Study On The Breeding Of High Daptomycin Producing Strains And Fermentation Conditions Optimization

Daptomycin is a new lipopeptide antibiotic which contains a ring with teen amino acids and three amino acids with a 10-carbon decanoic acid attached to the terminal L-tryptophan. Daptomycin is produced by Streptomyces roseosporus, and its activity limited to gram-positive organisms, especially the antibiotic-resistant bacteria such as Staphvlococcus aureus (MRSA), vancomycin-resistant enterococci (VRE). Daptomycin was approved by FDA in 2003, and it was used in skin and soft tissue infection. However, the internal research of daptomycin production is rare, so the application of daptomycin is limited severely. Therefore, it is of great importance to enhance the research and development of daptomycin production process. In this work, the high productivity strain was selected for daptomycin production and the medium composition and fermentation conditions were optimized. The major research contents and results are as follows:Streptomyces roseosporus LD-1 was used as an original strain. After treated with diethylsulfate and laser irradiation in series, a mutant strain LD-54 was screened out by using the medium with selective pressure of streptomycin. Under the fermentation condition without being optimized, the production of daptomycin was 34.29 mg/L, which was 1.94 times higher than that of the parental strain LD-1. The strain is genetically stable.The batch fermentation of daptomycin production with the strain LC-54 in shake flask was studied. The optimal seed age and the fermentation medium for daptomycin production were designed by using SAS. Also, the effects of cultivation conditions such as inoculum, rotate speed, initial pH value, and culture medium on daptomycin production by Streptomyces roseosporus were evaluated. Under the optimum conditions, strain LC-54 could produce daptomycin 54.5mg/L by shaking-flask batch fermentation, which was 58.9% higher than that of initial conditions.The effect of precursor on daptomycin production was studied. A 7.5L bioreactor was used to study the daptomycin fermentation, the production of daptomycin reached 126.6 mg/L in the optimized operating condition, which was 133% higher than that in shaking-flask fermentation. A logistic model was adopted to simulate the experiment data from 7.5 L bioreactor batch fermentation. The model simulations for cell growth, daptomycin formation and total carbohydrate consumption were in good agreement with the experiment data.