# Patent

## Title
Genes for the biosynthesis of epothilones

## Abstract

Nucleic acid molecules are isolated from Sorangium cellulosum that encode polypeptides necessary for the biosynthesis of epothilone. Disclosed are methods for the production of epothilone in recombinant hosts transformed with the genes of the invention. In this manner, epothilone can be produced in quantities large enough to enable their purification and use in pharmaceutical formulations such as those for the treatment of cancer.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 09/568,472, filed May 10, 2000, now issued as U.S. Pat. No. 6,358,719, which is a divisional of U.S. application Ser. No. 09/335,409, filed Jun. 17, 1999, now issued as U.S. Pat. No. 6,121,029, which claims benefit of U.S. Provisional Applications 60/155,183, filed Jun. 18, 1998, now abandoned 60/101,631, filed Sep. 24, 1998, and 60/118,906, filed Feb. 5, 1999.


FIELD OF THE INVENTION
The present invention relates generally to polyketides and genes for their synthesis. In particular, the present invention relates to the isolation and characterization of novel polyketide synthase and nonribosomal peptide synthetase genes from Sorangium cellulosum that are necessary for the biosynthesis of epothilones A and B.
BACKGROUND OF THE INVENTION
Polyketides are compounds synthesized from two-carbon building blocks, the β-carbon of which always carries a keto group, thus the name polyketide. These compounds include many important antibiotics, immunosuppressants, cancer chemotherapeutic agents, and other compounds possessing a broad range of biological properties. The tremendous structural diversity derives from the different lengths of the polyketide chain, the different side-chains introduced (either as part of the two-carbon building blocks or after the polyketide backbone is formed), and the stereochemistry of such groups. The keto groups may also be reduced to hydroxyls, enoyls, or removed altogether. Each round of two-carbon addition is carried out by a complex of enzymes called the polyketide synthase (PKS) in a manner similar to fatty acid biosynthesis.
The biosynthetic genes for an increasing number of polyketides have been isolated and sequenced. For example, see U.S. Pat. Nos. 5,639,949, 5,693,774, and 5,716,849, all of which are incorporated herein by reference, which describe genes for the biosynthesis of soraphen. See also, Schupp et al., FEMS Microbiology Letters 159: 201-207 (1998) and WO 98/07868, which describe genes for the biosynthesis of rifamycin, and U.S. Pat. No. 5,876,991, which describes genes for the biosynthesis of tylactone, all of which are incorporated herein by reference. The encoded proteins generally fall into two types: type I and type II. Type I proteins are polyfunctional, with several catalytic domains carrying out different enzymatic steps covalently linked together (e.g. PKS for erythromycin, soraphen, rifamycin, and avermectin (MacNeil et al., in Industrial Microorganisms: Basic and Applied Molecular Genetics, (ed.: Baltz et al.), American Society for Microbiology, Washington D.C. pp. 245-256 (1993)); whereas type II proteins are monofunctional (Hutchinson et al., in Industrial Microorganisms: Basic and Applied Molecular Genetics, (ed.: Baltz et al.), American Society for Microbiology, Washington D.C. pp. 203-216 (1993)).
For the simpler polyketides such as actinorhodin (produced by Streptomyces coelicolor), the several rounds of two-carbon additions are carried out iteratively on PKS enzymes encoded by one set of PKS genes. In contrast, synthesis of the more complicated compounds such as erythromycin and soraphen involves PKS enzymes that are organized into modules, whereby each module carries out one round of two-carbon addition (for review, see Hopwood et al., in Industrial Microorganisms: Basic and Applied Molecular Genetics, (ed.: Baltz et al), American Society for Microbiology, Washington D.C., pp. 267-275 (1993)).
Complex polyketides and secondary metabolites in general may contain substructures that are derived from amino acids instead of simple carboxylic acids. Incorporations of these building blocks are accomplished by non-ribosomal polypeptide synthetases (NRPSs). NRPSs are multienzymes that are organized in modules. Each module is responsible for the addition (and the additional processing, if required) of one amino acid building block. NRPSs activate amino acids by forming aminoacyl-adenylates, and capture the activated amino acids on thiol groups of phophopantheteinyl prosthetic groups on peptidyl carrier protein domains. Further, NRPSs modify the amino acids by epimerization, N-methylation, or cyclization if necessary, and catalyse the formation of peptide bonds between the enzyme-bound amino acids. NRPSs are responsible for the biosynthesis of peptide secondary metabolites like cyclosporin, could provide polyketide chain terminator units as in rapamycin, or form mixed systems with PKSs as in yersiniabactin biosynthesis.
Epothilones A and B are 16-membered macrocyclic polyketides with an acylcysteine-derived starter unit that are produced by the bacterium Sorangium cellulosum strain So ce90 (Gerth et al., J. Antibiotics 49: 560-563 (1996), incorporated herein by reference). The structure of epothilone A and B wherein R signifies hydrogen (epothilone A) or methyl (epothilone B) is: 




