Marcel·lí del Olmo

Analysis of the stress resistance of commercial wine yeast strains

Abstract. Alcoholic fermentation is an essential step in wine production that is usually conducted by yeasts belonging to the species Saccharomyces cerevisiae. The ability to carry out vinification is largely influenced by the response of yeast cells to the stress conditions that affect them during this process. In this work, we present a systematic analysis of the resistance of 14 commercial S. cerevisiae wine yeast strains to heat shock, ethanol, oxidative, osmotic and glucose starvation stresses. Significant differences were found between these yeast strains under certain severe conditions, Vitilevure Pris Mouse and Lalvin T73 being the most resistant strains, while Fermiblanc arom SM102 and UCLM S235 were the most sensitive ones. Induction of the expression of the HSP12 and HSP104 genes was analyzed. These genes are reported to be involved in the tolerance to several stress conditions in laboratory yeast strains. Our results indicate that each commercial strain shows a unique pattern of gene expression, and no clear correlation between the induction levels of either gene and stress resistance under the conditions tested was found. However, the increase in mRNA levels in both genes under heat shock indicates that the molecular mechanisms involved in the regulation of their expression by stress function in all of the strains.

Transcriptomic and Proteomic Approach for Understanding the Molecular Basis of Adaptation of Saccharomyces cerevisiae to Wine Fermentation

ABSTRACT Throughout alcoholic fermentation, Saccharomyces cerevisiae cells have to cope with several stress conditions that could affect their growth and viability. In addition, the metabolic activity of yeast cells during this process leads to the production of secondary compounds that contribute to the organoleptic properties of the resulting wine. Commercial strains have been selected during the last decades for inoculation into the must to carry out the alcoholic fermentation on the basis of physiological traits, but little is known about the molecular basis of the fermentative behavior of these strains. In this work, we present the first transcriptomic and proteomic comparison between two commercial strains with different fermentative behaviors. Our results indicate that some physiological differences between the fermentative behaviors of these two strains could be related to differences in the mRNA and protein profiles. In this sense, at the level of gene expression, we have found differences related to carbohydrate metabolism, nitrogen catabolite repression, and response to stimuli, among other factors. In addition, we have detected a relative increase in the abundance of proteins involved in stress responses (the heat shock protein Hsp26p, for instance) and in fermentation (in particular, the major cytosolic aldehyde dehydrogenase Ald6p) in the strain with better behavior during vinification. Moreover, in the case of the other strain, higher levels of enzymes required for sulfur metabolism (Cys4p, Hom6p, and Met22p) are observed, which could be related to the production of particular organoleptic compounds or to detoxification processes.

Response to acetaldehyde stress in the yeast Saccharomyces cerevisiae involves a strain-dependent regulation of several ALD genes and is mediated by the general stress response pathway.

One of the stress conditions that yeast may encounter is the presence of acetaldehyde. In a previous study we identified that, in response to this stress, several HSP genes are induced that are also involved in the response to other forms of stress. Aldehyde dehydrogenases (ALDH) play an important role in yeast acetaldehyde metabolism (e.g. when cells are growing in ethanol). In this work we analyse the expression of the genes encoding these enzymes (ALD) and also the corresponding enzymatic activities under several growth conditions. We investigate three kinds of yeast strains: laboratory strains, strains involved in the alcoholic fermentation stage of wine production and flor yeasts (responsible for the biological ageing of sherry wines). The latter are very important to consider because they grow in media containing high ethanol concentrations, and produce important amounts of acetaldehyde. Under several growth conditions, further addition of acetaldehyde or ethanol in flor yeasts induced the expression of some ALD genes and led to an increase in ALDH activity. This result is consistent with their need to obtain energy from ethanol during biological ageing processes. Our data also suggest that post‐transcriptional and/or post‐translational mechanisms are involved in regulating the activity of these enzymes. Finally, analyses indicate that the Msn2/4p and Hsf1p transcription factors are necessary for HSP26, ALD2/3 and ALD4 gene expression under acetaldehyde stress, while PKA represses the expression of these genes. Copyright

Exposure of Saccharomyces cerevisiae to acetaldehyde induces sulfur amino acid metabolism and polyamine transporter genes, which depend on Met4p and Haa1p transcription factors, respectively.

