Expression Levels of the Yeast Alcohol Acetyltransferase Genes<i>ATF1</i>,<i>Lg-ATF1</i>, and<i>ATF2</i>Control the Formation of a Broad Range of Volatile Esters

TLDR

Volatile esters, produced by yeast enzymes during fermentation, give beer and wine their fruity aroma. The study aimed to compare the roles of Atf1p, Atf2p, and Lg‑Atf1p in ester production by deleting or overexpressing their genes in laboratory and brewing yeast strains. Ester production was quantified using headspace gas chromatography and GC–MS. Expression levels of ATF1 and ATF2 strongly influence ethyl acetate and isoamyl acetate production; Atf1p and Atf2p also synthesize many other esters, with Atf2p playing a minor role, and double deletion eliminates isoamyl acetate but leaves other esters, indicating additional ester synthases, while overexpression of different ATF alleles alters aroma profiles.

Abstract

ABSTRACT Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1 Δ atf2 Δ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.