any synthetic oligonucleotide process, as are polynucleotides from which a region has been deleted.
  • a recombinant polynucleotide can also be a coding sequence that has been modified in vivo using a recombinant oligo or polynucleotide (such as a PKS in which a domain is inactivated by homologous recombination using a recombinant polynucleotide).
  • a "recombinant" polypeptide is one expressed from a recombinant polynucleotide.
  • recombinant polypeptides of the invention have a variety of uses, some of which are described in detail below, including but not limited to use as enzymes, or componants of enzymes, useful for the synthesis or modification of polyketides.
  • Recombinant polypeptides encoded by the chalcomycin PKS gene cluster are also useful as antigens for production of antibodies.
  • Such antibodies find use for purification of bacterial (e.g., Streptomyces bikiniensis) proteins, detection and typing of bacteria, and particularly, as tools for strain improvement (e.g., to assay PKS protein levels to identify "up-regulated” strains in which levels of polyketide producing or modifying proteins are elevated) or assessment of efficiency of expression of recombinant proteins.
  • Polyclonal and monoclonal antibodies can be made by well known and routine methods (see, e.g., Harlow and Lane, 1988, ANTIBODIES: A LABORATORY MANUAL, COLD SPRING HARBOR LABORATORY, New York; Koehler and Milstein 1075, Nature 256:495).
  • the protein fragment In selecting polypeptide sequences for antibody induction, it is not to retain biological activity; however, the protein fragment must be immunogenic, and preferably antigenic (as can be determined by routine methods). Generally the protein fragment is produced by recombinant expression of a DNA comprising at least about 60, more often at least about 200, or even at least about 500 or more base pairs of protein coding sequence, such as a polypeptide, module or domain derived from a chalcomycin polyketide synthase (PKS) gene cluster. Methods for expression of recombinant proteins are well known. (See, e.g., Ausubel et al., 2002, Current Protocols In Molecular Biology, Greene Publishing and Wiley-Interscience, New York.)
  • Further aspects of the invention include chimeric PKSs comprising a portion (in one embodiment at least a domain, optionally at least a module, or alternatively at least one polypeptide) from the chalcomycin PKS, and a portion (in one embodiment at least a domain, optionally at least a module, or alternatively at least a polypeptide) from one or more non- chalcomycin PKSs.
  • the invention provides (1) encoding DNA for a chimeric PKS that is substantially patterned on a non-chalcomycin producing enzyme, but which includes one or more functional domains or modules of chalcomycin PKS; (2) encoding DNA for a chimeric PKS that is substantially patterned on the chalcomycin PKS, but which includes one or more functional domains or modules of another PKS or NRPS; and (3) methods for making chalcomycin analogs and derivatives.
  • examples include chimeric PKS enzymes wherein the genes for the erythromycin PKS, rapamycin PKS, tylosin PKS, and spiramycin PKS, or another PKS function as accepting genes, and one or more of the above-identified coding sequences for chalcomycin domains or modules are inserted as replacements for domains or modules of comparable function.
  • examples include chimeric PKS enzymes wherein the chalcomycin PKS serves as an accepting gene, and genes for the erythromycin PKS, rapamycin PKS, tylosin PKS, and spiramycin PKS, or another PKS function as accepting genes, and one or more of the above-identified coding sequences for chalcomycin domains or modules are inserted as replacements for domains or modules of comparable function.
  • a partial list of sources of PKS sequences for use in making chimeric molecules includes Avermectin (U.S. Pat. No.
  • Nemadectin MacNeil et al., 1993, supra
  • Niddamycin Kakavas et al., 1997, J Bacteriol 179:7515-22
  • Oleandomycin Swan et al., 1994, Mol. Gen. Genet. 242:358-62; U.S. Pat. No. 6,388,099; Olano et al., 1998, Mol. Gen. Genet. 259:299-308
  • Platenolide ⁇ P Pat. App. 791,656
  • Rapamycin Rosecke et al., 1995, Proc. Natl. Acad. Sci.
  • appropriate encoding DNAs for construction of such chimeric PKS include those that encode at least 10, 15, 20 or more amino acids of a selected chalcomycin domain or module.
  • Recombinant methods for manipulating modular PKS genes to make chimeric PKS enzymes are described in U.S. Patent Nos. 5,672,491 ; 5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos. 98/49315 and 97/02358.
  • a number of genetic engineering strategies have been used with DEBS to demonstrate that the structures of polyketides can be manipulated to produce novel natural products, primarily analogs of the erythromycins (see the patent publications referenced supra and Hutchinson, 1998, Curr Opin Microbiol.
  • the invention methods may be directed to the preparation of an individual polyketide.
  • the polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it.
  • the resulting polyketides may be further modified to convert them to other useful compounds. Examples of chemical structures of sixteen-membered macrolides that can be made using the materials and methods of the present invention are described in PCT Patent Publication WO 02/32916; U.S. Patent Application US20020128213A (app. no. 09/969,177); and copending U.S. provisional patent application no. 60/493,966.
  • the recombinant DNAs and DNA vectors of the inventions can also be used to make "libraries" of polyketides.
  • members of these polyketide libraries may themselves be novel compounds, and the invention further includes novel polyketide members of these libraries.
  • the invention provides libraries of polyketides by generating modifications in, or using a portion of, the chalcomycin PKS so that the protein complexes produced have altered activities in one or more respects, and thus produce polyketides other than the natural product of the PKS. Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method.
  • the invention provides recombinant PKS wherein at least 10, 15, 20, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules of chalcomycin polyketide synthase.
  • a polyketide synthase "derived from" a naturally occurring PKS contains the scaffolding encoded by all the portion employed of the naturally occurring synthase gene, contains at least two modules that are functional, and contains mutations, deletions, or replacements of one or more of the activities of these functional modules so that the nature of the resulting polyketide is altered. This definition applies both at the protein and genetic levels.
  • Particular embodiments include those wherein a KS, AT, KR, DH, or ER has been deleted or replaced by a version of the activity from a different PKS or from another location within the same PKS, and derivatives where at least one noncondensation cycle enzymatic activity (KR, DH, or ER) has been deleted or wherein any of these activities has been added or mutated so as to change the ultimate polyketide synthesized.
  • KR, DH, or ER noncondensation cycle enzymatic activity
  • the nature of the carbon skeleton of the PKS will be determined by the specificities of the acyl transferases which determine the nature of the extender units at each position ⁇ e.g., malonyl, methyl malonyl, methoxy . malonyl, or ethyl malonyl, etc.
  • the loading domain specificity will also have an effect on the resulting carbon skeleton of the polyketide.
  • the oxidation state at various positions of the polyketide will be determined by the dehydratase and reductase portions of the modules. This will determine the presence and location of ketone, alcohol, alkene or alkane substituents at particular locations in the polyketide.
  • the stereochemistry of the resulting polyketide is a function of three aspects of the synthase.
  • the first aspect is related to the AT/KS specificity associated with substituted malonyls as extender units, which affects stereochemistry only when the reductive cycle is missing or when it contains only a ketoreductase since the dehydratase would abolish chirality.
  • the specificity of the ketoreductase will determine the chirality of the corresponding hydroxyl group.