ABSTRACT Acetaldehyde is a toxic compound produced by Saccharomyces cerevisiae cells under several growth conditions. The adverse effects of this molecule are important, as significant amounts accumulate inside the cells. By means of global gene expression analyses, we have detected the effects of acetaldehyde addition in the expression of about 400 genes. Repressed genes include many genes involved in cell cycle control, cell polarity, and the mitochondrial protein biosynthesis machinery. Increased expression is displayed in many stress response genes, as well as other families of genes, such as those encoding vitamin B1 biosynthesis machinery and proteins for aryl alcohol metabolism. The induction of genes involved in sulfur metabolism is dependent on Met4p and other well-known factors involved in the transcription of MET genes under nonrepressing conditions of sulfur metabolism. Moreover, the deletion of MET4 leads to increased acetaldehyde sensitivity. TPO genes encoding polyamine transporters are also induced by acetaldehyde; in this case, the regulation is dependent on the Haa1p transcription factor. In this paper, we discuss the connections between acetaldehyde and the processes affected by this compound in yeast cells with reference to the microarray data.

Analyses of stress resistance under laboratory conditions constitute a suitable criterion for wine yeast selection

During wine production, yeast cells are affected by several conditions that are adverse to growth (oxidative, osmotic and ethanol stress among others) and they should detect and respond to these conditions, otherwise alcoholic fermentation can be negatively affected. In this work we have analyzed the fermentative behaviour of 14 commercial and non-commercial strains in several synthetic musts. According to the data obtained these strains have been classified into three groups depending on whether or not vinification was completed (and on the amount of residual sugar remaining in the must if it was not). Moreover, we have determined the resistance of these strains to several stress situations under laboratory growth conditions. We have been able to establish a correlation between the groups based on fermentative behaviour and resistance to several stress conditions (especially oxidative and ethanol stress), by applying discriminant analysis to the data obtained in these experiments. Our results indicate a clear relationship between stress resistance and fermentative behaviour and this opens up the possibility of using this information as a criterion for the future selection of wine yeasts.

The nature of the nitrogen source added to nitrogen depleted vinifications conducted by a Saccharomyces cerevisiae strain in synthetic must affects gene expression and the levels of several volatile compounds

Nitrogen starvation may lead to stuck and sluggish fermentations. These undesirable situations result in wines with high residual sugar, longer vinification times, and risks of microbial contamination. The typical oenological method to prevent these problems is the early addition of ammonium salts to the grape juice, although excessive levels of these compounds may lead to negative consequences for the final product. This addition reduces the overall fermentation time, regardless of the time of addition, but the effect is more significant when nitrogen is added during the yeast exponential phase. In this work we analysed the effect of adding different nitrogen sources (ammonia, amino acids or a combination of both) under nitrogen depletion in order to understand yeast metabolic changes that lead to the adaptation to the new conditions. These studies were carried out in a synthetic must that mimics the composition of the natural must. Furthermore, we studied how this addition affects fermentative behaviour, the levels of several yeast volatile compounds in the final product, arginase activity, and the expression of several genes involved in stress response and nitrogen metabolism during vinification. We found that the nature of the nitrogen source added during yeast late exponential growth phase introduces changes to the volatile compounds profile and to the gene expression. On the other hand, arginase activity and the expression of the stress response gene ACA1 are useful to monitor nitrogen depletion/addition during growth of the wine yeast considered under our vinification conditions.

