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

Abstract

(2S,3S)-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 (2S,3S)-BDO directly from glucose by using an efficient (S)-acetoin-producing strain, which was generated by introducing Bacillus subtilis genes encoding alpha-acetolactate synthase (Bs.AlsS) and a mutant alpha-acetolactate decarboxylase (Bs.AlsDWP) with reversed stereospecificity toward (S)-acetoin formation, alongside targeted deletions of genes involved in racemic acetoin production and competing pathways. (2S,3S)-BDO production in this strain was enabled by introducing Cg.butA from Corynebacterium glutamicum, encoding an L-butanediol dehydrogenase that reduced (S)-acetoin to (2S,3S)-BDO with high efficiency and stereospecificity. The engineered genome-integrated strains co-expressing Bs.alsS, Bs.alsDWP, and Cg.butA produced 27.9 g/L (2S,3S)-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<middle dot>h. This represents the first demonstration of high-purity (2S,3S)-BDO biosynthesis directly from glucose without relying on spontaneous diacetyl-formation pathway, positioning S. cerevisiae as a versatile platform for stereoselective diol production.