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.     

Ascorbate and thiol antioxidants abolish sensitivity of yeast Saccharomyces cerevisiae to disulfiram

Sensitivity of baker’s yeast to disulfiram (DSF) and hypersensitivity of a mutant devoid of Cu, Zn-superoxide dismutase to this compound is reported, demonstrating that yeast may be a simple convenient eukaryotic model to study the mechanism of DSF toxicity. DSF was found to induce oxidative stress in yeast cells demonstrated by increased superoxide production and decrease of cellular glutathione content. Anoxic atmosphere and hydrophilic antioxidants (ascorbate, glutathione, dithiothreitol, cysteine, and N-acetylcysteine) ameliorated DSF toxicity to yeast indicating that oxidative stress plays a critical role in the cellular action of DSF.     

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2–40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO₃, in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, ³¹P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.     

Cyclic variations in sensitivity to X-irradiation during meiosis in Saccharomyces cerevisiae

Cyclic variation in mutation induction and lethality was found following X-irradiation during meiosis in Saccharomyces cerevisiae. An enhanced mutagenic response was found in meiotic G1 phase cells in comparison to cells later in meiosis, similar to the response shown during mitosis, but meiotic G1 phase cells appeared more resistant to the lethal effects of X-irradiation than mitotic G1 phase cells. Resistance to the lethal effects of X-rays was found during meiotic DNA synthesis in the strain SK1, which may indicate the operation of a sister-chromatid exchange repair mechanism. A difference was found between gene conversion which appeared to be at a maximum by the end of meiotic DNA synthesis and reciprocal recombination, which could be induced up to prophase I.     

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD⁺-dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.The traditional use of baker''s yeast, Saccharomyces cerevisiae, for ethanol production has resulted in the accumulation of substantial information about its genetics, metabolism, and process development. Consequently, the collection of compounds that are produced using S. cerevisiae has expanded to include organic acids and even secondary metabolites (1, 25, 28). Unlike ethanol, many of these products are not redox neutral relative to commonly used substrates such as glucose. Therefore, in addition to stoichiometry, redox constraints play an important role in the formation of the products. Additional reducing power has to be supplied to produce compounds whose degree of reduction is higher than that of the substrate. On the other hand, producing compounds with a degree of reduction lower than that of the substrate will force the synthesis of other compounds with higher degrees of reduction to compensate for excess reducing power generated from substrate oxidation. These constraints may decrease the product yield substantially.The catabolic currency that balances the degree of reduction between the substrate and the products is usually NADH. In S. cerevisiae, NADH is produced in the cytosol by mainly glyceraldehyde-3-phosphate dehydrogenase and other assimilatory reaction enzymes (35). In the mitochondria, NADH is formed in the tricarboxylic acid (TCA) cycle and the reaction of the pyruvate dehydrogenase complex. Cytosolic NADH is oxidized by the glycerol-3-phosphate shuttle or the external cytosolic NADH dehydrogenases, which are part of the electron transport chain (21). NADH can be transported across the outer mitochondrial membrane (18, 19) but not across the inner mitochondrial membrane (39). Therefore, a dedicated internal mitochondrial NADH dehydrogenase is required to oxidize mitochondrial NADH as part of the electron transport chain (22). The compartmental restriction of NADH oxidation has important ramifications for metabolism and electron transport. The electrons originating from cytosolic NADH are preferred over those originating from mitochondrial NADH (6) for entrance into the electron transport chain. The direct consequence of preferential utilization of cytosolic NADH is a higher redox potential (NADH/NAD⁺) in the mitochondria than in the cytosol. Consequently, during rapid NADH synthesis, as during exponential growth, the TCA cycle ceases to operate as a cycle and branches into oxidative and reducing pathways (12).Metabolic consequences of the compartmentalization of NADH homeostasis were evident from the difference in the product formation profile upon lowering of cytosolic or mitochondrial NADH. Lowering cytosolic NADH by overexpressing bacterial NADH oxidase lowered the production of glycerol and biomass by S. cerevisiae (14, 36). On the other hand, decreasing the mitochondrial NADH level decreased ethanol production and increased the biomass yield (36). These results are likely to be a combination of effects from alleviating the feedback inhibition of the TCA cycle by mitochondrial NADH and increasing respiratory capacity due to improved efficiency of oxidative phosphorylation, as quantified by the P/O ratio (15). There are no reports that describe the effect of increasing NADH in S. cerevisiae, although formate has been used previously as a source of additional reducing power in S. cerevisiae (2, 4, 11, 23, 24, 27). Formate (HCOO⁻) is efficiently oxidized to CO₂ by NAD⁺-dependent formate dehydrogenase (27) and, therefore, cannot be used as a carbon source for biomass synthesis. Thus, using formate as an auxiliary substrate for the generation of NADH to study the effect of increased NADH may be a feasible option. Given the compartment-dependent regulation of NADH homeostasis in S. cerevisiae (36), increasing the NADH level in the cytosol is likely to elicit a response different from that obtained by increasing the NADH level in the mitochondria.The aim of the present study is to differentiate between the metabolic consequences of increasing NADH in the cytosol and those of increasing NADH in the mitochondria of S. cerevisiae. Toward this aim, we either overexpressed the native Fdh1 (NAD⁺-dependent formate dehydrogenase) in the cytosol or directed it into the mitochondria in a strain background that is otherwise devoid of formate metabolism. We present our understanding of the physiological characteristics of the mutant strains under steady-state or dynamic conditions in the presence of different levels of formate.     

