Efficient production of optically pure (2S,3S)-butanediol in Saccharomyces cerevisiae

Abstract (2 S ,3 S )-Butanediol (BDO) is a valuable chiral diol with growing applications in pharmaceuticals and agriculture, but microbial production is hindered by inefficient ( S )-acetoin production. Here, we engineered Saccharomyces cerevisiae to synthesize (2 S ,3 S )-BDO directly from glucose by using an efficient ( S )-acetoin-producing strain, which was generated by introducing Bacillus subtilis genes encoding α-acetolactate synthase ( Bs .AlsS) and a mutant α-acetolactate decarboxylase ( Bs. AlsD WP ) with reversed stereospecificity toward ( S )-acetoin formation, alongside targeted deletions of genes involved in racemic acetoin production and competing pathways. (2 S ,3 S )-BDO production in this strain was enabled by introducing Cg . butA from Corynebacterium glutamicum , encoding an L-butanediol dehydrogenase that reduced ( S )-acetoin to (2 S ,3 S )-BDO with high efficiency and stereospecificity. The engineered genome-integrated strains co-expressing Bs.alsS , Bs.alsD WP , and Cg.butA produced 27.9 g/L (2 S ,3 S )-BDO in fed-batch fermentation with high optical purity, an overall yield of 0.20 g/g glucose, and an average volumetric productivity of 0.17 g/L·h. This represents the first demonstration of high-purity (2 S ,3 S )-BDO biosynthesis directly from glucose without relying on spontaneous diacetyl-formation pathway, positioning S. cerevisiae as a versatile platform for stereoselective diol production.