Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains.

For more than 20 years, Saccharomyces cerevisiae has served as a model organism for genetic studies and molecular biology, as well as a platform for biotechnology (e.g., wine production). One of the important ecological niches of this yeast that has been extensively studied is wine fermentation, a complex microbiological process in which S. cerevisiae faces various stresses such as limited availability of nitrogen. Nitrogen deficiencies in grape juice impair fermentation rate and yeast biomass production, leading to sluggish or stuck fermentations, resulting in considerable economic losses for the wine industry. In the present work, we took advantage of the "1002 Yeast Genomes Project" population, the most complete catalogue of the genetic variation in the species and a powerful resource for genotype-phenotype correlations, to study the adaptation to nitrogen limitation in wild and domesticated yeast strains in the context of wine fermentation. We found that wild and domesticated yeast strains have different adaptations to nitrogen limitation, corroborating their different evolutionary trajectories. Using a combination of state-of-the-art bioinformatic (GWAS) and molecular biology (CRISPR-Cas9) methodologies, we validated that PNP1, RRT5 and PDR12 are implicated in wine fermentation, where RRT5 and PDR12 are also involved in yeast adaptation to nitrogen limitation. In addition, we validated SNPs in these genes leading to differences in fermentative capacities and adaptation to nitrogen limitation. Altogether, the mapped genetic variants have potential applications for the genetic improvement of industrial yeast strains.

Differential Gene Expression and Allele Frequency Changes Favour Adaptation of a Heterogeneous Yeast Population to Nitrogen-Limited Fermentations.

Alcoholic fermentation is fundamentally an adaptation process, in which the yeast Saccharomyces cerevisiae outperforms its competitors and takes over the fermentation process itself. Although wine yeast strains appear to be adapted to the stressful conditions of alcoholic fermentation, nitrogen limitations in grape must cause stuck or slow fermentations, generating significant economic losses for the wine industry. One way to discover the genetic bases that promote yeast adaptation to nitrogen-deficient environments are selection experiments, where a yeast population undergoes selection under conditions of nitrogen restriction for a number of generations, to then identify by sequencing the molecular characteristics that promote this adaptation. In this work, we carried out selection experiments in bioreactors imitating wine fermentation under nitrogen-limited fermentation conditions (SM60), using the heterogeneous SGRP-4X yeast population, to then sequence the transcriptome and the genome of the population at different time points of the selection process. The transcriptomic results showed an overexpression of genes from the NA strain (North American/YPS128), a wild, non-domesticated isolate. In addition, genome sequencing and allele frequency results allowed several QTLs to be mapped for adaptation to nitrogen-limited fermentation. Finally, we validated the ECM38 allele of NA strain as responsible for higher growth efficiency under nitrogen-limited conditions. Taken together, our results revealed a complex pattern of molecular signatures favouring adaptation of the yeast population to nitrogen-limited fermentations, including differential gene expression, allele frequency changes and loss of the mitochondrial genome. Finally, the results suggest that wild alleles from a non-domesticated isolate (NA) may have a relevant role in the adaptation to the assayed fermentation conditions, with the consequent potential of these alleles for the genetic improvement of wine yeast strains.

Transcriptional Activity and Protein Levels of Horizontally Acquired Genes in Yeast Reveal Hallmarks of Adaptation to Fermentative Environments.

In the past decade, the sequencing of large cohorts of Saccharomyces cerevisiae strains has revealed a landscape of genomic regions acquired by Horizontal Gene Transfer (HGT). The genes acquired by HGT play important roles in yeast adaptation to the fermentation process, improving nitrogen and carbon source utilization. However, the functional characterization of these genes at the molecular level has been poorly attended. In this work, we carried out a systematic analysis of the promoter activity and protein level of 30 genes contained in three horizontally acquired regions commonly known as regions A, B, and C. In three strains (one for each region), we used the luciferase reporter gene and the mCherry fluorescent protein to quantify the transcriptional and translational activity of these genes, respectively. We assayed the strains generated in four different culture conditions; all showed low levels of transcriptional and translational activity across these environments. However, we observed an increase in protein levels under low nitrogen culture conditions, suggesting a possible role of the horizontally acquired genes in the adaptation to nitrogen-limited environments. Furthermore, since the strains carrying the luciferase reporter gene are null mutants for the horizontally acquired genes, we assayed growth parameters (latency time, growth rate, and efficiency) and the fermentation kinetics in this set of deletion strains. The results showed that single deletion of 20 horizontally acquired genes modified the growth parameters, whereas the deletion of five of them altered the maximal CO2 production rate (Vmax). Interestingly, we observed a correlation between growth parameters and Vmax for an ORF within region A, encoding an ortholog to a thiamine (vitamin B1) transporter whose deletion decreased the growth rate, growth efficiency, and CO2 production. Altogether, our results provided molecular and phenotypic evidence highlighting the importance of horizontally acquired genes in yeast adaptation to fermentative environments.