  • the enoyl reductase specificity for substituted malonyls as extender units will influence the result when there is a complete KR DH/ER available.
  • polyketide biosynthesis can be manipulated to make a product other than the product of a naturally occurring PKS biosynthetic cluster.
  • AT domains can be altered or replaced to change specificity.
  • the AT domain of chalcomycin module 0 (loading domain) can be replaced by an AT with specificity for methylmalonyl-CoA to produce chalcomycin derivatives with a C-15 ethyl group in place of the C-15 methyl group.
  • the variable domains within a module can be deleted and or inactivated or replaced with other variable domains found in other modules of the same PKS or from another PKS.
  • chalcomycin can be produced using a modified chalcomycin PKS in which module 7 of the chalcomycin PKS is replaced by module 7 of the tylosin PKS (optionally with appropriate linker modifications).
  • protein subunits of different PKSs also can be mixed and matched to make compounds having the desired backbone and modifications.
  • subunits of 1 and 2 (encoding modules 1-4) of the pikromycin PKS were combined with the DEBS3 subunit to make a hybrid PKS product (see Tang et al., Science, 287: 640 (2001), WO 00/26349 and WO 99/6159). Also see Examples, below.
  • Mutations can be introduced into PKS genes such that polypeptides with altered activity are encoded.
  • Polypeptides with "altered activity” include those in which one or more domains are inactivated or deleted, or in which a mutation changes the substrate specificity of a domain, as well as other alterations in activity. Mutations can be made to the native sequences using conventional techniques.
  • the substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes. Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion.
  • the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex.
  • the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. (See Zoller and Smith, Methods in Enzymology (1983) 100:468).
  • Primer extension is effected using DNA polymerase.
  • the product of the extension reaction is cloned, and those clones containing the mutated DNA are selected. Selection can be accomplished using the mutant primer as a hybridization probe.
  • the technique is also applicable for generating multiple point mutations. (See, e.g., Dalbie-McFarland et al. Proc Natl Acad Sci USA (1982) 79:6409). PCR mutagenesis can also be used for effecting the desired mutations.
  • Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, and 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.
  • regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS synthase can be recovered, for example, using PCR techniques with appropriate primers.
  • corresponding activity encoding regions is meant those regions encoding the same general type of activity ⁇ e.g., a ketoreductase activity in one location of a gene cluster would "correspond" to a ketoreductase-encoding activity in another location in the gene cluster or in a different gene cluster; similarly, a complete reductase cycle could be considered corresponding -- e.g., KR DH/ ⁇ R could correspond to KR alone.
  • the method is related to ⁇ T cloning methods (see, Datansko & Wanner, 2000, Proc. Natl. Acad. Sci. U.S.A. 97, 6640-45; Muyrers et al, 2000, Genetic Engineering 22:77-98).
  • the RED/ ⁇ T cloning procedure is used to introduce a unique restriction site in the recipient plasmid at the location of the targeted domain. This restriction site is used to subsequently linearize the recipient plasmid in a subsequent ET cloning step to introduce the modification. This linearization step is necessary in the absence of a selectable marker, which cannot be used for domain substitutions.
  • An advantage of using this method for PKS engineering is that restriction sites do not have to be introduced in the recipient plasmid in order to construct the swap, which makes it faster and more powerful because boundary junctions can be altered more easily.
  • the invention provides a chimeric PKS in which one of more polypeptides are derived from a chalcomycin PKS polypeptide, and one or more peptides are ⁇ derived from one or more non-chalcomycin PKS(s) that, like the chalcomysin PKS, produces a 16-membered macrolide.
  • PKS(s) that produces a 16-membered macrolide include, for example, the tylosin PKS, the spiramycin PKS, the niddamycin PKS, and the mycinamicin PKS.
  • All the currently known PKSs for 16-membered macrolides consists of five large polypeptides encoded by colinear genes in a single operon. The arrangement of modules on these polypeptides is conserved.
  • the first polypeptide has a loading module and two extender modules, the second a single extender module, the third two extender modules, the fourth a single extender module, and the fifth a single extender module followed by a thioesterase domain.
  • the different aglycone core structures produced by different 16-membered macrolide PKSs is due to differences in the catalytic domains within each of these modules.
  • new hybrid 16-membered macrolides can be made by expressing combinations of PKS polypeptides from different sources in a suitable host.
  • the hybrid PKS produces hybrid polyketides that, optionally can be further modified by the post-PKS tailoring enzymes present within the host. See Examples, infra.
  • selection of particular combinations of polypeptides provides a level of predictability as to the products formed by the hybrid PKS, the invention is not limited to any particular combinations or structures "predicted" by the table.
  • the components of the chimeric PKS are arranged onto polypeptides having interpolypeptide linkers that direct the assembly of the polypeptides into the functional PKS protein, such that it is not required that the PKS have the same arrangement of modules in the polypeptides as observed in natural PKSs. Suitable interpolypeptide linkers to join polypeptides and intrapolypeptide linkers to join modules within a polypeptide are described in PCT publication WO 00/47724. [0054] In one embodiment of the invention, the components of the PKS are arranged into five polypeptides similarly to natural PKS proteins involved in the biosynthesis of tylactone, platenolide, and the like.
  • the first polypeptide comprises the loading domain, first and second extender modules, and a C-terminal interpolypeptide linker region suitable for interaction with the second polypeptide.
  • the second polypeptide comprises an N-terminal interpolypeptide linker region suitable for interaction with the first polypeptide, the third extender module, and a C-terminal interpolypeptide linker region suitable for interaction with the third polypeptide.
  • the third polypeptide comprises an N-terminal interpolypeptide linker region suitable for interaction with the second polypeptide, the fourth and fifth extender modules, and a C-terminal interpolypeptide linker region suitable for interaction with the fourth polypeptide.
  • the fourth polypeptide comprises an N-terminal interpolypeptide linker region suitable for interaction with the third polypeptide, the sixth extender module, and a C-terminal interpolypeptide linker region suitable for interaction with the fifth polypeptide.
  • the fifth polypeptide comprises an N-terminal interpolypeptide linker region suitable for interaction with the fourth polypeptide, the seventh extender module, and the terminal thioesterase domain.
  • the components of the PKS residing on any given polypeptide are derived from the same source, and are naturally contiguous in that source, but the intrapolypeptide linkers are changed to allow proper assembly across heterologous polypeptide junctions to form a functional PKS.
  • the first polypeptide is the intact first polypeptide of the chalcomycin PKS, encoded by chmGl, and comprises the loading domain and first and second extender modules from the chalcomycin PKS together with the native C-terminal interpolypeptide linker region that directs interaction of the first polypeptide with the second polypeptide of the chalcomycin PKS.
  • the second polypeptide comprises the N-terminal interpolypeptide linker and module 3 of the chalcomycin PKS, encoded by chmGII, but with the C-terminal interpolypeptide linker replaced by the C-terminal interpolypeptide linker from the second polypeptide of the spiramycin PKS, encoded by srmG2.
  • This replaced C- terminal interpolypeptide linker directs the second polypeptide to interact with the third polypeptide, taken from the spiramycin PKS and encoded by the srmG3 gene.
