Engineering Escherichia coli BL21 genome to improve the heptanoic acid tolerance by using CRISPR-Cas9 system

Acid tolerance is one of the critical factors to determine the quality of the industrial production strains. Therefore, we have investigated the introduction of the acid tolerance genes into the genome of Escherichia coli BL21 by using CRISPR-Cas9 system. The dsrA and rcsB genes of E. coli K-12, which are involved in the heptanoic acid tolerance, were inserted into the genome of E. coli BL21 without scar. The native transcription unit (TU) of dsrA and the synthetic TU of rcsB were integrated in E. coli BL21 genome. We found that the position of genomic coordinate of 1,300,270 was more efficient to integrate dsrA and rcsB than genomic coordinate of 3,876,428. Furthermore, the rcsB was successfully expressed in the resulting engineered strains (i.e., rcsB⁺ or dsrA⁺rcsB⁺ strains). The engineered strains expressing dsrA and/or rcsB showed the higher survival rate and specific growth rate under n-heptanoic acid stress than wild-type E. coli BL21. These results indicate that the newly introduced acid-tolerance systems were active in the E. coli BL21 strain.