4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) is considered a key flavor compound in soy sauce. The compound has a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. Here, we report the identification and characterization of an enzyme involved in the biosynthesis of HEMF in yeast. We fractionated yeast cell-free extract from Saccharomyces cerevisiae using column chromatography and partially purified a fraction with HEMF-forming activity. Peptide mass fingerprinting analysis showed that the partially purified fraction contains aldehyde reductase encoded by YNL134C. This reductase shares low sequence identity with enone oxidoreductase, which is responsible for the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and HEMF in plants. YNL134C was expressed heterologously in Escherichia coli, and the purified protein catalyzed the formation of HEMF from the mixture of Maillard reaction products, acetaldehydes, and NADPH. Multicopy expression in S. cerevisiae resulted in increased HEMF productivity, and gene knockout of YNL134C in S. cerevisiae resulted in decreased HEMF productivity. These data suggest that the translation product of YNL134C is the HEMF-producing enzyme in yeast. Detailed analyses of an intermediate in the enzymatic reaction mixture revealed that HEMF is synthesized from (2E)-2-ethylidene-4-hydroxy-5-methy1-3(2H)-furanone, which formed via Knoevenagel condensation between the acetaldehyde and 4-hydroxy-5-methyl-3(2H)-furanone derived from the Maillard reaction based on ribose and glycine, by YNL134Cp in an NADPH dependent manner. Overall, this study shed light on the molecular basis for the improvement of soy sauce flavor and the biotechnological production of HEMF. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.