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

Abstract

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. 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 P45 , Ptrc , and PH36 , were used to replace PglyA and to expand the application of the CRISPR-Cas9 system. By using this system, a C. glutamicum strain producing L-homoserine to 22.1g per L in a 5-L bioreactor after 96 h was obtained. 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.