Construction And Fermentation Optimization Of High-Yield Natamycin Genetic Engineering Strains

Natamycin, also known as pimaricin, is a widely used polyene antifungal antibiotic. It is highly effective at low doses against yeast and molds but displays low toxicity to mammalian cells. Natamycin is one of the very few antibiotics that have been recommended as food additives and belongs to the Generally Regarded As Safe (GRAS) list of compounds by the US Food and Drugs Administration (FDA), being legally used in over 50 countries including the US, for the safe preservation of a wide variety of food products, such as cheese, fermented meat, yogurt, beverages, wine and baked foods, against yeasts and molds. Globally, natamycin is also a widely permitted food additive. For these reasons, it has a huge commercial value.Natamycin is produced in submerged culture under aerobic conditions by Streptomyces natalensis, Streptomyces chattanoogensis and Streptomyces gilvosporeus. Current industrial concerns include the low natamycin yields and the high cost of production. Recently, the natamycin biosynthetic gene cluster was characterized, and it is feasible that the genetic manipulations can be used as a good approach to significantly improve the production of polyene antibiotics, especially deletion of a negative regulator as well as alterations in the levels and activities of pathway-specific regulators.Cholesterol oxidase was confirmed to act as signaling proteins in the biosynthesis of natamycin. In this study, we investigated the effects of the heterogenous cholesterol oxidase on the natamycin biosynthesis firstly, and the results showed that the addition of heterogenous cholesterol oxidase in S.gilvosporeus 712 culture triggered the biosynthesis of natamycin.To develop the positive effects of the cholesterol oxidase on the natamycin biosynthesis, an additional copy of gene pimE was inserted into the genome of the initial strain S.gilvosporeus 712 by intergeneric conjugation. This strain overexpressed pimE stably, generating the engineering strain S.gilvosporeus swjs-801, which improved the antibiotic yield to 1.74 g/L, increasing by 72% than S.gilvosporeus 712 in the shake flask level. And the cell growth was not influenced by the gene pimE. Further improvement in the antibiotic production was achieved in a 1-L fermenter to 7.0 g/L, which was a 153% improvement after 120 h cultivation.The research also optimized carbon sources, nitrogen sources and other medium components in the flask level, recpectively, using S.gilvosporeus swjs-801 as the initial strain. The optimal condition was:soya bean meal 15 g/L, yeast extract 3.5 g/L, NaCl 1 g/L, MgSO4·7H2O 1 g/L, glycerol 60 g/L, initial pH 7.5, loading volume 30 mL, temperature of 29 ℃, rotate speed 220 rpm. The natamycin yield reached 7.98 g/L, increasing by 14.1% than that before optimization in the 1-L fermenter.