STUDY OF THE VOLATILE PHENOL METABOLISM AND ROLE OF SO2 AS STRESS AGENT IN BRETTANOMYCES/DEKKERA BRUXELLENSIS UNDER WINE CONDITIONS

Biological spoilage of wine arises from yeasts and bacteria metabolic activity. Brettanomyces/Dekkera bruxellensis, one of the main contaminating yeast, is able to produce unpleasant compounds, such as vinyl and ethyl phenols (VPs). These off-flavors define the so called “Brett” character. Two enzymes, the cinnamate decarboxylase (CD) and the vinylphenol reductase (VPR), are involved in the spoilage activity. Sulphur dioxide (SO2) is the most common additive used to prevent and/or control microbial contamination in many foods, but decreasing its use is advisable both for limiting the detrimental cumulative effects on human health and improving the sustainability in winemaking. The first aim of this study was to provide a certain identification of the VPR enzyme, by cloning the gene in a species not producing ethyl phenols, such as Saccharomyces cerevisiae. The role of this enzyme in the conversion of 4-vinyl guaiacol into 4-ethyl guaiacol was proven by the expression, of a biologically active form of the heterologous protein. A VPR specific activity of 9 ± 0.6 mU/mg is found in crude extracts of transformed clones of S. cerevisiae. A his-tag purification approach allowed to confirm the results in activity trials carried out in the enriched fraction of the protein purified from recombinant cells of S. cerevisiae; in particular, a VPR specific activity of 1.83 ± 0.03 U/mg at pH 6.0 is measured. Furthermore, the strain-dependent character of the species regarding the VP production was investigated at sequence level in 17 different D. bruxellensis strains. Since the observed polymorphism (2.3%) and the allelic heterozygosity state of the gene do not correlate with the different release in off-flavors, this could indicate that transcriptional/post-translational mechanisms might affect the final production. In addition, the expression of the two genes involved in VP production was investigated as a three-factor variation response using a Response Surface Methodology approach. As first, a proper house-keeping-gene was identified to allow the analysis of different SO2, pH and ethanol concentrations on VP production under oenological conditions. While statistical irrelevance as far SO2 lead this to not be commented as main factor affecting CD expression, the linear interaction with pH and ethanol concurr to define a significant effect (p < 0.05) on it. Considering the permissive growth condition (0 mg/L mol.SO2, pH 4.5 and 5% v/v EtOH), CD is generally downregulated. The combination of factor levels maximizing (0.83 fold-change) CD expression is: 0.25 mg/L mol. SO2, pH 4.5 and 12.5% v/v EtOH. Contrariwise, VPR expression does not seem to be influenced by any main factor nor by their interactions, but its expression is maximized (1.80 fold-change) at the same conditions calculated for CD gene. Finally, the study of the genetic mechanisms involved in the SO2 stress response of two B./D. bruxellensis strains (AWRI1499 and CBS2499) was carried out. In particular, a RNA-Seq based approach was applied on cells grew in a wine-model environment. Results confirm the ability of the species of growing in such severe conditions and suggest that the environmental adaptation observed might be due to a detoxification activity that include the sulphur metabolic process. Indeed, this metabolic strategy has been observed to be one of the main mechanisms of resistance to sulphur dioxide stress in S. cerevisiae. A relative up-regulation of genes involved in the last step of the Sequence of Reduction of Sulfate (SRS) and a high up-regulation of SSU1 gene (up to 4-fold and 47-fold in the CBS2499 and AWRI1499 strains, respectively), encoding a plasmamembrane sulfite pump, are observed.