WO2005118797A2 - Biosynthetic gene cluster for tautomycetin - Google Patents

Abstract

Domains of tautomycetin polyketide synthase and modification enzymes and polynucleotides encoding them are provided. Methods to prepare tautomycetin in pharmaceutically useful quantities are described, as are methods to prepare tautomycetin analogs and other polyketides using the polynucleotides encoding tautomycetin synthase domains or modifying enzymes.

Description

BIOSYNTHETIC GENE CLUSTER FOR TAUTOMYCETIN

FIELD OF THE INVENTION

[1 ] This invention relates to nucleic acid encoding polypeptides capable of synthesizing compounds having immunosuppressive activity, methods for their preparation, and methods for their use. DESCRIPTION OF RELATED ART [2] Polyketides are complex natural products that are produced by microorganisms such as fungi and mycelial bacteria. There are about 10,000 known polyketides, from which numerous pharmaceutical products in many therapeutic areas have been derived, including: adriamycin, epothilone, erythromycin, mevacor, rapamycin, tacrolimus, tetracycline, rapamycin, and many others. However, polyketides are made in very small amounts in microorganisms and are difficult to make or modify chemically. For this and other reasons, biosynthetic methods are preferred for production of therapeutically active polyketides. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; WO 97/02358; and WO 98/27203; U.S. Patent Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146 and 6,410,301; Fu et al. (1994), Biochemistry 33:9321- 26; McDaniel et al. (1993) Science 262: 1546-1550; Kao et al. (1994) Science, 265:509- 12, and Rohr (1995) Angew. Chem. Int. Ed. Engl. 34: 881-88, each of which is incoφorated herein by reference.

[3] Biosynthesis of polyketides may be accomplished by heterologous expression of Type I or modular polyketide synthase enzymes (PKSs). Type I PKSs are large multifunctional protein complexes, the protein components of which are encoded by multiple open reading frames (ORF) of PKS gene clusters. Each ORF of a Type I PKS gene cluster can encode one, two, or more modules of ketosynthase activity. Each module activates and incorporates a two-carbon (ketide) unit into the polyketide backbone. Each module also contains multiple ketide-modifying enzymatic activities, or domains. In classical Type I PKSs, the number and order of modules, and the types of ketide-modifying domains within each module, determine the structure ofthe resulting product. Recently, variants of Type I PKSs have been found in which single modules may be used in an iterative fashion to add more than one two-carbon unit to the growing polyketide chain (see, for example, Mϋller (2004) Chem. Biol. 1 l(l):4-6). Polyketide synthesis may also involve the activity of nonribosomal peptide synthetases (NRPSs) to catalyze incorporation of an amino acid-derived building block into the polyketide, as well as post-synthesis modification, or tailoring enzymes. The modification enzymes modify the polyketide by oxidation or reduction, addition of carbohydrate groups or methyl groups, or other modifications.

[4] In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker regions. These regions collectively can be considered to define boundaries ofthe various domains. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) from other PKSs by various available methodologies. Using this method, new polyketide synthases (which produce novel polyketides) can be produced. It will be recognized from the foregoing that genetic manipulation of PKS genes and heterologous expression of PKSs can be used for the efficient production of known polyketides, and for production of novel polyketides structurally related to, but distinct from, known polyketides (see references above, and Hutchinson (1998) Curr. Opin. Microbiol. 1 :319-29; Carreras and Santi (1998) Curr. Opin. Biotech. 9:403-11; and U.S. Patent Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). [5] One valuable class of polyketides includes tautomycetin and analogs (Figure 1), first isolated from the actinomycete Streptomyces griseochromogenus. Tautomycetin comprises a 2-(l-hydroxy-2-carboxyethyl)-3-methylmaleic anhydride moiety and is related in structure to tautomycin, isolated from Streptomyces spiroverticillatus . Tautomycetin has been found to specifically inhibit serine/threonine protein phosphatase type 1, or "PP1" (Mitsuhashi et al. 2001, "Tautomycetin is a novel and specific inhibitor of serine/threonine protein phosphatase type 1, PP1," Biochem. Biophys. Res. Comm. 287:328-331, incorporated herein by reference). Tautomycetin further shows immunosuppressive effects (Shim et al. 2002, "Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction," Proc. Natl. Acad. Sci. USA, 99:10617-10622, incoφorated herein by reference). [6] The biosynthesis of tautomycetin and tautomycin have been studied (Ubukata et al. 1995, "Biosynthesis ofthe dialkylmaleic anhydride-containing antibiotics, tautomycin and tautomycetin," J. Chem. Soc, Perkin Trans. I, 2399-2404, incoφorated herein by reference). As shown in Figure 2, the right half of tautomycetin is a polyketide derived from condensation of groups derived from acetate, propionate, and butyrate. The terminal carbon appears to derive from acetate, suggesting a decarboxylation to introduce the terminal alkene unit. The dialkylmaleic anhydride unit appears to derive from condensation of a propionate unit with alpha-ketoglutarate. The biosynthesis of tautomycin appears similar, as shown.

