Abstract

The alcohol‐O‐acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl‐coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol‐O‐acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl‐CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium‐chain‐length acyl‐CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

Highlights

  • Volatile esters are secondary metabolites produced by yeast and fungi during fermentation (Saerens et al, 2010; Mason and Dufour, 2000) and in plants during fruit ripening (El Hadi et al, 2013)

  • Atf1p belongs to a large and functionally diverse group of enzymes that act as coenzyme A (CoA)-dependent Oacyltransferases

  • As a subset of this broader grouping, the AATs (EC 2.3.1.84) are found in fungi and plants with the plant enzymes being classified as members of the BAHD superfamily (D’Auria, 2006)

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Summary

Introduction

Volatile esters are secondary metabolites produced by yeast and fungi during fermentation (Saerens et al, 2010; Mason and Dufour, 2000) and in plants during fruit ripening (El Hadi et al, 2013). The most important acetate esters from the perspective of fermented foods and beverages are probably ethyl acetate (fruity, solvent aroma), isobutyl acetate (sweet, fruit), isoamyl acetate (banana) and 2-phenylethyl acetate (rose) Homologues of these two enzymes are found in other yeasts (Van Laere et al, 2008). Of these two proteins, Atf1p plays the major role in acetate ester synthesis (Verstrepen et al, 2003b); there is evidence that Atf2p is important for sterol detoxification (Tiwari et al, 2007) These previous reports provide compelling evidence for the importance of AATs in yeast metabolism, they reveal little about the detailed structure and function of Atf1p and Atf2p. Such an understanding could be provided by studies of purified proteins in vitro

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