LAUSR

Abstract Interest in microbial 2,3-butanediol (2,3-BD) production has increased significantly because of its wide range of industrial applications. Bacillus subtilis is a potent 2,3-BD producer with GRAS status. However, 2,3-BD yields need to be improved to increase its potential in industrial-scale production. In this study, the 2,3-BD pathway in B. subtilis is engineered to improve 2,3-BD synthesis. The results showed that a 13.5-fold increase in the regulator ALsR transcriptional level resulted in the highest improvement 17.1 % of the total amount of acetoin and 2,3-BD. An extra NADH-dependent 2,3-BD dehydrogenase and an exogenous NADPH-dependent 2,3-BD dehydrogenase are both introduced into B. subtilis to improve the intracellular cofactors regeneration and enhance 2,3-BD production. This decreased the titer of acetoin by 28.9 % and increased 2,3-BD by 26.9 %. In addition, ldhA is knocked out to further drive carbon flux and NADH to 2,3-BD; this increased 2,3-BD yield by 37.7 %, and decreased lactate formation by 86.2 %. Finally, to meet different requirements of oxygen supply for cell growth and 2,3-BD formation, a three-stage oxygen supply control strategy is employed based on the characteristic of 2,3-BD fermentation, and a high titer (102.6 g/L) and productivity (0.93 g/L/h) of 2,3-BD were achieved at 110 h. The strategies proposed in this study have proven to result in an efficient 2,3-BD production in B. subtilis.