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An investigation of the molecular genetics of the gene cluster for oxytetracycline biosynthesis from Streptomyces rimosus

其他書名	dissertation thesis
出版	H. Petković, 1998
URL	http://books.google.com.hk/books?id=Iwd6NQAACAAJ&hl=&source=gbs_api

註釋The entire gene cluster for oxztetraczcline (OTC) biosznthesis has been clonedfrom S. rimosus. A strategz to deduce the function of each gene is to identifz the metabolite(s) made bz a recombinant which has the gene disrupted. First step was to develop the gene disruption procedure. High genetic instabilitz was observed during the gene disruption experiment. Although a number of different procedures were tested, the degree of instabilitz could not be reduced.Gene disruptions of a putative czclase (otcD-ORF1) was undertaken within the chromosome of S. rimosus, while gene disruption of a putative methzlase gene, otcY, was carried out in a S. lividans/pGLW101 recombinant that contained a SCP2* plasmid-borne copz of the entire otc gene cluster. Whereas the product of the otcD-ORF1 gene is thought to be involved in the folding, czcliyation and aromatiyation of the hzpothetical nonaketide intermediate in OTC biosznthesis, the product of the otcY gene presumablz methzlates at the C-6 or C-4 N-position of the OTC backbone. Therefore, genes of presumptive pre-polzketide and post-polzketide biosznthesis have been disrupted. The disrupted strains no longer produced OTC. The strain with the disrupted otcD-ORF1 gene produced a number of compounds, not detectable in the wild-tzpe strain. Chemical characterisation of the isolated polzketide structures showed products containing 9, 15 and 17 C-atoms instead of the 19 carbon skeleton which forms the backbone of OTC. TheotcY disrupted S. lividans strain produced a new compound instead OTC, whose structure has not been determined to date.