Construction Of Efficient Biosynthesis System Of Nugamycin

Natamycin is a kind of polyene macrolide antibiotic mainly used to treat keratitis caused by fungal infection.However,due to it’s hemolytic toxicity,it’s application in human therapy is limited.Therefore,it is of great significance to obtain new derivatives with higher efficiency and lower toxicity through the reconstruction of biosynthesis pathway.The mutant strain L140 was obtained by knocking out the scn G gene in S.chattanogenes L10 in the early stage of our laboratory.Through activity screening,it was found that the strain could produce a compound with stronger antifungal activity(no pure product).We further confirmed the activity of the compound and named it as nugamycin.In this study,the high-efficient synthesis system of nugamycin was successfully constructed,and the fermentation process of nugamycin was basically established.First of all,high expression rate limiting enzyme,module replacement and gene multi copy strategies were adopted to construct a high-efficiency synthesis system of nugamycin.The fermentation level of the high-yield nugamycin producing strain ZJU3 in shake flask was 835 mg/L.The culture medium was optimized by response surface analysis,and the culture components were preliminarily determined to be soluble starch 6%,soybean cake powder 2.9% and yeast extract 1.05%.The shaking flask fermentation level of nugamycin was increased to 927 mg/L.In the 15 L tank test,the key process parameters including p H of natural,dissolved oxygen ≥ 30%,reducing sugar content≈ 2% were controlled and fermented for 164 hours.The fermentation level of nugamycin reached 1257 mg/L.Meanwhile,the separation and purification process of nugamycin was established,and the structure and activity of nugamycin were identified.The separation and purification process of nugamycin was studied by methanol extraction,macroporousadsorption resin adsorption and reversed-phase column separation.The purity of the product was greater than 95%.Then,the structure of nugamycin was identified by NMR.The results showed that compared with natamycin,only the carboxyl group of C12 changed to methyl group.Bioactivity studies showed that nugamycin was more effective than natamycin in inhibiting fungi.