[7] Given the promise of tautomycetin in the treatment of disease, there exists an unmet need for a production system that can provide large quantities of tautomycetin or provide analogs thereof. The present invention meets this need by providing biosynthetic genes responsible for the production of tautomycetin and providing for their engineering and expression in heterologous hosts. SUMMARY OF THE INVENTION [8] The present invention provides recombinant nucleic acids encoding polyketide synthases and polyketide modification enzymes. The recombinant nucleic acids ofthe invention are useful in the production of polyketides, including but not limited to ambruticins and ambruticin analogs and derivatives in recombinant host cells. The biosynthesis of tautomycetin is performed by a modular PKS together with polyketide modification enzymes. The tautomycetin synthase is made up of several proteins, each having one or more modules. The modules have domains with specific synthetic functions.

[9] The present invention also provides domains and modules ofthe tautomycetin PKS and corresponding nucleic acid sequences encoding them and/or parts thereof. Such compounds are useful in the production of hybrid PKS enzymes and the recombinant genes that encode them.

[10] The present invention also provides modifying genes ofthe tautomycetin biosynthetic gene cluster, including but not limited to isolated and recombinant forms and forms incoφorated into a vector or the chromosomal DNA of a host cell. [11] The present invention also provides recombinant host cells that contain the nucleic acids ofthe invention. In one embodiment, the host cell provided by the invention is a Streptomyces host cell that produces a tautomycetin modification enzyme and/or a domain, module, or protein ofthe tautomycetin PKS. Methods for the genetic manipulation of Streptomyces are described in Kieser et al, "Practical Streptomyces Genetics," The John Innes Foundation, Norwich (2000), which is incoφorated herein by reference in its entirety. In other embodiments, the host cells provided by the invention are eubacterial cells such as Escherichia coli, yeast cells such as Saccharomyces cerevisiae, or myxobacterial cells such as Myxococcus xanthus.

[12] 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 ofthe tautomycetin polyketide synthase. Preferably at least an entire domain of a module ofthe tautomycetin synthase is included. Representative tautomycetin PKS domains useful in this aspect of the invention include, for example, KR, DH, ER, AT, ACP and KS domains. In one embodiment ofthe invention, the PKS is assembled from polypeptides encoded by DNA molecules that comprise coding sequences for PKS domains, wherein at least one encoded domain corresponds to a domain of tautomycetin PKS. In such DNA molecules, the coding sequences are operably linked to control sequences so that expression therefrom in host cells is effective. In this manner, tautomycetin PKS coding sequences or modules and/or domains can be made to encode PKS to biosynthesize compounds having antibiotic or other useful bioactivity other than tautomycetin.

[13] These and other aspects ofthe present invention are described in more detail in the Detailed Description ofthe Invention, below. BRIEF DESCRIPTION OF THE DRAWINGS [14] Figure 1 shows the structure of tautomycetin and tautomycin. [15] Figure 2 shows the biosynthetic origins of tautomycetin, as deduced by Ubukata et al, 1995.

[16] Figure 3 shows the organization of the portions ofthe tautomycetin biosynthetic cluster as deduced from SEQ ED NO 1 comprises contains the tautoycetin biosynthetic gene cluster. DETAILED DESCRIPTION OF THE INVENTION [17] The present invention provides recombinant materials for the production of polyketides. In an aspect, the invention provides recombinant nucleic acids encoding at least one domain of a polyketide synthase required for tautomycetin biosynthesis. Methods and host cells for using these genes to produce a polyketide in recombinant host cells are also provided.

[18] The nucleotide sequences encoding tautomycetin PKS domains, modules and polypeptides ofthe present invention were isolated from Streptomyces griseochromogenus as described in Example 1. Given the valuable properties of tautomycetin and its derivatives and analogs, means to produce useful quantities of these molecules in a highly pure form is of great potential value. The compounds produced may be used as antitumor agents or for other therapeutic uses, and/or intermediates for further enzymatic or chemical modification. The nucleotide sequences ofthe tautomycetin biosynthetic gene cluster encoding domains, modules and polypeptides of tautomycetin synthase, and modifying enzymes, and other polypeptides can be used, for example, to make both known and novel polyketides.

[19] In one aspect ofthe invention, purified and isolated DNA molecules are provided that comprise one or more coding sequences for one or more domains or modules of tautomycetin synthase. Examples of such encoded domains include tautomycetin synthase β-ketoreductase (KR), dehydratase (DH), enoylreductase (ER), acyltransferase (AT), acyl carrier protein (ACP), and β-ketoacylsynthase (KS) domains. Domains will herein be referred to according to the module in which they are found as "domain(module)"; for example, the module 1 AT domain will be referred to as "AT(1)." In one aspect, the invention provides DNA molecules in which sequences encoding one or more polypeptides of tautomycetin synthase are operably linked to expression control sequences that are effective in suitable host cells to produce tautomycetin, its analogs or derivatives, or novel polyketides.

[20] The sequence of fragments ofthe tautomycetin gene cluster were assembled from sequences deduced from cosmids 01C11, 04C5, 03F6, and 12H9, and are shown as SEQ ID NO:l (see Figures 3). SEQ ED NO:l was found to encode the complete sequences of TauA (modules 0-5), TauB (modules 6-9), TauC, TauD, TauE (ccr), TauF, and TauG (TEII).

