Recombinant polynucleotides encoding pro-geldanamycin producing polyketide synthases and accessory proteins, and uses thereof

This application claims the benefit of U.S. provisional patent application Nos. 60/389,255 (filed Jun. 14, 2002), 60/393,929 (filed Jul. 3, 2002), 60/395,275 (filed Jul. 12, 2002), 60/415,326 (filed Sep. 30, 2002), 60/420,820 (filed Oct. 24, 2002), 60/433,130 (filed Dec. 13, 2002), and is a Continuation-in-Part of U.S. patent application Ser. No. 10/212,962 (filed Aug. 5, 2002). The entire contents of each of these applications is incorporated herein by reference.

This invention was made in part with government support under SBIR Grant no. 1 R43 CA/GM96262-01, awarded by the National Institutes of Health, Department of Health and Human Services. The United States government may have certain rights in the invention.

The appended sequence listing is part of, and incorporated into, the specification.

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 International Patent Specification WO 95/08548; U.S. Pat. Nos. 5,672,491 and 6,303,342; Fu et al., 1994 , Biochemistry, 33:9321-26; McDaniel et al., 1993, Science, 262:1546-50; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34:881-88.

Polyketides are synthesized in nature by polyketide synthases (“PKS”). These synthase enzymes are complexes of multiple enzyme activities. Two major types of PKS are known and differ in their mode of synthesis. These are commonly referred to as Type I or “modular” and Type II “iterative.” The Type I or modular PKSs, as commonly found in bacteria but not in fungi, comprise a set of separate catalytic active sites; the portion of the protein that encompasses each active site region is termed a “domain”, and a set thereof is termed a “module”. One module exists for each cycle of carbon chain elongation and modification. FIG. 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. Six separate modules, each catalyzing a round of condensation and modification of a 3-carbon unit, are present in DEBS. 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., 1992, FEBS Letters, 304:205. DEBS provides a representative example of a modular Type I PKS. In DEBS, 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, 3, or more -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”) and a methyltransferases activity. With respect to DEBS-1, the first module thereof also contains additional AT and ACP domains because that module catalyzes the initial condensation, and so begins with a “loading di 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.

In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker or “scaffold”-encoding regions. These scaffold regions encode amino acid sequences that space the enzymatic activities (domains) at the appropriate distances and assure the correct order of modules in the PKS. Thus, these linker regions collectively can be considered to encode a scaffold into which the various domains (and thus modules) are placed in a particular order and spatial arrangement. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) from other PKS by various available methodologies. Thus, there is considerable flexibility in the design of a new PKS to produce a novel polyketide. An additional level of structural complexity in the resultant polyketides may be introduced by subsequent P450 oxidation, methylation, glycosylation or other enzymes that catalyze post-PKS reactions.

Geldanamycin is a polyketide produced by a modular PKS and was the first of four benzoquinone ansamycins isolated from microorganisms (see FIG. 1 ) to have been evaluated extensively as an antitumor drug. Although originally discovered by screening microbial extracts for antibacterial and antiviral activity [DeBoer, et al. (1970), Sasaki, et al. (1970); full citations of all references cited herein by the author and year of publication are provided below], geldanamycin was later found to be cytotoxic to tumor cells in vitro [Sasaki et al. (1979)] and to reverse the morphology of cells transformed by the Rous sarcoma virus to the normal state [Uehara et al. (1986)]. Subsequent discoveries of the herbimycins [Omura et al. (1979)], macbecins [Muroi et al. (1980)] and TAN 420A [Shibata et al. (1986)] expanded this class of antitumor natural products. Ansamycins like the ansamitocins are usually included in this class of microbial products. Reblastatin, isolated from the geldanamycin producer, was recently found to have interesting biological activities [Takatsu et al. (2000), Stead et al. (2001)].

Geldanamycin's nanomolar potency and apparent specificity for aberrant protein kinase dependent tumor cells, as well as the discovery that its primary target in mammalian cells is the ubiquitous Hsp90 protein chaperone, has stimulated interest in its development as an antitumor drug [Neckers et al. (2002); Blagosklonny, 2002]. Severe hepatotoxicity [Supko et al. (1995)] led to its withdrawal from Phase I clinical trials in 1995. Nonetheless, during the 1990's considerable information was obtained about the structure-activity relationships (SAR) of geldanamycin, herbimycin and reblastatin [Neckers et al. (2002), Schnur et al. (1995)]. In late 1999, 17-allylamino-17-desmethoxygeldanamycin entered Phase I clinical trials [Egorin et al. (2001), Wilson et al. (2001), Erlichman et al. (2001)] sponsored by the National Cancer Institute in the US and the Cancer Research Campaign in the UK because this analog had exhibited good in vivo activity [Wilson, et al. (2001), Erlichman, et al. (2001)], better pharmacokinetics and lower toxicity than geldanamycin [Egorin et al. (2001)] during preclinical development. The maximum tolerated dose is 40 mg/m 2 [Wilson et al. (2001)], and micromolar serum concentrations are achieved without overt toxicity. Efficacy in inhibiting signal transduction pathways has been demonstrated in peripheral blood lymphocytes.

There is therefore a need for recombinant nucleic acids, host cells, and methods of expressing those nucleic acids in host cells to produce geldanamycin at a commercially useful scale and to make geldanamycin analogs. These and other needs are met by the materials and methods provided by the present invention.

The following articles provide additional background information relating to the invention and are incorporated herein by reference. DeBoer et al. “Geldanamycin, a new antibiotic” J Antibiot ( Tokyo ) (1970) 23:442-7. Sasaki et al. “Geldanamycin. I. Structure assignment” J Am Chem Soc (1970) 92:7591-3. Blagosklonny, 2002, “Hsp-90-associated oncoproteins: multiple targets of geldanamycin and its analogs” Leukemia 16:455-62. Sasaki et al. “Growth inhibition of virus transformed cells in vitro and antitumor activity in vivo of geldanamycin and its derivatives” J Antibiot ( Tokyo ) (1979) 32:849-51. Uehara et al. “Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus” Mol Cell Biol (1986) 6:2198-206. Omura et al. “Herbimycin, a new antibiotic produced by a strain of Streptomyces.” J Antibiot ( Tokyo ) (1979) 32:255-61. Iwai et al. “Herbimycin B, a new benzoquinonoid ansamycin with anti-TMV and herbicidal activities” J. Antibiot ( Tokyo ) (1980) 33:1114-9. Muroi et al. “Macbecins I and II, new antitumor antibiotics. II. Isolation and characterization” J Antibiot ( Tokyo ) (1980) 33:205-12. Shibata et al. “The structure and cytocidal activity of herbimycin C.” J Antibiot ( Tokyo ) (1986) 39:1630-3. Takatsu et al. “Eblastatin, a novel benzenoid ansamycin-type cell cycle inhibitor.” J. Antibiot . (2000) 53:1310-1312. Stead et al. “Discovery of novel ansamycins possessing potent inhibitory activity in a cell-based oncostatin M signalling assay. J. Antibiot ( Tokyo ) 53:657-663. Neckers, L. “Hsp90 inhibitors as novel cancer chemotherapy agents.” Tr. Molec. Med . (2002) 8:S55-S61. Supko et al. “Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent.” Cancer Chemother Pharmacol (1995) 36:305-15. Schnur, et al. “erbB-2 Oncogene inhibition by geldanamycin derivatives: synthesis, mechanism of action, and structure-activity relationships.” J. Med. Chem . (1995) 38:3813-20. Egorinet et al. “Plasma pharmacokinetics and tissue distribution of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) in CD2F1 mice.” Cancer Chemother Pharmacol (2001) 47:291-302. Wilson et al. “Phase I pharmacologic study of 17-AAG in adult patients with advanced solid tumors.” Amer Soc of Clin Oncol , (2001) Abstract 325; Erlichman et al. “A phase I trial of 17-AAG in patients with advanced cancer.” Proceedings of the AACR (2001) Abstract. Guo J, Frost J W. “Biosynthesis of 1-deoxy-1-imino-D-erythrose 4-phosphate: (2002). A defining metabolite in the aminoshikimate pathway.” (2002) J Am Chem Soc. 124, 528-9. Yu et al. (2002). “The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum.” Proc Natl Acad Sci USA. 99, 7968-73. August et al. (1998). “Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699.” Chem Biol 5, 69-79; Leistner E (1999). “Biosynthesis of ansatrienin (mycotrienin) and naphthomycin. Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tu1892.” Eur J Biochem 261, 98-107; DeBoer C, Dietz A. (1976). “The description and antibiotic production of Streptomyces hygoscopicus var. geldanus.” J Antibiot 29, 1182-8. Kunkel, T. A. Proc Natl Acad Sci USA (1985) 82:448. Geisselsoder et al. BioTechniques (1987) 5:786. Zoller and Smith, Methods in Enzymology (1983) 100:468. Dalbie-McFarland et al. Proc Natl Acad Sci USA (1982) 79:6409.