  • the remaining polypeptides are the third, fourth, and fifth polypeptides of the spiramycin PKS, encoded by srmG3, srmG4, and srmG5, respectively.
  • the first polypeptide comprises the loading domain and first, second and third extender modules from the chalcomycin PKS, together with a C-terminal interpolypeptide linker region derived from the C-terminus of the first polypeptide of the tylosin PKS.
  • the remaining polypeptides are the third, fourth, and fifth polypeptides of the tylosin PKS.
  • the use of the appropriate interpolypeptide linkers directs the proper assembly of the PKS, thereby improving the catalytic activity of the resulting hybrid PKS.
  • the DNA compounds of the invention can be expressed in host cells for production of known and novel compounds.
  • Preferred hosts include fungal systems such as yeast and procaryotic hosts, but single cell cultures of, for example, mammalian cells could also be used.
  • a variety of methods for heterologous expression of PKS genes and host cells suitable for expression of these genes and production of polyketides are described, for example, in U.S. Patent Nos. 5,843,718 and 5,830,750; WO 01/31035, WO 01/27306, and WO 02/068613; and U.S. patent application nos. 10/087,451 (published as US2002000087451); 60/355,211; and 60/396,513 (corresponding to published application 20020045220).
  • Appropriate host cells for the expression of the hybrid PKS genes include those organisms capable of producing the needed precursors, such as malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-ACP, and having phosphopantotheinylation systems capable of activating the ACP domains of modular PKSs. See, for example, US Patent 6,579,695. However, as disclosed in U.S. Patent No. 6,033,883, a wide variety of hosts can be used, even though some hosts natively do not contain the appropriate post-translational mechanisms to activate the acyl carrier proteins of the synthases. Also see WO 97/13845 and WO 98/27203.
  • the host cell may natively produce none, some, or all of the required polyketide precursors, and may be genetically engineered so as to produce the required polyketide precursors. Such hosts can be modified with the appropriate recombinant enzymes to effect these modifications. Suitable host cells include Streptomyces, E. coli, yeast, and other procaryotic hosts which use control sequences compatible with Streptomyces spp.
  • suitable hosts that either natively produce modular polyketides or have been engineered so as to produce modular polyketides include but are not limited to actinomycetes such as Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces fradiae, Streptomyces ambofaciens, and Saccharopolyspora erythraea, eubacteria such as Escherichia coli, myxobacteria such as Myxococcus xanthus, and yeasts such as Saccharomyces cerevisiae.
  • actinomycetes such as Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces fradiae, Streptomyces ambofaciens
  • Saccharopolyspora erythraea eubacteria such as Escherichia coli
  • myxobacteria such as Myxococcus xant
  • any native modular PKS genes in the host cell have been deleted to produce a "clean host," as described in US Patent 5,672,491, incorporated herein by reference.
  • the construction of the clean host S. fradiae KI 59-1 , and the clean host S. fradiae K159-l/244-17a that produces methoxymalonyl-ACP are described below in Examples 2 and 3.
  • Other organisms can be engineered using similar methods.
  • the host cell expresses, or is engineered to express, a polyketide "tailoring" or "modifying" enzyme. Once a PKS product is released, it is subject to post-PKS tailoring reactions.
  • Tailoring enzymes normally associated with polyketide biosynthesis include oxygenases, glycosyl- and methyltransferases, acyltransferases, halogenases, cyclases, aminotransferases, and hydroxylases.
  • Tailoring enzymes for modification of a product of the chalcomycin PKS, a non-chalcomycin PKS, or a chimeric PKS can be those normally associated with chalcomycin biosynthesis (including, but not limited to, proteins described in Table 2) or "heterologous" tailoring enzymes.
  • the P450 hydrolases encoded by the chmHI, chmPIand chmPII genes are of particular interest for production of polyketides having hydroxy groups well suited for subsequent chemical modification.
  • tailoring enzymes can be expressed in the organism in which they are naturally produced, or as recombinant proteins in heterologous hosts.
  • a hybrid PKSs having elements from the chalcomycin and spiramycin PKSs, or from the tylosin and chalcomycin PKSs were expressed in an engineered host derived from a tylosin producing strain of S. fradiae in which all or most of the post-PKS tailoring reactions of the tylosin biosynthetic pathway (see Baltz and Seno, 1988, "Genetics of Streptomyces fradiae and tylosin biosynthesis” Annu Rev Microbiol.
  • the structure produced by the heterologous or hybrid PKS may be modified with different efficiencies by post-PKS tailoring enzymes from different sources.
  • post-PKS tailoring enzymes can be recruited from other pathways to obtain the desired compound.
  • a chmH. gene has been used to modify the product of a chalcomycin-spiramycin hybrid PKS.
  • host cells can be selected, or engineered, for expression of a glycosylatation apparatus (discussed below), amide synthases, (see, for example, U.S. patent publication 20020045220 "Biosynthesis of Polyketide Synthase Substrates").
  • the host cell can contain the desosamine, megosamine, and/or mycarose biosynthetic genes, corresponding glycosyl transferase genes, and hydroxylase genes (e.g., picK, megK, eryK, megF, and/or eryF).
  • hydroxylase genes e.g., picK, megK, eryK, megF, and/or eryF.
  • glycosylation is effected in accordance with the methods of the invention in recombinant host cells provided by the invention.
  • glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosylases.
  • synthetic chemical methods may be employed.
  • the aglycone compounds can be produced in the recombinant host cell, and the desired modification (e.g., glycosylation and hydroxylation) steps carried out in vitro (e.g., using purified enzymes, isolated from native sources or recombinantly produced) or in vivo in a converting cell different from the host cell (e.g., by supplying the converting cell with the aglycone).
  • desired modification e.g., glycosylation and hydroxylation
  • steps carried out in vitro (e.g., using purified enzymes, isolated from native sources or recombinantly produced) or in vivo in a converting cell different from the host cell (e.g., by supplying the converting cell with the aglycone).
  • vector refers to polynucleotide elements that are used to introduce recombinant nucleic acid into cells for either expression or replication. Selection and use of such vehicles is routine in the art.
  • An "expression vector” includes vectors capable of expressing DNAs that are operatively linked with regulatory sequences, such as promoter regions.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.
  • Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • the vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes may be chosen to contain control sequences operably linked to the resulting coding sequences in a manner that expression of the coding sequences may be effected in an appropriate host. Suitable control sequences include those which function in eucaryotic and procaryotic host cells. If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This can be done individually, or using a pool of isolated encoding nucleotide sequences, which can be inserted into host vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies.
  • control sequences for single cell cultures of various types of organisms are well known in the art.
  • Control systems for expression in yeast are widely available and are routinely used.
  • Control elements include promoters, optionally containing operator sequences, and other elements depending on the nature of the host, such as ribosome binding sites.
  • Particularly useful promoters for procaryotic hosts include those from PKS gene clusters which result in the production of polyketides as secondary metabolites, including those from Type I or aromatic (Type II) PKS gene clusters.
  • tcm promoters examples are act promoters, tcm promoters, spiramycin promoters, tylosin promoter (e.g., tylGIp, see Rodriguez et al., "Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains” J Ind Microbiol Biotechnol. 2003 Apr 16; and DeHoff et al., "Streptomyces fradiae tylactone synthase, starter module and modules 1-7, (tylG) gene, complete eds" Genbank Accession No. U78289), and other promoters.
