Project description:The yeast Dekkera bruxellensis is as ethanol tolerant as Saccharomyces cerevisiae and may be found in bottled wine. It causes the spoilage of wine, beer, cider and soft drinks. In wines, the metabolic products responsible for spoilage by Dekkera bruxellensis are mainly volatile phenols. These chemical compounds are responsible for the taints described as M-bM-^@M-^XM-bM-^@M-^XmedicinalM-bM-^@M-^YM-bM-^@M-^Y in white wines (due to vinyl phenols) and as M-bM-^@M-^XM-bM-^@M-^XleatherM-bM-^@M-^YM-bM-^@M-^Y, M-bM-^@M-^XM-bM-^@M-^Xhorse sweatM-bM-^@M-^YM-bM-^@M-^Y and M-bM-^@M-^XM-bM-^@M-^XstableM-bM-^@M-^YM-bM-^@M-^Y in red wines (due to ethyl phenols mainly 4-ethylphenol). Apart from the negative aroma nuances imparted by these yeasts, positive aromas such as M-bM-^@M-^XsmokyM-bM-^@M-^Y, M-bM-^@M-^XspicyM-bM-^@M-^Y and M-bM-^@M-^XtoffeeM-bM-^@M-^Y are also cited. Our goal was to identify the impact that the wine spoilage yeast Dekkera bruxellensis has on fermenting S. cerevisiae cells, especially on its gene expression level. To this end we co-inoculated both yeast species at the start of fermentation in a synthetic wine must, using S. cerevisiae-only fermentations without Dekkera bruxellensis as a control. All fermentations were employed in special membrane reactors (50 KDa pore size cut-off) physically separating Dekkera bruxellensis from wine yeast S. cerevisiae. Biomass separation with this membrane was done to abolish the possibility of hybridizing also D. bruxellensis probes on Agilent V2 (8x15K format) G4813 DNA microarrays designed just for S. cerevisiae ORF targets. The 50 KDa pore membrane separating both yeasts allowed the exchange of ethanol, metabolites and sugars during the fermentation. Fermentations were carried out in synthetic wine must in duplicate for both the control S. cerevisiae (strain Lalvin EC1118) and mixed fermentation. Sampling of yeast S. cerevisiae for RNA extractions were performed at 22 h of fermentation, during the exponential growth phase of S. cerevisiae, at 92 h and 144 h of fermentation, during its early and late stationary growth phase and at 187 h of fermentation, during its phase of growth decline.

Project description:The yeast Saccharomyces cerevisiae is a model for biology and is also one of the most important microorganisms for food and drink production. Surprisingly, only a few genes involved in the adaptation to anthropic niches have been described until now. Wine fermentation and flor aging, which are performed by strains from two closely related groups of yeast, are two technologies that have opposite approaches toward oxygen, which results in contrasting lifestyles for yeast: fermentation growth on grape for wine yeast, and biofilm aerobic growth on ethanol and glycerol contained in wine for flor strains. This pair of environments and the associated yeast populations can be a model for studying adaptation to anthropic environments. In this project, we have obtained high-quality genome sequences of 20 yeast strains from 9 flor yeast, 9 wine yeast as well as EC1118 and haploid derivative 59A. Phylogeny and population structure analysis, based on GATK genotyping, enable us to characterize a group of flor yeast that is clearly different from wine yeast. A comparison of the genomes of wine and flor yeasts using various methods (PCA, nucleotidic diversity, McDonald Kreitman test, potentially impacted genes according to SIFT) enabled us to note divergent regions, or genes, with potential non-neutral evolution, and highly variant genes. The results of these genomic comparisons are echoed by the comparison of a wine and a flor yeast transcriptome. These methods, as expected, highlight key genes that are involved in FLO11 regulation as well as in biofilm growth, but they also revealed the presence of many allelic variations in genes that are involved in the sensing and regulation of osmotic pressure (such as SLN1, HKR1, SSK22, AQY2) and specific metabolic traits, such as the fructophily of flor yeast, which carry a fructophile allele of HXT3. More remarkable is the accumulation of mutations in multiple genes, which creates a pattern of convergent mutations in regulatory networks, as seen in FLO11 regulation or the HOG MAP kinase pathway. The rewiring of these regulatory networks is clearly one of the hallmarks of domestication for the flor yeast genome. Data presented here correspond to the comparison of Flor yeast P3-D5 and wine yeast K1-280-2B transcriptomes under conditions potentially enabling the production of a biofilm.

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Kluyveromyces dobzhanskii

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