Genome-Wide Identification and Biochemical Characterization of Alcohol Acyltransferases for Aroma Generation in Wickerhamomyces subpelliculosus Isolates from Fermented Food

The importance of nonconventional yeasts has increasingly been highlighted, particularly for aroma formation in fermented foods. Here, we performed de novo whole-genome sequencing of Wickerhamomyces subpelliculosus, which produces a variety of volatile flavor compounds, leading to the identification of the alcohol acyltransferase (AATase) family of genes. The genome of W. subpelliculosus contains seven AATase genes, encoding alcohol-O-acetyltransferases (ATFs) and ethanol acetyltransferase 1 (EAT1) for acetate ester formation, along with ethanol hexanoyl transferase 1 (EHT1) for ethyl ester formation. Among five ATF homologues, only WsATF5 showed acetyltransferase activity toward myriocin, a structural analogue of sphingosine. In contrast, heterologous expression of WsEHT1 and WsEAT1 in Saccharomyces cerevisiae promoted the production of ethyl decanoate and ethyl acetate, respectively, supporting their AATase activity. The enzymatic activity analyses revealed the additional alcoholysis activity of WsEAT1 and the thioesterase activity of WsEHT1. Subcellular localization analysis indicated that WsEAT1 was localized in the mitochondria, WsEHT1 in the endoplasmic reticulum and lipid droplets (LDs), and WsATF5 in the LDs. The novel W. subpelliculosus AATases could be usefully applied to produce flavor components in various food industries.