DRAGON: Harnessing the power of DNA repair for accelerating genome evolution in Corynebacterium glutamicum.

Hypermutation is a robust phenotype characterized by high elevation of spontaneous mutation rates, which has been shown to facilitate rapid adaptation to the stressful environments by hitchhiking with favorable mutations. Accumulating evidence argues that deficient DNA repair can give rise to hypermutation events in bacteria. Here, we provided a comprehensive survey of DNA repair systems to identify promising targets ensuring high DNA fidelity in Corynebacterium glutamicum. Four effective DNA repair factors, including nucS, tag, xpb, and dinP, were found to be strongly associated with the occurrence of hypermutable phenotypes, and these targets were then engineered to establish a CRISPRi-based all-in-one plasmid system for genome mutagenesis. On the basis of these findings, we presented a novel evolutionary engineering method named "DNA repair-assisted genome evolution (DRAGON)". As a proof-of-concept, DRAGON strategy was successfully applied to facilitate rapid acquisition of microbial robustness in C. glutamicum, such as increased tolerances towards kanamycin, acidic pH and high L-serine, showing its promise and potential for rapid strain improvement. Overall, our study will offer new insights into the understanding of DNA repair and evolutionary adaptation in C. glutamicum.