Abstract
In wine fermentation, nitrogen is an essential nutrient for yeasts and its relative abundance is an important modulator of fermentation kinetics. The main sources of nitrogen in grape musts are ammonium and free amino acids; however, secondary sources such as oligopeptides are also important contributors to the nitrogen supply. Recently, the novel family of Fungal Oligopeptide Transporters (Fot) has been identified in Saccharomyces cerevisiae wine strains, while being absent in strains from other ecological niches. Presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. In the S. cerevisiae wine strain EC1118, members Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison to strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. The main objective of this thesis is the characterization of Fot family members to understand their biological significance, considering their high sequence identity. We provide evidence that FOT gene expression is dependent on the strain, stage of enological fermentation and composition of the yeast assimilable nitrogen. Moreover, we demonstrated that Fot1 is localized in the yeast plasma membrane through co-localization studies with Gfp labelling. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. Results of oligopeptide consumption showed that Fot counterparts have different oligopeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. Combining Fot protein structure models obtained by a threading strategy together with molecular docking experiments, we have predicted the amino acid residues in Fot potentially involved in substrate binding. Additionally, we inspected the distribution of Fot homologous sequences among fungal species. Putative Fot sequences found in ascomycetous yeasts and filamentous fungi showed a patchy distribution that did not follow the general taxonomy, suggesting a complex evolution trajectory that may include several gene duplications and HGT events. Overall, this work contributes to a better understanding of the Fot family, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation. In a second focus of this work, we have evaluated the impact of an adaptive laboratory evolution (ALE) strategy designed to increase the utilization of oligopeptides as nitrogen source in yeast fermentative performance. Populations derived from our ALE strategy showed higher fermentation rates and shorter times to complete fermentation in comparison to their ancestors. These new phenotypes constitute an improvement in the fermentative capacities, which opens the gate to a further characterization of our ALE-derived populations for their potential exploitation as commercial strains in winemaking.