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.

中文翻译：

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CRISPR-Cas9 介导的地衣芽孢杆菌工程用于 (2R,3S)-丁二醇的工业生产

地衣芽孢杆菌是一种工业上有前途的公认安全 (GRAS) 菌株，可用于生产有价值的化学品 2,3-丁二醇 (BDO)。传统的基因缺失载体和/或方法耗时且效率低下。因此，成簇的规则间隔短回文重复序列 (CRISPR)-Cas9 介导的同源重组被用于设计一种新分离和紫外线诱变的地衣芽孢杆菌4071-15 菌株。借助 CRISPR-Cas9 系统，这一一步过程可在 4 天内完成ldh基因的删除，效率超过 60%。此外，还评估了用于工程研究的目标基因的顺序缺失，并证实了三重突变菌株（ldh、dgp和acoR ) 可以通过重复的一步循环获得。此外，使用 CRISPR-Cas9 系统进行了一项实用的代谢工程研究，用于立体特异性生产 (2R,3S)-BDO。选择由gdh基因编码的预测的(2R,3R)-丁二醇脱氢酶作为产生(2R,3S)-BDO的靶标，并成功获得突变体。结果表明，(2R,3S)-BDO 的立体特异性生产可能与gdh缺失突变体一起产生，而 4071-15 宿主菌株仍产生 26% 的 (2R,3R)-BDO。还表明，4071-15 Δgdh通过两阶段补料分批发酵，突变体可在 64 小时内产生 115 g/L 的 (2R,3S)-BDO。本研究表明，基于 CRISPR-Cas9 和发酵技术，可有效开发产生 (2R,3S)-BDO的地衣芽孢杆菌菌株。