BACKGROUND Acetoin and xylitol are widely used as high-value platform chemicals with numerous potential applications. The chiral enantiomers L-(+)- and D-(-)-acetoin are used as pharmaceutical intermediates. Cell-free biosynthesis has many applications in chemicals production, but efficient methods for production of optically pure acetoin were rarely reported. RESULTS A novel two-enzyme system composed of meso-2,3-butanediol dehydrogenase (BDH) and xylose reductase was constructed to co-produce acetoin and xylitol with NAD(+) regeneration. Four BDHs from four candidates, as well as xylose reductase from Candida tenuis were first purified and characterized. The best BDH was then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, the ratios of meso-2,3-butanediol to xylose and BDH to xylose reductase, 28.5 g L-1 D-(-)-acetoin with an optical purity of 95.2% was produced in 6 h. The yield and productivity of acetoin was 0.97 g g(-1) and 4.75 g L-1 h(-1). The titer of co-product xylitol was 40.29 g L-1, and the yield and productivity of xylitol reached 0.98 g g(-1) and 6.72 g L-1 h(-1). CONCLUSIONS To our best knowledge, this is the first report on the production of optically pure D-(-)-acetoin using xylose reductase to regenerate NAD(+), as well as the highest acetoin titer among enzymatic synthesis methods. This work therefore provides an economical and green alternative for the in vitro production of D-(-)-acetoin. (c) 2018 Society of Chemical Industry