In one aspect, the invention relates to polyketide synthases (PKSs) that produce progeldanamycin, and polynucleotides encoding domains, modules and proteins of such synthases. The structure, sequences and characteristics of the geldanamycin PKS gene cluster and herbimycin PKS gene cluster are disclosed, along with other genes and proteins that participate in polyketide biosynthesis or have other functions. The geldanmycin PKS gene cluster was cloned from S. hygroscopicus var. geldanus NRRL 3602 and, in one embodiment of the invention, is encoded in SEQ ID NO:1. The herbimycin PKS gene cluster sequence was cloned from S. hygroscopicus AM-3672 and, in one embodiment of the invention, is encoded in SEQ ID NO:2.

In one aspect, the invention provides a method of producing a polyketide by culturing a cell under conditions under which the cell produces the polyketide, wherein the cell comprises a recombinant polynucleotide that hybridises under stringent conditions to the polyketide synthase-encoding region of SEQ ID NO:1 and/or SEQ ID NO:2 and encodes at least one core polyketide synthase protein, and where the cell is unable to make the polyketide in the absence of the recombinant polynucleotide. The recombinant polynucleotide can be an expression vector. In one embodiment the polyketide is pro-geldanamycin, and in related embodiments the cell produces geldanamycin or herbimycin. In one embodiment, the cell is not a Streptomyces cell. In a related aspect, the invention provides a recombinant host cell (e.g., which may be a other than a Streptomyces cell) comprising one or more expression vectors that drive expression of polyketide synthase enzymes capable of making pro-geldanamycin in the cell, where the host cell produces progeldanamycin and where the host cell does not produce progeldanamycin in the absence of the expression vector(s).

In another aspect the invention provides a recombinant DNA molecule encoding a domain of a geldanamycin polyketide synthase (PKS) or a herbimycin PKS. In one embodiment, the recombinant DNA molecule encodes one or more modules or polypeptides (open reading frames) of a chimeric PKS. The recombinant DNA molecule can encode a module of geldanamycin PKS and may comprise one or more open reading frames (ORFs) selected from gdmaAI, gdmAII and gdmAIII. In an embodiment, the recombinant DNA molecule differs from the corresponding region of native geldanamycin PKS by inactivation of at least one geldanamycin PKS domain. The recombinant DNA molecule can encode a module of a herbimycin PKS and may comprise one or more ORFs selected from hbmAI, hbmAII and hbmAIII. In an embodiment, the recombinant DNA molecule differs from the corresponding region of native herbimycin PKS by inactivation of at least one herbimycin PKS domain. In one embodiment, the recombinant DNA molecule hybridizes under stringent conditions to a nucleic acid having a nucleotide sequence of SEQ. ID NO:1 and/or SEQ. ID NO:2. In related embodiments, a recombinant DNA expression vector comprising the DNA molecule operably linked to a promoter (which can be a promoter is derived from a cell other than Streptomyces ) is provided.

In another aspects, a recombinant DNA molecule encoding a geldanamycin modification enzyme involved in the conversion of progeldanamycin to geldanamycin or a herbimycin modification enzyme involved in the conversion of proherbimycin to herbimycin is provided.

The invention also provides a host cell comprising a recombinant DNA molecule or vector described above or elsewhere herein. In one embodiment, the host cell is a S. hygroscopicus cell. In other embodiments, the host cell is not a S. hygroscopicus cell or is not an S. hygroscopicus var. geldanus NRRL 3602 cell.

The invention further provides a method of producing a polyketide by growing a host cell, as described above or elsewhere herein, under conditions where a polyketide synthesized by a PKS comprising a protein encoded by the recombinant DNA molecule is produced in the cell, optionally, recovering the synthesized polyketide, and optionally chemically modifying the polyketide and/or formulating the polyketide for administration to a mammal.

The invention further provides an isolated polypeptide encoded by a recombinant DNA molecule described above or elsewhere herein, as well as (1) a chimeric PKS that is composed of at least a portion of a geldanamycin PKS and at least a portion of a second PKS for a polyketide other than geldanamycin and (2) a chimeric PKS that is composed of at least a portion of a herbimycin PKS and at least a portion of a second PKS for a polyketide other than herbimycin. In one embodiment, the second PKS is from a narbonolide PKS, an oleandolide PKS, a DEBS PKS or a rapamycin PKS.

In another aspect, the invention provides a method of producing a polyketide comprising by recombinantly modifying a gene in the geldanamycin PKS gene cluster of a Streptomyces cell that comprises the gene cluster to produce a recombinant cell, or obtaining a progeny of the recombinant cell, and growing the recombinant cell or progeny under conditions whereby a polyketide other than geldanamycin is synthesized by the cell, optionally, recovering the synthesized polyketide and, optionally, chemically modifying the polyketide and/or formulating the polyketide for administration to a mammal. In one embodiment of this method, the cell is S. hygroscopicus var. geldanus NRRL 3602. In one embodiment the cell does not produce geldanamycin.

In another aspect, the invention provides a method of producing a polyketide by recombinantly modifying a gene in the herbimycin PKS gene cluster of a Streptomyces cell that comprises the gene cluster to produce a recombinant cell, or obtaining a progeny of the recombinant cell, and growing the recombinant cell or progeny under conditions whereby a polyketide other than herbimycin is synthesized by the cell, optionally, recovering the synthesized polyketide cell, and, optionally, chemically modifying the polyketide and/or formulating the polyketide for administration to a mammal. In one embodiment of this method, the cell is S. hygroscopicus AM-3672. In one embodiment the cell does not produce herbimycin.

In various embodiments of these methods, the modifying involves (1) substitution of a geldanamycin AT domain with an AT domain having a different specificity; (2) inactivation of a domain, wherein the domain is selected from the group consisting of a KS domain, an AT domain, an ACP domain, a KR domain, a DH domain, and an ER domain; and/or (3) substitution of KS domain, an ACP domain, a KR domain, a DH domain, or an ER domain with a domain having a different specificity.

In another aspect, the invention provides a recombinant DNA molecule comprising one or more open reading frames (ORFs) of SEQ ID NO:3 as well as a host cell comprising the DNA. In one embodiment, the ORF comprises basepairs 5263-6345; 6575-7270; 2427-3224; 1364-2413; 3397-3846; 4058-5224; and 428-1252. In a related embodiment, the invention provides a recombinant DNA expression vector comprising the above-described DNA molecule operably linked to a promoter as well as a host cell comprising the vector.

In one aspect, the present invention provides recombinant nucleic acids encoding polyketide synthases that produce geldanamycin or geldanamycin analogs in host cells.

In an embodiment of the present invention, there are provided polynucleotides that comprise a coding sequence for one or more domains of geldanamycin polyketide synthase. In another embodiment, the polynucleotide also comprises a coding sequence for one or more domains of another polyketide synthase. In another embodiment, a coding sequence for a domain (or portion thereof) of geldanamycin synthase is combined with coding sequence from another PKS to make a novel PKS that produces a polyketide. Expression of such DNAs, in suitable host cells leads to the production of synthases capable of producing useful polyketides.

Accordingly, there is provided 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 geldanamycin polyketide synthase. Preferably at least an entire domain of a module of geldanamycin synthase is included. Representative geldanamycin PKS domains useful in this aspect of the invention include, for example, KR, DH, ER, AT, ACP and KS domains. In one embodiment of the invention, the PKS is assembled from polypeptides encoded by DNA molecules that comprise coding sequences for polyketide synthase domains, wherein at least one encoded domain corresponds to a domain of geldanamycin polyketide synthase. In such DNA molecules, the coding sequences are operably linked to control sequences so that expression therefrom in host cells is effective.

In another embodiment of the present invention, there is provided a PCR based method to rapidly query the genomic DNA for the presence of type I modular PKS genes, then the number of these genes and their individual characteristics can be established by DNA sequence and bioinformatics analysis of short PKS gene amplimers. This method of the present invention is more reliable and informative than methods involving DNA hybridization, and much less costly than approaches based on whole genome sequencing. This method of the present invention was applied to identify the PKS and tailoring enzymes of the geldanamycin PKS and the genes responsible for the biosynthesis of the ansamycin and geldanamycin starter unit AHBA (3-amino-5-hydroxy benzoic acid).

Accordingly there are provided recombinant polynucleotides that comprise a coding sequence for one or more domains of the geldanamycin starter unit AHBA synthetic enzymes. Expression of such DNAs, in suitable host cells leads to the production of the AHBA starter unit.

FIG. 1 shows the structure of naturally occurring benzoquinone ansamycins, including geldanamycin and herbimycins A-C.

FIG. 2 shows the functions and products of the geldanamycin and herbimycin synthases. FIG. 2A shows the predicted functions and product of the PKS for geldanamycin biosynthesis. The schematic shows the enzyme-bound product assembled from AHBA and carbon chain extender substrates malonyl-CoA, 2-methoxymalonate and 2-methylmalonyl-CoA by the PKS modules indicated beneath the product structure. Progeldanamycin, produced by the PKS from the 3-amino-5-hydroxybenzoic acid starter unit (AHBA) is converted to geldanamycin by three oxidations, O-methylation, and O-carbamoylation. Panel A shows reduction of the unusual α-methoxy C═C during the second carbon chain extension cycle. Panel B shows creation of the 4,5 cis C═C by oxidation of the saturated system after formation of the ansamycin framework. FIG. 2B shows the predicted functions and product of the PKS effecting herbimycin biosynthesis. Proherbimycin, produced by the PKS from the AHBA starter unit, is converted to herbimycin by hydroxylation at C15, O-methylation at C15 and C11, oxidation at C21, and O-carbamoylation at C7.

