RESUMO
BACKGROUND: Epothilones are produced by the myxobacterium Sorangium cellulosum So ce90, and, like paclitaxel (Taxol((R))), they inhibit microtubule depolymerisation and arrest the cell cycle at the G2-M phase. They are effective against P-glycoprotein-expressing multiple-drug-resistant tumor cell lines and are more water soluble than paclitaxel. The total synthesis of epothilones has been achieved, but has not provided an economically viable alternative to fermentation. We set out to clone, sequence and analyze the gene cluster responsible for the biosynthesis of the epothilones in S. cellulosum So ce90. RESULTS: A cluster of 22 open reading frames spanning 68,750 base pairs of the S. cellulosum So ce90 genome has been sequenced and found to encode nine modules of a polyketide synthase (PKS), one module of a nonribosomal peptide synthetase (NRPS), a cytochrome P450, and two putative antibiotic transport proteins. Disruptions in the genes encoding the PKS abolished epothilone production. The first PKS module and the NRPS module are proposed to co-operate in forming the thiazole heterocycle of epothilone from an acetate and a cysteine by condensation, cyclodehydration and subsequent dehydrogenation. The remaining eight PKS modules are responsible for the elaboration of the rest of the epothilone carbon skeleton. CONCLUSIONS: The overall architecture of the gene cluster responsible for epothilone biosynthesis has been determined. The availability of the cluster should facilitate the generation of designer epothilones by combinatorial biosynthesis approaches, and the heterologous expression of epothilones in surrogate microbial hosts.
Assuntos
Epotilonas , Compostos de Epóxi/metabolismo , Família Multigênica/genética , Myxococcales/química , Myxococcales/genética , Tiazóis/metabolismo , Antibacterianos , Antineoplásicos/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Biblioteca Gênica , Genes Bacterianos , Macrolídeos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Fases de Leitura Aberta , Peptídeo Sintases/genética , Biossíntese de Proteínas/genéticaRESUMO
A method for estimating DNA strand breakage and subsequent repair based on alkaline gel electrophoresis was developed and tested with isogenic strains of Escherichia coli deficient in DNA repair enzymes. Samples from a cell suspension were removed at 2 min intervals following a 15 min exposure to 20 mmol l-1 H2O2. Catalase was added and the cells were embedded in blocks of low-melting point agarose and lysed. After alkaline gel electrophoresis, photographs of the gels were taken and the relative lengths of the distributions of DNA fragments were measured with a scanner and computer. The lengths were correlated with survival of the cells exposed to H2O2 and with the importance of particular DNA repair enzymes. Alkaline gel electrophoresis appears to be a relatively simple method for analysing the level of H2O2-caused DNA damage and repair in E. coli.
