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

Abstract

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 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 22.1 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.

O-Acetyl-L-homoserine production enhanced by pathway strengthening and acetate supplementation in Corynebacterium glutamicum

“…The plasmids pEC-XK99E and pXMJ19 were used to express the genes. The plasmid pKHAsgRNA was used for genome editing [ 18 ]. The detailed information is listed in Table 1 and Additional file 1 : Table S1, respectively.…”

“…The analytical method is the same as our previous study [ 17 , 18 ]. A biophotometer D30 (Eppendorf) was used to determine OD 600 .…”

“…Reports have shown that L-homoserine and OAH have been produced efficiently in E. coli [12][13][14][15][16]. However, thus far, C. glutamicum has only achieved efficient production of L-homoserine [17,18]. L-Homoserine and acetyl-CoA are the substrates for OAH biosynthesis, whereas the production of OAH in C. glutamicum has not been reported.…”

“…L-Homoserine can be efficiently accumulated in C. glutamicum , indicating that OAH should also be efficiently accumulated through efficient acetyl transfer [ 19 ]. Unfortunately, the engineered C. glutamicum strain only efficiently accumulated L-homoserine but not OAH without knock-out of the metX gene in our previous studies [ 17 , 18 ], suggesting that some problems need to be solved to achieve OAH accumulation. These problems may include the total enzyme activity, specific enzyme activity, heat resistance of L-homoserine acetyltransferase (MetX), and even the supply of acetyl-CoA [ 20 – 22 ].…”

“…In this study, an efficient OAH-producing strain was constructed via metabolic engineering based on an efficient L-homoserine-producing C. glutamicum strain reported in our previous study [ 18 ]. First, various L-homoserine acetyltransferase genes were individually introduced into the efficient L-homoserine-producing C. glutamicum strain.…”