Science.gov

Sample records for saccharomyces cerevisiae determines

  1. Yeast (Saccharomyces cerevisiae).

    PubMed

    Hooykaas, Paul J J; den Dulk-Ras, Amke; Bundock, Paul; Soltani, Jalal; van Attikum, Haico; van Heusden, G Paul H

    2006-01-01

    The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic organisms. This species has enabled a detailed study of the (genetic) requirements for Agrobacterium-mediated DNA transformation. For instance research with this yeast has led to the recognition that the transforming DNA molecules integrate into the eukaryotic chromosomes either by homologous recombination, which is the preferred pathway in S. cerevisiae, or by nonhomologous end-joining. Based on the protocol for Agrobacterium-mediated transformation of S. cerevisiae methodology has been developed for the transformation of many other yeast and fungal species.

  2. The toxic potential of an industrial effluent determined with the Saccharomyces cerevisiae-based assay.

    PubMed

    Schmitt, Marcel; Gellert, Georg; Lichtenberg-Fraté, Hella

    2005-09-01

    Increasing levels of environmental pollution and the continuous monitoring of water quality both request specific and sensitive methods for the detection of detrimental water contents. On a regulatory basis genotoxicity is assessed by the standard umu-test (ISO 13829) that responds to DNA damage induced by chemicals. The focus of this study was the examination of the toxic potential of samples taken from the wastewater treatment plant of a refinery factory to explore the applicability of the Saccharomyces cerevisiae (bakers yeast) test for the detection of bio-available genotoxic activity in complex matrices. The toxic potential of samples without pre-treatment and following centrifugation was determined with the eukaryotic Saccharomyces cerevisiae bioassay based on the transcriptional activation of the green fluorescent protein (gfp) fused to the DNA damage inducible RAD54 promoter and general growth inhibition. Primary effluent samples were taken as qualified sterile spot samples from the final effluent of the purification plant. The Saccharomyces cerevisiae assay yielded geno- and cytotoxic responses in all complex untreated and centrifuged samples with high reproducibility. The obtained results suggest that the yeast assay is suited as a screening tool to monitor genotoxic potential of wastewater.

  3. Saccharomyces cerevisiae Shuttle vectors.

    PubMed

    Gnügge, Robert; Rudolf, Fabian

    2017-01-10

    Yeast shuttle vectors are indispensable tools in yeast research. They enable cloning of defined DNA sequences in Escherichia coli and their direct transfer into Saccharomyces cerevisiae cells. There are three types of commonly used yeast shuttle vectors: centromeric plasmids, episomal plasmids and integrating plasmids. In this review, we discuss the different plasmid systems and their characteristic features. We focus on their segregational stability and copy number and indicate how to modify these properties. Copyright © 2017 John Wiley & Sons, Ltd.

  4. Determination of biological activity from fluorescence-lifetime measurements in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Rudek, F.; Baselt, T.; Lempe, B.; Taudt, C.; Hartmann, P.

    2015-03-01

    The importance of fluorescence lifetime measurement as an optical analysis tool is growing. Many applications already exist in order to determine the fluorescence lifetime, but the majority of these require the addition of fluorescence-active substances to enable measurements. Every usage of such foreign materials has an associated risk. This paper investigates the use of auto-fluorescing substances in Saccharomyces cerevisiae (Baker's yeast) as a risk free alternative to fluorescence-active substance enabled measurements. The experimental setup uses a nitrogen laser with a pulse length of 350 ps and a wavelength of 337 nm. The excited sample emits light due to fluorescence of NADH/NADPH and collagen. A fast photodiode collects the light at the output of an appropriate high-pass edge-filter at 400 nm. Fluorescence lifetimes can be determined from the decay of the measurement signals, which in turn characterizes the individual materials and their surrounding environment. Information about the quantity of the fluorescence active substances can also be measured based on the received signal intensity. The correlation between the fluorescence lifetime and the metabolic state of Saccharomyces cerevisiae was investigated and is presented here.

  5. Transcriptome structure variability in Saccharomyces cerevisiae strains determined with a newly developed assembly software.

    PubMed

    Sardu, Alessandro; Treu, Laura; Campanaro, Stefano

    2014-12-01

    RNA-seq studies have an important role for both large-scale analysis of gene expression and for transcriptome reconstruction. However, the lack of software specifically developed for the analysis of the transcriptome structure in lower eukaryotes, has so far limited the comparative studies among different species and strains. In order to fill this gap, an innovative software called ORA (Overlapped Reads Assembler) was developed. This software allows a simple and reliable analysis of the transcriptome structure in organisms with a low number of introns. It can also determine the size and the position of the untranslated regions (UTR) and of polycistronic transcripts. As a case study, we analyzed the transcriptional landscape of six S. cerevisiae strains in two different key steps of the fermentation process. This comparative analysis revealed differences in the UTR regions of transcripts. By extending the transcriptome analysis to yeast species belonging to the Saccharomyces genus, it was possible to examine the conservation level of unknown non-coding RNAs and their putative functional role. By comparing the results obtained using ORA with previous studies and with the transcriptome structure determined with other software, it was proven that ORA has a remarkable reliability. The results obtained from the training set made it possible to detect the presence of transcripts with variable UTRs between S. cerevisiae strains. Finally, we propose a regulatory role for some non-coding transcripts conserved within the Saccharomyces genus and localized in the antisense strand to genes involved in meiosis and cell wall biosynthesis.

  6. Saccharomyces cerevisiae aldolase mutants.

    PubMed Central

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo. PMID:6384192

  7. Diversification of Transcriptional Regulation Determines Subfunctionalization of Paralogous Branched Chain Aminotransferases in the Yeast Saccharomyces cerevisiae.

    PubMed

    González, James; López, Geovani; Argueta, Stefany; Escalera-Fanjul, Ximena; El Hafidi, Mohamed; Campero-Basaldua, Carlos; Strauss, Joseph; Riego-Ruiz, Lina; González, Alicia

    2017-09-14

    Saccharomyces cerevisiae harbors BAT1 and BAT2 paralogous genes encoding branched chain aminotransferases (BCATs), showing opposed expression profiles and physiological role. Accordingly, in primary nitrogen sources such as glutamine, BAT1 expression is induced, supporting Bat1-dependent valine-isoleucine-leucine (VIL) biosynthesis, while BAT2 expression is repressed. Conversely, in the presence of VIL as sole nitrogen source, BAT1 expression is hindered while that of BAT2 is activated resulting in Bat2-dependent VIL catabolism. Presented results confirm that BAT1 expression is determined by transcriptional activation through the action of the Leu3-α-IPM active isoform, and uncovers the existence of a novel α-IPM biosynthetic pathway operating in a put3Δ mutant grown on VIL, through Bat2-Leu2-Leu1 consecutive action. The classic α-IPM biosynthetic route operates in glutamine through the action of the leucine sensitive α-isopropylmalate synthases (α-IPMS). Presented results also show that BAT2 repression in glutamine can be alleviated in an ure2Δ mutant or through Gcn4-dependent transcriptional activation. Thus, when S. cerevisiae is grown on glutamine, VIL biosynthesis is predominant and is preferentially achieved through BAT1, while on VIL as sole nitrogen source, catabolism prevails and is mainly afforded by BAT2. Copyright © 2017, Genetics.

  8. PET genes of Saccharomyces cerevisiae.

    PubMed Central

    Tzagoloff, A; Dieckmann, C L

    1990-01-01

    We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae. PMID:2215420

  9. Nucleosome Positioning in Saccharomyces cerevisiae

    PubMed Central

    Jansen, An; Verstrepen, Kevin J.

    2011-01-01

    Summary: The DNA of eukaryotic cells is spooled around large histone protein complexes, forming nucleosomes that make up the basis for a high-order packaging structure called chromatin. Compared to naked DNA, nucleosomal DNA is less accessible to regulatory proteins and regulatory processes. The exact positions of nucleosomes therefore influence several cellular processes, including gene expression, chromosome segregation, recombination, replication, and DNA repair. Here, we review recent technological advances enabling the genome-wide mapping of nucleosome positions in the model eukaryote Saccharomyces cerevisiae. We discuss the various parameters that determine nucleosome positioning in vivo, including cis factors like AT content, variable tandem repeats, and poly(dA:dT) tracts that function as chromatin barriers and trans factors such as chromatin remodeling complexes, transcription factors, histone-modifying enzymes, and RNA polymerases. In the last section, we review the biological role of chromatin in gene transcription, the evolution of gene regulation, and epigenetic phenomena. PMID:21646431

  10. Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance

    NASA Technical Reports Server (NTRS)

    D'Elia, Riccardo; Allen, Patricia L.; Johanson, Kelly; Nickerson, Cheryl A.; Hammond, Timothy G.

    2005-01-01

    This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

  11. Haploid deletion strains of Saccharomyces cerevisiae that determine survival during space flight

    NASA Astrophysics Data System (ADS)

    Johanson, Kelly; Allen, Patricia L.; Gonzalez-Villalobos, Romer A.; Nesbit, Jacqueline; Nickerson, Cheryl A.; Höner zu Bentrup, Kerstin; Wilson, James W.; Ramamurthy, Rajee; D'Elia, Riccardo; Muse, Kenneth E.; Hammond, Jeffrey; Freeman, Jake; Stodieck, Louis S.; Hammond, Timothy G.

    2007-02-01

    This study identifies genes that determine survival during a space flight, using the model eukaryotic organism, Saccharomyces cerevisiae. Select strains of a haploid yeast deletion series grew during storage in distilled water in space, but not in ground based static or clinorotation controls. The survival advantages in space in distilled water include a 133-fold advantage for the deletion of PEX19, a chaperone and import receptor for newly- synthesized class I peroxisomal membrane proteins, to 77-40 fold for deletion strains lacking elements of aerobic respiration, isocitrate metabolism, and mitochondrial electron transport. Following automated addition of rich growth media, the space flight was associated with a marked survival advantage of strains with deletions in catalytically active genes including hydrolases, oxidoreductases and transferases. When compared to static controls, space flight was associated with a marked survival disadvantage of deletion strains lacking transporter, antioxidant and catalytic activity. This study identifies yeast deletion strains with a survival advantage during storage in distilled water and space flight, and amplifies our understanding of the genes critical for survival in space.

  12. Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance

    NASA Technical Reports Server (NTRS)

    D'Elia, Riccardo; Allen, Patricia L.; Johanson, Kelly; Nickerson, Cheryl A.; Hammond, Timothy G.

    2005-01-01

    This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

  13. Cellular effects and epistasis among three determinants of adaptation in experimental populations of Saccharomyces cerevisiae.

    PubMed

    Parreiras, Lucas S; Kohn, Linda M; Anderson, James B

    2011-10-01

    Epistatic interactions in which the phenotypic effect of an allele is conditional on its genetic background have been shown to play a central part in various evolutionary processes. In a previous study (J. B. Anderson et al., Curr. Biol. 20:1383-1388, 2010; J. R. Dettman, C. Sirjusingh, L. M. Kohn, and J. B. Anderson, Nature 447:585-588, 2007), beginning with a common ancestor, we identified three determinants of fitness as mutant alleles (each designated with the letter "e") that arose in replicate Saccharomyces cerevisiae populations propagated in two different environments, a low-glucose and a high-salt environment. In a low-glucose environment, MDS3e and MKT1e interacted positively to confer a fitness advantage. Also, PMA1e from a high-salt environment interacted negatively with MKT1e in a low-glucose environment, an example of a Dobzhansky-Muller incompatibility that confers reproductive isolation. Here we showed that the negative interaction between PMA1e and MKT1e is mediated by alterations in intracellular pH, while the positive interaction between MDS3e and MKT1e is mediated by changes in gene expression affecting glucose transporter genes. We specifically addressed the evolutionary significance of the positive interaction by showing that the presence of the MDS3 mutation is a necessary condition for the spread and fixation of the new mutations at the identical site in MKT1. The expected mutations in MKT1 rose to high frequencies in two of three experimental populations carrying MDS3e but not in any of three populations carrying the ancestral allele. These data show how positive and negative epistasis can contribute to adaptation and reproductive isolation.

  14. The rate of metabolism as a factor determining longevity of the Saccharomyces cerevisiae yeast.

    PubMed

    Molon, Mateusz; Szajwaj, Monika; Tchorzewski, Marek; Skoczowski, Andrzej; Niewiadomska, Ewa; Zadrag-Tecza, Renata

    2016-02-01

    Despite many controversies, the yeast Saccharomyces cerevisiae continues to be used as a model organism for the study of aging. Numerous theories and hypotheses have been created for several decades, yet basic mechanisms of aging have remained unclear. Therefore, the principal aim of this work is to propose a possible mechanism leading to increased longevity in yeast. In this paper, we suggest for the first time that there is a link between decreased metabolic activity, fertility and longevity expressed as time of life in yeast. Determination of reproductive potential and total lifespan with the use of fob1Δ and sfp1Δ mutants allows us to compare the "longevity" presented as the number of produced daughters with the longevity expressed as the time of life. The results of analyses presented in this paper suggest the need for a change in the definition of longevity of yeast by taking into consideration the time parameter. The mutants that have been described as "long-lived" in the literature, such as the fob1Δ mutant, have an increased reproductive potential but live no longer than their standard counterparts. On the other hand, the sfp1Δ mutant and the wild-type strain produce a similar number of daughter cells, but the former lives much longer. Our results demonstrate a correlation between the decreased efficiency of the translational apparatus and the longevity of the sfp1Δ mutant. We suggest that a possible factor regulating the lifespan is the rate of cell metabolism. To measure the basic metabolism of the yeast cells, we used the isothermal microcalorimetry method. In the case of sfp1Δ, the flow of energy, ATP concentration, polysome profile and translational fitness are significantly lower in comparison with the wild-type strain and the fob1Δ mutant.

  15. Examining the Role of Membrane Lipid Composition in Determining the Ethanol Tolerance of Saccharomyces cerevisiae

    PubMed Central

    Henderson, Clark M.

    2014-01-01

    Yeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance in Saccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated. PMID:24610851

  16. Examining the role of membrane lipid composition in determining the ethanol tolerance of Saccharomyces cerevisiae.

    PubMed

    Henderson, Clark M; Block, David E

    2014-05-01

    Yeast (Saccharomyces cerevisiae) has an innate ability to withstand high levels of ethanol that would prove lethal to or severely impair the physiology of other organisms. Significant efforts have been undertaken to elucidate the biochemical and biophysical mechanisms of how ethanol interacts with lipid bilayers and cellular membranes. This research has implicated the yeast cellular membrane as the primary target of the toxic effects of ethanol. Analysis of model membrane systems exposed to ethanol has demonstrated ethanol's perturbing effect on lipid bilayers, and altering the lipid composition of these model bilayers can mitigate the effect of ethanol. In addition, cell membrane composition has been correlated with the ethanol tolerance of yeast cells. However, the physical phenomena behind this correlation are likely to be complex. Previous work based on often divergent experimental conditions and time-consuming low-resolution methodologies that limit large-scale analysis of yeast fermentations has fallen short of revealing shared mechanisms of alcohol tolerance in Saccharomyces cerevisiae. Lipidomics, a modern mass spectrometry-based approach to analyze the complex physiological regulation of lipid composition in yeast and other organisms, has helped to uncover potential mechanisms for alcohol tolerance in yeast. Recent experimental work utilizing lipidomics methodologies has provided a more detailed molecular picture of the relationship between lipid composition and ethanol tolerance. While it has become clear that the yeast cell membrane composition affects its ability to tolerate ethanol, the molecular mechanisms of yeast alcohol tolerance remain to be elucidated.

  17. Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Ostergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

    2000-01-01

    Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production. PMID:10704473

  18. Genetic determinants of mitochondrial response to arsenic in yeast Saccharomyces cerevisiae.

    PubMed

    Vujcic, Marija; Shroff, Meghna; Singh, Keshav K

    2007-10-15

    We have used yeast Saccharomyces cerevisiae as a tool to identify the importance of mitochondrial processes involved in arsenic-induced carcinogenicity in humans. We screened 466 single-gene knockout strains of yeast S. cerevisiae known to be involved in biogenesis of mitochondria for sodium arsenite (AsIII) and sodium arsenate (AsV) sensitivity. We identified 72 arsenite-sensitive and 81 arsenate-sensitive mutants. We categorized the identified mutants based on the various mitochondrial processes, including nucleic acid metabolism, oxidative phosphorylation, protein synthesis, and vacuolar acidification. We have identified 65 human orthologues to proteins involved in arsenite sensitivity and 3 human orthologues to arsenite resistance. Furthermore, 23 human orthologues to arsenate sensitivity and 20 human orthologues to arsenate-resistant proteins, including MSH3, COX10, GCSH, PPOX, and MTHFD1, were also identified. Using PathwayAssist software, we did cellular network analysis between identified mitochondrial proteins. Three types of interactions, (a) protein-protein interactions, (b) common transcriptional regulators, and (c) common target genes, were identified. We found that RTG (retrograde) genes involved in mitochondria-to-nucleus signaling regulate both arsenite sensitivity and resistance. Furthermore, our study revealed that ABF1, a multifunctional transcriptional factor, regulates genes involved in both arsenite and arsenate sensitivity and resistance. However, REB1 and RAP1 transcriptional regulators were common to only arsenate- and arsenite-sensitive genes, respectively. These studies indicate that multiple pathways involved in mitochondrial biogenesis protect yeast S. cerevisiae from arsenic-induced toxicity. Together, our studies suggest that evolutionary conserved mitochondrial networks identified in yeast S. cerevisiae must play an important role in arsenic-induced carcinogenesis in humans.

  19. Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae.

    PubMed

    Gonzalez-Ramos, Daniel; Gonzalez, Ramon

    2006-12-13

    Cell wall mannoproteins released by Saccharomyces cerevisiae during wine fermentation and aging have recently attracted the attention of enologists and researchers in enology due to their positive effect over a number of technological and quality properties of the wines, including protein and tartaric stability, aroma and color stability, astringency, mouthfeel, malolactic fermentation, or foam properties of sparkling wines. This work has investigated the effect of deletions involving genes related to cell wall biogenesis on the release of mannoproteins, as well as the effect of the released mannoproteins on wine protein stability. When available, the phenotypes have been studied in different genetic backgrounds, in haploid or diploid strains, and in homo- or heterozygosis. Strains deleted for GAS1, GPI7, or KNR4 release higher amounts of mannoproteins and polysaccharides to the medium. These increased amounts of mannoproteins and polysaccharides lead to a stronger stability of Sauvignon Blanc wines against protein haze.

  20. Pyruvate metabolism in Saccharomyces cerevisiae.

    PubMed

    Pronk, J T; Yde Steensma, H; Van Dijken, J P

    1996-12-01

    In yeasts, pyruvate is located at a major junction of assimilatory and dissimilatory reactions as well as at the branch-point between respiratory dissimilation of sugars and alcoholic fermentation. This review deals with the enzymology, physiological function and regulation of three key reactions occurring at the pyruvate branch-point in the yeast Saccharomyces cerevisiae: (i) the direct oxidative decarboxylation of pyruvate to acetyl-CoA, catalysed by the pyruvate dehydrogenase complex, (ii) decarboxylation of pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase, and (iii) the anaplerotic carboxylation of pyruvate to oxaloacetate, catalysed by pyruvate carboxylase. Special attention is devoted to physiological studies on S. cerevisiae strains in which structural genes encoding these key enzymes have been inactivated by gene disruption.

  1. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system is...

  2. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system is...

  3. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system is...

  4. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system is...

  5. Growth-based determination and biochemical confirmation of genetic requirements for protein degradation in Saccharomyces cerevisiae.

    PubMed

    Watts, Sheldon G; Crowder, Justin J; Coffey, Samuel Z; Rubenstein, Eric M

    2015-02-16

    Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for

  6. Genetic Determinants of Volatile-Thiol Release by Saccharomyces cerevisiae during Wine Fermentation

    PubMed Central

    Howell, Kate S.; Klein, Mathias; Swiegers, Jan H.; Hayasaka, Yoji; Elsey, Gordon M.; Fleet, Graham H.; Høj, Peter B.; Pretorius, Isak S.; de Barros Lopes, Miguel A.

    2005-01-01

    Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens. PMID:16151133

  7. Chromosome Duplication in Saccharomyces cerevisiae

    PubMed Central

    Bell, Stephen P.; Labib, Karim

    2016-01-01

    The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. PMID:27384026

  8. Postreplication repair in Saccharomyces cerevisiae

    SciTech Connect

    Resnick, M.A.; Boyce, J.; Cox, B.

    1981-04-01

    Postreplication events in logarithmically growing excision-defective mutants of Saccharomyces cerevisiae were examined after low doses of ultraviolet light. Pulse-labeled deoxyribonucleic acid had interruptions, and when the cells were chased, the interruptions were no longer detected. Since the loss of interruptions was not associated with an exchange of pyrimidine dimers at a detection level of 10 to 20% of the induced dimers, it was concluded that postreplication repair in excision-defective mutants does not involve molecular recombination. Pyrimidine dimers were assayed by utilizing the ultraviolet-endonuclease activity in extracts of Micrococcus luteus and newly developed alkaline sucrose gradient techniques, which yielded chromosomal-size deoxyribonucleic acid after treatment of irradiated cells.

  9. Glucose repression in Saccharomyces cerevisiae

    PubMed Central

    Kayikci, Ömur; Nielsen, Jens

    2015-01-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression. PMID:26205245

  10. Whole cell immobilized amperometric biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1 (thiamine).

    PubMed

    Akyilmaz, Erol; Yaşa, Ihsan; Dinçkaya, Erhan

    2006-07-01

    A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated.

  11. [Thermoresistance in Saccharomyces cerevisiae yeasts].

    PubMed

    Kaliuzhin, V A

    2011-01-01

    Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.

  12. Translational thermotolerance in Saccharomyces cerevisiae

    PubMed Central

    Hallberg, Elizabeth M.; Hallberg, Richard L.

    1996-01-01

    While protein synthesis is rapidly inactivated in Saccharomyces cerevisiae, cells shifted from log growth at 30°C to 43°C, a 1-h 37°C treatment given to cells just prior to the shift to 43°C partially blocks this inactivation. By contrast, such a pre-heat shock treament has no protective effect on translational inactivation at 45°C or higher. Cells allowed to approach stationary phase not only develop an enhanced thermotolerance relative to log cells but also exhibit a pronounced resistance to inactivation of protein synthesis at 43°C as well as at 45°C. We have found that this ‘translational thermotolerance’ can also be induced in S. cerevisiae by briefly treating log phase cells at 30°C with cycloheximide. Using such a procedure to induce stabilization of protein synthesis at 43°C, we have been able to show that heat shock-induced proteins are not responsible for the establishment of this protective effect. This work shows that enhanced thermotolerance can be induced in log cells even after a shift to 43°C, as long as a prior translational thermotolerance has been established. Futhermore, we show that the capacity of plateau cells to maintain translation at 43°C contributes significantly to their state of enhanced thermotolerance. PMID:9222591

  13. Cell Wall Assembly in Saccharomyces cerevisiae

    PubMed Central

    Lesage, Guillaume; Bussey, Howard

    2006-01-01

    An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls. PMID:16760306

  14. 2μ plasmid in Saccharomyces species and in Saccharomyces cerevisiae

    PubMed Central

    Strope, Pooja K.; Kozmin, Stanislav G.; Skelly, Daniel A.; Magwene, Paul M.; Dietrich, Fred S.; McCusker, John H.

    2015-01-01

    We determined that extrachromosomal 2μ plasmid was present in 67 of the Saccharomyces cerevisiae 100-genome strains; in addition to variation in the size and copy number of 2μ, we identified three distinct classes of 2μ. We identified 2μ presence/absence and class associations with populations, clinical origin and nuclear genotypes. We also screened genome sequences of S. paradoxus, S. kudriavzevii, S. uvarum, S. eubayanus, S. mikatae, S. arboricolus and S. bayanus strains for both integrated and extrachromosomal 2μ. Similar to S. cerevisiae, we found no integrated 2μ sequences in any S. paradoxus strains. However, we identified part of 2μ integrated into the genomes of some S. uvarum, S. kudriavzevii, S. mikatae and S. bayanus strains, which were distinct from each other and from all extrachromosomal 2μ. We identified extrachromosomal 2μ in one S. paradoxus, one S. eubayanus, two S. bayanus and 13 S. uvarum strains. The extrachromosomal 2μ in S. paradoxus, S. eubayanus and S. cerevisiae were distinct from each other. In contrast, the extrachromosomal 2μ in S. bayanus and S. uvarum strains were identical with each other and with one of the three classes of S. cerevisiae 2μ, consistent with interspecific transfer. PMID:26463005

  15. 2μ plasmid in Saccharomyces species and in Saccharomyces cerevisiae.

    PubMed

    Strope, Pooja K; Kozmin, Stanislav G; Skelly, Daniel A; Magwene, Paul M; Dietrich, Fred S; McCusker, John H

    2015-12-01

    We determined that extrachromosomal 2μ plasmid was present in 67 of the Saccharomyces cerevisiae 100-genome strains; in addition to variation in the size and copy number of 2μ, we identified three distinct classes of 2μ. We identified 2μ presence/absence and class associations with populations, clinical origin and nuclear genotypes. We also screened genome sequences of S. paradoxus, S. kudriavzevii, S. uvarum, S. eubayanus, S. mikatae, S. arboricolus and S. bayanus strains for both integrated and extrachromosomal 2μ. Similar to S. cerevisiae, we found no integrated 2μ sequences in any S. paradoxus strains. However, we identified part of 2μ integrated into the genomes of some S. uvarum, S. kudriavzevii, S. mikatae and S. bayanus strains, which were distinct from each other and from all extrachromosomal 2μ. We identified extrachromosomal 2μ in one S. paradoxus, one S. eubayanus, two S. bayanus and 13 S. uvarum strains. The extrachromosomal 2μ in S. paradoxus, S. eubayanus and S. cerevisiae were distinct from each other. In contrast, the extrachromosomal 2μ in S. bayanus and S. uvarum strains were identical with each other and with one of the three classes of S. cerevisiae 2μ, consistent with interspecific transfer. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

    NASA Technical Reports Server (NTRS)

    Meyer, Michael (Technical Monitor); Smith, Davey; Metzgar, David; Wills, Christopher; Fierer, Joshua

    2002-01-01

    Saccharomyces cerevisiae is a yeast commonly used in baking and a frequent colonizer of human mucosal surfaces. It is considered relatively nonpathogenic in immunocompetent adults. We present a case of S. cerevisiae fungemia and aortic graft infection in an immunocompetent adult. This is the first reported case of S. cerevisiue fungemia where the identity of the pathogen was confirmed by rRNA sequencing.

  17. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

    NASA Technical Reports Server (NTRS)

    Meyer, Michael (Technical Monitor); Smith, Davey; Metzgar, David; Wills, Christopher; Fierer, Joshua

    2002-01-01

    Saccharomyces cerevisiae is a yeast commonly used in baking and a frequent colonizer of human mucosal surfaces. It is considered relatively nonpathogenic in immunocompetent adults. We present a case of S. cerevisiae fungemia and aortic graft infection in an immunocompetent adult. This is the first reported case of S. cerevisiue fungemia where the identity of the pathogen was confirmed by rRNA sequencing.

  18. A flow injection analysis system with encapsulated high-density Saccharomyces cerevisiae cells for rapid determination of biochemical oxygen demand.

    PubMed

    Seo, Kyo Seong; Choo, Kwang Ho; Chang, Ho Nam; Park, Joong Kon

    2009-05-01

    The biochemical oxygen demand (BOD) determination was studied using a novel flow injection analysis (FIA) system with encapsulated Saccharomyces cerevisiae cells and an oxygen electrode and was compared with conventional 5-day BOD tests. S. cerevisiae cells were packed in a calcium alginate capsule at a dry cell weight of 250 g/l of capsule core. The level of dissolved oxygen (DO) was reduced due to the enhanced respiratory activity of the microbial cells when the injected nutrient passed through the bioreactor. The decrease in DO (DeltaDO) was intensified with the amount of microbial cells packed in the bioreactor. However, the specific DeltaDO decreased as the amount of cells loaded in the bioreactor increased. The DeltaDO value was dependent on the pH and temperature of the mobile phase and reached its maximum value at 35 degrees C and pH 7-8. Also, DeltaDO became larger at longer response times as the flow rate of the mobile phase decreased. The measurement of DeltaDO was repeated more than six times consecutively using a 20-ppm standard glucose and glutamic acid solution, which confirmed the reproducibility with a standard deviation of 0.95%. A strong linear correlation between DeltaDO and BOD was also observed. The 5-day BOD values of actual water and wastewater samples were in accordance with the BOD values obtained by this FIA method using encapsulated S. cerevisiae cells. Unlike the cell-immobilized bead system, there was no contamination of the bioreactor resulting from any leak of yeast cells from the sensor capsules during BOD measurements.

