Rapid identification of unknown carboxyl esterase activity in Corynebacterium glutamicum using RNA-guided CRISPR interference.

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.