Department of Food Science and Biotechnology, Sungkyunkwan University (SKKU), 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea.
Clean Energy Research Center, Korea Institute of Science and Technology, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea.
Enzyme Microb Technol. 2018 Jul;114:63-68. doi: 10.1016/j.enzmictec.2018.04.004. Epub 2018 Apr 9.
RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance production of value-added chemicals in Corynebacterium glutamicum as an industrial host. In this study, the RNA-guided CRISPR interference (CRISPRi) was applied to rapidly identify of unknown genes for native esterase activity in C. glutamicum. Combining with the carboxyl esterase (MekB) protein sequence alignment, two target genes (the cg0961 and cg0754) were selected for the CRISPRi application to investigate the possible native esterase in C. glutamicum. The recombinant strain with repressed expression of the cg0961 gene exhibited almost no capability on degradation of methyl acetate as a substrate of carboxyl esterase. This result was also confirmed in the cg0961 gene deletion mutant. Thus, we concluded that Cg0961 plays a major role of the native carboxyl esterase activity in C. glutamicum. In addition, CRISPRi demonstrated an application for gene identification and its function as another genetic tool for metabolic engineering in C. glutamicum.
RNA 引导的基因组编辑技术已被开发用于微生物细胞的高级代谢工程，以提高谷氨酸棒杆菌作为工业宿主生产有价值化学品的能力。在这项研究中，RNA 引导的 CRISPR 干扰（CRISPRi）被应用于快速鉴定谷氨酸棒杆菌中天然酯酶活性的未知基因。结合羧基酯酶（MekB）蛋白序列比对，选择了两个靶基因（cg0961 和 cg0754）用于 CRISPRi 应用，以研究谷氨酸棒杆菌中可能存在的天然酯酶。抑制 cg0961 基因表达的重组菌株几乎没有降解甲基乙酸盐（羧基酯酶的底物）的能力。这一结果在 cg0961 基因缺失突变体中也得到了证实。因此，我们得出结论，Cg0961 在谷氨酸棒杆菌中发挥主要的天然羧基酯酶活性作用。此外，CRISPRi 展示了一种用于基因鉴定及其功能的应用，作为谷氨酸棒杆菌代谢工程的另一种遗传工具。