  19. Dissection of Saccharomyces cerevisiae asci.

    PubMed

    Morin, Audrey; Moores, Adrian W; Sacher, Michael

    2009-05-19

    Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

  20. Lead toxicity in Saccharomyces cerevisiae.

    PubMed

    Van der Heggen, Maarten; Martins, Sara; Flores, Gisela; Soares, Eduardo V

    2010-12-01

    The effect of Pb on Saccharomyces cerevisiae cell structure and function was examined. Membrane integrity was assessed by the release of UV-absorbing compounds and by the intracellular K(+) efflux. No leakage of UV(260)-absorbing compounds or loss of K(+) were observed in Pb (until 1,000 μmol/l) treated cells up to 30 min; these results suggest that plasma membrane seems not to be the immediate and primary target of Pb toxicity. The effect of Pb on yeast metabolism was examined using the fluorescent probe FUN-1 and compared with the ability to reproduce, evaluated by colony-forming units counting. The exposition of yeast cells, during 60 min to 1,000 μmol/l Pb, induces a decrease in the ability to process FUN-1 although the cells retain its proliferation capacity. A more prolonged contact time (120 min) of yeast cells with Pb induces a marked (> 50%) loss of yeast cells metabolic activity and replication competence through a mechanism which most likely requires protein synthesis.

  1. Proteomics of Saccharomyces cerevisiae Organelles*

    PubMed Central

    Wiederhold, Elena; Veenhoff, Liesbeth M.; Poolman, Bert; Slotboom, Dirk Jan

    2010-01-01

    Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them with other large scale studies on protein localization and abundance. We evaluate the completeness and reliability of the organelle proteomics studies. Reliability depends on the purity of the organelle preparations, which unavoidably contain (small) amounts of contaminants from different locations. Quantitative proteomics methods can be used to distinguish between true organellar constituents and contaminants. Completeness is compromised when loosely or dynamically associated proteins are lost during organelle preparation and also depends on the sensitivity of the analytical methods for protein detection. There is a clear trend in the data from the 18 organelle proteomics studies showing that proteins of low abundance frequently escape detection. Proteins with unknown function or cellular abundance are also infrequently detected, indicating that these proteins may not be expressed under the conditions used. We discuss that the yeast organelle proteomics studies provide powerful lead data for further detailed studies and that methodological advances in organelle preparation and in protein detection may help to improve the completeness and reliability of the data. PMID:19955081

  2. Strengths and weaknesses in the determination of Saccharomyces cerevisiae cell viability by ATP-based bioluminescence assay.

    PubMed

    Paciello, Lucia; Falco, Francesco Cristino; Landi, Carmine; Parascandola, Palma

    2013-03-05

    Due to its sensitivity and speed of execution, detection of ATP by luciferin-luciferase reaction is a widely spread system to highlight cell viability. The paper describes the methodology followed to successfully run the assay in the presence of yeast cells of two strains of the yeast Saccharomyces cerevisiae, BY4741 and CEN.PK2-1C and emphasizes the importance of correctly determining the contact time between the lysing agent and the yeast cells. Once this was established, luciferin-luciferase reaction was exploited to determine the maximum specific rate of growth, as well as cell viability in a series of routine tests. The results obtained in this preliminary study highlighted that using luciferin-luciferase can imply an over-estimation of maximum specific growth rate with respect to that determined by optical density and/or viable count. On the contrary, the bioluminescence assay gave the possibility to highlight, if employed together with viable count, physiological changes occurring in yeast cells as response to stressful environmental conditions such as those deriving from exposure of yeast cells to high temperature or those depending on the operative conditions applied during fed-batch operations.

  3. Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe.

    PubMed

    Rostron, Kerry A; Rolph, Carole E; Lawrence, Clare L

    2015-07-01

    Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321-328, 2012), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses.

  4. Sterol methylation in Saccharomyces cerevisiae.

    PubMed Central

    McCammon, M T; Hartmann, M A; Bottema, C D; Parks, L W

    1984-01-01

    Various nystatin-resistant mutants defective in S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) were shown to possess alleles of the same gene, erg6. The genetic map location of erg6 was shown to be close to trp1 on chromosome 4. Despite the single locus for erg6, S-adenosylmethionine: delta 24-sterol-C-methyltransferase enzyme activity was found in three separate fractions: mitochondria, microsomes, and the "floating lipid layer." The amount of activity in each fraction could be manipulated by assay conditions. The lipids and lipid synthesis of mutants of Saccharomyces cerevisiae defective in the delta 24-sterol-C-methyltransferase were compared with a C5(6) desaturase mutant and parental wild types. No ergosterol (C28 sterol) could be detected in whole-cell sterol extracts of the erg6 mutants, the limits of detection being less than 10(-11) mol of ergosterol per 10(8) cells. The distribution of accumulated sterols by these mutants varied with growth phase and between free and esterified fractions. The steryl ester concentrations of the mutants were eight times higher than those of the wild type from exponential growth samples. However, the concentration of the ester accumulated by the mutants was not as great in stationary-phase cells. Whereas the head group phospholipid composition was the same between parental and mutant strains, strain-dependent changes in fatty acids were observed, most notably a 40% increase in the oleic acid content of phosphatidylethanolamine of one erg6 mutant, JR5. PMID:6363386

  5. The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates

    PubMed Central

    2014-01-01

    We investigated the severity of the inhibitory effects of 13 phenolic compounds usually found in spruce hydrolysates (4-hydroxy-3-methoxycinnamaldehyde, homovanilyl alcohol, vanillin, syringic acid, vanillic acid, gallic acid, dihydroferulic acid, p-coumaric acid, hydroquinone, ferulic acid, homovanillic acid, 4-hydroxybenzoic acid and vanillylidenacetone). The effects of the selected compounds on cell growth, biomass yield and ethanol yield were studied and the toxic concentration threshold was defined for each compound. Using Ethanol Red, the popular industrial strain of Saccharomyces cerevisiae, we found the most toxic compound to be 4-hydroxy-3-methoxycinnamaldehyde which inhibited growth at a concentration of 1.8 mM. We also observed that toxicity did not generally follow a trend based on the aldehyde, acid, ketone or alcohol classification of phenolic compounds, but rather that other structural properties such as additional functional groups attached to the compound may determine its toxicity. Three distinctive growth patterns that effectively clustered all the compounds involved in the screening into three categories. We suggest that the compounds have different cellular targets, and that. We suggest that the compounds have different cellular targets and inhibitory mechanisms in the cells, also compounds who share similar pattern on cell growth may have similar inhibitory effect and mechanisms of inhibition. PMID:24949277

  6. Structural and functional mapping of Rtg2p determinants involved in retrograde signaling and aging of Saccharomyces cerevisiae

    PubMed Central

    Rios-Anjos, Rafaela Maria; Camandona, Vittoria de Lima; Bleicher, Lucas

    2017-01-01

    In Saccharomyces cerevisiae mitochondrial dysfunction induces retrograde signaling, a pathway of communication from mitochondria to the nucleus that promotes a metabolic remodeling to ensure sufficient biosynthetic precursors for replication. Rtg2p is a positive modulator of this pathway that is also required for cellular longevity. This protein belongs to the ASKHA superfamily, and contains a putative N-terminal ATP-binding domain, but there is no detailed structural and functional map of the residues in this domain that accounts for their contribution to retrograde signaling and aging. Here we use Decomposition of Residue Correlation Networks and site-directed mutagenesis to identify Rtg2p structural determinants of retrograde signaling and longevity. We found that most of the residues involved in retrograde signaling surround the ATP-binding loops, and that Rtg2p N-terminus is divided in three regions whose mutants have different aging phenotypes. We also identified E137, D158 and S163 as possible residues involved in stabilization of ATP at the active site. The mutants shown here may be used to map other Rtg2p activities that crosstalk to other pathways of the cell related to genomic stability and aging. PMID:28472157

  7. Structural and functional mapping of Rtg2p determinants involved in retrograde signaling and aging of Saccharomyces cerevisiae.

    PubMed

    Rios-Anjos, Rafaela Maria; Camandona, Vittoria de Lima; Bleicher, Lucas; Ferreira-Junior, Jose Ribamar

    2017-01-01

    In Saccharomyces cerevisiae mitochondrial dysfunction induces retrograde signaling, a pathway of communication from mitochondria to the nucleus that promotes a metabolic remodeling to ensure sufficient biosynthetic precursors for replication. Rtg2p is a positive modulator of this pathway that is also required for cellular longevity. This protein belongs to the ASKHA superfamily, and contains a putative N-terminal ATP-binding domain, but there is no detailed structural and functional map of the residues in this domain that accounts for their contribution to retrograde signaling and aging. Here we use Decomposition of Residue Correlation Networks and site-directed mutagenesis to identify Rtg2p structural determinants of retrograde signaling and longevity. We found that most of the residues involved in retrograde signaling surround the ATP-binding loops, and that Rtg2p N-terminus is divided in three regions whose mutants have different aging phenotypes. We also identified E137, D158 and S163 as possible residues involved in stabilization of ATP at the active site. The mutants shown here may be used to map other Rtg2p activities that crosstalk to other pathways of the cell related to genomic stability and aging.

  8. Phylogenetic relationship and Fourier-transform infrared spectroscopy-derived lipid determinants of lifespan parameters in the Saccharomyces cerevisiae yeast.

    PubMed

    Molon, Mateusz; Zebrowski, Jacek

    2017-06-01

    Yeast ageing has been gaining much attention in gerontology research, yet the process itself is still not entirely clear. One of the constraints related to the use of the Saccharomyces cerevisiae yeast in studies is the ambiguity of the results concerning ageing determinants for different genetic backgrounds. In this paper, we compare reproductive potentials and lifespans of seven widely used haploid laboratory strains differing in daughter cells production capabilities and highlight the importance of choosing an appropriate genotype for the studies on ageing. Moreover, we show here links between post-reproductive lifespan and lipid metabolism, as well as between reproductive potential, reproductive lifespan and phylogenetic relationship. Using FTIR spectroscopy that generated a biochemical fingerprint of cells, coupled with chemometrics, we found that the band of carbonyl (C = O) stretching vibration discriminates the strains according to post-reproductive lifespan. The results indicated that prolonged post-reproductive lifespan was associated with relatively lower amount of fatty acids esterified to phospholipids compared to a free acid pool, thus implying phospholipid metabolism for the post-reproductive lifespan of yeast. In addition, phylogenetic analysis showed a correlation between nucleotide similarity and the reproductive potential or reproductive lifespan, but not to the longevity expressed in time units. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. The chemical nature of phenolic compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates.

    PubMed

    Adeboye, Peter Temitope; Bettiga, Maurizio; Olsson, Lisbeth

    2014-01-01

    We investigated the severity of the inhibitory effects of 13 phenolic compounds usually found in spruce hydrolysates (4-hydroxy-3-methoxycinnamaldehyde, homovanilyl alcohol, vanillin, syringic acid, vanillic acid, gallic acid, dihydroferulic acid, p-coumaric acid, hydroquinone, ferulic acid, homovanillic acid, 4-hydroxybenzoic acid and vanillylidenacetone). The effects of the selected compounds on cell growth, biomass yield and ethanol yield were studied and the toxic concentration threshold was defined for each compound. Using Ethanol Red, the popular industrial strain of Saccharomyces cerevisiae, we found the most toxic compound to be 4-hydroxy-3-methoxycinnamaldehyde which inhibited growth at a concentration of 1.8 mM. We also observed that toxicity did not generally follow a trend based on the aldehyde, acid, ketone or alcohol classification of phenolic compounds, but rather that other structural properties such as additional functional groups attached to the compound may determine its toxicity. Three distinctive growth patterns that effectively clustered all the compounds involved in the screening into three categories. We suggest that the compounds have different cellular targets, and that. We suggest that the compounds have different cellular targets and inhibitory mechanisms in the cells, also compounds who share similar pattern on cell growth may have similar inhibitory effect and mechanisms of inhibition.

  10. Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

    ERIC Educational Resources Information Center

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

  11. Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

    ERIC Educational Resources Information Center

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

  12. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    ERIC Educational Resources Information Center

    Silva, Carlos M.; Neves, Patricia S.; Da Silva, Francisco A.; Xavier, Ana M. R. B.; Eusebio, M. F. J.

    2008-01-01

    Experimental work on ultrafiltration is presented to illustrate the practical and theoretical principles of this separation technique. The laboratory exercise comprises experiments with pure water and with aqueous "Saccharomyces cerevisiae" (from commercial Baker's yeast) suspensions. With this work students detect the characteristic phenomena…

  13. Regulation of Mitotic Exit in Saccharomyces cerevisiae.

    PubMed

    Baro, Bàrbara; Queralt, Ethel; Monje-Casas, Fernando

    2017-01-01

    The Mitotic Exit Network (MEN) is an essential signaling pathway, closely related to the Hippo pathway in mammals, which promotes mitotic exit and initiates cytokinesis in the budding yeast Saccharomyces cerevisiae. Here, we summarize the current knowledge about the MEN components and their regulation.

  14. Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae

    USDA-ARS?s Scientific Manuscript database

    Saccharomyces cerevisiae is a superb ethanol producer, yet is also sensitive to higher ethanol concentrations especially under high gravity or very high gravity fermentation conditions. Ethanol tolerance is associated with interplay of complex networks at the genome level. Although significant eff...

  15. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    ERIC Educational Resources Information Center

    Silva, Carlos M.; Neves, Patricia S.; Da Silva, Francisco A.; Xavier, Ana M. R. B.; Eusebio, M. F. J.

    2008-01-01

    Experimental work on ultrafiltration is presented to illustrate the practical and theoretical principles of this separation technique. The laboratory exercise comprises experiments with pure water and with aqueous "Saccharomyces cerevisiae" (from commercial Baker's yeast) suspensions. With this work students detect the characteristic phenomena…

  16. A halotolerant mutant of Saccharomyces cerevisiae.

    PubMed Central

    Gaxiola, R; Corona, M; Zinker, S

    1996-01-01

    FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression. PMID:8631691

  17. Determination of Adenosine A1 Receptor Agonist and Antagonist Pharmacology Using Saccharomyces cerevisiae: Implications for Ligand Screening and Functional Selectivity

    PubMed Central

    Stewart, Gregory D.; Valant, Celine; Dowell, Simon J.; Mijaljica, Dalibor; Devenish, Rodney J.; Scammells, Peter J.; Sexton, Patrick M.

    2009-01-01

    The budding yeast, Saccharomyces cerevisiae, is a convenient system for coupling heterologous G protein-coupled receptors (GPCRs) to the pheromone response pathway to facilitate empirical ligand screening and/or GPCR mutagenesis studies. However, few studies have applied this system to define GPCR-G protein-coupling preferences and furnish information on ligand affinities, efficacies, and functional selectivity. We thus used different S. cerevisiae strains, each expressing a specific human Gα/yeast Gpa1 protein chimera, and determined the pharmacology of various ligands of the coexpressed human adenosine A1 receptor. These assays, in conjunction with the application of quantitative models of agonism and antagonism, revealed that (−)-N6-(2-phenylisopropyl)adenosine was a high-efficacy agonist that selectively coupled to Gpa/1Gαo, Gpa1/Gαi1/2, and Gpa1/Gαi3, whereas the novel compound, 5′-deoxy-N6-(endo-norborn-2-yl)-5′-(2-fluorophenylthio)adenosine (VCP-189), was a lower-efficacy agonist that selectively coupled to Gpa1/Gαi proteins; the latter finding suggested that VCP-189 might be functionally selective. The affinity of the antagonist, 8-cyclopentyl-1,3-dipropylxanthine, was also determined at the various strains. Subsequent experiments performed in mammalian Chinese hamster ovary cells monitoring cAMP formation/inhibition, intracellular calcium mobilization, phosphorylation of extracellular signal-regulated kinase 1 and 2 or 35S-labeled guanosine 5′-(γ-thio)triphosphate binding, were in general agreement with the yeast data regarding agonist efficacy estimation and antagonist affinity estimation, but revealed that the apparent functional selectivity of VCP-189 could be explained by differences in stimulus-response coupling between yeast and mammalian cells. Our results suggest that this yeast system is a useful tool for quantifying ligand affinity and relative efficacy, but it may lack the sensitivity required to detect functional selectivity of

  18. Determining if an mRNA is a Substrate of Nonsense-Mediated mRNA Decay in Saccharomyces cerevisiae.

    PubMed

    Johansson, Marcus J O

    2017-01-01

    Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control mechanism which triggers decay of mRNAs harboring premature translation termination codons. In this chapter, I describe methods for monitoring the influence of NMD on mRNA abundance and decay rates in Saccharomyces cerevisiae. The descriptions include detailed methods for growing yeast cells, total RNA isolation, and Northern blotting. Although the chapter focuses on NMD, the methods can be easily adapted to assess the effect of other mRNA decay pathways.

  19. Kinetics of phosphomevalonate kinase from Saccharomyces cerevisiae.

    PubMed

    Garcia, David E; Keasling, Jay D

    2014-01-01

    The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni²⁺ column. The KM of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K(M) of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V(max) was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.

  20. Rad5 Template Switch Pathway of DNA Damage Tolerance Determines Synergism between Cisplatin and NSC109268 in Saccharomyces cerevisiae

    PubMed Central

    Jain, Dilip; Siede, Wolfram

    2013-01-01

    The success of cisplatin (CP) based therapy is often hindered by acquisition of CP resistance. We isolated NSC109268 as a compound altering cellular sensitivity to DNA damaging agents. Previous investigation revealed an enhancement of CP sensitivity by NSC109268 in wild-type Saccharomyces cerevisiae and CP-sensitive and -resistant cancer cell lines that correlated with a slower S phase traversal. Here, we extended these studies to determine the target pathway(s) of NSC109268 in mediating CP sensitization, using yeast as a model. We reasoned that mutants defective in the relevant target of NSC109268 should be hypersensitive to CP and the sensitization effect by NSC109268 should be absent or strongly reduced. A survey of various yeast deletion mutants converged on the Rad5 pathway of DNA damage tolerance by template switching as the likely target pathway of NSC109268 in mediating cellular sensitization to CP. Additionally, cell cycle delays following CP treatment were not synergistically influenced by NSC109268 in the CP hypersensitive rad5Δ mutant. The involvement of the known inhibitory activities of NSC109268 on 20S proteasome and phosphatases 2Cα and 2A was tested. In the CP hypersensitive ptc2Δptc3Δpph3Δ yeast strain, deficient for 2C and 2A-type phosphatases, cellular sensitization to CP by NSC109268 was greatly reduced. It is therefore suggested that NSC109268 affects CP sensitivity by inhibiting the activity of unknown protein(s) whose dephosphorylation is required for the template switch pathway. PMID:24130896

  1. Replicative and chronological aging in Saccharomyces cerevisiae.

    PubMed

    Longo, Valter D; Shadel, Gerald S; Kaeberlein, Matt; Kennedy, Brian

    2012-07-03

    Saccharomyces cerevisiae has directly or indirectly contributed to the identification of arguably more mammalian genes that affect aging than any other model organism. Aging in yeast is assayed primarily by measurement of replicative or chronological life span. Here, we review the genes and mechanisms implicated in these two aging model systems and key remaining issues that need to be addressed for their optimization. Because of its well-characterized genome that is remarkably amenable to genetic manipulation and high-throughput screening procedures, S. cerevisiae will continue to serve as a leading model organism for studying pathways relevant to human aging and disease.

  2. Biotechnological implications of filamentation in Saccharomyces cerevisiae.

    PubMed

    Ceccato-Antonini, Sandra Regina

    2008-07-01

    The genetics governing the morphological switch from round or ovoid cells to filamentous growth in Saccharomyces cerevisiae has received significant interest in relation to sensing and signaling pathways as well as the control of cell processes including budding, elongation and adhesion. Little is known about the environmental signals which trigger these morphological changes from a biotechnological point of view. This review aims to highlight the main causes of filamentous growth in S. cerevisiae in its industrial setting with the purpose of stimulating additional studies within this field.

  3. Biosynthesis of silver nanoparticles using Saccharomyces cerevisiae.

    PubMed

    Korbekandi, Hassan; Mohseni, Soudabeh; Mardani Jouneghani, Rasoul; Pourhossein, Meraj; Iravani, Siavash

    2016-01-01

    The objectives of this study were the biosynthesis of silver nanoparticles (NPs) by biotransformations using Saccharomyces cerevisiae and analysis of the sizes and shapes of the NPs produced. Dried and freshly cultured S. cerevisiae were used as the biocatalyst. Dried yeast synthesized few NPs, but freshly cultured yeast produced a large amount of them. Silver NPs were spherical, 2-20 nm in diameter, and the NPs with the size of 5.4 nm were the most frequent ones. NPs were seen inside the cells, within the cell membrane, attached to the cell membrane during the exocytosis, and outside of the cells.

  4. In vivo effects of fenpropimorph on the yeast Saccharomyces cerevisiae and determination of the molecular basis of the antifungal property.

    PubMed Central

    Marcireau, C; Guilloton, M; Karst, F

    1990-01-01

    The effects of fenpropimorph on sterol biosynthesis and growth of Saccharomyces cerevisiae were examined to pinpoint the mode of action of fungicides that inhibit ergosterol biosynthesis. Taking advantage of sterol auxotrophy and sterol permeability in mutant strains, we show that growth inhibition is strongly correlated with inhibition of sterol biosynthesis. We confirm that in vivo and at low concentrations, fenpropimorph inhibits delta 8----delta 7-sterol isomerase, and in addition, when it is used at higher concentrations, it inhibits delta 14-sterol reductase. We show also that the fungistatic effect of fenpropimorph is not due to the accumulation of abnormal sterols in treated cells but is linked to the specific inhibition of ergosterol biosynthesis, leading to the arrest of cell proliferation in the unbudded G1 phase of the cell cycle. PMID:2203312

  5. "Malonate uptake and metabolism in Saccharomyces cerevisiae".

    PubMed

    Chen, Wei Ning; Tan, Kee Yang

    2013-09-01

    Malonyl-CoA plays an important role in the synthesis and elongation of fatty acids in yeast Saccharomyces cerevisiae. Malonyl-CoA is at a low concentration inside the cell and is produced mainly from acetyl-CoA through the enzyme acetyl-CoA carboxylase. It would be beneficial to find an alternative source of malonyl-CoA to increase its intracellular concentration and overall synthesis of the fatty acids. MatB gene from the bacteria Rhizobium leguminosarium bv. trifolii encodes for a malonyl-CoA synthetase which catalyzes the formation of the malonyl-CoA directly from malonate and CoA. However, results from high-performance liquid chromatography (HPLC) proved that Saccharomyces cerevisiae itself does not contain enough cytoplasmic malonate within them and is unable to uptake exogenously supplied malonate in the form of malonic acid. A dicarboxylic acid plasma membrane transporter with the ability to uptake exogenous malonic acid was identified from another species of yeast known as Schizosaccharomyces pombe and the gene encoding this transporter is identified as the mae1 gene. From the experiments thus far, the mae1 gene had been successfully cloned and transformed into Saccharomyces cerevisiae. The expression and functional ability of the encoded plasma membrane dicarboxylic acid transporter were also demonstrated and verified using specialized technologies such as RT-PCR, yeast immunofluorescence, HPLC, and LC-MS.

  6. Progress in metabolic engineering of Saccharomyces cerevisiae.

    PubMed

    Nevoigt, Elke

    2008-09-01

    The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial ("white") biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

  7. [The ABC transporters of Saccharomyces cerevisiae].

    PubMed

    Wawrzycka, Donata

    2011-01-01

    The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.

  8. [Urinary infection by Saccharomyces cerevisiae: Emerging yeast?].

    PubMed

    Elkhihal, B; Elhalimi, M; Ghfir, B; Mostachi, A; Lyagoubi, M; Aoufi, S

    2015-12-01

    Saccharomyces cerevisiae is a commensal yeast of the digestive, respiratory and genito-urinary tract. It is widely used as a probiotic for the treatment of post-antibiotic diarrhea. It most often occurs in immunocompromised patients frequently causing fungemia. We report the case of an adult diabetic patient who had a urinary tract infection due to S. cerevisiae. The disease started with urination associated with urinary frequency burns without fever. The diagnosis was established by the presence of yeasts on direct examination and positivity of culture on Sabouraud-chloramphenicol three times. The auxanogramme gallery (Auxacolor BioRad(®)) allowed the identification of S. cerevisiae. The patient was put on fluconazole with good outcome. This observation points out that this is an opportunistic yeast in immunocompromised patients. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  9. Saccharomyces cerevisiae metabolism in ecological context.

    PubMed

    Jouhten, Paula; Ponomarova, Olga; Gonzalez, Ramon; Patil, Kiran R

    2016-11-01

    The architecture and regulation of Saccharomyces cerevisiae metabolic network are among the best studied owing to its widespread use in both basic research and industry. Yet, several recent studies have revealed notable limitations in explaining genotype-metabolic phenotype relations in this yeast, especially when concerning multiple genetic/environmental perturbations. Apparently unexpected genotype-phenotype relations may originate in the evolutionarily shaped cellular operating principles being hidden in common laboratory conditions. Predecessors of laboratory S. cerevisiae strains, the wild and the domesticated yeasts, have been evolutionarily shaped by highly variable environments, very distinct from laboratory conditions, and most interestingly by social life within microbial communities. Here we present a brief review of the genotypic and phenotypic peculiarities of S. cerevisiae in the context of its social lifestyle beyond laboratory environments. Accounting for this ecological context and the origin of the laboratory strains in experimental design and data analysis would be essential in improving the understanding of genotype-environment-phenotype relationships.

  10. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  11. Limited proteolysis of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.

    PubMed

    Herrera, L; Encinas, M V; Jabalquinto, A M; Cardemil, E

    1993-08-01

    Incubation of Saccharomyces 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.

  12. Functional profiling of the Saccharomyces cerevisiae genome.

    PubMed

    Giaever, Guri; Chu, Angela M; Ni, Li; Connelly, Carla; Riles, Linda; Véronneau, Steeve; Dow, Sally; Lucau-Danila, Ankuta; Anderson, Keith; André, Bruno; Arkin, Adam P; Astromoff, Anna; El-Bakkoury, Mohamed; Bangham, Rhonda; Benito, Rocio; Brachat, Sophie; Campanaro, Stefano; Curtiss, Matt; Davis, Karen; Deutschbauer, Adam; Entian, Karl-Dieter; Flaherty, Patrick; Foury, Francoise; Garfinkel, David J; Gerstein, Mark; Gotte, Deanna; Güldener, Ulrich; Hegemann, Johannes H; Hempel, Svenja; Herman, Zelek; Jaramillo, Daniel F; Kelly, Diane E; Kelly, Steven L; Kötter, Peter; LaBonte, Darlene; Lamb, David C; Lan, Ning; Liang, Hong; Liao, Hong; Liu, Lucy; Luo, Chuanyun; Lussier, Marc; Mao, Rong; Menard, Patrice; Ooi, Siew Loon; Revuelta, Jose L; Roberts, Christopher J; Rose, Matthias; Ross-Macdonald, Petra; Scherens, Bart; Schimmack, Greg; Shafer, Brenda; Shoemaker, Daniel D; Sookhai-Mahadeo, Sharon; Storms, Reginald K; Strathern, Jeffrey N; Valle, Giorgio; Voet, Marleen; Volckaert, Guido; Wang, Ching-yun; Ward, Teresa R; Wilhelmy, Julie; Winzeler, Elizabeth A; Yang, Yonghong; Yen, Grace; Youngman, Elaine; Yu, Kexin; Bussey, Howard; Boeke, Jef D; Snyder, Michael; Philippsen, Peter; Davis, Ronald W; Johnston, Mark

    2002-07-25

    Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

  13. The reference genome sequence of Saccharomyces cerevisiae: then and now.

    PubMed

    Engel, Stacia R; Dietrich, Fred S; Fisk, Dianna G; Binkley, Gail; Balakrishnan, Rama; Costanzo, Maria C; Dwight, Selina S; Hitz, Benjamin C; Karra, Kalpana; Nash, Robert S; Weng, Shuai; Wong, Edith D; Lloyd, Paul; Skrzypek, Marek S; Miyasato, Stuart R; Simison, Matt; Cherry, J Michael

    2014-03-20

    The genome of the budding yeast Saccharomyces cerevisiae was the first completely sequenced from a eukaryote. It was released in 1996 as the work of a worldwide effort of hundreds of researchers. In the time since, the yeast genome has been intensively studied by geneticists, molecular biologists, and computational scientists all over the world. Maintenance and annotation of the genome sequence have long been provided by the Saccharomyces Genome Database, one of the original model organism databases. To deepen our understanding of the eukaryotic genome, the S. cerevisiae strain S288C reference genome sequence was updated recently in its first major update since 1996. The new version, called "S288C 2010," was determined from a single yeast colony using modern sequencing technologies and serves as the anchor for further innovations in yeast genomic science.

