Sterol dependent growth and ethanol tolerance of a sterol-auxotrophicerg9::HIS3 mutant ofSaccharomyces cerevisiae

Summary A 3-fold rise in the ergosterol level of growing and stationary cells of theerg9::HIS3 Saccharomyces cerevisiae mutant grown at various concentrations of ergosterol resulted in a higher ethanol tolerance measured as the survival rate at 5 or 15% (V/V) ethanol. A similar protective effect was observed with cholesterol.     

Effect of some aldoses on growth ofSaccharomyces cerevisiae inhibited with molybdenum

The inhibitory effect of molybdenum ions on growth of yeasts at pH 5.5 was found to be decreased by aldoses in the following order: D-talose greater than L-mannose greater than L-ribose greater than D-lyxose greater than L-galactose greater than L-arabinose greater than L-glucose greater than L-xylose. Increased concentrations of molybdenum brought about morphological changes of yeast cells. Cells grown under these conditions were smaller, had thicker walls and formed clusters.     

Temperature-sensitive flocculation during growth ofSaccharomyces cerevisiae

Summary Cell wall surface proteins were extracted from a temperature-sensitive flocculent strain ofSaccharomyces cerevisiae. Electrophoretic analysis identified two protein bands (28 and 43 kDa) present when grown at 21°C. These proteins were initially absent when the strain was grown at 37°C, but intensified after 6 days of growth concomitant with the onset of flocculation.     

Inhibitory action of palmitic acid on the growth ofSaccharomyces cerevisiae

High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.     

Effect of 5,7-unsaturated sterols on ethanol tolerance in Saccharomyces cerevisiae.

Structural membrane lipids are known to contribute to the high ethanol resistance of Saccharomyces cerevisiae (2, 4, 17). By manipulating the yeast cellular sterol level by changing the carbon-to-nitrogen source ratio in the chemostat growth medium, high delta 5,7-sterol levels were found to increase the resistance of yeast populations to ethanol-induced death. The resistance of the erg2 (delta 8----delta 7-sterol isomerase) mutant to ethanol-induced death was generally comparable with that of the delta 5,7-sterol-synthesizing strain. In contrast, the sensitivity of anaerobic growth to inhibition by ethanol was higher in the erg2 mutant in comparison with the delta 5,7-sterol-synthesizing strains but a high level of those sterols increased the vulnerability of anaerobic growth to ethanol inhibition.     

The application of dynamic calorimetry for monitoring growth ofSaccharomyces cerevisiae

Summary A dynamic calorimetric technique was investigated to determine the feasibility of monitoring cell growth by thermal measurements. Theoretical analysis of growth ofSaccharomyces cerevisiae on glucose showed that the correlation depends on cellular yield values but not on ethanol formation. Experiments withS. cerevisiae on a molasses-mineral salts medium resulted in a thermal yield of 4.4 kcal/g cells, consistent with our theoretical expectations.     

The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae

Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy‐demanding growth‐related processes. Studies using genome‐wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial.     

Potassium requirements ofSaccharomyces cerevisiae

The growth rate ofSaccharomyces cerevisiae was dependent on K⁺ content in culture medium in a certain range of K⁺ concentrations. Above the upper limit of the range, growth did not respond to K⁺ increase, and below the lower limit, yeast died. Rb⁺ and Na⁺ enhanced growth in the range of K⁺ dependence and decreased the K⁺ concentration below which cells died. Both Rb⁺ and Na⁺ became toxic above a certain Rb⁺/K⁺ and Na⁺/K⁺ cellular ratio.     

The chitin-glucan complex ofSaccharomyces cerevisiae

Differentiation of the cell wall ofSaccharomyces cerevisiae at the site of the future bud was followed. A lentil-like structure originates on the inner side of the cell wall during the first phase. At the same time, an electron-dense layer occurs at the boundary between the inner layer of the cell wall and the lentil-like structure. During the second phase granular material is accumulated at the lower side of the lentil-like structure. During the third phase the lentil-like structure is split apart due to proliferation of the granular material resulting in formation of the base of the encircling region. The marked electron-dense layer observed from the first phase is attached to the surface of the encircling region during differentiation of the latter. During the budding proper the outer layers of the cell wall protrude and the end of the encircling region, together with the adjacent electron-dense layer, acquire their definitive appearance of rings, observed as marked electron-transparent and electron-dense tears on ultrathin sections.     

