Effect of CUP1 copy number and pH on copper resistance of Saccharomyces cerevisiae enological strains

Abstract

The widespread use of copper-based pesticides in winemaking can affect wine fermentation. Therefore, it is crucial to assess the resistance levels of Saccharomyces cerevisiae wine strains in enological growth conditions. In the context of winemaking, grape juice is a complex environment capable of chelating copper and is characterized by a distinctly acidic pH. In this work, the effects of copper concentration on the growth of 10 S. cerevisiae strains, isolated from an enological environment, and one commercial starter were tested in YNB minimal medium and synthetic must, mimicking enological conditions. In minimal medium, resistance to copper varied among yeasts (50-600 μM), revealing the presence of three resistance levels (high, intermediate, and low). Representative strains of the three groups were tested at a pH range from 5.2 to 3.0 at the copper concentration that showed a 20-25 % growth reduction. At pH range 5.2-4.5, a growth reduction was observed, while, conversely, a strain-specific recovery was observed at pH range 3.2-3.0. In synthetic must, the strains showed higher copper resistance levels than in minimal medium (50-4000 μM). In both synthetic must and minimal medium, a significant logarithmic correlation was found between copper resistance and CUP1 gene copy number. The copy number tended to better explain resistance in minimal medium compared to synthetic must. The results shed light on the role of CUP1 copy number within an enological environment.

Keywords: Cu detoxification system; Grape pomace; Metallothionein; Real-time PCR; Wine.

Fig. 1

a) Growth of S. cerevisiae strains under different copper doses in minimal medium. Data shows the average of triplicates growth curves ± standard error (shaded area around the curve). b) Normalized area under the growth curve (AUC) trend of S. cerevisiae strains over increasing copper concentration, in minimal medium. Each strain's trend is indicated with a different color, as shown by the legend in the figure. Average value points are plotted with standard error bars.

Fig. 2

a) Growth of S. cerevisiae strains in minimal medium at different pH values at the copper concentration that inhibits growth by 20–25 % (600 μM for R008, 200 μM for GF, 50 μM for P283). Data shows the average of triplicates growth curves ± standard error (shaded area around the curve). b) Normalized area under the growth curve (AUC) trend over decreasing pH, at the copper concentration that inhibits growth by 20–25 %, in minimal medium. Each strain's trend is indicated with a different color, as shown by the legend in the figure. Average value points are plotted with standard error bars.

Fig. 3

a) Growth of S. cerevisiae strains under different copper doses in synthetic must. Data shows the average of triplicates growth curves ± standard error (shaded area around the curve). b) Normalized area under the growth curve (AUC) trend over increasing copper concentration in synthetic must. Each strain's trend is indicated with a different color, as shown by the legend in the figure. Average value points are plotted with standard error bars.

Fig. 4

Relationship between normalized copper resistance measured in minimal medium and in synthetic must. Data were fitted with a linear regression model (red line). Dashed line represents the identity line. Each dot represents the average value calculated for the strain.

Fig. 5

Relationship between normalized copper resistance and CUP1 copy number, in minimal medium (a) and in synthetic must (b). Data were fitted with a generalized linear regression model for gamma distribution and with ‘log’ link function (blue line). Each dot represents the average value calculated for the strain.