[21] The TauA gene encodes modules 0-5 ofthe tautomycetin PKS. Module 0, or the loading module, comprises the KS^Q(0) domain, the AT(0) domain specific for malonyl- CoA, and the ACP(0) domain, and serves to prime the tautomycetic PKS with a propionyl starter unit. Module 1, the first extender module, comprises KS(1), AT(1) specific for methylmalonyl-CoA, DH(1) which is apparently inactive, KR(1), and ACP(l). Module 2 comprises KS(2), AT(2) specific for malonyl-CoA, KR(2), and ACP(2). Module 3 comprises KS(3), AT(3) specific for methylmalonyl-CoA, DH(3), an apparently inactive KR(3), and ACP(3). Module 4 comprises KS(4), AT(4) specific for malonyl-CoA, DH(4), KR(4), and ACP(4). Module 5 comprises KS(5), AT(5) having specificity for methylmalonyl-CoA, DH(5), ER(5), KR(5), and ACP(5). [22] The TauB gene encodes modules 6-9 ofthe tautomycetic PKS. Module 6 comprises KS(6), AT(6) specific for methylmalonyl-CoA, DH(6), ER(6), KR(6), and ACP(6). Module 7 comprises KS(7), AT(7) specific for malonyl-CoA, DH(7), ER(7), KR(7), and ACP(7). Module 8 comprises KS(8), AT(8) specific for ethylmalonyl-CoA, DH(8), KR(8), and ACP(8). Module 9 covered by SEQ ED NO:l comprises KS(9), AT(9) specific for malonyl-CoA, KR(9), and ACP (9). [23] The TauC gene encodes a flavoproein decarboxylase (Ubix-type).

[24] The TauD gene encodes a non-oxidative decarboxylase.

[25] The TauE gene encodes crotonyl-CoA reductase (ccr), and enzyme involved in the formation ofthe ethylmalonyl-CoA extender unit used by module 8.

[26] The TauF gene encodes a transcriptional regulator (LuxR family).

[27] The TauG gene encodes a type-II thioesterase ("TEH").

[28] The amino acid sequences encoded by ORFl, ORF3, ORF4, ORF5, TauA, TauB,

TauC, TauD, TauE, TauF, TauG, and ORF6 are depicted in SEQ ED NOs:2-13, respectively.

[29] Table 1 provides a description ofthe genes, modules, and domains/activities ofthe tautomycetin biosynthetic proteins.

[30] Table 1. Genes, modules, and domains/activities ofthe tautomycetin PKS determined from the nucleotide sequence given in SEQ D NO:l.

Gene Module Domain/i ^.ctivity boundaries

ORFl 5279 - • 5836

ORF3 citrate lyase beta suburitits 6999 - • 8090

ORF4 dehydratase 9013 - ^■ 10164

ORF5 carboxylesterases 10183 - 11580

TauA 11664 - 40448 Module 0 11871 - 14708 KS^q(0) 11871 - 13133 AT(0) 13425 - 14423 ACP(O) 14451 - 14708 Module 1 14748 - 19724 KS(1) 14748 - 15998 AT(1) 16356 - 17366 DH ( 1 ) x 17400 - 18036 KR(1) 18669 - 19454 ACP ( 1 ) 19467 - 19724 Module 2 19770 - 24212 KS(2) 19770 - 21047 AT (2) 21375 - 22373 KR(2) 23142 - 23837 ACP (2) 23955 - 24212 Module 3 24270 - 29099 KS(3) 24270 - 25550 AT (3) 25854 - 26855 DH(3) 26889 - 27470 ACP (3) 28842 - 29099 Module 4 29169 - 34190 KS(4) 29169 - 30434 AT (4) 30732 - 31730 DH(4) 31764 - 32342 KR(4) 33141 - 33899 ACP (4) 33933 - 34190 Module 5 34248 - 40301 KS(5) 34248 - 35525 AT (5) 35826 - 36836 DH(5) 36870 _ 37454 ER(5) 38322 - 39197 KR(5) 39234 - 40004 ACP (5) 40044 - 40301

TauB 40468 - 63204 Module 6 40573 - 46608 KS(6) 40573 - 41847 AT (6) 42208 - 43218 DH(6) 43252 - 43842 ER(6) 44656 - 45519 KR(6) 45544 - 46311 ACP ( 6 ) 46351 - 46608 Module 7 46666 - 52644 KS(7) 46666 - 47937 AT (7) 48223 - 49221 DH(7) 49255 - 49842 ER(7) 50725 - 51591 KR(7) 51616 - 52380 ACP ( 7 ) 52387 - 52644 Module 8 52699 - 57771 KS(8) 52699 - 53976 AT (8) 54271 - 55311 DH(8) 55345 - 55911 KR(8) 56716 - 57474 ACP ( 8 ) 57514 - 57771 Module 9 57829 - 62286 KS(9) 57829 - 59103 AT (9) 59401 - 60399 KR(9) 61216 - 61980 ACP (9) 62029 - 62286 TE 62296 - 63201

TauC decarboxylase 63210 - 63803

TauD decarboxylase 63835 - 65292

TauE crotonyl CoA reiductase 65344 - 66654

ORF6 transferases/dehydratase 67602 - 69014)

TauF transcriptional regulator 69136 - 72177

TauG thioesterase 72336 - 73115

[31 ] As suggested by Ubukata et al. , 1995, incoφorated herein by reference, tautomycetin is apparently formed by production of a typical modular polyketide chain which is processed by elimination ofthe terminal carboxylate and 3-OH groups to form a terminal alkene unit, and esterification ofthe 19-OH with a dialkylmaleic anhydride unit derived from alpha-ketoglutarate and propionate.

