The development of yeasts for the optimal production of flavor-active esters and higher alcohols in wine and distillates

[摘要] ENGLISH ABSTRACT: Yeasts produce a broad range of aroma-active volatile esters and higher alcohols duringalcoholic fermentation. Some of these esters and higher alcohols are important for the fruityflavors and therefore the final quality of wine and other fermented beverages. Esters areproduced and hydrolyzed by alcohol acetyltransferases and esterases, respectively. In yeast,ester-synthesizing activities are represented by two alcohol acetyltransferases encoded by theATFI and ATF2 genes, and by an ethanol hexanoyl transferase encoded by the EHTI gene.Atfl p and Atf2p appear responsible for the production of ethyl acetate and isoamyl acetate,while Ehtl p synthesizes ethyl hexanoate from ethanol and hexanoyl-CoA. Although a fairamount of information is available regarding the ATF 1 gene, limited information is availableon the remaining alcohol acetyltransferases. Only two genes that code for esterases have beenidentified in yeast, namely lAHI and TIPI. It has also been shown that the balance betweenalcohol acetyltransferases and esterases is important for the net rate of ester accumulation.Higher alcohols are synthesized from the a-keto-acids in the branched-chain amino acidmetabolic pathway by decarboxylation and reduction. The transamination of the amino acid tothe respective a-keto-acid is catalyzed by mitochondrial and cytosolic branched-chain aminoacid transferases, which are encoded by the BATI and BAT2 genes, respectively.In recent years, a strong scientific and industrial interest in the metabolism of flavoractivecompounds has emerged, but information regarding the roles of specific enzymes andthe physiological relevance of their metabolism remains limited. The aim of this project wasto investigate the physiological and metabolic consequences of changes in the expressionlevels of some of the key enzymes involved in aroma compound production. Theconsequences of these changes on the chemical composition and the fermentation bouquet ofwines and distillates were also investigated.The first part of the section on the results in this dissertation reports on the role andrelative importance of the Saccharomyces cerevisiae enzymes involved in ester metabolism,namely Atflp, Atf2p, Ehtlp, Iahlp and Tiplp. The corresponding genes were overexpressedin a laboratory strain of S. cerevisiae, BY4742, and in a widely used commercial wine yeaststrain, VIN13. Table wine and base wines for distillation were prepared with these VIN13transformed strains. The ester concentrations and aroma profiles of the wines and distillateswere analyzed and compared. The data indicated that the overexpression of ATF 1 and ATF2increased the concentrations of ethyl acetate, isoamyl acetate, 2-pheylethyl acetate and ethylcaproate, while the overexpression of JAHI resulted in a significant decrease in theconcentrations of ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. Theoverexpression of EHTI resulted in a marked increase in the concentrations of ethyl caproate,ethyl caprylate and ethyl caprate, while the overexpression of TJP1 did not decrease theconcentrations of any of the esters. In most cases, there was a correlation between the increasein esters and the decrease in higher alcohols. The data suggest that yeast balances the amountof different esters produced through alcohol acetyltransferases and esterases, and that, in somecases, these enzymes appear to overlap in function and/or influence each other's activity. In the second part of the results section, the consequences of the deletion and theoverexpression of two genes, BATl and BAT2, which encode transaminases that contribute tothe metabolism of higher alcohols, were investigated. The genes were both disrupted in aS. cerevisiae BY4742, and overexpressed in both this laboratory strain and in the VIN13 wineyeast strain. The effects of these modifications on the general physiology of the correspondingyeast strains and on higher alcohol metabolism were assessed in a range of growth conditions,including aerobic and anaerobic growth conditions, in the presence of glucose or raffinose assole carbon source and growth in the presence of various concentrations of amino acids. Tablewine and base wines for distillation were prepared with the modified industrial strains and theconcentrations of the higher alcohols and the aroma profiles of the wine and distillates wereanalyzed and compared. Batl deletion seemed to be lethal under the conditions that werecreated, and therefore only the bat2!:!.strain, together with the BATI and BAT2 overexpressionstrains, were investigated. These modifications did not appear to significantly affect thegeneral physiology of the strains. The results obtained indicated that the overexpression ofBATI increased the concentrations of isoamyl alcohol and isoamyl acetate, and, to a lesserextent, the concentrations of isobutanol and isobutyric acid. The overexpression of the BAT2gene resulted in a substantial increase in the levels of isobutanol, isobutyric acid andpropionic acid production, and a modest increase in the level of propanol and isovaleric acid.Interestingly, the overexpression of BAT2 led to a decrease in isoamyl alcohol and isoamylacetate concentrations. Sensory analyses indicated that the wines and distillates produced withthe strains in which the BATl and BAT2 genes were overexpressed had more fruitycharacteristics (peach and apricot aromas) than the wines produced by the wild-type strains.This study offers new prospects for the development of wine yeast starter strains withoptimized ester and higher alcohol-producing capability that could assist winemakers in theirefforts to consistently produce wine to definable specifications and styles and a predeterminedflavor profile.

[发布机构] Stellenbosch University