  • tylosin promoter e.g., tylGIp, see Rodriguez et al., "Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains” J Ind Microbiol Biotechnol. 2003 Apr 16; and DeHoff et al., "Streptomyces fradiae tylactone
  • bacterial promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose
  • additional examples include promoters derived from biosynthetic enzymes such as for tryptophan (trp), the ⁇ -lactamase (bid), bacteriophage lambda PL, and T5.
  • synthetic promoters such as the tac promoter (U.S. Patent No. 4,551,433), can be used.
  • particularly useful control sequences are those which themselves, or with suitable regulatory systems, activate expression during transition from growth to stationary phase in the vegetative mycelium.
  • the system contained in the plasmid identified as pCK7 i.e., the actl/actlll promoter pair and the ⁇ ctII-ORF4 (an activator gene), is particularly preferred.
  • Particularly preferred hosts are those which lack their own means for producing polyketides so that a cleaner result is obtained.
  • Illustrative control sequences, vectors, and host cells of these types include the modified S. coelicolor CH999 and vectors described in PCT publication WO 96/40968 and similar strains of S. lividans. See U.S. Patent Nos. 5,672,491; 5,830,750, 5,843,718; and 6,177,262, each of which is inco ⁇ orated herein by reference.
  • regulatory sequences may also be desirable which allow for regulation of expression of the PKS sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • 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.
  • markers include, for example, genes which confer antibiotic resistance or sensitivity to the plasmid.
  • several polyketides are naturally colored, and this characteristic provides a built-in marker for screening cells successfully transformed by the present constructs.
  • the various PKS nucleotide sequences, or a mixture of such sequences can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements or under the control of a single promoter.
  • the PKS subunits or components can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunits so that hybrid or chimeric PKSs can be generated.
  • the design of such 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 for introducing the recombinant vectors of the present invention into suitable hosts are known to those of skill in the art and typically include the use of CaCl 2 or other agents, such as divalent cations, lipofection, DMSO, protoplast transformation, conjugation, and electroporation.
  • Expression vectors containing nucleotide sequences encoding a variety of PKS systems for the production of different polyketides can be transformed into the appropriate host cells to construct a polyketide library.
  • a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected for successful transformants.
  • Each individual colony has the ability to produce a particular PKS and ultimately a particular polyketide.
  • the expression vectors can be used individually to transform hosts, which transformed hosts are then assembled into a library.
  • a variety of strategies might be devised to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide.
  • the number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products.
  • the number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large.
  • the polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity.
  • the libraries can thus be considered at four levels: (1) a multiplicity of colonies each with a different PKS encoding sequence encoding a different PKS cluster but all derived from a naturally occurring PKS cluster; (2) colonies which contain the proteins that are members of the PKS produced by the coding sequences; (3) the polyketides produced; and (4) compounds derived from the polyketides.
  • combination libraries can also be constructed wherein members of a library derived, for example, from the erythromycin PKS can be considered as a part of the same library as those derived from, for example, the rapamycin PKS cluster.
  • Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of candidate polyketides.
  • the polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like.
  • the supernatants per se can be used for screening, or partial or complete purification of the polyketides can first be effected.
  • screening methods involve detecting the binding of each member of the library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art.
  • individual polyketide members of the library can be tested against a desired target.
  • the present invention provides recombinant DNA molecules and vectors comprising those recombinant DNA molecules that encode all or a portion of the chalcomycin PKS and/or chalcomycin modification enzymes and that, when transformed into a host cell and the host cell is cultured under conditions that lead to the expression of said chalcomycin PKS and/or modification enzymes, results in the production of polyketides including but not limited to chalcomycin and/or analogs or derivatives thereof in useful quantities.
  • the present invention also provides recombinant host cells comprising those recombinant vectors.
  • Suitable culture conditions for production of polyketides using the cells of the invention will vary according to the host cell and the nature of the polyketide being produced, but will be know to those of skill in the art. See, for example, the examples below and WO 98/27203 "Production of Polyketides in Bacteria and Yeast” and WO 01/83803 "Overproduction Hosts for Biosynthesis of Polyketides.”
  • the polyketide product produced by host cells of the invention can be recovered (i.e., separated from the producing cells and at least partially purified) using routine techniques (e.g., extraction from broth followed by chromatography).
  • routine techniques e.g., extraction from broth followed by chromatography.
  • the compositions, cells and methods of the invention may be directed to the preparation of an individual polyketide or a number of polyketides.
  • the polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it. It will be understood that the resulting polyketides may be further modified to convert them to other useful compounds.
  • an ester linkage may be added to produce a "pharmaceutically acceptable ester" (i.e., an ester that hydrolyzes under physiologically relevant conditions to produce a compound or a salt thereof).
  • a pharmaceutically acceptable ester i.e., an ester that hydrolyzes under physiologically relevant conditions to produce a compound or a salt thereof.
  • suitable ester groups include but are not limited to formates, acetates, propionates, butyrates, succinates, and ethylsuccinates.
  • the polyketide product produced by recombinant cells can be chemically modified in a variety of ways. For example , for example by addition of a protecting group, for example to produce prodrug forms.
  • a variety of protecting groups are disclosed, for example, in T.H. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, New York (1999).
  • Prodrugs are in general functional derivatives of the compounds that are readily convertible in vivo into the required compound. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs," H. Bundgaard ed., Elsevier, 1985.
  • solubilizing functionalities include, but are not limited to: 2-(dimethylaminoethyl)amino, piperidinyl, N-alkylpiperidinyl, hexahydropyranyl, furfuryl, tefrahydrofurfuryl, pyrrolidinyl, N-alkylpyrrolidinyl, piperazinylamino, N- alkylpiperazinyl, morpholinyl, N-alkylaziridinylmethyl, (l-azabicyclo[1.3.0]hex-l-yl)ethyl, 2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazoly
  • solubilizing groups can be added by reaction with amines, which results in the displacement of the 17-methoxy group by the amine (see, Schnur et al., 1995, “Inhibition of the Oncogene Product pl85 erbB”2 in Vitro and in Vivo by Geldanamycin and Dihydrogeldanamycin Derivatives,", J. Med. Chem. 38, 3806-3812; Schnur et al, 1995 "erbB-2 Oncogene Inhibition by Geldanamycin Derivatives: Synthesis, Mechanism of Action, and Structure-Activity relationships," J. Med. Chem.
  • Typical amines containing solubilizing functionalities include 2-(dimethylamino)-ethylamine, 4-aminopiperidine, 4-amino-l- methylpiperidine, 4-aminohexahydropyran, fifffurylamine, tetrahydrofurfurylamine, 3- (- ⁇ minomethyl)-tefrahydrofuran, 2-(amino-methyl)pyrrolidine, 2-(aminomethyl)-l - methylpyrrolidine, 1-methylpiperazine, morpholine, l-methyl-2(aminomethyl)aziridine, l-(2- aminoethyl)-l-azabicyclo-[l .3.0]hexane, l-(2-a
  • polyketide forms or compositions can be produced, including but not limited to mixtures of polyketides, enantiomers, diastereomers, geometrical isomers, polymorphic crystalline forms and solvates, and combinations and mixtures thereof can be produced [0080] Many other modifications of polyketides produced according to the invention will be apparent to those of skill, and can be accomplished using techniques of pharmaceutical chemistry.