The epothilones have a narrow antifungal spectrum and especially show a high cytotoxicity in animal cell cultures (see, Höfle et al., Patent DE 4138042 (1993), incorporated herein by reference). Of significant importance, epothilones mimic the biological effects of taxol, both in vivo and in cultured cells (Bollag et al., Cancer Research 55: 2325-2333 (1995), incorporated herein by reference). Taxol and taxotere, which stabilize cellular microtubules, are cancer chemotherapeutic agents with significant activity against various human solid tumors (Rowinsky et al., J. Natl. Cancer Inst. 83: 1778-1781 (1991)). Competition studies have revealed that epothilones act as competitive inhibitors of taxol binding to microtubules, consistent with the interpretation that they share the same microtubule-binding site and possess a similar microtubule affinity as taxol. However, epothilones enjoy a significant advantage over taxol in that epothilones exhibit a much lower drop in potency compared to taxol against a multiple drug-resistant cell line (Bollag et al. (1995)). Furthermore, epothilones are considerably less efficiently exported from the cells by P-glycoprotein than is taxol (Gerth et al. (1996)). In addition, several epothilone analogs have been synthesized that have a superior cytotoxic activity as compared to epothilone A or epothilone B as demonstrated by their enhanced ability to induce the polymerization and stabilization of microtubules (WO 98/25929, incorporated herein by reference).
Despite the promise shown by the epothilones as anticancer agents, problems pertaining to the production of these compounds presently limit their commercial potential. The compounds are too complex for industrial-scale chemical synthesis and so must be produced by fermentation. Techniques for the genetic manipulation of myxobacteria such as Sorangium cellulosum are described in U.S. Pat. No. 5,686,295, incorporated herein by reference. However, Sorangium cellulosum is notoriously difficult to ferment and production levels of epothilones are therefore low. Recombinant production of epothilones in heterologous hosts that are more amenable to fermentation could solve current production problems. However, the genes that encode the polypeptides responsible for epothilone biosynthesis have heretofore not been isolated. Furthermore, the strain that produces epothilones, i.e. So ce90, also produces at least one additional polyketide, spirangien, which would be expected to greatly complicate the isolation of the genes particularly responsible for epothilone biosynthesis.
Therefore, in view of the foregoing, one object of the present invention is to isolate the genes that are involved in the synthesis of epothilones, particularly the genes that are involved in the synthesis of epothilones A and B in myxobacteria of the Sorangium/Polyangium group, i.e., Sorangium cellulosum strain So ce90. A further object of the invention is to provide a method for the recombinant production of epothilones for application in anticancer formulations.
SUMMARY OF THE INVENTION
In furtherance of the aforementioned and other objects, the present invention unexpectedly overcomes the difficulties set forth above to provide for the first time a nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of epothilone. In a preferred embodiment, the nucleotide sequence is isolated from a species belonging to Myxobacteria, most preferably Sorangium cellulosum. 
In another preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said polypeptide comprises an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: SEQ ID NO:2, amino acids 11-437 of SEQ ID NO:2, amino acids 543-864 of SEQ ID NO:2, amino acids 974-1273 of SEQ ID NO:2, amino acids 1314-1385 of SEQ ID NO:2, SEQ ID NO:3, amino acids 72-81 of SEQ ID NO:3, amino acids 118-125 of SEQ ID NO:3, amino acids 199-212 of SEQ ID NO:3, amino acids 353-363 of SEQ ID NO:3, amino acids 549-565 of SEQ ID NO:3, amino acids 588-603 of SEQ ID NO:3, amino acids 669-684 of SEQ ID NO:3, amino acids 815-821 of SEQ ID NO:3, amino acids 868-892 of SEQ ID NO:3, amino acids 903-912 of SEQ ID NO:3, amino acids 918-940 of SEQ ID NO:3, amino acids 1268-1274 of SEQ ID NO:3, amino acids 1285-1297 of SEQ ID NO:3, amino acids 973-1256 of SEQ ID NO:3, amino acids 1344-1351 of SEQ ID NO:3, SEQ ID NO:4, amino acids 7-432 of SEQ ID NO:4, amino acids 539-859 of SEQ ID NO:4, amino acids 869-1037 of SEQ ID NO:4, amino acids 1439-1684 of SEQ ID NO:4, amino acids 1722-1792 of SEQ ID NO:4, SEQ ID NO:5, amino acids 39-457 of SEQ ID NO:5, amino acids 563-884 of SEQ ID NO:5, amino acids 1147-1399 of SEQ ID NO:5, amino acids 1434-1506 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 3886-4048 of SEQ ID NO:5, amino acids 4433-4719 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, SEQ ID NO:6, amino acids 35-454 of SEQ ID NO:6, amino acids 561-881 of SEQ ID NO:6, amino acids 1143-1393 of SEQ ID NO:6, amino acids 1430-1503 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, amino acids 2053-2373 of SEQ ID NO:6, amino acids 2383-2551 of SEQ ID NO:6, amino acids 2671-3045 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, SEQ ID NO:7, amino acids 32-450 of SEQ ID NO:7, amino acids 556-877 of SEQ ID NO:7, amino acids 887-1051 of SEQ ID NO:7, amino acids 1478-1790 of SEQ ID NO:7, amino acids 1810-2055 of SEQ ID NO:7, amino acids 2093-2164 of SEQ ID NO:7, amino acids 2165-2439 of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:22.
In a more preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, amino acids 11-437 of SEQ ID NO:2, amino acids 543-864 of SEQ ID NO:2, amino acids 974-1273 of SEQ ID NO:2, amino acids 1314-1385 of SEQ ID NO:2, SEQ ID NO:3, amino acids 72-81 of SEQ ID NO:3, amino acids 118-125 of SEQ ID NO:3, amino acids 199-212 of SEQ ID NO:3, amino acids 353-363 of SEQ ID NO:3, amino acids 549-565 of SEQ ID NO:3, amino acids 588-603 of SEQ ID NO:3, amino acids 669-684 of SEQ ID NO:3, amino acids 815-821 of SEQ ID NO:3, amino acids 868-892 of SEQ ID NO:3, amino acids 903-912 of SEQ ID NO:3, amino acids 918-940 of SEQ ID NO:3, amino acids 1268-1274 of SEQ ID NO:3, amino acids 1285-1297 of SEQ ID NO:3, amino acids 973-1256 of SEQ ID NO:3, amino acids 1344-1351 of SEQ ID NO:3, SEQ ID NO:4, amino acids 7-432 of SEQ ID NO:4, amino acids 539-859 of SEQ ID NO:4, amino acids 869-1037 of SEQ ID NO:4, amino acids 1439-1684 of SEQ ID NO:4, amino acids 1722-1792 of SEQ ID NO:4, SEQ ID NO:5, amino acids 39-457 of SEQ ID NO:5, amino acids 563-884 of SEQ ID NO:5, amino acids 1147-1399 of SEQ ID NO:5, amino acids 1434-1506 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 3886-4048 of SEQ ID NO:5, amino acids 4433-4719 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, SEQ ID NO:6, amino acids 35-454 of SEQ ID NO:6, amino acids 561-881 of SEQ ID NO:6, amino acids 1143-1393 of SEQ ID NO:6, amino acids 1430-1503 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, amino acids 2053-2373 of SEQ ID NO:6, amino acids 2383-2551 of SEQ ID NO:6, amino acids 2671-3045 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, SEQ ID NO:7, amino acids 32-450 of SEQ ID NO:7, amino acids 556-877 of SEQ ID NO:7, amino acids 887-1051 of SEQ ID NO:7, amino acids 1478-1790 of SEQ ID NO:7, amino acids 1810-2055 of SEQ ID NO:7, amino acids 2093-2164 of SEQ ID NO:7, amino acids 2165-2439 of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:22.
In yet another preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said nucleotide sequence is substantially similar to a nucleotide sequence selected from the group consisting of: the complement of nucleotides 1900-3171 of SEQ ID NO:1, nucleotides 3415-5556 of SEQ ID NO:1, nucleotides 7610-11875 of SEQ ID NO:1, nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, nucleotides 15901-15924 of SEQ ID NO:1, nucleotides 16251-21749 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 21746-43519 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 43524-54920 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, nucleotides 51534-52657 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, nucleotides 54935-62254 of SEQ ID NO:1, nucleotides 55028-56284 of SEQ ID NO:1, nucleotides 56600-57565 of SEQ ID NO:1, nucleotides 57593-58087 of SEQ ID NO:1, nucleotides 59366-60304 of SEQ ID NO:1, nucleotides 60362-61099 of SEQ ID NO:1, nucleotides 61211-61426 of SEQ ID NO:1, nucleotides 61427-62254 of SEQ ID NO:1, nucleotides 62369-63628 of SEQ ID NO:1, nucleotides 67334-68251 of SEQ ID NO:1, and nucleotides 1-68750 SEQ ID NO:1.
In an especially preferred embodiment, the present invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said nucleotide sequence is selected from the group consisting of: the complement of nucleotides 1900-3171 of SEQ ID NO:1, nucleotides 3415-5556 of SEQ ID NO:1, nucleotides 7610-11875 of SEQ ID NO:1, nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, nucleotides 15901-15924 of SEQ ID NO:1, nucleotides 16251-21749 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 21746-43519 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 43524-54920 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, nucleotides 51534-52657 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, nucleotides 54935-62254 of SEQ ID NO:1, nucleotides 55028-56284 of SEQ ID NO:1, nucleotides 56600-57565 of SEQ ID NO:1, nucleotides 57593-58087 of SEQ ID NO:1, nucleotides 59366-60304 of SEQ ID NO:1, nucleotides 60362-61099 of SEQ ID NO:1, nucleotides 61211-61426 of SEQ ID NO:1, nucleotides 61427-62254 of SEQ ID NO:1, nucleotides 62369-63628 of SEQ ID NO:1, nucleotides 67334-68251 of SEQ ID NO:1, and nucleotides 1-68750 SEQ ID NO:1.
In yet another preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of an epothilone, wherein said nucleotide sequence comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: the complement of nucleotides 1900-3171 of SEQ ID NO:1, nucleotides 3415-5556 of SEQ ID NO:1, nucleotides 7610-11875 of SEQ ID NO:1, nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, nucleotides 15901-15924 of SEQ ID NO:1, nucleotides 16251-21749 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 21746-43519 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 43524-54920 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, nucleotides 51534-52657 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, nucleotides 54935-62254 of SEQ ID NO:1, nucleotides 55028-56284 of SEQ ID NO:1, nucleotides 56600-57565 of SEQ ID NO:1, nucleotides 57593-58087 of SEQ ID NO:1, nucleotides 59366-60304 of SEQ ID NO:1, nucleotides 60362-61099 of SEQ ID NO:1, nucleotides 61211-61426 of SEQ ID NO:1, nucleotides 61427-62254 of SEQ ID NO:1, nucleotides 62369-63628 of SEQ ID NO:1, nucleotides 67334-68251 of SEQ ID NO:1, and nucleotides 1-68750 SEQ ID NO:1.
The present invention also provides a chimeric gene comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule of the invention. Further, the present invention provides a recombinant vector comprising such a chimeric gene, wherein the vector is capable of being stably transformed into a host cell. Still further, the present invention provides a recombinant host cell comprising such a chimeric gene, wherein the host cell is capable of expressing the nucleotide sequence that encodes at least one polypeptide necessary for the biosynthesis of an epothilone. In a preferred embodiment, the recombinant host cell is a bacterium belonging to the order Actinomycetales, and in a more preferred embodiment the recombinant host cell is a strain of Streptomyces. In other embodiments, the recombinant host cell is any other bacterium amenable to fermentation, such as a pseudomonad or E. coli. Even further, the present invention provides a Bac clone comprising a nucleic acid molecule of the invention, preferably Bac clone pEPO15.
In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes an epothilone synthase domain.
According to one embodiment, the epothilone synthase domain is a β-ketoacyl-synthase (KS) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 11-437 of SEQ ID NO:2, amino acids 7-432 of SEQ ID NO:4, amino acids 39-457 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 35-454 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO:6, and amino acids 32-450 of SEQ ID NO:7. According to this embodiment, said KS domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 11-437 of SEQ ID NO:2, amino acids 7-432 of SEQ ID NO:4, amino acids 39-457 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 35-454 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, and amino acids 32-450 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, and nucleotides 55028-56284 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, and nucleotides 55028-56284 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 7643-8920 of SEQ ID NO:1, nucleotides 16269-17546 of SEQ ID NO:1, nucleotides 21860-23116 of SEQ ID NO:1, nucleotides 26318-27595 of SEQ ID NO:1, nucleotides 30815-32092 of SEQ ID NO:1, nucleotides 37052-38320 of SEQ ID NO:1, nucleotides 43626-44885 of SEQ ID NO:1, nucleotides 48087-49361 of SEQ ID NO:1, and nucleotides 55028-56284 of SEQ ID NO:1.
According to another embodiment, the epothilone synthase domain is an acyltransferase (AT) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 543-864 of SEQ ID NO:2, amino acids 539-859 of SEQ ID NO:4, amino acids 563-884 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 561-881 of SEQ ID NO:6, amino acids 2053-2373 of SEQ ID NO:6, and amino acids 556-877 of SEQ ID NO:7. According to this embodiment, said AT domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 543-864 of SEQ ID NO:2, amino acids 539-859 of SEQ ID NO:4, amino acids 563-884 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 561-881 of SEQ ID NO:6, amino acids 2053-2373 of SEQ ID NO:6, and amino acids 556-877 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, and nucleotides 56600-57565 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, and nucleotides 56600-57565 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 9236-10201 of SEQ ID NO:1, nucleotides 17865-18827 of SEQ ID NO:1, nucleotides 23431-24397 of SEQ ID NO:1, nucleotides 27911-28876 of SEQ ID NO:1, nucleotides 32408-33373 of SEQ ID NO:1, nucleotides 38636-39598 of SEQ ID NO:1, nucleotides 45204-46166 of SEQ ID NO:1, nucleotides 49680-50642 of SEQ ID NO:1, and nucleotides 56600-57565 of SEQ ID NO:1.
According to still another embodiment, the epothilone synthase domain is an enoyl reductase (ER) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 974-1273 of SEQ ID NO:2, amino acids 4433-4719 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, and amino acids 1478-1790 of SEQ ID NO:7. According to this embodiment, said ER domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 974-1273 of SEQ ID NO:2, amino acids 4433-4719 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, and amino acids 1478-1790 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, and nucleotides 59366-60304 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, and nucleotides 59366-60304 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 10529-11428 of SEQ ID NO:1, nucleotides 35042-35902 of SEQ ID NO:1, nucleotides 41369-42256 of SEQ ID NO:1, and nucleotides 59366-60304 of SEQ ID NO:1.
According to another embodiment, the epothilone synthase domain is an acyl carrier protein (ACP) domain, wherein said polypeptide comprises an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 1314-1385 of SEQ ID NO:2, amino acids 1722-1792 of SEQ ID NO:4, amino acids 1434-1506 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, amino acids 1430-1503 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, and amino acids 2093-2164 of SEQ ID NO:7. According to this embodiment, said ACP domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 1314-1385 of SEQ ID NO:2, amino acids 1722-1792 of SEQ ID NO:4, amino acids 1434-1506 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, amino acids 1430-1503 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, and amino acids 2093-2164 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, and nucleotides 61211-61426 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, and nucleotides 61211-61426 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 11549-11764 of SEQ ID NO:1, nucleotides 21414-21626 of SEQ ID NO:1, nucleotides 26045-26263 of SEQ ID NO:1, nucleotides 30539-30759 of SEQ ID NO:1, nucleotides 36773-36991 of SEQ ID NO:1, nucleotides 43163-43378 of SEQ ID NO:1, nucleotides 47811-48032 of SEQ ID NO:1, nucleotides 54540-54758 of SEQ ID NO:1, and nucleotides 61211-61426 of SEQ ID NO:1.
According to another embodiment, the epothilone synthase domain is a dehydratase (DH) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 869-1037 of SEQ ID NO:4, amino acids 3886-4048 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 2383-2551 of SEQ ID NO:6, and amino acids 887-1051 of SEQ ID NO:7. According to this embodiment, said DH domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 869-1037 of SEQ ID NO:4, amino acids 3886-4048 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 2383-2551 of SEQ ID NO:6, and amino acids 887-1051 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, and nucleotides 57593-58087 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, and nucleotides 57593-58087 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 18855-19361 of SEQ ID NO:1, nucleotides 33401-33889 of SEQ ID NO:1, nucleotides 39635-40141 of SEQ ID NO:1, nucleotides 50670-51176 of SEQ ID NO:1, and nucleotides 57593-58087 of SEQ ID NO:1.
According to yet another embodiment, the epothilone synthase domain is a β-keto-reductase (KR) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 1439-1684 of SEQ ID NO:4, amino acids 1147-1399 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 1143-1393 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, and amino acids 1810-2055 of SEQ ID NO:7. According to this embodiment, said KR domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 1439-1684 of SEQ ID NO:4, amino acids 1147-1399 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 1143-1393 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, and amino acids 1810-2055 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, and nucleotides 60362-61099 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, and nucleotides 60362-61099 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 20565-21302 of SEQ ID NO:1, nucleotides 25184-25942 of SEQ ID NO:1, nucleotides 29678-30429 of SEQ ID NO:1, nucleotides 35930-36667 of SEQ ID NO:1, nucleotides 42314-43048 of SEQ ID NO:1, nucleotides 46950-47702 of SEQ ID NO:1, nucleotides 53697-54431 of SEQ ID NO:1, and nucleotides 60362-61099 of SEQ ID NO:1.
According to an additional embodiment, the epothilone synthase domain is a methyltransferase (MT) domain comprising an amino acid sequence substantially similar to amino acids 2671-3045 of SEQ ID NO:6. According to this embodiment, said MT domain preferably comprises amino acids 2671-3045 of SEQ ID NO:6. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to nucleotides 51534-52657 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of nucleotides 51534-52657 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is nucleotides 51534-52657 of SEQ ID NO:1.
According to another embodiment, the epothilone synthase domain is a thioesterase (TE) domain comprising an amino acid sequence substantially similar to amino acids 2165-2439 of SEQ ID NO:7. According to this embodiment, said TE domain preferably comprises amino acids 2165-2439 of SEQ ID NO:7. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to nucleotides 61427-62254 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of nucleotides 61427-62254 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is nucleotides 61427-62254 of SEQ ID NO:1.
In still another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a non-ribosomal peptide synthetase, wherein said non-ribosomal peptide synthetase comprises an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: SEQ ID NO:3, amino acids 72-81 of SEQ ID NO:3, amino acids 118-125 of SEQ ID NO:3, amino acids 199-212 of SEQ ID NO:3, amino acids 353-363 of SEQ ID NO:3, amino acids 549-565 of SEQ ID NO:3, amino acids 588-603 of SEQ ID NO:3, amino acids 669-684 of SEQ ID NO:3, amino acids 815-821 of SEQ ID NO:3, amino acids 868-892 of SEQ ID NO:3, amino acids 903-912 of SEQ ID NO:3, amino acids 918-940 of SEQ ID NO:3, amino acids 1268-1274 of SEQ ID NO:3, amino acids 1285-1297 of SEQ ID NO:3, amino acids 973-1256 of SEQ ID NO:3, and amino acids 1344-1351 of SEQ ID NO:3. According to this embodiment, said non-ribosomal peptide synthetase preferably comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:3, amino acids 72-81 of SEQ ID NO:3, amino acids 118-125 of SEQ ID NO:3, amino acids 199-212 of SEQ ID NO:3, amino acids 353-363 of SEQ ID NO:3, amino acids 549-565 of SEQ ID NO:3, amino acids 588-603 of SEQ ID NO:3, amino acids 669-684 of SEQ ID NO:3, amino acids 815-821 of SEQ ID NO:3, amino acids 868-892 of SEQ ID NO:3, amino acids 903-912 of SEQ ID NO:3, amino acids 918-940 of SEQ ID NO:3, amino acids 1268-1274 of SEQ ID NO:3, amino acids 1285-1297 of SEQ ID NO:3, amino acids 973-1256 of SEQ ID NO:3, and amino acids 1344-1351 of SEQ ID NO:3. Also, according to this embodiment, said nucleotide sequence preferably is substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, and nucleotides 15901-15924 of SEQ ID NO:1. According to this embodiment, said nucleotide sequence more preferably comprises a consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair portion of a nucleotide sequence selected from the group consisting of: nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, and nucleotides 15901-15924 of SEQ ID NO:1. In addition, according to this embodiment, said nucleotide sequence most preferably is selected from the group consisting of: nucleotides 11872-16104 of SEQ ID NO:1, nucleotides 12085-12114 of SEQ ID NO:1, nucleotides 12223-12246 of SEQ ID NO:1, nucleotides 12466-12507 of SEQ ID NO:1, nucleotides 12928-12960 of SEQ ID NO:1, nucleotides 13516-13566 of SEQ ID NO:1, nucleotides 13633-13680 of SEQ ID NO:1, nucleotides 13876-13923 of SEQ ID NO:1, nucleotides 14313-14334 of SEQ ID NO:1, nucleotides 14473-14547 of SEQ ID NO:1, nucleotides 14578-14607 of SEQ ID NO:1, nucleotides 14623-14692 of SEQ ID NO:1, nucleotides 15673-15693 of SEQ ID NO:1, nucleotides 15724-15762 of SEQ ID NO:1, nucleotides 14788-15639 of SEQ ID NO:1, and nucleotides 15901-15924 of SEQ ID NO:1.
The present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2-23.
In accordance with another aspect, the present invention also provides methods for the recombinant production of polyketides such as epothilones in quantities large enough to enable their purification and use in pharmaceutical formulations such as those for the treatment of cancer. A specific advantage of these production methods is the chirality of the molecules produced; production in transgenic organisms avoids the generation of populations of racemic mixtures, within which some enantiomers may have reduced activity. In particular, the present invention provides a method for heterologous expression of epothilone in a recombinant host, comprising: (a) introducing into a host a chimeric gene comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule of the invention that comprises a nucleotide sequence that encodes at least one polypeptide involved in the biosynthesis of epothilone; and (b) growing the host in conditions that allow biosynthesis of epothilone in the host. The present invention also provides a method for producing epothilone, comprising: (a) expressing epothilone in a recombinant host by the aforementioned method; and (b) extracting epothilone from the recombinant host.
According to still another aspect, the present invention provides an isolated polypeptide comprising an amino acid sequence that consists of an epothilone synthase domain.
According to one embodiment, the epothilone synthase domain is a β-ketoacyl-synthase (KS) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 11-437 of SEQ ID NO:2, amino acids 7-432 of SEQ ID NO:4, amino acids 39-457 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 35-454 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, and amino acids 32-450 of SEQ ID NO:7. According to this embodiment, said KS domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 11-437 of SEQ ID NO:2, amino acids 7-432 of SEQ ID NO:4, amino acids 39-457 of SEQ ID NO:5, amino acids 1524-1950 of SEQ ID NO:5, amino acids 3024-3449 of SEQ ID NO:5, amino acids 5103-5525 of SEQ ID NO:5, amino acids 35-454 of SEQ ID NO:6, amino acids 1522-1946 of SEQ ID NO: 6, and amino acids 32-450 of SEQ ID NO:7.
According to another embodiment, the epothilone synthase domain is an acyltransferase (AT) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 543-864 of SEQ ID NO:2, amino acids 539-859 of SEQ ID NO:4, amino acids 563-884 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 561-881 of SEQ ID NO:6, amino acids 2053-2373 of SEQ ID NO:6, and amino acids 556-877 of SEQ ID NO:7. According to this embodiment, said AT domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 543-864 of SEQ ID NO:2, amino acids 539-859 of SEQ ID NO:4, amino acids 563-884 of SEQ ID NO:5, amino acids 2056-2377 of SEQ ID NO:5, amino acids 3555-3876 of SEQ ID NO:5, amino acids 5631-5951 of SEQ ID NO:5, amino acids 561-881 of SEQ ID NO:6, amino acids 2053-2373 of SEQ ID NO:6, and amino acids 556-877 of SEQ ID NO:7.
According to still another embodiment, the epothilone synthase domain is an enoyl reductase (ER) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 974-1273 of SEQ ID NO:2, amino acids 4433-4719 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, and amino acids 1478-1790 of SEQ ID NO:7. According to this embodiment, said ER domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 974-1273 of SEQ ID NO:2, amino acids 4433-4719 of SEQ ID NO:5, amino acids 6542-6837 of SEQ ID NO:5, and amino acids 1478-1790 of SEQ ID NO:7.
According to another embodiment, the epothilone synthase domain is an acyl carrier protein (ACP) domain, wherein said polypeptide comprises an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 1314-1385 of SEQ ID NO:2, amino acids 1722-1792 of SEQ ID NO:4, amino acids 1434-1506 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, amino acids 1430-1503 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, and amino acids 2093-2164 of SEQ ID NO:7. According to this embodiment, said ACP domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 1314-1385 of SEQ ID NO:2, amino acids 1722-1792 of SEQ ID NO:4, amino acids 1434-1506 of SEQ ID NO:5, amino acids 2932-3005 of SEQ ID NO:5, amino acids 5010-5082 of SEQ ID NO:5, amino acids 7140-7211 of SEQ ID NO:5, amino acids 1430-1503 of SEQ ID NO:6, amino acids 3673-3745 of SEQ ID NO:6, and amino acids 2093-2164 of SEQ ID NO:7.
According to another embodiment, the epothilone synthase domain is a dehydratase (DH) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 869-1037 of SEQ ID NO:4, amino acids 3886-4048 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 2383-2551 of SEQ ID NO:6, and amino acids 887-1051 of SEQ ID NO:7. According to this embodiment, said DH domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 869-1037 of SEQ ID NO:4, amino acids 3886-4048 of SEQ ID NO:5, amino acids 5964-6132 of SEQ ID NO:5, amino acids 2383-2551 of SEQ ID NO:6, and amino acids 887-1051 of SEQ ID NO:7.
According to yet another embodiment, the epothilone synthase domain is a β-keto-reductase (KR) domain comprising an amino acid sequence substantially similar to an amino acid sequence selected from the group consisting of: amino acids 1439-1684 of SEQ ID NO:4, amino acids 1147-1399 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 1143-1393 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, and amino acids 1810-2055 of SEQ ID NO:7. According to this embodiment, said KR domain preferably comprises an amino acid sequence selected from the group consisting of: amino acids 1439-1684 of SEQ ID NO:4, amino acids 1147-1399 of SEQ ID NO:5, amino acids 2645-2895 of SEQ ID NO:5, amino acids 4729-4974 of SEQ ID NO:5, amino acids 6857-7101 of SEQ ID NO:5, amino acids 1143-1393 of SEQ ID NO:6, amino acids 3392-3636 of SEQ ID NO:6, and amino acids 1810-2055 of SEQ ID NO:7.
According to an additional embodiment, the epothilone synthase domain is a methyl-transferase (MT) domain comprising an amino acid sequence substantially similar to amino acids 2671-3045 of SEQ ID NO:6. According to this embodiment, said MT domain preferably comprises amino acids 2671-3045 of SEQ ID NO:6.
According to another embodiment, the epothilone synthase domain is a thioesterase (TE) domain comprising an amino acid sequence substantially similar to amino acids 2165-2439 of SEQ ID NO:7. According to this embodiment, said TE domain preferably comprises amino acids 2165-2439 of SEQ ID NO:7.
Other aspects and advantages of the present invention will become apparent to those skilled in the art from a study of the following description of the invention and non-limiting examples.
DEFINITIONS
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
Associated With/Operatively Linked: Refers to two DNA sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
Chimeric Gene: A recombinant DNA sequence in which a promoter or regulatory DNA sequence is operatively linked to, or associated with, a DNA sequence that codes for an mRNA or which is expressed as a protein, such that the regulator DNA sequence is able to regulate transcription or expression of the associated DNA sequence. The regulator DNA sequence of the chimeric gene is not normally operatively linked to the associated DNA sequence as found in nature.
Coding DNA Sequence: A DNA sequence that is translated in an organism to produce a protein.
Domain: That part of a polyketide synthase necessary for a given distinct activity. Examples include acyl carrier protein (ACP), β-ketosynthase (KS), acyltransferase (AT), β-ketoreductase (KR), dehydratase (DH), enoylreductase (ER), and thioesterase (TE) domains.
Epothilones: 16-membered macrocyclic polyketides naturally produced by the bacterium Sorangium cellulosum strain So ce90, which mimic the biological effects of taxol. In this application, “epothilone” refers to the class of polyketides that includes epothilone A and epothilone B, as well as analogs thereof such as those described in WO 98/25929.
Epothilone Synthase: A polyketide synthase responsible for the biosynthesis of epothilone.
Gene: A defined region that is located within a genome and that, besides the aforementioned coding DNA sequence, comprises other, primarily regulatory, DNA sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion.
Heterologous DNA Sequence: A DNA sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring DNA sequence.
Homologous DNA Sequence: A DNA sequence naturally associated with a host cell into which it is introduced.
Homologous Recombination: Reciprocal exchange of DNA fragments between homologous DNA molecules.
Isolated: In the context of the present invention, an isolated nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
Module: A genetic element encoding all of the distinct activities required in a single round of polyketide biosynthesis, i.e., one condensation step and all the β-carbonyl processing steps associated therewith. Each module encodes an ACP, a KS, and an AT activity to accomplish the condensation portion of the biosynthesis, and selected post-condensation activities to effect the β-carbonyl processing.
NRPS: A non-ribosomal polypeptide synthetase, which is a complex of enzymatic activities responsible for the incorporation of amino acids into secondary metabolites including, for example, amino acid adenylation, epimerization, N-methylation, cyclization, peptidyl carrier protein, and condensation domains. A functional NRPS is one that catalyzes the incorporation of an amino acid into a secondary metabolite.
NRPS gene: One or more genes encoding NRPSs for producing functional secondary metabolites, e.g., epothilones A and B, when under the direction of one or more compatible control elements.
Nucleic Acid Molecule: A linear segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA.
ORF: Open Reading Frame.
PKS: A polyketide synthase, which is a complex of enzymatic activities (domains) responsible for the biosynthesis of polyketides including, for example, ketoreductase, dehydratase, acyl carrier protein, enoylreductase, ketoacyl ACP synthase, and acyltransferase. A functional PKS is one that catalyzes the synthesis of a polyketide.
PKS Genes: One or more genes encoding various polypeptides required for producing functional polyketides, e.g., epothilones A and B, when under the direction of one or more compatible control elements.
Substantially Similar: With respect to nucleic acids, a nucleic acid molecule that has at least 60 percent sequence identity with a reference nucleic acid molecule. In a preferred embodiment, a substantially similar DNA sequence is at least 80% identical to a reference DNA sequence; in a more preferred embodiment, a substantially similar DNA sequence is at least 90% identical to a reference DNA sequence; and in a most preferred embodiment, a substantially similar DNA sequence is at least 95% identical to a reference DNA sequence. A substantially similar DNA sequence preferably encodes a protein or peptide having substantially the same activity as the protein or peptide encoded by the reference DNA sequence. A substantially similar nucleotide sequence typically hybridizes to a reference nucleic acid molecule, or fragments thereof, under the following conditions: hybridization at 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4 pH 7.0, 1 mM EDTA at 50° C.; wash with 2×SSC, 1% SDS, at 50° C. With respect to proteins or peptides, a substantially similar amino acid sequence is an amino acid sequence that is at least 90% identical to the amino acid sequence of a reference protein or peptide and has substantially the same activity as the reference protein or peptide.
Transformation: A process for introducing heterologous nucleic acid into a host cell or organism.
Transformed/Transgenic/Recombinant: Refers to a host organism such as a bacterium into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A “non-transformed”, “non-transgenic”, or “non-recombinant” host refers to a wild-type organism, i.e., a bacterium, which does not contain the heterologous nucleic acid molecule.
Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise indicated by the following standard abbreviations: alanine (ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (IIe; I), leucine (Leu; L), lysine (lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Furthermore, (Xaa; X) represents any amino acid.
DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID NO:1 is the nucleotide sequence of a 68750 bp contig containing 22 open reading frames (ORFs), which comprises the epothilone biosynthesis genes.
SEQ ID NO:2 is the protein sequence of a type I polyketide synthase (EPOS A) encoded by epoA (nucleotides 7610-11875 of SEQ ID NO:1).
SEQ ID NO:3 is the protein sequence of a non-ribosomal peptide synthetase (EPOS P) encoded by epoP (nucleotides 11872-16104 of SEQ ID NO:1).
SEQ ID NO:4 is the protein sequence of a type I polyketide synthase (EPOS B) encoded by epoB (nucleotides 16251-21749 of SEQ ID NO:1).
SEQ ID NO:5 is the protein sequence of a type I polyketide synthase (EPOS C) encoded by epoC (nucleotides 21746-43519 of SEQ ID NO:1).
SEQ ID NO:6 is the protein sequence of a type I polyketide synthase (EPOS D) encoded by epoD (nucleotides 43524-54920 of SEQ ID NO:1).
SEQ ID NO:7 is the protein sequence of a type I polyketide synthase (EPOS E) encoded by epoE (nucleotides 54935-62254 of SEQ ID NO:1).
SEQ ID NO:8 is the protein sequence of a cytochrome P450 oxygenase homologue (EPOS F) encoded by epoF (nucleotides 62369-63628 of SEQ ID NO:1).
SEQ ID NO:9 is a partial protein sequence (partial Orf 1) encoded by orf1 (nucleotides 1-1826 of SEQ ID NO:1).
SEQ ID NO:10 is a protein sequence (Orf 2) encoded by orf2 (nucleotides 3171-1900 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:11 is a protein sequence (Orf 3) encoded by orf3 (nucleotides 3415-5556 of SEQ ID NO:1).
SEQ ID NO:12 is a protein sequence (Orf 4) encoded by orf4 (nucleotides 5992-5612 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:13 is a protein sequence (Orf 5) encoded by orf5 (nucleotides 6226-6675 of SEQ ID NO:1).
SEQ ID NO:14 is a protein sequence (Orf 6) encoded by orf6 (nucleotides 63779-64333 of SEQ ID NO:1).
SEQ ID NO:15 is a protein sequence (Orf 7) encoded by orf7 (nucleotides 64290-63853 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:16 is a protein sequence (Orf 8) encoded by orf8 (nucleotides 64363-64920 of SEQ ID NO:1).
SEQ ID NO:17 is a protein sequence (Orf 9) encoded by orf9 (nucleotides 64727-64287 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:18 is a protein sequence (Orf 10) encoded by orf10 (nucleotides 65063-65767 of SEQ ID NO:1).
SEQ ID NO:19 is a protein sequence (Orf 11) encoded by orf11 (nucleotides 65874-65008 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:20 is a protein sequence (Orf 12) encoded by orf12 (nucleotides 66338-65871 on the reverse complement strand of SEQ ID NO:1).
SEQ ID NO:21 is a protein sequence (Orf 13) encoded by orf13 (nucleotides 66667-67137 of SEQ ID NO:1).
SEQ ID NO:22 is a protein sequence (Orf 14) encoded by orf14 (nucleotides 67334-68251 of SEQ ID NO:1).
SEQ ID NO:23 is a partial protein sequence (partial Orf 15) encoded by orf15 (nucleotides 68346-68750 of SEQ ID NO:1).
SEQ ID NO:24 is the universal reverse PCR primer sequence.
SEQ ID NO:25 is the universal forward PCR primer sequence.
SEQ ID NO:26 is the NH24 end “B” PCR primer sequence.
SEQ ID NO:27 is the NH2 end “A” PCR primer sequence.
SEQ ID NO:28 is the NH2 end “B” PCR primer sequence.
SEQ ID NO:29 is the pEPO15-NH6 end “B” PCR primer sequence.
SEQ ID NO:30 is the pEPO15-H2.7 end “A” PCR primer sequence.
DEPOSIT INFORMATION
The following material has been deposited with the Agricultural Research Service, Patent Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon the granting of a patent.