FIG. 3 shows PKS gene clusters and flanking genes. FIG. 3A is a schematic of BAC clones pKOS256-154-1 (KS) and pKOS-256-107-3 (CT) encompassing the geldanamycin PKS gene cluster with flanking genes. FIG. 3B shows is a schematic of BAC clones pKOS279-78.04, pKOS279-78.17, and pKOS205-110.12, encompassing the herbimycin PKS gene cluster with flanking genes.

FIG. 4 is a schematic of the configuration of KS, CT, and LDD domains of geldanamycin PKS gene cluster and corresponding clones containing those domains.

FIG. 5 is a phylogenetic tree showing the two groups of DNA sequences encoding AHBA-B type and AHBA-N type AHBA synthases.

FIG. 6 is a schematic of pKOS-256-116-10 (“AH15”) showing the open reading frames and other structural motifs of the AHBA-B gene cluster of Streptomyces hygroscopicus NRRL3602.

FIG. 7 is a schematic of the geldanamycin PKS gene cluster showing ORFs and modifying genes. Abbreviations: ORF 16: efflux (SC3C8.01); gdmL: oxred. (rifl9); gdmF: amide synthase (riff); ORF 17: secreted protein (SC3C8.01); gdmX: unknown (homolog of JadX MmyY); gdmM: flavin-dependent monooxygenase (rifl9); ORF 18: hydrolase (SCF1.09); gdmA1: PKS modules 0-3; gdmN: carbamoyltransferase; ORF 19: transcriptional regulation (AraC family); gdmA2: PKS modules 4-5; gdmH: methoxymalonyl-ACP biosynthesis; ORF 20: transcriptional regulation (tetR); gdmA3: PKS modules 6-7; gdmI: Methoxymalonyl-ACP biosynthesis; gdmJ: Methoxymalonyl-ACP biosynthesis; gdmFdx: ferredoxin; gdmP: P450; gdmK: Methoxymalonyl-ACP biosynthesis; gdmRI: transcriptional regulation; gdmG: Methoxymalonyl-ACP biosynthesis; ORF 22: hydrolase ns; gdmRII: transcriptional regulation; gdmO: aminoDHQ synthase; (ahba3) gdmP: Regulation 450.

FIG. 8 is a schematic of the CT probe identified BAC showing overlap with KS probe hybridization sites of modules 4, 5 and 6.

FIG. 9 illustrates a recombinational cloning strategy for domain replacement in PKS genes.

Definitions and Methods

This section provides definitions of selected terms and abbreviations used in this disclosure, as well as resources useful in the practice of the invention. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

As used herein, the terms “tailoring enzyme” and “modification enzyme” are used interchangably and mean an enzyme that modifies the product of a PKS (e.g., progeldanamycin). Exemplary tailoring proteins include oxygenases, glycosyl- and methyltransferases, acyltransferases, halogenases, cyclases, aminotransferases, hydroxylases, and others known in the art.

As used herein, “core” polyketide synthase genes are genes encoding the loading and extendor modules of the PKS. The “core PKS” genes in the geldanamycin PKS cluster are gdmAI, gdmAII, and gdmAIII. The “core PKS” genes in the herbimycin PKS cluster are hbmAI, hbmAII, and hbmAIII. As used herein, a “core” polyketide synthase protein is a protein encoded by a core PKS gene. As used herein, a “polyketide synthase-encoding region” of a polynucleotide refers to the region encoding the core PKS genes.

As used herein, “polyketide synthase biosynthetic gene cluster” refers generally to section of the chromosome comprising the core PKS genes and other genes that play a role in polyketide biosynthesis.

As used herein, a PKS “accessory” protein is a protein, other than a PKS protein, that plays a role in the biosynthesis, modification, or activity of a polyketide. Exemplary accessory proteins include tailoring enzymes, enzymes involved in biosynthesis of polyketide starter units (e.g., AHBA) or extender units (e.g., malonate, 2-methymalonate and 2-methoxymalonate), CoA-ligases, and transcription regulatory proteins. In general, genes encoding accessory proteins are named “gdm —————— ” or “hdm —————— .”

As used herein, PKS “ancillary” proteins refers to proteins disclosed herein that are encoded in the S. hygroscopicus genome by genes located near the geldanamycin or herbimycin PKS gene clusters that are not accessory proteins or PKS proteins. In general, genes encoding ancillary proteins are named “ORF —————— .”

It will be appreciated that the terms “gene cluster,” “accessory protein,” and “ancillary proteins” are used for convenience and are not intended to precisely define the function of a gene or protein.

As used herein an “active fragment” of a polypeptide or domain (or a polynucleotide encoding a polypeptide) has the activity of polypeptide or domain from which it is derived, when intergrated into an appropriate PKS framework using methods known in the art.

As used herein the term “geldanamycin” sometimes refers to “progeldanamycin,” as will be clear from context.

As used herein the term “recombinant” 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. Thus, a “recombinant” polynucleotide is defined either by its method of production or its structure. In reference to its method of production, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, 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. Thus, for example, 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.

As used herein, “isolated” means that a substance is either present in a preparation at a concentration higher than that substance is found in nature or in its naturally occurring state or that the substance is present in a preparation that contains other materials with which the substance is not associated with in nature. As an example of the latter, an isolated geldanamycin PKS protein includes a geldanamycin PKS protein expressed in a Myxococus or Streptomyces lividans host cell.

“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. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g., Berger and Kimmel, 1987, Methods In Enzymology, Vol. 152: Guide To Molecular Cloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory). Typically, stringent hybridization conditions 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 60° C. for probes greater than 50 nucleotides. As noted, stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed.

The term substantially identical or homologous or similar varies with the context as understood by those skilled in the relevant art and generally means at least 70%, preferably means at least 80%, more preferably at least 90%, and most preferably at least 95% identity. To determine identity, optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, using the CLUSTAL W algorithm of Thompson et al., 1994, Nucleic Acids Res 22:467380, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis. The BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215:403-10) for which software may be obtained through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) can also be used. When using any of the aforementioned algorithms, the default parameters for “Window” length gap penalty, etc., are used.

As used herein, “substantially identical” to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.

As used herein, “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. Thus, 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 following abbreviations are used in this disclosure: 1) ACP, acyl carrier protein; 2) Aden, adenylation; 3) AT, acyltransferase; 4) DH, dehydratase; 5) ER, enoylreductase; 6) KR, ketoreductase; 7) KS, ketosynthase; 8) LDD, loading didomain; 9) NRPS, non-ribosomal peptide synthetase; 10) m, malonylCoA; 11) mm, 2-methylmalonylCoA; 12) moxm, 2-methoxymalonyl-ACP; 13) mod, module; 14) CT carbamoyltransferase; 15) PKS, polyketide synthase; 16) AHBA, 3-amino-5-hydroxy benzoic acid. The following convention is used to refer to domains in a PKS: the number following an abbreviation for a PKS domain refers to the module from which that domain originated. For example, “AT2” refers to the AT domain of module 2. When referring to plasmids, “periods” and “hyphens” are sometimes used interchangably (e.g., pKOS205-110-12 and pKOS205-110.12 are the same).

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2001); Molecular Cloning: A Laboratory Manual , third edition (Sambrook and Russel, 2001); PCR: The Polymerase Chain Reaction , (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1999, including supplements through 2001). Methods for the genetic manipulation of Streptomyces are described in Kieser et al, 2000, “Practical Streptomyces Genetics,” The John Innes Foundation, Norwich.

Geldanamycin and herbimycin are structurally related polyketides produced by Streptomyces hygroscopicus . Geldanamycin was originally identified as a product of S. hygroscopicus var. geldanus NRRL 3602, and herbimycin was first identified in S. hygroscopicus AM-3672. FIG. 2A and FIGURE B show the predicted synthetic pathways for geldanamycin and herbimycin. The geldanamycin polyketide synthase (in S. hygroscopicus var. geldanus NRRL 3602) and the herbimycin polyketide synthase (in S. hygroscopicus AM-3672) produce identical polyketide products, referred to as progeldanamycin or proherbimycin. As a result of post-PKS processing, herbimycin differs from geldanamycin by having a methoxy group at the C15 position instead of at the C17 position, and may also have a hydroxyl or methoxy group at the C11 position.

Given the valuable pharmaceutical properties of geldanamycin and other ansamycins, means to produce pharmaceutically useful quantities of this and related polyketides are useful. The genes encoding the geldanamycin and herbimycin polyketide synthases, as well as genes encoding tailoring enzymes, biosynthetic proteins, regulatory proteins, and other polypeptides have now been cloned, sequenced, and characterized. This information, along with the disclosure below, provides new methods for expressing PKS enzymes and polyketide modification enzymes derived in whole and in part from the geldanamycin and herbimycin gene clusters in recombinant host cells, resulting in the biosynthesis of progeldanamycin, geldanamycin, herbimycins, derivatives and analogs of progeldanamycin, geldanamycin and herbimycin, and other polyketides in host cells. Various aspects of the invention are described in detail in the following sections.

The geldanamycin and herbimycin PKS gene clusters are similar at both the sequence and organizational levels (see FIG. 3A and FIG. 3B ). TABLE 1, below, summarizes the organization of the gene clusters.