  • the polyketide products can be formulated as a "pharmaceutically acceptable salt.”
  • Suitable pharmaceutically acceptable salts of compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, benzoic acid, acetic acid, citric acid, tartaric acid, phosphoric acid, carbonic acid, or the like.
  • pharmaceutically acceptable salts may be formed by treatment of a solution of the compound with a solution of a pharmaceutically acceptable base, such as lithium hydroxide, sodium hydroxide, potassium hydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, ammonia, alkylamines, or the like.
  • a pharmaceutically acceptable base such as lithium hydroxide, sodium hydroxide, potassium hydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, ammonia, alkylamines, or the like.
  • the PKS product Prior to administration to a mammal the PKS product will be formulated as a pharmaceutical composition according to methods well known in the art, e.g., combination with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a medium that is used to prepare a desired dosage form of a compound.
  • a pharmaceutically acceptable carrier can include one or more solvents, diluents, or other liquid vehicles; dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents; preservatives; solid binders; lubricants; and the like.
  • compositions may be administered in any suitable form such as solid, semisolid, or liquid form. See Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 th edition, Lippicott Williams & Wilkins (1991).
  • the polyketide is combined in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application.
  • the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use.
  • the carriers that can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form.
  • auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.
  • the invention provides a recombinant DNA molecule that encodes a polypeptide, module or domain derived from a chalcomycin polyketide synthase (PKS) gene cluster.
  • PPS chalcomycin polyketide synthase
  • the DNA molecule (or its complement) has substantial sequence identity to SEQ ID NO: 1.
  • the DNA molecule hybridizes under stringent conditions to a nucleic acid having the sequence of SEQ ID NO: 1 or its complement.
  • the invention provides a recombinant DNA molecule, comprising a sequence of at least about 200, optionally at least about 500, basepairs with a sequence identical or substantially identical to a protein encoding region of SEQ ID NO: 1.
  • the DNA molecule encodes a polypeptide, module or domain derived from a chalcomycin polyketide synthase (PKS) gene cluster.
  • PKS chalcomycin polyketide synthase
  • the recombinant DNA molecule comprises a sequence encoding at least one module of a chalcomycin polyketide synthase.
  • the recombinant DNA molecule encodes a chalcomycin polyketide synthase polypeptide selected from the group consisting of ChmGI, ChmGII, ChmGIII, ChmGIV, and ChmV.
  • the recombinant DNA molecule includes a coding sequence for a chalcomycin modifying enzyme, such as a chalcomycin P450 hydrolase enzyme selected from the group consisting of ChmHI, ChmPI, and ChmPII.
  • the invention also provides vector that comprise the recombinant DNA molecules of the invention.
  • the invention provides a recombinant host cell comprising the vector.
  • the invention provides a recombinant host cell comprising a DNA molecule of the invention integrated into the cell chromosomal DNA.
  • a chimeric PKS that comprises at least one domain of a chalcomycin PKS, and a cell containing such a chimeric PKS.
  • the invention provides a modified functional chalcomycin PKS that differs from the S.
  • the invention provides a cell comprising a modified functional PKS.
  • the invention also provides a method to prepare an chalcomycin derivative which method comprises providing substrates including extender units to the cell.
  • the invention provides a recombinant expression system capable of producing a chalcomycin synthase domain in a host cell, said system comprising an encoding sequence for a chalcomycin polyketide synthase domain, and said encoding sequence being operably linked to control sequences effective in said cell to produce RNA that is translated into said domain, and a host cell modified to contain the recombinant expression system.
  • the invention provides an isolated polypeptide encoded by a recombinant chalcomycin polyketide synthase (PKS) gene, and a recombinant host cell containing or expressing such a polypeptide.
  • PPS recombinant chalcomycin polyketide synthase
  • the invention also provides a recombinant S. bikiniensis cell in which a chmGI, chmGII, chmGUl, chmGIY, or chm V is disrupted so as to reduce or eliminate production of chalcomycin.
  • the invention also provides a recombinant DNA molecule encoding a first protein comprising one or more modules of a chalcomycin PKS and a second protein comprising one or more modules of a tylosin PKS or spiramycin PKS, optionally one or more polypeptides of a chalcomycin PKS and one or more polypeptides of a tylosin PKS or spiramycin PKS.
  • the invention provides a recombinant host cell comprising a hybrid polyketide synthase comprising one or more modules of a chalcomycin PKS and one or more modules of a tylosin PKS or spiramycin PKS.
  • Streptomyces bikiniensis NRRL 2737 Growth of organism and extraction of genomic DNA.
  • genomic DNA extraction a spore stock of Streptomyces bikiniensis NRRL 2737 was used to prepare a seed culture. Spores were stored as a suspension in 25% (v/v) glycerol at -80°C and used to inoculate 5 ml of unbuffered Trypticase Soy Broth (TSB) liquid media. The entire seed culture was transferred into 50 ml of the same growth medium in a 250 ml baffled Erlenmeyer flask and incubated with shaking for 24 h at 28°C.
  • TTB Trypticase Soy Broth
  • the salt concentration was adjusted by adding 850 ⁇ l 5 M NaCl solution, then the mixture was extracted two times with phenol:chloroform:isoamylaclohol (25:24:1, vol/vol) with gentle agitation followed by centrifugation for 10 min at 3500 x g. After precipitation with 1 vol of isopropanol, the genomic DNA knot was spooled on a glass rod and redissolved in 200 ⁇ l of water. This method yielded about 0.5 mg total DNA.
  • PKS Probe design Five degenerate PCR primers were designed (degKSIF 5'- TTCGAY SCSGVSTTCTTCGSAT-3' [SEQ ID NO:44]; degKS2F 5'- GCSATGGAYCCSCARCARCGSVT-3' [SEQ ID O:45]; degKS3F 5'- SSCTSGTSGCSMTSCAYCWSGC-3' [SEQ ID NO:46]; degKS5R 5'- GTSCCSGTSCCRTGSSCYTCSAC-3' [SEQ ID NO:47]; degKS7R 5'- ASRTGSGCRTTSGTSCCSSWSA-3' [SEQ ID NO:48]) based on conserved regions of ketosynthase (KS) domains of type I PKS genes and codon bias of high G+C organisms.
  • KS ketosynthase
  • the primers were used in the following combinations: degKSlF/degKS5R, degKS2F/degKS5R and degKS3F/degKS7R.
  • the PCR conditions for the amplification of KS domains were as follows: A total reaction volume of 50 ⁇ l contained 100 ng of S. bikiniensis total DNA, 200 pmol of each primer, 0.2mM dNTP, 10% DMSO and 2.5 U Taq DNA polymerase (Roche Applied Science, Indianapolis, In). Thirty-five cycles of PCR were performed using the following steps: denaturation (94°C; 40 sec); annealing (55°C; 30 sec); extension (72°C; 60 sec), 35 cycles.
