Cell wall modifications in Saccharomyces cerevisiae wine yeast through adaptive laboratory evolution with Tebuconazole

Abstract

Saccharomyces cerevisiae is the most used yeast for wine production around the world. Several characteristics make this yeast the wine yeast of excellence; among them is a great tolerance to higher concentrations of sugar and alcohol. Nevertheless, some compounds could have detrimental effects on its development like pesticides. Tebuconazole is one of the most common fungicides used in agriculture, belonging to the largest group of fungicides, the triazoles chemical group, that act on sterol biosynthesis. Yeasts have different responses to compensate for stress, and changes in their cell wall are one of the main ones. This work aimed to obtain new variants of S. cerevisiae through adaptive laboratory evolution (ALE) using Tebuconazole as selection pressure and to evaluate changes in yeast cell wall structure, composition, and fermentative behavior. Three new variants of S. cerevisiae were obtained. Analysis of the relative expression of genes associated with cell wall components showed that the third variant obtained YCPUC209C, had overexpression of genes FKS1, FKS3, CHS3, and SED1 in comparison with the original strain, also morphological analysis through TEM microscopy showed that YCPUC209C had an increase of 22.2% of cell wall thickness and 19% increase in the amount of glucan in comparison to the original strain. These cell wall changes were accompanied by hypersensitive to β-1,3-glucanase activity. Increased tolerance to pesticides Buprofezin and Spirotetramat presence during alcoholic fermentation was achieved by YCPUC209C, improving fermentative efficiency parameter. Changes in cell wall structure and composition reported in this work open new lines of analysis like the evaluation of yeast pesticide dissipation capacity since it is reported that components, such as glucan and chitin, can bond to these contaminants, reducing their residues in the wine.

Keywords: ALE; Cell wall; Genetic; Pesticide; Yeast.

Fig. 1

Growth curves of S. cerevisiae YCPUC209, in the presence of pesticides Buprofezin 1 and 2 mg/L, Spirotetramat 2 and 4 mg/L, Tebuconazole 1 and 2 mg/L, and control without pesticides.

Fig. 2

Growth kinetic parameters, specific growth rate (h-1), lag phase (h), and generational time (h) of S. cerevisiae YCPUC209, in the presence of pesticides, Buprofezin 1 and 2 mg/L, Spirotetramat 2 and 4 mg/L, Tebuconazole 1 and 2 mg/L, and control without pesticides. Different letters above the bars represent significant differences between treatments according to the Tukey test.

Fig. 3

Fermentation curves measured as weight loss (g/L) of S. cerevisiae YCPUC209 (A) and YCPUC209C (B) in the presence of pesticides Buprofezin (1 mg/L), Spirotetramat (2 mg/L), Tebuconazole (1 mg/L), and control without pesticides.

Fig. 4

Final weight loss (g/L), and Fermentative efficiency measured as the area under the curve (AUC) of S. cerevisiae YCPUC209 and YCPUC209C, in the presence of pesticides Buprofezin (1 mg/L), Spirotetramat (2 mg/L), Tebuconazole (1 mg/L), and control without pesticides. Different letters above the bars represent significant differences between treatments according to the Tukey test.

Fig. 5

TEM images of YCPUC209 (A), YCPUC209C (B), and the cell wall (CW) thickness (nm) of YCPUC209 and YCPUC209C (C). *** indicate statistical differences according to the Tukey test p < 0.0001.

Fig. 6

Relative expression of selected genes related to the yeast cell wall, of YCPUC209C compared with original strain YCPUC209. Different letters showed significant relative expression differences for the different genes according to Tukey test.

Fig. 7

Effects on the cell wall of S. cerevisiae YCPUC209C variant. (A) Sensitivity to CFW or CR YCPUC209 strain and YCPUC209C variant. Cells were incubated in malt medium in the presence or absence of 25–50 µg/mL CFW or CR for 72 h at 20 °C. (B) Zymolyase sensitivity. YCPUC209 strain and YCPUC209C variant were incubated with 200 µg/mL zymolyase 20T at 25 ºC, and the decrease in OD600 nm was monitored over time (survival %). (C) The YCPUC209C strain presented a reduction in budding cell number.