Study On Screening Of Daptomycin Producing Strains And Fermentation Conditions

Nowadays, the resistance of pathogens to antibiotics is one of the severe challenges both in China and even the whole world. The fundamental way of solving this problem is to find the effective antibiotics that have great resistance to bacteria. Daptomycin (Daptomycin. LY146032) is the first approved lipopeptide antibiotic by the FDA, it could kill the most isolated Gram-positive bacteria, including anaerobic bacteria and the resistant enterococci, and it has broad application prospects. It has important scientific significance and economic benefits if the productivity of the strain could be enhanced.In order to improve the productivity of the daptomycin producing strain, the dissertation focuses on the screening of high-producing strain and on the optimization of 3 L fermentor batch fermentation conditions. The main research results are as follows:1. We take the method of screening to obtain a strain with high productivity, that is, we use the U.V. irradiation to mutate the spore and then spread them on the agar with the screening agents (decanoic acid and daptomycin). At last, a high production strain LC-C-58 with the productivity of 121.0 mg/L is obtained, and its stabilization test appears to be stable.2. The process of batch fermentation has been investigated in 3 L stirred tank reactor. The optimal culture conditions are 30℃, the fermentation medium volume 1.8 L/3 L, inoculation 1%, pH(0~30 h, 6.5; 30~96 h, 7.0), dissolved oxygen(12~45 h, 40%; 45 h~96 h, 30%).3. We have tried many methods of adding the precursors. And at last the result show that adding a sterile solution of 50% v/v decanoic acid and 50% methyl oleate at a rate of 0.1 mL/h at the 25th hour of the fermentation is the best method.4. The seed medium is composed of Dextrose 2.0%, Calcium Carbonate 0.2%, Soybean Flour 2.0%, Yeast Extract 0.1%, KCl 0.02%, MgSO4·7H2O 0.02%, FeSO4·7H2O 0.0004%, Sag471 (antifoam) 0.02% and Water 95.6396%. Fermentation medium is composed of Soybean Flour 2.2%, Fe2(NH4)3SO4 0.066%, Dextrose 0.825%, Sag471 0.022%, Potato Dextrin 3.3%, Molasses 0.275% and Water 93.312%。5. Two types of fed-batch processes have been studied. And the continuous fed-batch process is better than the intermissive fed-batch process. At last, the daptomycin yield can reach 130.02 mg/L.