Optimization of CRISPR-Cas9 through promoter replacement and efficient production of L-homoserine in Corynebacterium glutamicum

Background: Corynebacterium glutamicum is an important chassis for industrial applications. The low efficiency of commonly used genome editing methods for C. glutamicum limits the rapid multiple engineering of the bacterium. Main Methods and Major Results: In this study, chromosome-borne expression of cas9 and recET from Escherichia coli K12-MG1655 was achieved to avoid toxicity to the strain, increase the probability of homologous recombination, and reduce loss of viability caused by double-strand breaks. Constitutive strong promoters, such as P-45, P-trc, and P-H36, were used to replace P-glyA and to expand the application of the CRISPR-Cas9 system. By using this system, a C. glutamicum strain producing L-homoserine to 22.1 g per L in a 5-L bioreactor after 96 h was obtained. Conclusions and Implications: Through the application of visualized fluorescent protein, the process of plasmid curing was optimized, obtain a continuous and rapid CRISPR-Cas9 genome editing system. The method described here could be useful to construct C. glutamicum mutant rapidly.

Automated summary

The study successfully achieved chromosome-borne expression of cas9 and recET genes from E. coli in C. glutamicum, which increased the probability of homologous recombination and reduced loss of viability. By using strong promoters to replace P-glyA, a continuous and rapid CRISPR-Cas9 genome editing system was developed, leading to the high production of L-homoserine in the bacterium.