[32] In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one domain, alternatively at least one module, alternatively at least one polypeptide, involved in the biosynthesis of an tautomycetin.

[33] In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to SEQ ED NO: 1 or its complement. Hereinafter, each reference to a nucleic acid sequence is also intended to refer to and include the complementary sequence, unless otherwise stated or apparent from context. In an embodiment the subsequence comprises a sequence encoding a complete tautomycetin PKS domain, module or polypeptide.

[34] In one aspect, the present invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes an open reading frame, module or domain having an amino acid sequence identical or substantially similar to an ORF, module or domain encoded by SEQ ID NO: 1. Generally, a polypeptide, module or domain having a sequence substantially similar to a reference sequence has substantially the same activity as the reference protein, module or domain (e.g., when integrated into an appropriate PKS framework using methods known in the art). In certain embodiments, one or more activities of a substantially similar polypeptide, module or domain are modified or inactivated as described below.

[35] In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain encoded by SEQ ED NO:l, e.g., a polypeptide, module or domain involved in the biosynthesis of an tautomycetin, wherein said nucleotide sequence comprises at least 10, 20, 25, 30, 35, 40, 45, or 50 contiguous base pairs identical to a sequence of SEQ ED NO: 1. In one aspect, the invention provides an isolated or recombinant DNA molecule comprising a nucleotide sequence that encodes at least one polypeptide, module or domain encoded by SEQ ED NO: 1, e.g., a polypeptide, module or domain involved in the biosynthesis of a tautomycetin, wherein said polypeptide, module or domain comprises at least 10, 15, 20, 30, or 40 contiguous residues of a corresponding polypeptide, module or domain comprising a sequence of SEQ ID NO: 1.

[36] It will be understood that SEQ TD NO: 1 was determined using the inserts of cosmids 01C11, 04C5, 03F6, and 12H9. Accordingly, the invention provides an isolated or recombinant DNA molecule comprising a sequence identical or substantially similar to a sequence ofthe inserts of cosmids 01C11, 04C5, 03F6, and 12H9. While the nucleic acids ofthe invention may be obtained as described below from genomic DNA of any organism producing tautomycetin, modem methods of DNA and gene synthesis may also be used to generate the nucleic acids ofthe invention, as described for example in Santi et al., PCT publication WO2004/029220, incoφorated herein by reference in its entirety. [37] In another aspect, the invention provides the proteins encoded by the genes of SEQ ID NO:l in both purified and recombinant form, as well as proteins having amino acid sequences substantial identity to the proteins encoded by the genes of SEQ ID NO:l. The amino acid sequences of these proteins are provided in SEQ ID NOs:2-13. [38] Those of skill will recognize that, due to the degeneracy ofthe genetic code, a large number of DNA sequences encode the amino acid sequences ofthe domains, modules, and proteins ofthe tautomycetin PKS, the enzymes involved in tautomycetin modification and other polypeptides encoded by the genes ofthe tautomycetin biosynthetic gene cluster. The present invention contemplates all such DNAs. For example, it may be advantageous to optimize sequence to account for the codon preference of a host organism, as described for example in Santi et al., PCT publication WO2004/029220, incoφorated herein by reference in its entirety. The invention also contemplates naturally occurring genes encoding the tautomycetin PKS that are polymoφhic or other variants.

[39] As used herein, the terms "substantial identity," "substantial sequence identity," or "substantial similarity" in the context of nucleic acids, refers to a measure of sequence similarity between two polynucleotides. Substantial sequence identity can be determined by hybridization under stringent conditions, by direct comparison, or other means. For example, two polynucleotides can be identified as having substantial sequence identity if they are capable of specifically hybridizing to each other under stringent hybridization conditions. Other degrees of sequence identity (e.g., less than "substantial") can be characterized by hybridization under different conditions of stringency. "Stringent hybridization conditions" refers to conditions in a range from about 5°C to about 20°C or 25°C below the melting temperature (Tm) ofthe 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 for probes greater than 50 nucleotides are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 50°C, preferably at least about 60°C. 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. Exemplary conditions include hybridization at 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄ pH 7.0, 1 mM EDTA at 65° C; wash with 2xSSC, 1% SDS, at 50° C.

[40] Alternatively, substantial sequence identity can be described as a percentage identity between two nucleotide or amino acid sequences. Two nucleic acid sequences are considered substantially identical when they are at least about 70% identical, or at least about 80% identical, or at least about 90% identical, or at least about 95% or 98% identical. Two amino acid sequences are considered substantially identical when they are at least about 60%, sequence identical, more often at least about 70%, at least about 80%, or at least about 90% sequence identity to the reference sequence. Percentage sequence (nucleotide or amino acid) identity is typically calculated using art known means to determine the optimal alignment between two sequences and comparing the two sequences. Optimal alignment of sequences may be conducted using the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by the BLAST algorithm of Altschul (1990) J. Mol. Biol. 215: 403-410; and Shpaer (1996) Genomics 38:179-191, or by the algorithm of Needleham et al. (1970) J. Mol. Biol. 48: 443-453; and Sankoff et al., 1983, Time Warps, String Edits, and Macromolecules, The Theory and Practice of Sequence Comparison, Chapter One, Addison- Wesley, Reading, MA; generally by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI; BLAST from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). In each case default parameters are used (for example the BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=- 4, and a comparison of both strands).