  • the resulting PCR reactions were subjected to electrophoresis on 1% agarose gels and the PCR products of approximately 700 bp were extracted from the gels using the gel extraction kit from Quiagen (Valencia, CA) according to manufacturer's protocol.
  • the fragments were treated with Pfu DNA Polymerase (Stratagene, La Jolla, Ca) to remove the A overhangs and cloned into the plasmid vector pLitmus28 (New England Biolabs, Beverley, Ma) cut with EcRY.
  • Thirty-two "amplimers" (the ca. 700 bp PCR-amplified segment) for each primer pair were sequenced using standard protocols. Of the 96 inserts sequenced, 81 were found to be KS amplimers.
  • Genomic library preparation Approximately 10 ⁇ g of genomic DNA was partially digested with S ⁇ w3Al (1 hr incubation using dilutions of the enzyme) and the digested DNA was run on an agarose gel with DNA standards. One of the conditions used was found to have generated fragments of size 35-47 kb.
  • the DNA from this digestion was ligated with pSuperKos plasmid, a derivative of pSuperCos (Stratagene) digested wif Afel and self- ligated to eliminate the neo marker, pre-linearized with BamHI and b ⁇ l and the ligation mixture was packaged using a Gigapack XIII (Stragene) in vitro packaging Kit and the mixture was subsequently used for infection of Escherichia coli DH5 ⁇ employing protocols supplied by the manufacturer . Approximately 2000 of E.
  • coli transductants were probed by in-situ colony hybridization with DIG labeled Sb3/7-31 (KS q ), Sbl/5-75 (KS3) and and Sbl/5-78 (KS7). Plasmids from 15 colonies, which showed strong hybridization signals were isolated, digested with BamHI and subjected to Southern blotting employing the KS q or KS7 amplimers as probes. Ten plasmids showed strong hybridization with one or both amplimers. The ends of the insert in each of the 10 plasmids were sequenced using convergent primers for each (T7 promoter and T3 promoter).
  • Cycle steps were as follows: denaturation (94°C; 40 sec), annealing (55°C for KSq and KS3 specific primers, 65°C for KS5 specific primers; 30 sec), extension (72°C; 60 sec), 25 cycles.
  • Each primer set was found to amplify its cognate amplimer exclusively, with the exception of the primer set for KS7, which was also seen to give a small amount of amplification of non-cognate amplimers.
  • Each primer set was then used for PCR with cosmids pKOS146.185.1, pKOS146.185.10 and pKOS 146.185.11 employing the same conditions as described above but using cosmid DNA in place of the plasmid-containing amplimer DNA.
  • pKOS 146.185.1 gave correctly sized amplimers with KSq and KS3 primers but not with KS7 specific primers, whereas pKOS 146-185.10 gave a correctly sized amplimer with KS7 but not with KSq and KS3 specific primers, indicating that pKOS146.185.1 contained the 5' region of the chalcomycin PKS genes.
  • sequence of the insert of pKOS146.185.1 corresponds to bases 1 to 48,595 of SEQ ID No.l and the sequence of the insert of pKOS146.185.10 corresponds to bases 44,218 to 85,915 of SEQ ID No.1.
  • Table 2 below provides open reading frame (ORF) boundaries corresponding to the nucleotide position in SEQ ID NO:l (Table 3) of the chalcomycin PKS as well as the nucleotide sequences encoding enzymes involved in precursor synthesis and chalcomycin modification.
  • SEQ ID NO:l includes additional open reading frames of genes encoding proteins or domains thereof that may be useful in the biosynthesis of chalcomycin and/or analogs thereof in certain host cells.
  • the various open reading frames, module-coding sequences, and domain encoding sequences shown in Table 2 and the figures are sometimes referred to as "subsequences.”
  • sequences are sometimes referred to as "subsequences.”
  • Those of skill in the art will recognize, upon consideration of the sequence shown in Example 1, that the actual start locations of several of the genes could differ from the start locations shown in the table, for example due to the presence in-frame of codons utilizable by the initiator methionine tRNA in close proximity to the codon indicated as the start codon. The actual start codon can be confirmed by amino acid sequencing of the proteins expressed from the genes.
  • Genes listed in Table 2 that encode proteins with post-PKS polyketide- modifying activities include: chmPI, chmPII, chmHI (P450 homologs), chmN, chmCIII (glycosyltransferases) and chmU (polyketide ketoreductase).
  • Genes listed in Table 2 that encode proteins predicted to participate in the biosynthesis of sugar residue subunits of chalcomycin or modification of sugar residues after their addition to the polyketide include: chmCIV, chmMIII, chmCV, chmAII, chmAI, chmJ, chmMII, chmD, chmMI, and chmCII.
  • ChmCII, ChmCIV and ChmCV three are predicted to participate in D- chalcose residue biosynthesis (ChmCII, ChmCIV and ChmCV), two are predicted to participate in D-allose residue biosynthesis (ChmD and ChmJ) two are predicted to participate in conversion of the D-allose residue to D-mycinose residue after covalent linkage of the D-allose to the polyketide (ChmMI and ChmMII), two are predicted to provide precursors for both the allose and chalcose pathways (ChmAI and ChmAII), and one is predicted to O-methylate the chalcose residue (ChmMIII).
  • the invention also provides inter-polypeptide linker sequences, which can be identified by the skilled reader (e.g., comprinsing the sequences between the N- terminus of the polypeptide and the beginning of the first KS domain; or between the C- terminue of the polypeptide and the beginning of the last ACP domain) and polynucleotides encoding such linkers.
  • CAGCAGGCCG CAGATGACCG CCACGGGAAG GGGGACGGCG TAGTGGTCCA CGAGGTCGAC
  • Example 2 Construction of a "Clean" Host Strain, S. fradiae K159-1 [0102]
  • This example describes the preparation of the clean host, Streptomyces fradiae K159-1, a strain in which the tylGI, tylGII, tylGIII, tylGW, and tylGV genes have been deleted.
  • Plasmid pKOS 159-5 was first constructed as follows. Two fragments flanking the tylG genes were PCR amplified from S. fradiae genomic DNA using the following primers:
  • tylGI left flank forward 5'-TTTGCATGCGATGTTGACGATCTCCTCGTC [SEQ ID NO:_J; reverse 5'-GGAAGCTTCATATGTTCTCTCCGGAATGTG [SEQ ID NO:_J;
  • tylGVI right flank forward 5'-TTAAGCTTTCTAGAGAGGAGAGGCCGTGAAC [SEQ ID NO:_J; reverse 5'- AAAGAATTCGAACTCGAGCACGGACTCGTTG [SEQ ID NO:_J.
  • This plasmid no longer contains the t- ⁇ C31 gene and attP locus from pSET152 and therefore serves as a suicide vector for delivery by homologous recombination.
  • Spores of S. fradiae 99 ( Russia) were prepared by harvesting from strain grown on 1-2 AS-1 plates [see Wilson, V.T.W. and Cundliffe, E. (1998). Characterization and targeted disruption of a glycosyltransferase gene in the tylosin producer, Streptomyces fradiea. Gene 214: 95-100], filtering the spores through sterile cotton, and resuspending in 1 ml of 20% glycerol [see Hopwood, D.A., et al.
  • Streptomyces fradiae KI 59-1 was deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA, 20110-2209, on 12 March 2003, with accession number PTA-5060.
