Transcription profiling of S. cerevisiae industrial wine strains at three time points during allcoholic fermentation
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ABSTRACT: Our study involves a transcriptomic approach to the analysis of industrial yeast metabolism. Historically, among the hundreds of yeast species, Saccharomyces cerevisiae has played an important role in scientific investigations and industrial applications, and it is universally acknowledged as one of the model systems for eukaryotic organisms. Yeast is also an important component of the wine fermentation process and determines various attributes of the final product. Our research takes a holistic approach to the improvement of industrial yeast strains by integrating large data sets from various yeast strains during fermentation. This means that analysis can be done in such a way as to co-evaluate several parameters simultaneously to identify points of interest and target genes for metabolic engineering. Eventually we hope to construct an accurate information matrix and a more complete cellular map for the fermenting yeast. This will enable accurate model-building for industrial yeast and facilitated the design of intelligent yeast improvement strategies which can be applied via traditional avenues of molecular biology. Experiment Overall Design: Five different Saccharomyces cerevisiae strains used in industrial winemaking processes were used in synthetic must (MS300) fermentations. All fermentations were carried out in triplicate, so each sample is represented by three completely independent biological repeats. Samples for microarray analysis were taken at three different time points during fermentation, representative of the exponential (day2), early stationary (day5) and late stationary (day14) growth stages.
Project description:Our study involves a transcriptomic approach to the analysis of industrial yeast metabolism. Historically, among the hundreds of yeast species, Saccharomyces cerevisiae has played an important role in scientific investigations and industrial applications, and it is universally acknowledged as one of the model systems for eukaryotic organisms. Yeast is also an important component of the wine fermentation process and determines various attributes of the final product. Our research takes a holistic approach to the improvement of industrial yeast strains by integrating large data sets from various yeast strains during fermentation. This means that analysis can be done in such a way as to co-evaluate several parameters simultaneously to identify points of interest and target genes for metabolic engineering. Eventually we hope to construct an accurate information matrix and a more complete cellular map for the fermenting yeast. This will enable accurate model-building for industrial yeast and facilitated the design of intelligent yeast improvement strategies which can be applied via traditional avenues of molecular biology.
Project description:Transcriptomic analyses of fermenting yeast are increasingly being carried out under small scale simulated winemaking conditions. It is not known to what degree data generated from such experiments are a reflection of transcriptional processes in large-scale commercial fermentation tanks. In this experiment we set out to determine the effect of scale, or fermentation volume, on the transcriptional respone of wine yeast strains. Parallel fermentations were carried out in laboratory fermentation vials and commercial fermentation tanks using the same wine media and inoculated yeast strain. Comparative transcriptomic analyses were carried out at three time points during alcoholic fermentation. Fermentations were carried out in Chardonnay wine must in triplicate for both the lab-scale (80ml) and commercial scale (300L) fermentations. Sampling of yeast for RNA extractions were performed at day 2 of fermentation (during the exponential growth phase of the yeast cells), and again at day 5 (early stationary growth phase) and day 10 (late stationary growth phase) of fermentation.
Project description:Industrial wine yeast strains are geno- and phenotypically highly diversified, and have adapted to the ecological niches provided by industrial wine making environments. These strains have been selected for very specific and diverse purposes, and the adaptation of these strains to the oenological environment is a function of the specific expression profiles of their genomes. It has been proposed that some of the primary targets of yeast adaptation are functional binding sites of transcription factors (TF) and the transcription factors themselves. Sequence divergence or regulatory changes related to specific transcription factors would lead to far-reaching changes in overall gene expression patterns, which will in turn impact on specific phenotypic characteristics of different yeast species/ strains. Variations in transcriptional regulation between different wine yeast strains could thus be responsible for rapid adaptation to different fermentative requirements in the context of commercial wine-making. In this study, we compare the transcriptional profiles of five different wine yeast strains in simulated wine-making conditions: Comparative analyses of gene expression profiles in the context of TF regulatory networks provided new insights into the molecular basis for variations in gene expression in these industrial strains. We also show that the metabolic phenotype of one strain can indeed be shifted in the direction of another by modifying the expression of key transcription factors. SOK2 was one target transcription factor identified in this study. This expression factor was overexpressed in order to validate our hypotheses that altered expression levels of key transcription factors could shift metabolism in a directed, predicted manner. Fermentations were carried out in synthetic wine must in triplicate for both the control VIN13 strain and the SOK2 overexpressing strain. Sampling for RNA extractions were performed at day 2 of fermentation, during the exponential growth phase of the yeast cells.
