One-Step Multi-fragment Assembly and Targeted Genomic Integration in Corynebacterium glutamicum

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.