The geldanamycin PKS contains seven modules and produces progeldanamycin. As noted above, this ansamycin is formed from the starter unit 3-amino-5-hydroxybenzoic acid (AHBA) and three different α-carboxy acid chain extender units: malonate, 2-methymalonate and 2-methoxymalonate. (AHBA is formed by the products of AHBA-biosynthetic genes of the geldanamycin gene cluster, which are discussed below.) Module 1 of the geldanamycin PKS contains the loading didomain, which is homologous to the corresponding portion of the rifamycin and ansamitocin PKSs, and consists of a domain for activation of AHBA via formation of its thioester and an ACP domain for subsequent attachment of the activated starter unit to the PKS. This module also contains the six domains required for selection of the first chain extender substrate, 2-methylmalonyl-CoA, and its loading onto the ACP1 domain, followed by condensation of the starter and extender unit catalyzed by the KS1 domain, then reduction, dehydration and double bond reduction catalyzed by the KR1, DH1 and ER1 domains acting in sequo. The domain organization and functions of the six other modules in the geldanamycin PKS are listed in FIG. 2A . The AT2 and AT5 domains (of modules 2 and 5) recognize and load 2-methoxymalonate, a comparatively rare substrate in polyketide synthesis whose formation is governed by five genes in the geldanamycin gene cluster that are homologous to the corresponding FK520 and ansamitocin genes (see WO 00/20601). Modules 3, 4 and 7 utilize 2-methylmalonate, and module 6 uses methylmalonate. These six modules also contain KR, DH and/or ER domains that establish the functionality at positions 2, 4, 7, 9, 11 and 13 in the product of the PKS.

Modification of progeldanamycin to produce geldanamycin involves at least four and perhaps five or six enzymatic reactions: C7 carbamoylation; C17 hydroxylation; C17 O-methylation, C21 oxidation, and perhaps either C4,5 desaturation (oxidation) or C4 or C5 hydroxylation plus dehydration. C7 carbamoylation is believed to be carried out by the gdmN gene product. The oxidation steps, including the hydroxylation, are believed to involve the activities of a subset of the gene products of gdmL, gdmM, gdmP (with gdmFdx) and ORF4P450.

As noted above, the organization of the herbimycin PKS is similar to that of the geldanamycin gene cluster. The herbimycin PKS contains seven modules and produces progeldanamycin from an AHBA starter unit and malonate, 2-methymalonate and 2-methoxymalonate extender units. Module 1 of the herbimycin PKS contains the loading didomain, and consists of a domain for activation of AHBA via formation of its thioester and an ACP domain for subsequent attachment of the activated starter unit to the PKS. This module also contains the six domains required for selection of the first chain extender substrate, 2-methylmalonyl-CoA, and its loading onto the ACP1 domain, followed by condensation of the starter and extender unit catalyzed by the KS1 domain, then reduction, dehydration and double bond reduction catalyzed by the KR1, DH1 and ER1 domains acting in sequo. The domain organization and functions of the six other modules in the herbimycin PKS are shown in FIG. 2B . The AT2 and AT5 domains (of modules 2 and 5) recognize and load 2-methoxymalonate, a comparatively rare substrate in polyketide synthesis whose formation is governed by five non-PKS genes in the herbimycin gene cluster that are homologous to the corresponding FK520 and ansamitocin genes. Modules 3, 4 and 7 utilize 2-methylmalonate, and module 6 uses malonate. These six modules also contain KR, DH and/or ER domains that establish the functionality at positions 2, 4, 7, 9, 11 and 13 in the product of the PKS.

Modification of progeldanamycin to produce herbimycin involves five enzymatic reactions: C7 carbamoylation, C11 O-methylation, C15 hydroxylation, C15 O-methylation, C21 oxidation, and perhaps either C4,5 desaturation (oxidation) or C4 or C5 hydroxylation plus dehydration. C7 carbamoylation is believed to be carried out by the hbmN gene product. C11 and/or C15 O-methylation may involve the hbmG gene product. The oxidation steps, including the hydroxylation, are believed to involve the activities of a subset of the gene products of hbmL, hbmM, hbmP (with hbmFdx) and ORF4P450.

The reader skilled in the art of molecular biology and polyketide biosynthesis will understand, guided by this disclosure, that the polynucleotide sequences and other teachings of the specification and figures make possible a wide variety of applications. These applications include, but are not limited to, applications in which core PKS genes, accessory genes, and ancillary genes are modified and/or expressed using recombinant methods.

The present invention provides, for example, (1) recombinant polynucleotides that comprise sequences encoding a PKS protein, module, domain or fragment thereof, and/or encode an accessory protein or fragment thereof; (2) recombinant polypeptides comprising the sequence of a PKS protein, module, domain or fragment thereof or comprising the sequence of an accessory protein or fragment thereof; and (3) cells comprising a recombinant polynucleotide or polypeptide of the invention. The following sections describe these and other aspects of the invention. However, it will be understood that the embodiments discussed below are for illustration, and are not intended to limit the invention.

In one aspect, the invention provides recombinant polynucleotides that encode a PKS protein, module, domain or fragment thereof, and/or encode an accessory protein or fragment thereof. The polynucleotides of the invention are useful for expression of recombinant proteins (e.g., chimeric PKS proteins), as tools for manipulation of PKS and accessory genes (e.g., vectors for homologous recombination for mutation or deletion of PKS and accessory genes), as probes and primers, and a variety of other uses. It is contemplated that a polynucleotide of the invention can be in any of a variety of forms, depending on its intended function: e.g., integrated into a host cell genome (whether episomal or chromosomal), encoded by a recombinant vector (such as an expression vector), as a linear oligomer (such as a probe or primer), and other forms. In one aspect, the polynucleotide compounds of the invention are used in recombinant procedures for production of desired portions of the geldanamycin or herbimycin synthases. Optionally these portions are fused to, or expressed in conjunction with, all or a portion of a heterologous PKS protein(s), or are modified to change activity. Optionally, recombinant geldanamycin or herbimycin PKS protein, or a chimeric PKS of the invention, is co-expressed with one or more polyketide modification enzymes that modify the polyketide product of the geldanamycin or herbimycin or a chimeric PKS.

In one embodiment, the invention is directed to recombinant materials comprising nucleic acids with nucleotide sequences encoding at least one domain, at least one module, or least one polypeptide encoded by a geldanamycin or herbimycin PKS gene. In one embodiment, purified and isolated DNA molecules are provided that comprise one or more coding sequences for one or more domains or modules of geldanamycin synthase or herbimycin synthase. In one embodiment of the invention, the DNA compounds of the invention comprise a coding sequence for at least two, at least three, at least four, or more, of the domains of the loading module and extender modules 1 through 7, inclusive, of the geldanamycin/herbimycin PKS, or at least one, at least two, or three of the modules of the geldanamycin/herbimycin PKS gene. Examples of such encoded domains include geldanamycin synthase KR, DH, ER, AT, ACP, and KS domains and herbimycin synthase KR, DH, ER, AT, ACP, and KS domains. Examples of such modules include the PKS modules of the geldanamycin PKS and the PKS modules of the herbimycin PKS.

In one embodiment, the invention provides an isolated nucleic acid fragment which hybridizes to a nucleic acid having a nucleotide sequence set forth in the SEQ. ID NO:1, SEQ. ID NO:2, or SEQ. ID NO:3 under stringent conditions. In an embodiment, the nucleic acid fragment comprises, consists or consists essentially of a nucleic acid having a nucleotide sequence set forth in SEQ. ID NO:1, SEQ. ID NO:2, or SEQ. ID NO:3. Encoding sequences for geldanamycin and herbimycin polyketide synthase proteins and accessory proteins may comprise substitutions, additions or deletions relative to SEQ. ID NO:1, SEQ. ID NO:2, or SEQ. ID NO:3 that provide for functionally equivalent molecules. For example, the invention provides, due to the degeneracy of the genetic code, a large number of DNA sequences that encode the amino acid sequences of the domains, modules, and proteins of the geldanamycin and herbimycin PKSs as well as the accessory enzymes. The PKS and accessory genes include those with nucleotide sequences encoding substantially the same amino acid sequences as found in native PKS and accessory genes biosynthetic enzyme proteins, and those encoding amino acid sequences with functionally equivalent amino acids, as well as PKS and accessory genes biosynthetic enzyme derivatives or analogs as described herein. These include but are not limited to nucleotide sequences comprising all or portions of SEQ ID NO:1, 2 or 3 genes that are altered by the substitution of different codons that encode the amino acid residue within the sequence, thus producing a silent change, or in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In specific embodiments, the biosynthetic nucleic acids encoding PKS and accessory proteins comprise the sequence of SEQ. ID NO:1, SEQ. ID NO:2, or SEQ. ID NO:3, or the coding regions thereof, or nucleotide sequences encoding, in whole or in part, a PKS and accessory genes biosynthetic enzyme protein. The isolated nucleic acids typically consists of at least 25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of PKS and accessory genes biosynthetic nucleic acid sequence, or a full-length PKS and accessory genes biosynthetic coding sequence. In another embodiment, the nucleic acids are smaller than 35, 200, or 500 nucleotides in length. Nucleic acids can be single or double stranded. Nucleic acids that hybridize to or are complementary to the foregoing sequences, in particular the inverse complement to nucleic acids that hybridize to the foregoing sequences (i.e., the inverse complement of a nucleic acid strand has the complementary sequence running in reverse orientation to the strand so that the inverse complement would hybridize without mismatches to the nucleic acid strand) are also provided. In specific aspects, nucleic acids are provided which comprise a sequence complementary to (specifically are the inverse complement of) at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a PKS and accessory genes biosynthetic gene.