Project description:Comparative gene expression analysis of two wine yeast strains at three time points (days 2, 5 and 14) during fermentation of colombar must. In our study we conducted parallel fermentations with the VIN13 and BM45 wine yeast strains in two different media, namely MS300 (syntheticmust) and Colombar must. The intersection of transcriptome datasets from both MS300 (simulated wine must;GSE11651) and Colombar fermentations should help to delineate relevant and ânoisyâ changes in gene expression in response to experimental factors such as fermentation stage and strain identity. Experiment Overall Design: Two industrial wine yeast strains (BM45 and VIN13) grown micro-aerobically in Colombar must. Microarray analysis was performed at three time points during fermentation (days 2, 5 and 14), representing the exponential, early and late stationary growth phases respectively.
Project description:Furfural, phenol and acetic acid, generated during cellulosic material pretreatment, are the representative inhibitors to yeast used for ethanol production. The responses to these inhibitors in industrial yeast and the corresponding adapted strains were analyzed. Experiment Overall Design: We analyzed the transient response to inhibitors and the different transcriptions in industrial yeast and furfural-, phenol-, and acetic acid-adapted strains. Industrial yeast and the adapted strains were collected at 20 minutes after inhibitor addition. The reference samples for industrial yeast and adapted strains were collected at the same time without inhibitor addition. 2 replicates for each strain/treatment were analyzed.
Project description:Industrial wine yeast strains possess specific abilities to ferment under stressing conditions and give a suitable aromatic outcome. Although the fermentations properties of Saccharomyces cervisiae wine yeasts are well documented little is known on the genetic basis underlying the fermentation traits. Besides, although strain differences in gene expression has been reported, their relationships with gene expression variations and fermentation phenotypic variations is unknown. To both identify the genetic basis of fermentation traits and get insight on their relationships with gene expression variations, we combined fermentation traits QTL mapping and expression profiling in a segregating population from a cross between a wine yeast derivative and a laboratory strain.
Project description:The yeast Saccharomyces cerevisiae is an important component of the wine fermentation process and determines various attributes of the final product. However, lactic acid bacteria (LAB) are also an integral part of the microflora of any fermenting must. Various wine microorganism engineering projects have been endeavoured in the past in order to change certain wine characteristics, namely aroma compound composition, ethanol concentration, levels of toxic/ allergenic compounds etc. Most of these projects focus on a specific gene or pathway, whereas our approach aims to understand the genetically complex traits responsible for these phenotypes in a systematic manner by implementing a transcriptomic analysis of yeast in mixed fermentations with the LAB O. oeni. Our aim is to investigate interactions between yeast and LAB on a gene expression level to identify targets for modification of yeast and O. oeni in a directed manner. Our goal was to identify the impact that the common wine microorganism O. oeni (malolactic bacteria) has on fermenting yeast cells on a gene expression level. To this end we co-inoculated the yeast and bacteria at the start of fermentation in a synthetic wine must, using yeast-only fermentations witout O. oeni as a control. Fermentations were carried out in synthetic wine must in triplicate for both the control S. cerevisiae VIN13 strain and the mixed fermentation of VIN13 and O. oeni (strain S5). Sampling of yeast for RNA extractions were performed at day 3 of fermentation, during the exponential growth phase of the yeast cells, and again at day 7 of fermentation, during the early stationary growth phase.
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:Comparative gene expression analysis of two wine yeast strains at three time points (days 2, 5 and 14) during fermentation of colombar must. In our study we conducted parallel fermentations with the VIN13 and BM45 wine yeast strains in two different media, namely MS300 (syntheticmust) and Colombar must. The intersection of transcriptome datasets from both MS300 (simulated wine must;GSE11651) and Colombar fermentations should help to delineate relevant and ‘noisy’ changes in gene expression in response to experimental factors such as fermentation stage and strain identity.
Project description:Comparative genome-wide gene expression analysis between two industrial wine yeast hybrid strains belonging to the species S. cerevisiae x S. Kudriavzevii, in natural must fermentations.