[41 ] The invention methods may be directed to the preparation of an individual polyketide. The polyketide may or may not be novel, but the method of preparation permits a more convenient or alternative method of preparing it. The resulting polyketides may be further modified to convert them to other useful compounds. Examples of chemical structures of that can be made using the materials and methods of the present invention include known analogs, such as those described in Kalesse and Christmann, 2002, "The Chemistry and Biology ofthe Tautomycetin Family" Synthesis (8):981-1003 and the references cited therein, and novel molecules produced by modified or chimeric PKSs comprising a portion ofthe tautomycetin PKS sequence, molecules produced by the action of polyketide modifying enzymes from the tautomycetin PKS cluster on products of other PKSs, molecules produced by the action on products ofthe tautomycetin PKS of polyketide modifying enzymes from other PKSs, and the like. As noted, in one aspect the invention provides recombinant PKS wherein at least 10, 15, 20, or more consecutive amino acids in one or more domains of one or more modules thereof are derived from one or more domains of one or more modules ofthe tautomycetin polyketide synthase. A polyketide synthase "derived from" a naturally occurring PKS contains the scaffolding encoded by all the portion employed ofthe naturally occurring synthase gene, contains at least two modules that are functional, and contains mutations, deletions, or replacements of one or more ofthe activities of these functional modules so that the nature ofthe resulting polyketide is altered. This definition applies both at the protein and genetic levels. Particular embodiments include those wherein a KS, AT, KR, DH, or ER has been deleted or replaced by a version ofthe activity from a different PKS or from another location within the same PKS, and derivatives where at least one noncondensation cycle enzymatic activity (KR, DH, or ER) has been deleted or wherein any of these activities has been added or mutated so as to change the ultimate polyketide synthesized. There are at least five degrees of freedom for constructing a polyketide synthase in terms ofthe polyketide that will be produced. See, U.S. Patent No. 6,509,455. [42] As can be appreciated by those skilled in the art, polyketide biosynthesis can be manipulated to make a product other than the product of a naturally occurring PKS biosynthetic cluster. For example, AT domains can be altered or replaced to change specificity. The variable domains within a module can be deleted and or inactivated or replaced with other variable domains found in other modules ofthe same PKS or from another PKS. See e.g., Katz and McDaniel, Med. Res. Rev. 19: 543-558 (1999) and WO 98/49315. Similarly, entire modules can be deleted and/or replaced with other modules from the same PKS or another PKS. See e.g., Gokhale et al., Science 284: 482 (1999) and WO 00/47724 each of which are incoφorated herein by reference. Protein subunits of different PKSs also can be mixed and matched to make compounds having the desired backbone and modifications. For example, subunits of 1 and 2 (encoding modules 1-4) of the pikromycin PKS were combined with the DEBS3 subunit to make a hybrid PKS product (see Tang et al., Science, 287: 640 (2001), WO 00/26349 and WO 99/6159). [43] Mutations can be introduced into PKS genes such that polypeptides with altered activity are encoded. Polypeptides with "altered activity" include those in which one or more domains are inactivated or deleted, or in which a mutation changes the substrate specificity of a domain, as well as other alterations in activity. Mutations can be made to the native sequences using conventional techniques. The substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes. Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. (See, e.g., Kunkel, Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. BioTechniques (1987) 5:786.) Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature ofthe mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. (See Zoller and Smith, Methods in Enzymology (1983) 100:468). Primer extension is effected using DNA polymerase. The product of the extension reaction is cloned, and those clones containing the mutated DNA are selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. (See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA (1982) 79:6409). PCR mutagenesis can also be used for effecting the desired mutations. Random mutagenesis of selected portions ofthe nucleotide sequences encoding enzymatic activities can be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid, by chemical mutagenesis, or by irradiation. In addition to providing mutated forms of regions encoding enzymatic activity, regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS synthase can be recovered, for example, using PCR techniques with appropriate primers. 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. [44] 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 PCT application WO 96/40968. Another 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 and Wanner, 2000, Proc. Natl. Acad. Sci. USA, 97, 6640-45; Muyrers et al, 2000, Genetic Engineering 22:77-98). The RED/ET cloning procedure is used to introduce a unique restriction site in the recipient plasmid at the ' location ofthe 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 carmot 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.

[45] In a further aspect, the invention provides methods for expressing chimeric or hybrid PKSs and products of such PKSs. For example, the invention provides (1) encoding DNA for a chimeric PKS that is substantially patterned on a non-tautomycetin producing enzyme, but which includes one or more functional domains, modules or polypeptides of tautomycetin PKS; and (2) encoding DNA for a chimeric PKS that is substantially patterned on the tautomycetin PKS, but which includes one or more functional domains, modules, or polypeptides of another PKS or NRPS. [46] With respect to item (1) above, in one embodiment, the invention provides chimeric PKS enzymes in which the genes for a non-tautomycetin PKS function as accepting genes, and one or more ofthe above-identified coding sequences for tautomycetin domains or modules are inserted as replacements for one or more domains or modules of comparable function. Construction of chimeric molecules is most effectively achieved by construction of appropriate encoding polynucleotides. In making a chimeric molecule, it is not necessary to replace an entire domain or module accepting ofthe PKS with an entire domain or module of tautomycetin PKS: 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 10, 15, 20 or more amino acids of a selected tautomycetin domain or module.

