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Genome-wide study of the adaptation of Saccharomyces cerevisiae to the proliferative stages of wine fermentation

ABSTRACT: This work was designed to identify yeast cellular functions specifically affected by the stress factors predominating during the first stages of wine fermentation and genes required for optimal growth under these conditions. The main experimental method used was quantitative fitness analysis by means of competition experiments of whole genome barcoded yeast knock-out collections in continuous culture. This methodology allowed the identification of haploinsufficient genes and homozygous deletions resulting in growth impairment in synthetic must. However, genes identified as haploproficient or homozygous deletions resulting in fitness advantage were of little predictive power concerning optimal growth in this medium. The relevance of these functions for enological performance of yeast was assessed in batch cultures with single strains. Previous studies addressing yeast adaptation to winemaking conditions by quantitative fitness analysis, were not specifically focused on the proliferative stages, and their results were greatly dependent on the effects of gene deletions on yeast survival during stationary phase. Since biomass production has a great influence on the whole fermentation kinetics, focusing on the proliferative stages of the fermentation process has practical implications. In some instances our results highlight the importance of genes not previously linked to winemaking. In other cases our results are complementary to those reported in previous studies concerning, for example, the relevance of some genes involved in vacuolar, peroxisomal, or ribosomal functions. Transport processes and glucose signaling seem to be negatively affected by the stress factors encountered by yeast in synthetic must. Vacuolar activity is important for continued growth during the transition to stationary phase. Finally, reduced biogenesis of peroxisomes also seems to be advantageous. However, in contrast to what was described for later stages, reduced protein synthesis is not advantageous for the first stages of the fermentation, when most cell proliferation takes place.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE46145 | GEO | 2013/04/18

SECONDARY ACCESSION(S): PRJNA197364

REPOSITORIES: GEO

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Project description:This work was designed to identify yeast cellular functions specifically affected by the stress factors predominating during the first stages of wine fermentation and genes required for optimal growth under these conditions. The main experimental method used was quantitative fitness analysis by means of competition experiments of whole genome barcoded yeast knock-out collections in continuous culture. This methodology allowed the identification of haploinsufficient genes and homozygous deletions resulting in growth impairment in synthetic must. However, genes identified as haploproficient or homozygous deletions resulting in fitness advantage were of little predictive power concerning optimal growth in this medium. The relevance of these functions for enological performance of yeast was assessed in batch cultures with single strains. Previous studies addressing yeast adaptation to winemaking conditions by quantitative fitness analysis, were not specifically focused on the proliferative stages, and their results were greatly dependent on the effects of gene deletions on yeast survival during stationary phase. Since biomass production has a great influence on the whole fermentation kinetics, focusing on the proliferative stages of the fermentation process has practical implications. In some instances our results highlight the importance of genes not previously linked to winemaking. In other cases our results are complementary to those reported in previous studies concerning, for example, the relevance of some genes involved in vacuolar, peroxisomal, or ribosomal functions. Transport processes and glucose signaling seem to be negatively affected by the stress factors encountered by yeast in synthetic must. Vacuolar activity is important for continued growth during the transition to stationary phase. Finally, reduced biogenesis of peroxisomes also seems to be advantageous. However, in contrast to what was described for later stages, reduced protein synthesis is not advantageous for the first stages of the fermentation, when most cell proliferation takes place. Competition experiments of the genome-wide collections of mutants were performed in triplicate using conditions that mimicked Phases I or II of a batch fermentation (equivalent to around 14 and 22 hours after inoculation of a batch fermentation, respectively), as well as on YPD (complete medium) for reference purposes. To this end, different feed formulations were used. One mL of either the homozygote or heterozygote pool stored at -80 ºC was added to 50 mL of YPD broth supplemented with 200 µg/mL G418 and incubated overnight at 28 °C and 150 rpm to serve as inoculum for the competition experiments. Samples were taken before the onset of the continuous culture as t=0 (preculture) references. Variations in pool composition were always estimated against the cognate preculture (most often a single preculture served as inoculum for several competitions). After 10 or 20 generations in continuous culture, samples of cells were used for the genomic DNA extraction. lification of the barcodes (uptags and downtags), hybridization, and scanning were performed according to the protocol described elsewhere [Pierce SE, Davis RW, Nislow C, Giaever G (2007) Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures. Nat Protocols 2: 2958-2974]. Slight modifications concerning the hybridization of Tag4 microarrays by using reagents and protocols from the GeneChip Hybridization, Wash and Stain Kit (Affymetrix) were made.

Project description:Second fermentation in a bottle supposes such specific conditions that undergo yeasts to a set of stress situations like high ethanol, low nitrogen, low pH or sub-optimal temperature. Also, yeast have to grow until 1 or 2 generations and ferment all sugar available while they resist increasing CO2 pressure produced along with fermentation. Because of this, yeast for second fermentation must be selected depending on different technological criteria such as resistance to ethanol, pressure, high flocculation capacity, and good autolytic and foaming properties. All of these stress factors appear sequentially or simultaneously, and their superposition could amplify their inhibitory effects over yeast growth. Considering all of the above, it has supposed interesting to characterize the adaptive response of commercial yeast strain EC1118 during second-fermentation experiments under oenological/industrial conditions by transcriptomic profiling. We have pointed ethanol as the most relevant environmental condition in the induction of genes involved in respiratory metabolism, oxidative stress, autophagy, vacuolar and peroxisomal function, after comparison between time-course transcriptomic analysis in alcoholic fermentation and transcriptomic profiling in second fermentation. Other examples of parallelism include overexpression of cellular homeostasis and sugar metabolism genes. Finally, this study brings out the role of low-temperature on yeast physiology during second-fermentation. S. cerevisiae EC1118 pre-adapted to ethanol cells and sucrose (20 g/L) were added to 20 L of base wine (Cavas Freixenet, Sant Sadurní D’Anoia, Spain). Complete volume was bottled with 350 mL each one. All were sealed and incubated in static conditions at 16ºC for approximately 40 days after tirage. Three samples were taken during the process for transcriptional study of the physiological adaptation of yeast cells to industrial second fermentation conditions. A sample corresponding to exponential-growth phase under unstressed conditions (in YPD at 28ºC) was used as an external reference. Three timepoints from second-fermentation were monitored and three biological replicates from each timepoint were analyzed.

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Genome-wide study of the adaptation of Saccharomyces cerevisiae to the proliferative stages of wine fermentation

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