Yao Chengzhen, Hu Xiaoqing, Wang Xiaoyuan
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
AMB Express. 2021 May 19;11(1):70. doi: 10.1186/s13568-021-01231-7.
Corynebacterium glutamicum is widely used as microbial cell factory for various bioproducts, but its genomic editing efficiency needs to be improved. In this study, a highly efficient CRISPR/Cas9-assisted genomic editing system for C. glutamicum was constructed. This system mainly involves a plasmid and can be used for both gene insertion and deletion in the chromosome of C. glutamicum. The recombinant plasmid for the target gene containing all the editing elements, and first constructed it in E. coli, then purified and transformed it into C. glutamicum. This temperature-sensitive plasmid was cured at high temperature after the genomic editing was completed in C. glutamicum. Using this genetic editing system, the genetic editing efficiency in C. glutamicum ATCC 13032 could reach 95%. The whole work of editing could be done in 8-9 days and showed most time-saving compared to the reported. Using this system, the native promoter of gdhA1 in ATCC 13032 has been replaced with the strong promoter PtacM, and more than 10 genes in ATCC 13032 have been deleted. The results demonstrate that this CRISPR/Cas9-assisted system is highly efficient and very suitable for genome editing in C. glutamicum.
谷氨酸棒杆菌被广泛用作生产各种生物产品的微生物细胞工厂，但其基因组编辑效率有待提高。在本研究中，构建了一种用于谷氨酸棒杆菌的高效CRISPR/Cas9辅助基因组编辑系统。该系统主要涉及一种质粒，可用于谷氨酸棒杆菌染色体中的基因插入和缺失。包含所有编辑元件的靶向基因重组质粒，先在大肠杆菌中构建，然后纯化并转化到谷氨酸棒杆菌中。在谷氨酸棒杆菌中完成基因组编辑后，这种温度敏感型质粒在高温下被消除。使用这种基因编辑系统，谷氨酸棒杆菌ATCC 13032中的基因编辑效率可达95%。整个编辑工作可在8-9天内完成，与已报道的方法相比最为省时。利用该系统，ATCC 13032中gdhA1的天然启动子已被强启动子PtacM取代，并且ATCC 13032中的10多个基因已被删除。结果表明，这种CRISPR/Cas9辅助系统效率高，非常适合谷氨酸棒杆菌的基因组编辑。
