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.     

Carbon starvation can induce energy deprivation and loss of fermentative capacity in Saccharomyces cerevisiae

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 micro mol/g, while nitrogen-starved cells still contained approximately 6 micro mol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.     

Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae

The synthesis of isocitrate lyase was induced by the presence of ethanol in the chemostat reaching a specific activity of 200 mU·mg^-1 at this induced state. In glucoselimited, derepressed cells, 20 mU·mg^-1 were detected and under repressed conditions isocitrate lyase activity was not detected.The sensitivity of gluconeogenic enzymes: cytoplasmic malate dehydrogenase; fructose 1,6-bisphosphatase and isocitrate lyase as well as the mitochondrial enzymes NADH dehydrogenase and succinate cytochrome c oxidase to glucose and galactose repression were studied in chemostat cultures. Our results show that galactose was less effective as a repressor than glucose. Malate dehydrogenase was completely inactivated by glucose, whereas galactose only produced a 78% decrease of specific activity. Fructose 1,6-bisphosphatase and isocitrate lyase were completely inactivated by both sugars but at different rate. Glucose produced an 85% decrease of specific activity of the mitochondrial enzymes whereas galactose only decrease an 67%.     

The effect of respiratory inhibitors on the fermentative ability of Pichia stipitis,Pachysolen tannophilus and Saccharomyces cerevisiae under various conditions of aerobiosis

Summary The growth and ethanol production by the d-xylose-fermenting yeasts Pichia stipitis and Pachysolen tannophilus under various conditions of aerobiosis responded similarly to the addition of the respiratory inhibitors potassium cyanide (KCN), antimycin A (AA), sodium azide and rotenone. However, the d-glucose-fermenting yeast Saccharomyces cerevisiae differed markedly from these yeasts in response to the inhibitors. In general the growth of the d-xylose-fermenting yeasts was inhibited by the respiratory inhibitors while ethanol production was either stimulated (especially when oxygen was available) or unaffected or inhibited by rotenone or AA or KCN and sodium azide, respectively. However, by exception KCN and AA stimulated ethanol production under aerobic conditions by Pichia stipitis and Pachysolen tannophilus respectively. Stimulatory or inhibitory effects by respiratory inhibitors were less marked in S. cerevisiae. These data suggest that unimpaired mitochondrial function is necessary for growth on d-xylose and optimal d-xylose fermentation. A requirement for membrane generated energy during d-xylose utilisation is indicated by 2,4-dinitrophenol inhibition of growth and fermentation.     

Increased sensitivity of heat-stressed Saccharomyces cerevisiae cells to food-grade antioxidants.

Unheated and heat-stressed Saccharomyces cerevisiae cells were examined for their relative sensitivities to butylated hydroxyanisole (BHA), tertiary butylhydroquinone (TBHQ), and propyl gallate. Heated cells had significant (P less than or equal to 0.05) increases in sensitivity to 50 micrograms of BHA, 100 micrograms of TBHQ, and 1,000 micrograms of propyl gallate per ml as compared with unheated cells when surface plated on antioxidant-supplemented recovery agar. The rate of increase in size of colonies developed by heated cells was slower than that of unheated cells, and the presence of antioxidants in recovery agar enhanced this effect. Heat-stressed cells also had increased sensitivity to ethanol. Incubation temperatures of 15, 21, 30, and 37 degrees C for enumerating unheated cells had no significant effect on the numbers of colonies formed on unsupplemented recovery agar; however, incorporation of 100 micrograms of BHA, 200 micrograms of TBHQ, or 1,000 micrograms of propyl gallate per ml into agar resulted in significant decreases in the number of colonies formed by heated cells at various incubation temperatures. The detrimental effects of TBHQ and propyl gallate on repair of heat-injured cells are apparently expressed at a temperature higher than that observed for BHA. It is suggested that the adverse effects of antioxidants on repair of heat-injured S. cerevisiae cells may be associated with oxygen availability.     

