Optimization Of Fermentation Medium And Strain Breeding For Titer Improvement Of Daptomycin By Streptomyces Coelicolor

Daptomycin,a new type of cyclic lipopeptide antibiotic,was originally isolated from Streptomyces roseosporus NRRL 11379.It was named Cubicin and marketed in the United States by Cubist Pharmaceuticals in 2003.Its distinct mechanism of action makes it useful in treating complicated skin and skin structure infections caused by high pathogenic Gram-positive pathogens including methicillin-resistant Staphylococcus aureus(MRSA).As a result,compared to vancomycin,daptomycin has become the best alternative and has a broad application prospect in medicine.However,the titer of daptomycin is too low to meet the industry needs.Therefore,it is of great significance to improve the yield of daptomycin.In this paper,Streptomyces coelicolor K10,a heterologous expression strain of daptomycin,was used as the producing strain of daptomycin.The medium formula for daptomycin improvement was optimized using response surface methodology(RSM)preliminarily.To further increase the yield of daptomycin,we used UV and NTG mutagenesis methods in combination with multiple rounds of genome shuffling to breed the high producing strain of daptomycin..The major results are as follows:1)An optimal medium formula for daptomycin was determined.We optimized carbon sources,nitrogen sources and trace elements in medium using single factor test.Dextrin and maltose as composite carbon sources were substituted for soluble starch and glucose in original medium,and L-asparagine was added as one of medium composition.Afterwards,the key ingredients of medium components were found out using PB design experiment,and their optimal concentrations were determined by RSM.The results of PB design experiment revealed that dextrin,yeast extract and casein were the crucial ingredients for titer improvement of daptomycin..A prediction model has been built according to the experiments of central composite design.By Design Expert software 8.0 version,the optimal medium composition was calculated(g/L): dextrin 25.11,maltose 16.00,yeast extract 2.20,casein2.00,L-asparagine 1.00,MOPS 10.00 and p H 7.0.Under the optimum medium,the yields of daptomycin was 15.30 ± 1.23 mg/L,which was 2.17 times higher than that of initial medium.2)Breeding strain for titer improvement of daptomycin was carried out using various mutagenesis approaches.Taked the S.coelicolor K10 as the original strain,we first obtained six mutants UV-1,UV-19,UV-23,NTG-1,NTG-5 and NTG-9 with high yields of daptomycin.Two rounds of genome shuffling mutagenesis were then performed in sequence to continue for breeding high yielding mutants.As anticipated,we obtained one high producing mutant GS-2-30 with the yields of 39.66 ± 0.99 mg/L,which was 1.59-fold higher of that for the parent strain of K10.In addition,the strain GS-2-30 is genetically stable.