Optimizing recombineering in Corynebacterium glutamicum

Owing to the increasing demand for amino acids and valuable commodities that can be produced by Corynebacterium glutamicum, there is a pressing need for new rapid genome engineering tools that improve the speed and efficiency of genomic insertions, deletions, and mutations. Recombineering using the λ Red system in Escherichia coli has proven very successful at genetically modifying this organism in a quick and efficient manner, suggesting that optimizing a recombineering system for C. glutamicum will also improve the speed for genomic modifications. Here, we maximized the recombineering efficiency in C. glutamicum by testing the efficacy of seven different recombinase/exonuclease pairs for integrating single‐stranded DNA and double‐stranded DNA (dsDNA) into the genome. By optimizing the homologous arm length and the amount of dsDNA transformed, as well as eliminating codon bias, a dsDNA recombineering efficiency of 13,250 transformed colonies/109 viable cells was achieved, the highest efficiency currently reported in the literature. Using this optimized system, over 40,000 bp could be deleted in one transformation step. This recombineering strategy will greatly improve the speed of genetic modifications in C. glutamicum and assist other systems, such as clustered regularly interspaced short palindromic repeats and multiplexed automated genome engineering, in improving targeted genome editing. Recombinase and exonuclease pairs have proven to be very successful at genetically modifying microbes in a quick and efficient manner. Here, we maximized recombineering efficiency in Corynebacterium glutamicum using the RecET system by optimizing the homologous arm length and the amount of dsDNA transformed, as well as eliminating codon bias. By multiplexing this recombineering system, we obtained three distinct genomic modifications, demonstrating this necessary tool to advance genome engineering in C. glutamicum.