Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, the People's Republic of China.
Can J Microbiol. 2011 May;57(5):427-36. doi: 10.1139/w11-035. Epub 2011 May 5.
This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.
本研究描述了一种在枯草芽孢杆菌 168 中重复基因失活的新方法。通过用 Pspac 启动子替换 PlysA 启动子，获得了条件性赖氨酸营养缺陷型枯草芽孢杆菌菌株（BS-PS）。构建了同源重组整合载体 PLC-T，其中包含编码 Pspac 启动子抑制剂的 lacI 和氯霉素抗性基因。通过在 PLC-T 中生成带有两个同源臂和靶基因的插入序列来操纵靶基因，从而创建了一个特定的整合载体。整合到 BS-PS 染色体中是通过两个同源臂中的任何一个发生单交换完成的。由此产生的过渡菌株（BS-PS-PI）对氯霉素具有抗性且对赖氨酸有营养缺陷，由于重复出现不稳定的基因组结构。通过在重复处的第二次单交换实现了 lacI 和氯霉素抗性基因的切除。通过 PCR 鉴定了 nprE 和 aprE 两个蛋白酶基因的缺失，nprE 和 aprE 是枯草芽孢杆菌 168 分泌的两种蛋白酶基因，缺失过程中没有选择标记。
