Homing endonuclease I-SceI-mediated Corynebacterium glutamicum ATCC 13032 genome engineering

Corynebacterium glutamicum is widely used to produce amino acids and is a chassis for the production of value-added compounds. Effective genome engineering methods are crucial to metabolic engineering and synthetic biology studies of C . glutamicum . Herein, a homing endonuclease I-SceI-mediated genome engineering strategy was established for the model strain C . glutamicum ATCC 13032. A vegetative R6K replicon-based, suicide plasmid was employed. The plasmid, pLS3661, contains both tightly regulated, IPTG (isopropyl-β-D-1-thiogalactopyranoside)-inducible I-SceI expression elements and two I-SceI recognition sites. Following cloning of the homologous arms into pLS3661 and transfer the recombinant vector into C . glutamicum ATCC 13032, through the homologous recombination between the cloned fragment and its chromosomal allele, a merodiploid was selected under kanamycin selection. Subsequently, a merodiploid was resolved by double-stranded break repair stimulated by IPTG-stimulated I-SceI expression, generating desired mutants. The protocol obviates a pre-generated strain, transfer of a second I-SceI expression plasmid, and there is not any strain, medium, and temperature restrictions. We validated the approach via deletions of five genes (up to ~ 13.0 kb) and knock-in of one DNA fragment. Furthermore, through kanamycin resistance repair, the ssDNA recombineering parameters were optimized. We hope the highly efficient method will be helpful for the studies of C. glutamicum , and potentially, to other bacteria. Key Points • Counterselection marker I-SceI-mediated C. glutamicum genome engineering • A suicide vector contains I-SceI expression elements and its recognition sites • Gene deletions and knock-in were conducted; efficiency was as high as 90% • Through antibiotic resistance repair, ssDNA recombineering parameters were optimized