  • Streptomyces fradiae KI 59- 1/244- 17a is derived from strain K159-1 (Example 2) by addition of the fkbGHIJK genes from Streptomyces hygroscopicus var. ascomyceticus ATCC 14891, which encode proteins catalyzing the biosynthesis of methoxymalonyl-ACP.
  • hygroscopicus ATCC 14891 (fkbGHIJK) are arranged with the 3' end of fkbG (encoding an O-methyl transferase) overlapping by 6 codons the 3' end of flbH (encoding an unknown function), which is the last gene of a convergent operon that begins with fkbB (one of the PKS genes) and ends with the genes fkbK, J, I and H.
  • an operon was constructed beginning v ⁇ fhfkbK and ending vAOufkbG, all in the same direction.
  • Example 4 Construction of an operon containing all five chalcomycin PKS genes and expression in S. fradiae [0110]
  • a construct comprising the genes encoding chmGI-Vvtas constructed as follows: The 3' end of ChmGV was obtained by PCR with pKOS146-l 85.10 as the template and the following primers (Chalco-1 A: GACACGGCCGGTGAGAGCAGC [SEQ ID NO:_
  • the 942 bp PCR product was digested with Ncol and Xbal, the 309 bp fragment was gel isolated, and the fragment ligated into the same sites of Litmus29 to give pKOS342-33. That plasmid was cut with Ncol and Xhol and ligated to a 2.4 kb Ncol-Xhol fragment from pKOS146-185.10 to give pKOS342-35.
  • That plasmid was digested with Bglll and Xhol and ligated with a 6.4 kb Bglll-Xhol fragment (including chmGIVand the 5' region of chmGV) from pKOS146-185.10 to create pKOS342-36 (containing chmGIVand GV).
  • a 5.4 kb Hindlll/ Pstl fragment containing the 5' half of chmGIII was isolated from pKOS146-185.1 and a 6.3 kb Pstl/Bglll fragment containing the 3' half of chmGIII was isolated from pKOS146-185.10. These two pieces were ligated into Litmus28 cut with Hindlll and Bglll to obtain pKOS342-38. Plasmid pKOS342-36 was cut with Bglll and Spel, the 9 kb fragment was gel isolated and the fragment ligated to the Bglll and Spel sites of pKOS342-38 to obtain pKOS342-39.
  • Plasmid pKOS232- 172 (described in Example 5), containing chmGI and Gil was cut with Ndel and Hindlll and the 19 kb fragment was isolated. Plasmid pKOS342-39 was digested with Hindlll and Spel and the 20 kb fragment was isolated. These two fragments were then ligated into the vector portion of an expression cosmid, pKOS244-20 (gel isolated 8 kb Ndel-Spel fragment). The resulting plasmid (pKOS342-45) was recovered using in vitro ⁇ phage packaging (Stratagene) and infection of E. coli DH5 . The correct clone was identified by restriction enzyme analysis and the plasmid was moved into E. coli DH5 ⁇ /pUB307 and conjugated into S. fradiae.
  • the culture broth was analyzed for 16-membered macrolide production by HPLC (Metachem Metasil Basic column, 4.6x150 mm, 5 ⁇ m particle) using linear gradient from 15 to 100% organic phase (56% methanol, 5mM ammonium acetate) at 1 ml/min over 7 min.
  • HPLC used simultaneous detection by electrospray mass spectrometry (Turbo Ionspray source) and UV absorption at 282 nm.
  • LC-MS analysis of the broth showed that several chalconolide derivatives were produced. The most abundant compounds were purified and shown to have the structures below.
  • the 3- keto also forms the enol tautomer.
  • Chalcomycin-Spiramycin PKS [0115] Streptomyces fradiae K232-192 is derived from strain K159-l/244-17a (Example 3) by addition of hybrid chalcomycin-spiramycin PKS genes, which encode proteins catalyzing the biosynthesis of 14-methylplatenolide.
  • the chalcomycin genes were obtained from cosmid pKOS 146- 185.1, which was deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA, 20110- 2209, on 19 February 2003, with accession number PTA-4961.
  • the first two genes of the chalcomycin PKS were isolated from the cosmid pKOS146-185.1 as EcoRI/XhoI and XhoI/BspHI fragments, and a coding sequence for a spiramycin PKS C-terminal linker attached to 3' end.
  • the EcoRI site is near the 5' end of chmGl.
  • the EcoRI/XhoI fragment was cloned into a modified Litmus28 with a synthetic linker inserted in order to create an appropriate translation start sequence.
  • the altered region of the Litmus28 polylinker between the Aflll and EcoRI sites in this plasmid (pKOS232-165) is given below.
  • the plasmid with the chmG fragment was pKOS232-168A.
  • the PCR product was cut with Mlul and Avrll, gel isolated and ligated into a Litmus-based vector (pKOS232-75B) between the same sites to give pKOS231 - 118 A.
  • the 7 kb BamHI/MluI fragment from cosmid pKC 1306 was subcloned in Litmus38 (New England Biolabs) to give pKOS231-113A.
  • the 3.8 kb BamHI/MluI fragment of pKOS231-l 18A was gel isolated and ligated with the 7 kb BamHI/MluI fragment of pKOS231-l 13A to give pKOS231-120.
  • the 7 kb BsrGI/BamHI fragment from pKC1306 was subcloned in Litmus38 to give pKOS231-l 13B.
  • the 6.2 kb Pstl/BamHI fragment from pKOS231-l 13B was cloned into Litmus28 to give pKOS231-122.
  • the 7.5 kb BamHI/Avrll fragment was isolated from pKOS231-120 and ligated with pKOS231-122, which was cut with BamHI and Avrll and dephosphorylated, to give pKOS231-124.
  • the 3.1 kb BamHI/Spel fragment from pKOS231-l 18B (which contained a PCR fragment that created a 5' end for srmGS) and the 7.5 kb BamHI/ Avrll fragment from pKOS231-120 were isolated and ligated to give pKOS231-130.
  • the 14 kb BamHI fragment was isolated from ⁇ KC1306 and subcloned in Litmus28 to give pKOS231-l 1 IB.
  • the 14 kb BamHI fragment was isolated from ⁇ KOS231-11 IB and ligated to pKOS231-130 cut with BamHI and dephosphorylated, to give pKOS231-132.
  • Hindlll site was introduced at the 3' end of srmG2 using PCR with pKOS231- 112B as template.
  • the engineered Hindlll site was positioned with respect to the reading frame to match that of the natural Hindlll site in the chalcomycin chmGII gene.
  • the resulting PCR product was cut with Hindlll and BamHI (a natural site) and ligated into the same sites ofpKOS231-114A to give pKOS232-178.
  • pKOS232-l 82 This was then joined to pKOS231-132 at the BsrGI site to give pKOS232-l 82.
  • the chmGl,2 cassette was isolated from pKOS232- 172 as an 18 kb Ndel/Hindlll fragment and the srmG3,4,5 cassette was isolated from pKOS232-182 as a 20 kb Hindlll/Avrll fragment.
