Unknown,Transcriptomics,Genomics,Proteomics

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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. 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.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Ramon Gonzalez

PROVIDER: E-GEOD-46145 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description:In many animals living in groups the reproductivestatus of individuals is determined by their social status. In specieswith social hierarchies, the death of dominant individuals typicallyupheaves the social hierarchy and provides an opportunity for subordinateindividuals to improve their social status. Such a phenomenon occursin the monogyne form of the fire ant\emph{, Solenopsis invicta, }wherecolonies typically contain a single wingless reproductive queen, thousandsof workers and hundreds of winged non-reproductive virgin queens.Upon the death of the mother queen, many virgin queens shed theirwings and initiate reproductive development instead of departing ona mating flight. Workers progressively execute almost all of themover the following weeks. The workers base their collective decisionon pheromonal cues associated with the onset of reproductive developmentof the virgin queens which occurs after orphaning. To examine the factors that determine which virgin queens are executed and which survive, we set up artificial competitions between queens from different colonies. Using microarrays, we found that queens from winning colonies showed higher mitochondrial as well as organ development activities 24 hours after orphaning than did queens from colonies that lost the competitions. Furthermore, queens from colonies where queens shed their wings faster after orphaning were more likely to survive competitions. Finally, higher wing shedding speed is linked to higher mitochondrial activity. Eight competitions were initially conducted between queens form pairs of colonies. Six competitions clearly identified one winning colony and one losing colony. We thus have microarray data for six competitions (ie biological replicates), with one winning and one losing colony within each competition. Thus 12 colonies total. For each colony, RNA extracted from five queens was pooled, and hybridized against an unrelated common reference RNA that had been made from a pool of many S. invicta individuals of all castes and developmental stages. Lab work was conducted in blocks (by competition) with randomized order within competition to avoid introducing bias. No technical replication was performed.

Project description:Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes. To this end, we used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 ºC. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins. Thus we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms. The first aim of this study was to assess the most competitive strains that grow under wine fermentation conditions at low temperature. To this end, we performed a growth competition assay with 27 commercial wine strains inoculated at equal population size in synthetic grape must. In spite of the economical and industrial importance of these strains, their phenotypic variation in the main enological traits, particularly those related to optimum growth temperature, and their ability to adapt to low temperature fermentation have been poorly investigated. The second goal was to obtain an improved strain to grow and ferment at low temperature by evolutionary engineering. For this purpose, we maintained growth competition in synthetic grape must during 200 generations to select for the mutations that produce phenotypes with improved growth in this medium. One of these evolved cultures was previously treated with ethyl methanesulfonate (EMS) to increase the mutation rate. Finally, we aimed to decipher the molecular basis underlying this improvement by analyzing the genomic and transcriptional differences between the parental strain and the strain evolved at low temperature.

Project description:Gene expression profiles of bakerâs yeast during initial dough-fermentation were investigated using liquid fermentation media to obtain insights at the molecular level into rapid adaptation mechanisms of bakerâs yeast. Results showed that onset of fermentation caused drastic changes in gene expression profiles within 15 min. Genes involved in the tricarboxylic acid (TCA) cycle were down-regulated and genes involved in glycolysis were up-regulated, indicating a metabolic shift from respiration to fermentation. Genes involved in ethanol production (PDC genes and ADH1), in glycerol synthesis (GPD1 and HOR2), and in low-affinity hexose transporters (HXT1 and HXT3) were up-regulated at the beginning of model dough-fermentation. Among genes up-regulated at 15 min, several genes classified as transcription were down-regulated within 30 min. These down-regulated genes are involved in messenger RNA splicing and ribosomal protein biogenesis, in zinc finger transcription factor proteins, and in transcriptional regulator (SRB8, MIG1). In contrast, genes involved in amino acid metabolism and in vitamin metabolism, such as arginine biosynthesis, riboflavin biosynthesis, and thiamin biosynthesis, were subsequently up-regulated after 30 min. Interestingly, the genes involved in the unfolded protein response (UPR) pathway were also subsequently up-regulated. Our study presents the first overall description of the transcriptional response of bakerâs yeast during dough-fermentation, and will thus help clarify genomic responses to various stresses during commercial fermentation processes. Experiment Overall Design: Saccharomyces cerevisiae T128 was used as a model of typical commercial bakerâs yeast used in Japan. After 18 h cultivation, cells in stationary phase were collected by centrifugation (2,700 ï g for 5 min). Some of the cell pellets were suspended in 900 ml of sterilized water. Cells for no-fermentation control were harvested after the fed-butch cultivation and stored until RNA extraction. Cell pellets (11,700 OD units) were suspended in 390 ml of lequid fermentation (LF) medium in a 500-ml flask and then fermented for 300 min. To investigate gene expression profiles during initial stages of dough-fermentation, cell samples for DNA microarray analysis were obtained from each culture medium at 15 min, 30 min, and 60 min. Cells in stationary phase were then collected by centrifugation (2,700ï g for 5 min), and stored until RNA extraction.

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

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