Time-controlled dual-stage strategy for bioproduction of high-purity (R-R) 2,3-butanediol and acetoin in Bacillus licheniformis

(R-R) 2,3-butanediol (BDO) is a chemical compound of significant medical value, and there is a desire to biologically produce high-purity (R-R) 2,3-BDO instead of a mixture of various configurations. Acetoin, a key downstream metabolite, gains attention for food industry applications. Simultaneous production through a single fermentation holds great potential, but the absence of a convenient and efficient separation strategy remains a source of disappointment. Here, we meticulously chose non-pathogenic Bacillus licheniformis as cell factories with specific genetic regulation on this metabolic pathway. Firstly, genome engineering was performed to attain 100% purity in the production of (R-R) 2,3-BDO. Subsequently, by harnessing the inherent regulation of the glyoxylate cycle within the strain, a pioneering two-stage production strategy was developed. This strategy enables selective production of high-purity (R-R) 2,3-BDO or acetoin based on production preferences, simply by terminating fermentation at the appropriate time. When using 80 g/L of glucose as the substrate, it is feasible to obtain 22.0 g/L of (R-R) 2,3-BDO with 100% purity or 26.0 g/L of acetoin with 90.0% purity. In summary, this work presents an inspiring strategy that addresses dual production for a better product separation, which can serve as a source of inspiration for other cell factories in the field of biomanufacturing.