星形孢菌素产生菌电转化条件的优化及其高产菌株的构建  

Optimization of electrotransformation of staurosporine producing Streptomyces sp.and construction of high-yield mutants

作　　者：黄浩龙 田勋 刘佳乐 张梦莹 胡海峰[1,2] Huang Haolong;Tian Xun;Liu Jiale;Zhang Mengying;Hu Haifeng(Shanghai Institute of Pharmaceutical Industry,China State Institute of Pharmaceutical Industry,Shanghai 200040;Sinopharm Health Industry Institute Co.,Ltd.,Shanghai 201203)

机构地区：[1]中国医药工业研究总院上海医药工业研究院,上海200040 [2]国药集团健康产业研究院有限公司,上海201203

出　　处：《中国抗生素杂志》2023年第9期1024-1030,共7页Chinese Journal of Antibiotics

摘　　要：目的提高星形孢菌素的产量。方法本研究以星形孢菌素产生菌(Streptomyces sp.ST-07)为出发菌株,采用电转化方法和应用PCR扩增星形孢菌素生物合成的调控基因staR,成功构建含不同启动子(kasO*p和ermE*p)的加强表达staR基因的重组工程菌,分别命名为ST-D-5和ST-H-23。结果通过对电转化条件的甘氨酸浓度、电场强度、菌丝体浓度、质粒浓度、孵育时间和孵育温度等关键参数优化,使电转化的效率从4×10^(6)提高至9.6×10^(8)(CFU/μg DNA);工程菌摇瓶发酵结果表明,与星孢菌素原始菌株ST-07相比,采用kasO*p启动子构建的工程菌ST-D-5星形孢菌素的产量提高了17%,最高单位可达923 mg/L。结论本文系统摸索了星形孢菌素产生菌的电转化条件,显著提高了电转化效率,不但为该菌株的高产遗传改造奠定了基础,而且对其他放线菌的遗传操作体系建立提供了有益借鉴。Objective To improve the production of staurosporine.Methods In this study,Streptomyces sp.ST-07 was used as the starting strain,its staR gene was amplified by electrotransformation and PCR.Engineered strains containing the staR gene overexpressed under the control of different promoters(kasO*pand ermE*p)were successfully constructed and named ST-D-5 and ST-H-23.Results The electrotransformation method was used to improve the efficiency of transformation from 4×10^(6) to 9.6×10^(8)(CFU/μg DNA)by optimizing key parameters such as the glycine concentration,the electric field intensity,the mycelia concentration,the plasmid concentration,incubation time and temperature.Shaking flask fermentation showed that the yield of staurosporine of engineered strain ST-D-5 with the kasO*p promoter was increased by 17%,the highest titer reached 923 mg/L.Conclusion In this paper,the electrotransformation conditions of the bacteria producing staurosporine were explored systematically,the electrotransformation efficiency was significantly improved.It not only laid a foundation for the genetic modification of high yield of this strain,but also provided a useful reference for the establishment of genetic manipulation system of other actinomyces.

关 键 词：星形孢菌素 电转化 staR基因 kasO*p 启动子 高产菌 

分 类 号：R9[医药卫生—药学]