BACKGROUND Acetoin, especially the chiral l-(+)- or d-(-)-enantiomer, is a high-value industrial product that can be used as a pharmaceutical intermediate. However, the production of optically pure acetoin via conventional chemical synthesis is expensive. Classical microbial fermentations satisfy the criteria of green chemistry, but have numerous disadvantages, such as complicated process engineering, unsatisfactory titers or productivities, and difficult product separation due to the inevitable generation of by-products and overly complex media. By contrast, enzymatic biocatalysis in cell-free biosystems has numerous advantages in production of chemicals with high purity, whereas efficient enzymatic methods for the production of optically pure acetoin have rarely been reported. RESULTS An efficient coupled two-enzyme system composed of 2,3-butanediol dehydrogenase and H2O-forming NADH oxidase was constructed to produce optically pure acetoin without introducing any substrate or by-product. A 2,3-butanediol dehydrogenase and three NADH oxidases were purified, characterized and assembled into different two-enzyme systems. The best system, composed of LL-NOX and CG-BDH, was then selected according to the acetoin titer. After optimization of the reaction conditions, 94.0% of meso-2,3-butanediol was converted to d-(-)-acetoin. Finally, 399.7 mmol L-1 d-(-)-acetoin with an enantiomeric excess of 91.2% was produced in 6 h without any detected by-products. CONCLUSIONS To our best knowledge, this is the first report on the production of optically pure d-(-)-acetoin from meso-2,3-butanediol in vitro using NADH oxidase to regenerate NAD(+), as well as the best substrate conversion and acetoin titer among enzymatic biosynthesis. This work therefore provides an economical and green alternative for the in vitro production of d-(-)-acetoin. (c) 2019 Society of Chemical Industry