Abstract
Degenerate PCR primers were used to amplify cytochrome P450 gene fragments from the high-GC gram-negative bacteria Amycolatopsis orientalis, which catalyzes the hydroxylation of epothilone B to produce epothilone F. The amplified fragments were used as hybridization probes to identify and clone two intact cytochrome P450 genes. The expression of one of the cloned genes in a Streptomyces lividans transformant resulted in the biotransformation of epothilone B to epothilone F. The conversion of epothilone B to epothilone F by the S. lividans transformant was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.
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References
Altenbuchner J, Schmid K, Schmitt R (1983) Tn1721-encoded tetracycline resistance: mapping of structural and regulatory genes mediating resistance. J Bacteriol 153:116–123
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Basch J, Franceschini T, Tonzi S, Chiang S-JD (2004) Expression of a cephalosporin C esterase gene in Acremonium chrysogenum for the direct production of deacetylcephalosporin C. J Ind Microbiol Biotechnol 31:531–539
Bollag DM, McQueney PA, Zhu J, Hensens O, Koupal L, Liesch J, Goetz M, Lazarides E, Woods CM (1995) Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Res 55:2325-2333
Brautaset T, Sekurova ON, Sletta H, Ellingsen TE, StrLm AR, Valla S, Zotchev SB (2000) Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway. Chem Biol 7:395–403
Bruntner C, Lauer B, Schwarz W, Mohrle V, Bormann C (1999) Molecular characterization of co-transcribed genes from Streptomyces tendae Tu901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. Mol Gen Genet 262:102–114
Gerth K, Schummer D, Höfle G, Irschik H, Reichenbach H (1995) A new antifungal compound from Sorangium cellulosum (Myxobacteria), production, physico-chemical and biological properties. J Antibiot 48:973–976
Gerth K, Steinmetz H, Höfle G, Reichenbach H (2002) Studies on the biosynthesis of epothilones: hydroxylation of Epo A and B to epothilones E and F. J Antibiot 55:41–45
Hardt IH, Steinmetz H, Gerth K, Sasse F, Reichenbach H, Höfle G (2001) New natural epothilones from Sorangium cellulosum, strains So ce90/B2 and So ce90/D13: isolation, structure elucidation, and SAR studies. J Nat Prod 64:847–856
Höfle G, Bedrof N, Gerth K, Reichenbach H (1993) Epothilons, process for preparing the same and their use as medicaments and as plant protecting agents. WO 93/10121
Holton TA, Lester DR (1996) Cloning of novel cytochrome P450 gene sequences via polymerase chain reaction amplification. Meth Enzymol 272:275–283
Hopwood DA, Keiser T, Bibb MJ, Buttner MJ, Chater KF (2000) Practical Streptomyces Genetics. The John Innes Foundation, Norwich
Inouye M, Takada Y, Muto N, Beppu T, Horinouchi S (1994) Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector. Mol Gen Genet 245:456–464
Lee FY, Vite G, Gerhard H, Kim S-H, Clark J, Fager K, Kennedy K, Smykla R, Wen M-L, Leavitt K, Johnston K, Peterson R, Kamath A, Franchini M, Schulze G, Fairchild C, Raghavan K, Long B, Kramer R (2002) The discovery of BMS 310705: a water-soluble and chemically stable semisynthetic epothilone possessing potent parenteral and oral antitumor activity against models of taxane-sensitive and taxane-resistant human tumor in vivo. Proc AACR 43:792–793
Li W, Matson J, Huang X, Lam KS, McClure G (2000) Microbial transformation method for the preparation of an epothilone. WO 00/39276
Matsuoka M, Sasaki T (2004) Inactivation of macrolides by producers and pathogens. Curr Drug Targets Infect Disord 4:217–240
Meyer RP, Hagemeyer CE, Knoth R, Kurz G, Volk B (2001) Oxidative hydrolysis of scoparone by cytochrome p450 CYP2C29 reveals a novel metabolite. Biochem Biophys Res Commun 285:32–39
O’Keefe DP, Gibson KJ, Emptage MH, Lenstra R, Romesser JA, Litle PJ, Omer CA (1991) Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus. Biochemistry 30:447–455
Parajuli N, Basnet DB, Chan Lee H, Sohng JK, Liou K (2004) Genome analyses of Streptomyces peucetius ATCC 27952 for the identification and comparison of cytochrome P450 complement with other Streptomyces. Arch Biochem Biophys 425:233–241
Watanabe I, Nara F, Serizawa N (1995) Cloning, characterization and expression of the gene encoding cytochrome P-450sca-2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. Gene 163:81–85
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We thank Daniel Carazzato and Chantal Jean for assistance with the LC/MS analysis of the bioconversion broths.
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An erratum to this article can be found at http://dx.doi.org/10.1007/s10295-006-0182-4
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Basch, J., Chiang, SJ. Cloning and expression of a cytochrome P450 hydroxylase gene from Amycolatopsis orientalis: hydroxylation of epothilone B for the production of epothilone F. J Ind Microbiol Biotechnol 34, 171–176 (2007). https://doi.org/10.1007/s10295-006-0173-5
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DOI: https://doi.org/10.1007/s10295-006-0173-5