In one important aspect, the invention provides a modified and/or chimeric (also called “hybrid”) polyketide synthases. A “modified” PKS is a PKS in which a domain or module has been deleted (including deletion by replacement with a different domain) or mutated to change or eliminate the enzymatic activity of the domain (e.g., inactivation of the domain). Further, reference herein to an “inactivated” domain is intended to encompass a domain that does not function in a PKS because it is partially or completely deleted. As will be apparent to the reader, “modifying” polynucleotides or proteins, as used herein, refers to recombinantly modifying said polynucleotides or proteins, in contrast to, for example, random changes induced by radiation, chemical mutagens, or the like.

A “chimeric” PKS is a PKS protein (or encoding gene) that expresses modules, domains, or portions of domains from two different PKS proteins (either as a fusion protein or by coexpression). Recombinant methods for manipulating modular PKS genes to make hybrid PKS enzymes are described in U.S. Pat. Nos. 5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in WO 98/49315 and WO 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. 1:319-329, and Baltz, 1998, Trends Microbiol. 6:76-83).

It will be appreciated that a PKS that is chimeric is also modified and, moreover, that these characterizations are used for convenience and not limitation.

In constructing novel PKS proteins, a number of general principles are known, some of which are summarized here. There are at least six degrees of freedom for constructing a polyketide synthase in terms of the polyketide that will be produced. First, the polyketide chain length will be determined by the number of modules in the PKS. Second, 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, ethyl malonyl, etc. Third, the loading domain specificity will also have an effect on the resulting carbon skeleton of the polyketide. Thus, the loading domain may use a different starter unit, such as acetyl, propionyl, ahba, and the like. Fourth, 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. Fifth, 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. Also, the specificity of the ketoreductase will determine the chirality of the corresponding hydroxyl group. Also, the enoyl reductase specificity for substituted malonyls as extender units will influence the result when there is a complete KR/DH/ER available. Sixth, the presence and position of PKS methyl transferase domain(s) in PKS module(s) will determine the presence of methyl functions in the polyketide.

Recombinant methods for manipulating modular PKS genes to make chimeric PKS enzymes are described in U.S. Pat. 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. 1:319-329, and Baltz, 1998, Trends Microbiol. 6:76-83). In general, these techniques include: (i) deletion or insertion of modules to control chain length, (ii) inactivation of reduction/dehydration domains to bypass beta-carbon processing steps, (iii) substitution of AT domains to alter starter and extender units, (iv) addition of reduction/dehydration domains to introduce catalytic activities, and (v) substitution of ketoreductase KR domains to control hydroxyl stereochemistry. In addition, engineered blocked mutants of DEBS have been used for precursor directed biosynthesis of analogs that incorporate synthetically derived starter units and it is contemplated that the analogous biosynthesis is carried out by a geldanamycin/herbimycin based PKS.

Thus, further aspects of the invention include: (1) encoding DNA for a chimeric PKS that is substantially patterned on a non-geldanamycin producing enzyme, but which includes one or more functional domains or modules of geldanamycin PKS; (2) encoding DNA for a chimeric PKS that is substantially patterned on the geldanamycin PKS, but which includes one or more functional domains or modules of another PKS or NRPS; (3) encoding DNA for a modified PKS that is substantially patterned on a geldanamycin producing enzyme, but in which one or more domains or modules has been deleted or inactivated; (4) methods for making geldanamycin analogs and derivatives; (5) encoding DNA for a chimeric PKS that is substantially patterned on a non-herbimycin producing enzyme, but which includes one or more functional domains or modules of herbimycin PKS; (6) encoding DNA for a chimeric PKS that is substantially patterned on the herbimycin PKS, but which includes one or more functional domains or modules of another PKS or NRPS; (7) encoding DNA for a modified PKS that is substantially patterned on a herbimycin producing enzyme, but in which one or more domains or modules has been deleted or inactivated; and (8) methods for making herbimycin analogs and derivatives.

With respect to items (1) and (5) above, preferred examples include chimeric PKS enzymes wherein the genes for the erythromycin PKS or rapamycin PKS function as accepting genes, and one or more of the above-identified coding sequences for geldanamycin or herbimycin PKS domains or modules are inserted as replacements for domains or modules of comparable function. With respect for (2) and (6) above, a number of other PKS coding sequences that can be used to prepare chimeric domains or molecules are known which are can be used in conjunction with geldanamycin and/or herbimycin PKS encoding sequences to construct a chimeric molecule. A partial list, for illustration and not limitation, includes Avermectin (U.S. Pat. No. 5,252,474; MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256; MacNeil et al., 1992, Gene 115: 119-25); Candicidin (FRO008) (Hu et al., 1994, Mol. Microbiol. 14: 163-72); Epothilone (U.S. Pat. No. 6,303,342); Erythromycin (WO 93/13663; U.S. Pat. No. 5,824,513; Donadio et al., 1991, Science 252:675-79; Cortes et al., 1990, Nature 348:176-8); FK-506 (Motamedi et al., 1998, Eur. J. Biochem. 256:528-34; Motamedi et al., 1997, Eur. J. Biochem. 244:74-80); FK-520 (U.S. Pat. No. 6,503,737; see also Nielsen et al., 1991, Biochem. 30:5789-96); Lovastatin (U.S. Pat. No. 5,744,350); 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 (EP Pat. App. 791,656); Rapamycin (Schwecke et al., 1995, Proc. Natl. Acad. Sci. USA 92:7839-43); Aparicio et al., 1996, Gene 169:9-16); Rifamycin (August et al., 1998, Chemistry & Biology, 5: 69-79); Soraphen (U.S. Pat. No. 5,716,849; Schupp et al., 1995, J. Bacteriology 177: 3673-79); Spiramycin (U.S. Pat. No. 5,098,837); Tylosin (EP 0 791,655; Kuhstoss et al., 1996, Gene 183:231-36; U.S. Pat. No. 5,876,991). Additional suitable PKS coding sequences remain to be discovered and characterized, but will be available to those of skill (e.g., by reference to GenBank).

In a related embodiment, a domain in a PKS gene is replaced with a domain or domains from a different location (e.g., different module) from same PKS gene. In another embodiment, portions of more than two or more than three PKS genes are combined to produce a chimeric gene and protein.

As noted, construction of such enzymes is most effectively achieved by construction of appropriate encoding polynucleotides. In this example of the invention, it is not necessary to replace an entire domain or module accepting of the PKS with an entire domain or module of geldanamycin PKS, rather peptide subsequences of a PKS domain or module that correspond to a peptide subsequence in an accepting domain or module, or which otherwise provide useful function, may be used as replacements. Accordingly, appropriate encoding DNAs for construction of such chimeric PKS include those that encode at least 5, 10, 15, 20 or more amino acids of a selected geldanamycin domain or module. Those of skill in the art will recognize that all or part of a PKS sequence in a chimeric PKS of the invention need not be isolated from a naturally occurring source. For example, only a small portion of an AT domain determines its specificity. See WO US99/15047, and Lau et al., Biochemistry 38:1643-51. The state of the art in DNA synthesis allows the artisan to construct de novo DNA compounds of size sufficient to construct a useful portion of a PKS module or domain. Thus, the desired derivative coding sequences can be synthesized using standard solid phase synthesis methods such as those described by Jaye et al., 1984, J. Biol. Chem. 259: 6331, and instruments for automated synthesis are available commercially from, for example, Applied Biosystems, Inc. For purposes of the invention, such synthetic DNA compounds are deemed to be a portion of a PKS.

In addition to providing mutated forms of regions encoding enzymatic activity, regions encoding corresponding activities from different PKS or from different locations in the same PKS can be recovered, for example, using PCR techniques with appropriate primers. By “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/ER could correspond to KR alone.

If replacement of a particular target region in a host polyketide synthase is to be made, this replacement can be conducted in vitro using suitable restriction enzymes or can be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene. One such system involving plasmids of differing temperature sensitivities is described in WO 96/40968.

A particularly useful method for modifying a PKS gene (e.g., making domain substitutions or “swaps”) is a RED/ET cloning procedure developed for constructing domain swaps or modifications in an expression plasmid without first introducing restriction sites. The method is related to ET cloning methods (see, Datansko & Wanner, 2000, Proc. Natl. Acad. Sci. U.S.A. 97, 664045; Muyrers et al, 2000, Genetic Engineering 22:77-98) and is described in Example 8, infra. The RED/ET 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.

As noted supra, mutations can be introduced into PKS genes such that polypeptides with altered activity are encoded. Polypeptides with “altered activity” include those in which 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 any number of 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 (see, e.g., Kunkel, 1985, Proc Natl Acad Sci USA 82:448; Geisselsoder et al., 1987, BioTechniques 5:786). Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA 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, 1983, Methods in Enzymology 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., 1982, Proc Natl Acad Sci USA 79:6409). PCR mutagenesis can also be used for effecting the desired mutations.

