Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture.

This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.     

ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression

We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Deltaace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Deltaace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.     

Transcriptional regulation of fermentative and respiratory metabolism in Saccharomyces cerevisiae industrial bakers' strains

Bakers' yeast-producing companies grow cells under respiratory conditions, at a very high growth rate. Some desirable properties of bakers' yeast may be altered if fermentation rather than respiration occurs during biomass production. That is why differences in gene expression patterns that take place when industrial bakers' yeasts are grown under fermentative, rather than respiratory conditions, were examined. Macroarray analysis of V1 strain indicated changes in gene expression similar to those already described in laboratory Saccharomyces cerevisiae strains: repression of most genes related to respiration and oxidative metabolism and derepression of genes related to ribosome biogenesis and stress resistance in fermentation. Under respiratory conditions, genes related to the glyoxylate and Krebs cycles, respiration, gluconeogenesis, and energy production are activated. DOG21 strain, a partly catabolite-derepressed mutant derived from V1, displayed gene expression patterns quite similar to those of V1, although lower levels of gene expression and changes in fewer number of genes as compared to V1 were both detected in all cases. However, under fermentative conditions, DOG21 mutant significantly increased the expression of SNF1 -controlled genes and other genes involved in stress resistance, whereas the expression of the HXK2 gene, involved in catabolite repression, was considerably reduced, according to the pleiotropic stress-resistant phenotype of this mutant. These results also seemed to suggest that stress-resistant genes control desirable bakers' yeast qualities.     

MGA2 is involved in the low-oxygen response element-dependent hypoxic induction of genes in Saccharomyces cerevisiae

Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example, OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1 expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-induced OLE1 expression, we examined OLE1 expression under normoxia, hypoxia, and cobalt treatment conditions in Deltamga2 or Deltaspt23 deletion strains. The results of OLE1 promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia- and cobalt-induced OLE1 expression was dramatically decreased in a Deltamga2 strain but not in a Deltaspt23 strain. Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression. When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S. cerevisiae Deltamga2 strain. Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex. Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the Deltamga2 strain. These results indicate that MGA2 is required for the LORE-dependent hypoxic gene induction in S. cerevisiae.     

Bat2p is essential in Saccharomyces cerevisiae for fusel alcohol production on the non-fermentable carbon source ethanol

Branched-chain amino acids (BCAAs) are key substrates in the formation of fusel alcohols, important flavour components in fermented foods. The first step in the catabolic BCAA degradation is a transaminase step, catalyzed by a branched-chain amino acid transaminase (BCAAT). Saccharomyces cerevisiae possesses a mitochondrial and a cytosolic BCAAT, Bat1p and Bat2p, respectively. In order to study the impact of the BCAATs on fusel alcohol production derived from the BCAA metabolism, S. cerevisiae BCAAT-deletion mutants were constructed. The BCAA l-leucine was exogenously supplied during cultivations with mutants of S. cerevisiae. BAT1 deletion is not essential for fusel alcohol production, neither under glucose nor under ethanol growth conditions. The 3-methyl-1-butanol production rate of bat1Delta-cells on ethanol was decreased in comparison with that of wild-type cells, but the cells were still able to produce 3-methyl-1-butanol. However, drastic effects in fusel alcohol production were obtained in cells lacking BAT2. Although the constructed bat2Delta-single deletion strain and the bat1Deltabat2Delta-double deletion strain were still able to produce 3-methyl-1-butanol when grown on glucose, they were incapable of producing any 3-methyl-1-butanol when ethanol was the sole carbon source available. In the circumstances used, gene expression analysis revealed a strong upregulation of BAT2 gene activity in the wild type, when cells grew on ethanol as carbon source. Apparently, the carbon metabolism is able to influence the expression of BCAATs and interferes with the nitrogen metabolism. Furthermore, analysis of gene expression profiles shows that the expression of genes coding for other transaminases present in S. cerevisiae was influenced by the deletion of one or both BCAATs. Several transaminases were upregulated when a BCAAT was deleted. Strikingly, none of the known transaminases was significantly upregulated when BAT2 was deleted. Therefore we conclude that the expression of BAT2 is essential for 3-methyl-1-butanol formation on the non-fermentable carbon source, ethanol.     

Saccharomyces cerevisiae Hsp31p, a stress response protein conferring protection against reactive oxygen species

The Saccharomyces cerevisiae HSP31 (YDR533c) gene encodes a protein that belongs to the DJ-1/PfpI family and its function is unknown. Homologs to Hsp31p polypeptide can be found in organisms from all systematic groups of eukaryotes and prokaryotes, and the functions of the vast majority of them are also hypothetical. One of the homologs is human protein DJ-1. Various amino acid substitutions within this protein correlate with early onset hereditary Parkinson's disease. The deletion of the HSP31 gene displays no apparent phenotype under standard growth conditions, but a thorough functional analysis of S. cerevisiae revealed that its absence makes the cells sensitive to a subset of reactive oxygen species (ROS) generators. HSP31 is induced under conditions of oxidative stress in a YAP1-dependent manner. Similar to other stress response genes, it is also induced in the postdiauxic phase of growth and this induction is YAP1-independent. The patterns of sensitivities to various ROS generators of the hsp31Delta strain and the strain with the deletion of SOD1, another gene defending the cell against ROS, are different. We postulate that Hsp31p protects the cell against oxidative stress and complements other stress protection systems within the cell.     

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.     

Analysis of the stress resistance of commercial wine yeast strains

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.     

Yeast genome-wide expression analysis identifies a strong ergosterol and oxidative stress response during the initial stages of an industrial lager fermentation

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.     

Gene expression analysis of cold and freeze stress in Baker's yeast

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10 degrees C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.