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

Abstract

Basic research with laboratory strains of the yeast Saccharomyces cerevisiae has 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 of ATF1 encoding 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.

Contribution of Eat1 and Other Alcohol Acyltransferases to Ester Production in Saccharomyces cerevisiae

“…We have previously shown that overexpression of the S. cerevisiae EAT1 increased ethyl acetate production in S. cerevisiae (Kruis et al, 2017). These results were confirmed in a study where EAT1 was identified as a source of ethyl acetate synthesis in S. cerevisiae Δ atf1 (Holt et al, 2018).…”

“…This was the most apparent in the case of ethyl acetate and isoamyl acetate, where more than 50% ethyl acetate production remained in some cases (Supplementary Figure S4). Imo32p was previously associated with ester production in the absence of Atf1p (Holt et al, 2018). The single deletion strain CEN.PK2-1D Δ imo32 produced 10% less ethyl acetate in both YSg and YPD-80 media.…”

“…This may explain why Eat1p seems to be unspecific toward alcohols but prefers short chain acyl-CoAs. Curiously, a study where the S. cerevisiae EAT1 was overexpressed found that Eat1p was mostly limited to the production of ethyl acetate (Holt et al, 2018). The substrate preferences of the enzymes should be determined in vitro .…”

“…However, when the gene was disrupted in various combinations with the remaining four AATs, it caused a decrease in acetate ester production. This indicates that in some cases, the effect of a single AAT disruption may be masked by the remaining AATs, which is plausible as they compete for the same substrates; although not all AATs have the same cellular location (Huh et al, 2003; Lin and Wheeldon, 2014; Holt et al, 2018). There is a significant knowledge gap on the expression, function and interaction of yeast AATs, especially under industrially relevant conditions.…”

Yeast Strain Optimization for Enological Applications

“…The obtained strains are tested for their phenotypes. Thanks to those approaches, genes, mutations, or even translocations have been validated for diverse wine yeast properties, such as lag phase duration, fermentation capacity under nitrogen starvation, and ester production [42,51,52].…”