Huang Kai, Zhang Tao, Jiang Bo, Yan Xin, Mu Wanmeng, Miao Ming
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, 210095, China.
Appl Microbiol Biotechnol. 2017 Aug;101(15):6039-6048. doi: 10.1007/s00253-017-8355-9. Epub 2017 Jun 29.
A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg cassettes, in which the R. pycnus arginase gene was fused with the strong promoter P43, were inserted into the recipient chromosome. These multiple insertions were completed via step-by-step integrations into designed (2 copies) and random (9 copies) sites, respectively. A strategy for random site integration was developed based on the construction of the arg cassette sandwiched between "front" and "back" homologous arms which were randomly restricted from chromosomal DNA. An antibiotic resistance marker was applied in transformant selection and was eliminated via the Cre/lox system. Performance showed that the highest enzyme activity (14.5 U/mL) was obtained after culture in flasks, and this segregation stable strain could efficiently hydrolyze L-arginine with a 97.2% molar yield, showing potential application in the food industry.
以枯草芽孢杆菌168为宿主，构建了无质粒、无标记且含有来自致密鲁梅利芽孢杆菌精氨酸酶基因多拷贝整合的菌株。总共九个精氨酸盒拷贝被插入到受体染色体中，其中致密鲁梅利芽孢杆菌精氨酸酶基因与强启动子P43融合。这些多拷贝插入分别通过逐步整合到设计位点（2个拷贝）和随机位点（9个拷贝）完成。基于构建夹在从染色体DNA随机切割得到的“前”“后”同源臂之间的精氨酸盒，开发了一种随机位点整合策略。在转化体筛选中应用了抗生素抗性标记，并通过Cre/lox系统将其去除。性能显示，摇瓶培养后获得了最高酶活性（14.5 U/mL），且该遗传稳定菌株能够高效水解L-精氨酸，摩尔产率达97.2%，在食品工业中显示出潜在应用价值。