It will be apparent that, as described above, a large number of other chimeric and/or modified PKSs can be made. Examples of chimeric polyketide synthases are provided in Examples 6 and 7, below. Example 6 shows substitution of the geldanamycin AT7domain with the AT2 domain of the rapamycin PKS, and Example 7 shows substitution of geldanamycin AT5 domain with the rapamycin AT2 domain. For illustration (and not limitation) several additional examples are provided in the paragraphs A-H, below.

A. Substitution of the Geldanamycin PKS AT1 Domain with an AT Domain Specific for Malonyl-CoA

One illustrative recombinant host cell provided by the present invention expresses a recombinant geldanamycin PKS in which the acyltransferase domain in module 1 of the geldanamycin PKS gene is replaced with an AT domain specific for malonyl-CoA instead of 2-methylmalonyl-CoA. The domain substitution is created by introducing a malonyl-CoA specific acyltransferase domain from a heterologous PKS gene, for example from the rapamycin, tylosin, or FK520 PKS genes or the like, into the geldanamycin PKS locus by homologous recombination into a geldanamycin-producing strain, aided by a selectable antibiotic resistance gene, then isolating the recombinants resulting from double crossover events in which the wild-type acyltransferase domain is replaced with one specific for malonyl-CoA. The AT domain of module 1 is encoded by nucleotides 27864 through 28908, approximately, of SEQ ID-NO:1. This sequence information together with the methods described in U.S. Pat. Nos. 6,399,789; 6,403,775; and 5,962,290 allows one skilled in the art to construct recombination vectors that result in replacement of the native AT domain of module 1 with an AT domain having a specificity for malonyl-CoA. Suitable examples of AT domains with specificity for malonyl-CoA may be found in the rapamycin PKS genes (modules 2, 5, 8, 9, 11, 12, and 14), as described in U.S. Pat. No. 6,399,789, as well as the tylosin PKS genes (modules 3 and 7) as described in U.S. Pat. No. 5,876,991; the spiramycin genes (modules 1-3 and 7), as described in U.S. Pat. No. 5,945,320; the FK520 genes (modules 3 and 10), as described in WO 00/20601; the pikromycin genes (module 2) as described in WO 99/61599; the narbomycin genes (module 2), as described in U.S. Pat. No. 6,303,767; the avermectin genes (module 2), and others. Fermentation of a host cell comprising the resulting hybrid PKS together with the remaining geldanamycin biosynthetic genes under conditions wherein the native strain produces geldanamycin, provides novel compounds.

B. Mutagenesis of Geldanamycin AT1

One illustrative recombinant host cell provided by the present invention expresses a recombinant geldanamycin PKS in which the AT1 domain of the geldanamycin PKS gene is mutagenized by site-directed mutagenesis to alter the substrate specificity of the AT domain. The AT1 domain can be mutagenized by art-known methods, such as methods described in Reeves et al., “Alteration of the substrate specificity of a modular polyketide synthase acyltranserase domain through site-directed mutagenesis,” Biochemistry 2001, 40: 15464-70, and in WO 03/014312. The amino acid sequence Tyr-Ala-Ser-His (SEQ ID NO:81), encoded by nucleotide sequence TAC-GCC-TCC-CAC (SEQ ID NO:82) at positions 56052 to 56063 in SEQ ID NO:1, is mutagenized using methods known to one skilled in the art to generate the mutant amino acid sequence His-Ala-Phe-His (SEQ ID NO:83), for example by mutagensis of the nucleotide sequence to CAC-GCC-TTC-CAC (SEQ ID NO:84) as described in the Reeves et al. reference cited above. Fermentation of a host cell comprising the resulting mutagenized PKS together with the remaining geldanamycin biosynthetic genes under conditions wherein the native strain produces geldanamycin, followed by extraction of the broth and purification provides novel compounds.

C. Substitution of KR Domain for the Reduction Cassette of Geldanamycin PKS Module 6 (DH6+KR6)

One illustrative recombinant host cell provided by the present invention expresses a recombinant geldanamycin PKS in which the coding sequence for the reduction cassette of module 6, which has both DH and KR domains, is replaced with a coding sequence for a reduction cassette that has only a KR domain.

The coding sequence for the reduction cassette of module 6, which has both DH and KR domains, is replaced with a coding sequence for a reduction cassette that has only a KR domain. The reduction cassette is contained in the sequence between the end of the AT domain, at approximately nucleotide position 56663 of SEQ ID NO:1, and the beginning of the ACP domain, at approximately nucleotide position 59886 of SEQ ID NO:1. This sequence information together with the methods described in U.S. Pat. Nos. 6,399,789; 6,403,775; and 5,962,290 allows one skilled in the art to construct recombination vectors that result in replacement of the native reduction cassette of module 6 with a cassette encoding only a KR domain. Suitable examples of cassettes encoding only a KR domain may be found in the erythromycin and rapamycin PKS genes, as described in U.S. Pat. No. 6,399,789. Fermentation of a host cell comprising the resulting hybrid PKS together with the remaining geldanamycin biosynthetic genes under conditions wherein the native strain produces geldanamycin, followed by extraction of the broth and purification provides 4,5-dihydro-5-hydroxy-geldanamycin.

D. Inactivation of DH6

One illustrative recombinant host cell provided by the present invention expresses a recombinant geldanamycin PKS in which the dehydratase domain in module 6 is inactivated by site-specific mutation.

Inactivation of the dehydratase domain in module 6 of the geldanamycin PKS gene by site-specific mutation of the wild-type domain results in production of 4,5-dihydro-5-hydroxygeldanamycin. The DH domain of module 6 is encoded by nucleotides 56663 to 59886, approximately, of SEQ ID NO:1. Two particular sequences may be targeted for mutational inactivation of the DH domain. In one embodiment, the DNA sequence encoding the DH peptide motif His-Val-Ile-Ser-Gly-Ala-Val-Leu-Val-Pro (SEQ ID NO:7), nucleotides 56814 to 56843 of SEQ ID NO:1, is mutated so as to produce a peptide having an amino acid other than histidine at the first position. The CAC codon encoding histidine is mutated, for example to CAA or CAG to encode a glutamine. Fermentation of a host cell comprising the resulting mutagenized PKS together with the remaining geldanamycin biosynthetic genes under conditions wherein the native strain produces geldanamycin, followed by extraction of the broth and purification, provides 4,5-dihydro-5-hydroxy-geldanamycin.

E. Deletion of DH6+ER6

One illustrative recombinant host cell provided by the present invention expresses a recombinant geldanamycin PKS in which a substantial portion of the nucleotide sequence between the end of the AT6 and KR6 domain is deleted.

A portion of the nucleotide sequence in module 6 between the end of the AT domain (approximately nucleotide 56663 of SEQ ID NO:1) and the start of the KR domain (approximately nucleotide 57128 of SEQ ID NO:1) is deleted, resulting in deletion of the dehydratase and enol-reductase domains. This leaves a linker region between the AT and KR domains of approximately 465 amino acids.

F. Reductive Domain Swap

One illustrative recombinant host cell provided by the present invention expresses a recombinant geldanamycin PKS in which the dehydratase domain of module 1 is replaced or inactivated by site-specific mutation.

The reduction cassette in module 1 is encoded by the sequence between the end of the AT domain, at approximately nucleotide position 28908 of SEQ ID NO:1, and the beginning of the ACP domain, at approximately nucleotide position 32133 of SEQ ID NO:1. This sequence information together with the methods described in U.S. Pat. Nos. 6,399,789; 6,403,775; and 5,962,290 allows one skilled in the art to construct recombination vectors that result in replacement of the native reduction cassette of module 1 with a cassette encoding only a KR domain. Suitable examples of cassettes encoding only a KR domain may be found in the erythromycin and rapamycin PKS genes, as described in U.S. Pat. No. 6,399,789. Fermentation of a host cell comprising the resulting hybrid PKS together with the remaining geldanamycin biosynthetic genes under conditions wherein the native strain produces geldanamycin, followed by extraction of the broth and purification, provides 15-hydroxy-geldanamycin.

G. Inactivation of DH1

One illustrative recombinant host cell provided by the present invention expresses a recombinant geldanamycin PKS in which the dehydratase domain of module 1 is inactivated by site-specific mutation of the wild-type domain.

Inactivation of the dehydratase domain in module 1 of the geldanamycin PKS gene by site-specific mutation of the wild-type domain results in production of 15-hydroxygeldanamycin. The DH domain of module 1 is encoded by nucleotides 28908 to 30378 approximately, of SEQ ID NO:1. Two particular sequences may be targeted for mutational inactivation of the DH domain. In one embodiment, the DNA sequence encoding the DH peptide motif His-Ala-Val-Ser-Gly-Thr-Val-Leu-Leu-Pro (SEQ ID NO:9), nucleotides 29088 through 29059 of SEQ ID NO:1, is mutated so as to produce a peptide having an amino acid other than histidine at the first position. The CAC codon encoding histidine is mutated, for example to CAA or CAG to encode a glutamine. Fermentation of a host cell comprising the resulting mutagenized PKS together with the remaining geldanamycin biosynthetic genes under conditions wherein the native strain produces geldanamycin, followed by extraction of the broth and purification provides 15-hydroxy-geldanamycin.

