An update of the suicide plasmid‐mediated genome editing system in Corynebacterium glutamicum

Summary Corynebacterium glutamicum is an important industrial microorganism, but the availability of tools for its genetic modification has lagged compared to other model microorganisms such as Escherichia coli. Despite great progress in CRISPR‐based technologies, the most feasible genome editing method in C. glutamicum is suicide plasmid‐mediated, the editing efficiency of which is low due to high false‐positive rates of sacB counter selection, and the requirement for tedious two‐round selection and verification of rare double‐cross‐over events. In this study, an rpsL mutant conferring streptomycin resistance was harnessed for counter selection, significantly increasing the positive selection rate. More importantly, with the aid of high selection efficiencies through the use of antibiotics, namely kanamycin and streptomycin, the two‐step verification strategy can be simplified to just one‐step verification of the final edited strain. As proof of concept, a 2.5‐kb DNA fragment comprising aroGfbrpheAfbr expressing cassettes was integrated into the genome of C. glutamicum, with an efficiency of 20% out of the theoretical 50%. The resulting strain produced 110 mg l−1 l‐tyrosine in shake‐flask fermentation. This updated suicide plasmid‐mediated genome editing system will greatly facilitate genetic manipulations including single nucleotide mutation, gene deletion and gene insertion in C. glutamicum and can be easily applied to other microbes. Mutated rpsL gene encoding small ribosomal protein S12P enables Corynebacterium glutamicum resistance to streptomycin. The second‐crossover strains can be high‐efficiently selected by harnessing rpsL counter‐selection in the suicide plasmid‐mediated genome editing method for C. glutamicum. Taking advantage of the biotics, kanamycin and streptomycin selection, the procedure can be simplified to one‐step verification of edited strains.