Study On Production Of Staurosporine By Strain Modification And Optimization Of Fermentation Process

Staurosporine can induce apoptosis of many kinds of cells as a strong protein kinase inhibitor,and thus expected to be used as antitumor drug.But it was limited in clinical trials as anticancer drug due to its poor selectivity and strong toxicity.Now it is mainly used as a leading compound to develop a series of selective protein kinase inhibitors,which could be used in the medical research of tumor and cancer treatment.Staurosporine is obtained by microbial fermentation,but it is difficult to meet the demand of industrial production owing to low production of reported strain.Firstly,we optimized the fermentation mediums and conditions of original strain Streptomyces sp.SIPI 506(145 mg/L of staurosporine),and obtained the suitable fermentation parameters as followings:32 h of seeds incubation time,5%inoculum,5%glycerol as carbon source,and peanut meal 1%+hot pressed soybean cake meal 2%as nitrogen source.After the optimization of process,the yield of staurosporine in shake flask culture reached 289 mg/L.In addition,the original strain Streptomyces sp.SIPI 506 was mutated by different methods,such as UV mutagenesis,NTG mutagenesis,ARTP mutagenesis and DES mutagenesis;A high-yielding mutant strain Streptomyces sp.SIPI D-79(599 mg/L of staurosporine)was obtained.Secondly,the fed-batch culture of Streptomyces sp.SIPI D-79 was carried out in a 5 L fermenter.The titer was improved by optimizing the carbon source feeding,and the final yield of staurosporine reached 527 mg/L.Thirdly,the genomic of the producing strain was sequenced,and the staurosporine biosynthetic gene cluster and a regulatory gene were obtained by bioinformatics analysis.And then a genetic operating method by electrotransformation was established and optimized.Finally,the gene cluster of staurosporine biosynthesis was over-expressed in Streptomyces sp.SIPI D-79 to construct the engineering strain Streptomyces sp.SIPI ST-D-79,and staurosporine production was increased to 814 mg/L.And an engineering strain Streptomyces sp.SIPI StaR-D-79 was constructed by overexpressed staR gene,which had a 27%(766 mg/L)increase in fermentation titer,suggesting that the staR gene was a positive regulatory gene.These results indicated that it is feasible to improve the production of staurosporine by strain modification with classic strain breeding or genetic engineering.