Deposited Material
Accession Number
Deposit Date









pEPO15
NRRL B-30033
Jun. 11, 1998



pEPO32
NRRL B-30119
Apr. 16, 1999










DETAILED DESCRIPTION OF THE INVENTION
The genes involved in the biosynthesis of epothilones can be isolated using the techniques according to the present invention. The preferable procedure for the isolation of epothilone biosynthesis genes requires the isolation of genomic DNA from an organism identified as producing epothilones A and B, and the transfer of the isolated DNA on a suitable plasmid or vector to a host organism that does not normally produce the polyketide, followed by the identification of transformed host colonies to which the epothilone-producing ability has been conferred. Using a technique such as λ::Tn5 transposon mutagenesis (de Bruijn & Lupski, Gene 27: 131-149 (1984)), the exact region of the transforming epothilone-conferring DNA can be more precisely defined. Alternatively or additionally, the transforming epothilone-conferring DNA can be cleaved into smaller fragments and the smallest that maintains the epothilone-conferring ability further characterized. Whereas the host organism lacking the ability to produce epothilone may be a different species from the organism from which the polyketide derives, a variation of this technique involves the transformation of host DNA into the same host that has had its epothilone-producing ability disrupted by mutagenesis. In this method, an epothilone-producing organism is mutated and non-epothilone-producing mutants are isolated. These are then complemented by genomic DNA isolated from the epothilone-producing parent strain.
A further example of a technique that can be used to isolate genes required for epothilone biosynthesis is the use of transposon mutagenesis to generate mutants of an epothilone-producing organism that, after mutagenesis, fails to produce the polyketide. Thus, the region of the host genome responsible for epothilone production is tagged by the transposon and can be recovered and used as a probe to isolate the native genes from the parent strain. PKS genes that are required for the synthesis of polyketides and that are similar to known PKS genes may be isolated by virtue of their sequence homology to the biosynthetic genes for which the sequence is known, such as those for the biosynthesis of rifamycin or soraphen. Techniques suitable for isolation by homology include standard library screening by DNA hybridization.
Preferred for use as a probe molecule is a DNA fragment that is obtainable from a gene or another DNA sequence that plays a part in the synthesis of a known polyketide. A preferred probe molecule comprises a 1.2 kb Smal DNA fragment encoding the ketosynthase domain of the fourth module of the soraphen PKS (U.S. Pat. No. 5,716,849), and a more preferred probe molecule comprises the β-ketoacyl synthase domains from the first and second modules of the rifamycin PKS (Schupp et al., FEMS Microbiology Letters 159: 201-207 (1998)). These can be used to probe a gene library of an epothilone-producing microorganism to isolate the PKS genes responsible for epothilone biosynthesis.
Despite the well-known difficulties with PKS gene isolation in general and despite the difficulties expected to be encountered with the isolation of epothilone biosynthesis genes in particular, by using the methods described in the instant specification, biosynthetic genes for epothilones A and B can surprisingly be cloned from a microorganism that produces that polyketide. Using the methods of gene manipulation and recombinant production described in this specification, the cloned PKS genes can be modified and expressed in transgenic host organisms.
The isolated epothilone biosynthetic genes can be expressed in heterologous hosts to enable the production of the polyketide with greater efficiency than might be possible from native hosts. Techniques for these genetic manipulations are specific for the different available hosts and are known in the art. For example, heterologous genes can be expressed in Streptomyces and other actinomycetes using techniques such as those described in McDaniel et al., Science 262: 1546-1550 (1993) and Kao et al., Science 265: 509-512 (1994), both of which are incorporated herein by reference. See also, Rowe et al., Gene 216: 215-223 (1998); Holmes et al., EMBO Journal 12(8): 3183-3191 (1993) and Bibb et al., Gene 38: 215-226 (1985), all of which are incorporated herein by reference.
Alternately, genes responsible for polyketide biosynthesis, i.e., epothilone biosynthetic genes, can also be expressed in other host organisms such as pseudomonads and E. coli. Techniques for these genetic manipulations are specific for the different available hosts and are known in the art. For example, PKS genes have been sucessfully expressed in E. coli using the pT7-7 vector, which uses the T7 promoter. See, Tabor et al., Proc. Natl. Acad. Sci. USA 82: 1074-1078 (1985), incorporated herein by reference. In addition, the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coli, either in transcriptional or translational fusion, behind the tac or trc promoter. For the expression of operons encoding multiple ORFs, the simplest procedure is to insert the operon into a vector such as pKK223-3 in transcriptional fusion, allowing the cognate ribosome binding site of the heterologous genes to be used. Techniques for overexpression in gram-positive species such as Bacillus are also known in the art and can be used in the context of this invention (Quax et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et al., American Society for Microbiology, Washington (1993)).
Other expression systems that may be used with the epothilone biosynthetic genes of the invention include yeast and baculovirus expression systems. See, for example, “The Expression of Recombinant Proteins in Yeasts,” Sudbery, P. E., Curr. Opin. Biotechnol. 7(5): 517-524 (1996); “Methods for Expressing Recombinant Proteins in Yeast,” Mackay, et al., Editor(s): Carey, Paul R., Protein Eng. Des. 105-153, Publisher: Academic, San Diego, Calif. (1996); “Expression of heterologous gene products in yeast,” Pichuantes, et al., Editor(s): Cleland, J. L., Craik, C. S., Protein Eng. 129-161, Publisher: Wiley-Liss, New York, N.Y. (1996); WO 98/27203; Kealey et al., Proc. Natl. Acad. Sci. USA 95: 505-509 (1998); “Insect Cell Culture: Recent Advances, Bioengineering Challenges And Implications In Protein Production,” Palomares, et al., Editor(s): Galindo, Enrique; Ramirez, Octavio T., Adv. Bioprocess Eng. Vol. II, Invited Pap. Int. Symp., 2nd (1998) 25-52, Publisher: Kluwer, Dordrecht, Neth; “Baculovirus Expression Vectors,” Jarvis, Donald L., Editor(s): Miller, Lois K., Baculoviruses 389-431, Publisher: Plenum, New York, N.Y. (1997); “Production Of Heterologous Proteins Using The Baculovirus/Insect Expression System,” Grittiths, et al., Methods Mol. Biol. (Totowa, N.J.) 75 (Basic Cell Culture Protocols (2nd Edition)) 427-440 (1997); and “Insect Cell Expression Technology,” Luckow, Verne A., Protein Eng. 183-218, Publisher: Wiley-Liss, New York, N.Y. (1996); all of which are incorporated herein by reference.
Another consideration for expression of PKS genes in heterologous hosts is the requirement of enzymes for posttranslational modification of PKS enzymes by phosphopante-theinylation before they can synthesize polyketides. However, the enzymes responsible for this modification of type I PKS enzymes, phosphopantetheinyl (P-pant) transferases are not normally present in many hosts such as E. coli. This problem can be solved by coexpression of a P-pant transferase with the PKS genes in the heterologous host, as described by Kealey et al., Proc. Natl. Acad. Sci. USA 95: 505-509 (1998), incorporated herein by reference.
Therefore, for the purposes of polyketide production, the significant criteria in the choice of host organism are its ease of manipulation, rapidity of growth (i.e. fermentation), possession or the proper molecular machinery for processes such as posttranslational modification, and its lack of susceptibility to the polyketide being overproduced. Most preferred host organisms are actinomycetes such as strains of Streptomyces. Other preferred host organisms are pseudomonads and E. coli. The above-described methods of polyketide production have significant advantages over the technology currently used in the preparation of the compounds. These advantages include the cheaper cost of production, the ability to produce greater quantities of the compounds, and the ability to produce compounds of a preferred biological enantiomer, as opposed to racemic mixtures inevitably generated by organic synthesis. Compounds produced by heterologous hosts can be used in medical (e.g. cancer treatment in the case of epothilones) as well as agricultural applications.
EXPERIMENTAL
The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989); and by T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984).