[47] Recombinant methods for manipulating modular PKS genes to make chimeric PKS enzymes are described in U.S. Patent Nos. 5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in PCT publications 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 ofthe 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 one embodiment, the components ofthe chimeric PKS are arranged onto polypeptides having inteφolypeptide linkers that direct the assembly ofthe polypeptides into the functional PKS protein, such that it is not required that the PKS have the same arrangement of modules in the polypeptides as observed in natural PKSs. Suitable inteφolypeptide linkers to join polypeptides and intrapolypeptide linkers to join modules within a polypeptide are described in PCT publication WO 00/47724. [48] A partial list of sources of PKS sequences for use in making chimeric molecules, for illustration and not limitation, includes Avermectin (U.S. Patent No. 5,252,474; MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, and 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. Patent No. 6,303,342); Erythromycin (WO 93/13663; U.S. Patent 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. Patent No. 6,503,737; see also Nielsen et al., 1991, Biochem. 30:5789-96 ); Lovastatin (U.S. Patent 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. Patent 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 and Biology, 5: 69-79); Soraphen (U.S. Patent No. 5,716,849; Schupp et al., 1995, J. Bacteriol. 177: 3673-79); Spiramycin (U.S. Patent No. 5,098,837); Tylosin (EP 0 791,655; Kuhstoss et al., 1996, Gene 183:231-36; U.S. Patent 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). [49] The tautomycetin PKS-encoding polynucleotides ofthe invention may also be used in the production of libraries of PKSs (i.e., modified and chimeric PKSs comprising at least a portion ofthe tautomycetin PKS sequence. The invention provides libraries of polyketides by generating modifications in, or using a portion of, the tautomycetin 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 tautomycetin product ofthe 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 PKS cluster, an effectively combinatorial library of polyketides can be produced as a result ofthe multiple variations in these activities. Expression vectors containing nucleotide sequences encoding a variety of PKS systems for the production of different polyketides can be transformed into the appropriate host cells to construct a polyketide library. 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. A variety of strategies can 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 leasst 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products. The number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large. The polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity. [50] 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 er se can be used for screening, or partial or complete purification ofthe polyketides can first be effected. Typically, such screening methods involve detecting the binding of each member ofthe library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art. Alternatively, individual polyketide members ofthe library can be tested against a desired target. In this event, screens wherein the biological response ofthe target is measured can be included.

[51] As noted above, the DNA compounds ofthe invention can be expressed in host cells for production of proteins and of known and novel compounds. Preferred hosts include fungal systems such as yeast and procaryotic hosts, but single cell cultures of, for example, mammalian cells could also be used. A variety of methods for heterologous expression of PKS genes and host cells suitable for expression of these genes and production of polyketides are described, for example, in U.S. Patent Nos. 5,843,718; 5,830,750 and 6,262,340; WO 01/31035, WO 01/27306, and WO 02/068613; and U.S. Patent Application Pub. Nos. 20020192767 and 20020045220

[52] Appropriate host cells for the expression ofthe hybrid PKS genes include those organisms capable of producing the needed precursors, such as malonyl-CoA, methylmalonyl-CoA, efhylmalonyl-CoA, and methoxymalonyl-ACP, and having phosphopantotheinylation systems capable of activating the ACP domains of modular PKSs. See, for example, U.S. Patent No. 6,579,695. However, as disclosed in U.S. Patent No. 6,033,883, a wide variety of hosts can be used, even though some hosts natively do not contain the appropriate post-translational mechanisms to activate the acyl carrier proteins ofthe synthases. Also see WO 97/13845 and WO 98/27203. The host cell may natively produce none, some, or all ofthe required polyketide precursors, and may be genetically engineered so as to produce the required polyketide precursors. Such hosts can be modified with the appropriate recombinant enzymes to effect these modifications. Suitable host cells include Streptomyces, E. coli, yeast, and other procaryotic hosts which use control sequences compatible with Streptomyces spp. Examples of suitable hosts that either natively produce modular polyketides or have been engineered so as to produce modular polyketides include but are not limited to actinomyctes such as Streptomyces coelicolor, Streptomyces venezuelae, Streptomyces fradiae, Streptomyces ambofaciens, and Saccharopolyspora erythraea, eubacteria such as Escherichia coli, myxobacteria such as Myxococcus xanthus, and yeasts such as Saccharomyces cerevisiae.

[53] In one embodiment, any native modular PKS genes in the host cell have been deleted to produce a "clean host," as described in U.S. Patent No. 5,672,491, incoφorated herein by reference.

[54] In some embodiments, the host cell expresses, or is engineered to express, a polyketide "tailoring" or "modifying" enzyme. Once a PKS product is released, it is subject to post-PKS tailoring reactions. These reactions are important for biological activity and for the diversity seen among polyketides. Tailoring enzymes normally associated with polyketide biosynthesis include oxygenases, glycosyl- and mefhyl- transferases, acyltransferases, halogenases, cyclases, aminotransferases, and hydroxylases. In addition to biosynthetic accessory activities, secondary metabolite clusters often code for activities such as transport.

