中文摘要：

枯草芽孢杆菌是一种革兰氏阳性好氧细菌,因其安全性和高分泌特性,已被广泛用作异源蛋白的表达宿主。然而,相比大肠杆菌,枯草芽孢杆菌的转化效率低,限制了其作为宿主的异源蛋白的定向进化。本文通过改变培养基、诱导剂浓度、质粒类型等参数优化感受态制备的条件,利用常规质粒进行转化,结果表明,用营养丰富的YN培养基替代常规LB培养基制备感受态,可以使转化效率提高4倍左右;加入1.5%的木糖诱导2 h,感受态的转化效率又提高2倍左右;用大肠杆菌Escherichia coli GM272来源的质粒又可进一步提高转化效率3倍左右。综合最优条件制备SCK6的感受态,转化整合型质粒p DG1730,效率可以达到10~6 CFU/μg,相对未优化的条件提高了2个数量级,为基于枯草芽孢杆菌的酶的定向进化和代谢工程奠定了基础。

英文摘要：

Bacillus subtilis is Gram-positive aerobic bacterium and widely used as a heterologous protein expression host because of its safety and high protein secretion property. However, comparing to Escherichia coli, the low transformation efficiency limits the application of B. subtilis as a host cell for directed evolution of heterologous enzymes. Therefore, we optimized the competent cell preparation conditions for conventional plasmid, including the alteration of the medium, the concentration of inducer, the plasmid type, and other parameters. Compared with the original LB medium, YN medium improved the transformation efficiency by about 4 folds. The transformation efficiency enhanced by about 2 folds under induction with 1.5% xylose for 2 h. In addition, with plasmids prepared from E. coli GM272 strain the transformation efficiency increased by about 3 folds. Combining all these findings, the transformation efficiency of pDG1730 plasmid under the optimized conditions could reach 106 CFU/~tg, which was 2 orders of magnitude higher than that the original. Our findings provide references for directed evolution of enzymes and metabolic engineering in Bacillus subtilis.