EXAMPLE 1
Cultivation of an Epothilone-Producing Strain of Sorangium cellulosum 
Sorangium cellulosum strain 90 (DSM 6773, Deutsche Sammlung von Mikroorganis-men und Zellkulturen, Braunschweig) is streaked out and grown (30° C.) on an agar plate of SolE medium (0.35% glucose, 0.05% tryptone, 0.15% MgSO4×7H2O, 0.05% ammonium sulfate, 0.1% CaCl2, 0.006% K2HPO4, 0.01% sodium dithionite, 0.0008% Fe-EDTA, 1.2% HEPES, 3.5% [vol/vol] supernatant of sterilized stationary S. cellulosum culture) pH ad. 7.4. Cells from about 1 square cm are picked and inoculated into 5 mls of G51t liquid medium (0.2% glucose, 0.5% starch, 0.2% tryptone, 0.1% probion S, 0.05% CaCl2×2H2O, 0.05% MgSO4×7H2O, 1.2% HEPES, pH ad. 7.4) and incubated at 30° C. with shaking at 225 rpm. After 4 days, the culture is transferred into 50 mls of G51t and incubated as above for 5 days. This culture is used to inoculate 500 mls of G51t and incubated as above for 6 days. The culture is centrifuged for 10 minutes at 4000 rpm and the cell pellet is resuspended in 50 mls of G51t.
EXAMPLE 2
Generation of a Bacterial Artificial Chromosome (Bac) Library
To generate a Bac library, S. cellulosum cells cultivated as described in Example 1 above are embedded into agarose blocks, lysed, and the liberated genomic DNA is partially digested by the restriction enzyme HindIII. The digested DNA is separated on an agarose gel by pulsed-field electrophoresis. Large (approximately 90-150 kb) DNA fragments are isolated from the agarose gel and ligated into the vector pBelobacll. pBelobacll contains a gene encoding chloramphenicol resistance, a multiple cloning site in the lacZ gene providing for blue/white selection on appropriate medium, as well as the genes required for the replication and maintenance of the plasmid at one or two copies per cell. The ligation mixture is used to transform Escherichia coli DH10B electrocompetent cells using standard electroporation techniques. Chloramphenicol-resistant recombinant (white, lacZ mutant) colonies are transferred to a positively charged nylon membrane filter in 384 3×3 grid format. The clones are lysed and the DNA is cross-linked to the filters. The same clones are also preserved as liquid cultures at −80° C.
EXAMPLE 3
Screening the Bac Library of Sorangium cellulosum 90 for the Presence of Type I Polyketide Synthase-Related Sequences
The Bac library filters are probed by standard Southern hybridization procedures. The DNA probes used encode β-ketoacyl synthase domains from the first and second modules of the rifamycin polyketide synthase (Schupp et al., FEMS Microbiology Letters 159: 201-207 (1998)). The probe DNAs are generated by PCR with primers flanking each ketosynthase domain using the plasmid pNE95 as the template (pNE95 equals cosmid 2 described in Schupp et al. (1998)). 25 ng of PCR-amplified DNA is isolated from a 0.5% agarose gel and labeled with 32P-dCTP using a random primer labeling kit (Gibco-BRL, Bethesda Md., USA) according to the manufacturer's instructions. Hybridization is at 65° C. for 36 hours and membranes are washed at high stringency (3 times with 0.1×SSC and 0.5% SDS for 20 min at 65° C.). The labeled blot is exposed on a phosphorescent screen and the signals are detected on a PhosphoImager 445SI (screen and 445SI from Molecular Dynamics). This results in strong hybridization of certain Bac clones to the probes. These clones are selected and cultured overnight in 5 mls of Luria broth (LB) at 37° C. Bac DNA from the Bac clones of interest is isolated by a typical miniprep procedure. The cells are resuspended in 200 μl lysozyme solution (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, 5 mg/ml lysozyme), lysed in 400 μl lysis solution (0.2 N NaOH and 2% SDS), the proteins are precipitated (3.0 M potassium acetate, adjusted to pH5.2 with acetic acid), and the Bac DNA is precipitated with isopropanol. The DNA is resuspended in 20 μl of nuclease-free distilled water, restricted with BamHI (New England Biolabs, Inc.) and separated on a 0.7% agarose gel. The gel is blotted by Southern hybridization as described above and probed under conditions described above, with a 1.2 kb Smal DNA fragment encoding the ketosynthase domain of the fourth module of the soraphen polyketide synthase as the probe (see, U.S. Pat. No. 5,716,849). Five different hybridization patterns are observed. One clone representing each of the five patterns is selected and named pEPO15, pEPO20, pEPO30, pEPO31, and pEPO33, respectively.
EXAMPLE 4
Subcloning of BamHI Fragments from pEPO15, pEPO20, pEPO30, pEPO31, and pEPO33
The DNA of the five selected Bac clones is digested with BamHI and random fragments are subcloned into pBluescript II SK+(Stratagene) at the BamHI site. Subclones carrying inserts between 2 and 10 kb in size are selected for sequencing of the flanking ends of the inserts and also probed with the 1.2 Smal probe as described above. Subclones that show a high degree of sequence homology to known polyketide synthases and/or strong hybridization to the soraphen ketosynthase domain are used for gene disruption experiments.
EXAMPLE 5
Preparation of Streptomycin-Resistant Spontaneous Mutants of Sorangium cellulosum strain So ce90
0.1 ml of a three day old culture of Sorangium cellulosum strain So ce90, which is raised in liquid medium G52-H (0.2% yeast extract, 0.2% soyameal defatted, 0.8% potato starch, 0.2% glucose, 0.1% MgSO4×7H2O, 0.1% CaCl2×2H2O, 0.008% Fe-EDTA, pH ad 7.4 with KOH), is plated out on agar plates with SolE medium supplemented with 100 μg/ml streptomycin. The plates are incubated at 30° C. for 2 weeks. The colonies growing on this medium are streptomycin-resistant mutants, which are streaked out and cultivated once more on the same agar medium with streptomycin for purification. One of these streptomycin-resistant mutants is selected and is called BCE28/2.
EXAMPLE 6
Gene Disruptions in Sorangium cellulosum BCE28/2 Using the Subcloned BamHI Fragments
The BamHI inserts of the subclones generated from the five selected Bac clones as described above are isolated and ligated into the unique BamHI site of plasmid pCIB132 (see, U.S. Pat. No. 5,716,849). The pCIB132 derivatives carrying the inserts are transformed into Escherichia coli ED8767 containing the helper plasmid pUZ8 (Hedges and Matthew, Plasmid 2: 269-278 (1979). The transformants are used as donors in conjugation experiments with Sorangium cellulosum BCE28/2 as recipient. For the conjugation, 5-10×109 cells of Sorangium cellulosum BCE28/2 from an early stationary phase culture (reaching about 5×108 cells/ml) grown at 30° C. in liquid medium G51b (G51b equals medium G51t with tryptone replaced by peptone) are mixed in a 1:1 cellular ratio with a late-log phase culture (in LB liquid medium) of E. coli ED8767 containing pCIB132 derivatives carrying the subcloned BamHI fragments and the helper plasmid pUZ8. The mixed cells are then centrifuged at 4000 rpm for 10 minutes and resuspended in 0.5 ml G51b medium. This cell suspension is then plated as a drop in the center of a plate with So1E agar containg 50 mg/l kanamycin. The cells obtained after incubation for 24 hours at 30° C. are harvested and resuspended in 0.8 ml of G51b medium, and 0.1 to 0.3 ml of this suspension is plated out on a selective So1E solid medium containing phleomycin (30 mg/l), streptomycin (300 mg/l), and kanamycin (50 mg/l). The counterselection of the donor Escherichia coli strain takes place with the aid of streptomycin. The colonies that grow on this selective medium after an incubation time of 8-12 days at a temperature of 30° C. are isolated with a plastic loop and streaked out and cultivated on the same agar medium for a second round of selection and purification. The colony-derived cultures that grow on this selective agar medium after 7 days at a temperature of 30° C. are transconjugants of Sorangium cellulosum BCE28/2 that have acquired phleomycin resistance by conjugative transfer of the pCIB132 derivatives carrying the subcloned BamHI fragments.
Integration of the pCIB132-derived plasmids into the chromosome of Sorangium cellulosum BCE28/2 by homologous recombination is verified by Southern hybridization. For this experiment, complete DNA from 5-10 tranconjugants per transferred BamHI fragment is isolated (from 10 ml cultures grown in medium G52-H for three days) applying the method described by Pospiech and Neumann, Trends Genet. 11: 217 (1995). For the Southern blot, the DNA isolated as described above is cleaved either with the restriction enzymes Bg/II, ClaI, or NotI, and the respective BamHI inserts or pCIB132 are used as 32P labelled probes.
EXAMPLE 7
Analysis of the Effect of the Integrated BamHI Fragments on Epothilone Production by Sorangium cellulosum After Gene Disruption
Transconjugant cells grown on about 1 square cm surface of the selective So1E plates of the second round of selection (see Example 6) are transferred by a sterile plastic loop into 10 ml of medium G52-H in an 50 ml Erlenmeyer flask. After incubation at 30° C. and 180 rpm for 3 days, the culture is transfered into 50 ml of medium G52-H in an 200 ml Erlenmeyer flask. After incubation at 30° C. and 180 rpm for 4-5 days, 10 ml of this culture is transfered into 50 ml of medium 23B3 (0.2% glucose, 2% potato starch, 1.6% soya meal defatted, 0.0008% Fe-EDTA Sodium salt, 0.5% HEPES (4-(2-hydroxyethyl)-piperazine-1-ethane-sulfonic-acid), 2% vol/vol polysterole resin XAD16 (Rohm & Haas), pH adjusted to 7.8 with NaOH) in an 200 ml Erlenmeyer flask.
Quantitative determination of the epothilone produced takes place after incubation of the cultures at 30° C. and 180 rpm for 7 days. The complete culture broth is filtered by suction through a 150 μm nylon filter. The resin remaining on the filter is then resuspended in 10 ml isopropanol and extracted by shaking the suspension at 180 rpm for 1 hour. 1 ml is removed from this suspension and centrifuged at 12,000 rpm in an Eppendorff Microfuge. The amount of epothilones A and B therein is determined by means of an HPLC and detection at 250 nm with a UV_DAD detector (HPLC with Waters-Symetry C18 column and a gradient of 0.02% phosphoric acid 60%-0% and acetonitril 40%-100%).
Transconjugants with three different integrated BamHI fragments subcloned from pEPO15, namely transconjugants with the BamHI fragment of plasmid pEPO15-21, transconjugants with the BamHI fragment of plasmid pEPO15-4-5, and transconjugants with the BamHI fragment of plasmid pEPO15-4-1, are tested in the manner described above. HPLC analysis reveals that all transconjugants no longer produce epothilone A or B. By contrast, epothilone A and B are detectable in a concentration of 2-4 mg/l in transconjugants with BamHI fragments integrated that are derived from pEPO20, pEPO30, pEPO31, pEPO33, and in the parental strain BCE28/2.
EXAMPLE 8
Nucleotide Sequence Determination of the Cloned Fragments and Construction of Contigs
A. BamHI Insert of Plasmid pEPO15-21
Plasmid DNA is isolated from the strain Escherichia coli DH10B [pEPO15-21], and the nucleotide sequence of the 2.3-kb BamHI insert in pEPO15-21 is determined. Automated DNA sequencing is done on the double-stranded DNA template by the dideoxynucleotide chain termination method, using Applied Biosystems model 377 sequencers. The primers used are the universal reverse primer (5′ GGA AAC AGC TAT GAC CAT G 3′ (SEQ ID NO:24)) and the universal forward primer (5′ GTA AAA CGA CGG CCA GT 3′ (SEQ ID NO:25)). In subsequent rounds of sequencing reactions, custom-synthesized oligonucleotides, designed for the 3′ ends of the previously determined sequences, are used to extend and join contigs. Both strands are entirely sequenced, and every nucleotide is sequenced at least two times. The nucleotide sequence is compiled using the program Sequencher vers. 3.0 (Gene Codes Corporation), and analyzed using the University of Wisconsin Genetics Computer Group programs. The nucleotide sequence of the 2213-bp insert corresponds to nucleotides 20779-22991 of SEQ ID NO:1.
B. BamHI Insert of Plasmid pEPO15-4-1
Plasmid DNA is isolated from the strain Escherichia coli DH10B [pEPO15-4-1], and the nucleotide sequence of the 3.9-kb BamHI insert in pEPO15-4-1 is determined as described in (A) above. The nucleotide sequence of the 3909-bp insert corresponds to nucleotides 16876-20784 of SEQ ID NO:1.
C. BamHI Insert of Plasmid pEPO15-4-5
Plasmid DNA is isolated from the strain Escherichia coli DH10B [pEPO15-4-5], and the nucleotide sequence of the 2.3-kb BamHI insert in pEPO15-4-5 is determined as described in (A) above. The nucleotide sequence of the 2233-bp insert corresponds to nucleotides 42528-44760 of SEQ ID NO:1.
EXAMPLE 9
Subcloning and Ordering of DNA Fragments from pEPO15 Containing Epothilone Biosynthesis Genes
pEPO15 is digested to completion with the restriction enzyme HindIII and the resulting fragments are subcloned into pBluescript II SK−or pNEB193 (New England Biolabs) that has been cut with HindIII and dephosphorylated with calf intestinal alkaline phosphatase. Six different clones are generated and named pEPO15-NH1, pEPO15-NH2, pEPO15-NH6, pEPO15-NH24 (all based on pNEB193), and pEPO15-H2.7 and pEPO15-H3.0 (both based on pBluescript II SK−).
The BamHI insert of pEPO15-21 is isolated and DIG-labeled (Non-radioactive DNA labeling and detection system, Boehringer Mannheim), and used as a probe in DNA hybridization experiments at high stringency against pEPO15-NH1, pEPO15-NH2, pEPO15-NH6, pEPO15-NH24, pEPO15-H2.7 and pEPO15-H3.0. Strong hybridization signal is detected for pEPO15-NH24, indicating that pEPO15-21 is contained within pEPO15-NH24.
The BamHI insert of pEPO15-4-1 is isolated and DIG-labeled as above, and used as a probe in DNA hybridization experiments at high stringency against pEPO15-NH1, pEPO15-NH2, pEPO15-NH6, pEPO15-NH24, pEPO15-H2.7 and pEPO15-H3.0. Strong hybridization signals are detected for pEPO15-NH24 and pEPO15-H2.7. Nucleotide sequence data generated from one end each of pEPO15-NH24 and pEPO15-H2.7 are also in complete agreement with the previously determined sequence of the BamHI insert of pEPO15-4-1. These experiments demonstrate that pEPO15-4-1 (which contains one internal HindIII site) overlaps pEPO15-H2.7 and pEPO15-NH24, and that pEPO15-H2.7 and pEPO15-NH24, in this order, are contiguous.
The BamHI insert of pEPO15-4-5 is isolated and DIG-labeled as above, and used as a probe in DNA hybridization experiments at high stringency against pEPO15-NH1, pEPO15-NH2, pEPO15-NH6, pEPO15-NH24, pEPO15-H2.7 and pEPO15-H3.0. Strong hybridization signal is detected for pEPO15-NH2, indicating that pEPO15-21 is contained within pEPO15-NH2.
Nucleotide sequence data is generated from both ends of pEPO15-NH2 and from the end of pEPO15-NH24 that does not overlap with pEPO15-4-1. PCR primers NH24 end “B”: GTGACTGGCGCCTGGAATCTGCATGAGC (SEQ ID NO:26), NH2 end “A”: AGCGGGAGCTTGCTAGACATTCTGTTTC (SEQ ID NO:27), and NH2 end “B”: GACGCGCCTCGGGCAGCGCCCCAA (SEQ ID NO:28), pointing towards the HindIII sites, are designed based on these sequences and used in amplification reactions with pEPO15 and, in separate experiments, with Sorangium cellulosum So ce90 genomic DNA as the templates. Specific amplification is found with primer pair NH24 end “B” and NH2 end “A” with both templates. The amplimers are cloned into pBluescript II SK− and completely sequenced. The sequences of the amplimers are identical, and also agree completely with the end sequences of pEPO15-NH24 and pEPO15-NH2, fused at the HindIII site, establishing that the HindIII fragments of pEPO15-NH2 and pEPO15-NH24 are, in this order, contiguous.
The HindIII insert of pEPO15-H2.7 is isolated and DIG-labeled as above, and used as a probe in a DNA hybridization experiment at high stringency against pEPO15 digested by NotI. A NotI fragment of about 9 kb in size shows a strong a hybridization, and is further subcloned into pBluescript II SK− that has been digested with NotI and dephosphorylated with calf intestinal alkaline phosphatase, to yield pEPO15-N9-16. The NotI insert of pEPO15-N9-16 is isolated and DIG-labeled as above, and used as a probe in DNA hybridization experiments at high stringency against pEPO15-NH1, pEPO15-NH2, pEPO15-NH6, pEPO15-NH24, pEPO15-H2.7 and pEPO15-H3.0. Strong hybridization signals are detected for pEPO15-NH6, and also for the expected clones pEPO15-H2.7 and pEPO15-NH24. Nucleotide sequence data is generated from both ends of pEPO15-NH6 and from the end of pEPO15-H2.7 that does not overlap with pEPO15-4-1. PCR primers are designed pointing towards the HindIII sites and used in amplification reactions with pEPO15 and, in separate experiments, with Sorangium cellulosum So ce90 genomic DNA as the templates. Specific amplification is found with primer pair pEPO15-NH6 end “B”: CACCGAAGCGTCGATCTGGTCCATC (SEQ ID NO:29) and pEPO15-H2.7 end “A”: CGGTCAGATCGACGACGGGCTTTCC (SEQ ID NO:30) with both templates. The amplimers are cloned into pBluescript II SK− and completely sequenced. The sequences of the amplimers are identical, and also agree completely with the end sequences of pEPO15-NH6 and pEPO15-H2.7, fused at the HindIII site, establishing that the HindIII fragments of pEPO15-NH6 and pEPO15-H2.7 are, in this order, contiguous.
All of these experiments, taken together, establish a contig of HindIII fragments covering a region of about 55 kb and consisting of the HindIII inserts of pEPO15-NH6, pEPO15-H2.7, pEPO15-NH24, and pEPO15-NH2, in this order. The inserts of the remaining two HindIII subclones, namely pEPO15-NH1 and pEPO15-H3.0, are not found to be parts of this contig.
EXAMPLE 10
Further Extension of the Subclone Contig Covering the Epothilone Biosynthesis Genes
An approximately 2.2 kb BamHI-HindIII fragment derived from the downstream end of the insert of pEPO15-NH2 and thus representing the downstream end of the subclone contig described in Example 9 is isolated, DIG-labeled, and used in Southern hybridization experiments against pEPO15 and pEPO15-NH2 DNAs digested with several enzymes. The strongly hybridizing bands are always found to be the same in size between the two target DNAs indicating that the Sorangium cellulosum So ce90 genomic DNA fragment cloned into pEPO15 ends with the HindIII site at the downstream end of pEPO15-NH2.
A cosmid DNA library of Sorangium cellulosum So ce90 is generated, using established procedures, in pScosTriplex-II (Ji, et al., Genomics 31: 185-192 (1996)). Briefly, high-molecular weight genomic DNA of Sorangium cellulosum So ce90 is partially digested with the restriction enzyme Sau3Al to provide fragments with average sizes of about 40 kb, and ligated to BamHI and XbaI digested pScosTriplex-II. The ligation mix is packaged with Gigapack III XL (Stratagene) and used to transfect E. coliXL1 Blue MR cells.
The cosmid library is screened with the approximately 2.2 kb BamHI-HindIII fragment, derived from the downstream end of the insert of pEPO15-NH2, used as a probe in colony hybridization. A strongly hybridizing clone, named pEPO4E7 is selected.
pEPO4E7 DNA is isolated, digested with several restriction endonucleases, and probed in Southern hybridization experiments with the 2.2 kb BamHI-HindIII fragment. A strongly hybridizing NotI fragment of approximately 9 kb in size is selected and subcloned into pBluescript II SK− to yield pEPO4E7-N9-8. Further Southern hybridization experiments reveal that the approximately 9 kb NotI insert of pEPO4E7-N9-8 overlaps pEPO15-NH2 over 6 kb in a NotI-HindIII fragment, while the remaining approximately 3 kb HindIII-NotI fragment would extend the subclone contig described in Example 9. End sequencing reveals, however, that the downstream end of the insert of pEPO4E7-N9-8 contains the BamHI-NotI polylinker of pScosTriplex-II, thereby indicating that the genomic DNA insert of pEPO4E7 ends at a Sau3AI site within the extending HindIII-NotI fragment and that the NotI site is derived from pScosTriplex-II.
An approximately 1.6 kb PstI—SalI fragment derived from the approximately 3 kb extending HindIII-NotI subfragment of pEPO4E7-N9-8, containing only Sorangium cellulosum So ce90-derived sequences free of vector, is used as a probe against the bacterial artificial chromosome library described in Example 2. Besides the previously-isolated EPO15, a Bac clone, named EPO32, is found to strongly hybridize to the probe. pEPO32 is isolated, digested with several restriction endonucleases, and hybridized with the approximately 1.6 kb PstI—SalI probe. A HindIII—EcoRV fragment of about 13 kb in size is found to strongly hybridize to the probe, and is subcloned into pBluescript II SK− digested with HindIII and HincII to yield pEPO32-HEV15.
Oligonucleotide primers are designed based on the downstream end sequence of pEPO15-NH2 and on the upstream (HindIII) end sequence derived from pEPO32-HEV15, and used in sequencing reactions with pEPO4E7-N9-8 as the template. The sequences reveal the existence of a small HindIII fragment (EPO4E7-H0.02) of 24 bp, undetectable in standard restriction analysis, separating the HindIII site at the downstream end of pEPO15-NH2 from the HindIII site at the upstream end of pEPO32-HEV15.
Thus, the subclone contig described in Example 9 is extended to include the HindIII fragment EPO4E7-H0.02 and the insert of pEPO32-HEV15, and constitutes the inserts of: pEPO15-NH6, pEPO15-H2.7, pEPO15-NH24, pEPO15-NH2, EPO4E7-H0.02 and pEPO32-HEV15, in this order.
EXAMPLE 11
Nucleotide Sequence Determination of the Subclone Contig Covering the Epothilone Biosynthesis Genes
The nucleotide sequence of the subclone contig described in Example 10 is determined as follows.
pEPO15-H2.7. Plasmid DNA is isolated from the strain Escherichia coliDH10B [pEPO15-H2.7], and the nucleotide sequence of the 2.7-kb BamHI insert in pEPO15-H2.7 is determined. Automated DNA sequencing is done on the double-stranded DNA template by the dideoxynucleotide chain termination method, using Applied Biosystems model 377 sequencers. The primers used are the universal reverse primer (5′ GGA AAC AGC TAT GAC CAT G 3′ (SEQ ID NO:24)) and the universal forward primer (5′ GTA AAA CGA CGG CCA GT 3′ (SEQ ID NO:25)). In subsequent rounds of sequencing reactions, custom-synthesized oligonucleotides, designed for the 3′ ends of the previously determined sequences, are used to extend and join contigs.
pEPO15-NH6, pEPO15-NH24 and pEPO15-NH2. The HindIII inserts of these plasmids are isolated, and subjected to random fragmentation using a Hydroshear apparatus (Genomic Instrumentation Services, Inc.) to yield an average fragment size of 1-2 kb. The fragments are end-repaired using T4 DNA Polymerase and Klenow DNA Polymerase enzymes in the presence of desoxynucleotide triphosphates, and phosphorylated with T4 DNA Kinase in the presence of ribo-ATP. Fragments in the size range of 1.5-2.2 kb are isolated from agarose gels, and ligated into pBluescript II SK− that has been cut with EcoRV and dephosphorylated. Random subclones are sequenced using the universal reverse and the universal forward primers.
pEPO32-HEV15. pEPO32-HEV15 is digested with HindIII and SspI, the approximately 13.3 kb fragment containing the  ̃13 kb HindIII—EcoRV insert from So. cellulosum So ce90 and a 0.3 kb HincII—SspI fragment from pBluescript II SK− is isolated, and partially digested with HaeIII to yield fragments with an average size of 1-2 kb. Fragments in the size range of 1.5-2.2 kb are isolated from agarose gels, and ligated into pBluescript II SK− that has been cut with EcoRV and dephosphorylated. Random subclones are sequenced using the universal reverse and the universal forward primers.
The chromatograms are analyzed and assembled into contigs with the Phred, Phrap and Consed programs (Ewing, et al., Genome Res. 8(3): 175-185 (1998); Ewing, et al., Genome Res. 8(3): 186-194 (1998); Gordon, et al, Genome Res.8(3): 195-202 (1998)). Contig gaps are filled, sequence discrepancies are resolved, and low-quality regions are resequenced using custom-designed oligonucleotide primers for sequencing on either the original subclones or selected clones from the random subclone libraries. Both strands are completely sequenced, and every basepair is covered with at least a minimum aggregated Phred score of 40 (confidence level of 99.99%).
The nucleotide sequence of the 68750 bp contig is shown as SEQ ID NO:1.
EXAMPLE 12
Nucleotide Sequence Analysis of the Epothilone Biosynthesis Genes
SEQ ID NO:1 is found to contain 22 ORFs as detailed below in Table 1:











TABLE 1






Start
Stop
Homology of
Proposed function


ORF
codon
codon
deduced protein
of deduced protein







orf1
outside of
 1826





sequenced



range


orf2 *
 3171
 1900
Hypothetical





protein





SP: Q11037;





DD-peptidase





SP: P15555


orf3
 3415
 5556
Na/H antiporter
Transport





PID: D1017724


orf4 *
 5992
 5612


orf5
 6226
 6675


epoA
 7610
11875
Type I polyketide
Epothilone synthase:





synthase
Thiazole ring






formation


epoP
11872
16104
Non-ribosomal
Epothilone synthase:





peptide synthetase
Thiazole ring






formation


epoB
16251
21749
Type I polyketide
Epothilone synthase:





synthase
Polyketide backbone






formation


epoC
21746
43519
Type I polyketide
Epothilone synthase:





synthase
Polyketide backbone






formation


epoD
43524
54920
Type I polyketide
Epothilone synthase:





synthase
Polyketide backbone






formation


epoE
54935
62254
Type I polyketide
Epothilone synthase:





synthase
Polyketide backbone






formation


epoF
62369
63628
Cytochrome P450
Epothilone






macrolactone






oxidase


orf6
63779
64333


orf7 *
64290
63853


orf8
64363
64920


orf9 *
64727
64287


orf10
65063
65767


orf11 *
65874
65008


orf12 *
66338
65871


orf13
66667
67137


orf14
67334
68251
Hypothetical
Transport





protein





GI: 3293544;





