RAGATH-Associated DNA Nuclease Assisted DNA Insertion in Corynebacterium glutamicum

Corynebacterium glutamicum serves
as a key microbial chassis for the industrial production of feed and
food ingredients. While long DNA fragment insertion technologies have
advanced strain engineering capabilities, previous approaches such
as utilizing a chromosome-integrated Cas9-RecET system were constrained
by a maximum insertion fragment size of 7.5 kb. Through systematic
evaluation of Cas9, gRNA, and recombinase expression driven by five
distinct promoters and their implementation on 1 or 2 plasmids with
compatible replicons (resulting in a total of 17 combinations), we
developed an optimized genome editing vector capable of inserting
DNA fragments of up to 8.0 kb in C. glutamicum. Parallel implementation of the Cpf1 system also successfully achieved
8.0 kb of DNA insertions. However, the construction of plasmids carrying
insertion sequences larger than 8.0 kb was hindered by the plasmid
vector capacity. To address this limitation, we screened six smaller
RAGATH-associated DNA nucleases, ultimately identifying two with high
cleavage activity in C. glutamicum. These nucleases
demonstrated superior editing efficiencies compared to both Cas9 and
Cpf1, enabling the integration of DNA fragments up to 11.3 kbsurpassing
previously reported size limitations for C. glutamicum. These RAGATH-associated DNA nuclease-based systems effectively
overcome the previous size constraints for long fragment insertions,
thereby advancing metabolic engineering and fundamental research applications.