Resumen de Filamentous growth in wine yeast: signal triggering and genetic factors involved
The present work to obtain a PhD degree has been performed from 2013 to 2017 in the University Rovira I Virgili. This work has been developed in the Oenological Biotechnology research group, at the Department of Biochemistry and Biotechnology. During these years I held a pre-doctoral fellowship from University Rovira i Virgili. The work that I carried out was part of the project “Production of bioactive compounds derived from aromatic amino acids during alcoholic fermentation” supported by the Ministry of Economy and Competitiveness of Spain (Grant no. AGL2013-47300-C3). The project had as main objective to understand the metabolism of aromatic amino acid to control the concentration of bioactive compounds in fermented beverages. During alcoholic fermentation yeast are able to synthetize some compounds derived from aromatic amino acids (tryptophan, phenylalanine and tyrosine) as tryptophol, phenylethanol and tyrosol, which have been associated to quorum sensing activities in yeast. Quorum sensing seems to control cell density and morphogenesis in yeast. The hypothesis of this thesis: Aromatic amino acid metabolism generates compounds with quorum sensing activity that modulates growth and morphological changes in wine yeast species. This general objective was divided in the following specific objectives.
The first objective was to assess the production of aromatic alcohols during alcoholic fermentation under various nutrient conditions by different wine yeast species (Chapter 1). Fermentations with different nutrient content were performed, using five wine yeast species (Saccharomyces cerevisiae, Hanseniaspora uvarum, Starmerella bacillaris, Metschnikowia pulcherrima and Torulaspora delbrueckii), and the final concentration of aromatic alcohols was determined. The results showed that the synthesis pattern of tryptophol, phenylethanol and tyrosol is controlled by nutrient availability. Nitrogen deprivation triggers an elevated aromatic alcohol production, whereas sugar limitation has the opposite effect. Non-Saccharomyces strains are also able to produce the three aromatic alcohols, nevertheless, at lower concentrations than S. cerevisiae. Additionally, S. bacillaris produce very low amounts of tryptophol, phenylethanol and tyrosol compared to other strains. The second objective was to study the effect of compounds derived from aromatic amino acids on the physiological response of different wine yeast species, both on their growth and on their morphological changes (Chapters 2 and 3). Aromatic alcohols and ethanol have been described to modulate quorum sensing activities and cell growth in wine yeast. Other aromatic amino acid-derived compounds are produced by yeast with unknown role in yeast. The effect of these compounds was studied in a broad collection of wine yeast. Our results showed that compounds derived from aromatic amino acids differently affect growth in the tested species. Tryptamine and serotonin only inhibit yeast growth at high concentrations, which are not common in wine. Moreover, aromatic alcohols at levels easy found in wines are able to affect yeast growth: tryptophol reduces growth in all tested yeast species, while phenylethanol and tyrosol mainly in non-Saccharomyces species. Additionally, aromatic alcohols and ethanol have signaling roles, affecting yeast morphology in both Saccharomyces and non-Saccharomyces. However, this effect depends on nutrient availability.
The third objective of this thesis was to decipher the regulation and genetic basis of the filamentous growth on S. cerevisiae (Chapter 3 and 4). Firstly, we performed different analysis with mutants in order to identify the genetic basis involved in filamentous growth inducible by ethanol. Consequently, we found that ethanol-inducible filamentous growth is controlled by the RTG pathway, which requires polarisome function and occurs by a Flo11p-dependent transcriptional induction. Moreover, a QTL approach was used to unravel genetic basis involved in filamentous growth. Through the analysis of the progeny generating by crossing parental strains with opposite phenotype, seven genes were identified as candidates to induce filamentous growth in natural wine strains: MEP2, NGR1, PHD1, SRV2, ELM1, SER33, YNL234W. All of them are involved in regulatory sensing pathways.