Cation efflux





system protein





GI: 2623026


orf15
68346
outside of




sequenced




range





* On the reverse complementer strand. Numbering according to SEQ ID NO:1. 






epoA (nucleotides 7610-11875 of SEQ ID NO:1) codes for EPOS A (SEQ ID NO:2), a type I polyketide synthase consisting of a single module, and harboring the following domains: β-ketoacyl-synthase (KS) (nucleotides 7643-8920 of SEQ ID NO:1, amino acids 11-437 of SEQ ID NO:2); acyltransferase (AT) (nucleotides 9236-10201 of SEQ ID NO:1, amino acids 543-864 of SEQ ID NO:2); enoyl reductase (ER) (nucleotides 10529-11428 of SEQ ID NO:1, amino acids 974-1273 of SEQ ID NO:2); and acyl carrier protein homologous domain (ACP) (nucleotides 11549-11764 of SEQ ID NO:1, amino acids 1314-1385 of SEQ ID NO:2). Sequence comparisons and motif analysis (Haydock, et al. FEBS Lett. 374: 246-248 (1995); Tang, et al., Gene 216: 255-265 (1998)) reveal that the AT encoded by EPOS A is specific for malonyl-CoA. EPOS A should be involved in the initiation of epothilone biosynthesis by loading the acetate unit to the multienzyme complex that will eventually form part of the 2-methylthiazole ring (C26 and C20).
epoP (nucleotides 11872-16104 of SEQ ID NO:1) codes for EPOS P (SEQ ID NO:3), a non-ribosomal peptide synthetase containing one module. EPOS P harbors the following domains:

    
    
        peptide bond formation domain, as delineated by motif K (amino acids 72-81 [FPLTDIQESY] of SEQ ID NO:3, corresponding to nucleotide positions 12085-12114 of SEQ ID NO:1); motif L (amino acids 118-125 [VVARHDML] of SEQ ID NO:3, corresponding to nucleotide positions 12223-12246 of SEQ ID NO:1); motif M (amino acids 199-212 [SIDLINVDLGSLSI] of SEQ ID NO:3, corresponding to nucleotide positions 12466-12507 of SEQ ID NO:1); and motif O (amino acids 353-363 [GDFTSMVLLDI] of SEQ ID NO:3, corresponding to nucleotide positions 12928-12960 of SEQ ID NO:1);
        aminoacyl adenylate formation domain, as delineated by motif A (amino acids 549-565 [LTYEELSRRSRRLGARL] of SEQ ID NO:3, corresponding to nucleotide positions 13516-13566 of SEQ ID NO:1); motif B (amino acids 588-603 [VAVLAVLESGAAYVPI] of SEQ ID NO:3, corresponding to nucleotide positions 13633-13680 of SEQ ID NO:1); motif C (amino acids 669-684 [AYVIYTSGSTGLPKGV] of SEQ ID NO:3, corresponding to nucleotide positions 13876-13923 of SEQ ID NO:1); motif D (amino acids 815-821 [SLGGATE] of SEQ ID NO:3, corresponding to nucleotide positions 14313-14334 of SEQ ID NO:1); motif E (amino acids 868-892 [GQLYIGGVGLALGYWRDEEKTRKSF] of SEQ ID NO:3, corresponding to nucleotide positions 14473-14547 of SEQ ID NO:1); motif F (amino acids 903-912 [YKTGDLGRYL] of SEQ ID NO:3, corresponding to nucleotide positions 14578-14607 of SEQ ID NO:1); motif G (amino acids 918-940 [EFMGREDNQIKLRGYRVELGEIE] of SEQ ID NO:3, corresponding to nucleotide positions 14623-14692 of SEQ ID NO:1); motif H (amino acids 1268-1274 [LPEYMVP] of SEQ ID NO:3, corresponding to nucleotide positions 15673-15693 of SEQ ID NO:1); and motif I (amino acids 1285-1297 [LTSNGKVDRKALR] of SEQ ID NO:3, corresponding to nucleotide positions 15724-15762 of SEQ ID NO:1);
        an unknown domain, inserted between motifs G and H of the aminoacyl adenylate formation domain (amino acids 973-1256 of SEQ ID NO:3, corresponding to nucleotide positions 14788-15639 of SEQ ID NO:1); and
        a peptidyl carrier protein homologous domain (PCP), delineated by motif J (amino acids 1344-1351 [GATSIHIV] of SEQ ID NO:3, corresponding to nucleotide positions 15901-15924 of SEQ ID NO:1).

It is proposed that EPOS P is involved in the activation of a cysteine by adenylation, binding the activated cysteine as an aminoacyl-S-PCP, forming a peptide bond between the enzyme-bound cysteine and the acetyl-S-ACP supplied by EPOS A, and the formation of the initial thiazoline ring by intramolecular heterocyclization. The unknown domain of EPOS P displays very weak homologies to NAD(P)H oxidases and reductases from Bacillus species. Thus, this unknown domain and/or the ER domain of EPOS A may be involved in the oxidation of the initial 2-methylthiazoline ring to a 2-methylthiazole.

    
    


epoB (nucleotides 16251-21749 of SEQ ID NO:1) codes for EPOS B (SEQ ID NO:4), a type I polyketide synthase consisting of a single module, and harboring the following domains: KS (nucleotides 16269-17546 of SEQ ID NO:1, amino acids 7-432 of SEQ ID NO:4); AT (nucleotides 17865-18827 of SEQ ID NO:1, amino acids 539-859 of SEQ ID NO:4); dehydratase (DH) (nucleotides 18855-19361 of SEQ ID NO:1, amino acids 869-1037 of SEQ ID NO:4); β-ketoreductase (KR) (nucleotides 20565-21302 of SEQ ID NO:1, amino acids 1439-1684 of SEQ ID NO:4); and ACP (nucleotides 21414-21626 of SEQ ID NO:1, amino acids 1722-1792 of SEQ ID NO:4). Sequence comparisons and motif analysis reveal that the AT encoded by EPOS B is specific for methylmalonyl-CoA. EPOS A should be involved in the first polyketide chain extension by catalysing the Claisen-like condensation of the 2-methyl-4-thiazolecarboxyl-S-PCP starter group with the methylmalonyl-S-ACP, and the concomitant reduction of the b-keto group of C17 to an enoyl.
epoC (nucleotides 21746-43519 of SEQ ID NO:1) codes for EPOS C (SEQ ID NO:5), a type I polyketide synthase consisting of 4 modules. The first module harbors a KS (nucleotides 21860-23116 of SEQ ID NO:1, amino acids 39-457 of SEQ ID NO:5); a malonyl CoA-specific AT (nucleotides 23431-24397 of SEQ ID NO:1, amino acids 563-884 of SEQ ID NO:5); a KR (nucleotides 25184-25942 of SEQ ID NO:1, amino acids 1147-1399 of SEQ ID NO:5); and an ACP (nucleotides 26045-26263 of SEQ ID NO:1, amino acids 1434-1506 of SEQ ID NO:5). This module incorporates an acetate extender unit (C14-C13) and reduces the β-keto group at C15 to the hydroxyl group that takes part in the final lactonization of the epothilone macrolactone ring. The second module of EPOS C harbors a KS (nucleotides 26318-27595 of SEQ ID NO:1, amino acids 1524-1950 of SEQ ID NO:5); a malonyl CoA-specific AT (nucleotides 27911-28876 of SEQ ID NO:1, amino acids 2056-2377 of SEQ ID NO:5); a KR (nucleotides 29678-30429 of SEQ ID NO:1, amino acids 2645-2895 of SEQ ID NO:5); and an ACP (nucleotides 30539-30759 of SEQ ID NO:1, amino acids 2932-3005 of SEQ ID NO:5). This module incorporates an acetate extender unit (C12-C11) and reduces the β-keto group at C13 to a hydroxyl group. Thus, the nascent polyketide chain of epothilone corresponds to epothilone A, and the incorporation of the methyl side chain at C12 in epothilone B would require a post-PKS C-methyltransferase activity. The formation of the epoxi ring at C13-C12 would also require a post-PKS oxidation step. The third module of EPOS C harbors a KS (nucleotides 30815-32092 of SEQ ID NO:1, amino acids 3024-3449 of SEQ ID NO:5); a malonyl CoA-specific AT (nucleotides 32408-33373 of SEQ ID NO:1, amino acids 3555-3876 of SEQ ID NO:5); a DH (nucleotides 33401-33889 of SEQ ID NO:1, amino acids 3886-4048 of SEQ ID NO:5); an ER (nucleotides 35042-35902 of SEQ ID NO:1, amino acids 4433-4719 of SEQ ID NO:5); a KR (nucleotides 35930-36667 of SEQ ID NO:1, amino acids 4729-4974 of SEQ ID NO:5); and an ACP (nucleotides 36773-36991 of SEQ ID. NO:1, amino acids 5010-5082 of SEQ ID NO:5). This module incorporates an acetate extender unit (C10-C9) and fully reduces the β-keto group at C11. The fourth module of EPOS C harbors a KS (nucleotides 37052-38320 of SEQ ID NO:1, amino acids 5103-5525 of SEQ ID NO:5); a methylmalonyl CoA-specific AT (nucleotides 38636-39598 of SEQ ID NO:1, amino acids 5631-5951 of SEQ ID NO:5); a DH (nucleotides 39635-40141 of SEQ ID NO:1, amino acids 5964-6132 of SEQ ID NO:5); an ER (nucleotides 41369-42256 of SEQ ID NO:1, amino acids 6542-6837 of SEQ ID NO:5); a KR (nucleotides 42314-43048 of SEQ ID NO:1, amino acids 6857-7101 of SEQ ID NO:5); and an ACP (nucleotides 43163-43378 of SEQ ID NO:1, amino acids 7140-7211 of SEQ ID NO:5). This module incorporates a propionate extender unit (C24 and C8-C7) and fully reduces the β-keto group at C9.
epoD (nucleotides 43524-54920 of SEQ ID NO:1) codes for EPOS D (SEQ ID NO:6), a type I polyketide synthase consisting of 2 modules. The first module harbors a KS (nucleotides 43626-44885 of SEQ ID NO:1, amino acids 35-454 of SEQ ID NO:6); a methylmalonyl CoA-specific AT (nucleotides 45204-46166 of SEQ ID NO:1, amino acids 561-881 of SEQ ID NO:6); a KR (nucleotides 46950-47702 of SEQ ID NO:1, amino acids 1143-1393 of SEQ ID NO:6); and an ACP (nucleotides 47811-48032 of SEQ ID NO:1, amino acids 1430-1503 of SEQ ID NO:6). This module incorporates a propionate extender unit (C23 and C6-C5) and reduces the β-keto group at C7 to a hydoxyl group. The second module harbors a KS (nucleotides 48087-49361 of SEQ ID NO:1, amino acids 1522-1946 of SEQ ID NO: 6); a methylmalonyl CoA-specific AT (nucleotides 49680-50642 of SEQ ID NO:1, amino acids 2053-2373 of SEQ ID NO:6); a DH (nucleotides 50670-51176 of SEQ ID NO:1, amino acids 2383-2551 of SEQ ID NO:6); a methyltransferase (MT, nucleotides 51534-52657 of SEQ ID NO:1, amino acids 2671-3045 of SEQ ID NO:6); a KR (nucleotides 53697-54431 of SEQ ID NO:1, amino acids 3392-3636 of SEQ ID NO:6); and an ACP (nucleotides 54540-54758 of SEQ ID NO:1, amino acids 3673-3745 of SEQ ID NO:6). This module incorporates a propionate extender unit (C21 or C22 and C4-C3) and reduces the β-keto group at C5 to a hydoxyl group. This reduction is somewhat unexpected, since epothilones contain a keto group at C5. Discrepancies of this kind between the deduced reductive capabilities of PKS modules and the redox state of the corresponding positions in the final polyketide products have been, however, reported in the literature (see, for example, Schwecke, et al., Proc. Nat. Acad. Sci. USA 92: 7839-7843 (1995) and Schupp, et al., FEMS Microbiology Letters 159: 201-207 (1998)). An important feature of epothilones is the presence of gem-methyl side groups at C4 (C21 and C22). The second module of EPOS D is predicted to incorporate a propionate unit into the growing polyketide chain, providing one methyl side chain at C4. This module also contains a methyltransferase domain integrated into the PKS between the DH and the KR domains, in an arrangement similar to the one seen in the HMWP1 yersiniabactin synthase (Gehring, A. M., DeMoll, E., Fetherston, J. D., Mori, I., Mayhew, G. F., Blattner, F. R., Walsh, C. T., and Perry, R. D.: Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis. Chem. Biol. 5, 573-586, 1998). This MT domain in EPOS D is proposed to be responsible for the incorporation of the second methyl side group (C21 or C22) at C4.
epoE (nucleotides 54935-62254 of SEQ ID NO:1) codes for EPOS E (SEQ ID NO:7), a type I polyketide synthase consisting of one module, harboring a KS (nucleotides 55028-56284 of SEQ ID NO:1, amino acids 32-450 of SEQ ID NO:7); a malonyl CoA-specific AT (nucleotides 56600-57565 of SEQ ID NO:1, amino acids 556-877 of SEQ ID NO:7); a DH (nucleotides 57593-58087 of SEQ ID NO:1, amino acids 887-1051 of SEQ ID NO:7); a probably nonfunctional ER (nucleotides 59366-60304 of SEQ ID NO:1, amino acids 1478-1790 of SEQ ID NO:7); a KR (nucleotides 60362-61099 of SEQ ID NO:1, amino acids 1810-2055 of SEQ ID NO:7); an ACP (nucleotides 61211-61426 of SEQ ID NO:1, amino acids 2093-2164 of SEQ ID NO:7); and a thioesterase (TE) (nucleotides 61427-62254 of SEQ ID NO:1, amino acids 2165-2439 of SEQ ID NO:7). The ER domain in this module harbors an active site motif with some highly unusual amino acid substitutions that probably render this domain inactive. The module incorporates an acetate extender unit (C2-C1), and reduces the β-keto at C3 to an enoyl group. Epothilones contain a hydroxyl group at C3, so this reduction also appears to be excessive as discussed for the second module of EPOS D. The TE domain of EPOS E takes part in the release and cyclization of the grown polyketide chain via lactonization between the carboxyl group of C1 and the hydroxyl group of C15.
Five ORFs are detected upstream of epoA in the sequenced region. The partially sequenced orf1 has no homologues in the sequence databanks. The deduced protein product (Orf 2, SEQ ID NO:10) of orf2 (nucleotides 3171-1900 on the reverse complement strand of SEQ ID NO:1) shows strong similarities to hypothetical ORFs from Mycobacterium and Streptomyces coelicolor, and more distant similarities to carboxypeptidases and DD-peptidases of different bacteria. The deduced protein product of orf3 (nucleotides 3415-5556 of SEQ ID NO:1), Orf 3 (SEQ ID NO:11), shows homologies to Na/H antiporters of different bacteria. Orf 3 might take part in the export of epothilones from the producer strain. orf4 and orf5 have no homologues in the sequence databanks.
Eleven ORFs are found downstream of epoE in the sequenced region. epoF (nucleotides 62369-63628 of SEQ ID NO:1) codes for EPOS F (SEQ ID NO:8), a deduced protein; with strong sequence similarities to cytochrome P450 oxygenases. EPOS F may take part in the adjustment of the redox state of the carbons C12, C5, and/or C3. The deduced protein product of orf14 (nucleotides 67334-68251 of SEQ ID NO:1), Orf 14 (SEQ ID NO:22) shows strong similarities to GI:3293544, a hypothetic protein with no proposed function from Streptomyces coelicolor, and also to GI:2654559, the human embrionic lung protein. It is also more distantly related to cation efflux system proteins like GI:2623026 from Methano-bacterium thermoautotrophicum, so it might also take part in the export of epothilones from the producing cells. The remaining ORFs (orf6-orf3 and orf15) show no homologies to entries in the sequence databanks.
EXAMPLE 13
Recombinant Expression of Epothilone Biosynthesis Genes
Epothilone synthase genes according to the present invention are expressed in heterologous organisms for the purposes of epothilone production at greater quantities than can be accomplished by fermentation of Sorangium cellulosum. A preferable host for heterologous expression is Streptomyces, e.g. Streptomyces coelicolor, which natively produces the polyketide actinorhodin. Techniques for recombinant PKS gene expression in this host are described in McDaniel et al., Science 262: 1546-1550 (1993) and Kao et al., Science 265: 509-512 (1994). See also, Holmes et al., EMBO Journal 12(8): 3183-3191 (1993) and Bibb et al., Gene 38: 215-226 (1985), as well as U.S. Pat. Nos. 5,521,077, 5,672,491, and 5,712,146, which are incorporated herein by reference.
According to one method, the heterologous host strain is engineered to contain a chromosomal deletion of the actinorhodin (act) gene cluster. Expression plasmids containing the epothilone synthase genes of the invention are constructed by transferring DNA from a temperature-sensitive donor plasmid to a recipient shuttle vector in E. coil (McDaniel et al. (1993) and Kao et al. (1994)), such that the synthase genes are built-up by homologous recombination within the vector. Alternatively, the epothilone synthase gene cluster is introduced into the vector by restriction fragment ligation. Following selection, e.g. as described in Kao et al. (1994), DNA from the vector is introduced into the act-minus Streptomyces coelicolor strain according to protocols set forth in Hopwood et al., Genetic Manipulation of Streptomyces. A Laboratory Manual (John Innes Foundation, Norwich, United Kingdom, 1985), incorporated herein by reference. The recombinant Streptomyces strain is grown on R2YE medium (Hopwood et al. (1985)) and produces epothilones. Alternatively, the epothilone synthase genes according to the present invention are expressed in other host organisms such as pseudomonads, Bacillus, yeast, insect cells and/or E. coli. PKS and NRPS genes are preferably expressed in E. coli using the pT7-7 vector, which uses the T7 promoter. See, Tabor et al., Proc. Natl. Acad. Sci. USA 82: 1074-1078 (1985). In another embodiment, the expression vectors pKK223-3 and pKK223-2 are used to express PKS and NRPS genes in E. coli, either in transcriptional or translational fusion, behind the tac or trc promoter. Expression of PKS and NRPS genes in heterologous hosts, which do not naturally have the phosphopantetheinyl (P-pant) transferases needed for post-translational modification of PKS enzymes, requires the coexpression in the host of a P-pant transferase, as described by Kealey et al., Proc. Natl. Acad. Sci. USA 95: 505-509 (1998).
EXAMPLE 14
Isolation of Epothilones from Producing Strains
Examples of cultivation, fermentation, and extraction procedures for polyketide isolation, which are useful for extracting epothilones from both native and recombinant hosts according to the present invention, are given in WO 93/10121, incorporated herein by reference, in Example 57 of U.S. Pat. No. 5,639,949, in Gerth et al., J. Antibiotics 49: 560-563 (1996), and in Swiss patent application no. 396/98, filed Feb. 19, 1998, and U.S. patent application Ser. No. 09/248,910 (that discloses also preferred mutant strains of Sorangium cellulosum), both of which are incorporated herein by reference. The following are procedures that are useful for isolating epothilones from cultured Sorangium cellulosum strains, e.g., So ce90, and may also be used for the isolation of epothilone from recombinant hosts.
A: Cultivation of Epothilone-producing Strains
Strain: Sorangium cellulosum Soce-90 or a Recombinant Host Strain According to the Present Invention
Preservation of the Strain: In Liquid N2.
Media: Precultures and Intermediate Cultures: G52 Main Culture: 1B12














