George van der Merwe

Genome‐wide expression analyses: Metabolic adaptation of Saccharomyces cerevisiae to high sugar stress

The transcriptional response of laboratory strains of Saccharomyces cerevisiae to salt or sorbitol stress has been well studied. These studies have yielded valuable data on how the yeast adapts to these stress conditions. However, S. cerevisiae is a saccharophilic fungus and in its natural environment this yeast encounters high concentrations of sugars. For the production of dessert wines, the sugar concentration may be as high as 50% (w/v). The metabolic pathways in S. cerevisiae under these fermentation conditions have not been studied and the transcriptional response of this yeast to sugar stress has not been investigated. High-density DNA microarrays showed that the transcription of 589 genes in an industrial strain of S. cerevisiae were affected more than two-fold in grape juice containing 40% (w/v) sugars (equimolar amounts of glucose and fructose). High sugar stress up-regulated the glycolytic and pentose phosphate pathway genes. The PDC6 gene, previously thought to encode a minor isozyme of pyruvate decarboxylase, was highly induced under these conditions. Gene expression profiles indicate that the oxidative and non-oxidative branches of the pentose phosphate pathway were up-regulated and might be used to shunt more glucose-6-phosphate and fructose-6-phosphate, respectively, from the glycolytic pathway into the pentose phosphate pathway. Structural genes involved in the formation of acetic acid from acetaldehyde, and succinic acid from glutamate, were also up-regulated. Genes involved in de novo biosynthesis of purines, pyrimidines, histidine and lysine were down-regulated by sugar stress.

Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response

In this study, genome-wide expression analyses were used to study the response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation. Forty per cent of the yeast genome significantly changed expression levels to mediate long-term adaptation to fermenting grape must. Among the genes that changed expression levels, a group of 223 genes was identified, which was designated as fermentation stress response (FSR) genes that were dramatically induced at various points during fermentation. FSR genes sustain high levels of induction up to the final time point and exhibited changes in expression levels ranging from four- to 80-fold. The FSR is novel; 62% of the genes involved have not been implicated in global stress responses and 28% of the FSR genes have no functional annotation. Genes involved in respiratory metabolism and gluconeogenesis were expressed during fermentation despite the presence of high concentrations of glucose. Ethanol, rather than nutrient depletion, seems to be responsible for entry of yeast cells into the stationary phase.

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate.

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.

ETP1/YHL010c is a novel gene needed for the adaptation of Saccharomyces cerevisiae to ethanol

Saccharomyces cerevisiae has the ability to use a variety of different carbon sources to support its growth. Abundant fermentable sugars such as glucose and fructose are metabolized to ethanol that accumulates in the environment. Upon glucose depletion, nonfermentable carbon sources, such as ethanol and glycerol, are sufficient to support growth. However, high ethanol concentrations inhibit yeast growth and can become toxic to the cell. Here we show that YHL010c, a previously uncharacterized gene of S. cerevisiae, is needed by the yeast to adapt to ethanol, either as a sole carbon source or as a stressor. We named the gene ETP1 (Ethanol Tolerance Protein 1) and show that the etp1Delta strain has a growth defect in the presence of ethanol, ETP1 is needed for the ethanol-induced transcriptional activation of the ENA1 promoter and heat shock protein genes (HSP12 and HSP26), and plays a role in ethanol-induced turnover of the low-affinity hexose transporter Hxt3p. In addition, the hypersensitivity of etp1Delta to ethanol stress is partly due to the inability of the mutant to control the level of the cation/H(+) antiporter Nha1p in the cell.

Nsf1/Ypl230w participates in transcriptional activation during non-fermentative growth and in response to salt stress in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, fermentable carbon sources such as glucose and fructose are preferred and elicit glucose repression of genes needed to metabolize non-fermentable carbon sources such as glycerol, ethanol and acetate. Different sets of transcription factors are needed to adjust to specific carbon conditions. For example, Mig1 and Mig2 repress the transcription of gluconeogenic and respiratory genes in the presence of abundant glucose, while the transcriptional activation of these genes depends on transcription factors such as Adr1 and Cat8. Here we show that Ypl230w, which we renamed to Nsf1 (nutrient and stress factor 1), is expressed and localizes to the nucleus under non-fermentable carbon conditions to activate gene transcription. Specifically, the transcriptional activation of ACS1, CIT2 and IDH1 is shown to be partially dependent on intact NSF1. Similarly, the transcriptional activation of ENA1 is impaired in the nsf1Delta mutant in response to high concentrations of NaCl, implying that NSF1 is also needed for the yeast response to sodium stress. The carbon- and NaCl-mediated transcriptional activation of ENA1 is dependent on Nsf1. This finding implies that the yeast response to non-fermentable carbon and salt stress is at least partially dependent on NSF1.