H. Inactivation of KS Domain

One illustrative recombinant host cell provided by the present invention expresses a recombinant geldanamycin PKS in which the module 1 KS domain is inactivated by deletion or other mutation. In one version, the inactivation results from a change in the KS domain that renders it incapable of binding substrate (called a KS1 o mutation). This inactivation can be accomplished by a mutation in the codon for the active site cysteine that changes the codon to another codon, such as an alanine codon. Preferably the modified KS domain is in translational reading frame with extender modules 1 and 2 of the PKS. The host cells expressing a PKS comprising the protein encoded thereby can be fed or supplied with N-acylcysteamine thioesters of precursor molecules to prepare a polyketide of interest. See U.S. patent application Ser. No. 09/492,773 (published as U.S. Pat. No. 6,492,562) and WO 00/44717.

Examples of compounds that can be produced using geldanamycin-based chimeric PKSs, for illustration and not limitation, are described in WO 03/013430 (“Benzoquinone Ansamycins,” published Feb. 20, 2003).

In an aspect, the invention provides chimeric and/or modified polyketide synthases based on the geldanamycin or herbimycin PKSs or containing a portion (e.g., domain) of geldanamycin or herbimycin PKS. Regardless of the naturally occurring PKS gene used as an acceptor, the invention provides libraries of polyketides by generating modifications in, or using a portion of, the geldanamycin or herbimycin PKS so that the protein complexes produced by the cluster 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. By providing a large number of different genes or gene clusters derived from a naturally occurring PKS gene cluster, each of which has been modified in a different way from the native cluster, an effectively combinatorial library of polyketides can be produced as a result of the multiple variations in these activities.

As used herein, a polyketide synthase “derived from” a naturally occurring PKS contains the scaffolding encoded by all the portion of the naturally occurring synthase gene used, 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. Particularly preferred embodiments include those wherein a KS, AT, KR, DH, NRPS, 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. Also preferred are derivatives where at least one noncondensation cycle enzymatic activity (KR, DH, or ER) has been deleted or wherein any of these activities has been mutated so as to change the ultimate polyketide synthesized.

In one aspect, the invention provides libraries of recombinant cells producing polyketides wherein the polyketides are synthesized by a PKS derived from naturally occurring PKSs. Generally, many members of these polyketide libraries may themselves be novel compounds, and the invention further includes novel polyketide members of these libraries.

Expression vectors containing nucleotide sequences encoding a variety of PKS systems for the production of different polyketides can be introduced by transformation into the appropriate host cells to construct a polyketide library. In one approach, 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 synthase and ultimately a particular polyketide. Typically, there will be duplications in some of the colonies; the subset of the transformed colonies that contains a different PKS in each member colony can be considered the library. Alternatively, 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 some kind of chemical or biological 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. Of course, 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. Typically, such screening methods involve detecting the binding of each member of the library to a receptor or other target molecule or complex of molecules. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art. Alternatively, individual polyketide members of the library can be tested against a desired target. In this event, screens wherein the biological response of the target is measured can be included.

In one version, libraries of polyketides are produced by cloning PKS genes as a set of three or more mutually selectable plasmids, each carrying a different wild-type or mutant PKS gene, then introducing all possible combinations of the plasmids with wild-type, mutant, and hybrid PKS coding sequences into the same host (see WO 00/63361 and WO 98/27203).

In aspects of the invention, accessory genes and proteins disclosed herein are used for production of novel polyketides (e.g., by post-PKS tailoring of polyketides), more efficient production of known polyketides (e.g., increased and/or heterologous biosynthesis of a desired polyketide), increased and/or heterologous biosynthesis of PKS substrates (such as AHBA, malonyl-CoA, 2-methoxymalonate and 2-methylmalonyl-CoA), regulation of protein biosynthesis (e.g., transcriptional regulation of genes encoding PKS and accessory proteins, increased and/or heterologous transport of polyketides), drug resistance (e.g., resistance to geldanamycin and/or herbimycin), and other uses. These and other results are accomplished by heterologous expression of one or more accessory proteins and/or inactivation of one or more accessory proteins and/or modification of one or more accessory proteins. Similarly, genes denoted as ancillary genes encode useful proteins and can be expressed and/or modified in a host cell, used for targeting, and the like.

One useful set of accessory proteins are the AHBA biosynthetic proteins described in EXAMPLE 4, infra. The genes encoding these proteins can be expressed alone or in combination with AHBA biosynthetic genes from other sources (see, e.g., Yu et al., 2002 Proc Natl Acad Sci USA. 99:7968-73; August et al., 1998 Chem Biol 5: 69-79; and Kim et al., 1998 , J. Biol. Chem. 273:6030-40) to produce AHBA in a heterologous cell. Alternatively, one or more genes in the AHBA synthetic pathway can be inactivated by recombinant means. Such inactivation can be employed, for example, to facilitate production of polyketides modified or that use starter units other than AHBA, such as modified AHBA derivatives or diketides, including polyketides expressed by modified or chimeric PKSs.

Sequences of the geldanamycin (or herbimycin) gene cluster or mutated versions of the geldanamycin gene cluster prepared according to the methods of the invention can be expressed in the native geldanamycin (or herbimycin) producer or in heterologous systems. 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. Pat. Nos. 5,843,718 and 5,830,750; WO 01/31035, WO 01/27306, and WO 02/068613; and U.S. patent application Ser. Nos. 10/087,451; 60/355,211; and 60/396,513.

Particularly preferred host cells for purposes of the present invention are Streptomyces, Myxococcus , and Saccharopolyspora host cells. Preferred hosts include fungal systems such as yeast, and procaryotic hosts; mammalian cells could also be used. As disclosed in U.S. Pat. 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. These hosts can be modified with the appropriate recombinant enzymes to effect these modifications. Suitable host cells include Streptomyces spp., E. coli , yeast, and other procaryotic hosts which use control sequences compatible with Streptomyces spp.

Similarly, host cells can be selected, or engineered, for expression of polyketide biosynthetic activities, such as glycosylatation apparatus (discussed below), amide synthases, (see, for example, U.S. provisional patent application 60/396,513 “Metabolic Pathways For Starter Units in Polyketide Biosynthesis in E. coli ”). In one embodiment herbimycin PKS genes are co-expressed with a heterologous amide synthase, such as the synthase encoded by gdmF. In a related embodiment, gdmF is expressed in the herbimycin producer S. hygroscopicus AM-3672.

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. If the cloning vectors employed to obtain PKS genes encoding a derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This need not be done individually, but a pool of isolated encoding nucleotide sequences can be inserted into host vectors, the resulting vectors transformed or transfected into host cells and the resulting cells plated out into individual colonies.

Preferred host cells for purposes of selecting vector components for expression vectors of the present invention include fungal host cells such as yeast and procaryotic host cells such as E. coli and Streptomyces , but mammalian host cells can also be used. Suitable control sequences include those which function in eucaryotic and procaryotic host cells.

Suitable 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. Examples are act promoters, tcm promoters, spiramycin promoters, and the like. However, other bacterial promoters, such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, are also useful. Additional examples include promoters derived from biosynthetic enzymes such as for tryptophan (trp), the β-lactamase (bla) bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433) can be used.

As noted, 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 actI/actIII promoter pair and the actII-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. Pat. Nos. 5,672,491; 5,830,750, 5,843,718; and 6,177,262.

Other 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 marker genes are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes which confer antibiotic resistance or sensitivity to the plasmid. Alternatively, 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, and electroporation.

When such DNA molecules are introduced into a host cell and the host cell is cultured under conditions that lead to the expression of the geldanamycin (or herbimycin), or chimeric PKS proteins, geldanamycin (or herbimycin) and/or its analogs or derivatives may be produced. In one embodiment, the expression control sequences are those normally associated with a module of the S. hygroscopicus geldanamycin or herbimycin polyketide synthase gene cluster.

The native, chimeric or modified PKS genes can be expressed in a cell that also expresses other proteins involved in polyketide biosynthesis or modification. These other proteins can be endogenous proteins (normally expressed in the host cell), heterologous recombinant proteins (encoded by a sequence not normally expressed in the host cell), or combinations of both.

In hosts such as yeasts, plants, or mammalian cells that ordinarily do not produce polyketides, it may be necessary to provide, also typically by recombinant means, suitable holo-ACP synthases to convert the recombinantly produced PKS to functionality. Provision of such enzymes is described, for example, in WO 97/13845 and WO 98/27203.

For example and not limitation, 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). Methods for glycosylating polyketides are generally known in the art and can be applied in accordance with the methods of the present invention; the glycosylation may be effected intracellularly by providing the appropriate glycosylation enzymes or may be effected in vitro using chemical synthetic means as described herein and in WO 98/49315, incorporated herein by reference. Glycosylation with desosamine, mycarose, and/or megosamine is effected in accordance with the methods of the invention in recombinant host cells provided by the invention. Alternatively and as noted, glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosylases. In addition, synthetic chemical methods may be employed.

Alternatively, 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).

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, 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).

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. For example, 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). Illustrative examples of suitable ester groups include but are not limited to formates, acetates, propionates, butyrates, succinates, and ethylsuccinates.

The polyketide product can be modified 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.