  • pSET152-based vector having the tylG promoter (the vector portion gel isolated from pKOS244-20) were joined in a three-piece ligation and recombinants were recovered by in vitro lambda phage packaging and infection of E. coli. Correct constructs were identified by restriction analysis (pKOS232-184A) and transferred into E. coli DH5 ⁇ /pUB307.
  • Streptomyces fradiae K232-192 was deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA, 20110-2209, on 12 March 2003, with accession number PTA-5052. Conjugation was performed as described in Practical Streptomyces Genetics (Kieser et al., 2000) except that plates were left overnight at 37°C before overlaying with the selective agent (apramycin and naladixic acid).
  • Apramycin resistant exconjugants were streaked for single colonies and a set of clones were patched onto R5 plates and inoculated into tryptic soy broth (40 ml in 250 ml shake flasks). Both the solid and liquid media contained apramycin (to select for pKOS232-184A) and kanamycin (to select for pKOS244-17A). Liquid and solid cultures were grown at 30°C. Agar plugs taken from most patches on R5 showed bioactivity when placed on an luteus test lawn. The agar was extracted with ethyl acetate and found to contain a compound of 730 amu.
  • TSB seed cultures at 2-3 days were used to inoculate fermentation media and these cultures were grown for 7-10 days at 28°C.
  • the 730 amu compound (730-1) was isolated and its structure verified by NMR as shown below.
  • LC-MS analysis of the filtered culture broth showed abundant production of a 586 amu and a 730 amu (730- II) compound, and a 714 amu compound and a smaller amount of a 904 amu compound (most likely representing 14-methyl-platenolide with all three sugars attached).
  • the chalcomycin-spiramycin hybrid PKS synthesized the predicted 14-methyl platenolide.
  • the unique Fsel site in pKOS344-022B was changed to an Xbal site with a synthetic linker and the chmH gene plus ferredoxin gene were excised withNdel and Xbal and ligated into the expression vector, pKOS342-108D, between the Ndel and Avrll sites to give pKOS344-037B.
  • This vector was transferred into DH5 ⁇ /pUB307, and conjugated into K232-192 (Example 5). Exconjugants were selected with thiostrepton and streaked for single colonies to yield S. fr ⁇ di ⁇ e K344-51.
  • the vector for integration of chmH, pKOS342-108D uses the int and ⁇ tt functions of Streptomyces phage ⁇ BTl . All S. fr ⁇ di ⁇ e strains were plated on AS1 agar for sporulation, R5 agar for solid media production, or grown in liquid TSB for vegetative growth and Russia medium for production. All appropriate antibiotics for selection of integrated markers were added to the media, except for the production stage.
  • Figure 2 shows proposed pathways for post-PKS modification of the chalcomycin-spiramycin hybrid PKS macrolide product in the absence or presence of ChmH.
  • ChmH the post-PKS reaction sequence from the Chm Srm hybrid essentially follows that for tylosin and gives the 904 amu structure, which is converted by reduction of the aldehyde to a 906 amu compound. This reduction of the aldehyde has been described for tylosin (to give relomycin).
  • Knockout of genes for allose biosynthesis or its transfer (tylJ) would give the demycinosyl compound of 730 amu (with the 14-hydroxymethyl and the aldehyde).
  • Example 7 Expression of a chmGI-GII oneron with the tylG2 C-terminal linker in S. fradiae K105-2
  • the tylD knockout plasmid was constructed from two PCR products encompassing 1.8 kb regions upstream and downstream of the tylD gene using PCR primers that introduced new restriction sites.
  • the upstream PCR product was cut with EcoRI and Pstl and the downstream product was cut with Pstl and Sphl. These were then ligated together between the EcoRI and Sphl sites of pUC19 and the sequence was verified.
  • the resulting plasmid, pKOS168-106 has about 80% of the tylD gene deleted between the artificial Pstl sites. This plasmid was introduced into S.
  • fradiae by conjugation from E. coli DH5 /pUB307 and apramycin resistant exconjugants were obtained. Three were found by PCR to be the result of homologous recombination at the expected tylD locus and these were grown in the absence of selection and screened for the second crossover. Apramycin sensitive clones were isolated and some were found that produced demycinosyltylosin (DMT) by LC-MS analysis of the fermentation broths. The strain was designated S. fradiae K168-173.
  • DMT demycinosyltylosin
  • the S. fradiae DMT (demycinosyltylosin) producer (KI 68- 173) described above was used to introduce a KS-1 null mutation in the tylosin PKS.
  • the plasmids pKOS168-190 and pKOS268-145 were digested with EcoRI and EcoRV and the 6.2kb and 2.6kb fragments, respectively, were gel isolated and ligated together to give pKOS264-65.
  • a mutation was introduced into pKOS264-65 using PCR to change the active site cysteine of the tylosin KS1 to alanine, with the simultaneous introduction of an Nhel site, to give pKOS325-8.
  • ⁇ KOS325-8 and pKOS241-52 were digested with PvuII and Xbal and ligated together to give pKOS264-76.
  • Plasmid pKOS264-76 was conjugated into the DMT producer strain S. fradiae K168-173 (Example 5) from E. coli DH5oc/pUB307 and exconjugants were selected for apramycin resistance. Clones that underwent the correct first crossover event were identified by Southern blot analysis and one of these was propagated without selection to allow a second crossover. DNA from clones that had become apramycin sensitive was digested with Xmal/Nhel and analyzed by Southern blot.
  • the expression vector pKOS342-84 was transferred to E. coli DH5 ⁇ /pUB307 and conjugated into S. fradiae K105-2 (this example, above) .
  • Apramycin resistant colonies were isolated and fermented in production medium.
  • the broth was analyzed by LC-MS and found to contain the compound shown below.
  • the chm tyl hybrids differ from the chm/srm hybrids only by having a 4-methyl in place of a 4-methoxy, apparently making the chm/tyl good substrates for the TylH hydroxylase.

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Abstract

L'invention porte sur des domaines des synthases de la chalcomycine et des polycétides et sur les enzymes de modification et sur les polynucléotides les codant. L'invention porte également sur des procédés de préparation de la chalcomycine dans des quantités utiles d'un point de vue pharmaceutique et sur des procédés de préparation des analogues de la chalcomycine et autres polycétides utilisant les polynucléotides codant les domaines des synthases de la chalcomycine et des polycétides ou les enzymes les modifiant.
PCT/US2003/026569 2002-08-21 2003-08-21 Synthase par recombinaison de la chalcomycine et des polycetides et genes les modifiant WO2004018703A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3065137A (en) * 1959-02-02 1962-11-20 Parke Davis & Co Chalcomycin and its fermentative production
US5672491A (en) * 1993-09-20 1997-09-30 The Leland Stanford Junior University Recombinant production of novel polyketides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3065137A (en) * 1959-02-02 1962-11-20 Parke Davis & Co Chalcomycin and its fermentative production
US5672491A (en) * 1993-09-20 1997-09-30 The Leland Stanford Junior University Recombinant production of novel polyketides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
REEVES ET AL.: 'Production of Hybrid 16-Membered Macrolides by Expressing Combinations of Polyketide Synthase Genes in Engineered Streptomyces fradiae Hosts' CHEMISTRY & BIOLOGY vol. 11, 2004, pages 1465 - 1472, XP004601880 *

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