Role of mitochondrial NADH kinase and NADPH supply in the respiratory chain activity of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the mitochondrial nicotinamide adenine dinucleotide hydride kinase Pos5p is required for a variety of essential cellular pathways, most importantly respiration. The Pos5p knockout strain pos5Δ grows poorly in non-fermentable media. A potential relationship between this respiratory deficiency and the ability of the cells to supply nicotinamide adenine dinucleotide phosphate (NADPH) was examined by analyzing the respiratory chain activity of pos5Δ and two NADP(+)-specific dehydrogenase mutants, idp1Δ and zwf1Δ. All of the respiratory chain complexes of pos5Δ exhibited poor relative activity of <26% at the middle-log phase and 62% at the stationary phase. The respiratory chain activity levels of idp1Δ and zwf1Δ also reduced to 22%-37% and 28%-84% at the middle-log phase, and 73%-81% and 67%-88% at the stationary phase, not as robustly as those of pos5Δ. The double-mutant idp1pos5Δ exhibited even lower activities of <20% at the middle-log phase, but zwf1pos5Δ showed similar activities with pos5Δ. The complemented strain POS5/pos5Δ exhibited 1.05- to 3-fold higher activities than pos5Δ. These data showed that Pos5p contributes to the maintenance of respiratory chain complex activities, with other NADPH sources, such as Idp1p and Zwf1p, making a smaller contribution. These contributions were partly related to the ability of the cells to supply NADPH, especially in the mitochondria.     

Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments

The yeast Saccharomyces cerevisiae is an attractive cell factory, but in many cases there are constraints related with balancing the formation and consumption of redox cofactors. In this work, we studied the effect of having an additional source of NADPH in the cell. In order to do this, two strains were engineered by overexpression of malic enzyme. In one of them, malic enzyme was overexpressed as its wild-type mitochondrial form, and in the other strain a short form lacking the mitochondrial targeting sequence was overexpressed. The recombinant strains were analyzed in aerobic batch and continuous cultivations, and the basic growth characteristics were generally not affected to a great extent, even though pleiotropic effects of the manipulations could be seen by the altered in vitro activities of selected enzymes of the central metabolism. Moreover, the decreased pentose-phosphate pathway flux and the ratios of redox cofactors showed that a net transhydrogenase effect was obtained, which can be directed to the cytosol or the mitochondria. This may find application in redirecting fluxes for improving specific biotechnological applications.     

Effects of acetic acid on growth and fermentative activity of Saccharomyces cerevisiae

The effect of acetic acid on the growth and the fermentative activity of S. cerevisiae was analysed comparatively with the pH. This study showed that the pH does not affect these two activities. On the contrary, the acetic acid has an inhibition effect. This effect was modelised by the relation of Levenspiel. Finally, it was shown that the quantities of acetic acid produced by Brettanomyces were not sufficient to explain the inhibition of Saccharomyces.     

Interactions between carbon and nitrogen sources depend on RIM15 and determine fermentative or respiratory growth in Saccharomyces cerevisiae

Applied Microbiology and Biotechnology - Nutritional homeostasis is fundamental for alcoholic fermentation in Saccharomyces cerevisiae. Carbon and nitrogen have been related to this metabolic...     

Differential repair of UV damage in Saccharomyces cerevisiae.

Preferential repair of UV-induced damage is a phenomenon by which mammalian cells might enhance their survival. This paper presents the first evidence that preferential repair occurs in the lower eukaryote Saccharomyces cerevisiae. Moreover an unique approach is reported to compare identical sequences present on the same chromosome and only differing in expression. We determined the removal of pyrimidine dimers from two identical alpha-mating type loci and we were able to show that the active MAT alpha locus is repaired preferentially to the inactive HML alpha locus. In a sir-3 mutant, in which both loci are active this preference is not observed.     

Possible role of superoxide dismutases in the yeast Saccharomyces cerevisiae under respiratory conditions

We have analyzed the activity of antioxidant and tricarboxylic acid cycle enzymes as well as protein carbonyl content in budding yeast Saccharomyces cerevisiae cells grown in medium with glycerol using wild-strain cells and defective mutants in superoxide dismutases (SODs). The present report demonstrates that the activity of catalase, glucose-6-phosphate dehydrogenase, glutathione reductase, isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase, on average, was lower in the strains lacking SODs than that in the parental strain. On the other hand, under conditions used in this study, the content of carbonyl groups in proteins was relatively higher in the wild type as compared with SOD-defective strains. It may be suggested that in vivo SOD can demonstrate protective as well as pro-oxidant properties, and the final result depends on particular conditions.     

