CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum

- Development of CRISPR/Cas9-based knockout in C. glutamicum.
- CRISPR/Cas9-coupled recombineering efficiency up to 100% for gene knockout.
- Developed a curable plasmid based on pCC1 origin for orthogonal plasmid curing.
- Demonstration of simultaneous, coupled deletion of two genes with one sgRNA.
- Generated and screened for C. glutamicum mutants producing high titers of GABA.

Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study the combinatorial deletion effects of three different genes on the production of γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum.

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