Systems biology approaches for the design of novel Saccharomyces cerevisiae winemaking strains for enhanced flavour compounds synthesis

Abstract

Wine flavour and aroma are the result of yeast metabolism and must compounds interactions. During must fermentation thousands of volatile aroma compounds are formed, with higher alcohols, acetate esters and ethyl esters being the main aromatic compounds contributing to a floral and fruity aroma. The action of yeast, in particular of Saccharomyces cerevisiae strains, on the must components will build the architecture of the wine flavour and their fermentation bouquet. Only a holistic approach based on systems biology, an inter-disciplinary area combining the study of biology, chemistry, physics and mathematics, based on data-driven and model-based elucidation of complex biological systems, will allow the complete understanding on the vast and dynamical relationships between genomics, phenomics and metabolomics. In this work a S. cerevisiae collection was constituded comprising 172 strains of worldwide geographical origins and different technological applications. This collection was characterized regarding 30 physiological traits that are important mostly from an oenological point of view. From the different traits studied, growth in the presence of potassium bisulphite, growth at 40 ºC, and resistance to ethanol contributed the most to strain variability, as shown by the principal component analysis. Computational methods were developed in order to assess the importance of phenotypic features to identify candidate strains to be used commercially in winemaking. In particular, the probability of a strain to be assigned to the group of commercial strains was 27% using the entire phenotypic profile and increased to 95%, when only results from the three tests suggested by the model were considered. Results show the usefulness of the mentioned approaches to simplify strain selection procedures. In addition to the phenotypic characterization, we undertook genetic typing of the 172 S. cerevisiae strains, using 11 polymorphic microsatellites. We found 280 alleles, whereas microsatellite ScAAT1 contributed the most to intra-strain variability, together with the alleles 20, 9 and 16, from microsatellites ScAAT4, ScAAT5 and ScAAT6, respectively. These microsatellite allelic profiles are characteristic for both the phenotype and origin of yeast strains. Data were computationally related with the previously obtained results of the 30 phenotypic tests, and the phenotypes associated with higher number of alleles were the capacity to resist to sulphur dioxide (tested by the capacity to grow in the presence of potassium bisulphite) and the presence of galactosidase activity. Our study demonstrated the utility of computational modelling to estimate a strain technological group and phenotype from microsatellite allelic combinations as tools for preliminary yeast strain selection. To better understand the molecular and metabolic bases of aroma production during a fermentation process, we used comparative transcriptomic and metabolic analysis of four yeast strains from different origins and/or technological applications (cachaça, sake, wine, and laboratory), to rationally identify new targets for improving aroma production. Results showed that strains from cachaça, sake and wine presented a higher production of acetate esters, ethyl esters, acids and higher alcohols, in comparison with the laboratory strain S288c. At fermentation time T1 (5 g/L of CO2 released), comparative transcriptomics of these three S. cerevisiae strains from different fermentative environments in comparison with the laboratory yeast S288c, showed an increased expression of genes related with tetracyclic and pentacyclic triterpenes metabolism, involved in sterol synthesis. Sake strain showed also an upregulation of genes ADH7 and AAD6, involved in the formation of higher alcohols in the Ehrlich pathway. For fermentation time point T2 (50 g/L CO2 released), again sake strain, but also wine strain, showed an increased expression of genes involved in formation of higher alcohols in the Ehrlich pathway, namely ADH7, ADH6 and AAD6, which is in accordance with the higher levels of methionol, isobutanol, isoamyl alcohol and phenylethanol observed. Our approach revealed successful to integrate data from several technologies (HPLC, GC-MS and microarrays) and using different data analysis methods (PCA, MFA). The results obtained, increased our knowledge on the association of genes with the formation of metabolic compounds that contribute to the wine aroma and flavour, and showed differences in the metabolism of cachaça, sake and wine strains not yet addressed, and mainly explained by the production of fatty acids, and ethyl and acetate esters.