Development of a genetically engineered Escherichia coli strain for plasmid transformation in Corynebacterium glutamicum

- A novel strain E. coli AU1 was developed by chromosomally integrating the methyltransferase gene cglIM from C. glutamicum.
- The plasmid isolated from E. coli AU1 is able to resist the cglIR-cglIIR restriction system in C. glutamicum.
- E. coli AU1 is a useful intermediate host for efficient transformation of C. glutamicum.

Gene disruption and replacement in Corynebacterium glutamicum is dependent upon a high transformation efficiency. The cglIR-cgIIR restriction system is a major barrier to introduction of foreign DNA into Corynebacterium glutamicum cells. To improve the transformation efficiency of C. glutamicum, the cglIM gene encoding methyltransferase in the cglIR-cglIIR-cglIM restriction-modification system of C. glutamicum ATCC 13032 was chromosomally integrated and expressed in Escherichia coli, resulting in an engineered strain E. coli AU1. The electro-transformation experiments of C. glutamicum ATCC 13032 with the E. coli-C. glutamicum shuttle plasmid pAU4 showed that the transformation efficiency of C. glutamicum with pAU4 DNA extracted from E. coli TG1/pAU4 was 1.80 ± 0.21 × 102 cfu/μg plasmid DNA, while using pAU4 DNA extracted from E. coli AU1/pAU4, the transformation efficiency reached up to 5.22 ± 0.33 × 106 cfu/μg plasmid DNA. The results demonstrated that E. coli AU1 is able to confer the cglIM-specific DNA methylation pattern to its resident plasmid, which makes the plasmid resistant to the cglIR-cglIIR restriction and efficiently transferred into C. glutamicum. E. coli AU1 is a useful intermediate host for efficient transformation of C. glutamicum.