地中海拟无枝酸菌S699电转化方法的优化  

Optimizing Electroporation of Amycolatopsis mediterranei S699

作　　者：王明艳[1] 何璟[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《湖北农业科学》2014年第4期920-924,共5页Hubei Agricultural Sciences

基　　金：国家自然科学基金项目(30800020;31270136);教育部留学回国人员科研启动基金项目([2009]1590);教育部新世纪人才支持计划项目(NCET-08-0779)

摘　　要：地中海拟无枝酸菌S699(Amycolatopsis mediterranei S699)能够产生具有重要经济价值的抗生素———利福霉素B。在该菌株中建立高效的遗传操作系统是研究利福霉素生物合成机制以及构建基因工程高产菌株的基础。研究利用单因素试验探索了供体质粒的构型、甘氨酸溶度、MgCl2浓度及电转化参数对地中海拟无枝酸菌S699电转化效率的影响。结果表明,使用添加了0.05 g/L甘氨酸和2.5 mmol/L MgCl2的34%YEME培养基培养菌丝体,在9 kV/cm、25μF、550Ω的电转化条件下,以双链DNA为供体,转化效率最高可达到3.37×102个转化子/μg DNA。利用优化后的电转化方法,成功地获得了利福霉素生物合成基因簇中细胞色素P450氧化酶基因orf5的同源缺失突变菌株,证实了优化后的电转化方法可以很好地用于后期遗传学及利福霉素生物合成基因的研究。Amycolatopsis mediterranei S699 produced an important antibiotic, rifamycin B having high economic value. To investigate biosynthetic pathway of rifamycins and construct high-yield strains by genetic engineering, establishment of a highly efficient genetic manipulation system was critically required in this strain. The effects of dsDNA and ssDNA, concentration of glycine and MgCl2in culture medium, and electroporation parameters on electroporation efficiency were studied by single factor test. Using 34% YEME medium supplemented with 0.05 g / L glycine and 2.5 mmol / L MgCl2to cultivate mycelia, electroporation parameters of 9 kV / cm, 25 μF and 550 Ω, double-stranded DNA as donor, a highly efficient electroporation method for A. mediterranei S699 was established with the transformation efficiency of 3.37×102transformant / μg DNA. The homologous deletion mutant strain of the cytochrome P450 oxygenase gene orf5 from the rifamycin biosynthetic gene cluster was obtained by using the optimized method. It is demonstrated that the optimized eletroporation could be employed for genetic study on the rifamycin biosynthetic pathway.

关 键 词：地中海拟无枝酸菌S699(Amycolatopsis MEDITERRANEI S699) 利福霉素B 电转化 同源缺失 

分 类 号：Q784[生物学—分子生物学]