Deciphering <i>D</i>-2,3-Butanediol Metabolic Pathways in a Newly Isolated <i>Bacillus velezensis</i> and Rationally Engineering <i>Bacillus subtilis</i> for Efficient Production of Optically Pure <i>D</i>-2,3-Butanediol

2,3-Butanediol (2,3-BD) is a versatile platform chemical with diverse applications spanning polymers, solvents, and fuel additives. Developing biobased processes for producing this compound with high enantioselectivity and minimal waste generation is crucial for advancing sustainable biomanufacturing from renewable resources. In this work, an environmentally friendly strategy is reported to produce optically pure <i>D</i>-2,3-BD by harnessing and engineering <i>Bacillus</i> strains under mild fermentation conditions. First, an isolate of <i>Bacillus velezensis</i> FJ-4, observed to generate high titers of <i>D</i>-2,3-BD, underwent genome sequencing, and the biosynthetic pathway of <i>D-</i>2,3BD was elucidated. Genes associated with <i>D</i>-2,3-BD synthesis were subsequently introduced into genetically tractable <i>Bacillus subtilis</i> 168 to create an efficient host for <i>D</i>-2,3-BD production. Rational metabolic engineering approaches, including deletion of byproduct pathways (<i>ldh</i> and <i>pta</i>), manipulation of redox balance via <i>nadC</i> knockout, and overexpression of core biosynthetic genes (<i>bdhA</i> and <i>alsSD</i>), led to a near-theoretical yield (92%) and a titer of 34.1 g/L <i>D</i>-2,3-BD. Further tuning of NADH regeneration by integrating <i>fdh</i> and supplementing formate improved <i>D</i>-2,3-BD volumetric productivity by 10%. Overall, this work elucidates the biochemistry of <i>D</i>-2,3-BD biosynthesis in <i>Bacillus</i> species and demonstrates an effective route toward sustainable production of <i>D</i>-2,3-BD from bioresources. The strategies developed herein provide a blueprint for environmentally responsible biomanufacturing of value-added chemicals, supporting the transition toward a circular and sustainable chemical industry.