  14. The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now

    PubMed Central

    Engel, Stacia R.; Dietrich, Fred S.; Fisk, Dianna G.; Binkley, Gail; Balakrishnan, Rama; Costanzo, Maria C.; Dwight, Selina S.; Hitz, Benjamin C.; Karra, Kalpana; Nash, Robert S.; Weng, Shuai; Wong, Edith D.; Lloyd, Paul; Skrzypek, Marek S.; Miyasato, Stuart R.; Simison, Matt; Cherry, J. Michael

    2014-01-01

    The genome of the budding yeast Saccharomyces cerevisiae was the first completely sequenced from a eukaryote. It was released in 1996 as the work of a worldwide effort of hundreds of researchers. In the time since, the yeast genome has been intensively studied by geneticists, molecular biologists, and computational scientists all over the world. Maintenance and annotation of the genome sequence have long been provided by the Saccharomyces Genome Database, one of the original model organism databases. To deepen our understanding of the eukaryotic genome, the S. cerevisiae strain S288C reference genome sequence was updated recently in its first major update since 1996. The new version, called “S288C 2010,” was determined from a single yeast colony using modern sequencing technologies and serves as the anchor for further innovations in yeast genomic science. PMID:24374639

  15. Determination of physical characteristics, chemical composition and digestion coefficients of treated lemon pulp with Saccharomyces cerevisiae in goat diet.

    PubMed

    Dadvar, P; Dayani, O; Mehdipour, M; Morovat, M

    2015-02-01

    The aim of this study was to evaluate the effects of processing of lemon pulp with Saccharomyces cerevisiae on physical properties, chemical composition, digestion coefficients and blood parameters. Eight adult male Raeini goats were used in a 28-day period. The experimental design was a completely randomised design with two treatments and four replicates. The first 21 days were for adaptation, and the last 7 days were for collecting samples. The animals were housed in individual metabolic cages equipped with a urine-faeces separator and were fed with diet containing alfalfa hay (60%) and lemon pulp (40%) at the maintenance level. Collected data were subjected to analysis of completely randomised design. With diet containing processed lemon pulp, functional specific gravity, bulk density, soluble dry matter, percentage of crude protein, neutral detergent fibre (NDF), acid detergent fibre and crude ash were significantly increased and water-holding capacity, insoluble dry matter, insoluble ash percentage of dry matter, organic matter, crude fat, non-fibrous carbohydrates and nitrogen-free extract were significantly decreased (p < 0.05). Digestion coefficient of organic matter in dry matter and metabolisable energy were also decreased in treated lemon pulp (p < 0.05). Compared with untreated lemon pulp, digestibility of crude protein and NDF was higher (p < 0.05), but digestibility of crude fat was lower (p < 0.05). In general, processing of lemon pulp with S. cerevisiae improved the physical characteristics and increased the percentage of crude protein and the digestion coefficients of protein and NDF. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

  16. Determining the effects of inositol supplementation and the opi1 mutation on ethanol tolerance of Saccharomyces cerevisiae.

    PubMed

    Krause, Erin L; Villa-García, Manuel J; Henry, Susan A; Walker, Larry P

    2007-11-07

    The yeast Saccharomyces cerevisiae is an important microorganism for the ethanol fuel industry. As with many microorganisms, the production and accumulation of certain metabolites, such as ethanol, can have a detrimental effect on cell growth and productivity. Yeast cells containing a higher concentration of phosphatidylinositol (PI) in the cellular membrane, due to inositol supplementation in the growth media, have been shown to tolerate and produce higher concentrations of ethanol. The specific goal of our research was to assess the effects of inositol supplementation in the growth media as well as to compare the ethanol tolerance of the wild-type S. cerevisiae to a mutant, the opi1 strain (opi=overproduction of inositol). The OPI1 gene product is a negative regulatory factor that controls the transcription of the INO1 structural gene, which encodes the enzyme catalyzing the limiting step in the biosynthesis of inositol, that is, the conversion of glucose-6-phosphate to inositol-3-phosphate. Upon the deletion of the OPI1 gene, the cell will constitutively produce inositol, regardless of the extracellular inositol concentration. Inositol supplementation in cultures of wild-type cells increased ethanol tolerance in terms of cell viability. Cells grown in -I media had a 20% higher specific death rate than cells grown in +I media when exposed to 15% ethanol. The opi1 strain, with the ability to constitutively produce inositol regardless of media composition, showed less inhibition of cell growth in the presence of ethanol than did the wild-type strain, particularly in inositol-free media. We conclude that the introduction of an opi1 mutation in yeast results in an inherent increase in PI levels and constitutive biosynthesis of inositol that, in turn, will reduce the cost of supplementing inositol into the media to achieve a higher ethanol tolerance.

  17. Sporulation in the Budding Yeast Saccharomyces cerevisiae

    PubMed Central

    Neiman, Aaron M.

    2011-01-01

    In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae. PMID:22084423

  18. Transformation of Saccharomyces cerevisiae and other fungi

    PubMed Central

    Kawai, Shigeyuki; Hashimoto, Wataru

    2010-01-01

    Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall. PMID:21468206

  19. [Engineering Saccharomyces cerevisiae for sclareol production].

    PubMed

    Yang, Wei; Zhou, Yongjin; Liu, Wujun; Shen, Hongwei; Zhao, Zongbao K

    2013-08-01

    Sclareol is a member of labdane type diterpenes mostly used as fragrance ingredient. To enable microbial production of sclareol, synthetic pathways were constructed by incorporating labdenediol diphosphate synthase (LPPS) and terpene synthase (TPS) of the plant Salvia sclarea into Saccharomyces cerevisiae. It was found that sclareol production could be benefited by overexpression of key enzyme for precursor biosynthesis, construction of fusion protein for substrate channeling, and removal of signal peptides from LPPS and TPS. Under optimal shake flask culture conditions, strain S6 produced 8.96 mg/L sclareol. These results provided useful information for development of heterologous hosts for production of terpenoids.

  20. Mitochondrial fission facilitates mitophagy in Saccharomyces cerevisiae.

    PubMed

    Mao, Kai; Klionsky, Daniel J

    2013-11-01

    As a highly dynamic organelle, mitochondria undergo constitutive fusion and fission as well as biogenesis and degradation. Mitophagy, selective mitochondrial degradation through autophagy, is a conserved cellular process used for the elimination of excessive and damaged mitochondria in eukaryotes. Despite the significance of mitophagy in cellular physiology and pathophysiologies, the underlying mechanism of this process is far from clear. In this report, we studied the role of mitochondrial fission during mitophagy, and uncover a direct link between the fission complex and mitophagy machinery in Saccharomyces cerevisiae.

  1. Components of microtubular structures in Saccharomyces cerevisiae.

    PubMed Central

    Pillus, L; Solomon, F

    1986-01-01

    Most studies of cytoskeletal organelles have concentrated on molecular analyses of abundant and biochemically accessible structures. In many of the classical cases, however, the nature of the system chosen has precluded a concurrent genetic analysis. The mitotic spindle of the yeast Saccharomyces cerevisiae is one example of an organelle that can be studied by both classical and molecular genetics. We show here that this microtubule structure also can be examined biochemically. The spindle can be isolated by selective extractions of yeast cells by using adaptations of methods successfully applied to animal cells. In this way, microtubule-associated proteins of the yeast spindle are identified. Images PMID:3517870

  2. Fatty Acid Synthetase of Saccharomyces cerevisiae

    PubMed Central

    Klein, Harold P.; Volkmann, Carol M.; Chao, Fu-Chuan

    1967-01-01

    A light particle fraction of Saccharomyces cerevisiae, obtained from the crude ribosomal material, and containing the fatty acid synthetase, consisted primarily of 27S and 47S components. This fraction has a protein-ribonucleic acid ratio of about 13. Electron micrographs showed particles ranging in diameter between 100 and 300 A in this material. By use of density gradient analysis, the fatty acid synthetase was found in the 47S component. This component contained particles which were predominantly 300 A in diameter and which were considerably flatter than ribosomes, and it consisted almost entirely of protein. Images PMID:6025308

  3. Nitrogen and carbon source balance determines longevity, independently of fermentative or respiratory metabolism in the yeast Saccharomyces cerevisiae.

    PubMed

    Santos, Júlia; Leitão-Correia, Fernanda; Sousa, Maria João; Leão, Cecília

    2016-04-26

    Dietary regimens have proven to delay aging and age-associated diseases in several eukaryotic model organisms but the input of nutritional balance to longevity regulation is still poorly understood. Here, we present data on the role of single carbon and nitrogen sources and their interplay in yeast longevity. Data demonstrate that ammonium, a rich nitrogen source, decreases chronological life span (CLS) of the prototrophic Saccharomyces cerevisiae strain PYCC 4072 in a concentration-dependent manner and, accordingly, that CLS can be extended through ammonium restriction, even in conditions of initial glucose abundance. We further show that CLS extension depends on initial ammonium and glucose concentrations in the growth medium, as long as other nutrients are not limiting. Glutamine, another rich nitrogen source, induced CLS shortening similarly to ammonium, but this effect was not observed with the poor nitrogen source urea. Ammonium decreased yeast CLS independently of the metabolic process activated during aging, either respiration or fermentation, and induced replication stress inhibiting a proper cell cycle arrest in G0/G1 phase. The present results shade new light on the nutritional equilibrium as a key factor on cell longevity and may contribute for the definition of interventions to promote life span and healthy aging.

  4. Mobilomics in Saccharomyces cerevisiae strains

    PubMed Central

    2013-01-01

    Background Mobile Genetic Elements (MGEs) are selfish DNA integrated in the genomes. Their detection is mainly based on consensus–like searches by scanning the investigated genome against the sequence of an already identified MGE. Mobilomics aims at discovering all the MGEs in a genome and understanding their dynamic behavior: The data for this kind of investigation can be provided by comparative genomics of closely related organisms. The amount of data thus involved requires a strong computational effort, which should be alleviated. Results Our approach proposes to exploit the high similarity among homologous chromosomes of different strains of the same species, following a progressive comparative genomics philosophy. We introduce a software tool based on our new fast algorithm, called regender, which is able to identify the conserved regions between chromosomes. Our case study is represented by a unique recently available dataset of 39 different strains of S.cerevisiae, which regender is able to compare in few minutes. By exploring the non–conserved regions, where MGEs are mainly retrotransposons called Tys, and marking the candidate Tys based on their length, we are able to locate a priori and automatically all the already known Tys and map all the putative Tys in all the strains. The remaining putative mobile elements (PMEs) emerging from this intra–specific comparison are sharp markers of inter–specific evolution: indeed, many events of non–conservation among different yeast strains correspond to PMEs. A clustering based on the presence/absence of the candidate Tys in the strains suggests an evolutionary interconnection that is very similar to classic phylogenetic trees based on SNPs analysis, even though it is computed without using phylogenetic information. Conclusions The case study indicates that the proposed methodology brings two major advantages: (a) it does not require any template sequence for the wanted MGEs and (b) it can be applied to

  5. Saccharomyces cerevisiae engineered for xylose metabolism exhibits a respiratory response

    Treesearch

    Yong-Su Jin; Jose M. Laplaza; Thomas W. Jeffries

    2004-01-01

    Native strains of Saccharomyces cerevisiae do not assimilate xylose. S. cerevisiae engineered for D-xylose utilization through the heterologous expression of genes for aldose reductase ( XYL1), xylitol dehydrogenase (XYL2), and D-xylulokinase ( XYL3 or XKS1) produce only limited amounts of ethanol in xylose medium. In recombinant S. cerevisiae expressing XYL1, XYL2,...

  6. Glycerol stress in Saccharomyces cerevisiae: Cellular responses and evolved adaptations.

    PubMed

    Mattenberger, Florian; Sabater-Muñoz, Beatriz; Hallsworth, John E; Fares, Mario A

    2017-03-01

    Glycerol synthesis is key to central metabolism and stress biology in Saccharomyces cerevisiae, yet the cellular adjustments needed to respond and adapt to glycerol stress are little understood. Here, we determined impacts of acute and chronic exposures to glycerol stress in S. cerevisiae. Glycerol stress can result from an increase of glycerol concentration in the medium due to the S. cerevisiae fermenting activity or other metabolic activities. Acute glycerol-stress led to a 50% decline in growth rate and altered transcription of more than 40% of genes. The increased genetic diversity in S. cerevisiae population, which had evolved in the standard nutrient medium for hundreds of generations, led to an increase in growth rate and altered transcriptome when such population was transferred to stressful media containing a high concentration of glycerol; 0.41 M (0.990 water activity). Evolution of S. cerevisiae populations during a 10-day period in the glycerol-containing medium led to transcriptome changes and readjustments to improve control of glycerol flux across the membrane, regulation of cell cycle, and more robust stress response; and a remarkable increase of growth rate under glycerol stress. Most of the observed regulatory changes arose in duplicated genes. These findings elucidate the physiological mechanisms, which underlie glycerol-stress response, and longer-term adaptations, in S. cerevisiae; they also have implications for enigmatic aspects of the ecology of this otherwise well-characterized yeast.

  7. Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae

    PubMed Central

    Ryzhova, Tatyana A.; Inge-Vechtomov, Sergey G.; Galkin, Alexey P.

    2016-01-01

    The concept of "protein-based inheritance" defines prions as epigenetic determinants that cause several heritable traits in eukaryotic microorganisms, such as Saccharomyces cerevisiae and Podospora anserina. Previously, we discovered a non-chromosomal factor, [NSI+], which possesses the main features of yeast prions, including cytoplasmic infectivity, reversible curability, dominance, and non-Mendelian inheritance in meiosis. This factor causes omnipotent suppression of nonsense mutations in strains of S. cerevisiae bearing a deleted or modified Sup35 N-terminal domain. In this work, we identified protein determinants of [NSI+] using an original method of proteomic screening for prions. The suppression of nonsense mutations in [NSI+] strains is determined by the interaction between [SWI+] and [PIN+] prions. Using genetic and biochemical methods, we showed that [SWI+] is the key determinant of this nonsense suppression, whereas [PIN+] does not cause nonsense suppression by itself but strongly enhances the effect of [SWI+]. We demonstrated that interaction of [SWI+] and [PIN+] causes inactivation of SUP45 gene that leads to nonsense suppression. Our data show that prion interactions may cause heritable traits in Saccharomyces cerevisiae. PMID:28027291

  8. Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae.

    PubMed

    Nizhnikov, Anton A; Ryzhova, Tatyana A; Volkov, Kirill V; Zadorsky, Sergey P; Sopova, Julia V; Inge-Vechtomov, Sergey G; Galkin, Alexey P

    2016-12-01

    The concept of "protein-based inheritance" defines prions as epigenetic determinants that cause several heritable traits in eukaryotic microorganisms, such as Saccharomyces cerevisiae and Podospora anserina. Previously, we discovered a non-chromosomal factor, [NSI+], which possesses the main features of yeast prions, including cytoplasmic infectivity, reversible curability, dominance, and non-Mendelian inheritance in meiosis. This factor causes omnipotent suppression of nonsense mutations in strains of S. cerevisiae bearing a deleted or modified Sup35 N-terminal domain. In this work, we identified protein determinants of [NSI+] using an original method of proteomic screening for prions. The suppression of nonsense mutations in [NSI+] strains is determined by the interaction between [SWI+] and [PIN+] prions. Using genetic and biochemical methods, we showed that [SWI+] is the key determinant of this nonsense suppression, whereas [PIN+] does not cause nonsense suppression by itself but strongly enhances the effect of [SWI+]. We demonstrated that interaction of [SWI+] and [PIN+] causes inactivation of SUP45 gene that leads to nonsense suppression. Our data show that prion interactions may cause heritable traits in Saccharomyces cerevisiae.

  9. Saccharomyces cerevisiae metabolism in ecological context

    PubMed Central

    Jouhten, Paula; Ponomarova, Olga; Gonzalez, Ramon; Patil, Kiran R.

    2016-01-01

    The architecture and regulation of Saccharomyces cerevisiae metabolic network are among the best studied owing to its widespread use in both basic research and industry. Yet, several recent studies have revealed notable limitations in explaining genotype–metabolic phenotype relations in this yeast, especially when concerning multiple genetic/environmental perturbations. Apparently unexpected genotype–phenotype relations may originate in the evolutionarily shaped cellular operating principles being hidden in common laboratory conditions. Predecessors of laboratory S. cerevisiae strains, the wild and the domesticated yeasts, have been evolutionarily shaped by highly variable environments, very distinct from laboratory conditions, and most interestingly by social life within microbial communities. Here we present a brief review of the genotypic and phenotypic peculiarities of S. cerevisiae in the context of its social lifestyle beyond laboratory environments. Accounting for this ecological context and the origin of the laboratory strains in experimental design and data analysis would be essential in improving the understanding of genotype–environment–phenotype relationships. PMID:27634775

  10. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.

    PubMed

    Fossati, Elena; Narcross, Lauren; Ekins, Andrew; Falgueyret, Jean-Pierre; Martin, Vincent J J

    2015-01-01

    Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-reticuline. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes.

  11. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Nevoigt, Elke

    2008-01-01

    Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate. PMID:18772282

  12. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae

    PubMed Central

    Fossati, Elena; Narcross, Lauren; Ekins, Andrew; Falgueyret, Jean-Pierre; Martin, Vincent J. J.

    2015-01-01

    Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-reticuline. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes. PMID:25905794

  13. Genetic mapping of quantitative phenotypic traits in Saccharomyces cerevisiae.

    PubMed

    Swinnen, Steve; Thevelein, Johan M; Nevoigt, Elke

    2012-03-01

    Saccharomyces cerevisiae has become a favorite production organism in industrial biotechnology presenting new challenges to yeast engineers in terms of introducing advantageous traits such as stress tolerances. Exploring subspecies diversity of S. cerevisiae has identified strains that bear industrially relevant phenotypic traits. Provided that the genetic basis of such phenotypic traits can be identified inverse engineering allows the targeted modification of production strains. Most phenotypic traits of interest in S. cerevisiae strains are quantitative, meaning that they are controlled by multiple genetic loci referred to as quantitative trait loci (QTL). A straightforward approach to identify the genetic basis of quantitative traits is QTL mapping which aims at the allocation of the genetic determinants to regions in the genome. The application of high-density oligonucleotide arrays and whole-genome re-sequencing to detect genetic variations between strains has facilitated the detection of large numbers of molecular markers thus allowing high-resolution QTL mapping over the entire genome. This review focuses on the basic principle and state of the art of QTL mapping in S. cerevisiae. Furthermore we discuss several approaches developed during the last decade that allow down-scaling of the regions identified by QTL mapping to the gene level. We also emphasize the particular challenges of QTL mapping in nonlaboratory strains of S. cerevisiae.

  14. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. 866.5785 Section 866.5785 Food and Drugs FOOD AND DRUG ADMINISTRATION... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S...

  15. Viruses and prions of Saccharomyces cerevisiae.

    PubMed

    Wickner, Reed B; Fujimura, Tsutomu; Esteban, Rosa

    2013-01-01

    Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses, and prions. Studies of the mechanisms of virus and prion replication, virus structure, and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular components: chromosomally encoded proteins necessary for blocking the propagation of the viruses and prions, and proteins involved in the expression of viral components. Here, we emphasize the L-A dsRNA virus and its killer-toxin-encoding satellites, the 20S and 23S ssRNA naked viruses, and the several infectious proteins (prions) of yeast.

  16. Microautophagy in the yeast Saccharomyces cerevisiae.

    PubMed

    Uttenweiler, Andreas; Mayer, Andreas

    2008-01-01

    Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar membrane invagination. At the tip of an autophagic tube vesicles (autophagic bodies) pinch off into thevacuolar lumen for degradation. Formation of autophagic tubes is topologically equivalent to other budding processes directed away from the cytosolic environment, e.g., the invagination of multivesicular endosomes, retroviral budding, piecemeal microautophagy of the nucleus and micropexophagy. This clearly distinguishes microautophagy from other membrane fission events following budding toward the cytosol. Such processes are implicated in transport between organelles like the plasma membrane, the endoplasmic reticulum (ER), and the Golgi. Over many years microautophagy only could be characterized microscopically. Recent studies provided the possibility to study the process in vitro and have identified the first molecules that are involved in microautophagy.

  17. Synchronization of the Budding Yeast Saccharomyces cerevisiae.

    PubMed

    Foltman, Magdalena; Molist, Iago; Sanchez-Diaz, Alberto

    2016-01-01

    A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle.

  18. Viruses and prions of Saccharomyces cerevisiae

    PubMed Central

    Wickner, Reed B.; Fujimura, Tsutomu; Esteban, Rosa

    2014-01-01

    Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses and prions. Studies of the mechanisms of virus and prion replication, virus structure and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular components: chromosomally encoded proteins necessary for or blocking the propagation of the viruses and prions, and proteins involved in expression of viral components. Here we emphasize the L-A dsRNA virus and its killer-toxin-encoding satellites, the 20S and 23S ssRNA naked viruses, and the several infectious proteins (prions) of yeast. PMID:23498901

  19. Saccharomyces cerevisiae var. boulardii fungemia following probiotic treatment.

    PubMed

    Appel-da-Silva, Marcelo C; Narvaez, Gabriel A; Perez, Leandro R R; Drehmer, Laura; Lewgoy, Jairo

    2017-12-01

    Probiotics are commonly prescribed as an adjuvant in the treatment of antibiotic-associated diarrhea caused by Clostridium difficile. We report the case of an immunocompromised 73-year-old patient on chemotherapy who developed Saccharomyces cerevisiae var. boulardii fungemia in a central venous catheter during treatment of antibiotic-associated pseudomembranous colitis with the probiotic Saccharomyces cerevisiae var. boulardii. Fungemia was resolved after interruption of probiotic administration without the need to replace the central venous line.

  20. Cell wall construction in Saccharomyces cerevisiae.

    PubMed

    Klis, Frans M; Boorsma, Andre; De Groot, Piet W J

    2006-02-01

    In this review, we discuss new insights in cell wall architecture and cell wall construction in the ascomycetous yeast Saccharomyces cerevisiae. Transcriptional profiling studies combined with biochemical work have provided ample evidence that the cell wall is a highly adaptable organelle. In particular, the protein population that is anchored to the stress-bearing polysaccharides of the cell wall, and forms the interface with the outside world, is highly diverse. This diversity is believed to play an important role in adaptation of the cell to environmental conditions, in growth mode and in survival. Cell wall construction is tightly controlled and strictly coordinated with progression of the cell cycle. This is reflected in the usage of specific cell wall proteins during consecutive phases of the cell cycle and in the recent discovery of a cell wall integrity checkpoint. When the cell is challenged with stress conditions that affect the cell wall, a specific transcriptional response is observed that includes the general stress response, the cell wall integrity pathway and the calcineurin pathway. This salvage mechanism includes increased expression of putative cell wall assemblases and some potential cross-linking cell wall proteins, and crucial changes in cell wall architecture. We discuss some more enzymes involved in cell wall construction and also potential inhibitors of these enzymes. Finally, we use both biochemical and genomic data to infer that the architectural principles used by S. cerevisiae to build its cell wall are also used by many other ascomycetous yeasts and also by some mycelial ascomycetous fungi.

  1. Mechanisms of ethanol tolerance in Saccharomyces cerevisiae.

    PubMed

    Ma, Menggen; Liu, Z Lewis

    2010-07-01

    Saccharomyces cerevisiae is a superb ethanol producer, yet is also sensitive to higher ethanol concentrations especially under high gravity or very high gravity fermentation conditions. Ethanol tolerance is associated with interplay of complex networks at the genome level. Although significant efforts have been made to study ethanol stress response in past decades, mechanisms of ethanol tolerance are not well known. With developments of genome sequencing and genomic technologies, our understanding of yeast biology has been revolutionarily advanced. More evidence of mechanisms of ethanol tolerance have been discovered involving multiple loci, multi-stress, and complex interactions as well as signal transduction pathways and regulatory networks. Transcription dynamics and profiling studies of key gene sets including heat shock proteins provided insight into tolerance mechanisms. A transient gene expression response or a stress response to ethanol does not necessarily lead to ethanol tolerance in yeast. Reprogrammed pathways and interactions of cofactor regeneration and redox balance observed from studies of tolerant yeast demonstrated the significant importance of a time-course study for ethanol tolerance. In this review, we focus on current advances of our understanding for ethanol-tolerance mechanisms of S. cerevisiae including gene expression responses, pathway-based analysis, signal transduction and regulatory networks. A prototype of global system model for mechanisms of ethanol tolerance is presented.

  2. A Saccharomyces cerevisiae mutant with increased virulence.

    PubMed

    Wheeler, Robert T; Kupiec, Martin; Magnelli, Paula; Abeijon, Claudia; Fink, Gerald R

    2003-03-04

    Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of S. cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent ssd1 Delta/ssd1 Delta yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response.

  3. Killer systems of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Nesterova, G.F.

    1989-01-01

    The killer systems of Saccharomyces cerevisiae are an unusual class of cytoplasmic symbionts of primitive eukaryotes. The genetic material of these symbionts is double-stranded RNA. They are characterized by the linearity of the genome, its fragmentation into a major and a minor fraction, which replicate separately, and their ability to control the synthesis of secretory mycocin proteins possessing a toxic action on closely related strains. The secretion of mycocins at the same time ensures acquiring of resistance to them. Strains containing killer symbionts are toxigenic and resistant to the action of their own toxin, but strains that are free of killer double-stranded RNAs are sensitive to the action of mycocins. The killer systems of S. cerevisiae have retained features relating them to viruses and are apparently the result of evolution of infectious viruses. The occurrences of such systems among monocellular eukaryotic organisms is an example of complication of the genome by means of its assembly from virus-like components. We discuss the unusual features of replication and the expression of killer systems and their utilization in the construction of vector molecules.

  4. Fluid-phase endocytosis in yeasts other than Saccharomyces cerevisiae.

    PubMed

    Fernandez, N; Puente, P; Leal, F

    1990-05-01

    A FITC-dextran internalization assay with Saccharomyces cerevisiae as positive control was used to determine whether fluid-phase endocytosis is a general characteristic of yeasts. Schizosaccharomyces pombe, Pichia polymorpha, Kluyveromyces phaseolosporus, Yarrowia lipolytica and Candida albicans were clearly positive, whereas results obtained with Debaryomyces marama were inconclusive. In all cases internalized FITC-dextran was found to be localized in the vacuoles and the process was always time- and temperature-dependent. Lower eucaryotes, particularly yeasts, appear to have the ability to incorporate substances from the extracellular medium through fluid-phase endocytosis.

  5. Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae

    Treesearch

    Yong-Su Jin; Thomas W. Jeffries

    2004-01-01

    Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast raditionally...

  6. The SUP35 omnipotent suppressor gene is involved in the maintenance of the non-Mendelian determinant [psi+] in the yeast Saccharomyces cerevisiae.

    PubMed

    Ter-Avanesyan, M D; Dagkesamanskaya, A R; Kushnirov, V V; Smirnov, V N

    1994-07-01

    The SUP35 gene of yeast Saccharomyces cerevisiae encodes a 76.5-kD ribosome-associated protein (Sup35p), the C-terminal part of which exhibits a high degree of similarity to EF-1 alpha elongation factor, while its N-terminal region is unique. Mutations in or overexpression of the SUP35 gene can generate an omnipotent suppressor effect. In the present study the SUP35 wild-type gene was replaced with deletion alleles generated in vitro that encode Sup35p lacking all or a part of the unique N-terminal region. These 5'-deletion alleles lead, in a haploid strain, simultaneously to an antisuppressor effect and to loss of the non-Mendelian determinant [psi+]. The antisuppressor effect is dominant while the elimination of the [psi+] determinant is a recessive trait. A set of the plasmid-borne deletion alleles of the SUP35 gene was tested for the ability to maintain [psi+]. It was shown that the first 114 amino acids of Sup35p are sufficient to maintain the [psi+] determinant. We propose that the Sup35p serves as a trans-acting factor required for the maintenance of [psi+].

