RAGATH-Associated DNA Nuclease Assisted DNA Insertion in Corynebacterium glutamicum

• DOI: 10.1021/acssynbio.5c00022
• Publication Date: 2025-05-08T13:50:46Z
• Authors: Xiaoyu Wang; Siqi Yang; Fenghui Qian; Feng Dong; Xiaojie Zhou; Mingyu Yin; Ying Zhang; Zhiwei Huang; Yu Jiang; Sheng Yang
• Keywords: Corynebacterium glutamicum; Nuclease

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 utilizing chromosome-integrated Cas9-RecET system were constrained by maximum size 7.5 kb. Through systematic evaluation Cas9, gRNA, recombinase expression driven five distinct promoters their implementation on 1 or 2 plasmids with compatible replicons (resulting in total 17 combinations), we developed an optimized genome editing vector capable inserting fragments up to 8.0 kb C. glutamicum. Parallel Cpf1 also successfully achieved insertions. However, construction carrying sequences larger than was hindered plasmid capacity. To address this limitation, screened six smaller RAGATH-associated nucleases, ultimately identifying two high cleavage activity These nucleases demonstrated superior efficiencies compared both Cas9 Cpf1, enabling integration 11.3 kb─surpassing previously reported limitations nuclease-based systems effectively overcome constraints insertions, thereby advancing metabolic fundamental research applications.