A combined genetic and genomic approach uncovers molecular basis of wine yeast fermentation traits
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ABSTRACT: 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. 40 samples are analysed with 2 technical replicates, using a unique reference named pool of the 30 segregants. The transcriptome of each segregant is compared to the transcriptome of the pool. The transcriptome of 5 biologic replicates of each parental strain is also compared to this reference. An haploid derivative of the commercialized wine yeast EC1118 which sequence is available (Novo et al. 2009. PNAS, 106:16333-16338) called 59A was used as industrial wine yeast. It is a prototroph strain and has a MATa sexual type. The haploid laboratory strain S288C (MATa) was used for crossing.
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: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: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:EKD-13 is among the recently developed mannoprotein overproducing strains. It is a recombinant derivative of the well known EC1118 commercial strain, in which the ORF of all the alleles of KNR4/SMI1 was replaced by different integration cassettes. Lack of Knr4p resulted in a negligible impairment in fermentation kinetics and a net reduction in bentonite requirements to attain complete protein stabilization of white wines made out of natural grape must. In order to improve the technological characterization of this strain, and to better understand the mechanisms underlying mannoprotein oversecretion, we have analyzed kinetics of mannoprotein release and autolysis during fermentation and aging, and we have performed a genome wide expression analysis in two different steps of the fermentation process. The results give some additional clues on KNR4 function in S. cerevisiae as well as on the best application conditions for this recombinant strain. A comparative transcriptome analysis between the S. cerevisiae recombinant strain (EKD-13) defective for KNR4/SMI1 and the wild-type strain (EC1118) was implemented. At every step of the fermentation (mid-exponential growth phase of fermentation advancement as 10 g of released CO2 and stationary-growth phase as 70 g of CO2 released) a recombinant and a WT yeast samples are put on a slide. There are three biological replicates that are labeled in dye-switch way (sometimes called biological dye-swap) for each step of fermentation leading to 3 independent intensity values for each gene (spots) on the microarrays. This experimental design minimizes the intrinsic biological noise between identical culture conditions and the technical variations inherent to the DNA microarray technology. In summary, there are six samples, each corresponding to comparisons between independent fermentations of both strains.
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:Comparison between two commercial wine yeast strains (UCD522 and P29) differing in their production of H2S during wine fermentation. Due to the characteristics of the strains (commercial, non-standard wine strains), the experiment was duplicated using two completely different platforms and techniques (cDNA-based and in situ synthesized oligonucleotide-based). UCD522 and P29 wine microfermentations were performed in parallel and yeast samples were taken at the stage of fastest fermentation rate. Two biological replicates per yeast strain.
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: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.
Project description:The goal of this study was to compare the expression level of the whole genome of two wine yeast strains highly differing in their sulfite production (High producer strain: 10281A; Low producer strain: 1764A). Conditions maximizing SO2 production were selected: nitrogen rich media (425 mg/l assimilable nitrogen) and low temperature (16°C). This transcriptomic analysis was performed during the sulfite production phase, just after the entry in stationary phase. This analysis is part of a global work, aiming at the identification of the molecular basis of sulfite production by wine yeasts through physiologic, transcriptomic and genetic studies. Two strains compared in the same conditions with biological replicates following a dye-swap design.
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.