Influence of stressful fermentation conditions on neutral lipids of a Saccharomyces cerevisiae brewing strain

The neutral lipid fraction of the aerobically grown starter yeast culture of a Saccharomyces cerevisiae brewing strain, and three-first recycled yeast generations exposed to multiple stress factors during beer fermentation was studied. No pronounced changes in the cellular neutral lipid content between the non-stressed starter and stressed recycled cells were found. However, it was found that recycled yeast generations modulate their neutral lipid composition during fermentation. The ergosterol content was increased at the expense of steryl esters (SEs) and squalene, which resulted in a higher ergosterol/SEs molar ratio and a slightly higher ergosterol/squalene molar ratio. In addition, the proportion of unsaturated fatty acids, mainly palmitoleic acid increased in the neutral lipid fraction of the stressed recycled yeast generations. These results suggest that some specific neutral lipid species and fatty acids stored in the neutral lipid fraction are involved in the adaptive response of the brewer’s yeast to stressful fermentation conditions. The striking finding was a high squalene content in the neutral lipid fraction of both the starter yeast culture and recycled yeast generations (22.4 vs. 19–20%, respectively), implying a possible biotechnological exploitation of this biologically active molecule from the yeast biomass.     

Cadmium accumulation and its effects on intracellular ion pools in a brewing strain of Saccharomyces cerevisiae

The presence of glucose resulted in a two- to three-fold increase in levels of Cd²⁺accumulated by Saccharomyces cerevisiae after 5 h compared with those observed in the absence of glucose. However, time-dependent Cd²⁺ uptake continued in the absence of glucose over 5 h, resulting in an appreciable increase in cellular Cd²⁺levels. Substantial K⁺ efflux but little Mg²⁺ and negligible Ca²⁺release was observed. Cell fractionation revealed that the bulk of intracellular Cd²⁺ was located in the vacuolar (25%) and bound (60%) fractions. Accumulation of Cd²⁺ ions impacted most noticeably on K⁺ rather than Mg²⁺ levels in intracellular compartments. Cytoplasmic and particularly vacuolar K⁺ levels decreased as Cd²⁺ sequestration continued resulting in increased extracellular levels. In contrast, corresponding intracellular Mg²⁺ pools were only modestly affected with a slight increase and decrease observed in the cytoplasmic and vacuolar fractions respectively. However, levels of bound Mg²⁺ decreased in response to continued Cd²⁺ accumulation. Received 07 March 1999/ Accepted in revised form 26 June 1999     

The role of fructose 2,6-bisphosphate in glycolytic oscillations in extracts and cells of Saccharomyces cerevisiae

Fructose 2,6-bisphosphate is physiologically one of the most potent activators of yeast 6-phosphofructo-1-kinase. The glycolytic oscillation observed in cell-free cytoplasmic extracts of the yeast Saccharomyces cerevisiae responds to the addition of fructose 2,6-bisphosphate in micromolar concentrations by showing a pronounced decrease of both the amplitude and the period. The oscillations can be suppressed completely by 10 microM and above of this activator but recovers almost fully (95%) to the unperturbed state after 3 h. Fructose 2,6-bisphosphate shifts the phases of the oscillations by a maximal +/- 60 degrees. Oscillations in concentration of endogenous fructose 2,6-bisphosphate in the extract were also observed. Fructose 2,6-bisphosphate alters the dynamic properties of 6-phosphofructo-1-kinase which are vital for its role as the 'oscillophore'. However, the minute amount (approximately 0.3 microM) of endogenous fructose 2,6-bisphosphate and the phase relationship of its oscillations compared with other metabolites indicate that this activator is not an essential component of the oscillatory mechanism. Further support for this conclusion is the observation of sustained oscillations in both the extracts and a population of intact cells of a mutant strain (YFA) of S. cerevisiae with no detectable fructose 2,6-bisphosphate (less than 5 nM).     

Spheroplast fusion of a brewing yeast strain with Saccharomyces diastaticus

Summary One haploid and one diploid strain of Saccharomyces diastaticus carrying genes responsible for glucoamylase synthesis were fused with a brewing polyploid Saccharomyces uvarum lager strain. With the spheroplast fusion technique, the ability to use dextrin and starch was introduced in the brewing yeast. Spheroplasts of the strains to be used were obtained by enzymatic digestion of the cell walls. Fusion took place in polyethylene glycol; complete cells were then regenerated in hypertonic medium containing 3% agar at 37°C. In the first fusion experiment melibiose was used as carbon source; in the second fusion experiment glycerol was employed as carbon source, for the parental Saccharomyces diastaticus diploid strain was a petite mutant. Fusion products were capable of utilizing melibiose and dextrin as carbon sources.     