[55] Tailoring enzymes for modification of a product ofthe tautomycetin PKS, a non- tautomycetin PKS, or a chimeric PKS, can be those normally associated with tautomycetin biosynthesis or "heterologous" tailoring enzymes. Examples of tailoring enzymes normally associated with tautomycetin biosynthesis include those associated with the formation ofthe dialkylmaleic anhydride segment of tautomycetin or with decarboxylation ofthe tautomycetin polyketide chain. Tailoring enzymes can be expressed in the organism in which they are naturally produced, or as recombinant proteins in heterologous hosts. In some cases, the structure produced by the heterologous or hybrid PKS may be modified with different efficiencies by post-PKS tailoring enzymes from different sources. In such cases, post-PKS tailoring enzymes can be recruited from other pathways to obtain the desired compound. For example, the tailoring enzymes of the tautomycetin PKS gene cluster can be expressed heterologously to modify polyketides produced by non-tautomycetin synthases or can be inactivated in the Tautomycetin producer.

[56] Alternatively, the unmodified polyketide compounds can be produced in the recombinant host cell, and the desired modification (e.g., oxidation) 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 unmodified polyketide). [57] It will be apparent to one of skill in the art that a variety of recombinant vectors can be utilized in the practice of aspects ofthe invention. 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 ofthe 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 that integrate into the host cell genome.

[58] 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 ofthe host PKS genes may be chosen to contain control sequences operably linked to the resulting coding sequences in a manner that expression ofthe coding sequences may be effected in an appropriate host. Suitable control sequences include those that function in eucaryotic and procaryotic host cells. If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This can be done individually, or using a pool of isolated encoding nucleotide sequences, which can be inserted into host vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies. 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 ofthe host, such as ribosome binding sites. Particularly useful promoters for procaryotic hosts include those from PKS gene clusters that 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 (bid), bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Patent No. 4,551,433), can be used.

[59] 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 actVactlϊl promoter pair and the αctII-ORF4 (an activator gene), is particularly preferred. Particularly preferred hosts are those that lack their own means for producing polyketides so that a cleaner result is obtained. Illustrative control sequences, vectors, and host cells of these types include the modified S. coelicolor CH999 and vectors described in PCT publication WO 96/40968 and similar strains of S. lividans. See U.S. Patent Nos. 5,672,491; 5,830,750, 5,843,718; and 6,177,262, each of which is incoφorated herein by reference.

[60] Other regulatory sequences may also be desirable which allow for regulation of expression ofthe PKS sequences relative to the growth ofthe host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences. Selectable markers can also be included in the recombinant expression vectors. A variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid. [61 ] Alternatively, several polyketides are naturally colored, and this characteristic provides a built-in marker for screening cells successfully transformed by the present constructs.

[62] 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 ofthe present invention into suitable hosts are known to those of skill in the art and typically include the use of CaCl₂ or other agents, such as divalent cations, lipofection, DMSO, protoplast transformation, conjugation, and electroporation.

Thus, the present invention provides recombinant DNA molecules and vectors comprising those recombinant DNA molecules that encode at least a portion ofthe tautomycetin PKS and that, when transformed into a host cell and the host cell is cultured under conditions that lead to the expression of said tautomycetin PKS enzymes, results in the production of polyketides including but not limited to tautomycetin and/or analogs or derivatives thereof in useful quantities. The present invention also provides recombinant host cells comprising those recombinant vectors.

[63] Suitable culture conditions for production of polyketides using the cells ofthe invention will vary according to the host cell and the nature ofthe polyketide being produced, but will be know to those of skill in the art. See, for example, the examples below and WO 98/27203 "Production of Polyketides in Bacteria and Yeast" and WO

01/83803 "Oveφroduction Hosts For Biosynthesis of Polyketides."

[64] The polyketide product produced by host cells ofthe 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).

[65] The compositions, cells and methods ofthe 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.

[66] The following Examples are intended to illustrate, but not limit, the scope ofthe invention. EXAMPLE 1 Preparation of Genomic DNA library [67] For genomic DNA preparation, a spore stock of Streptomyces sp. K276-35 was used to inoculate 35 ml of Tryptone Soy Broth (TSB) liquid media. After two days growth at 30 °C, the culture was centrifuged (10,000 x g). The pellet was suspended in 12 ml of buffer 1 (Tris, 50 mM, pH7.5; 20 mM EDTA, 150 μg/ml RNase (Sigma-Aldrich) and lmg/ml of lysozyme (Sigma)). After incubation ofthe mixture at 37 °C for 30 min, the salt concentration was adjusted by adding 3 ml of 5 M NaCl solution, then the mixture was extracted two times with phenol :chloroform:isoamyl alcohol (25:24:1, v/v) with gentle agitation followed by centrifugation for 10 min at 3500 x g. After precipitation with 1 volume of isopropanol, the genomic DNA knot was spooled on a glass rod and redissolved in 1ml of water

