Construction and application of a CRISPR/Cas9-assisted genomic editing system for Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum is widely used as microbial cell factory for various bioproducts, but its genomic editing efficiency needs to be improved. In this study, a highly efficient CRISPR/Cas9-assisted genomic editing system for C. glutamicum was constructed. This system mainly involves a plasmid and can be used for both gene insertion and deletion in the chromosome of C. glutamicum. The recombinant plasmid for the target gene containing all the editing elements, and first constructed it in E. coli, then purified and transformed it into C. glutamicum. This temperature-sensitive plasmid was cured at high temperature after the genomic editing was completed in C. glutamicum. Using this genetic editing system, the genetic editing efficiency in C. glutamicum ATCC 13032 could reach 95%. The whole work of editing could be done in 8–9 days and showed most time-saving compared to the reported. Using this system, the native promoter of gdhA1 in ATCC 13032 has been replaced with the strong promoter PtacM, and more than 10 genes in ATCC 13032 have been deleted. The results demonstrate that this CRISPR/Cas9-assisted system is highly efficient and very suitable for genome editing in C. glutamicum.

Chromosomal editing of Corynebacterium glutamicum ATCC 13032 to produce gamma‐aminobutyric acid

“…The strains and plasmids mainly used in this study are listed in Table 1. The other plasmids were shown in the previous reported 48 . C. glutamicum ATCC 13032 is wild type, NCBI Reference Sequence: NC_003450.2.…”

“…The CRISPR/Cas9‐based method 48 was used to edit the genome of C. glutamicum ATCC 13032 in this study. The primers used in this study are listed in Table 2.…”

“…Electroporation was used to introduce plasmids into C. glutamicum . Electroporation‐competent cells of C. glutamicum were prepared as previously reported 48 . For genomic editing, 1 μg plasmid was mixed with 80 μL competent cells and added the mixture into precooled 1 mm gene pulser cuvette (Bio‐Rad, USA), then keep on ice for 10–15 min.…”

“…Electroporation-competent cells of C. glutamicum were prepared as previously reported. 48 For genomic editing, 1 μg plasmid was mixed with 80 μL competent cells and added the mixture into precooled 1 mm gene pulser cuvette (Bio-Rad, USA), then keep on ice for 10-15 min. Electroporation in a micropulser (Bio-Rad, USA) at 1.8 kV for twice, the cells were resuspended immediately in 0.9 mL precooled liquid BHIS medium and recovered at 30 • C, 150 rpm for 1-1.5 h in a shaker, then the cells were spread on BHIS agar (supplementing 0.01 mM IPTG, 20 mg/L kanamycin) and cultured for 2-3 days at 28 • C. The transformants were verified by colony PCR.…”

Synthetic Biology Toolkits and Metabolic Engineering Applied in Corynebacterium glutamicum for Biomanufacturing

“…This system was suitable for both small and large (>3 kb) target gene modification with a 50–95% editing efficiency in C. glutamicum ATCC 13032. However, this system was still required to further improve the editing efficiency in other C. glutamicum strains except ATCC 13032 and ATCC 14067 . In addition, the CRISPR/Cas9 systems combined with recombinase RecT were successfully established.…”