The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates.

In Saccharomyces cerevisiae, branched-chain amino acid transaminases (BCAATases) are encoded by the BAT1 and BAT2 genes. BCAATases catalyse the transfer of amino groups between those amino acids and alpha-keto-acids. alpha-Keto-acids are precursors for the biosynthesis of higher alcohols, which significantly influence the aroma and flavour of yeast-derived fermentation products. The objective of this study was to investigate the influence of BAT-gene expression on general yeast physiology, on aroma and flavour compound formation and on the sensory characteristics of wines and distillates. For this purpose, the genes were overexpressed and deleted in a laboratory strain, BY4742, and overexpressed in an industrial wine yeast strain, VIN13. The data show that, with the exception of a slow growth phenotype observed for the BAT1 deletion strain, the fermentation behaviour of the strains was unaffected by the modifications. The chemical and sensory analysis of fermentation products revealed a strong correction between BAT gene expression and the formation of many aroma compounds. The data suggest that the adjustment of BAT gene expression could play an important role in assisting winemakers in their endeavour to produce wines with specific flavour profiles.

The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates.

The fruity odours of wine are largely derived from the synthesis of esters and higher alcohols during yeast fermentation. The ATF1- and ATF2-encoded alcohol acetyltransferases of S. cerevisiae are responsible for the synthesis of ethyl acetate and isoamyl acetate esters, while the EHT1-encoded ethanol hexanoyl transferase is responsible for synthesizing ethyl caproate. However, esters such as these might be degraded by the IAH1-encoded esterase. The objectives of this study were: (a) to overexpress the genes encoding ester-synthesizing and ester-degrading enzymes in wine yeast; (b) to prepare Colombard table wines and base wines for distillation using these modified strains; and (c) to analyse and compare the ester concentrations and aroma profiles of these wines and distillates. The overexpression of ATF1 significantly increased the concentrations of ethyl acetate, isoamyl acetate, 2-phenylethyl acetate and ethyl caproate, while the overexpression of ATF2 affected the concentrations of ethyl acetate and isoamyl acetate to a lesser degree. The overexpression of IAH1 resulted in a significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHT1 resulted in a marked increase in ethyl caproate, ethyl caprylate and ethyl caprate. The flavour profile of the wines and distillates prepared using the modified strains were also significantly altered as indicated by formal sensory analysis. This study offers prospects for the development of wine yeast starter strains with optimized ester-producing capability that could assist winemakers in their effort to consistently produce wine and distillates such as brandy to definable flavour specifications and styles.

Metabolic engineering of Saccharomyces cerevisiae for the synthesis of the wine-related antioxidant resveratrol.

The stilbene resveratrol is a stress metabolite produced by Vitis vinifera grapevines during fungal infection, wounding or UV radiation. Resveratrol is synthesised particularly in the skins of grape berries and only trace amounts are present in the fruit flesh. Red wine contains a much higher resveratrol concentration than white wine, due to skin contact during fermentation. Apart from its antifungal characteristics, resveratrol has also been shown to have cancer chemopreventive activity and to reduce the risk of coronary heart disease. It acts as an antioxidant and anti-mutagen and has the ability to induce specific enzymes that metabolise carcinogenic substances. The objective of this pilot study was to investigate the feasibility of developing wine yeasts with the ability to produce resveratrol during fermentation in both red and white wines, thereby increasing the wholesomeness of the product. To achieve this goal, the phenylpropanoid pathway in Saccharomyces cerevisiae would have to be introduced to produce p-coumaroyl-CoA, one of the substrates required for resveratrol synthesis. The other substrate for resveratrol synthase, malonyl-CoA, is already found in yeast and is involved in de novo fatty-acid biosynthesis. We hypothesised that production of p-coumaroyl-CoA and resveratrol can be achieved by co-expressing the coenzyme-A ligase-encoding gene (4CL216) from a hybrid poplar and the grapevine resveratrol synthase gene (vst1) in laboratory strains of S. cerevisiae. This yeast has the ability to metabolise p-coumaric acid, a substance already present in grape must. This compound was therefore added to the synthetic media used for the growth of laboratory cultures. Transformants expressing both the 4CL216 and vst1 genes were obtained and tested for production of resveratrol. Following beta-glucosidase treatment of organic extracts for removal of glucose moieties that are typically bound to resveratrol, the results showed that the yeast transformants had produced the resveratrol beta-glucoside, piceid. This is the first report of the reconstruction of a biochemical pathway in a heterologous host to produce resveratrol.

Enhancing volatile phenol concentrations in wine by expressing various phenolic acid decarboxylase genes in Saccharomyces cerevisiae.

Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1(P)()) and terminator (PGK1(T)()) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Sigma1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Sigma1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future.