A Genome-Wide Phenotypic Analysis of Saccharomyces cerevisiae's Adaptive Response and Tolerance to Chitosan in Conditions Relevant for Winemaking

In the wine industry, the use of chitosan, a non-toxic biodegradable polysaccharide with antimicrobial properties, has been gaining interest with respect to envisaging the reduction in the use of sulfur dioxide (SO2). Although the mechanisms of toxicity of chitosan against fungal cells have been addressed before, most of the studies undertaken used other sources of chitosan and/or used conditions to solubilize the polymer that were not compatible with winemaking. Herein, the effect of a commercial formulation of chitosan approved for use in winemaking over the growth of the spoilage yeast species Dekkera anomala, Saccharomycodes ludwigii, Zygosaccharomyces bailii, and Pichia anomala was assessed. At the legally allowed concentration of 0.1 g/L, chitosan inhibited the growth of all spoilage yeasts, except for the tested Pichia anomala strains. Interestingly, the highly SO2-tolerant yeasts S. ludwigii and Z. bailii were highly susceptible to chitosan. The growth of commercial Saccharomyces cerevisiae was also impacted by chitosan, in a strain-dependent manner, albeit at higher concentrations. To dissect this differential inhibitory potential and gain further insight into the interaction of chitosan over fungal cells, we explored a chemogenomic analysis to identify all of the S. cerevisiae genes conferring protection against or increasing susceptibility to the commercial formulation of chitosan. Among the genes found to confer protection against chitosan, a high proportion was found to encode proteins required for the assembly and structuring of the cell wall, enzymes involved in the synthesis of plasma membrane lipids, and components of signaling pathways that respond to damages in the plasma membrane (e.g., the Rim101 pathway). The data obtained also suggest that the fungal ribosome and the vacuolar V-ATPase could be directly targeted by chitosan, since the deletion of genes encoding proteins required for the structure and function of these organelles was found to increase tolerance to chitosan. We also demonstrated, for the first time, that the deletion of ITR1, AGP2 and FPS1, encoding plasma membrane transporters, prominently increased the tolerance of S. cerevisiae to chitosan, suggesting that they can serve as carriers for chitosan. Besides providing new insights into the mode of action of chitosan against wine yeasts, this study adds relevant information for its rational use as a substitute/complementary preservative to SO2.