Similarly, improvements in water solubility of a polyketide compound can be achieved by addition of groups containing solubilizing functionalities to the compound or by removal of hydrophobic groups from the compound, so as to decrease the lipophilicity of the compound. Typical groups containing solubilizing functionalities include, but are not limited to: 2-(dimethylaminoethyl)amino, piperidinyl, N-alkylpiperidinyl, hexahydropyranyl, furfuryl, tetrahydrofurfuryl, pyrrolidinyl, N-alkylpyrrolidinyl, piperazinylamino, N-alkylpiperazinyl, morpholinyl, N-alkylaziridinylmethyl, (1-azabicyclo[1.3.0]hex-1-yl)ethyl, 2-(N-methylpyrrolidin-2-yl)ethyl, 2-(4-imidazolyl)ethyl, 2-(1-methyl-4-imidazolyl)ethyl, 2-(1-methyl-5-imidazolyl)ethyl, 2-(4-pyridyl)ethyl, and 3-(4-morpholino)-1-propyl. In the case of geldanamycin analogs, 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 p 185 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. 38, 3813-3820; Schnur et al., “Ansamycin Derivatives as Antioncogene and Anticancer Agents,” U.S. Pat. No. 5,932,655; all of which are incorporated herein by reference). Typical amines containing solubilizing functionalities include 2-(dimethylamino)-ethylamine, 4-aminopiperidine, 4-amino-1-methylpiperidine, 4-aminohexahydropyran, furfurylamine, tetrahydrofurfurylamine, 3-(aminomethyl)-tetrahydrofuran, 2-(amino-methyl)pyrrolidine, 2-(aminomethyl)-1-methylpyrrolidine, 1-methylpiperazine, morpholine, 1-methyl-2(aminomethyl)aziridine, 1-(2-aminoethyl)-1-azabicyclo-[1.3.0]hexane, 1-(2-aminoethyl)piperazine, 4-(2-aminoethyl)morpholine, 1-(2-amino-ethyl)pyrrolidine, 2-(2-aminoethyl)pyridine, 2-fluoroethylamine, 2,2-difluoroethylamine, and the like.

In addition to post synthesis chemical or biosynthetic modifications, various 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

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.

Prior to use the PKS product (whether modified or not) can be formulated for storage, stability or administration. For example, 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. Where the compounds carry one or more acidic moieties, 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.

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. The term “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. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) and Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe ed. (American Pharmaceutical Assoc. 2000), disclose various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

The composition 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). In an embodiment, for illustration and not limitation, 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. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.

In one aspect, the invention provides recombinant DNA molecules. In some embodiments, the invention provides a recombinant DNA molecule that contains an open reading frame of a polyketide synthase that includes an encoding sequence for a polyketide synthase domain, where the encoding sequence is SEQ ID NO:1-3, (or no:1′ or 1″) or a fraction thereof that encodes at least 10 consecutive amino acids of the polyketide synthase. In some embodiments, the invention provides a recombinant DNA molecule that comprises an encoding sequence for a geldanamycin synthase domain. In some of theses embodiments, the sequence is SEQ ID NO:1-3 (or no:1′ or 1″). In some embodiments, the invention provides a recombinant DNA molecule encoding a geldanamycin PKS domain of at least 10 amino acids, where the DNA molecule includes a sequence contained in a sequence of SEQ ID NO:1-3 (or no:1′ or 1″).

In another aspect, the invention provides a recombinant expression system capable of producing a polyketide synthase domain in a host cell, where the system includes an encoding sequence for a geldanamycin polyketide synthase domain that is operably linked to control sequences effective in the host cell to produce RNA that is translated into the polyketide synthase domain. In a further aspect, the invention provides a host cell modified to contain the recombinant expression system.

In another aspect, the invention provides vectors. In some embodiments, the invention provides a vector containing geldanamycin PKS genes, where the vector is pKOS-256-144-1, pKOS-256-144-2, pKOS-256-144-3, pKOS-256-144-4, pKOS-256-154-1, pKOS-256-154-2, pKOS-256-154-3, pKOS-256-154-4, pKOS-256-154-5, pKOS-256-154-6, pKOS-256-154-7, pKOS-256-163-1, pKOS-256-163-2, pKOS-256-163-3, pKOS-256-107-1, pKOS-256-107-2, pKOS256-107-3, pKOS-256-107-4, pKOS-256-107-5, pKOS-256-107-6, or pKOS-256-107-7. In some embodiments, the invention provides a vector that includes an open reading frame of SEQ ID NO:1 (or no: 1′ or 1″). In some embodiments, the invention provides a vector that comprises an open reading frame of SEQ ID NO:3. In some embodiments, the invention provides a pKOS256-116-10 vector.

In yet another aspect, the invention provides an isolated and purified nucleic acid encoding a geldanamycin PKS domain, where the sequence of the nucleic acid is one of the following sequences: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In another aspect, the invention provides an isolated and purified geldanamycin LDD domain comprising the amino acid sequence of SEQ ID NO:12.

In another aspect, the invention provides a recombinant DNA molecule that comprises an open reading frame of a polyketide synthase, where the open reading frame includes an encoding sequence for a polyketide synthase domain, where the encoding sequence contains a sequence that is the sequence one of SEQ ID NO:1-3 and 22-38 (or no:1′ or 1″), or a fraction thereof that encodes at least 10 consecutive amino acids of the polyketide synthase.

In another aspect, the invention provides recombinant DNA molecules. In some embodiments, the invention provides a recombinant DNA molecule that contains an encoding sequence for a herbimycin synthase domain. In some embodiments, the recombinant DNA molecule of contains a sequence of SEQ ID NO:1-3 and 22-38 (or no:1′ or 1″). In some embodiments, the invention provides a recombinant DNA molecule encoding a herbimycin PKS domain that includes at least 10 amino acids, where the DNA molecule contains a sequence contained in a sequence from the following group of sequences: SEQ ID NO:1-3 and 22-38 (or no:1′ or 1″).

In a further aspect, the invention provides a recombinant expression system capable of producing a polyketide synthase domain in a host cell, where the system contains an encoding sequence for a herbimycin polyketide synthase domain, and where the encoding sequence is operably linked to control sequences effective in the host cell to produce RNA that is translated into the polyketide synthase domain. In another aspect, the invention provides a host cell modified to contain this recombinant expression system.

In a yet further aspect, the invention provides vectors. In some embodiments, the invention provides a vector containing herbimycin PKS gene. In some embodiments, the invention provides a vector that contains an open reading frame of a herbimycin PKS clone, where the sequence of the open reading frame is one of SEQ ID NO:1-3 and 22-38 (or no:1′ or 1″). In yet other embodiments, the invention provides a vector that contains an open reading frame of a herbimycin PKS cluster.

In another aspect, the invention provides an isolated and purified nucleic acid encoding a herbimycin PKS domain, where the sequence of the nucleic acid is from one of SEQ ID NO:1-3 and 22-38 (or no:1′ or 1″).

In a further aspect, the invention provides an isolated and purified herbimycin domain expressed from an isolated and purified nucleic acid whose sequence is from one of SEQ ID NO:1-3 and 22-38 (or no:1′ or 1″).

In one aspect, the invention provides recombinant DNA molecules, and vectors comprising those recombinant DNA molecules, that encode all or a portion of the geldanamycin PKS and that, when transformed into a host cell which is then cultured under conditions that lead to the expression of said geldanamycin PKS proteins, results in the production of geldanamycin and/or analogs or derivatives thereof in useful quantities. The present invention also provides recombinant host cells comprising those recombinant vectors. In a related aspect, the invention provides recombinant DNA molecules, and vectors comprising those recombinant DNA molecules, that encode all or a portion of the herbimycin PKS and that, when transformed into a host cell which is then cultured under conditions that lead to the expression of said herbimycin PKS proteins, results in the production of geldanamycin and/or analogs or derivatives thereof in useful quantities. The present invention also provides recombinant host cells comprising those recombinant vectors.

The invention provides the coding sequences for the proteins of the geldanamycin synthase complex, and DNA molecules in which the complete set of appropriately arranged geldanamycin PKS-encoding sequences are operably linked to expression control sequences that are effective in suitable host cells of the invention to produce geldanamycin and/or its analogs or derivatives. The invention similarly provides the coding sequences for the proteins of the herbimycin synthase complex, and DNA molecules in which the complete set of appropriately arranged herbimycin PKS-encoding sequences are operably linked to expression control sequences that are effective in suitable host cells of the invention to produce a herbimycin and/or its analogs or derivatives.

In a further aspect, the invention provides a method of identifying and isolating a PKS gene cluster from the genome of a host cell by: constructing a genomic library of the host cell into a vector, where the vector is a plasmid, cosmid, bacterial artificial chromosome, or yeast artificial chromosome; designing degenerate PCR primers homologous to a conserved region of the PKS gene cluster; performing PCR amplification using these degenerate primers and the host cell genome as template; cloning and sequencing the resulting PCR amplimers; comparing nucleotide sequences of the PCR amplimers to known homologs of the PKS gene cluster to create a sequence homology phylogenetic tree; analyzing the sequence homology data to deduce a most likely candidate PKS gene cluster; probing the host cell genomic library with the PCR amplimer as a probe; isolating genomic library clones that hybridize with the amplimer probe; and sequencing these positive genomic library clones.