  7. Regulation of Phosphatidylcholine Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Waechter, Charles J.; Lester, Robert L.

    1971-01-01

    Evidence is presented which indicates that the biosynthesis of phosphatidylcholine by the methylation pathway in growing cultures of Saccharomyces cerevisiae is repressed by the presence of choline in the growth medium. This result, obtained previously for glucose-grown cells, was also observed for lactate-grown cells, of which half of the phosphatidylcholine is mitochondrial. A respiration-deficient mutant of the parent wild-type strain has been studied, and its inability to form functional mitochondria cannot be due to an impaired methylation pathway, as it has been shown to incorporate 14C-CH3-methionine into all of the methylated glycerophosphatides. The incorporation rate is depressed by the inclusion of 1 mm choline in the growth medium, suggesting a regulatory effect similar to that demonstrated for the wild-type strain. The effects of choline on the glycerophospholipid composition of lactate and glucose-grown cells is presented. The repressive effects of the two related bases, mono- and dimethylethanolamine, were examined, and reduced levels of 14C-CH3-methionine incorporation were found for cells grown in the presence of these bases. The effect of choline on the methylation rates is reversible and glucosegrown cells regain the nonrepressed level of methylation activity in 60 to 80 min after removal of choline from the growth medium. Images PMID:5547992

  8. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.

    PubMed

    Weinert, Brian T; Iesmantavicius, Vytautas; Moustafa, Tarek; Schölz, Christian; Wagner, Sebastian A; Magnes, Christoph; Zechner, Rudolf; Choudhary, Chunaram

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation accumulated in growth-arrested cells in a manner that depended on acetyl-CoA generation in distinct subcellular compartments. Mitochondrial acetylation levels correlated with acetyl-CoA concentration in vivo and acetyl-CoA acetylated lysine residues nonenzymatically in vitro. We developed a method to estimate acetylation stoichiometry and found that the vast majority of mitochondrial and cytoplasmic acetylation had a very low stoichiometry. However, mitochondrial acetylation occurred at a significantly higher basal level than cytoplasmic acetylation, consistent with the distinct acetylation dynamics and higher acetyl-CoA concentration in mitochondria. High stoichiometry acetylation occurred mostly on histones, proteins present in histone acetyltransferase and deacetylase complexes, and on transcription factors. These data show that a majority of acetylation occurs at very low levels in exponentially growing yeast and is uniformly affected by exposure to acetyl-CoA.

  9. A biochemically structured model for Saccharomyces cerevisiae.

    PubMed

    Lei, F; Rotbøll, M; Jørgensen, S B

    2001-07-12

    A biochemically structured model for the aerobic growth of Saccharomyces cerevisiae on glucose and ethanol is presented. The model focuses on the pyruvate and acetaldehyde branch points where overflow metabolism occurs when the growth changes from oxidative to oxido-reductive. The model is designed to describe the onset of aerobic alcoholic fermentation during steady-state as well as under dynamical conditions, by triggering an increase in the glycolytic flux using a key signalling component which is assumed to be closely related to acetaldehyde. An investigation of the modelled process dynamics in a continuous cultivation revealed multiple steady states in a region of dilution rates around the transition between oxidative and oxido-reductive growth. A bifurcation analysis using the two external variables, the dilution rate, D, and the inlet concentration of glucose, S(f), as parameters, showed that a fold bifurcation occurs close to the critical dilution rate resulting in multiple steady-states. The region of dilution rates within which multiple steady states may occur depends strongly on the substrate feed concentration. Consequently a single steady state may prevail at low feed concentrations, whereas multiple steady states may occur over a relatively wide range of dilution rates at higher feed concentrations.

  10. Cold Osmotic Shock in Saccharomyces cerevisiae

    PubMed Central

    Patching, J. W.; Rose, A. H.

    1971-01-01

    Saccharomyces cerevisiae NCYC 366 is susceptible to cold osmotic shock. Exponentially growing cells from batch cultures grown in defined medium at 30 C, after being suspended in 0.8 m mannitol containing 10 mm ethylenedia-minetetraacetic acid and then resuspended in ice-cold 0.5 mm MgCl2, accumulated the nonmetabolizable solutes d-glucosamine-hydrochloride and 2-aminoisobutyrate at slower rates than unshocked cells; shocked cells retained their viability. Storage of unshocked batch-grown cells in buffer at 10 C led to an increase in ability to accumulate glucosamine, and further experiments were confined to cells grown in a chemostat under conditions of glucose limitation, thereby obviating the need for storing cells before use. A study was made of the effect of the different stages in the cold osmotic shock procedure, including the osmotic stress, the chelating agent, and the cold Mg2+-containing diluent, on viability and solute-accumulating ability. Growth of shocked cells in defined medium resembled that of unshocked cells; however, in malt extract-yeast extract-glucose-peptone medium, the shocked cells had a longer lag phase of growth and initially grew at a slower rate. Cold osmotic shock caused the release of low-molecular-weight compounds and about 6 to 8% of the cell protein. Neither the cell envelope enzymes, invertase, acid phosphatase and l-leucine-β-naphthylamidase, nor the cytoplasmic enzyme, alkaline phosphatase, were released when yeast cells were subjected to cold osmotic shock. PMID:5001201

  11. Methylamine and ammonia transport in Saccharomyces cerevisiae.

    PubMed Central

    Roon, R J; Even, H L; Dunlop, P; Larimore, F L

    1975-01-01

    Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the vmax for methylamine transport was similar 17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 muM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70 percent when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (Mea-R) simultaneously exhibited a decreased ability to transport ammonia. PMID:236281

  12. Stationary phase in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Werner-Washburne, M; Braun, E; Johnston, G C; Singer, R A

    1993-01-01

    Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion, (vi) membrane biosynthesis, and (vii) cell polarity. The exact nature of the relationship between these processes and survival during stationary phase remains to be elucidated. We propose that cell cycle arrest coordinated with the ability to remain viable in the absence of additional nutrients provides a good operational definition of starvation-induced stationary phase. PMID:8393130

  13. Omics analysis of acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Geng, Peng; Zhang, Liang; Shi, Gui Yang

    2017-05-01

    Acetic acid is an inhibitor in industrial processes such as wine making and bioethanol production from cellulosic hydrolysate. It causes energy depletion, inhibition of metabolic enzyme activity, growth arrest and ethanol productivity losses in Saccharomyces cerevisiae. Therefore, understanding the mechanisms of the yeast responses to acetic acid stress is essential for improving acetic acid tolerance and ethanol production. Although 329 genes associated with acetic acid tolerance have been identified in the Saccharomyces genome and included in the database ( http://www.yeastgenome.org/observable/resistance_to_acetic_acid/overview ), the cellular mechanistic responses to acetic acid remain unclear in this organism. Post-genomic approaches such as transcriptomics, proteomics, metabolomics and chemogenomics are being applied to yeast and are providing insight into the mechanisms and interactions of genes, proteins and other components that together determine complex quantitative phenotypic traits such as acetic acid tolerance. This review focuses on these omics approaches in the response to acetic acid in S. cerevisiae. Additionally, several novel strains with improved acetic acid tolerance have been engineered by modifying key genes, and the application of these strains and recently acquired knowledge to industrial processes is also discussed.

  14. Investigation of the Best Saccharomyces cerevisiae Growth Condition.

    PubMed

    Salari, Roshanak; Salari, Rosita

    2017-01-01

    Saccharomyces cerevisiae is known as one of the useful yeasts which are utilized in baking and other industries. It can be easily cultured at an economic price. Today the introduction of safe and efficient carriers is being considered. Due to its generally round shape, and the volume that is enclosed by its membrane and cell wall, it is used to encapsulate active materials to protect them from degradation or to introduce a sustained release drug delivery system. Providing the best conditions in order to achieve the best morphological properties of Saccharomyces cerevisiae as a carrier. In this research, the most suitable growth condition of yeast cells which provides the best size for use as drug carriers was found by a bioreactor in a synthetic culture medium. Yeast cell reproduction and growth curves were obtained, based on pour plate colony counting data and UV/Visible sample absorption at 600 nm. Yeast cell growth patterns and growth rates were determined by Matlab mathematical software. Results showed that pH=4 and dissolving oxygen (DO) 5% was the best condition for yeast cells to grow and reproduce. This condition also provided the largest size (2 × 3 μ) yeast cells. Owing to the yeast cells' low-cost production and their structural characteristics, they could be used as potent drug carriers. This work was supported by a grant from the Vice Chancellor of Research of Mashhad University of Medical Sciences.

  15. Investigation of the Best Saccharomyces cerevisiae Growth Condition

    PubMed Central

    Salari, Roshanak; Salari, Rosita

    2017-01-01

    Introduction Saccharomyces cerevisiae is known as one of the useful yeasts which are utilized in baking and other industries. It can be easily cultured at an economic price. Today the introduction of safe and efficient carriers is being considered. Due to its generally round shape, and the volume that is enclosed by its membrane and cell wall, it is used to encapsulate active materials to protect them from degradation or to introduce a sustained release drug delivery system. Providing the best conditions in order to achieve the best morphological properties of Saccharomyces cerevisiae as a carrier. Methods In this research, the most suitable growth condition of yeast cells which provides the best size for use as drug carriers was found by a bioreactor in a synthetic culture medium. Yeast cell reproduction and growth curves were obtained, based on pour plate colony counting data and UV/Visible sample absorption at 600 nm. Yeast cell growth patterns and growth rates were determined by Matlab mathematical software. Results Results showed that pH=4 and dissolving oxygen (DO) 5% was the best condition for yeast cells to grow and reproduce. This condition also provided the largest size (2 × 3 μ) yeast cells. Conclusion Owing to the yeast cells’ low-cost production and their structural characteristics, they could be used as potent drug carriers. Funding This work was supported by a grant from the Vice Chancellor of Research of Mashhad University of Medical Sciences. PMID:28243411

  16. Antimicrobial action of palmarosa oil (Cymbopogon martinii) on Saccharomyces cerevisiae.

    PubMed

    Prashar, Anjali; Hili, Pauline; Veness, Robert G; Evans, Christine S

    2003-07-01

    The essential oil extracted from palmarosa (Cymbopogon martinii) has proven anti-microbial properties against cells of Saccharomyces cerevisiae. Low concentrations of the oil (0.1%) inhibited the growth of S. cerevisiae cells completely. The composition of the sample of palmarosa oil was determined as 65% geraniol and 20% geranyl acetate as confirmed by GC-FTIR. The effect of palmarosa oil in causing K(+) leakage from yeast cells was attributed mainly to geraniol. Some leakage of magnesium ions was also observed. Blocking potassium membrane channels with caesium ions before addition of palmarosa oil did not change the extent of K(+) ion leakage, which was equal to the total sequestered K(+) in the cells. Palmarosa oil led to changes in the composition of the yeast cell membrane, with more saturated and less unsaturated fatty acids in the membrane after exposure of S. cerevisiae cells to the oil. Some of the palmarosa oil was lost by volatilization during incubation of the oil with the yeast cells. The actual concentration of the oil components affecting the yeast cells could not therefore be accurately determined.

  17. Ciclohexadespipeptide beauvericin degradation by different strains of Saccharomyces cerevisiae.

    PubMed

    Meca, G; Zhou, T; Li, X Z; Ritieni, A; Mañes, J

    2013-09-01

    The interaction between the mycotoxin beauvericin (BEA) and 9 yeast strains of Saccharomyces cerevisiae named LO9, YE-2, YE5, YE-6, YE-4, A34, A17, A42 and A08 was studied. The biological degradations were carried out under aerobic conditions in the liquid medium of Potato Dextrose Broth (PDB) at 25°C for 48 h and in a food/feed system composed of corn flour at 37°C for 3 days, respectively. BEA present in fermented medium and corn flour was determined using liquid chromatography coupled to the mass spectrometry detector in tandem (LC-MS/MS) and the BEA degradation products produced during the fermentations were determined using the technique of the liquid chromatography coupled to a linear ion trap (LIT). Results showed that the S. cerevisiae strains reduced meanly the concentration of the BEA present in PDB by 86.2% and in a food system by 71.1%. All the S. cerevisiae strains used in this study showed a significant BEA reduction during the fermentation process employed.

  18. Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae

    SciTech Connect

    Yannai, S.; Berdicevsky, I.; Duek, L. )

    1991-01-01

    Saccharomyces cerevisiae and Candida albicans were incubated with 0.25, 0.5, or 0.75 {mu}g of Hg (as HgCl{sub 2}) per ml of Nelson's medium in the presence of trace amounts of oxygen at 28{degree}C for 12 days. Two control media were used, one without added Hg and one without yeast inoculum. Yeast cell growth was estimated after 1, 2, 3, and 8 days of incubation. The contents of organomercury in the system and of elemental mercury released from the media and collected in traps were determined at the end of the experiments. The results were as follows: (1) C. albicans was the more mercury-resistant species, but both yeast species failed to grown in the media containing 0.75 {mu}g of Hg per ml.; (2) The amounts of organomercury produced by the two species were proportional to the amount of HgCl{sub 2} added to the medium. In all cases C. albicans produced considerably larger amounts of methylmercury than S. cerevisiae; (3) The amounts of elemental Hg produced were inversely proportional to the HgCl{sub 2} level added in the case of S. cerevisiae but were all similar in the case of C. albicans;and (4) Neither organomercury nor elemental Hg was produced in any of the control media.

  19. Effects of cyclohexane, an industrial solvent, on the yeast Saccharomyces cerevisiae and on isolated yeast mitochondria

    SciTech Connect

    Uribe, S.; Rangel, P.; Espinola, G.; Aguirre, G. )

    1990-07-01

    Little information on the effects of cyclohexane at the cellular or subcellular level is available. In Saccharomyces cerevisiae, cyclohexane inhibited respiration and diverse energy-dependent processes. In mitochondria isolated from S. cerevisiae, oxygen uptake and ATP synthesis were inhibited, although ATPase activity was not affected. Cyclohexane effects were similar to those reported for beta-pinene and limonene, suggesting that the cyclohexane ring in these monoterpenes may be a determinant for their biological activities.

  20. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae.

    PubMed

    Comitini, Francesca; Gobbi, Mirko; Domizio, Paola; Romani, Cristina; Lencioni, Livio; Mannazzu, Ilaria; Ciani, Maurizio

    2011-08-01

    Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation). Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Regulation of Cation Balance in Saccharomyces cerevisiae

    PubMed Central

    Cyert, Martha S.; Philpott, Caroline C.

    2013-01-01

    All living organisms require nutrient minerals for growth and have developed mechanisms to acquire, utilize, and store nutrient minerals effectively. In the aqueous cellular environment, these elements exist as charged ions that, together with protons and hydroxide ions, facilitate biochemical reactions and establish the electrochemical gradients across membranes that drive cellular processes such as transport and ATP synthesis. Metal ions serve as essential enzyme cofactors and perform both structural and signaling roles within cells. However, because these ions can also be toxic, cells have developed sophisticated homeostatic mechanisms to regulate their levels and avoid toxicity. Studies in Saccharomyces cerevisiae have characterized many of the gene products and processes responsible for acquiring, utilizing, storing, and regulating levels of these ions. Findings in this model organism have often allowed the corresponding machinery in humans to be identified and have provided insights into diseases that result from defects in ion homeostasis. This review summarizes our current understanding of how cation balance is achieved and modulated in baker’s yeast. Control of intracellular pH is discussed, as well as uptake, storage, and efflux mechanisms for the alkali metal cations, Na+ and K+, the divalent cations, Ca2+ and Mg2+, and the trace metal ions, Fe2+, Zn2+, Cu2+, and Mn2+. Signal transduction pathways that are regulated by pH and Ca2+ are reviewed, as well as the mechanisms that allow cells to maintain appropriate intracellular cation concentrations when challenged by extreme conditions, i.e., either limited availability or toxic levels in the environment. PMID:23463800

  2. Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions, using alcohol dehydrogenase as sensor reaction.

    PubMed

    Bekers, K M; Heijnen, J J; van Gulik, W M

    2015-08-01

    With the current quantitative metabolomics techniques, only whole-cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH-catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose-limited chemostats under steady-state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose-limited steady-state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction.

  3. Molecular and enological characterization of a natural Saccharomyces uvarum and Saccharomyces cerevisiae hybrid.

    PubMed

    Pérez-Torrado, Roberto; González, Sara Susana; Combina, Mariana; Barrio, Eladio; Querol, Amparo

    2015-07-02

    Saccharomyces cerevisiae plays a main role in the winemaking process, although other species, like Saccharomyces uvarum or Saccharomyces paradoxus, have been associated with must fermentations. It has been reported in recent years, that yeast hybrids of different Saccharomyces species might be responsible for wine productions. Although S. cerevisiae×Saccharomyces kudriavzevii hybrids have been well studied, very little attention has been paid to S. cerevisiae×S. uvarum hybrids. In this work we characterized the genomic composition of S6U, a widely used commercial S. cerevisiae×S. uvarum yeast hybrid isolated in wine fermentations containing one copy of the genome of each parental species, which suggests a relatively recent hybridization event. We also studied its performance under diverse enological conditions. The results show enhanced performance under low temperature enological conditions, increased glycerol production, lower acetic acid production and increased production of interesting aroma compounds. We also examined the transcriptomic response of the S6U hybrid strain compared with the reference species under enological conditions. The results show that although the hybrid strain transcriptome is more similar to S. uvarum than to S. cerevisiae, it presents specifically regulated genes involved in stress response, lipids and amino acid metabolism. The enological performance and aroma profile of this S. cerevisiae×S. uvarum hybrid makes it a good candidate for participating in winemaking, especially at low temperatures.

  4. A global topology map of the Saccharomyces cerevisiae membrane proteome

    NASA Astrophysics Data System (ADS)

    Kim, Hyun; Melén, Karin; Österberg, Marie; von Heijne, Gunnar

    2006-07-01

    The yeast Saccharomyces cerevisiae is, arguably, the best understood eukaryotic model organism, yet comparatively little is known about its membrane proteome. Here, we report the cloning and expression of 617 S. cerevisiae membrane proteins as fusions to a C-terminal topology reporter and present experimentally constrained topology models for 546 proteins. By homology, the experimental topology information can be extended to 15,000 membrane proteins from 38 fully sequenced eukaryotic genomes. membrane proteins | membrane proteomics | yeast

  5. Saccharomyces cerevisiae vaginitis: microbiology and in vitro antifungal susceptibility.

    PubMed

    Echeverría-Irigoyen, María Julia; Eraso, Elena; Cano, Josep; Gomáriz, María; Guarro, Josep; Quindós, Guillermo

    2011-09-01

    Genitourinary infections by Saccharomyces cerevisiae are rare. Here, we describe eight S. cerevisiae vulvovaginitis episodes where molecular (Affirm VPIII) and conventional microbiological methods (culture and carbohydrate assimilation) have proven to be inadequate for diagnostic purposes. DNA sequencing allowed the correct identification of the pathogen. All isolates were susceptible to most antifungal agents, with two of them also found to be susceptible-dose-dependent to itraconazole.

  6. Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women.

    PubMed

    Papaemmanouil, V; Georgogiannis, N; Plega, M; Lalaki, J; Lydakis, D; Dimitriou, M; Papadimitriou, A

    2011-12-01

    Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare. The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus. Vaginal samples were collected from a total of 262 (asymptomatic and symptomatic) women with vaginitis attending the centre of family planning of General hospital of Piraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptibility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae. A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker's yeast. Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Aging and senescence of the budding yeast Saccharomyces cerevisiae.

    PubMed

    Jazwinski, S M

    1990-03-01

    The budding yeast Saccharomyces cerevisiae has a limited life span, defined by the number of times an individual cell divides. Longevity in this organism involves a genetic component. Several morphological and physiological changes are associated with yeast aging and senescence. One of these, an increase in generation time with age, provides a 'biomarker' for the aging process. This increase in generation time has revealed the operation of a 'senescence factor(s)', which is likely to be a product of age-specific gene expression. The Cell Spiral Model indicates coordination of successive cell cycles to be inherent in the determination of life span. It is proposed that life expectancy depends on the function of a stochastic trigger during aging that sets in motion a programme leading to cell senescence and death.

  8. Response of Saccharomyces cerevisiae strains to antineoplastic agents.

    PubMed

    Delitheos, A; Karavokyros, I; Tiligada, E

    1995-10-01

    The effect of several antineoplastic agents on Saccharomyces cerevisiae strains has been investigated. Minimum inhibitory concentration (MIC), minimum cytotoxic concentration (MCC) and median effective concentration (EC50) were determined to identify strains with inherent sensitivity to the agents tested. Several strains proved to be sensitive to the antimetabolites 5-fluorouracil and methotrexate as well as to doxorubicin and cis-platine. On the contrary m-amsacrine, procarbazine, vinca alcaloids, melphalan and hydroxyurea were inactive at concentrations up to 400 micrograms ml-1. The strain ATCC 2366, the most relatively sensitive to the agents tested, was used for studying the effect of treatment duration and of drug concentration on cell survival. Methotrexate and cis-platine, which according to MIC and MCC tests seemed ineffective for this strain, reduced survival significantly after 6 h of treatment. A correlation of the shape of the survival curves with MIC and MCC values was attempted.

  9. The HOG MAP kinase pathway is required for the induction of methylglyoxal-responsive genes and determines methylglyoxal resistance in Saccharomyces cerevisiae.

    PubMed

    Aguilera, Jaime; Rodríguez-Vargas, Sonia; Prieto, Jose A

    2005-04-01

    A sudden overaccumulation of methylglyoxal (MG) induces, in Saccharomyces cerevisiae, the expression of MG-protective genes, including GPD1, GLO1 and GRE3. The response is partially dependent on the transcriptional factors Msn2p/Msn4p, but unrelated with the general stress response mechanism. Here, we show that the high-osmolarity glycerol (HOG)-pathway controls the genetic response to MG and determines the yeast growth capacity upon MG exposure. Strains lacking the MAPK Hog1p, the upstream component Ssk1p or the HOG-dependent nuclear factor Msn1p, showed a reduction in the mRNA accumulation of MG-responsive genes after MG addition. Moreover, hyperactivation of Hog1p by deletion of protein phosphatase PTP2 enhanced the response, while blocking the pathway by deletion of the MAPKK PBS2 had a negative effect. In addition, the activity of Hog1p affected the basal level of GPD1 mRNA under non-inducing conditions. These effects had a great influence on MG resistance, as hog1Delta and other HOG-pathway mutants with impaired MG-specific expression displayed MG sensitivity, whereas those with enhanced expression exhibited MG resistance as compared with the wild-type. However, MG does not trigger the overphosphorylation of Hog1p or its nuclear import in the parental strain. Moreover, dual phosphorylation of Hog1p appears to be dispensable in the triggering of the transcriptional response, although a phosphorylable form of Hog1p is fundamental for the transcriptional activity. Overall, our results suggest that the basal activity of the HOG-pathway serves to amplify the expression of MG-responsive genes under non-inducing and inducing conditions, ensuring cell protection against this toxic glycolytic by-product.

  10. Phosphate uptake in Saccharomyces cerevisiae Hansen wild type and phenotypes exposed to space flight irradiation.

    PubMed Central

    Berry, D; Volz, P A

    1979-01-01

    Rates of phosphate uptake were approximately twice as great for Saccharomyces cerevisiae single-cell phenotypic isolates exposed to space parameters as for the wild-type ground control. Quantitative determination of 32P was performed by liquid scintillation spectrometry utilizing Cerenkov radiation counting techniques. PMID:395899

  11. Modulation of the acute phase response in feedlot steers supplemented with Saccharomyces cerevisiae

    USDA-ARS?s Scientific Manuscript database

    This study was designed to determine the effect of supplementing feedlot steers with Saccharomyces cerevisiae CNCM I-1079 (SC) on the acute phase response to a lipopolysaccharide (LPS) challenge. Steers (n = 18; 266 ± 4 kilograms body weight) were separated into three treatment groups (n = 6/treatm...

  12. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

    The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins. It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules. It is carried out efficiently and is exquisitely sensitive to the needs of the cell. Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed. The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies. This tandem array has led to unique ways of carrying out a number of functions. Replication is asymmetric and does not initiate from every autonomously replicating sequence. Recombination is suppressed. Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit. Genes for many ribosomal proteins have been cloned and sequenced. Few are linked; most are duplicated; most have an intron. There is extensive homology between yeast ribosomal proteins and those of other species. Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor. This factor binds also to many other places in the genome, including the telomeres. There is coordinated transcription of the ribosomal protein genes under a variety of conditions. However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein. A deficiency causes slow growth. Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min. Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins. However, in the case of ribosomal protein L32

  13. Interaction between Hanseniaspora uvarum and Saccharomyces cerevisiae during alcoholic fermentation.

    PubMed

    Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio

    2015-08-03

    During wine fermentation, Saccharomyces clearly dominate over non-Saccharomyces wine yeasts, and several factors could be related to this dominance. However, the main factor causing the reduction of cultivable non-Saccharomyces populations has not yet been fully established. In the present study, various single and mixed fermentations were performed to evaluate some of the factors likely responsible for the interaction between Saccharomyces cerevisiae and Hanseniaspora uvarum. Alcoholic fermentation was performed in compartmented experimental set ups with ratios of 1:1 and 1:9 and the cultivable population of both species was followed. The cultivable H. uvarum population decreased sharply at late stages when S. cerevisiae was present in the other compartment, similarly to alcoholic fermentations in non-compartmented vessels. Thus, cell-to-cell contact did not seem to be the main cause for the lack of cultivability of H. uvarum. Other compounds related to fermentation performance (such as sugar and ethanol) and/or certain metabolites secreted by S. cerevisiae could be related to the sharp decrease in H. uvarum cultivability. When these factors were analyzed, it was confirmed that metabolites from S. cerevisiae induced lack of cultivability in H. uvarum, however ethanol and other possible compounds did not seem to induce this effect but played some role during the process. This study contributes to a new understanding of the lack of cultivability of H. uvarum populations during the late stages of wine fermentation.

  14. Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae

    USDA-ARS?s Scientific Manuscript database

    The yeast Saccharomyces cerevisiae is a superb ethanol producer, yet sensitive to ethanol at higher concentrations especially under high gravity or very high gravity fermentation conditions. Although significant efforts have been made to study ethanol-stress response in past decades, molecular mecha...

  15. Improving biomass sugar utilization by engineered Saccharomyces cerevisiae

    USDA-ARS?s Scientific Manuscript database

    The efficient utilization of all available sugars in lignocellulosic biomass, which is more abundant than available commodity crops and starch, represents one of the most difficult technological challenges for the production of bioethanol. The well-studied yeast Saccharomyces cerevisiae has played a...

  16. Global mapping of DNA conformational flexibility on Saccharomyces cerevisiae.

    PubMed

    Menconi, Giulia; Bedini, Andrea; Barale, Roberto; Sbrana, Isabella

    2015-04-01

    In this study we provide the first comprehensive map of DNA conformational flexibility in Saccharomyces cerevisiae complete genome. Flexibility plays a key role in DNA supercoiling and DNA/protein binding, regulating DNA transcription, replication or repair. Specific interest in flexibility analysis concerns its relationship with human genome instability. Enrichment in flexible sequences has been detected in unstable regions of human genome defined fragile sites, where genes map and carry frequent deletions and rearrangements in cancer. Flexible sequences have been suggested to be the determinants of fragile gene proneness to breakage; however, their actual role and properties remain elusive. Our in silico analysis carried out genome-wide via the StabFlex algorithm, shows the conserved presence of highly flexible regions in budding yeast genome as well as in genomes of other Saccharomyces sensu stricto species. Flexibile peaks in S. cerevisiae identify 175 ORFs mapping on their 3'UTR, a region affecting mRNA translation, localization and stability. (TA)n repeats of different extension shape the central structure of peaks and co-localize with polyadenylation efficiency element (EE) signals. ORFs with flexible peaks share common features. Transcripts are characterized by decreased half-life: this is considered peculiar of genes involved in regulatory systems with high turnover; consistently, their function affects biological processes such as cell cycle regulation or stress response. Our findings support the functional importance of flexibility peaks, suggesting that the flexible sequence may be derived by an expansion of canonical TAYRTA polyadenylation efficiency element. The flexible (TA)n repeat amplification could be the outcome of an evolutionary neofunctionalization leading to a differential 3'-end processing and expression regulation in genes with peculiar function. Our study provides a new support to the functional role of flexibility in genomes and a

  17. Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

    PubMed Central

    Liu, Ling-ling; Jia, Bo; Zhao, Fang; Huang, Wei-dong; Zhan, Ji-cheng

    2015-01-01

    At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu2+ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu2+ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu2+ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress. PMID:26030864

  18. Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation.

    PubMed

    Sun, Xiang-Yu; Zhao, Yu; Liu, Ling-Ling; Jia, Bo; Zhao, Fang; Huang, Wei-Dong; Zhan, Ji-Cheng

    2015-01-01

    At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu2+ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu2+ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu2+ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China's stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.

  19. Effects of a Saccharomyces cerevisiae culture on in vitro mixed ruminal microorganism fermentation.

    PubMed

    Sullivan, H M; Martin, S A

    1999-09-01

    Previous research has shown that Saccharomyces cerevisiae culture increases lactate utilization and cellulose digestion by pure cultures of ruminal bacteria. Based on these pure culture results, in vitro mixed ruminal microorganism fermentations were conducted to determine the effects of 0.35 and 0.73 g/L of Sacc. cerevisiae culture on the fermentation of ground corn, maltose, alfalfa hay, bermudagrass hay, and lactate. In addition, experiments were performed to evaluate the effects of Sacc. cerevisiae culture and monensin on the mixed ruminal microorganism fermentation. In the presence of ground corn, both concentrations of Sacc. cerevisiae culture had little effect on final pH or fermentation products, except the 0.35 g/L treatment increased valerate concentration. Saccharomyces cerevisiae culture had little effect on final pH or fermentation products in maltose or lactate fermentations. When alfalfa hay was the substrate, 0.73 g/L of Sacc. cerevisiae culture increased propionate concentration and both treatments decreased the acetate to propionate ratio. In the case of Coastal bermudagrass hay, 0.73 g/L Sacc. cerevisiae culture increased concentrations of acetate, propionate, CH4, butyrate, isovalerate, valerate, and decreased the acetate to propionate ratio, whereas both treatments increased total volatile fatty acid concentrations. Similar to alfalfa hay, in vitro dry matter disappearance of Coastal bermudagrass hay was numerically increased in the presence of Sacc. cerevisiae culture. Monensin altered the fermentation by decreasing concentrations of CH4 and lactate and increasing concentrations of propionate. There was no interaction between Sacc. cerevisiae culture and monensin. In conclusion, the incorporation of Sacc. cerevisiae culture into mixed ruminal microorganism fermentations of ground corn, maltose, or lactate had little effect on final pH and fermentation products. However, in the presence of alfalfa hay or Coastal bermudagrass hay Sacc

  20. Regulation of phosphatidylserine synthase from Saccharomyces cerevisiae by phospholipid precursors.

    PubMed Central

    Poole, M A; Homann, M J; Bae-Lee, M S; Carman, G M

    1986-01-01

    The addition of ethanolamine or choline to inositol-containing growth medium of Saccharomyces cerevisiae wild-type cells resulted in a reduction of membrane-associated phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activity in cell extracts. The reduction of activity did not occur when inositol was absent from the growth medium. Under the growth conditions where a reduction of enzyme activity occurred, there was a corresponding qualitative reduction of enzyme subunit as determined by immunoblotting with antiserum raised against purified phosphatidylserine synthase. Water-soluble phospholipid precursors did not effect purified phosphatidylserine synthase activity. Phosphatidylserine synthase (activity and enzyme subunit) was not regulated by the availability of water-soluble phospholipid precursors in S. cerevisiae VAL2C(YEp CHO1) and the opi1 mutant. VAL2C(YEp CHO1) is a plasmid-bearing strain that over produces phosphatidylserine synthase activity, and the opi1 mutant is an inositol biosynthesis regulatory mutant. The results of this study suggest that the regulation of phosphatidylserine synthase by the availability of phospholipid precursors occurs at the level of enzyme formation and not at the enzyme activity level. Furthermore, the regulation of phosphatidylserine synthase is coupled to inositol synthesis. Images PMID:3023284

  1. Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae.

    PubMed

    Billingsley, John M; DeNicola, Anthony B; Barber, Joyann S; Tang, Man-Cheng; Horecka, Joe; Chu, Angela; Garg, Neil K; Tang, Yi

    2017-09-20

    Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous 'ene'-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  2. Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

    PubMed Central

    THOMSEN, RUNE; LIBRI, DOMENICO; BOULAY, JOCELYNE; ROSBASH, MICHAEL; JENSEN, TORBEN HEICK

    2003-01-01

    In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retaining RNAs in these foci; on deletion of the exosome component Rrp6p, the RNA is released. To determine the exact nuclear location of retained as well as released mRNAs, we have used mRNA export mutant strains to analyze the spatial relationship between newly synthesized heat shock mRNA, the chromosomal site of transcription, and known S. cerevisiae nuclear structures such as the nucleolus and the nucleolar body. Our results show that retained SSA4 RNA localizes to an area in close proximity to the SSA4 locus. On deletion of Rrp6p and release from the genomic locus, heat shock mRNAs produced in the rat7–1 strain colocalize predominantly with nucleolar antigens. Bulk poly(A)+ RNA, on the other hand, is localized primarily to the nuclear rim. Interestingly, the RNA binding nucleocytoplasmic shuttle protein Npl3p shows strong colocalization with bulk poly(A)+ RNA, regardless of its nuclear location. Taken together, our data show that retention occurs close to the gene and indicate distinct nuclear fates of different mRNAs. PMID:12923254

  3. Electrochemical insights into the ethanol tolerance of Saccharomyces cerevisiae.

    PubMed

    Wang, Min; Zhao, Jinsheng; Yang, Zhenyu; Du, Zhankui; Yang, Zhengyu

    2007-11-01

    It is expected that intracellular redox activity may closely related to catabolic states of living cells, based on which a mediated electrochemical method has been proposed to measure the ethanol tolerance of the yeast Saccharomyces cerevisiae AS 3800. The couple menadione/ferricyanide was employed as a carrier mediator system, sensing intracellular redox activity. Microelectrode voltammetric method was introduced to assay the ferrocyanide accumulations arising from menadione mediated reduction of ferricyanide by the yeast. The mediated electrochemical study show that the maximal ethanol tolerance limit of S. cerevisiae is about 25% (v/v) ethanol, which is consistent with the result obtained by the conventional fermentative ability measurement. Moreover, the electrochemical method for the first time confirmed that the specific activities of the glycolytic and alcohologenic enzymes within intact living cells remained high by the presence of sublethal ethanol, which was only predicted by in vitro enzymatic assay and cannot be measured by conventional method. The new method can be used as an easy and rapid method to determine the maximal ethanol tolerance of yeast cells.

  4. Intracellular metabolite profiling of Saccharomyces cerevisiae evolved under furfural.

    PubMed

    Jung, Young Hoon; Kim, Sooah; Yang, Jungwoo; Seo, Jin-Ho; Kim, Kyoung Heon

    2017-03-01

    Furfural, one of the most common inhibitors in pre-treatment hydrolysates, reduces the cell growth and ethanol production of yeast. Evolutionary engineering has been used as a selection scheme to obtain yeast strains that exhibit furfural tolerance. However, the response of Saccharomyces cerevisiae to furfural at the metabolite level during evolution remains unknown. In this study, evolutionary engineering and metabolomic analyses were applied to determine the effects of furfural on yeasts and their metabolic response to continuous exposure to furfural. After 50 serial transfers of cultures in the presence of furfural, the evolved strains acquired the ability to stably manage its physiological status under the furfural stress. A total of 98 metabolites were identified, and their abundance profiles implied that yeast metabolism was globally regulated. Under the furfural stress, stress-protective molecules and cofactor-related mechanisms were mainly induced in the parental strain. However, during evolution under the furfural stress, S. cerevisiae underwent global metabolic allocations to quickly overcome the stress, particularly by maintaining higher levels of metabolites related to energy generation, cofactor regeneration and recovery from cellular damage. Mapping the mechanisms of furfural tolerance conferred by evolutionary engineering in the present study will be led to rational design of metabolically engineered yeasts. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  5. Genomic Evolution of Saccharomyces cerevisiae under Chinese Rice Wine Fermentation

    PubMed Central

    Li, Yudong; Zhang, Weiping; Zheng, Daoqiong; Zhou, Zhan; Yu, Wenwen; Zhang, Lei; Feng, Lifang; Liang, Xinle; Guan, Wenjun; Zhou, Jingwen; Chen, Jian; Lin, Zhenguo

    2014-01-01

    Rice wine fermentation represents a unique environment for the evolution of the budding yeast, Saccharomyces cerevisiae. To understand how the selection pressure shaped the yeast genome and gene regulation, we determined the genome sequence and transcriptome of a S. cerevisiae strain YHJ7 isolated from Chinese rice wine (Huangjiu), a popular traditional alcoholic beverage in China. By comparing the genome of YHJ7 to the lab strain S288c, a Japanese sake strain K7, and a Chinese industrial bioethanol strain YJSH1, we identified many genomic sequence and structural variations in YHJ7, which are mainly located in subtelomeric regions, suggesting that these regions play an important role in genomic evolution between strains. In addition, our comparative transcriptome analysis between YHJ7 and S288c revealed a set of differentially expressed genes, including those involved in glucose transport (e.g., HXT2, HXT7) and oxidoredutase activity (e.g., AAD10, ADH7). Interestingly, many of these genomic and transcriptional variations are directly or indirectly associated with the adaptation of YHJ7 strain to its specific niches. Our molecular evolution analysis suggested that Japanese sake strains (K7/UC5) were derived from Chinese rice wine strains (YHJ7) at least approximately 2,300 years ago, providing the first molecular evidence elucidating the origin of Japanese sake strains. Our results depict interesting insights regarding the evolution of yeast during rice wine fermentation, and provided a valuable resource for genetic engineering to improve industrial wine-making strains. PMID:25212861

  6. Potential immobilized Saccharomyces cerevisiae as heavy metal removal

    NASA Astrophysics Data System (ADS)

    Raffar, Nur Izzati Abdul; Rahman, Nadhratul Nur Ain Abdul; Alrozi, Rasyidah; Senusi, Faraziehan; Chang, Siu Hua

    2015-05-01

    Biosorption of copper ion using treated and untreated immobilized Saccharomyces cerevisiae from aqueous solution was investigate in this study. S.cerevisiae has been choosing as biosorbent due to low cost, easy and continuously available from various industries. In this study, the ability of treated and untreated immobilized S.cerevisiae in removing copper ion influence by the effect of pH solution, and initial concentration of copper ion with contact time. Besides, adsorption isotherm and kinetic model also studied. The result indicated that the copper ion uptake on treated and untreated immobilized S.cerevisiae was increased with increasing of contact time and initial concentration of copper ion. The optimum pH for copper ion uptake on untreated and treated immobilized S.cerevisiae at 4 and 6. From the data obtained of copper ion uptake, the adsorption isotherm was fitted well by Freundlich model for treated immobilized S.cerevisiae and Langmuir model for untreated immobilized S.cerevisiae according to high correlation coefficient. Meanwhile, the pseudo second order was described as suitable model present according to high correlation coefficient. Since the application of biosorption process has been received more attention from numerous researchers as a potential process to be applied in the industry, future study will be conducted to investigate the potential of immobilized S.cerevisiae in continuous process.

  7. The Saccharomyces cerevisiae enolase-related regions encode proteins that are active enolases.

    PubMed

    Kornblatt, M J; Richard Albert, J; Mattie, S; Zakaib, J; Dayanandan, S; Hanic-Joyce, P J; Joyce, P B M

    2013-02-01

    In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase-related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2-phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double-null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase-related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality ▿

    PubMed Central

    Weigert, Claudia; Steffler, Fabian; Kurz, Tomas; Shellhammer, Thomas H.; Methner, Frank-Jürgen

    2009-01-01

    The measurement of yeast's intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good (r2 = 0.898, n = 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases (P = 0.433), ICP via flow cytometry did (P = 0.012). Yeast in an exponential phase has a unimodal ICP distribution, reflective of a homogeneous population; however, yeast in a stationary phase displays a broader ICP distribution, and subpopulations could be defined by using the flow cytometry method. In conclusion, flow cytometry yielded specific evidence of the heterogeneity in vitality of a yeast population as measured via ICP. In contrast to photometry, flow cytometry increases information about the yeast population's vitality via a short measurement, which is suitable for routine analysis. PMID:19581482

  9. ULTRAVIOLET MICROSCOPY OF THE VACUOLE OF SACCHAROMYCES CEREVISIAE DURING SPORULATION

    PubMed Central

    Svihla, G.; Dainko, J. L.; Schlenk, F.

    1964-01-01

    Svihla, G. (Argonne National Laboratory, Argonne, Ill.), J. L. Dainko, and F. Schlenk. Ultraviolet microscopy of the vacuole of Saccharomyces cerevisiae during sporulation. J. Bacteriol. 88:449–456. 1964.—Normal cells of Saccharomyces cerevisiae and cells containing, in their vacuoles, large quantities of S-adenosylmethionine were induced to sporulate. In the latter case, the strong ultraviolet absorption of the compound permitted photomicrographic observation of cytological detail. Chromatographic and spectrophotometric analyses of cell extracts supplemented the cytological studies. The vacuole is abolished at the onset of sporulation, and its contents may be observed temporarily in the intersporular space. As sporulation progresses, the material is discharged into the culture medium. Sporulation of both types of cells also leads to a release of nucleic acid fragments into the culture medium. Images PMID:14203363

  10. SOME FACTORS AFFECTING STEROL FORMATION IN SACCHAROMYCES CEREVISIAE1

    PubMed Central

    Starr, Patricia R.; Parks, L. W.

    1962-01-01

    Starr, Patricia R. (Oregon State University, Corvallis) and L. W. Parks. Some factors affecting sterol formation in Saccharomyces cerevisiae. J. Bacteriol. 83:1042–1046. 1962.—A wild-type diploid strain of Saccharomyces cerevisiae was used in a study of factors that influence sterol synthesis. Maltose, glucose, sodium acetate, and ethanol were shown to be readily available for sterol synthesis in growing cultures of yeast. In cells grown anaerobically and then exposed to various substrates in aerobic resting-cell suspension, only glucose and ethanol stimulated ergosterol formation. Under these conditions, sterol synthesis was directly proportional to the amount of glucose provided. Sulfanilamide decreased the yield of sterol in growing cells, but had no effect on sterol synthesis by resting cultures. PMID:13916377

  11. Efficient Extraction of Thioreodoxin from Saccharomyces cerevisiae by Ethanol▿

    PubMed Central

    Inoue, Yoshiharu; Nomura, Wataru; Takeuchi, Yoko; Ohdate, Takumi; Tamasu, Shogo; Kitaoka, Atsushi; Kiyokawa, Yoshifumi; Masutani, Hiroshi; Murata, Kazuo; Wakai, Yoshinori; Izawa, Shingo; Yodoi, Junji

    2007-01-01

    Thioredoxin, an antioxidant protein, is a promising molecule for development of functional foods because it protects the gastric mucosa and reduces the allergenicity of allergens. To establish a method for obtaining an ample amount of yeast thioredoxin, we found here that thioredoxin is released from Saccharomyces cerevisiae by treatment with 20% ethanol. We also found that Japanese sake contains a considerable amount of thioredoxin. PMID:17209065

  12. Indentation with atomic force microscope, Saccharomyces cerevisiae cell gains elasticity under ethanol stress.

    PubMed

    Niu, Yuan-Pu; Lin, Xiang-Hua; Dong, Shi-Jun; Yuan, Qi-Peng; Li, Hao

    2016-10-01

    During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane is the first target to be attacked by the accumulated ethanol. In such a prominent position, S. cerevisiae cell membrane could reversely provide protection through changing fluidity or elasticity secondary to remodeled membrane components or structure during the fermentation process. However, there is yet to be a direct observation of the real effect of the membrane compositional change. In this study, atomic force microscope-based strategy was performed to determine Young's modulus of S. cerevisiae to directly clarify ethanol stress-associated changes and roles of S. cerevisiae cell membrane fluidity and elasticity. Cell survival rate decreased while the cell swelling rate and membrane permeability increased as ethanol concentration increased from 0% to 20% v/v. Young's modulus decreased continuously from 3.76MPa to 1.53MPa while ethanol stress increased from 0% to 20% v/v, indicating that ethanol stress induced the S. cerevisiae membrane fluidity and elasticity changes. Combined with the fact that membrane composition varies under ethanol stress, to some extent, this could be considered as a forced defensive act to the ethanol stress by S. cerevisiae cells. On the other hand, the ethanol stress induced loosening of cell membrane also caused S. cerevisiae cell to proactively remodel membrane to make cell membrane more agreeable to the increase of environmental threat. Increased ethanol stress made S. cerevisiae cell membrane more fluidized and elastic, and eventually further facilitated yeast cell's survival. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific.

    PubMed

    Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio

    2016-01-01

    The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae.

  14. The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific

    PubMed Central

    Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio

    2016-01-01

    The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae. PMID:27148191

  15. Efficient screening of environmental isolates for Saccharomyces cerevisiae strains that are suitable for brewing.

    PubMed

    Fujihara, Hidehiko; Hino, Mika; Takashita, Hideharu; Kajiwara, Yasuhiro; Okamoto, Keiko; Furukawa, Kensuke

    2014-01-01

    We developed an efficient screening method for Saccharomyces cerevisiae strains from environmental isolates. MultiPlex PCR was performed targeting four brewing S. cerevisiae genes (SSU1, AWA1, BIO6, and FLO1). At least three genes among the four were amplified from all S. cerevisiae strains. The use of this method allowed us to successfully obtain S. cerevisiae strains.

  16. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains.

    PubMed

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines.

  17. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

    PubMed Central

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

  18. [Saccharomyces cerevisiae invasive infection: The first reported case in Morocco].

    PubMed

    Maleb, A; Sebbar, E; Frikh, M; Boubker, S; Moussaoui, A; El Mekkaoui, A; Khannoussi, W; Kharrasse, G; Belefquih, B; Lemnouer, A; Ismaili, Z; Elouennass, M

    2017-02-07

    Saccharomyces cerevisiae is a cosmopolitan yeast, widely used in agro-alimentary and pharmaceutical industry. Its impact in human pathology is rare, but maybe still underestimated compared to the real situation. This yeast is currently considered as an emerging and opportunistic pathogen. Risk factors are immunosuppression and intravascular device carrying. Fungemias are the most frequent clinical forms. We report the first case of S. cerevisiae invasive infection described in Morocco, and to propose a review of the literature cases of S. cerevisiae infections described worldwide. A 77-year-old patient, with no notable medical history, who was hospitalized for a upper gastrointestinal stenosis secondary to impassable metastatic gastric tumor. Its history was marked by the onset of septic shock, with S. cerevisiae in his urine and in his blood, with arguments for confirmation of invasion: the presence of several risk factors in the patient, positive direct microbiological examination, abundant and exclusive culture of S. cerevisiae from clinical samples. Species identification was confirmed by the study of biochemical characteristics of the isolated yeast. Confirmation of S. cerevisiae infection requires a clinical suspicion in patients with risk factors, but also a correct microbiological diagnosis.

  19. Redox interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in mixed culture under enological conditions.

    PubMed

    Cheraiti, Naoufel; Guezenec, Stéphane; Salmon, Jean-Michel

    2005-01-01

    Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.

  20. Growth of non-Saccharomyces yeasts affects nutrient availability for Saccharomyces cerevisiae during wine fermentation.

    PubMed

    Medina, Karina; Boido, Eduardo; Dellacassa, Eduardo; Carrau, Francisco

    2012-07-02

    Yeast produces numerous secondary metabolites during fermentation that impact final wine quality. Although it is widely recognized that growth of diverse non-Saccharomyces (NS) yeast can positively affect flavor complexity during Saccharomyces cerevisiae wine fermentation, the inability to control spontaneous or co-fermentation processes by NS yeast has restricted their use in winemaking. We selected two NS yeasts from our Uruguayan native collection to study NS-S. cerevisiae interactions during wine fermentation. The selected strains of Hanseniaspora vineae and Metschnikowia pulcherrima had different yeast assimilable nitrogen consumption profiles and had different effects on S. cerevisiae fermentation and growth kinetics. Studies in which we varied inoculum size and using either simultaneous or sequential inoculation of NS yeast and S. cerevisiae suggested that competition for nutrients had a significant effect on fermentation kinetics. Sluggish fermentations were more pronounced when S. cerevisiae was inoculated 24h after the initial stage of fermentation with a NS strain compared to co-inoculation. Monitoring strain populations using differential WL nutrient agar medium and fermentation kinetics of mixed cultures allowed for a better understanding of strain interactions and nutrient addition effects. Limitation of nutrient availability for S. cerevisiae was shown to result in stuck fermentations as well as to reduce sensory desirability of the resulting wine. Addition of diammonium phosphate (DAP) and a vitamin mix to a defined medium allowed for a comparison of nutrient competition between strains. Addition of DAP and the vitamin mix was most effective in preventing stuck fermentations.

  1. Overproduction of threonine by Saccharomyces cerevisiae mutants resistant to hydroxynorvaline.

    PubMed Central

    Ramos, C; Calderon, I L

    1992-01-01

    In this work, we isolated and characterized mutants that overproduce threonine from Saccharomyces cerevisiae. The mutants were selected for resistance to the threonine analog alpha-amino-beta-hydroxynorvalerate (hydroxynorvaline), and, of these, the ones able to excrete threonine to the medium were chosen. The mutant strains produce between 15 and 30 times more threonine than the wild type does, and, to a lesser degree, they also accumulate isoleucine. Genetic and biochemical studies have revealed that the threonine overproduction is, in all cases studied, associated with the presence in the strain of a HOM3 allele coding for a mutant aspartate kinase that is totally or partially insensitive to feedback inhibition by threonine. This enzyme seems, therefore, to be crucial in the regulation of threonine biosynthesis in S. cerevisiae. The results obtained suggest that this strategy could be efficiently applied to the isolation of threonine-overproducing strains of yeasts other than S. cerevisiae, even those used industrially. PMID:1622238

  2. Antimutagenic and antioxidant activity of Lisosan G in Saccharomyces cerevisiae.

    PubMed

    Frassinetti, Stefania; Della Croce, Clara Maria; Caltavuturo, Leonardo; Longo, Vincenzo

    2012-12-01

    In the present study the antimutagenic and antioxidant effects of a powder of grain (Lisosan G) in yeast Saccharomyces cerevisiae were studied. Results showed that Lisosan G treatment decreased significantly the intracellular ROS concentration and mutagenesis induced by hydrogen peroxide in S. cerevisiae D7 strain. The effect of Lisosan G was then evaluated by using superoxide dismutase (SOD) proficient and deficient strains of S. cerevisiae. Lisosan G showed protective activity in sod1Δ and sod2Δ mutant strains, indicating an in vivo antioxidant effect. A high radical scavenging activity of Lisosan G was also demonstrated in vitro using the oxygen radical absorbance capacity (ORAC) assay. The obtained results showed a protective effect of Lisosan G in yeast cells, indicating that its antioxidant capacity contributes to its antimutagenic action.

  3. Alternative Splicing in Next Generation Sequencing Data of Saccharomyces cerevisiae

    PubMed Central

    Schreiber, Konrad; Csaba, Gergely; Haslbeck, Martin; Zimmer, Ralf

    2015-01-01

    mRNA splicing is required in about 4% of protein coding genes in Saccharomyces cerevisiae. The gene structure of those genes is simple, generally comprising two exons and one intron. In order to characterize the impact of alternative splicing on the S. cerevisiae transcriptome, we perform a systematic analysis of mRNA sequencing data. We find evidence of a pervasive use of alternative splice sites and detect several novel introns both within and outside protein coding regions. We also find a predominance of alternative splicing on the 3’ side of introns, a finding which is consistent with existing knowledge on conservation of exon-intron boundaries in S. cerevisiae. Some of the alternatively spliced transcripts allow for a translation into different protein products. PMID:26469855

  4. Genetic engineering of industrial strains of Saccharomyces cerevisiae.

    PubMed

    Le Borgne, Sylvie

    2012-01-01

    Genetic engineering has been successfully applied to Saccharomyces cerevisiae laboratory strains for different purposes: extension of substrate range, improvement of productivity and yield, elimination of by-products, improvement of process performance and cellular properties, and extension of product range. The potential of genetically engineered yeasts for the massive production of biofuels as bioethanol and other nonfuel products from renewable resources as lignocellulosic biomass hydrolysates has been recognized. For such applications, robust industrial strains of S. cerevisiae have to be used. Here, some relevant genetic and genomic characteristics of industrial strains are discussed in relation to the problematic of the genetic engineering of such strains. General molecular tools applicable to the manipulation of S. cerevisiae industrial strains are presented and examples of genetically engineered industrial strains developed for the production of bioethanol from lignocellulosic biomass are given.

  5. Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation

    PubMed Central

    Guimarães, Pedro MR; Oliveira, Carla

    2010-01-01

    Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, does not have a lactose metabolization system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisiae strains, particularly involving the expression of the lactose genes of the phylogenetically related yeast Kluyveromyces lactis, but also the lactose genes from Escherichia coli and Aspergillus niger, as reviewed here. Due to the existing large amounts of whey, the production of bio-ethanol from lactose by engineered S. cerevisiae has been considered as a possible route for whey surplus. Emphasis is given in the present review on strain improvement for lactose-to-ethanol bioprocesses, namely flocculent yeast strains for continuous high-cell-density systems with enhanced ethanol productivity. PMID:21326922

  6. Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation.

    PubMed

    Domingues, Lucília; Guimarães, Pedro M R; Oliveira, Carla

    2010-01-01

    Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, does not have a lactose metabolization system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisiae strains, particularly involving the expression of the lactose genes of the phylogenetically related yeast Kluyveromyces lactis, but also the lactose genes from Escherichia coli and Aspergillus niger, as reviewed here. Due to the existing large amounts of whey, the production of bio-ethanol from lactose by engineered S. cerevisiae has been considered as a possible route for whey surplus. Emphasis is given in the present review on strain improvement for lactose-to-ethanol bioprocesses, namely flocculent yeast strains for continuous high-cell-density systems with enhanced ethanol productivity.

  7. Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae.

    PubMed

    van Zyl, Willem H; Lynd, Lee R; den Haan, Riaan; McBride, John E

    2007-01-01

    Consolidated bioprocessing (CBP) of lignocellulose to bioethanol refers to the combining of the four biological events required for this conversion process (production of saccharolytic enzymes, hydrolysis of the polysaccharides present in pretreated biomass, fermentation of hexose sugars, and fermentation of pentose sugars) in one reactor. CBP is gaining increasing recognition as a potential breakthrough for low-cost biomass processing. Although no natural microorganism exhibits all the features desired for CBP, a number of microorganisms, both bacteria and fungi, possess some of the desirable properties. This review focuses on progress made toward the development of baker's yeast (Saccharomyces cerevisiae) for CBP. The current status of saccharolytic enzyme (cellulases and hemicellulases) expression in S. cerevisiae to complement its natural fermentative ability is highlighted. Attention is also devoted to the challenges ahead to integrate all required enzymatic activities in an industrial S. cerevisiae strain(s) and the need for molecular and selection strategies pursuant to developing a yeast capable of CBP.

  8. Genomic evolution of Saccharomyces cerevisiae under Chinese rice wine fermentation.

    PubMed

    Li, Yudong; Zhang, Weiping; Zheng, Daoqiong; Zhou, Zhan; Yu, Wenwen; Zhang, Lei; Feng, Lifang; Liang, Xinle; Guan, Wenjun; Zhou, Jingwen; Chen, Jian; Lin, Zhenguo

    2014-09-10

    Rice wine fermentation represents a unique environment for the evolution of the budding yeast, Saccharomyces cerevisiae. To understand how the selection pressure shaped the yeast genome and gene regulation, we determined the genome sequence and transcriptome of a S. cerevisiae strain YHJ7 isolated from Chinese rice wine (Huangjiu), a popular traditional alcoholic beverage in China. By comparing the genome of YHJ7 to the lab strain S288c, a Japanese sake strain K7, and a Chinese industrial bioethanol strain YJSH1, we identified many genomic sequence and structural variations in YHJ7, which are mainly located in subtelomeric regions, suggesting that these regions play an important role in genomic evolution between strains. In addition, our comparative transcriptome analysis between YHJ7 and S288c revealed a set of differentially expressed genes, including those involved in glucose transport (e.g., HXT2, HXT7) and oxidoredutase activity (e.g., AAD10, ADH7). Interestingly, many of these genomic and transcriptional variations are directly or indirectly associated with the adaptation of YHJ7 strain to its specific niches. Our molecular evolution analysis suggested that Japanese sake strains (K7/UC5) were derived from Chinese rice wine strains (YHJ7) at least approximately 2,300 years ago, providing the first molecular evidence elucidating the origin of Japanese sake strains. Our results depict interesting insights regarding the evolution of yeast during rice wine fermentation, and provided a valuable resource for genetic engineering to improve industrial wine-making strains. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Genomic expression program of Saccharomyces cerevisiae along a mixed-culture wine fermentation with Hanseniaspora guilliermondii.

