Improvement Of Hygrocin A Based On The Optimization Of Fermentation Medium And Microbial Metabolomics Methods

A variety of secondary metabolites with the characteristic of anti-microbial, mould proof, antivirus, antitumor activity, hypotensive activity and immunosuppressants are produced in Streptomyces, therefore Streptomyces is regarded as “the synthesis factory of natural medicine”. hygrocin A is one of the secondary metabolites of Streptomyces hygroscopicus ATCC 29253 with significant anti-bacterial activity, and hygrocin C, D and F, the degradation products of which are found to be toxic to human breast cancer MDA-MB-431 cells and prostate cancer PC3 cells. It is vital significance to improve the hygrocin A fermentation productivity for later application research. The work of this paper is focused on the optimization of hygrocin A productivity based on the optimization of fermentation process and microbial metabolomics respectively. The main research contents and conclusions as follows:1) In this study, we optimizes the hygrocin A fermentation culture medium as 4 g/L glucose, 8 g/L soybean cake meal, 10 g/L malt extract, 1.5 g/L K2HPO4, 1.5 g/L KH2PO4, 1 g/L Mg Cl2 and 1.5 g/L Na Cl with and single factor experiments orthogonal methodology. The highest hygrocin A productivity can be realized with 10 days as fermentation time and 48 h as activation time. The hygrocin A productivity in the optimized medium can be up to 6 folds compares to that in the original culture medium M10. Furthermore, Rapamycin is found in the fermentation broth, and the productivity can reach to 62.78% compares to that in M2 medium which is the original Rapamycin culture medium.2) With the aid of the combination of the methods of microbial metabolomics and exogenous additives, we optimize hygrocin A fermentation process to increase the hygrocin A productivity and compare with the method of optimizing fermentation medium. A microbial metabolomics method which is fit for S.hygroscopicus ATCC 29253 is established. To the normal metabolites, fast filtration is used for quenching, 50% Me OH at-40℃ is used as metabolite extraction solvent and 50% Me OH is used as redissolve solvent. To the intracellular short-chain Co A-esters, fast filtration is used for quenching, 15% TCA is used as metabolite extraction solvent with Waters Oasis® HLB extraction cartridges and 0.1M NH4CO2H/H2O(95:5) mixture is used as redissolve solvent. Detecting the exchange of intracellular metabolites which are related to the biosynthesis of Rapamycin and hygrocin A during the fermentation process, and on the base of the exchenge, the proposed relationship of metabolites is conjectured. The Rapamycin productivity in the added mediums in which Shikimic acid, lysine and sodium succinate are added into the fermentation broth during the fermentation process are much higher than that in the blanks, and the productivity in the added M10 medium is much higher than that in the added M3 medium. The hygrocin A productivity in the added mediums are much higher than that in the blanks, however the productivity in the added M10 medium is much lower than that in the added M3 medium. The Rapamycin and hygrocin A productivities can be enhanced at the same time using the combination of the methods of microbial metabolomics and exogenous additives.