Regulation of Hxt3 and Hxt7 Turnover Converges on the Vid30 Complex and Requires Inactivation of the Ras/cAMP/PKA Pathway in Saccharomyces cerevisiae

Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA) pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c), an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.

Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p

ABSTRACT The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.

Functional Analyses of NSF1 in Wine Yeast Using Interconnected Correlation Clustering and Molecular Analyses

Analyzing time-course expression data captured in microarray datasets is a complex undertaking as the vast and complex data space is represented by a relatively low number of samples as compared to thousands of available genes. Here, we developed the Interdependent Correlation Clustering (ICC) method to analyze relationships that exist among genes conditioned on the expression of a specific target gene in microarray data. Based on Correlation Clustering, the ICC method analyzes a large set of correlation values related to gene expression profiles extracted from given microarray datasets. ICC can be applied to any microarray dataset and any target gene. We applied this method to microarray data generated from wine fermentations and selected NSF1, which encodes a C2H2 zinc finger-type transcription factor, as the target gene. The validity of the method was verified by accurate identifications of the previously known functional roles of NSF1. In addition, we identified and verified potential new functions for this gene; specifically, NSF1 is a negative regulator for the expression of sulfur metabolism genes, the nuclear localization of Nsf1 protein (Nsf1p) is controlled in a sulfur-dependent manner, and the transcription of NSF1 is regulated by Met4p, an important transcriptional activator of sulfur metabolism genes. The inter-disciplinary approach adopted here highlighted the accuracy and relevancy of the ICC method in mining for novel gene functions using complex microarray datasets with a limited number of samples.

Traditional Norwegian Kveik Are a Genetically Distinct Group of Domesticated Saccharomyces cerevisiae Brewing Yeasts

The widespread production of fermented food and beverages has resulted in the domestication of Saccharomyces cerevisiae yeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a farmhouse brewing culture in western Norway which has passed down yeasts referred to as kveik for generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavor negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast. Additionally, kveik yeasts are reportedly high-temperature tolerant, likely due to the traditional practice of pitching yeast into warm (>28°C) wort. Here, we characterize kveik yeasts from 9 different Norwegian sources via PCR fingerprinting, whole genome sequencing of selected strains, phenotypic screens, and lab-scale fermentations. Phylogenetic analysis suggests that kveik yeasts form a distinct group among beer yeasts. Additionally, we identify a novel POF- loss-of-function mutation, as well as SNPs and CNVs potentially relevant to the thermotolerance, high ethanol tolerance, and high fermentation rate phenotypes of kveik strains. We also identify domestication markers related to flocculation in kveik. Taken together, the results suggest that Norwegian kveik yeasts are a genetically distinct group of domesticated beer yeasts with properties highly relevant to the brewing sector.

Traditional Norwegian Kveik Yeasts: Underexplored Domesticated Saccharomyces cerevisiae Yeasts

Human activity has resulted in the domestication of Saccharomyces cerevisiae yeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a home-brewing culture in western Norway which has passed down yeasts referred to as kveik for generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavour negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast. Additionally, kveik yeasts are highly temperature tolerant, likely due to the traditional practice of pitching yeast into warm (>30 °C) wort. Here, we characterize kveik yeasts from 9 different Norwegian sources via PCR fingerprinting, phenotypic screens, lab-scale fermentations, and flavour metabolite analysis using HS-SPME-GC-MS. Genetic fingerprinting via interdelta PCR suggests that kveik yeasts form a lineage distinct from other domesticated yeasts. Our analyses confirm that kveik yeasts display hallmarks of domestication such as loss of 4-vinylguaiacol production and high flocculation, and show superior thermotolerance, ethanol tolerance, fermentation rate, and unique flavour metabolite production profiles in comparison to other ale strains, suggesting a broad industrial potential for this group of yeasts.