    PubMed

    Barbosa, Catarina; Mendes-Faia, Arlete; Lage, Patrícia; Mira, Nuno P; Mendes-Ferreira, Ana

    2015-08-28

    The introduction of yeast starter cultures consisting in a blend of Saccharomyces cerevisiae and non-Saccharomyces yeast strains is emerging for production of wines with improved complexity of flavor. The rational use of this approach is, however, dependent on knowing the impact that co-inoculation has in the physiology of S. cerevisiae. In this work the transcriptome of S. cerevisiae was monitored throughout a wine fermentation, carried out in single culture or in a consortium with Hanseniaspora guilliermondii, this being the first time that this relevant yeast-yeast interaction is examined at a genomic scale. Co-inoculation with H. guilliermondii reduced the overall genome-wide transcriptional response of S. cerevisiae throughout the fermentation, which was attributable to a lower fermentative activity of S. cerevisiae while in the mixed-fermentation. Approximately 350 genes S. cerevisiae genes were found to be differently expressed (FDR < 0.05) in response to the presence of H. guilliermondii in the fermentation medium. Genes involved in biosynthesis of vitamins were enriched among those up-regulated in the mixed-culture fermentation, while genes related with the uptake and biosynthesis of amino acids were enriched among those more expressed in the single-culture. The differences in the aromatic profiles of wines obtained in the single and in the mixed-fermentations correlated with the differential expression of S. cerevisiae genes encoding enzymes required for formation of aroma compounds. By integrating results obtained in the transcriptomic analysis performed with physiological data our study provided, for the first time, an integrated view into the adaptive responses of S. cerevisiae to the challenging environment of mixed culture fermentation. The availability of nutrients, in particular, of nitrogen and vitamins, stands out as a factor that may determine population dynamics, fermentative activity and by-product formation.

  10. Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.

    PubMed Central

    Gilon, T; Chomsky, O; Kulka, R G

    1998-01-01

    Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes. PMID:9582269

  11. Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae.

    PubMed

    Flis, Vid V; Fankl, Ariane; Ramprecht, Claudia; Zellnig, Günther; Leitner, Erich; Hermetter, Albin; Daum, Günther

    2015-01-01

    In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.

  12. Rapid identification of chemical genetic interactions in Saccharomyces cerevisiae.

    PubMed

    Dilworth, David; Nelson, Christopher J

    2015-04-05

    Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

  13. Anti-Saccharomyces cerevisiae antibodies in primary biliary cirrhosis.

    PubMed

    Sakly, Wahiba; Jeddi, Moncef; Ghedira, Ibtissem

    2008-07-01

    The aim of this study was to evaluate, retrospectively, the frequency of anti-Saccharomyces cerevisiae antibodies (ASCA) in patients with primary biliary cirrhosis (PBC). ASCA, IgG, and IgA, were determined by ELISA in sera of 95 PBC patients; 80 healthy blood donors served as controls. The frequency of ASCA (IgG or IgA) was significantly higher in PBC patients than in the control group (24.2% vs 3.7%, P = 0.0001). The frequency of ASCA IgG and ASCA IgA in PBC patients was also significantly higher than that found in the control group (18.9% vs 2.5%, P = 0.0006 and 11.6% vs 1.2%, P = 0.007, respectively). Six patients out of 95 (6.3%) had both ASCA IgG and ASCA IgA; in contrast, none of the control group had both isotypes (P = 0.02). There was no correlation between ASCA levels and mitochondrial autoantibodies (AMA) titres in PBC patients. We conclude that ASCA are common in patients with PBC.

  14. Identification of genes affecting vacuole membrane fragmentation in Saccharomyces cerevisiae.

    PubMed

    Michaillat, Lydie; Mayer, Andreas

    2013-01-01

    The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

  15. Four Acyltransferases Uniquely Contribute to Phospholipid Heterogeneity in Saccharomyces cerevisiae

    PubMed Central

    Oelkers, Peter; Pokhrel, Keshav

    2016-01-01

    Diverse acyl-CoA species and acyltransferase isoenzymes are components of a complex system that synthesizes glycerophospholipids and triacylglycerols. Saccharomyces cerevisiae has four main acyl-CoA species, two main glycerol-3-phosphate 1-O-acyltransferases (Gat1p, Gat2p), and two main 1-acylglycerol-3-phosphate O-acyltransferases (Lpt1p, Slc1p). The in vivo contribution of these isoenzymes to phospholipid heterogeneity was determined using haploids with compound mutations: gat1Δlpt1Δ, gat2Δlpt1Δ, gat1Δslc1Δ, and gat2Δslc1Δ. All mutations mildly reduced [3H]palmitic acid incorporation into phospholipids relative to triacylglycerol. Electrospray ionization tandem mass spectrometry identified few differences from wild type in gat1Δlpt1Δ, dramatic differences in gat2Δslc1Δ, and intermediate changes in gat2Δlpt1Δ and gat1Δslc1Δ. Yeast expressing Gat1p and Lpt1p had phospholipids enriched with acyl chains that were unsaturated, 18 carbons long, and paired for length. These alterations prevented growth at 18.5°C and in 10% ethanol. Therefore, Gat2p and Slc1p dictate phospholipid acyl chain composition in rich media at 30°C. Slc1p selectively pairs acyl chains of different lengths. PMID:27920551

  16. Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae

    PubMed Central

    Ramprecht, Claudia; Zellnig, Günther; Leitner, Erich; Hermetter, Albin; Daum, Günther

    2015-01-01

    In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity. PMID:26241051

  17. Polyphosphates as an energy source for growth of Saccharomyces cerevisiae.

    PubMed

    Trilisenko, L V; Kulakovskaya, T V

    2014-05-01

    Cells of the yeast Saccharomyces cerevisiae with a low content of polyphosphates (polyP) are characterized by disturbance of growth in medium with 0.5% glucose. The parent strain with polyP level reduced by phosphate starvation had a longer lag phase. The growth rate of strains with genetically determined low content of polyP due to their enhanced hydrolysis (CRN/pMB1_PPN1 Sc is a superproducer of exopolyphosphatase PPN1) or reduced synthesis (the BY4741 vma2Δ mutant with impaired vacuolar membrane energization) was lower in the exponential phase. The growth of cells with high content of polyP was accompanied by polyP consumption. In cells of strains with low content of polyP, CRN/pMB1_PPN1 Sc and BY4741 vma2Δ, their consumption was insignificant. These findings provide more evidence indicating the use of polyP as an extra energy source for maintaining high growth rate.

  18. Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

    PubMed Central

    Sumrada, R A; Chisholm, G; Cooper, T G

    1982-01-01

    Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein. PMID:6152837

  19. Repair of Oxidative DNA Damage in Saccharomyces cerevisiae.

    PubMed

    Chalissery, Jisha; Jalal, Deena; Al-Natour, Zeina; Hassan, Ahmed H

    2017-03-01

    Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Assessing compensation for loss of vacuolar function in Saccharomyces cerevisiae.

    PubMed

    Marshall, Pamela A; Netzel, Nicholas; Guintchev, Jillian Wisby

    2012-02-01

    We analyzed how Saccharomyces cerevisiae cells compensate for the lack of a functional vacuole, an acidic membrane-bound degradative and ion storage compartment. We hypothesized that cells lacking a functional vacuole would compensate for the loss of the functions of the vacuole by altering gene expression and (or) metabolic flux. We used gene expression profiling and Biolog phenotype microarray analysis to determine the compensatory mechanisms of cells lacking vacuolar function. In steady state, vps33 and vps41 cells changed the transcriptional profile of some genes, but no complete pathways were upregulated or downregulated. We treated vps41 cells with calcium to tease out cellular compensation for loss of vacuole function under ionic stress; however, changes in gene expression were not utilized to compensate for loss of vacuole function under stress either, as genes whose transcriptional profiles were changed did not function together in any one cellular process. Phenotype microarray analysis indicated that logarithmically growing vps33 or vps41 cells did not seem to compensate for loss of vacuolar function but instead demonstrated additional pleiotropic phenotypes due to the function of the vacuole. Under rich media conditions, yeast utilize the vacuole to regulate stress, ion response, and peptide degradation. However, loss of the vacuole does not lead to observable compensation mechanisms.

  1. Temperature-sensitive glucosamine auxotroph of Saccharomyces cerevisiae.

    PubMed Central

    Ballou, L; Grove, J R; Roon, R J; Wiggs, J; Ballou, C E

    1981-01-01

    Temperature-sensitive revertants were isolated from Saccharomyces cerevisiae D-glucosamine auxotrophs previously obtained in this laboratory (W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). The auxotrophs lack the enzyme 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (EC 5.3.1.19), and the revertants appear to be temperature sensitive in the formation of enzyme activity. The enzyme they produce under permissive conditions decays in activity at a rate comparable to that of the wild-type enzyme, and it has similar kinetic properties. The homozygous diploid mutant fails to sporulate at the nonpermissive temperature. Temperature shift experiments were carried out in an effort to determine what effect glucosamine deficiency had on mannoprotein secretion as reflected in the formation of external asparaginase. Although the results were complicated by the slow decay of the residual ketol-isomerase activity, they did show that mannoprotein synthesis or secretion was altered when the internal pool of D-glucosamine was depleted. PMID:6765596

  2. Mating-type genes and MAT switching in Saccharomyces cerevisiae.

    PubMed

    Haber, James E

    2012-05-01

    Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATα. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATα1, and MATα2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break.

  3. Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

    PubMed Central

    Dilworth, David; Nelson, Christopher J.

    2015-01-01

    Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described. PMID:25867090

  4. Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae

    PubMed Central

    Haber, James E.

    2012-01-01

    Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATα. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATα1, and MATα2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break. PMID:22555442

  5. Defects arising from whole-genome duplications in Saccharomyces cerevisiae.

    PubMed Central

    Andalis, Alex A; Storchova, Zuzana; Styles, Cora; Galitski, Timothy; Pellman, David; Fink, Gerald R

    2004-01-01

    Comparisons among closely related species have led to the proposal that the duplications found in many extant genomes are the remnants of an ancient polyploidization event, rather than a result of successive duplications of individual chromosomal segments. If this interpretation is correct, it would support Ohno's proposal that polyploidization drives evolution by generating the genetic material necessary for the creation of new genes. Paradoxically, analysis of contemporary polyploids suggests that increased ploidy is an inherently unstable state. To shed light on this apparent contradiction and to determine the effects of nascent duplications of the entire genome, we generated isogenic polyploid strains of the budding yeast Saccharomyces cerevisiae. Our data show that an increase in ploidy results in a marked decrease in a cell's ability to survive during stationary phase in growth medium. Tetraploid cells die rapidly, whereas isogenic haploids remain viable for weeks. Unlike haploid cells, which arrest growth as unbudded cells, tetraploid cells continue to bud and form mitotic spindles in stationary phase. The stationary-phase death of tetraploids can be prevented by mutations or conditions that result in growth arrest. These data show that whole-genome duplications are accompanied by defects that affect viability and subsequent survival of the new organism. PMID:15280227

  6. Oxygen requirements of yeasts. [Saccharomyces cerevisiae; Candida tropicalis

    SciTech Connect

    Visser, W.; Scheffers, W.A.; Batenburg-Van Der Vegte, W.H.; Van Dijken, J.P. )

    1990-12-01

    Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly ({mu}{sub max}, 0.03 and 0.05 h{sup {minus}1}, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.

  7. Dynamics of cell wall structure in Saccharomyces cerevisiae.

    PubMed

    Klis, Frans M; Mol, Pieternella; Hellingwerf, Klaas; Brul, Stanley

    2002-08-01

    The cell wall of Saccharomyces cerevisiae is an elastic structure that provides osmotic and physical protection and determines the shape of the cell. The inner layer of the wall is largely responsible for the mechanical strength of the wall and also provides the attachment sites for the proteins that form the outer layer of the wall. Here we find among others the sexual agglutinins and the flocculins. The outer protein layer also limits the permeability of the cell wall, thus shielding the plasma membrane from attack by foreign enzymes and membrane-perturbing compounds. The main features of the molecular organization of the yeast cell wall are now known. Importantly, the molecular composition and organization of the cell wall may vary considerably. For example, the incorporation of many cell wall proteins is temporally and spatially controlled and depends strongly on environmental conditions. Similarly, the formation of specific cell wall protein-polysaccharide complexes is strongly affected by external conditions. This points to a tight regulation of cell wall construction. Indeed, all five mitogen-activated protein kinase pathways in bakers' yeast affect the cell wall, and additional cell wall-related signaling routes have been identified. Finally, some potential targets for new antifungal compounds related to cell wall construction are discussed.

  8. Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae

    PubMed Central

    Michaillat, Lydie; Mayer, Andreas

    2013-01-01

    The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property. PMID:23383298

  9. Biodegradation of crude oil by Saccharomyces cerevisiae isolated from fermented zobo (locally fermented beverage in Nigeria).

    PubMed

    Abioye, O P; Akinsola, R O; Aransiola, S A; Damisa, D

    2013-12-15

    The increase in demand for crude oil as a source of energy and as a primary raw material for industries has resulted in an increase in its production, transportation and refining, which in turn has resulted in gross pollution of the environment. In this study, Saccharomyces cerevisiae isolated from a commercially prepared local fermented beverage 'zobo' (prepared from Hibiscus flower) was tested to determine its potential to degrade crude oil for a period of 28 days under aerobic condition. The percentage of oil biodegradation was determined using weight loss method and gas chromatography mass spectroscopy (GC/MS) of the residual crude oil after 28 days. At the end of 28 days 49.29% crude oil degradation was recorded. The result suggests the potential of Saccharomyces cerevisiae for bioremediation of oil polluted sites.

  10. Ultrastructural changes of Saccharomyces cerevisiae in response to ethanol stress.

    PubMed

    Ma, Manli; Han, Pei; Zhang, Ruimin; Li, Hao

    2013-09-01

    In the fermentative process using Saccharomyces cerevisiae to produce bioethanol, the performance of cells is often compromised by the accumulation of ethanol. However, the mechanism of how S. cerevisiae responds against ethanol stress remains elusive. In the current study, S. cerevisiae cells were cultured in YPD (yeast extract - peptone - dextrose) medium containing various concentrations of ethanol (0%, 2.5%, 5%, 7.5%, 10%, and 15% (v/v)). Compared with the control group without ethanol, the mean cell volume of S. cerevisiae decreased significantly in the presence of 7.5% and 10% ethanol after incubation for 16 h (P < 0.05), and in the presence of 15% ethanol at all 3 sampling time points (1, 8, and 16 h) (P < 0.05). The exposure of S. cerevisiae cells to ethanol also led to an increase in malonyldialdehyde content (P < 0.05) and a decrease in sulfhydryl group content (P < 0.05). Moreover, the observations through transmission electron microscopy enabled us to relate ultrastructural changes elicited by ethanol with the cellular stress physiology. Under ethanol stress, the integrity of the cell membrane was compromised. The swelling or distortion of mitochondria together with the occurrence of a single and large vacuole was correlated with the addition of ethanol. These results suggested that the cell membrane is one of the targets of ethanol, and the degeneration of mitochondria promoted the accumulation of intracellular reactive oxygen species.

  11. Saccharomyces cerevisiae S288C genome annotation: a working hypothesis

    PubMed Central

    Fisk, Dianna G.; Ball, Catherine A.; Dolinski, Kara; Engel, Stacia R.; Hong, Eurie L.; Issel-Tarver, Laurie; Schwartz, Katja; Sethuraman, Anand; Botstein, David; Cherry, J. Michael

    2011-01-01

    The S. cerevisiae genome is the most well-characterized eukaryotic genome and one of the simplest in terms of identifying open reading frames (ORFs), yet its primary annotation has been updated continually in the decade since its initial release in 1996 (Goffeau et al., 1996). The Saccharomyces Genome Database (SGD; www.yeastgenome.org) (Hirschman et al., 2006), the community-designated repository for this reference genome, strives to ensure that the S. cerevisiae annotation is as accurate and useful as possible. At SGD, the S. cerevisiae genome sequence and annotation are treated as a working hypothesis, which must be repeatedly tested and refined. In this paper, in celebration of the tenth anniversary of the completion of the S. cerevisiae genome sequence, we discuss the ways in which the S. cerevisiae sequence and annotation have changed, consider the multiple sources of experimental and comparative data on which these changes are based, and describe our methods for evaluating, incorporating and documenting these new data. PMID:17001629

  12. Saccharomyces cerevisiae: a nomadic yeast with no niche?

    PubMed Central

    Goddard, Matthew R.; Greig, Duncan

    2015-01-01

    Different species are usually thought to have specific adaptations, which allow them to occupy different ecological niches. But recent neutral ecology theory suggests that species diversity can simply be the result of random sampling, due to finite population sizes and limited dispersal. Neutral models predict that species are not necessarily adapted to specific niches, but are functionally equivalent across a range of habitats. Here, we evaluate the ecology of Saccharomyces cerevisiae, one of the most important microbial species in human history. The artificial collection, concentration and fermentation of large volumes of fruit for alcohol production produce an environment in which S. cerevisiae thrives, and therefore it is assumed that fruit is the ecological niche that S. cerevisiae inhabits and has adapted to. We find very little direct evidence that S. cerevisiae is adapted to fruit, or indeed to any other specific niche. We propose instead a neutral nomad model for S. cerevisiae, which we believe should be used as the starting hypothesis in attempting to unravel the ecology of this important microbe. PMID:25725024

  13. Saccharomyces cerevisiae as a starter culture in Mycella.

    PubMed

    Hansen, T K; Tempel, T V; Cantor, M D; Jakobsen, M

    2001-09-19

    The potential use of Saccharomyces cerevisiae FB7 as an additional starter culture for the production of Mycella, a Danish Gorgonzola type cheese, was investigated. Two dairy productions of Mycella, each containing batches of experimental cheeses with S. cerevisiae added and reference cheeses without yeast added were carried out. For both experimental and reference cheeses, chemical analysis (pH, a(w), NaCl, water and fat content) were carried out during the ripening period, but no significant differences were found. The evolution of lactic acid bacteria was almost identical in both the experimental and reference cheeses and similar results were found for the number of yeast. S. cerevisiae FB7 was found to be predominant in the core of the experimental cheeses throughout the ripening period, while Debaryomyces hansenii dominated in the reference cheese and on the surface of the experimental cheeses. In the cheeses with S. cerevisiae FB7, an earlier sporulation and an improved growth of Penicillium roqueforti was observed compared to the reference cheeses. Furthermore, in the experimental cheese, synergistic interactions were also found in the aroma analysis, the degradation of casein and by the sensory analysis. The observed differences indicate a positive contribution to the overall quality of Mycella by S. cerevisiae FB7.

  14. Identification of Two Saccharomyces cerevisiae Cell Wall Mannan Chemotypes

    PubMed Central

    Cawley, T. N.; Ballou, Clinton E.

    1972-01-01

    We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 → 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 → 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. α-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties. PMID:4559821

  15. [Mitochondria inheritance in yeast saccharomyces cerevisiae].

    PubMed

    Fizikova, A Iu

    2011-01-01

    The review is devoted to the main mechanisms of mitochondria inheritance in yeast Saccharonmyces cerevisiae. The genetic mechanisms of functionally active mitochondria inheritance in eukaryotic cells is one of the most relevant in modem researches. A great number of genetic diseases are associated with mitochondria dysfunction. Plasticity of eukaryotic cell metabolism according to the environmental changes is ensured by adequate mitochondria functioning by means of ATP synthesis coordination, reactive oxygen species accumulation, apoptosis regulation and is an important factor of cell adaptation to stress. Mitochondria participation in important for cell vitality processes masters the presence of accurate mechanisms of mitochondria functions regulation according to environment fluctuations. The mechanisms of mitochondria division and distribution are highly conserved. Baker yeast S. cerevisiae is an ideal model object for mitochondria researches due to energetic metabolism lability, ability to switch over respiration to fermentation, and petite-positive phenotype. Correction of metabolism according to the environmental changes is necessary for cell vitality. The influence of respiratory, carbon, amino acid and phosphate metabolism on mitochondria functions was shown. As far as the mechanisms that stabilize functions of mitochondria and mtDNA are highly conserve, we can project yeast regularities on higher eukaryotes systems. This makes it possible to approximate understanding the etiology and pathogenesis of a great number of human diseases.

  16. Mitochondrial inheritance and fermentative : oxidative balance in hybrids between Saccharomyces cerevisiae and Saccharomyces uvarum.

    PubMed

    Solieri, Lisa; Antúnez, Oreto; Pérez-Ortín, Josè Enrique; Barrio, Eladio; Giudici, Paolo

    2008-07-01

    Breeding between Saccharomyces species is a useful tool for obtaining improved wine yeast strains, combining fermentative features of parental species. In this work, 25 artificial Saccharomyces cerevisiae x Saccharomyces uvarum hybrids were constructed by spore conjugation. A multi-locus PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting six nuclear gene markers and the ribosomal region including the 5.8S rRNA gene and the two internal transcribed spacers, showed that the hybrid genome is the result of two chromosome sets, one coming from S. cerevisiae and the other from S. uvarum. Mitochondrial DNA (mtDNA) typing showed uniparental inheritance in all hybrids. Furthermore, sibling hybrids, obtained by repeated crosses between the same parental strains, showed the same mtDNA, suggesting that the mitochondrial transmission is not stochastic or species-specific, but dependent on the parental strains. Finally four hybrids, two of which with S. cerevisiae mtDNA and two with S. uvarum mtDNA, were subjected to transcriptome analysis. Our results showed that the hybrids bearing S. cerevisiae mtDNA exhibited less expression of genes involved in glycolysis/fermentation pathways and in hexose transport compared to hybrids with S. uvarum mtDNA. Respiration assay confirmed the increased respiratory activity of hybrids with the S. cerevisiae mtDNA genome. These findings suggest that mtDNA type and fermentative : respiratory performances are correlated in S. cerevisiae x S. uvarum hybrids and the mtDNA type is an important trait for constructing new improved hybrids for winemaking.

  17. The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA.

    PubMed

    Matot, Béatrice; Le Bihan, Yann-Vaï; Lescasse, Rachel; Pérez, Javier; Miron, Simona; David, Gabriel; Castaing, Bertrand; Weber, Patrick; Raynal, Bertrand; Zinn-Justin, Sophie; Gasparini, Sylvaine; Le Du, Marie-Hélène

    2012-04-01

    Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.

  18. Hydroxylation state of fatty acid and long-chain base moieties of sphingolipid determine the sensitivity to growth inhibition due to AUR1 repression in Saccharomyces cerevisiae.

    PubMed

    Tani, Motohiro; Kuge, Osamu

    2012-01-13

    The structures of ceramide found in the yeast Saccharomyces cerevisiae are classified into five groups according to the hydroxylation states of the long-chain base and fatty acid moieties. This diversity is created through the action of enzymes encoded by SUR2, SCS7, and as yet unidentified hydroxylation enzyme(s). Aur1p is an enzyme catalyzing the formation of inositol phosphorylceramide in the yeast, and the defect leads to strong growth inhibition due to accumulation of ceramide and reductions in complex sphingolipid levels. In this study, we found that the deletion of SCS7 results in the enhancement of growth inhibition due to repression of AUR1 expression under the control of a tetracycline-regulatable promoter, whereas the deletion of SUR2 attenuates the growth inhibition. Under AUR1-repressive conditions, SCS7 and SUR2 mutants showed reductions in the complex sphingolipid levels and the accumulation of ceramide, like wild-type cells. On the other hand, the deletion of SCS7 had no effect on the growth inhibition through reductions in the complex sphingolipid levels caused by repression of LIP1 encoding a ceramide synthase subunit. Furthermore, the deletion of SUR2 did not suppress the growth inhibition under LIP1-repressive conditions. Therefore, it is suggested that the deletion of sphingolipid hydroxylases changes the toxicity of ceramide under AUR1-repressive conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Loss of lager specific genes and subtelomeric regions define two different Saccharomyces cerevisiae lineages for Saccharomyces pastorianus Group I and II strains.

    PubMed

    Monerawela, Chandre; James, Tharappel C; Wolfe, Kenneth H; Bond, Ursula

    2015-03-01

    Lager yeasts, Saccharomyces pastorianus, are interspecies hybrids between S. cerevisiae and S. eubayanus and are classified into Group I and Group II clades. The genome of the Group II strain, Weihenstephan 34/70, contains eight so-called 'lager-specific' genes that are located in subtelomeric regions. We evaluated the origins of these genes through bioinformatic and PCR analyses of Saccharomyces genomes. We determined that four are of cerevisiae origin while four originate from S. eubayanus. The Group I yeasts contain all four S. eubayanus genes but individual strains contain only a subset of the cerevisiae genes. We identified S. cerevisiae strains that contain all four cerevisiae 'lager-specific' genes, and distinct patterns of loss of these genes in other strains. Analysis of the subtelomeric regions uncovered patterns of loss in different S. cerevisiae strains. We identify two classes of S. cerevisiae strains: ale yeasts (Foster O) and stout yeasts with patterns of 'lager-specific' genes and subtelomeric regions identical to Group I and II S. pastorianus yeasts, respectively. These findings lead us to propose that Group I and II S. pastorianus strains originate from separate hybridization events involving different S. cerevisiae lineages. Using the combined bioinformatic and PCR data, we describe a potential classification map for industrial yeasts. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  20. Overproduction of fatty acids in engineered Saccharomyces cerevisiae.

    PubMed

    Li, Xiaowei; Guo, Daoyi; Cheng, Yongbo; Zhu, Fayin; Deng, Zixin; Liu, Tiangang

    2014-09-01

    The long hydrocarbon fatty acyl chain is energy rich, making it an ideal precursor for liquid transportation fuels and high-value oleo chemicals. As Saccharomyces cerevisiae has many advantages for industrial production compared to Escherichia coli. Here, we attempted to engineer Saccharomyces cerevisiae for overproduction of fatty acids. First, disruption of the beta-oxidation pathway, elimination of the acyl-CoA synthetases, overexpression of different thioesterases and acetyl-CoA carboxylase ACC1, and engineering the supply of precursor acetyl-CoA. The engineered strain XL122 produced more than 120 mg/L of fatty acids. In parallel, we inactivated ADH1, the dominant gene for ethanol production, to redirect the metabolic flux to fatty acids synthesis. The engineered strain DG005 produced about 140 mg/L fatty acids. Additionally, Acetyl-CoA carboxylase was identified as a critical bottleneck of fatty acids synthesis in S. cerevisiae with a cell-free system. However, overexpression of ACC1 has little effect on fatty acids biosynthesis. As it has been reported that phosphorylation of ACC1 may influent its activity, so phosphorylation sites of ACC1 were further identified. Although the regulatory mechanisms remain unclear, our results provide rationale for future studies to target this critical step. All these efforts, particularly the discovery of the limiting step are critical for developing a "cell factory" for the overproduction of fatty acids by using type I fatty acids synthase in yeast or other fungi.

  1. Crystallization and Preliminary X-ray Diffraction Analysis of motif N from Saccharomyces cerevisiae Dbf4

    SciTech Connect

    Matthews, L.; Duong, A; Prasad, A; Duncker, B; Guarne, A

    2009-01-01

    The Cdc7-Dbf4 complex plays an instrumental role in the initiation of DNA replication and is a target of replication-checkpoint responses in Saccharomyces cerevisiae. Cdc7 is a conserved serine/threonine kinase whose activity depends on association with its regulatory subunit, Dbf4. A conserved sequence near the N-terminus of Dbf4 (motif N) is necessary for the interaction of Cdc7-Dbf4 with the checkpoint kinase Rad53. To understand the role of the Cdc7-Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. A complete native data set was collected at 100 K from crystals that diffracted X-rays to 2.75 {angstrom} resolution and structure determination is currently under way.

  2. Ethanol production from carob extract by using Saccharomyces cerevisiae.

    PubMed

    Turhan, Irfan; Bialka, Katherine L; Demirci, Ali; Karhan, Mustafa

    2010-07-01

    Carob has been widely grown in the Mediterranean region for a long time. It has been regarded as only a forest tree and has been neglected for other economical benefits. However, in recent years, this fruit has gained attention for several applications. As petroleum has become depleted, renewable energy production has started to gain attention all over the world; including the production of ethanol from underutilized agricultural products such as carob. In this project, the optimum extraction conditions were determined for the carob fruit by using the response surface design method. The obtained extract was utilized for production of ethanol by using suspended Saccharomyces cerevisiae fermentation. The effect of various fermentation parameters such as pH, media content and inoculum size were evaluated for ethanol fermentation in carob extract. Also, in order to determine economically appropriate nitrogen sources, four different nitrogen sources were evaluated. The optimum extraction condition for carob extract was determined to be 80 degrees C, 2h in 1:4 dilution rate (fruit: water ratio) according to the result of response surface analysis (115.3g/L). When the fermentation with pH at 5.5 was applied, the final ethanol concentration and production rates were 42.6g/L and 3.37 g/L/h, respectively, which were higher than using an uncontrolled pH. Among inoculum sizes of 1%, 3%, and 5%, 3% was determined as the best inoculum size. The maximum production rate and final ethanol concentration were 3.48 g/L/h and 44.51%, respectively, with an alternative nitrogen source of meat-bone meal. Overall, this study suggested that carob extract can be utilized for production of ethanol in order to meet the demands of renewable energy.

  3. Construction of Killer Industrial Yeast Saccharomyces Cerevisiae Hau-1 and its Fermentation Performance

    PubMed Central

    Bajaj, Bijender K.; Sharma, S.

    2010-01-01

    Saccharomyces cerevisiae HAU-1, a time tested industrial yeast possesses most of the desirable fermentation characteristics like fast growth and fermentation rate, osmotolerance, high ethanol tolerance, ability to ferment molasses, and to ferment at elevated temperatures etc. However, this yeast was found to be sensitive against the killer strains of Saccharomyces cerevisiae. In the present study, killer trait was introduced into Saccharomyces cerevisiae HAU-1 by protoplast fusion with Saccharomyces cerevisiae MTCC 475, a killer strain. The resultant fusants were characterized for desirable fermentation characteristics. All the technologically important characteristics of distillery yeast Saccharomyces cerevisiae HAU-1 were retained in the fusants, and in addition the killer trait was also introduced into them. Further, the killer activity was found to be stably maintained during hostile conditions of ethanol fermentations in dextrose or molasses, and even during biomass recycling. PMID:24031519

  4. Anti-oxidant effects of pomegranate juice on Saccharomyces cerevisiae cell growth.

    PubMed

    Aslan, Abdullah; Can, Muhammed İsmail; Boydak, Didem

    2014-01-01

    Pomegranate juice has a number of positive effects on both human and animal subjects. Four groups were used in this study. i: Control group, ii: H2O2 group, iii: Pomegranate juice (PJ) group and iv: PJ + H2O2 group. Following the sterilization method for pomegranate juice (10%) and H2O2 (6% v/v), Saccharomyces cerevisiae cultures were added and the cultivation incubated at 35°C for 72 hours. Fatty acids and vitamin concentrations were measured using HPLC and GC and the total protein bands profile were determined by SDS-PAGE. According to our results statistically significant differences have been determined among the study groups in terms of fatty acids and vitamin (p<0,05). Fatty acid synthesis, vitamin control and cell density increased in groups to which PJ was given in comparison with the control group (p<0,05). Pomegranate juice increased vitamins, fatty acids and total protein expression in Saccharomyces cerevisiae in comparison with the control. Pomegranate juice has a positive effect on fatty acid, vitamin and protein synthesis by Saccharomyces cerevisiae. Accordingly, we believe that it has significantly decreased oxidative damage thereby making a positive impact on yeast development.

  5. Sequential Inoculation of Native Non-Saccharomyces and Saccharomyces cerevisiae Strains for Wine Making

    PubMed Central

    Padilla, Beatriz; Zulian, Laura; Ferreres, Àngela; Pastor, Rosa; Esteve-Zarzoso, Braulio; Beltran, Gemma; Mas, Albert

    2017-01-01

    The use of non-Saccharomyces yeast for wine making is becoming a common trend in many innovative wineries. The application is normally aimed at increasing aromas, glycerol, reducing acidity, and other improvements. This manuscript focuses on the reproduction of the native microbiota from the vineyard in the inoculum. Thus, native selected yeasts (Hanseniaspora uvarum, Metschnikowia pulcherrima, Torulaspora delbrueckii, Starmerella bacillaris species and three different strains of Saccharomyces cerevisiae) were inoculated sequentially, or only S. cerevisiae (three native strains together or one commercial) was used. Inoculations were performed both in laboratory conditions with synthetic must (400 mL) as well as in industrial conditions (2000 kg of grapes) in red winemaking in two different varieties, Grenache and Carignan. The results showed that all the inoculated S. cerevisiae strains were found at the end of the vinifications, and when non-Saccharomyces yeasts were inoculated, they were found in appreciable populations at mid-fermentation. The final wines produced could be clearly differentiated by sensory analysis and were of similar quality, in terms of sensory analysis panelists’ appreciation. PMID:28769887

  6. Gains and Losses of Transcription Factor Binding Sites in Saccharomyces cerevisiae and Saccharomyces paradoxus.

    PubMed

    Schaefke, Bernhard; Wang, Tzi-Yuan; Wang, Chuen-Yi; Li, Wen-Hsiung

    2015-07-27

    Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains). © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae.

    PubMed

    Den Haan, Riaan; Rose, Shaunita H; Lynd, Lee R; van Zyl, Willem H

    2007-01-01

    In this study, we expressed two cellulase encoding genes, an endoglucanase of Trichoderma reesei (EGI) and the beta-glucosidase of Saccharomycopsis fibuligera (BGL1), in combination in Saccharomyces cerevisiae. The resulting strain was able to grow on phosphoric acid swollen cellulose (PASC) through simultaneous production of sufficient extracellular endoglucanase and beta-glucosidase activity. Anaerobic growth (0.03h(-1)) up to 0.27gl(-1) DCW was observed on medium containing 10gl(-1) PASC as sole carbohydrate source with concomitant ethanol production of up to 1.0gl(-1). We have thus demonstrated the construction of a yeast strain capable of growth on and one-step conversion of amorphous cellulose to ethanol, representing significant progress towards realization of one-step processing of cellulosic biomass in a consolidated bioprocessing configuration. To our knowledge, this is the first report of a recombinant strain of S. cerevisiae growing on pure cellulose.

  8. Pathways and Mechanisms that Prevent Genome Instability in Saccharomyces cerevisiae.

    PubMed

    Putnam, Christopher D; Kolodner, Richard D

    2017-07-01

    Genome rearrangements result in mutations that underlie many human diseases, and ongoing genome instability likely contributes to the development of many cancers. The tools for studying genome instability in mammalian cells are limited, whereas model organisms such as Saccharomyces cerevisiae are more amenable to these studies. Here, we discuss the many genetic assays developed to measure the rate of occurrence of Gross Chromosomal Rearrangements (called GCRs) in S. cerevisiae These genetic assays have been used to identify many types of GCRs, including translocations, interstitial deletions, and broken chromosomes healed by de novo telomere addition, and have identified genes that act in the suppression and formation of GCRs. Insights from these studies have contributed to the understanding of pathways and mechanisms that suppress genome instability and how these pathways cooperate with each other. Integrated models for the formation and suppression of GCRs are discussed. Copyright © 2017 by the Genetics Society of America.

  9. Saccharomyces cerevisiae thermal inactivation kinetics combined with ultrasound.

    PubMed

    López-Malo, A; Guerrero, S; Alzamora, S M

    1999-10-01

    Inactivation kinetics of Saccharomyces cerevisiae during thermal treatments at moderate temperatures (45.0, 47.5, 50.0, 52.5, or 55.0 degrees C) combined with application of 20 kHz of ultrasound were evaluated. S. cerevisiae inactivation under the combined effects of heat and ultrasound followed first-order reaction kinetics, with decimal reduction times (D) that varied from 22.3 to 0.8 min. D values in treatments that combined heat and ultrasound were significantly smaller (P < 0.05) than D values obtained for thermal treatments and were more noticeable at temperatures below 50 degrees C. The dependence of the D value on temperature had a significantly (P < 0.05) greater z value for combined treatments. Yeast heat inactivation kinetics revealed decreased thermal resistance caused by ultrasound.

  10. Advanced biofuel production by the yeast Saccharomyces cerevisiae.

    PubMed

    Buijs, Nicolaas A; Siewers, Verena; Nielsen, Jens

    2013-06-01

    Replacement of conventional transportation fuels with biofuels will require production of compounds that can cover the complete fuel spectrum, ranging from gasoline to kerosene. Advanced biofuels are expected to play an important role in replacing fossil fuels because they have improved properties compared with ethanol and some of these may have the energy density required for use in heavy duty vehicles, ships, and aviation. Moreover, advanced biofuels can be used as drop-in fuels in existing internal combustion engines. The yeast cell factory Saccharomyces cerevisiae can be turned into a producer of higher alcohols (1-butanol and isobutanol), sesquiterpenes (farnesene and bisabolene), and fatty acid ethyl esters (biodiesel), and here we discusses progress in metabolic engineering of S. cerevisiae for production of these advanced biofuels. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae

    SciTech Connect

    Batt, C.A.; Carvallo, S.; Easson, D.D.; Akedo, M.; Sinskey, A.J.

    1986-04-01

    Xylose transport, xylose reductase, and xylitol dehydrogenase activities are demonstrated in Saccharomyces cerevisiae. The enzymes in the xylose catabolic pathway necessary for the conversion of xylose xylulose are present, although S. cerevisiae cannot grow on xylose as a sole carbon source. Xylose transport is less efficient than glucose transport, and its rate is dependent upon aeration. Xylose reductase appears to be a xylose inducible enzyme and xylitol dehydrogenase activity is constitutive, although both are repressed by glucose. Both xylose reductase and xylitol dehydrogenase activities are five- to tenfold lower in S. cerevisie as compared to Candida utilis. In vivo conversion of /sup 14/C-xylose in S. cerevisiage is demonstrated and xylitol is detected, although no significant levels of any other /sup 14/C-labeled metabolites (e.g., ethanol) are observed. 22 references.

  12. Metabolic engineering of Saccharomyces cerevisiae for linalool production.

    PubMed

    Amiri, Pegah; Shahpiri, Azar; Asadollahi, Mohammad Ali; Momenbeik, Fariborz; Partow, Siavash

    2016-03-01

    To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool. Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l(-1) in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l(-1). Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

  13. Pathways and Mechanisms that Prevent Genome Instability in Saccharomyces cerevisiae

    PubMed Central

    Putnam, Christopher D.; Kolodner, Richard D.

    2017-01-01

    Genome rearrangements result in mutations that underlie many human diseases, and ongoing genome instability likely contributes to the development of many cancers. The tools for studying genome instability in mammalian cells are limited, whereas model organisms such as Saccharomyces cerevisiae are more amenable to these studies. Here, we discuss the many genetic assays developed to measure the rate of occurrence of Gross Chromosomal Rearrangements (called GCRs) in S. cerevisiae. These genetic assays have been used to identify many types of GCRs, including translocations, interstitial deletions, and broken chromosomes healed by de novo telomere addition, and have identified genes that act in the suppression and formation of GCRs. Insights from these studies have contributed to the understanding of pathways and mechanisms that suppress genome instability and how these pathways cooperate with each other. Integrated models for the formation and suppression of GCRs are discussed. PMID:28684602

  14. Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae.

    PubMed

    Young, M; Davies, M J; Bailey, D; Gradwell, M J; Smestad-Paulsen, B; Wold, J K; Barnes, R M; Hounsell, E F

    1998-08-01

    Mannans of the yeast Saccharomyces cerevisiae have been implicated as containing the allergens to which bakers and brewers are sensitive and also the antigen recognized by patients with Crohn's disease. A fraction of S. cerevisiae mannan, Sc500, having high affinity for antibodies in Crohn's patients has been characterized by NMR spectroscopy followed by fragmentation using alkaline elimination, partial acid hydrolysis and acetolysis. The released oligosaccharides were separated by gel filtration on a Biogel P4 column and analyzed by fluorescence labeling, HPLC and methylation analysis. The relationship between structure and antigen activity was measured by competitive ELISA. The antigenic activity of the original high molecular weight mannan could be ascribed to terminal Manalpha1-->3Manalpha1-->2 sequences which are rarely found in human glycoproteins but were over-represented in Sc500 compared to other yeast mannans.

  15. Glucose- and nitrogen sensing and regulatory mechanisms in Saccharomyces cerevisiae.

    PubMed

    Rødkaer, Steven V; Faergeman, Nils J

    2014-08-01

    Pro- and eukaryotic cells are constantly challenged by varying concentrations of nutrients in their environment. Perceiving and adapting to such changes are therefore crucial for cellular viability. Thus, numerous specialized cellular receptors continuously sense and react to the availability of nutrients such as glucose and nitrogen. When stimulated, these receptors initiate various cellular signaling pathways, which in concert constitute a complex regulatory network. To ensure a highly specific response, these pathways and networks cross-communicate with each other and are regulated at several steps and by numerous different regulators. As numerous of these regulating proteins, biochemical mechanisms, and cellular pathways are evolutionary conserved, complex biochemical information relevant to humans can be obtained by studying simple organisms. Thus, the yeast Saccharomyces cerevisiae has been recognized as a powerful model system to study fundamental biochemical processes. In the present review, we highlight central signaling pathways and molecular circuits conferring nitrogen- and glucose sensing in S. cerevisiae.

  16. Expression of acylphosphatase in Saccharomyces cerevisiae enhances ethanol fermentation rate

    SciTech Connect

    Raugei, G.; Modesti, A.; Magherini, F.

    1996-06-01

    Previous experiments in vitro have demonstrated the ability of acylphosphatase to increase the rate of glucose fermentation in yeast. To evaluate the possibility of increasing fermentation in vivo also, a chemically synthesized DNA sequence coding for human muscle acylphosphatase was expressed at high level in Saccharomyces cerevisiae. Ethanol production was measured in these engineered strains in comparison with a control. Acylphosphatase expression strongly increased the rate of ethanol production both in aerobic and anaerobic culture. This finding may be potentially important for the development of more efficient industrial fermentation processes. 20 refs., 5 figs.

  17. Flocculation of industrial and laboratory strains of Saccharomyces cerevisiae.

    PubMed

    Sieiro, C; Reboredo, N M; Villa, T G

    1995-06-01

    A comparative study has been made of different laboratory and industrial wild-type strains of Saccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flo1 or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic to FLO1 found in genetic strains.

  18. Isobutanol production from D-xylose by recombinant Saccharomyces cerevisiae.

    PubMed

    Brat, Dawid; Boles, Eckhard

    2013-03-01

    Simultaneous overexpression of an optimized, cytosolically localized valine biosynthesis pathway together with overexpression of xylose isomerase XylA from Clostridium phytofermentans, transaldolase Tal1 and xylulokinase Xks1 enabled recombinant Saccharomyces cerevisiae cells to complement the valine auxotrophy of ilv2,3,5 triple deletion mutants for growth on D-xylose as the sole carbon source. Moreover, after additional overexpression of ketoacid decarboxylase Aro10 and alcohol dehydrogenase Adh2, the cells were able to ferment D-xylose directly to isobutanol. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  19. High-throughput expression in microplate format in Saccharomyces cerevisiae.

    PubMed

    Holz, Caterina; Lang, Christine

    2004-01-01

    We have developed a high-throughput technology that allows parallel expression, purification, and analysis of large numbers of cloned cDNAs in the yeast Saccharomyces cerevisiae. The technology is based on a vector for intracellular protein expression under control of the inducible CUP1 promoter, where the gene products are fused to specific peptide sequences. These N-terminal and C-terminal epitope tags allow the immunological identification and purification of the gene products independent of the protein produced. By introducing the method of recombinational cloning we avoid time-consuming re-cloning steps and enable the easy switching between different expression vectors and host systems.

  20. A waterbath method for preparation of RNA from Saccharomyces cerevisiae.

    PubMed

    Li, Jing; Liu, Juan; Wang, Xin; Zhao, Lei; Chen, Qiang; Zhao, Weiming

    2009-01-01

    We have developed a simple and efficient method for the preparation of total RNA from Saccharomyces cerevisiae. Yeast cells were incubated at 65 degrees C for 5 min in yeast RNA isolation buffer (10 mM EDTA, 50mM Tris-HCl, 5% SDS, pH 6.0), and the RNA was isolated and purified. The yield and quality of the isolated RNA was consistently high, and the isolated RNA was suitable for downstream applications, such as Northern blot hybridization and reverse transcription PCR (RT-PCR).

  1. [Purification and properties of intercellular inorganic pyrophosphatase from Saccharomyces cerevisiae].

    PubMed

    Gou, P; Yang, S

    1998-06-01

    An inorganic pyrophosphatase (EC3.6.1.1) from Saccharomyces cerevisiae was purified to PAGE homogeneity by sonication disruption, (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The optimum pH and temperature of the enzyme were 7.4-7.8 and 60 degrees C, respectively. The Km was 19.3 mmol/L. The enzyme required Mg2+ as a cofactor for hydrolysis of pyrophosphate and was inhibited by Ca2+, Hg2+, Pb2+, Mn2+.

  2. Immobilized cell cross-flow reactor. [Saccharomyces cerevisiae

    SciTech Connect

    Chotani, G.K.; Constantinides, A.

    1984-01-01

    A cross-current flow reactor was operated using sodium alginate gel entrapped yeast cells (Saccharomyces cerevisiae) under growth conditions. Micron-sized silica, incorporated into the biocatalyst particles (1 mm mean diameter) improved mechanical strength and internal surface adhesion. The process showed decreased productivity and stability at 35/sup 0/C compared to the normal study done at 30/sup 0/C. The increased number of cross flows diminish the product inhibition effect. The residence time distribution shows that the cross-flow bioreactor system can be approximated to either a train of backmixed fermentors in series or a plug flow fermentor with moderate axial dispersion.

  3. Improved anaerobic use of arginine by Saccharomyces cerevisiae.

    PubMed

    Martin, Olga; Brandriss, Marjorie C; Schneider, Gisbert; Bakalinsky, Alan T

    2003-03-01

    Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Delta(1)-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.

  4. Identification of the mitochondrial pyruvate carrier in Saccharomyces cerevisiae.

    PubMed Central

    Hildyard, John C W; Halestrap, Andrew P

    2003-01-01

    Mitochondrial pyruvate transport is fundamental for metabolism and mediated by a specific inhibitable carrier. We have identified the yeast mitochondrial pyruvate carrier by measuring inhibitor-sensitive pyruvate uptake into mitochondria from 18 different Saccharomyces cerevisiae mutants, each lacking an unattributed member of the mitochondrial carrier family (MCF). Only mitochondria from the YIL006w deletion mutant exhibited no inhibitor-sensitive pyruvate transport, but otherwise behaved normally. YIL006w encodes a 41.9 kDa MCF member with homologous proteins present in both the human and mouse genomes. PMID:12887330

  5. The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae.

    PubMed

    Stanley, D; Bandara, A; Fraser, S; Chambers, P J; Stanley, G A

    2010-07-01

    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.

  6. Quantifying the complexities of Saccharomyces cerevisiae's ecosystem engineering via fermentation.

    PubMed

    Goddard, Matthew R

    2008-08-01

    The theory of niche construction suggests that organisms may engineer environments via their activities. Despite the potential of this phenomenon being realized by Darwin, the capability of niche construction to generally unite ecological and evolutionary biology has never been empirically quantified. Here I quantify the fitness effects of Saccharomyces cerevisiae's ecosystem engineering in a natural ferment in order to understand the interaction between ecological and evolutionary processes. I show that S. cerevisiae eventually dominates in fruit niches, where it is naturally initially rare, by modifying the environment through fermentation (the Crabtree effect) in ways which extend beyond just considering ethanol production. These data show that an additional cause of S. cerevisiae's competitive advantage over the other yeasts in the community is due to the production of heat via fermentation. Even though fermentation is less energetically efficient than respiration, it seems that this trait has been selected for because its net effect provides roughly a 7% fitness advantage over the other members of the community. These data provide an elegant example of niche construction because this trait clearly modifies the environment and therefore the selection pressures to which S. cerevisiae, and other organisms that access the fruit resource, including humans, are exposed to.

  7. Genetic stabilization of Saccharomyces cerevisiae oenological strains by using benomyl.

    PubMed

    Blasco, Lucía; Feijoo-Siota, Lucía; Veiga-Crespo, Patricia; Villa, Tomás G

    2008-06-01

    Wild-type oenological strains of Saccharomyces cerevisiae are usually aneuploid and heterozygotes; thus, when they are used as starters in must fermentation the resulting wine characteristics may vary from year to year. Treatment of a wild-type S. cerevisiae oenological strain with benomyl (methyl-l-butylcarbamoyl-2-benzimidazole carbamate), an antifungal agent shown to cause chromosome loss in yeasts, resulted in a stable starter strain in which the parental oenological traits were unchanged. The oenological S. cerevisiae strain was treated with benomyl in two different ways (A and B), and sporulation ability and spore viability were subsequently assayed. Treatment A resulted in both the highest numbers of tetrads and a reduction in DNA cell content, while treatment B increased spore viability. Fermentation assays were carried out with spore clones obtained from treatment A, and the concentrations of glycerol, lactic acid, acetic acid, and ethanol resulting from the treated strains were found to be similar to those of the parental strain. Benomyl treatment thus achieved stable, highly sporulating oenological S. cerevisiae strains of low ploidy, but preserved the desirable oenological properties of the parental strain.

  8. Information propagation within the Genetic Network of Saccharomyces cerevisiae.

    PubMed

    Chowdhury, Sharif; Lloyd-Price, Jason; Smolander, Olli-Pekka; Baici, Wayne C V; Hughes, Timothy R; Yli-Harja, Olli; Chua, Gordon; Ribeiro, Andre S

    2010-10-26

    A gene network's capacity to process information, so as to bind past events to future actions, depends on its structure and logic. From previous and new microarray measurements in Saccharomyces cerevisiae following gene deletions and overexpressions, we identify a core gene regulatory network (GRN) of functional interactions between 328 genes and the transfer functions of each gene. Inferred connections are verified by gene enrichment. We find that this core network has a generalized clustering coefficient that is much higher than chance. The inferred Boolean transfer functions have a mean p-bias of 0.41, and thus similar amounts of activation and repression interactions. However, the distribution of p-biases differs significantly from what is expected by chance that, along with the high mean connectivity, is found to cause the core GRN of S. cerevisiae's to have an overall sensitivity similar to critical Boolean networks. In agreement, we find that the amount of information propagated between nodes in finite time series is much higher in the inferred core GRN of S. cerevisiae than what is expected by chance. We suggest that S. cerevisiae is likely to have evolved a core GRN with enhanced information propagation among its genes.

  9. Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae.

    PubMed

    Van Hoek, P; Modesti, A; Ramponi, G; Kötter, P; van Dijken, J P; Pron, J T

    2001-10-01

    Human acylphosphatase (h-AP, EC 3.6.1.7) has been reported to catalyse the hydrolysis of the 1-phosphate group of 1,3-diphosphoglycerate. In vivo operation of this reaction in the yeast Saccharomyces cerevisiae would bypass phosphoglycerate kinase and thus reduce the ATP yield from glycolysis. To investigate whether h-AP can indeed replace the S. cerevisiae phosphoglycerate kinase, a multi-copy plasmid carrying the h-AP gene under control of the yeast TDH3 promoter was introduced into a pgk1 delta mutant of S. cerevisiae. A strain carrying the expression vector without the h-AP cassette was used as a reference. For both strains, steady-state carbon- and energy-limited chemostat cultures were obtained at a dilution rate of 0.10 h(-1) on a medium containing a mixture of glucose and ethanol (15% and 85% on a carbon basis, respectively). Although the h-AP strain exhibited a high acylphosphatase activity in cell extracts, switching to glucose as sole carbon and energy source resulted in a complete arrest of glucose consumption and growth. The lack of a functional glycolytic pathway was further evident from the absence of ethanol formation in the presence of excess glucose in the culture. As h-AP cannot replace yeast phosphoglycerate kinase in vivo, the enzyme is not a useful tool to modify the ATP yield of glycolysis in S. cerevisiae.

  10. Sucrose and Saccharomyces cerevisiae: a relationship most sweet.

    PubMed

    Marques, Wesley Leoricy; Raghavendran, Vijayendran; Stambuk, Boris Ugarte; Gombert, Andreas Karoly

    2016-02-01

    Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering.

  11. Ethanol production and maximum cell growth are highly correlated with membrane lipid composition during fermentation as determined by lipidomic analysis of 22 Saccharomyces cerevisiae strains.

    PubMed

    Henderson, Clark M; Lozada-Contreras, Michelle; Jiranek, Vladimir; Longo, Marjorie L; Block, David E

    2013-01-01

    Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R(2) = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R(2) = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems.

  12. Ethanol Production and Maximum Cell Growth Are Highly Correlated with Membrane Lipid Composition during Fermentation as Determined by Lipidomic Analysis of 22 Saccharomyces cerevisiae Strains

    PubMed Central

    Henderson, Clark M.; Lozada-Contreras, Michelle; Jiranek, Vladimir; Longo, Marjorie L.

    2013-01-01

    Optimizing ethanol yield during fermentation is important for efficient production of fuel alcohol, as well as wine and other alcoholic beverages. However, increasing ethanol concentrations during fermentation can create problems that result in arrested or sluggish sugar-to-ethanol conversion. The fundamental cellular basis for these problem fermentations, however, is not well understood. Small-scale fermentations were performed in a synthetic grape must using 22 industrial Saccharomyces cerevisiae strains (primarily wine strains) with various degrees of ethanol tolerance to assess the correlation between lipid composition and fermentation kinetic parameters. Lipids were extracted at several fermentation time points representing different growth phases of the yeast to quantitatively analyze phospholipids and ergosterol utilizing atmospheric pressure ionization-mass spectrometry methods. Lipid profiling of individual fermentations indicated that yeast lipid class profiles do not shift dramatically in composition over the course of fermentation. Multivariate statistical analysis of the data was performed using partial least-squares linear regression modeling to correlate lipid composition data with fermentation kinetic data. The results indicate a strong correlation (R2 = 0.91) between the overall lipid composition and the final ethanol concentration (wt/wt), an indicator of strain ethanol tolerance. One potential component of ethanol tolerance, the maximum yeast cell concentration, was also found to be a strong function of lipid composition (R2 = 0.97). Specifically, strains unable to complete fermentation were associated with high phosphatidylinositol levels early in fermentation. Yeast strains that achieved the highest cell densities and ethanol concentrations were positively correlated with phosphatidylcholine species similar to those known to decrease the perturbing effects of ethanol in model membrane systems. PMID:23064336

  13. A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae

    PubMed Central

    Chu, Daniel B.; Burgess, Sean M.

    2016-01-01

    Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions. PMID:26747203

  14. Diversity of Saccharomyces cerevisiae Strains Isolated from Two Italian Wine-Producing Regions

    PubMed Central

    Capece, Angela; Granchi, Lisa; Guerrini, Simona; Mangani, Silvia; Romaniello, Rossana; Vincenzini, Massimo; Romano, Patrizia

    2016-01-01

    Numerous studies, based on different molecular techniques analyzing DNA polymorphism, have provided evidence that indigenous Saccharomyces cerevisiae populations display biogeographic patterns. Since the differentiated populations of S. cerevisiae seem to be responsible for the regional identity of wine, the aim of this work was to assess a possible relationship between the diversity and the geographical origin of indigenous S. cerevisiae isolates from two different Italian wine-producing regions (Tuscany and Basilicata). For this purpose, sixty-three isolates from Aglianico del Vulture grape must (main cultivar in the Basilicata region) and from Sangiovese grape must (main cultivar in the Tuscany region) were characterized genotypically, by mitochondrial DNA restriction analysis and MSP-PCR by using (GTG)5 primers, and phenotypically, by determining technological properties and metabolic compounds of oenological interest after alcoholic fermentation. All the S. cerevisiae isolates from each region were inoculated both in must obtained from Aglianico grape and in must obtained from Sangiovese grape to carry out fermentations at laboratory-scale. Numerical analysis of DNA patterns resulting from both molecular methods and principal component analysis of phenotypic data demonstrated a high diversity among the S. cerevisiae strains. Moreover, a correlation between genotypic and phenotypic groups and geographical origin of the strains was found, supporting the concept that there can be a microbial aspect to terroir. Therefore, exploring the diversity of indigenous S. cerevisiae strains can allow developing tailored strategies to select wine yeast strains better adapted to each viticultural area. PMID:27446054

  15. The distribution of inverted repeat sequences in the Saccharomyces cerevisiae genome

    PubMed Central

    Benson, Gary; Gelfand, Yevgeniy; Benham, Craig J.

