Corynebacterium glutamicum Genome sequencing and assembly. Corynebacterium glutamicum

Genome editing technology is a powerful tool for programming microbial cell factories. Herein, we demonstrate the genome engineering of Corynebacterium glutamicum as a model bacterium by generating premature termination codons (PTCs) in desired genes using cytosine base editors (CBEs). Through this CBE-STOP approach of introducing specific cytosine conversions, we constructed several single gene-inactivated strains for three genes (ldh, idsA, and pyc) and ultimately developed a strain with five genes (ldh, actA, ackA, pqo, and pta) that were inactivated in a sequential manner for enhancing succinate production with high base editing efficiencies. Although these mutant strains showed the desired phenotypes, whole genome sequencing (WGS) data revealed that genome-wide point mutations occurred in each strain and further accumulated according to the duration of CBE plasmids. To increase precision of the base editor, high-fidelity CBEs (YE1-BE3 and BE3-R132E) employed for CBE-STOP, resulting in drastically reduced sgRNA-independent off-targets. Thus, we provide a precision bacterial genome engineering tool with high efficiency and precision.