Construction and application of an inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system in Corynebacterium glutamicum

Aiming at the problems of low efficiency of gene editing and time-consuming operation in Corynebacterium glutamicum, the Cpf1 was expressed using the inducible promoter PrpR-PrpD2, the recombination and cleavage events were separated, and a high-efficiency inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system was established. The gene editing efficiency of the dual-plasmid gene editing system was statistically calculated by comparing the expression effects of Cpf1 protein with constitutive and inducible promoters. Finally, using C. glutamicum ATCC 13032 without L-serine production as starting strain, a high-producing L-serine recombinant strain was constructed using this system. The results showed that the gene deletion efficiency of the inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system was (66.67±1.53)%, and the gene integration efficiency was (14.29±1.50)%, both of which were significantly higher than those of the constitutive promoter group (P<0.05). In addition, an high-producing L-serine recombinant strain SW-3 was successfully constructed using the inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system with an L-serine yield of 10.51 mg/L, which indicated that the inducible CRISPR-Cpf1/dsDNA dual-plasmid gene editing system was feasible in the metabolic engineering modification of C. glutamicum, and it would have far-reaching significance in accelerating the process of metabolic engineering modification of C. glutamicum and promoting its application in the research and development of amino acids and other high value-added products.