Screening And Breeding Of Daptomycin Producing Strains And Optimization Of Its Fermentation Conditions

Daptomycin,with broad-spectrum anti-Gram-positive bacteria activity,is a newly-approved antibacterial agent derived from Streptomyces Roseosporus.It is difficult to cause cross-resistance with other antibiotics for its unique mechanism of action,so it is usually used to treat antibiotic resistant bacteria in clinical trails.At present,little studies have been reported about the fermentation of daptomycin in china and it can't be applied in commercial production for the low yield.Therefore,it will bring great social and economic benefits to research and develop the technology of daptomycin fermentation.In order to improve the yeild of fermentation of daptomycin,the dissertation focuses on the ultraviolet mutagenesis breeding,optimization of fermentation medium,optimization of fermentation process of flask and 15 L fermenter.Besides,the preparation of the crude product of daptomycin is preliminary explored.The main research contents and results are as follows:The mutant strain YZ-378 was obtained by ultraviolet mutagenesis breeding of Streptomyces Roseosporus.Its genetic is stability;fermentation titer can reach to 84 mg/L,an increase of 133.33% compared with the original strain(36 mg/L).Response surface methodology was used for the optimization of the culture medium for daptomycin fermentation and a set of optimal ratio of medium was identified.In addition,the single factor of fermentation conditions such as the age of seed,initial pH,temperature,inoculum volume,dissolved oxygen,add the amount of precursors and so on was studied.On that basis,the fermentation process of shake flask was optimized using response surface methodology,and a set of optimum fermentation conditions was abtained.The fermentation titer of fermenter is 188 mg/L,an increase of 123.81% compared with original conditions(84 mg/L).The amount of precursors,residual glucose,and rotational speed for 15 L fermenter was established through three factors and three levels orthogonal test design and the optimum level of fermentation process technology wad obtained.The final titer is 785 mg/L,an increase of 69.18% compared with original conditions(464 mg/L).Daptomycin is determined to be an extracellular product(>90%)by studying the daptomycin distribution in the fermentation broth.The thermal stability,acid solution stability and the distribution of daptomycin in solution of butanol-water mixtures was investigated.The technology of crude product preparation was determined on that basis.The final purity of crude daptomycin is 10-15% and the final yield is about 90%.