Plasma-Membrane lipid composition and ethanol tolerance inSaccharomyces cerevisiae

Populations of cells suspended anaerobically in buffered (pH 4.5) M ethanol remained viable to a greater extent when their plasma membranes were enriched in linoleyl rather than oleyl residues irrespective of the nature of the sterol enrichment. However, populations with membranes enriched in ergosterol or stigmasterol and linoleyl residues were more resistant to ethanol than populations enriched in campesterol or cholesterol and linoleyl residues. Populations enriched in ergosterol and cetoleic acid lost viability at about the same rate as those enriched in oleyl residues, while populations grown in the presence of this sterol and palmitoleic acid were more resistant to ethanol. Suspending cells in buffered ethanol for up to 24 h did not lower the ethanol concentration.     

The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii

Summary The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii, at different times after feeding, has been examined by cytochemical techniques at electron microscope level and by measurement of yeast viability. The measurement of viability, combined with cytochemistry is presented as a novel method of examining the progress of digestion. Particular attention has been given to the temporal development of digestion.Vacuoles, probably primary lysosomes, have been identified containing acid phosphatase activity within minutes of feeding and these accumulate around and fuse with phagocytic vacuoles. Acid phosphatase levels in the digestive vacuoles appeared highest at 20 to 40 minutes. Yeast digestion was observed and yeast viability began to decline at this time. Mixing of autophagic and heterophagic material was also observed. At least half of the yeast population was still viable after 90 minutes.Our method (p-nitrophenyl phosphate) of enzyme localization has demonstrated plasma membrane associated acid phosphatase activity.     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

Genetic aspects of ethanol tolerance and production bySaccharomyces cerevisiae

We developed a method of hybrid selection between homothallic wild-type and heterothallic strains. The hybrids obtained were used to study the heredity of ethanol tolerance and production. Both characters segregated independently, but no ethanol-sensitive strains were able to produce high levels of ethanol. At least four genes are implicated in ethanol tolerance.     

Freeze-drying characteristics ofSaccharomyces cerevisiae andSaccharomyces carlsbergensis

The dispersal of yeast clumps to the unicellular state by certain sugars, does not increase the percentage survival after freeze-drying. Neither are those changes in cell-wall composition which occur upon ageing of the cell, and which are detectable by means of snail-gut enzymes, related to this cellular property. However, pre-treatment, with -mercaptoethanol, of a strain ofSaccharomyces carisbergensis increased the survival rate. This may be due to the reduction of certain sites in the cell wall. The oxygen consumption of yeast cultures before and after freeze-drying, agree with the hypothesis that low viabilities can arise from localized cellular damage which prevents cell reproduction by budding.     

Limited proteolysis ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Incubation ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments. Cleavage sites determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and sequence analyses identified protease-sensitive peptide bonds between amino acid residues at positions 9–10 and 76–77. Additional fragmentation sites were also detected in a region approximately 70–80 amino acids before the carboxyl end of the protein. These results suggest that the enzyme is formed by a central compact domain comprising more than two thirds of the whole protein structure. From proteolysis experiments carried out in the presence of substrates, it could be inferred that CO₂ binding specifically protects position 76–77 from trypsin action. Intrinsic fluorescence measurements demonstrated that CO₂ binding induces a protein conformational change, and a dissociation constant for the enzyme CO₂ complex of 8.2±0.6 mM was determined     

Transcriptional changes associated with ethanol tolerance in Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanol production; however, fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, Saccharomyces cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently: SM1 by adaptive evolution involving long-term exposure to ethanol stress and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of gene ontology categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and other energy-yielding pathways. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD⁺/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation.