Rapid identification of unknown carboxyl esterase activity in <i>Corynebacterium glutamicum</i> using RNA-guided CRISPR interference

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P>RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance production of value-added chemicals in <I>Corynebacterium glutamicum</I> 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 <I>C. glutamicum</I>. Combining with the carboxyl esterase (MekB) protein sequence alignment, two target genes (the <I>cg0961</I> and <I>cg0754</I>) were selected for the CRISPRi application to investigate the possible native esterase in <I>C. glutamicum</I>. The recombinant strain with repressed expression of the <I>cg0961</I> gene exhibited almost no capability on degradation of methyl acetate as a substrate of carboxyl esterase. This result was also confirmed in the <I>cg0961</I> gene deletion mutant. Thus, we concluded that Cg0961 plays a major role of the native carboxyl esterase activity in <I>C. glutamicum</I>. In addition, CRISPRi demonstrated an application for gene identification and its function as another genetic tool for metabolic engineering in <I>C. glutamicum.</I> </P> <P><B>Highlights</B></P> <P> <UL> <LI> RNA-guided genome engineering technology was applied for the gene identification. </LI> <LI> Cg0961 plays a major role of the native esterase activity in <I>C. glutamicum</I>. </LI> <LI> CRISPR interference saved time for the laborious work to delete the target gene. </LI> <LI> CRISPR interference has potentials used for genome-wide gene identifications. </LI> </UL> </P>