Effect Of FabD And AceA Gene Inactivation On Production Of Rifamycin In Amycolatopsis Mediterranei

The rifamycin is one of ansamycins antibiotic used for treatment of tuberculosis caused by mycobacterial infections.Amycolatopsis mediterranei is an important industrial strain for production of rifamycin,however,in most cases,wild type strains only produce limited amounts of the desired compounds and thus require genetic manipulations and improvement is required before they can be used in an industrial setting.This thesis used A.mediterranei as the research object to construct gene inactivation strain,in order to provide a feasible basis of building industrial high yield strain.S-malonyltransferase is transferred to acyl carrier protein(ACP)by malonyl–CoA/ACP transacylase(encoded by the fabD gene).The acyl–ACP thioesterase serves as the essential enzyme in the fatty acid elongation pathway.To acquire better producer of rifamycin,the S-malonyltransferase gene(fabD)homologous recombinant vector with apramycin resistance marker was constructed by using fusion PCR in this study.The suicide vector was transformed into A.mediterranei by electroporation and fabD inactivation mutant was obtained by screening.The rifamycin production in fermentation was measured with high performance liquid chromatographyin.The mutant strain A.mediterranei ?fabD produce 9.9% more rifamycin SV than the parental strain.To explore the influence of isocitrate lyase gene(aceA)on the production of rifamycin,an aceA suicide plasmid with resistance marker was constructed by using fusion PCR,plasmid without resistance marker was constructed by overlap extension PCR in this study.The vectors was transferred to the cells of A.mediterranei by electroporation in order to homologous with chromosome.As the result,The aceA inactivation mutant was obtained by screening with resistance maker.Compared with the original strain,rifamycin SV production of the mutant strain is decreased by 26.0%.