Isolation and characteristics of Saccharomyces cerevisiae strains with increased sensitivity to detergents

The yeast mutants possessing enhanced sensitivity to detergents were obtained after treatment with ethyl methanesulfonate. The whole set of mutants may be divided into three groups, according to sensitivity to: cetylthreemethylammonium chloride detergents and dyes of the ethidium bromide type, detergents, dyes and antibiotics (gramycidin C and actinomycin D). The genetic analysis performed indicated that more than one gene are responsible for sensitivity. On the basis of the test for allelism mutants were distributed into three groups. It was shown that ethidium bromide is far more potent inducer of cytoplasmic petites in detergent-sensitive than in wild-type strains.     

Increased DNA double strand breakage is responsible for sensitivity of the pso3-1 mutant of Saccharomyces cerevisiae to hydrogen peroxide

Escherichia coli endonuclease III (endo III) is the key repair enzyme essential for removal of oxidized pyrimidines and abasic sites. Although two homologues of endo III, Ntgl and Ntg2, were found in Saccharomyces cerevisiae, they do not significantly contribute to repair of oxidative DNA damage in vivo. This suggests that an additional activity(ies) or a regulatory pathway(s) involved in cellular response to oxidative DNA damage may exist in yeast. The pso3-1 mutant of S. cerevisiae was previously shown to be specifically sensitive to toxic effects of hydrogen peroxide (H2O2) and paraquat. Here, we show that increased DNA double strand breakage is very likely the basis of sensitivity of the pso3-1 mutant cells to H2O2. Our results, thus, indicate an involvement of the Pso3 protein in protection of yeast cells from oxidative stress presumably through its ability to prevent DNA double strand breakage. Furthermore, complementation of the repair defects of the pso3-1 mutant cells by E. coli endo III has been examined. It has been found that expression of the nth gene in the pso3-1 mutant cells recovers survival, decreases mutability and protects yeast genomic DNA from breakage following H2O2 treatment. This might suggest some degree of functional similarity between Pso3 and Nth.     

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride

Ribonucleotide reductase (RNR) of the yeast Saccharomyces cerevisiae is a tetrameric protein complex, consisting of two large and two small subunits. The small subunits Y2 and Y4 form a heterodimer and are encoded by yeast genes RNR2 and RNR4, respectively. Loss of Y4 in yeast mutant rnr4Delta can be compensated for by up-regulated expression of Y2, and the formation of a small subunit Y2Y2 homodimer that allows for a partially functional RNR. However, rnr4Delta mutants exhibit slower growth than wild-type (WT) cells and are sensitive to many mutagens, amongst them UVC and photo-activated mono- and bi-functional psoralens. Cells of the haploid rnr4Delta mutant also show a 3- to 4-fold higher sensitivity to the oxidative stress-inducing chemical stannous chloride than those of the isogenic WT. Both strains acquired increased resistance to SnCl2 with age of culture, i.e., 24-h cultures were more sensitive than cells grown for 2, 3, 4, and 5 days in liquid culture. However, the sensitivity factor of three to four (WT/mutant) did not change significantly. Cultures of the rnr4Delta mutant in stationary phase of growth always showed higher frequency of budding cells (budding index around 0.5) than those of the corresponding WT (budding index <0.1), pointing to a delay of mitosis/cytokinesis.     

