Genes involved in thiol release from cysteine and glutathione conjugates by Saccharomyces cerevisiae during wine fermentation

Abstract

The aroma of Sauvignon blanc is highly influenced by three volatile thiols: 4MMP, 3MH and 3MHA. Considerable effort has been made during the last decade in order to understand how these desirable thiols are released by Saccharomyces cerevisiae during wine fermentation. So far, three types of thiol precursors have been found in grape: cysteinylated-conjugates (C-4MMP and C-3MH), glutathionylated-conjugates (G-4MMP and G-3MH) and C6 compounds ((E)-2-hexen-1-ol and (E)-2-hexenal), providing a huge thiol potential. All these precursors have been shown to release their respective thiol by the action of yeast. However, their rate of conversion is low (less than 10%) and their relative contribution is unclear. Little is known about yeast genes that participate in thiol release. So far, the most important gene is IRC7F, which is necessary for 4MMP release. Additionally, the deletion of the transporters, GAP1 and OPT1, partially reduced 3MH release. The aim of this thesis is to identify genes involved in thiol release from cysteinylated and glutathionylated precursors. A full-length allele, IRC7F, was shown to be necessary and sufficient to release 4MMP from C-4MMP and 3MH from C-3MH and G-3MH in synthetic grape media. A wine yeast strain overexpressing IRC7F cleaved around 50% of C-4MMP and 10% of G-3MH into 4MMP and 3MH, respectively, in synthetic media. In addition, it was demonstrated that Irc7F cleaves L-cysteine in vitro (enzymatic assay with purified Irc7Fp) and in vivo (growth in L-cysteine in fermentative-like conditions), suggesting that it plays a biological role in yeast sulfur metabolism. Moreover, yeast selected for growth on L-cysteine showed improved 4MMP production. However, the uptake of C-4MMP does not occur via the known cysteine permeases, but it is controlled by the nitrogen catabolism repression. The uptake of G-3MH occurs through Opt1p and its cleavage needs the action of the γ-glutamyltransferase, Cis2p, to release 3MH. It was demonstrated that yeast can use G-3MH as both a sulfur and nitrogen source, pathways that may account for losses in thiol precursor. Interestingly, glutathione seems to play a role in 3MH release from precursors different than C-3MH or G-3MH. The results from this thesis contribute to the understanding and improvement of thiol production from cysteinylated and glutathionylated precursors. It also provides tools to search for novel 3MH precursors that may account for most of 3MH production.