Role of bestatin-sensitive exopeptidases in the intracellular degradation of hepatic proteins

Injection of bestatin into intact mice produces accumulation of di- and tripeptide intermediates in the degradation of short- and long-lived hepatic proteins, whereas lysosomal breakdown of endocytosed plasma asialoglycoproteins is not affected. The majority of the peptides are found in the liver cytosol, but a minor portion appears in a sedimentable fraction containing mitochondria and lysosomes (Botbol, V., and Scornik, O. A. (1983) J. Biol. Chem. 258, 1942-1949). We now report that (a) the primary location of the intermediates is the cytosol. The particulate fraction represents cytosolic peptides trapped within mitochondria, as evidenced by sedimentation equilibrium in sucrose gradients after loading lysosomes with Triton WR1339 and by the sensitivity of the particles to lysis by digitonin. (b) In isolated hepatocytes, where we can measure simultaneously protein breakdown and bestatin-induced peptides, the accumulation of intermediates parallels protein degradation of analog-containing, short- and long-lived proteins, even after stimulation of the latter by amino acid deprivation. These observations are consistent with the hypothesis that bestatin inhibits cytosolic exopeptidases that complete the intracellular breakdown to amino acids of the major classes of hepatic proteins. The role of cytosolic exopeptidases is expected in the rapid degradation of abnormal proteins, a demonstrated cytosolic process. In stimulated degradation of long-lived proteins, the importance of cytosolic exopeptidases implies either that this process is largely cytosolic or, more likely, that peptides escape from autophagic organelles.     

Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae.

Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain.     

Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae.

Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain.     

The essential and ancillary role of glutathione in Saccharomyces cerevisiae analysed using a grande gsh1 disruptant strain

A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain. This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome. The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation. The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound. GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells. This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth. This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress. Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis. These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress. Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant. Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.     

Isolation and improvement of a thermotolerant Saccharomyces cerevisiae strain

The ambient temperature is a drawback in industrial ethanol production in Jaffna due to heat killing of yeast during fermentation. Thus a search was initiated for thermotolerant organisms suitable for fermentation in hot climates. The screening of the best wild-type organisms was undertaken as the first step. Thermotolerant strains were selected from environments where there are chances of organisms being exposed to high temperature. The samples were enriched and screened for thermotolerant organisms which survived at 45 °C for 15 h. Among the yeast strains selected from different sources, thermotolerant strains with the capacity to withstand 45 °C for 15 h were found in samples collected from the compost heap and distillery environments. Three colonies from the distillery environment were selected for further studies and named p1, p2 and p3. Exponential phase (18 h) cultures of p1, p2 and p3 were subjected to 15 temperature treatment cycles (at 50 °C each for 3 h) and thermally adapted strains pt1, pt2 and pt3 were obtained, showing 100, 30 and 20% viability at 50 °C for 30 min respectively. The initial round of thermal adaptation cycles increased the duration of 100% viability from 20 h (p1) to 68 h (pt1) when incubated at 40 °C. Very little benefit was obtained when pt1 was treated with u.v. and ethyl methanesulphonate. The selected strain was identified and designated as Saccharomyces cerevisiae S1. The ethanol produced from 100 g glucose l^–1 by S. cerevisiae S1 was 46 g l^–1 (36 h), 38 g l^–1 (48 h) and 26 g l^–1 (48 h) at 40, 43 and 45 °C respectively in rich nutrient medium.     

Characterization of a Dio-9-resistant strain of Saccharomyces cerevisiae.

The ATPase inhibitor Dio-9 effectively suppressed a number of physiological processes in a wild-type strain of Saccharomyces cerevisiae, X2180-1A. Low levels of the antibiotic inhibited cell growth, amino acid transport, hydrogen ion efflux, and ATPase activity. In addition, Dio-9 acted as a permeabilizing agent for the yeast plasma membrane. A mutant yeast strain, XC24, was selected on the basis of its ability to grow on minimal medium containing 200 μg/ml of Dio-9. Strain XC24 had acquired a pH-conditional ability to resist the permeabilizing effects of Dio-9. In addition, amino acid transport and hydrogen ion pumping exhibited a reduced senstivity to Dio-9 at low pH in the mutant strain. Strain XC24 was also resistant to the permeabilizing effects of the basic polymers protamine and deacylated chitin.     