Protective effect of antioxidants against para-nonylphenol-induced inhibition of cell growth in Saccharomyces cerevisiae

The cell growth-modulating activity of an endocrine disruptor, p-nonylphenol (NP), was estimated using the yeast Saccharomyces cerevisiae as a simple model of eukaryotic cells. NP caused a dose-dependent suppressive effect on cell growth of S. cerevisiae at 10, 25 and 50 microM. The NP-induced cell growth inhibition was restored when concomitantly lipophilic antioxidants such as alpha-tocopherol and beta-carotene were supplied, but not the hydrophilic antioxidants ascorbic acid or (-)epigallocatechin gallate (EGCG). The cellular oxygen consumption of S. cerevisiae was also inhibited in a dose-dependent fashion by the extracellular addition of NP, and pretreatment with alpha-tocopherol and beta-carotene suppressed NP-induced inhibition of cellular oxygen consumption, but ascorbic acid and EGCG were not effective. Furthermore, NP caused a marked generation of radical oxygen species (ROS) in S. cerevisiae, which was suppressed by treatment with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG. However, NP did not show a significant inhibitory effect on cell growth and survival of mitochondria-deficient petite mutant cells and they showed a relatively weak ROS-generating activity compared with parent yeast cells. These results suggest that NP-induced inhibition of cell growth and oxygen consumption in S. cerevisiae might be possibly associated with ROS generation in yeast mitochondria. The significance of this finding is discussed from the viewpoint of NP-induced oxidative stress against eukaryotic cells.     

Effects of sterol alterations on nystatin sensitivity in Saccharomyces cerevisiae

A systematic study of the qualitative and quantitative effects of sterol on nystatin sensitivity has been made in a single organism. The use of a sterol auxotroph of Saccharomyces cerevisiae offered a convenient way to control the sterol content of the yeast cell. There was a correlation between the ergosterol content of the cell and sensitivity to nystatin, as monitored by both potassium leakage from the cell and viability. When the sterol auxotroph contained high levels of ergosterol, the cells were sensitive to the effects of nystatin. When the ergosterol content was low or when ergosterol was replaced by cholesterol or cholestanol, sensitivity to nystatin was markedly decreased. Although resistant to nystatin, cholestanol enriched cells showed an enhanced background of potassium ion loss.     

Effect of growth temperature upon heat sensitivity in Saccharomyces cerevisiae

The resistance of exponentially growing yeast cells to killing by exposure to 52°C increased markedly as the growth temperature was increased. Identical killing curves were obtained for cells suspended in growth medium or in 0.9% saline. Cells resistant to killing at 52°C were quite sensitive to killing at slightly higher temperatures. These results suggest a primary role for membrane damage in the mechanism of heat killing.     

Increased inactivation of Saccharomyces cerevisiae by protraction of UV irradiation.

The principle of equi-effectivity of the product of intensity and exposure time (principle of Bunsen-Roscoe) of UV irradiation has been assumed to be valid for the inactivation of microorganisms in general. Earlier studies claimed higher survival of Escherichia coli B/r with fractionated irradiation compared with single-exposure survival. However, data on the inactivation effect of protraction of UV irradiation are not available. By means of a specially designed UV irradiation apparatus which secured absolute UV dose measurements throughout the experiments, the effects of variation of UV irradiation intensities (253.7 nm) and exposure times were tested on the inactivation of a bacterial virus (Staphylococcus aureus phage A994), a vegetative bacterial strain (E. coli ATCC 25922), and bacterial spores (Bacillus subtilis ATCC 6633) as well as three haploid laboratory strains (RC43a, YNN281, and YNN282) and two diploid strains (commercial bakery yeast strain and laboratory strain YNN281 x YNN282) or yeast (Saccharomyces cerevisiae) and spores of the latter diploid yeast strain. Each test organism was exposed to three UV intensities (0.02, 0.2, and 2 W/m2), with corresponding exposure times resulting in three dose levels for each intensity. Differences in inactivation rates were tested by analyses of variance and Newman-Keuls tests. Virus and bacteria showed no differences in inactivation rates by variation of intensities and exposure times within selected UV doses; hence, the principle of Bunsen-Roscoe could not be rejected for these strains. However, in the eukaryotic test strains of S. cerevisiae longer exposure times with lower intensities led to enhanced inactivation in both haploid and diploid strains, with a more pronounced effect in the diploid yeast strains, whereas in yeast spores in this dose rate effect could not be observed.     

