Genetic Engineering Of Precursor Supply Pathway For The Overproduction Of Natamycin In Streptomyces Gilvosporeus

Natamycin is a food grade antifungal agent,which can effectively inhibit the growth of mold and yeast.It is widely used in the field of food preservation.Natamycin belongs to polyene macrolides with trehalose aminoglycosyl modification in structure.Streptomyces gilvosporeus is an important producer of natamycin.In the process of natamycin biosynthesis,acetyl CoA,malonyl COA and methylmalonyl COA are the direct precursors of lactone ring synthesis,and GDP-mannose is the direct precursor of trehalose amine.In this study,the key enzyme coding genes of the above precursor synthesis pathway were overexpressed,and their effects on the yield of natamycin were studied,in order to find the bottleneck step of natamycin precursor supply pathway.The main results are as follows:(1): Genetic modification of acetyl CoA supply pathway: overexpression of fatty acid degradation key node fatty acyl CoA synthase gene(fad D),no increase in natamycin production was observed.In the genetic transformation of acetic acid reflux pathway,the recombinant strain was constructed by overexpression of acetyl CoA synthase gene(acs),and the yield of natamycin increased by 60.91%..(2): Genetic modification of malonyl COA supply pathway: through co overexpression of malonyl COA synthase gene(mat B)and dicarboxylic acid transporter gene(mat C),the synthesis of malonic acid to malonyl COA was strengthened,and the yield of natamycin was increased by 26.76%.Overexpression of acetyl CoA carboxylase gene(acc)enhanced the synthesis of acetyl CoA to malonyl COA,and no increase in the yield of natamycin was observed.Therefore,malonyl coenzyme A may be the rate limiting precursor of natamycin synthesis.Strengthening malonic acid pathway can increase the yield of natamycin.(3): Genetic modification of methylmalonyl COA supply pathway: the methylmalonic acid synthesis pathway was strengthened by overexpression of methylmalonyl COA synthase gene(mat B)and isomerase gene(epi),and the yield of natamycin did not increase significantly.The synthesis of succinyl COA was enhanced by overexpression of methylmalonyl COA mutase gene(mcm),and the yield of natamycin was increased by21.35%.(4): GDP-mannose synthesis pathway genetically engineered: overexpression of S.coelicolor derived man A increased the yield of natamycin by 59.81% compared to the wildtype strain;Overexpression of the phosphomannomutase gene(man B)in a recombinant strain overexpressing S.coelicolor M145 derived man A improved the yield of 85.12%compared to the wild-type strain natamycin;Further overexpression of the GDP mannose pyrophosphorylase(manc)construct produced 1223.34 mg/L.Natamycin production was efficiently increased by strengthening the GDP-mannose pathway,and there was an additive effect of overexpression of multiple genes from the GDP-mannose pathway.By overexpressing the trehalosaminyltransferase gene(pimk),the yield of natamycin was improved by 21.62% compared to the wild-type strain.In this study,the key synthetic pathways to improve the efficient supply of each precursor species were discovered.The intracellular supply of acyl CoA and GDP-mannose precursor species can be enhanced by means of gene overexpression,which in turn enhances natamycin production.