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.