Study of the First Hours of Microvinification by the Use of Osmotic Stress-response Genes as Probes

When yeast cells are inoculated into grape must for vinification they find stress conditions because of osmolarity, which is due to very high sugar concentration, and pH lower than 4. In this work an analysis of the expression of three osmotic stress induced genes (GPD1, HSP12 and HSP104) under microvinification conditions is shown as a way to probe those stress situations and the regulatory mechanisms that control them. The results indicate that during the first hours of microvinification there is an increase in the GPDI mRNA levels with a maximum about one hour after inoculation, and a decrease in the amount of HSP12 and HSP104 mRNAs, although with differences between them. The RNA steady-state levels of all the genes considered, and in some cases the microvinification progress are significantly affected by the composition of the must (pH, nature of the osmotic agent and carbon source). These results point out the importance of the control of these parameters and the yeast molecular response during the first hours of vinification for an accurate winemaking process.

Arginase Activity is a Useful Marker of Nitrogen Limitation During Alcoholic Fermentations

Nitrogen deficiency in musts is one of the causes of sluggish or stuck fermentations. In this work we propose that arginase activity determination can be useful for detecting nitrogen starvation early in vinification. CAR1 and YGP1 genes are not specifically induced under conditions of nitrogen starvation. However, a significant increase in the enzymatic activity of arginase, the product of the CAR1 gene, is detected in vinifications carried out with musts containing limiting amounts of nitrogen. Moreover, on adding ammonia to a nitrogen-deficient vinification, even at late stages, this enzymatic activity is repressed, and growth rate is restored simultaneously. We also investigate the role of ethanol toxicity in nitrogen starvation. The results suggest that ethanol produced during vinification or exogenously added up to 8% (v/v) concentration does not cause nitrogen starvation under the conditions tested because arginase activity is not increased.

Towards an understanding of the adaptation of wine yeasts to must: relevance of the osmotic stress response

During the transformation of grape must sugars in ethanol, yeasts belonging to Saccharomyces cerevisiae strains are particularly involved. One of the stress conditions that yeast cells have to cope with during vinification, especially at the time of inoculation into must, is osmotic stress caused by high sugar concentrations. In this work, we compare several laboratory and wine yeast strains in terms of their ability to start growth in must. By means of transcriptomic approaches and the determination of glycerol intracellular content, we propose several clues for yeast strains to adapt to the wine production conditions: the high expression of genes involved in both biosynthetic processes and glycerol biosynthesis, and the appropriate levels of intracellular glycerol. Besides, we demonstrate that the pre-adaptation of the wine yeast strains showing growth problems at the beginning of vinification in a rehydration medium containing 2% or 5% glucose (depending on the yeast strain considered) may increase their vitality when inoculated into high sugar media.

ANALYSIS OF THE STRUCTURE OF A NATURAL ALTERNATING D(TA)N SEQUENCE IN YEAST CHROMATIN

We address here the question of the in vivo structure of a natural alternating d(TA)n sequence found at the 3′ region of the Saccharomyces cerevisiae FBP1 gene. This sequence consists of 13 TA pairs interrupted by a TT dinucleotide in the middle of the tract. Previous experiments with cruciform‐specific nucleases S1 and Endonuclease VII demonstrated the presence in vitro of a cruciform in this region. We also showed this region to be part of a nuclease hypersensitive site flanked by nucleosomes in yeast chromatin. Here we demonstrate, by means of S1 in vivo footprinting, that in yeast plasmids also adopts in vivo a non B‐DNA structure which is not a cruciform. A theoretical analysis of this region shows that it contains a site susceptible to superhelical stress duplex destabilization. The locations and conditions under which alternative structures form in the wild‐type sequence and in deletion mutants agree with these theoretical predictions, suggesting that some kind of denaturation is the alternative structure adopted by the sequence in vivo. This suggests that negative superhelical stress sufficient for local denaturation exists in nucleosomal DNA. We also demonstrate by micrococcal nuclease digestions that the deletion of the alternating d(TA)n sequence modifies the chromatin hypersensitive site but does not affect nucleosome positioning.