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S-acetoin (S-AC) is an important four-carbon chiral compound that has unique industrial applications in the asymmetric synthesis of valuable chiral specialty chemicals. However, previous studies showed that the usually low yield and optical purity of S-AC as well as the very high substrate cost have hindered the application of this compound. In the current work, a gene encoding diacetyl reductase (DAR) from a Paenibacillus polymyxa strain ZJ-9 was cloned and expressed in Escherichia coli. Whole cells of the recombinant E. coli were used to produce S-AC from diacetyl (DA). Under optimal conditions, S-AC with high optical purity (purity >99·9%) was obtained with a yield of 13·5 ± 0·24 and 39·4 ± 0·38 g l−1 under batch and fed-batch culture conditions, respectively. This process featured the biotransformation of DA into S-AC using whole cells of engineered E. coli. The result is a considerable increase in the yield and optical purity of S-AC, which in turn facilitated the practical application of the compound.Significance and Impact of the Study: This study demonstrated a highly efficient new method to produce S-acetoin with higher than 99·9% optical purity from diacetyl using whole cells of engineered Escherichia coli. It will therefore decrease the production cost of S-acetoin and highlight its application in asymmetric synthesis of highly valuable chiral compounds.