CRISPR‐Cas9 mediated engineering of Bacillus licheniformis for industrial production of (2R,3S)‐butanediol

Bacillus lichenformis is an industrially promising generally recognized as safe (GRAS) strain that can be used for the production of a valuable chemical, 2,3‐butanediol (BDO). Conventional gene deletion vectors and/or methods are time‐consuming and have poor efficiency. Therefore, clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas9 mediated homologous recombination was used to engineer a newly isolated and UV‐mutagenized B. licheniformis 4071–15 strain. With the help of a CRISPR‐Cas9 system, this one‐step process could be used for the deletion of ldh gene within 4 days with high‐efficiency exceeding 60%. In addition, the sequential deletion of target genes for engineering studies was evaluated, and it was confirmed that a triple mutant strain (ldh, dgp, and acoR) could be obtained by repeated one‐step cycles. Furthermore, a practical metabolic engineering study was carried out using a CRISPR‐Cas9 system for the stereospecific production of (2R,3S)‐BDO. The predicted (2R,3R)‐butanediol dehydrogenase encoded by the gdh gene was selected as a target for the production of (2R,3S)‐BDO, and the mutant was successfully obtained. The results show that the stereospecific production of (2R,3S)‐BDO was possible with the gdh deletion mutant, while the 4071–15 host strain still generated 26% of (2R,3R)‐BDO. It was also shown that the 4071–15 Δgdh mutant could produce 115 g/L of (2R,3S)‐BDO in 64 hr by two‐stage fed‐batch fermentation. This study has shown the efficient development of a (2R,3S)‐BDO producing B. licheniformis strain based on CRISPR‐Cas9 and fermentation technologies.