G52 Medium:




yeast extract, low in salt (BioSpringer, Maison Alfort, France)
2
g/l


MgSO4 (7 H2O)
1
g/l


CaCl2 (2 H2O)
1
g/l


soya meal defatted Soyamine 50T (Lucas Meyer, Hamburg,
2
g/l


Germany)


potato starch Noredux A-150 (Blattmann, Waedenswil,
8
g/l


Switzerland)


glucose anhydrous
2
g/l


EDTA-Fe(III)-Na salt (8 g/l)
1
ml/l


pH 7.4, corrected with KOH


Sterilisation: 20 mins. 120° C.


1B12 Medium:


potato starch Noredux A-150 (Blattmann, Waedenswil,
20
g/l


Switzerland)


soya meal defatted Soyamine 50T (Lucas Meyer, Hamburg,
11
g/l


Germany)


EDTA-Fe(III)-Na salt
8
mg/l


pH 7.8, corrected with KOH


Sterilisation: 20 mins. 120° C.









Addition of Cyclodextrins and Cyclodextrin Derivatives:

Cyclodextrins (Fluka, Buchs, Switzerland, or Wacker Chemie, Munich, Germany) in different concentrations are sterilised separately and added to the 1B12 medium prior to seeding.
Cultivation: 1 ml of the suspension of Sorangium cellulosum Soce-90 from a liquid N2 ampoule is transferred to 10 ml of G52 medium (in a 50 ml Erlenmeyer flask) and incubated for 3 days at 180 rpm in an agitator at 30° C., 25 mm displacement. 5 ml of this culture is added to 45 ml of G52 medium (in a 200 ml Erlenmeyer flask) and incubated for 3 days at 180 rpm in an agitator at 30° C., 25 mm displacement. 50 ml of this culture is then added to 450 ml of G52 medium (in a 2 liter Erlenmeyer flask) and incubated for 3 days at 180 rpm in an agitator at 30° C., 50 mm displacement.
Maintenance culture:
The culture is overseeded every 3-4 days, by adding 50 ml of culture to 450 ml of G52 medium (in a 2 liter Erlenmeyer flask). All experiments and fermentations are carried out by starting with this maintenance culture.
Tests in a Flask
(i) Preculture in an agitating flask
Starting with the 500 ml of maintenance culture, 1×450 ml of G52 medium are seeded with 50 ml of the maintenance culture and incubated for 4 days at 180 rpm in an agitator at 30° C., 50 mm displacement.
(ii) Main culture in the agitating flask
40 ml of 1B12 medium plus 5 g/l 4-morpholine-propane-sulfonic acid (=MOPS) powder (in a 200 ml Erlenmeyer flask) are mixed with 5 ml of a 10× concentrated cyclodextrin solution, seeded with 10 ml of preculture and incubated for 5 days at 180 rpm in an agitator at 30° C., 50 mm displacement.
Fermentation
Fermentations are carried out on a scale of 10 liters, 100 liters and 500 liters. 20 liter and 100 liter fermentations serve as an intermediate culture step. Whereas the precultures and intermediate cultures are seeded as the maintenance culture 10% (v/v), the main cultures are seeded with 20% (v/v) of the intermediate culture. In contrast to the agitating cultures, ingredients of the fermentation media are calculated on the final culture volume including the inoculum. If, for example, 18 liters of medium +2 liters of inoculum are combined, then substances for 20 liters are weighed in, but are only mixed with 18 liters.
Preculture in an Agitating Flask
Starting with the 500 ml maintenance culture, 4×450 ml of G52 medium (in a 2 liter Erlenmeyer flask) are each seeded with 50 ml thereof, and incubated for 4 days at 180 rpm in an agitator at 30° C., 50 mm displacement.
Intermediate Culture, 20 Liters or 100 Liters 20 liters
18 liters of G52 medium in a fermenter having a total volume of 30 liters are seeded with 2 liters of preculture. Cultivation lasts for 3-4 days, and the conditions are: 30° C., 250 rpm, 0.5 liters of air per liter liquid per min, 0.5 bars excess pressure, no pH control.
100 liters
90 liters of G52 medium in a fermenter having a total volume of 150 liters are seeded with 10 liters of the 20 liter intermediate culture. Cultivation lasts for 3-4 days, and the conditions are: 30° C., 150 rpm, 0.5 liters of air per liter liquid per min, 0.5 bars excess pressure, no pH control.
Main Culture, 10 Liters, 100 Liters or 500 Liters
10 liters
The media substances for 10 liters of 1B12 medium are sterilised in 7 liters of water, then 1 liter of a sterile 10% 2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 2 liters of a 20 liter intermediate culture. The duration of the main culture is 6-7 days, and the conditions are: 30° C., 250 rpm, 0.5 liters of air per liter of liquid per min, 0.5 bars excess pressure, pH control with H2SO4/KOH to pH 7.6+/−0.5 (i.e. no control between pH 7.1 and 8.1).
100 liters
The media substances for 100 liters of 1B12 medium are sterilised in 70 liters of water, then 10 liters of a sterile 10% 2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 20 liters of a 20 liter intermediate culture. The duration of the main culture is 6-7 days, and the conditions are: 30° C., 200 rpm, 0.5 liters air per liter liquid per min., 0.5 bars excess pressure, pH control with H2SO4/KOH to pH 7.6+/−0.5. The chain of seeding for a 100 liter fermentation is shown schematically as follows: maintenance culture (500 ml) 




500 liters

The media substances for 500 liters of 1B12 medium are sterilised in 350 liters of water, then 50 liters of a sterile 10% 2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 100 liters of a 100 liter intermediate culture. The duration of the main culture is 6-7 days, and the conditions are: 30° C., 120 rpm, 0.5 liters air per liter liquid per min., 0.5 bars excess pressure, pH control with H2SO4/KOH to pH 7.6+/−0.5.
Product Analysis
Preparation of the Sample
50 ml samples are mixed with 2 ml of polystyrene resin Amberlite XAD16 (Rohm+Haas, Frankfurt, Germany) and shaken at 180 rpm for one hour at 30° C. The resin is subsequently filtered using a 150 μm nylon sieve, washed with a little water and then added together with the filter to a 15 ml Nunc tube.
Elution of the Product from the Resin
10 ml of isopropanol (>99%) are added to the tube with the filter and the resin. Afterwards, the sealed tube is shaken for 30 minutes at room temperature on a Rota-Mixer (Labinco BV, Netherlands). Then, 2 ml of the liquid are centrifuged off and the supernatant is added using a pipette to HPLC tubes.
HPLC Analysis
















Column:
Waters-Symetry C18, 100 × 4 mm, 3.5 μm




WAT066220 + preliminary column 3.9 × 20 mm




WAT054225



Solvents:
A: 0.02% phosphoric acid




B: Acetonitrile (HPLC-Quality)



Gradient:
 41% B from 0 to 7 min.




100% B from 7.2 to 7.8 min.




 41% B from 8 to 12 min.



Oven temp.:
30° C.



Detection:
250 nm, UV-DAD detection



Injection vol.:
10 μl











Retention time:
Epo A: 4.30 min
Epo B: 5.38 min










B: Effect of the Addition of Cyclodextrin and Cyclodextrin Derivatives to the Epothilone Concentrations Attained

Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides of α-D-glucopyranose with a relatively hydrophobic central cavity and a hydrophilic external surface area.
The following are distinguished in particular (the figures in parenthesis give the number of glucose units per molecule): α-cyclodextrin (6), β-cyclodextrin (7), γ-cyclodextrin (8), δ-cyclodextrin (9), ε-cyclodextrin (10), ζ-cyclodextrin (11), η-cyclodextrin (12), and θ-cyclodextrin (13). Especially preferred are δ-cyclodextrin and in particular α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, or mixtures thereof.
Cyclodextrin derivatives are primarily derivatives of the above-mentioned cyclodextrins, especially of α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, primarily those in which one or more up to all of the hydroxy groups (3 per glucose radical) are etherified or esterified. Ethers are primarily alkyl ethers, especially lower alkyl, such as methyl or ethyl ether, also propyl or butyl ether; the aryl-hydroxyalkyl ethers, such as phenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; the hydroxyalkyl ethers, in particular hydroxy-lower-alkyl ethers, especially 2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or hydroxy-butyl such as 2-hydroxybutyl ether; the carboxyalkyl ethers, in particular carboxy-lower-alkyl ethers, especially carboxymethyl or carboxyethyl ether; derivatised carboxyalkyl ethers, in particular derivatised carboxy-lower-alkyl ether in which the derivatised carboxy is etherified or amidated carboxy (primarily aminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-, piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl), in particular lower alkoxycarbonyl-lower-alkyl ether, for example methyloxycarbonylpropyl ether or ethyloxycarbonylpropyl ether; the sulfoalkyl ethers, in particular sulfo-lower-alkyl ethers, especially sulfobutyl ether; cyclodextrins in which one or more OH groups are etherified with a radical of formula
 —O-[alk-O—]n—H
wherein alk is alkyl, especially lower alkyl, and n is a whole number from 2 to 12, especially 2 to 5, in particular 2 or 3; cyclodextrins in which one or more OH groups are etherified with a radical of formula 




wherein R′ is hydrogen, hydroxy, —O-(alk-O)z—H, —O-(alk(—R)—O—)p—H or —O-(alk(—R)—O—)q-alk-CO—Y; alk in all cases is alkyl, especially lower alkyl; m, n, p, q and z are a whole number from 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR1 or NR2R3, wherein R1, R2 and R3 independently of one another, are hydrogen or lower alkyl, or R2 and R3 combined together with the linking nitrogen signify morpholino, piperidino, pyrrolidino or piperazino; or branched cyclodextrins, in which etherifications or acetals with other sugar molecules are present, especially glucosyl-, diglucosyl-(G2-β-cyclodextrin), maltosyl- or di-maltosyl-cyclodextrin, or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- or galactosaminyl-cyclodextrin.
Esters are primarily alkanoyl esters, in particular lower alkanoyl esters, such as acetyl esters of cyclodextrins.
It is also possible to have cyclodextrins in which two or more different said ether and ester groups are present at the same time.
Mixtures of two or more of the said cyclodextrins and/or cyclodextrin derivatives may also exist.
Preference is given in particular to α-, β- or γ-cyclodextrins or the lower alkyl ethers thereof, such as methyl-β-cyclodextrin or in particular 2,6-di-O-methyl-β-cyclodextrin, or in particular the hydroxy lower alkyl ethers thereof, such as 2-hydroxypropyl-α-, 2-hydroxypropyl -β- or 2-hydroxypropyl-γ-cyclodextrin.
The cyclodextrins or cyclodextrin derivatives are added to the culture medium preferably in a concentration of 0.02 to 10, preferably 0.05 to 5, especially 0.1 to 4, for example 0.1 to 2 percent by weight (w/v).
Cyclodextrins or cyclodextrin derivatives are known or may be produced by known processes (see for example U.S. Pat. Nos. 3,459,731; 4,383,992; 4,535,152; 4,659,696; EP 0 094 157; EP 0 149 197; EP 0 197 571; EP 0 300 526; EP 0 320 032; EP 0 499 322; EP 0 503 710; EP 0 818 469; WO 90/12035; WO 91/11200; WO 93/19061; WO 95/08993; WO 96/14090; GB 2,189,245; DE 3,118,218; DE 3,317,064 and the references mentioned therein, which also refer to the synthesis of cyclodextrins or cyclodextrin derivatives, or also: T. Loftsson and M. E. Brewster (1996): Pharmaceutical Applications of Cyclodextrins: Drug Solubilization and Stabilisation: Journal of Pharmaceutical Science 85 (10):1017-1025; R. A. Rajewski and V. J. Stella(1996): Pharmaceutical Applications of Cyclodextrins: In Vivo Drug Delivery: Journal of Pharmaceutical Science 85 (11): 1142-1169).
All the cyclodextrin derivatives tested here are obtainable from the company Fluka, Buchs, CH. The tests are carried out in 200 ml agitating flasks with 50 ml culture volume. As controls, flasks with adsorber resin Amberlite XAD-16 (Rohm & Haas, Frankfurt, Germany) and without any adsorber addition are used. After incubation for 5 days, the following epothilone titres can be determined by HPLC:











