Construction Of High-yield Natamycin Genetic Engineering Strains

Natamycin is one kind of efficient,broad-spectrum and safe macrolide antifungal antibiotic which is highly effective to inhibit the growth of mould and yeast and displays low toxicity to mammalian cells,therefore,it has been widely used in food and medicine field.The market demand of natural preservative natamycin gradually becomes larger,but the low fermentation level of natamycin and the high cost of production at present limites the further development and use of natamycin.Recently,the natamycin biosynthetic gene cluster of Streptomyces natalensis ATCC 27448 and Streptomyces chatanoogensis L10 have been cloned and identified,so it is possible to use genetic engineering to improve the natamycin production of the Streptomycetes strain.Using molecular technology to investigate the effect of the pathway-specific regulatory gene(pimM)?cholesterol oxidase-encoding gene(pimE)in the natamycin biosynthesis gene cluster and the heterologous Vitreoscilla hemoglobin gene(vgb)on natamycin yield with Streptomyces gilvosporeus S139 as the starting strain in this study.The main results were as follows:(1)Based on the recombinant plasmid pIMEP which was integrated with an ermE*promoter(Perm)successfully built six kinds of expression vector:pIMEP::pimE,pIMEP::pimM,pIMEP::vgb,pIMEP::pimE::pimM,pIMEP::pimM::vgb and pIMEP::pimE:pimM::vgb in this paper.(2)The six integration recombinant vectors were introduced into the original strain S.gilvosporeus S139 by intergeneric conjugation,and by screening for apramycin resistance and the natamycin inhibition zone antibiotic and PCR verified obtained six genetic engineered strains S.gilvosporeus/pIMEP::pimE,S.gilvosporeus/pIMEP::pimM,S.gilvosporeus/pIMEP::vgb,S.gilvosporeus/pIMEP::pimE::pimM,S.gilvosporeus/pIMEP::pimM::vgb and S.gilvosporeus/pIMEP::pimE::pimM::vgb.The six engineering strains have a good genetic stability of descendiblity after being inoculated for seven subsequent generations on SS plate without apramycin resistance.(3)Compared to the original strain by shaking flask fermentation in this paper,the natamycin production of engineering strains had increased at different degrees while the growth curves were similar.when the engineering strains S.gilvosporeus/pIMEP::pimE,S.gilvosporeus/pIMEP::pimM and S.gilvosporeus/pIMEP::vgb compared to the original strain,the highest percentage of natamycin production increased 19.56%,17.77%and 24.08%respectively;when the engineering strains S.gilvosporeus/pIMEP::pimE::pimM and S.gilvosporeus/pIMEP::pimM::vgb compared to the original strain,the highest percentage of natamycin production increased 40.28%and 43.66%respectively;however,when the engineering strain S.gilvosporeus/pIMEP::pimE::pimM::vgb compared with the original strain,the highest percentage of natamycin production increased 55.69%.Through shake flask fermentation experiments,we knew that the over expression of pimE and pimM in the natamycin biosynthesis gene cluster and the heterologous expression of Vitreoscilla hemoglobin gene vgb all could successfully improve the natamycin production compared to the parent strain,and the co-expression of genes had superimposed strengthening effect for the biosynthesis of natamycin.