Inorganic polyphosphates and exopolyphosphatases in different cell compartments of Saccharomyces cerevisiae

The cytosol, nuclei, vacuoles, and mitochondria of the yeast Saccharomyces cerevisiae possess inorganic polyphosphates (polyPs). PolyP levels, spectra of polyP chain lengths, and their dependence on the growth phase are distinguished in the mentioned compartments. Inactivation of the PPX1 gene has no effect on the polyP metabolism under cultivation of the yeast in medium with glucose and 5–7 mM P_i. Inactivation of the PPN1 gene results in elimination of the high-molecular-mass exopolyphosphatases (∼120 to 830 kD) of the cytosol, nuclei, vacuoles, and mitochondria of S. cerevisiae suggesting that it is just PPN1 that encodes these enzymes. Expression of the low-molecular-mass exopolyphosphatase of ∼45 kD encoded by the PPX1 gene decreases under PPN1 inactivation as well. While PPN1 inactivation has negligible effect on polyP levels, it results in increase in the long-chain polyPs in all the compartments under study. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 11, pp. 1445–1450.     

Pressure response in the yeast Saccharomyces cerevisiae: from cellular to molecular approaches.

Yeasts are unicellular organisms that are exposed to a highly variable environment, concerning the availability of nutrients, temperature, pH, radiation, access to oxygen and, specially, water activity. Evolution has selected yeasts to tolerate, to a certain extent, these environmental stresses. High hydrostatic pressure (HHP) exerts a broad effect upon yeast cells, interfering with the cell membranes, cellular architecture and in processes ofpolymerisation and denaturation of proteins. Gene expression patterns in response to HHP revealed a stress response profile. The majority of the upregulated genes were involved in stress defence and carbohydrate metabolism while most of the repressed ones were in cell cycle progression and protein synthesis categories. In addition, in the present work it was seen that mild pressure induced cell cycle arrest and protection against severe stresses, such as high temperature, high pressure and ultra cold shock. Nevertheless, this protection was only significant if the cells were incubated at atmospheric pressure after the HHP treatment. Expression of genes that were upregulated by HHP and are related to resistance to this stresses were also analyzed, and, for the majority of them, higher induction was attained after 15 min post-pressurization. Taken together, the results imply an interconnection among stresses.     

Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.     

Increased sensitivity of heat-stressed Saccharomyces cerevisiae cells to food-grade antioxidants

Unheated and heat-stressed Saccharomyces cerevisiae cells were examined for their relative sensitivities to butylated hydroxyanisole (BHA), tertiary butylhydroquinone (TBHQ), and propyl gallate. Heated cells had significant (P less than or equal to 0.05) increases in sensitivity to 50 micrograms of BHA, 100 micrograms of TBHQ, and 1,000 micrograms of propyl gallate per ml as compared with unheated cells when surface plated on antioxidant-supplemented recovery agar. The rate of increase in size of colonies developed by heated cells was slower than that of unheated cells, and the presence of antioxidants in recovery agar enhanced this effect. Heat-stressed cells also had increased sensitivity to ethanol. Incubation temperatures of 15, 21, 30, and 37 degrees C for enumerating unheated cells had no significant effect on the numbers of colonies formed on unsupplemented recovery agar; however, incorporation of 100 micrograms of BHA, 200 micrograms of TBHQ, or 1,000 micrograms of propyl gallate per ml into agar resulted in significant decreases in the number of colonies formed by heated cells at various incubation temperatures. The detrimental effects of TBHQ and propyl gallate on repair of heat-injured cells are apparently expressed at a temperature higher than that observed for BHA. It is suggested that the adverse effects of antioxidants on repair of heat-injured S. cerevisiae cells may be associated with oxygen availability.     

Physiological conditions affecting the sensitivity of Saccharomyces cerevisiae to a Pichia kluyveri killer toxin and energy requirement for toxin action

The interaction between the killer toxin of Pichia kluyveri 1002 and cells of Saccharomyces cerevisiae SCF 1717 is strongly affected by the physiological state of sensitive cells. The killing effect is maximal for cells in the lag and early exponential phase of growth, whereas stationary cells are completely resistant. Furthermore, sensitivity is markedly enhanced by a rise of the pH (from 3.2 to 6.8) at which cells are cultured.Three successive stages can be distinguished in the killing process: (I) binding of the toxin to the primary binding site; (II) transmission of the toxin to its reactive site in the plasma membrane; (III) occurrence of functional damage (K⁺-leakage; decrease of intracellular pH). The transition from stage I to II is prevented in the absence of metabolic energy or at low temperature (below 10°C). Sensitive cells in stage I can be rescued from toxin-induced killing by a short incubation at pH 7.0, which treatment is not effective for cells in stage II. Cells in stage II are able to resume growth when plated in a rich medium containing suitable concentrations of potassium and hydrogen ions. Rescue was not observed for cells in stage III of the killing process.