Abstract

Corynebacterium glutamicum is a key industrial chassis for producing high-value chemicals, particularly amino acids. However, integration of large DNA fragments remains either inefficient or labor-intensive. Here, we optimized a RecET variant-assisted homologous recombination system to achieve single-step integration of DNA fragments larger than 11.3 kb by transforming donor DNA derived from either chromosome or linear DNA fragments. To further expand the size limit, we developed the One-Step Multi-Fragment Assembly Integration (OMAI) strategy, in which multiple overlapping PCR fragments are cotransformed and assembled in vivo, permitting integration of heterologous sequences with a length approximately 50% longer than the conventional single-fragment limit. Each editing cycle of OMAI is completed within 3 days, which is expected to be the most rapid method for large-fragment insertion in C. glutamicum.