Increased production of FAEEs for biodiesel with lipase enhanced Saccharomyces cerevisiae

In this study, we expressed lipase 2 from Candida sp. 99-125 in Saccharomyces cerevisiae, and tried direct biodiesel production. Driven by 3-phosphoglycerate kinase promoter, Lip2 showed high expression level in cytoplasm. SDS-PAGE analysis confirmed the successful lipase expression with a 40 kDa molecular weight. The enzyme assay indicated that lipase 2 had a specific activity of 12.12 μmol/min/mg toward p-nitrophenyl palmitate. Gas chromatography showed that the main fatty acids of S. cerevisiae lipids were palmitoleic acid (31.79%) and oleic acid (29.84%). By three-step addition of 4% ethanol to culture broth, the yield of fatty acid ethyl esters by recombinant S. cerevisiae reached 11.4 mg/g dry cell weight. This work proposed a novel pathway for S. cerevisiae that could be applied for producing biodiesel directly.     

Thiol Peroxidase Deficiency Leads to Increased Mutational Load and Decreased Fitness in Saccharomyces cerevisiae

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.     

Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids

We have found that the medium-chain fatty acids (MCFAs) undecanoic acid (11:0), 10-undecenoic acid (11:1 Delta 10), and lauric acid (12:0) can affect the growth of Saccharomyces cerevisiae in a dose-dependent manner. The principal effect was a longer lag phase in MCFA-containing medium, although higher concentrations of 11:1 Delta 10 inhibited growth. Their relative order of inhibitory action was 11:1 Delta 10>11:0>12:0. Cellular content with MCFA supplementation was dependent on the concentration and the particular species of fatty acid, with 12:0 showing the highest relative accumulation and 11:1 Delta 10 the lowest at all concentrations. We have isolated and characterized a mutant that is hypersensitive to MCFA supplementation and is unable to grow at the normally permissive condition of 1 mM 11:1 Delta 10. However, it does not appear to accumulate higher relative levels of the fed MCFA compared to wild-type cells. Complementation of the mutant revealed that the ERG4 gene, encoding the enzyme that catalyzes the last step in ergosterol biosynthesis, had been mutated. The fatty acid composition of the erg4 Delta mutant differs only slightly from wild-type cells, mainly involving an increase in the relative amount of 12:0. These results indicate that yeast require ergosterol for optimal growth on certain MCFAs. We discuss the role ergosterol may have in cells responding to exogenous MCFAs and in supporting optimal cell growth.     

Spontaneous duplications in diploid Saccharomyces cerevisiae cells

The duplication of DNA sequences is a powerful determinant of genomic plasticity and is known to be one of the key factors responsible for evolution. Recent genomic sequence data demonstrate the abundance of duplicated genes in all surveyed organisms. Over the past years, experimental systems were adequately designed to explore the molecular mechanisms involved in their formation in haploid Saccharomyces cerevisiae strains. To obtain a more global and accurate view of the events leading to DNA sequence duplications, we have selected and characterized duplication occurrences in diploid S. cerevisiae cells. The molecular analysis showed that two other predominant ways lead to duplication in this context: formation of extra chimeric chromosomes and non-reciprocal translocation events. Moreover, we demonstrated that these two types of rearrangements are RAD52 independent and therefore that homologous recombination plays no part in their formation. Finally, our results show the multiplicity of mechanisms involved in duplication events and provide the first experimental evidence that these mechanisms might be ploidy dependent.