One-Step Multi-fragment Assembly and Targeted Genomic Integration in <i>Corynebacterium glutamicum</i>

<i>Corynebacterium glutamicum</i> 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 <u>O</u>ne-Step <u>M</u>ulti-Fragment <u>A</u>ssembly <u>I</u>ntegration (OMAI) strategy, in which multiple overlapping PCR fragments are cotransformed and assembled <i>in vivo</i>, 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 <i>C. glutamicum</i>.