Studies On Daptomvcin Fermentation By Streptomyces Roseosporus

In order to improve the yield of the daptomycin produced by Streptomyces roseosporus, production medium and cultural conditions were optimized by single-factor experiment, orthogonal experiment, and response surface methodology, and the influences of glucose feeding and different amino acids feeding on daptomycin fermentation were also investigated. The results are as follows,HPLC analysis for daptomycin concentration in fermentation broth was established. The column packed with Hypersil ODS2(4.6mmx250mm,5μm) was developed with acetonitrile containing0.1%trifluoroacetic acid and ultrapure water containing0.1%trifluoroacetic acid (V/V=45:55) as the flow phase, and at a flow of1.0ml/min. An aliquot of20μL of the supernatant was injected, and the daptomycin concentration was detected in UV absorption at220nm.Single-factor experiment was used to optimize the fermentation cultural conditions of Streptomyces roseosporus, and the optimum conditions obtained were inoculum time,42h; inoculum size,2%; medium capacity,25ml/250ml; cultural temperature,28℃; and initial pH,7.5. The production medium was also optimized by orthogonal experiment. Under the optimum medium and conditions, the yield of daptomycin was increased by107.99%than that before optimization.The statistical method combined PB design, the steepest ascent method and CCD was used to optimize and evaluate the medium components and main external conditions for daptomycin production. The optimum medium was obtained, and the yield of daptomycin was191.99mg/L which was in agreement with predicted value (184.67mg/L), and2.25folds higher than that with the initial medium.The influences of glucose feeding on daptomycin fermentation process were investigated in shaking flask and2.5L bioreactor, respectively. In shaking flask, the optimal initial glucose concentration was40g/L for daptomycin fermentation. By adding20g/L glucose at the late-exponential phase, the yield of daptomycin was improved by39.77%. When four aliquots of20g/L glucose were added at4different time intervals during the exponential phase and stationary phase, the final yield was increased by62.06%. In a2.5L bioreactor, when30g/L glucose was added after12h of fermentation, not only the duration of daptomycin biosynthesis was prolonged effectively, but also the productivity of daptomycin was enhanced. The daptomycin production with glucose feeding in2.5L fermentor reached90.42mg/L, which was1.39folds higher than that of batch fermentation.Effect of adding amino acids on daptomycin production was studied, and the results showed that L-Trp and L-Asp can stimulate the synthesis of daptomycin. By adding1g/L L-Trp into initial medium, the yield of daptomycin was increased by2.5folds. When1.75g/L L-Trp was added at the exponential phase, the yield of daptomycin reached160.23mg/L,3.6folds improved compared with that without amino acid addition.