Polygenic Analysis in Absence of Major Effector ATF1 Unveils Novel Components in Yeast Flavor Ester Biosynthesis

Flavor production in yeast fermentation is of paramount importance for industrial production of alcoholic beverages. Although major enzymes of flavor compound biosynthesis have been identified, few specific mutations responsible for strain diversity in flavor production are known. The ATF1-encoded alcohol acetyl coenzyme A (acetyl-CoA) transferase (AATase) is responsible for the majority of acetate ester biosynthesis, but other components affecting strain diversity remain unknown. We have performed parallel polygenic analysis of low production of ethyl acetate, a compound with an undesirable solvent-like off-flavor, in strains with and without deletion of ATF1. We identified two unique causative mutations, eat1(K179fs) and snf8(E148)*, not present in any other sequenced yeast strain and responsible for most ethyl acetate produced in absence of ATF1. EAT1 encodes a putative mitochondrial ethanol acetyl-CoA transferase (EATase) and its overexpression, but not that of EAT1(K179fs), and strongly increases ethyl acetate without affecting other flavor acetate esters. Unexpectedly, a higher level of acetate esters (including ethyl acetate) was produced when eat1(K179fs) was present together with ATF1 in the same strain, suggesting that the Eat1 and Atf1 enzymes are intertwined. On the other hand, introduction of snf8(E148)* lowered ethyl acetate levels also in the presence of ATF1, and it affected other aroma compounds, growth, and fermentation as well. Engineering of snf8(E148)* in three industrial yeast strains (for production of wine, saké, and ale beer) and fermentation in an application-relevant medium showed a high but strain-dependent potential for flavor enhancement. Our work has identified EAT1 and SNF8 as new genetic elements determining ethyl acetate production diversity in yeast strains.