[68] Approximately 10 μg of genomic DNA was partially digested with Sα«3Al (10 min incubation using dilutions ofthe enzyme) and the digested DNA was run on an agarose gel with DNA standards to check that the size of fragments was about 30-45 kb. The DNA from this digestion was treated with shrimp alkaline phosphatase and ligated with pSuperCos -1 (Stratagene), pre-linearized with famHI and Xbal and the ligation mixture was packaged using a Gigapack XIII (Stragene) in vitro packaging kit and the mixture was subsequently used for infection of Escherichia coli DH5α employing protocols supplied by the manufacturer. EXAMPLE 2 Cloning of the tautomycetin gene cluster [69] To find the gene cluster for tautomycetin biosynthesis, cosmids from 10X 95 E. coli transductants resulting from the ligation mixmre of Example 1 were sequenced using convergent primersT7cos (5'-CATAATACGACTCACTATAGGG) and T3cos-l(5'- TTCCCCGAAAAGTGCCAC) (SEQ ED NOs:14 and 15, respectively). After BLAST analysis, the sequences revealed 22 cosmids carrying DNA fragments encoding type I PKS (polyketide synthetase) genes at one end. Basing on the sequence and restriction enzyme maps of those cosmids, we chose to sequence one of those cosmids, pKOS276- 46.1C11 (also refered to herein as "cosmid OlCl 1"), first. pKOS276-46.1Cl 1 was found to contain 21,148 bp encoding tautomycetin PKS domains, modules, and/or polypeptides. Cosmid 12H9 contained the sequences contained in the region ofthe tautomycetin gene cluster as depicted in Figure 3. Subsequent sequencing of cosmides 04C5, 03F6, and 12H9 allowed assembly ofthe 73,931 bp sequence shown in Figure 3 as SEQ ED NO:l.

[70] All publications and patent documents cited herein are incoφorated herein by reference as if each such publication or document were specifically and individually indicated to be incoφorated herein by reference.

[71 ] Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit ofthe invention. Citation of publications and patent documents is not intended as an admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date ofthe same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description are for puφoses of illustration and not limitation ofthe following claims.

Claims

What is claimed is:

1. A purified or recombinant nucleic acid comprising a nucleotide sequence that encodes at least one polypeptide required for the biosynthesis of tautomycetin, wherein the complement of said nucleotide sequence hybridizes to a nucleic acid comprising the nucleotide sequence given in SEQ ED NO:l, under conditions of hybridization at 65 °C for 36 hours and washing 3 times at high stringency with O.lxSSC and 0.5% SDS for 20 minutes at 65 °C.

2. A purified or recombinant nucleic acid a nucleotide sequence that encodes at least one module ofthe tautomycetin polyketide synthase, wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of nucleotides that encode modules ofthe tautomycetin PKS as listed in Table 1.

3. A purified or recombinant nucleic acid according to Claim 1, wherein said polypeptide comprises a β-ketoacylsynthase domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of β- ketoacylsynthase domains as listed in Table 1, under conditions of hybridization at 65 °C for 36 hours and washing 3 times at high stringency with O.lxSSC and 0.5% SDS for 20 minutes at 65 °C.

4. A purified or recombinant nucleic acid according to Claim 1 , wherein said polypeptide comprises an acyltransferase domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of acyltransferase domains as listed in Table 1, under conditions of hybridization at 65 °C for 36 hours and washing 3 times at high stringency with O.lxSSC and 0.5% SDS for 20 minutes at 65 °C.

5. A purified or recombinant nucleic acid according to Claim 1, wherein said polypeptide comprises a β-ketoreductase domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of β- ketoreductase domains as listed in Table 1, under conditions of hybridization at 65 °C for 36 hours and washing 3 times at high stringency with O.lxSSC and 0.5% SDS for 20 minutes at 65 °C.

6. A purified or recombinant nucleic acid according to Claim 1, wherein said polypeptide comprises a dehydratase domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of dehydratase domains as listed in Table 1, under conditions of hybridization at 65 °C for 36 hours and washing 3 times at high stringency with O.lxSSC and 0.5% SDS for 20 minutes at 65 °C.

7. A purified or recombinant nucleic acid according to Claim 1, wherein said polypeptide comprises an enoylreductase domain and wherein the complement of said nucleotide sequence hybridizes to enoylreductase domains as listed in Table 1 , under conditions of hybridization at 65 °C for 36 hours and washing 3 times at high stringency with O.lxSSC and 0.5% SDS for 20 minutes at 65 °C.

8. A purified or recombinant nucleic acid according to Claim 1, wherein said polypeptide comprises an acyl carrier protein domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of acyl carrier protein domains as listed in Table 1, under conditions of hybridization at 65 °C for 36 hours and washing 3 times at high stringency with O.lxSSC and 0.5% SDS for 20 minutes at 65 °C.

9. A purified or recombinant polypeptide involved in the biosynthesis of tautomycetin, wherein said polypeptide has an amino acid sequence that can be encoded by a nucleic acid sequence of Claim 1.

10. The polypetide of Claim 9 that can be encoded by the TauA gene.

11. The polypetide of Claim 9 that can be encoded by the TauB gene.

12. The polypetide of Claim 9 that can be encoded by the TauC gene.

13. The polypetide of Claim 9 that can be encoded by the TauD gene.

14. The polypetide of Claim 9 that can be encoded by the TauE gene.

15. The polypetide of Claim 9 that can be encoded by the TauF gene.

16. The polypetide of Claim 9 that can be encoded by the TauG gene.

17. A method of making polyketides, said method comprising expressing at least one recombinant gene, module, or domain of Claim 1 in a host cell capable of producing polyketides.

18. The method of Claim 17 wherein the polyketide is tautomycetin or a tautomycetin analog.