Polygenic Analysis in Absence of Major EffectorATF1Unveils Novel Components in Yeast Flavor Ester Biosynthesis

ABSTRACTFlavor 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. TheATF1-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 ofATF1. We identified two unique causative mutations,eat1K179fsandsnf8E148*, not present in any other sequenced yeast strain and responsible for most ethyl acetate produced in absence ofATF1.EAT1encodes a putative mitochondrial ethanol acetyl-CoA transferase (EATase) and its overexpression, but not that ofEAT1K179fs, and strongly increases ethyl acetate without affecting other flavor acetate esters. Unexpectedly, a higher level of acetate esters (including ethyl acetate) was produced wheneat1K179fswas present together withATF1in the same strain, suggesting that the Eat1 and Atf1 enzymes are intertwined. On the other hand, introduction ofsnf8E148* lowered ethyl acetate levels also in the presence ofATF1, and it affected other aroma compounds, growth, and fermentation as well. Engineering ofsnf8E148* 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 identifiedEAT1andSNF8as new genetic elements determining ethyl acetate production diversity in yeast strains.IMPORTANCEBasic research with laboratory strains of the yeastSaccharomyces cerevisiaehas identified the structural genes of most metabolic enzymes, as well as genes encoding major regulators of metabolism. On the other hand, more recent work on polygenic analysis of yeast biodiversity in natural and industrial yeast strains is revealing novel components of yeast metabolism. A major example is the metabolism of flavor compounds, a particularly important property of industrial yeast strains used for the production of alcoholic beverages. In this work, we have performed polygenic analysis of production of ethyl acetate, an important off-flavor compound in beer and other alcoholic beverages. To increase the chances of identifying novel components, we have used in parallel a wild-type strain and a strain with a deletion ofATF1encoding the main enzyme of acetate ester biosynthesis. This revealed a new structural gene,EAT1, encoding a putative mitochondrial enzyme, which was recently identified as an ethanol acetyl-CoA transferase in another yeast species. We also identified a novel regulatory gene,SNF8, which has not previously been linked to flavor production. Our results show that polygenic analysis of metabolic traits in the absence of major effector genes can reveal novel structural and regulatory genes. The mutant alleles identified can be used to affect the flavor profile in industrial yeast strains for production of alcoholic beverages in more subtle ways than by deletion or overexpression of the already known major effector genes and without significantly altering other industrially important traits. The effect of the novel variants was dependent on the genetic background, with a highly desirable outcome in the flavor profile of an ale brewing yeast.