The role of glutamate in modulating the synthesis and degradation of NADP-glutamate dehydrogenase from Saccharomyces cerevisiae

Abstract NADP-glutamate dehydrogenase (NADP-GDH) from Saccharomyces cerevisiae has a lower activity in yeast grown on glutamate as nitrogen source than when grown on ammonium. With the use of the immunotitration method, it was found that the difference in activity was parallel to the difference in immunoprecipitable material. By isotope incorporation studies, it was established that the decrease in NADP-glutamate dehydrogenase levels in glutamate-grown cells was brought about by an increase in the degradation rate and a decrease in the synthesis constant of the enzyme. The degradation rate of NADP-glutamate dehydrogenase is further increased in carbon-starved cells. The possible role of internal metabolites in modulating NADP-glutamate dehydrogenase degradation is discussed.     

The role of MEN (mitosis exit network) proteins in the cytokinesis of Saccharomyces cerevisiae.

At the latest stages of their cell cycle, cells carry out crucial processes for the correct segregation of their genetic and cytoplasmic material. In this work, we provide evidence demonstrating that the cell cycle arrest of some MEN (mitosis exit network) mutants in the anaphase-telophase transition is bypassed. In addition, the ability of cdc15 diploid mutant strains to develop non-septated chains of cells, supported by nuclear division, is shown. This phenotype is also displayed by haploid cdc15 mutant strains when cell lysis is prevented by osmotic protection, and shared by other MEN mutants. By contrast, anaphase-telophase arrest is strictly observed in double MEN-FEAR (fourteen early anaphase release) mutants. In this context, the overexpression of a FEAR component, SPO12, in a MEN mutant background enhances the ability of MEN mutants to bypass cell cycle arrest. Taken together, these data suggest a critical role of Cdc15 and other MEN proteins in cytokinesis, allowing a new model for their cellular function to be proposed.     

Base mismatch-specific endonuclease activity in extracts from Saccharomyces cerevisiae.

An endonuclease activity (called MS-nicking) for all possible base mismatches has been detected in the extracts of yeast, Saccharomyces cerevisiae. DNAs with twelve possible base mismatches at one defined position are cleaved at different efficiencies. DNA fragments with A/G, G/A, T/G, G/T, G/G, or A/A mismatches are nicked with greater efficiencies than C/T, T/C, C/A, and C/C. DNA with an A/C or T/T mismatch is nicked with an intermediate efficiency. The MS-nicking is only on one particular DNA strand, and this strand disparity is not controlled by methylation, strand break, or nature of the mismatch. The nicks have been mapped at 2-3 places at second, third, and fourth phosphodiester bonds 5' to the mispaired base; from the time course study, the fourth phosphodiester bond probably is the primary incision site. This activity may be involved in mismatch repair during genetic recombination.     

Ribosome activity and degradation in meiotic cells of Saccharomyces cerevisiae.

Summary The acetamidase of Aspergillus nidulans is induced by sources of acetyl CoA, benzoate and benzamide and by -alanine and other -amino acids. The effects of these groups of inducers are approximately additive. The cis-acting control site mutant, amdI9, affects induction by sources of acetyl-CoA specifically. Lesions in the amdR and gatA genes affect induction by -amino acids specifically. Mutations in the amdA gene can lead to elevated acetamidase levels which still respond to the various inducers. The induction controls act independently of repression control by nitrogen metabolites and are not altered by the areA102 mutation. The properties of double mutants with lesions affecting the different control mechanisms also indicate their independence of each other. It is suggested that the acetamidase is subject to complex control by multiple regulatory circuits and that functionally independent control sites adjacent to the structural gene occur.     

Molecular characterization of new natural hybrids of Saccharomyces cerevisiae and S. kudriavzevii in brewing

We analyzed 24 beer strains from different origins by using PCR-restriction fragment length polymorphism analysis of different gene regions, and six new Saccharomyces cerevisiae x Saccharomyces kudriavzevii hybrid strains were found. This is the first time that the presence in brewing of this new type of hybrid has been demonstrated. From the comparative molecular analysis of these natural hybrids with respect to those described in wines, it can be concluded that these originated from at least two hybridization events and that some brewing hybrids share a common origin with wine hybrids. Finally, a reduction of the S. kudriavzevii fraction of the hybrid genomes was observed, but this reduction was found to vary among hybrids regardless of the source of isolation. The fact that 25% of the strains analyzed were discovered to be S. cerevisiae x S. kudriavzevii hybrids suggests that an important fraction of brewing strains classified as S. cerevisiae may correspond to hybrids, contributing to the complexity of Saccharomyces diversity in brewing environments. The present study raises new questions about the prevalence of these new hybrids in brewing as well as their contribution to the properties of the final product.