Breeding High-Yield Ethyl Caproate-Producing Saccharomyces cerevisiae in Sake: Flux Regulation from Glycolytic Fermentation to the FAS Pathway and Alcohol Acyltransferase Overexpression.

Ethyl caproate is the characteristic aroma compound with an apple-like scent in Ginjoka sake. However, the medium-chain acyl-CoA flux of the fatty acid synthesis (FAS) pathway originating from glycolytic fermentation and the precursor-induced alcohol acyltransferase (AAT) activity by natural yeast limits the ethyl caproate content in sake. Here, we established combinatorial strategies involving genetic engineering and adaptive laboratory evolution (ALE) to increase the ethyl caproate production by Saccharomyces cerevisiae. In this study, we screened Saccharomyces cerevisiae YH-2, which exhibited high ethanol and ester yields , achieving a trade-off between FAS flux and energy metabolism. Subsequently, the cerulenin-resistant mutant strain YH-2-34, after 15 passages of adaptive domestication, produced 4.13 times more caproic acid than the wild type. This increase is attributed to the G1250S variation in the FAS2 sequences, which mediate acyl-CoA chain length in the FAS pathway, thereby producing more caproyl-CoA as the precursor. While AAT activity increased 2.40 times in the mutant YH-2-34, both EEB1 and EHT1 genes, which together encode AAT responsible for esterifying ethyl caproate, played critical roles. Although pEEB1s overexpression affected cell viability and ethyl caproate production, pEHT1s overexpression successfully increased the yield of ethyl caproate during post-fermentation. Finally, the yield of YH-2-34 with EHT1 overexpression achieved a significant increase from 1.21 to 7.40 mg/L in sake fermentation. By regulating the flux from glycolytic fermentation to the FAS pathway and overexpressing AAT, we constructed a high-yield ethyl-caproate-producing Saccharomyces cerevisiae strain. This may bring practical transformations to traditional brewing industries.