TABLE 2






order
Conc
Epo A
Epo B


Addition
No.
[% w/v]1
[mg/l]
[mg/l]



















Amberlite XAD-16 (v/v)

2.0
9.2
3.8




(% v/v)


2-hydroxypropyl-β-cyclodextrin
56332
0.1
2.7
1.7


2-hydroxypropyl-β-cyclodextrin
56332
0.5
4.7
3.3


2-hydroxypropyl-β-cyclodextrin
56332
1.0
4.7
3.4


2-hydroxypropyl-β-cyclodextrin
56332
2.0
4.7
4.1


2-hydroxypropyl-β-cyclodextrin
56332
5.0
1.7
0.5


2-hydroxypropyl-α-cyclodextrin
56330
0.5
1.2
1.2


2-hydroxypropyl-α-cyclodextrin
56330
1.0
1.2
1.2


2-hydroxypropyl-α-cyclodextrin
56330
5.0
2.5
2.3


β-cyclodextrin
28707
0.1
1.6
1.3


β-cyclodextrin
28707
0.5
3.6
2.5


β-cyclodextrin
28707
1.0
4.8
3.7


β-cyclodextrin
28707
2.0
4.8
2.9


β-cyclodextrin
28707
5.0
1.1
0.4


methyl-β-cyclodextrin
66292
0.5
0.8
<0.3


methyl-β-cyclodextrin
66292
1.0
<0.3
<0.3


methyl-β-cyclodextrin
66292
2.0
<0.3
<0.3


2,6 di-o-methyl-β-cyclodextrin
39915
1.0
<0.3
<0.3


2-hydroxypropyl-γ-cyclodextrin
56334
0.1
0.3
<0.3


2-hydroxypropyl-γ-cyclodextrin
56334
0.5
0.9
0.8


2-hydroxypropyl-γ-cyclodextrin
56334
1.0
1.1
0.7


2-hydroxypropyl-γ-cyclodextrin
56334
2.0
2.6
0.7


2-hydroxypropyl-γ-cyclodextrin
56334
5.0
5.0
1.1


no addition


0.5
0.5





1Apart from Amberlite (% v/v), all percentages are by weight (% w/v). 






Few of the cyclodextrins tested (2,6-di-o-methyl-β-cyclodextrin, methyl-β-cyclodextrin) displayed no effect or a negative effect on epothilone production at the concentrations used. 1-2% 2-hydroxy-propyl-βcyclodextrin and β-cyclodextrin increase epothilone production in the examples by 6 to 8 times compared with production using no cyclodextrins.
C: 10 Liter Fermentation with 1% 2-(hydroxypropyl)-β-cyclodextrin)
Fermentation is carried out in a 15 liter glass fermenter. The medium contains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin from Wacker Chemie, Munich, DE. Fermentation progress is illustrated in Table 3. Fermentation is ended after 6 days and working up takes place.







TABLE 3







Progress of a 10 liter fermentation









duration of culture [d]
Epothilone A [mg/l]
Epothilone B [mg/l]












0
0
0


1
0
0


2
0.5
0.3


3
1.8
2.5


4
3.0
5.1


5
3.7
5.9


6
3.6
5.7









D:100 Liter Fermentation with 1% 2-(hydroxypropyl)-β-cyclodextrin)

Fermentation is carried out in a 150 liter fermenter. The medium contains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin. The progress of fermentation is illustrated in Table 4. The fermentation is harvester 7 days and worked up.







TABLE 4







Progress of a 100 liter fermentation









duration of culture [d]
Epothilone A [mg/l]
Epothilone B [mg/l]












0
0
0


1
0
0


2
0.3
0


3
0.9
1.1


4
1.5
2.3


5
1.6
3.3


6
1.8
3.7


7
1.8
3.5









E: 500 Liter Fermentation with 1% 2-(hydroxypropyl)-β-cyclodextrin)

Fermentation is carried out in a 750 liter fermenter. The medium contains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin. The progress of fermentation is illustrated in Table 5. The fermentation is harvested after 7 days and worked up.







TABLE 5







Progress of a 500 liter fermentation









duration of culture [d]
Epothilone A [mg/l]
Epothilone B [mg/l]












0
0
0


1
0
0


2
0
0


3
0.6
0.6


4
1.7
2.2


5
3.1
4.5


6
3.1
5.1









F: Comparison Example 10 Liter Fermentation without Adding an Adsorber

Fermentation is carried out in a 15 liter glass fermenter. The medium does not contain any cyclodextrin or other adsorber. The progress of fermentation is illustrated in Table 6. The fermentation is not harvested and worked up.







TABLE 6







Progress of a 10 liter fermentation without adsorber.









duration of culture [d]
Epothilone A [mg/l]
Epothilone B [mg/l]












0
0
0


1
0
0


2
0
0


3
0
0


4
0.7
0.7


5
0.7
1.0


6
0.8
1.3









G: Working up of the Epothilones: Isolation from a 500 Liter Main Culture

The volume of harvest from the 500 liter main culture of example 2D is 450 liters and is separated using a Westfalia clarifying separator Type SA-20-06 (rpm=6500) into the liquid phase (centrifugate+rinsing water=650 liters) and solid phase (cells=ca. 15 kg). The main part of the epothilones are found in the centrifugate, The centrifuged cell pulp contains <15% of the epothilone portion and is not further processed. The 650 liter centrifugate is then placed in a 4000 liter stirring vessel, mixed with 10 liters of Amberlite XAD-16 (centrifugate:resin volume=65:1) and stirred. After a period of contact of ca. 2 hours, the resin is centrifuged away in a Heine overflow centrifuge (basket content 40 liters; rpm=2800). The resin is discharged from the centrifuge and washed with 10-15 liters of deionised water. Desorption is effected by stirring the resin twice, each time in portions with 30 liters of isopropanol in 30 liter glass stirring vessels for 30 minutes. Separation of the insopropanol phase from the resin takes place using a suction filter. The isopropanol is then removed from the combined isopropanol phases by adding 15-20 liters of water in a vacuum-operated circulating evaporator (Schmid-Verdampfer) and the resulting water phase of ca. 10 liters is extracted 3× each time with 10 liters of ethyl acetate. Extraction is effected in 30 liter glass stirring vessels. The ethyl acetate extract is concentrated to 3-5 liters in a vacuum-operated circulating evaporator (Schmid-Verdampfer) and afterwards concentrated to dryness in a rotary evaporator (Büchi type) under vacuum. The result is an ethyl acetate extract of 50.2 g. The ethyl ecetate extract is dissolved in 500 ml of methanol, the insoluble position filtered off using a folded filter, and the solution added to a 10 kg Sephadex LH 20 column (Pharmacia, Uppsala, Sweden) (column diameter 20 cm, filling level ca. 1.2 m). Elution is effected with methanol as eluant. Epothilone A and B is present predominantly in fractions 21-23 (at a fraction size of 1 liter). These fractions are concentrated to dryness in a vacuum on a rotary evaporator (total weight 9.0 g). These Sephadex peak fractions (9.0 g) are thereafter dissolved in 92 ml of acetonitrile:-water:-methylene chloride=50:40:2, the solution filtered through a folded filter and added to a RP column (equipment Prepbar 200, Merck; 2.0 kg LiChrospher RP-18 Merck, grain size 12 μm, column diameter 10 cm, filling level 42 cm; Merck, Darmstadt, Germany). Elution is effected with acetonitrile:water=3:7 (flow rate=500 ml/min.; retention time of epothilone A=ca. 51-59 mins.; retention time of epothilone B=ca. 60-69 mins.). Fractionation is monitored with a UV detector at 250 nm. The fractions are concentrated to dryness under vacuum on a Büchi-Rotavapor rotary evaporator. The weight of the epothilone A peak fraction is 700 mg, and according to HPLC (external standard) it has a content of 75.1%. That of the epothilone B peak fraction is 1980 mg, and the content according to HPLC (external standard) is 86.6%. Finally, the epothilone A fraction (700 mg) is crystallised from 5 ml of ethyl acetate:toluene=2:3, and yields 170 mg of epothilone A pure crystallisate [content according to HLPC (% of area)=94.3%]. Crystallisation of the epothilone B fraction (1980 mg) is effected from 18 ml of methanol and yields 1440 mg of epothilone B pure crystallisate [content according to HPLC (% of area)=99.2%]. m.p. (Epothilone B): e.g. 124-125° C.; 1H-NMR data for Epothilone B: 500 MHZ-NMR, solvent: DMSO-d6. Chemical displacement δ in ppm relative to TMS. s=singlet; d=doublet; m=multiplet














δ (Multiplicity)
Integral (number of H)




















7.34
(s)
1



6.50
(s)
1



5.28
(d)
1



5.08
(d)
1



4.46
(d)
1



4.08
(m)
1



3.47
(m)
1



3.11
(m)
1



2.83
(dd)
1



2.64
(s)
3



2.36
(m)
2



2.09
(s)
3



2.04
(m)
1



1.83
(m)
1



1.61
(m)
1



1.47-1.24
(m)
4



1.18
(s)
6



1.13
(m)
2



1.06
(d)
3



0.89
(d + s,
6




overlapping)





Σ = 41










EXAMPLE 15
Medical Uses of Recombinantly Produced Epothilones
Pharmaceutical preparations or compositions comprising epothilones are used for example in the treatment of cancerous diseases, such as various human solid tumors. Such anticancer formulations comprise, for example, an active amount of an epothilone together with one or more organic or inorganic, liquid or solid, pharmaceutically suitable carrier materials. Such formulations are delivered, for example, enterally, nasally, rectally, orally, or parenterally, particularly intramuscularly or intravenously. The dosage of the active ingredient is dependent upon the weight, age, and physical and pharmacokinetical condition of the patient and is further dependent upon the method of delivery. Because epothilones mimic the biological effects of taxol, epothilones may be substituted for taxol in compositions and methods utilizing taxol in the treatment of cancer. See, for example, U.S. Pat. Nos. 5,496,804, 5,565,478, and 5,641,803, all of which are incorporated herein by reference.
For example, for treatments, epothilone B is supplied in individual 2 ml glass vials formulated as 1 mg/1 ml of clear, colorless intravenous concentrate. The substance is formulated in polyethylene glycol 300 (PEG 300) and diluted with 50 or 100 ml 0.9% Sodium Chloride Injection, USP, to achieve the desired final concentration of the drug for infusion. It is administered as a single 30-minute intravenous infusion every 21 days (treatment three-weekly) for six cycles, or as a single 30-minute intravenous infusion every 7 days (weekly treatment).
Preferably, for weekly treatment, the dose is between about 0.1 and about 6, preferably about 0.1 and about 5 mg/m2, more preferably about 0.1 and about 3 mg/m2, even more preferably 0.1 and 1.7 mg/m2, most preferably about 0.3 and about 1 mg/m2; for three-weekly treatment (treatment every three weeks or every third week) the dose is between about 0.3 and about 18 mg/m2, preferably about 0.3 and about 15 mg/m2, more preferably about 0.3 and about 12 mg/m2, even more preferably about 0.3 and about 7.5 mg/m2, still more preferably about 0.3 and about 5 mg/m2, most preferably about 1.0 and about 3.0 mg/m2. This dose is preferably administered to the human by intravenous (i.v.) administration during 2 to 180 min, preferably 2 to 120 min, more preferably during about 5 to about 30 min, most preferably during about 10 to about 30 min, e.g. during about 30 min.
While the present invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications and embodiments are to be regarded as being within the spirit and scope of the present invention.

## Claims

1. A process for the preparation of an epothilone selected from epothilone A or epothilone B, which comprises
(a) preparing recombinant host by introducing into a host cell a group of nucleotide fragments that code for epothilone synthase proteins, which group of nucleotide fragments comprises an epoA nucleotide fragment, an epoP nucleotide fragment, an epoB nucleotide fragment, an epoC nucleotide fragment, an epoD nucleotide fragment, an epoE nucleotide fragment, and an epoF nucleotide fragment such that each nucleotide fragment is operatively linked to a promoter sequence; wherein 
(i) the complement of the epoA nucleotide fragment hybridizes to nucleotides 7610-11875 of SEQ ID NO:1; 
(ii) the complement of the epo P nucleotide fragment hybridizes to nucleotides 11872-16104 of SEQ ID NO:1 
(iii) the complement of the epoB nucleotide fragment hybridizes to nucleotides 16251-21749 of SEQ ID NO:1; 
(iv) the complement of the epoC nucleotide fragment hybridizes to nucleotides 21746-43519 of SEQ ID NO:1; 
(v) the complement of the epoD nucleotide fragment hybridizes to nucleotides 43524-54920 of SEQ ID NO:1; 
(vi) the complement of the epoE nucleotide fragment hybridizes to nucleotides 54935-62254 of SEQ ID NO:1; 
(vii) the complement of the epoF nucleotide fragment hybridizes to nucleotides 62369-63628 of SEQ ID NO:1; 
wherein the conditions of hybridization are at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minutes at 65° C.; 

(b) growing the recombinant host under conditions that allow biosynthesis of the epothilone, in the recombinant host; and 
(c) isolating the epothilone.

2. A process of claim 1 wherein each nucleotide fragment is linked to the same promoter sequence.

3. A process of claim 1 wherein the recombinant host is a heterologous host.

4. A process of claim 1 wherein the promoter is heterologous.

5. A process of claim 3 wherein the recombinant host is a member of the genus Streptomyces.

6. A process of claim 1 wherein a gene for a phosphopantetheinyl transferase is also introduced into the host.

7. A process of claim 6 wherein the recombinant host is E. coli.

8. A process of claim 1 wherein the recombinant host is grown in the presence of a cyclodextrin.

9. A process of claim 8 wherein the cyclodextrin is 2-(hydroxypropyl)-beta-cyclodextrin.

10. A process of claim 1 wherein the epothilone is epothilone B.