    2010-01-01

    Although a variety of possible functions have been proposed for inverted repeat sequences (IRs), it is not known which of them might occur in vivo. We investigate this question by assessing the distributions and properties of IRs in the Saccharomyces cerevisiae (SC) genome. Using the IRFinder algorithm we detect 100,514 IRs having copy length greater than 6 bp and spacer length less than 77 bp. To assess statistical significance we also determine the IR distributions in two types of randomization of the S. cerevisiae genome. We find that the S. cerevisiae genome is significantly enriched in IRs relative to random. The S. cerevisiae IRs are significantly longer and contain fewer imperfections than those from the randomized genomes, suggesting that processes to lengthen and/or correct errors in IRs may be operative in vivo. The S. cerevisiae IRs are highly clustered in intergenic regions, while their occurrence in coding sequences is consistent with random. Clustering is stronger in the 3′ flanks of genes than in their 5′ flanks. However, the S. cerevisiae genome is not enriched in those IRs that would extrude cruciforms, suggesting that this is not a common event. Various explanations for these results are considered. Electronic supplementary material The online version of this article (doi:10.1007/s00294-010-0302-6) contains supplementary material, which is available to authorized users. PMID:20446088

  16. Diversity of Saccharomyces cerevisiae Strains Isolated from Two Italian Wine-Producing Regions.

    PubMed

    Capece, Angela; Granchi, Lisa; Guerrini, Simona; Mangani, Silvia; Romaniello, Rossana; Vincenzini, Massimo; Romano, Patrizia

    2016-01-01

    Numerous studies, based on different molecular techniques analyzing DNA polymorphism, have provided evidence that indigenous Saccharomyces cerevisiae populations display biogeographic patterns. Since the differentiated populations of S. cerevisiae seem to be responsible for the regional identity of wine, the aim of this work was to assess a possible relationship between the diversity and the geographical origin of indigenous S. cerevisiae isolates from two different Italian wine-producing regions (Tuscany and Basilicata). For this purpose, sixty-three isolates from Aglianico del Vulture grape must (main cultivar in the Basilicata region) and from Sangiovese grape must (main cultivar in the Tuscany region) were characterized genotypically, by mitochondrial DNA restriction analysis and MSP-PCR by using (GTG)5 primers, and phenotypically, by determining technological properties and metabolic compounds of oenological interest after alcoholic fermentation. All the S. cerevisiae isolates from each region were inoculated both in must obtained from Aglianico grape and in must obtained from Sangiovese grape to carry out fermentations at laboratory-scale. Numerical analysis of DNA patterns resulting from both molecular methods and principal component analysis of phenotypic data demonstrated a high diversity among the S. cerevisiae strains. Moreover, a correlation between genotypic and phenotypic groups and geographical origin of the strains was found, supporting the concept that there can be a microbial aspect to terroir. Therefore, exploring the diversity of indigenous S. cerevisiae strains can allow developing tailored strategies to select wine yeast strains better adapted to each viticultural area.

  17. Effect of Saccharomyces cerevisiae strain UFMG A-905 in experimental model of inflammatory bowel disease.

    PubMed

    Tiago, F C P; Porto, B A A; Ribeiro, N S; Moreira, L M C; Arantes, R M E; Vieira, A T; Teixeira, M M; Generoso, S V; Nascimento, V N; Martins, F S; Nicoli, J R

    2015-01-01

    In the present study, the protective potential of Saccharomyces cerevisiae strain UFMG A-905 was evaluated in a murine model of acute ulcerative colitis (UC). Six groups of Balb/c mice were used: not treated with yeast and not challenged with dextran sulphate sodium (DSS) (control); treated with S. cerevisiae UFMG A-905 (905); treated with the non-probiotic S. cerevisiae W303 (W303); challenged with DSS (DSS); treated with S. cerevisiae UFMG A-905 and challenged with DSS (905 + DSS); and treated with S. cerevisiae W303 and challenged with DSS (W303 + DSS). Seven days after induction of UC, mice were euthanised to remove colon for enzymatic, immunological, and histopathological analysis. In vivo intestinal permeability was also evaluated. An improvement of clinical manifestations of experimental UC was observed only in mice of the 905 + DSS group when compared to animals from DSS and W303 + DSS groups. This observation was confirmed by histological and morphometrical data and determination of myeloperoxidase and eosinophil peroxidase activities, intestinal permeability and some pro-inflammatory cytokines. S. cerevisiae UFMG A-905 showed to be a potential alternative treatment for UC when used in an experimental animal model of the disease.

  18. Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations.

    PubMed

    Rantsiou, Kalliopi; Dolci, Paola; Giacosa, Simone; Torchio, Fabrizio; Tofalo, Rosanna; Torriani, Sandra; Suzzi, Giovanna; Rolle, Luca; Cocolin, Luca

    2012-03-01

    In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ~0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response.

  19. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae on alcoholic fermentation behaviour and wine aroma of cherry wines.

    PubMed

    Sun, Shu Yang; Gong, Han Sheng; Jiang, Xiao Man; Zhao, Yu Ping

    2014-12-01

    This study examined the effect of mixed fermentation of non-Saccharomyces (Torulaspora delbrueckii ZYMAFLORE Alpha(TD n. Sacch) and Metschnikowia pulcherrima JS22) and Saccharomyces cerevisiae yeasts (D254 and EC1118) on the production of cherry wines, in comparison with commonly used mono-culture. Results obtained during AF demonstrated that negligible inhibitory effect was observed in S. cerevisiae/Alpha pair, whereas a strong antagonistic effect was detected between MJS22 and S. cerevisiae strain, resulting in an early death of MJS22. For volatile components determined, S. cerevisiae/MJS22 couple was found to significantly boost the production of most detected compounds, more particularly in higher alcohols, esters, acids and terpenes; while the characteristic of S. cerevisiae/Alpha pair is an increase in fruity esters, higher alcohols and decrease in acid production. Sensory evaluation revealed that S. cerevisiae/MJS22 pair reinforced sweet, green and fatty notes to the cherry wines, and S. cerevisiae/Alpha trial enhanced the fruity odour and reduced green note. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Combined effect of the Saccharomyces cerevisiae lag phase and the non-Saccharomyces consortium to enhance wine fruitiness and complexity.

    PubMed

    Albertin, Warren; Zimmer, Adrien; Miot-Sertier, Cécile; Bernard, Margaux; Coulon, Joana; Moine, Virginie; Colonna-Ceccaldi, Benoit; Bely, Marina; Marullo, Philippe; Masneuf-Pomarede, Isabelle

    2017-09-14

    Non-Saccharomyces (NS) species that are either naturally present in grape must or added in mixed fermentation with S. cerevisiae may impact the wine's chemical composition and sensory properties. NS yeasts are prevailing during prefermentation and early stages of alcoholic fermentation. However, obtaining the correct balance between S. cerevisiae and NS species is still a critical issue: if S. cerevisiae outcompetes the non-Saccharomyces, it may minimize their impact, while conversely if NS take over S. cerevisiae, it may result in stuck or sluggish fermentations. Here, we propose an original strategy to promote the non-Saccharomyces consortium during the prefermentation stage while securing fermentation completion: the use of a long lag phase S. cerevisiae. Various fermentations in a Sauvignon Blanc with near isogenic S. cerevisiae displaying short or long lag phase were compared. Fermentations were performed with or without a consortium of five non-Saccharomyces yeasts (Hanseniaspora uvarum, Candida zemplinina, Metschnikowia spp., Torulaspora delbrueckii, and Pichia kluyveri), mimicking the composition of natural NS community in grape must. The sensorial analysis highlighted the positive impact of the long lag phase on the wine fruitiness and complexity. Surprisingly, the presence of NS modified only marginally the wine composition but significantly impacted the lag phase of S. cerevisiae. The underlying mechanisms are still unclear, but it is the first time that a study suggests that the wine composition can be affected by the lag phase duration per se. Further experiments should address the suitability of the use of long lag phase S. cerevisiae in winemaking.

  1. Flocculation onset in Saccharomyces cerevisiae: the role of nutrients.

    PubMed

    Sampermans, S; Mortier, J; Soares, E V

    2005-01-01

    To examine the role of the nutrients on the onset of flocculation in an ale-brewing strain, Saccharomyces cerevisiae NCYC 1195. Flocculation was evaluated using the method of Soares, E.V. and Vroman, A. [Journal of Applied Microbiology (2003) 95, 325]. For cells grown in chemically defined medium (yeast nitrogen base with glucose) or in rich medium (containing yeast extract, peptone and fermentable sugars: fructose or maltose), the onset of flocculation occurred after the end of exponential respiro-fermentative phase of growth being coincident with the attainment of the lower level of carbon source in the culture medium. Cells, in exponential respiro-fermentative phase of growth, transferred to a glucose-containing medium without nitrogen source, developed a flocculent phenotype, while these carbon source starved cells, in the presence of all other nutrients that support growth, did not flocculate. In addition, cells in exponential phase of growth, under catabolite repression, when transferred to a medium containing 0.2% (w/v) of fermentable sugar (fructose or maltose) or 2% (v/v) ethanol, showed a rapid triggering of flocculation, while when incubated in 2% (v/v) glycerol did not develop a flocculent phenotype. The onset of flocculation occurs when a low sugar and/or nitrogen concentration is reached in culture media. The triggering of flocculation is an energetic dependent process influenced by the carbon source metabolism. The presence of external nitrogen source is not necessary for developing a flocculent phenotype. This work contributes to the elucidation of the role of nutrients on the onset of flocculation in NewFlo phenotype yeast strains. This information might be useful to the brewing industry, in the control of yeast flocculation, as the time when the onset of flocculation occurs can determine the fermentation performance and the beer quality.

  2. [Rab GTPases networks in membrane traffic in Saccharomyces cerevisiae].

    PubMed

    Nagano, Makoto; Toshima, Junko Y; Toshima, Jiro

    2015-01-01

    Intracellular membrane trafficking between membranous compartments is essential for organelle biogenesis, structure, and identity. Rab/Ypt GTPases are well-characterized regulators of intracellular membrane trafficking, functioning as molecular switches that alternate between GTP- and GDP-bound forms. In Saccharomyces cerevisiae, 11 Rab/Ypt GTPases have been identified and their functions are known to be conserved in their mammalian counterparts. In yeast, the secretory pathway is regulated by sequential activation and inactivation (the so-called Rab cascade) of three types of yeast Rab protein -Ypt1p, Ypt31p/32p and Sec4p -via specific guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In addition to these Rabs, we and others have recently demonstrated that Ypt6p is predominantly localized to the early Golgi compartment, and functions as another regulator of anterograde transport for intra-Golgi trafficking in the secretory pathway. On the other hand, the endocytic pathway is known to be regulated by three yeast Rab5s (Vps21p, Ypt52p and Ypt53p) and one Rab7 (Ypt7p). Rab5 and Rab7 are key determinants of endosome identity, and the Rab5-Rab7 cascade is important for the progression from early to late endosome. Our recent study demonstrates that the endocytic pathway branches into two vacuolar targeting pathways, the Rab5-dependent vacuole protein sorting (VPS) pathway and the Rab5-independent pathway. In this review, we focus on recent advances in our understanding of molecular mechanisms that regulate the localization and activity of yeast Rab GTPases in intracellular membrane trafficking.

  3. Molecular Analysis of Maltotriose Transport and Utilization by Saccharomyces cerevisiae

    PubMed Central

    Day, Rachel E.; Rogers, Peter J.; Dawes, Ian W.; Higgins, Vincent J.

    2002-01-01

    Efficient fermentation of maltotriose is a desired property of Saccharomyces cerevisiae for brewing. In a standard wort, maltotriose is the second most abundant sugar, and slower uptake leads to residual maltotriose in the finished product. The limiting factor of sugar metabolism is its transport, and there are conflicting reports on whether a specific maltotriose permease exists or whether the mechanisms responsible for maltose uptake also carry out maltotriose transport. In this study, radiolabeled maltotriose was used to show that overexpression of the maltose permease gene, MAL61, in an industrial yeast strain resulted in an increase in the rate of transport of maltotriose as well as maltose. A strain derived from W303-1A and lacking any maltose or maltotriose transporter but carrying a functional maltose transport activator (MAL63) was developed. By complementing this strain with permeases encoded by MAL31, MAL61, and AGT1, it was possible to measure their specific transport kinetics by using maltotriose and maltose. All three permeases were capable of high-affinity transport of maltotriose and of allowing growth of the strain on the sugar. Maltotriose utilization from the permease encoded by AGT1 was regulated by the same genetic mechanisms as those involving the maltose transcriptional activator. Competition studies carried out with two industrial strains, one not containing any homologue of AGT1, showed that maltose uptake and maltotriose uptake were competitive and that maltose was the preferred substrate. These results indicate that the presence of residual maltotriose in beer is not due to a genetic or physiological inability of yeast cells to utilize the sugar but rather to the lower affinity for maltotriose uptake in conjunction with deteriorating conditions present at the later stages of fermentation. Here we identify molecular mechanisms regulating the uptake of maltotriose and determine the role of each of the transporter genes in the cells. PMID

  4. Dihydroxyacetone detoxification in Saccharomyces cerevisiae involves formaldehyde dissimilation.

    PubMed

    Molin, Mikael; Blomberg, Anders

    2006-05-01

    To investigate Saccharomyces cerevisiae physiology during growth on the conditionally toxic triose dihydroxyacetone (DHA), protein expression was studied in strains overexpressing either of the two dihydroxyacetone kinase isogenes, DAK1 or DAK2, that grow well utilizing DHA as a carbon and energy source. DHA metabolism was found mostly similar to ethanol utilization, involving a strong component of glucose derepression, but also involved DHA-specific regulatory changes. A specific and strong (10- to 30-fold induction of formaldehyde dehydrogenase, Fdhlp, indicated activation of the formaldehyde dissimilation pathway in DHA medium. The importance of this pathway was further supported by impaired adaptation to DHA growth and DHA survival in a glutathione-dependent formaldehyde dehydrogenase (SFA1) deletion mutant. Glutathione synthase (GSH1) deletion led to decreased DHA survival in agreement with the glutathione cofactor requirement for the SFA1-encoded activity. DHA toxicity did, however, not solely appear related to formaldehyde accumulation, because SFA1 overexpression only enhanced formaldehyde but not DHA tolerance. In further agreement with a low DHA-to-formaldehyde flux, GSH supplements in the low microM range also fully suppressed the DHA sensitivity of a gsh1Delta strain. Under growth reduction on high (100 mM) DHA medium we report increased levels of advanced glycation end-product (AGE) formation on total protein. Under these high-DHA conditions expression of several stress-related proteins, e.g. a heat-shock protein (Hsp104p) and the oxidative stress indicator, alkyl hydroperoxide reductase (Ahp1p) was also found induced. However, hallmark determinants of oxidative stress tolerance (e.g. YAP1, SKN7, HYR1/GPX3 and SOD2) were redundant for DHA tolerance, thus indicating mechanisms of DHA toxicity largely independent of central oxidative stress defence mechanisms. We conclude that mechanisms for DHA growth and detoxification appear complex and that the

  5. Mechanistic Analysis of the Saccharomyces cerevisiae Kinesin Kar3*S

    PubMed Central

    Mackey, Andrew T.; Sproul, Lisa R.; Sontag, Christopher A.; Satterwhite, Lisa L.; Correia, John J.; Gilbert, Susan P.

    2005-01-01

    Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended α-helical coiled-coil. The α-helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended α-helical domain NH2-terminal to the catalytic core provides the structural transitions in response to the ATPase cycle that are critical for motility and that dimerization is not specifically required. This study provides the foundation to define the mechanistic contributions of Cik1 and Vik1 for Kar3 force generation and function in vivo. PMID:15385545

  6. Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

    PubMed Central

    Adams, A K; Holm, C

    1996-01-01

    To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length. PMID:8756617

  7. Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Cid, V J; Durán, A; del Rey, F; Snyder, M P; Nombela, C; Sánchez, M

    1995-01-01

    In fungi and many other organisms, a thick outer cell wall is responsible for determining the shape of the cell and for maintaining its integrity. The budding yeast Saccharomyces cerevisiae has been a useful model organism for the study of cell wall synthesis, and over the past few decades, many aspects of the composition, structure, and enzymology of the cell wall have been elucidated. The cell wall of budding yeasts is a complex and dynamic structure; its arrangement alters as the cell grows, and its composition changes in response to different environmental conditions and at different times during the yeast life cycle. In the past few years, we have witnessed a profilic genetic and molecular characterization of some key aspects of cell wall polymer synthesis and hydrolysis in the budding yeast. Furthermore, this organism has been the target of numerous recent studies on the topic of morphogenesis, which have had an enormous impact on our understanding of the intracellular events that participate in directed cell wall synthesis. A number of components that direct polarized secretion, including those involved in assembly and organization of the actin cytoskeleton, secretory pathways, and a series of novel signal transduction systems and regulatory components have been identified. Analysis of these different components has suggested pathways by which polarized secretion is directed and controlled. Our aim is to offer an overall view of the current understanding of cell wall dynamics and of the complex network that controls polarized growth at particular stages of the budding yeast cell cycle and life cycle. PMID:7565410

  8. The anatomy of a hypoxic operator in Saccharomyces cerevisiae.

    PubMed Central

    Deckert, J; Torres, A M; Hwang, S M; Kastaniotis, A J; Zitomer, R S

    1998-01-01

    Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes. PMID:9832521

  9. Nutritional Control of Chronological Aging and Heterochromatin in Saccharomyces cerevisiae.

    PubMed

    McCleary, David F; Rine, Jasper

    2017-03-01

    Calorie restriction extends life span in organisms as diverse as yeast and mammals through incompletely understood mechanisms.The role of NAD(+)-dependent deacetylases known as Sirtuins in this process, particularly in the yeast Saccharomyces cerevisiae, is controversial. We measured chronological life span of wild-type and sir2Δ strains over a higher glucose range than typically used for studying yeast calorie restriction. sir2Δ extended life span in high glucose complete minimal medium and had little effect in low glucose medium, revealing a partial role for Sir2 in the calorie-restriction response under these conditions. Experiments performed on cells grown in rich medium with a newly developed genetic strategy revealed that sir2Δ shortened life span in low glucose while having little effect in high glucose, again revealing a partial role for Sir2 In complete minimal media, Sir2 shortened life span as glucose levels increased; whereas in rich media, Sir2 extended life span as glucose levels decreased. Using a genetic strategy to measure the strength of gene silencing at HML, we determined increasing glucose stabilized Sir2-based silencing during growth on complete minimal media. Conversely, increasing glucose destabilized Sir-based silencing during growth on rich media, specifically during late cell divisions. In rich medium, silencing was far less stable in high glucose than in low glucose during stationary phase. Therefore, Sir2 was involved in a response to nutrient cues including glucose that regulates chronological aging, possibly through Sir2-dependent modification of chromatin or deacetylation of a nonhistone protein. Copyright © 2017 by the Genetics Society of America.

  10. Effects of low-intensity ultrasound on the growth, cell membrane permeability and ethanol tolerance of Saccharomyces cerevisiae.

    PubMed

    Dai, Chunhua; Xiong, Feng; He, Ronghai; Zhang, Weiwei; Ma, Haile

    2017-05-01

    Effects of low-intensity ultrasound (at different frequency, treatment time and power) on Saccharomyces cerevisiae in different growth phase were evaluated by the biomass in the paper. In addition, the cell membrane permeability and ethanol tolerance of sonicated Saccharomyces cerevisiae were also researched. The results revealed that the biomass of Saccharomyces cerevisiae increased by 127.03% under the optimum ultrasonic conditions such as frequency 28kHz, power 140W/L and ultrasonic time 1h when Saccharomyces cerevisiae cultured to the latent anaphase. And the membrane permeability of Saccharomyces cerevisiae in latent anaphase enhanced by ultrasound, resulting in the augment of extracellular protein, nucleic acid and fructose-1,6-diphosphate (FDP) contents. In addition, sonication could accelerate the damage of high concentration alcohol to Saccharomyces cerevisiae although the ethanol tolerance of Saccharomyces cerevisiae was not affected significantly by ultrasound.

  11. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae.

    PubMed

    Brat, Dawid; Boles, Eckhard; Wiedemann, Beate

    2009-04-01

    In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

  12. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    PubMed

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions.

  13. Population structure of mitochondrial genomes in Saccharomyces cerevisiae.

    PubMed

    Wolters, John F; Chiu, Kenneth; Fiumera, Heather L

    2015-06-11

    Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences. To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae. Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

  14. Effects of supplementing Saccharomyces cerevisiae fermentation product in sow diets on performance of sows and nursing piglets

    USDA-ARS?s Scientific Manuscript database

    Forty-two sows were used to determine the effects of adding Saccharomyces cerevisiae fermentation product to gestation and lactation diets on performance of sows and their progeny. On 5 d before breeding, sows were allotted to 2 dietary treatments representing: (1) sows fed a diet with 12.0 g fermen...

  15. Effects of supplementing a Saccharomyces cerevisiae fermentation product in sow diets on performance of sows and nursing piglets

    USDA-ARS?s Scientific Manuscript database

    Forty-two sows (Camborough-22, PIC) were used to determine the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; Diamond V Original XPC) in gestation and lactation diets on performance of sows and their progeny. On 5 d before breeding, sows were allotted to 2 dietary tr...

  16. Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae.

    PubMed

    Vemuri, G N; Eiteman, M A; McEwen, J E; Olsson, L; Nielsen, J

    2007-02-13

    Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as "overflow metabolism" or "the Crabtree effect." The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.

  17. Polymorphisms of Saccharomyces cerevisiae genes involved in wine production.

    PubMed

    Vigentini, Ileana; Fracassetti, Daniela; Picozzi, Claudia; Foschino, Roberto

    2009-03-01

    The setting up of new molecular methods for Saccharomyces cerevisiae typing is valuable in enology. Actually, the ability to discriminate different strains in wine making can have a benefit both for the control of the fermentation process and for the preservation of wine typicity. This study focused on the screening of single-nucleotide polymorphisms in genes involved in wine production that could evolve rapidly considering the selective pressure of the isolation environment. Preliminary screening of 30 genes in silico was performed, followed by the selection of 10 loci belonging to 8 genes. The sequence analysis showed a low polymorphism and a degree of heterozygosity. However, a new potential molecular target was recognized in the TPS1 gene coding for the trehalose-6-phosphate synthase enzyme involved in the ethanol resistance mechanism. This gene showed a 1.42% sequence diversity with seven different nucleotide substitutions. Moreover, classic techniques were applied to a collection of 50 S. cerevisiae isolates, mostly with enologic origin. Our results confirmed that the wine making was not carried out only by the inoculated commercial starter because indigenous strains of S. cerevisiae present during fermentation were detected. In addition, a high genetic relationship among some commercial cultures was found, highlighting imprecision or fraudulent practices by starter manufacturers.

  18. An assay for functional xylose transporters in Saccharomyces cerevisiae.

    PubMed

    Wang, Chengqiang; Shen, Yu; Hou, Jin; Suo, Fan; Bao, Xiaoming

    2013-11-15

    It has been considered that more efficient uptake of xylose could promote increased xylose metabolic capacity of several microorganisms. In this study, an assay to screen xylose transporters was established in the Saccharomyces cerevisiae strain, which expresses the xylosidase gene of Bacillus pumilus intracellularly. The absorbed xylose analog p-nitrophenyl-β-d-xylopyranoside (pNPX) rapidly hydrolyzed to p-nitrophenol (pNP), which displayed a yellow tint when exposed to xylosidase in vivo. The xylose transporter activities of the strain were computed using the pNP production rate, which was detected extracellularly. This method could be used for both high-throughput screening and smaller scale investigations. AraEp, which is a pentose transporter of Corynebacterium glutamicum, was expressed in S. cerevisiae and exhibited better transport capacity than the endogenous transporters Hxt7p and Gal2p. Moreover, a mutant of AraEp with 103% greater transport capacity was screened out, and the computer simulation suggested that transmembrane domain 5 was an important factor for the transport capacity of AraEp in S. cerevisiae.

  19. Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production.

    PubMed

    Chen, Binbin; Lee, Dong-Yup; Chang, Matthew Wook

    2015-09-01

    Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

  20. The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability

    NASA Astrophysics Data System (ADS)

    Yi, Zong-Chun; Li, Xiao-Fei; Wang, Yan; Wang, Jie; Sun, Yan; Zhuang, Feng-Yuan

    2011-06-01

    The budding yeast Saccharomyces cerevisiae has been proposed as an ideal model organism for clarifying the biological effects caused by spaceflight conditions. The postmitotic S. cerevisiae cells onboard Practice eight recoverable satellite were subjected to spaceflight for 15 days. After recovery, the viability, the glycogen content, the activities of carbohydrate metabolism enzymes, the DNA content and the lipid peroxidation level in yeast cells were analyzed. The viability of the postmitotic yeast cells after spaceflight showed a three-fold increase as compared with that of the ground control cells. Compared to the ground control cells, the lipid peroxidation level in the spaceflight yeast cells markedly decreased. The spaceflight yeast cells also showed an increase in G2/M cell population and a decrease in Sub-G1 cell population. The glycogen content and the activities of hexokinase and succinate dehydrogenase significantly decreased in the yeast cells after spaceflight. In contrast, the activity of malate dehydrogenase showed an obvious increase after spaceflight. These results suggested that microgravity or spaceflight could promote the survival of postmitotic S. cerevisiae cells through regulating carbohydrate metabolism, ROS level and cell cycle progression.

  1. Early manifestations of replicative aging in the yeast Saccharomyces cerevisiae

    PubMed Central

    Sorokin, Maksim I.; Knorre, Dmitry A.; Severin, Fedor F.

    2014-01-01

    The yeast Saccharomyces cerevisiae is successfully used as a model organism to find genes responsible for lifespan control of higher organisms. As functional decline of higher eukaryotes can start as early as one quarter of the average lifespan, we asked whether S. cerevisiae can be used to model this manifestation of aging. While the average replicative lifespan of S. cerevisiae mother cells ranges between 15 and 30 division cycles, we found that resistances to certain stresses start to decrease much earlier. Looking into the mechanism, we found that knockouts of genes responsible for mitochondria-to-nucleus (retrograde) signaling, RTG1 or RTG3, significantly decrease the resistance of cells that generated more than four daughters, but not of the younger ones. We also found that even young mother cells frequently contain mitochondria with heterogeneous transmembrane potential and that the percentage of such cells correlates with replicative age. Together, these facts suggest that retrograde signaling starts to malfunction in relatively young cells, leading to accumulation of heterogeneous mitochondria within one cell. The latter may further contribute to a decline in stress resistances.

  2. Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties.

    PubMed

    Cakar, Z Petek; Turanli-Yildiz, Burcu; Alkim, Ceren; Yilmaz, Ulkü

    2012-03-01

    This article reviews evolutionary engineering of Saccharomyces cerevisiae. Following a brief introduction to the 'rational' metabolic engineering approach and its limitations such as extensive genetic and metabolic information requirement on the organism of interest, complexity of cellular physiological responses, and difficulties of cloning in industrial strains, evolutionary engineering is discussed as an alternative, inverse metabolic engineering strategy. Major evolutionary engineering applications with S. cerevisiae are then discussed in two general categories: (1) evolutionary engineering of substrate utilization and product formation and (2) evolutionary engineering of stress resistance. Recent developments in functional genomics methods allow rapid identification of the molecular basis of the desired phenotypes obtained by evolutionary engineering. To conclude, when used alone or in combination with rational metabolic engineering and/or computational methods to study and analyze processes of adaptive evolution, evolutionary engineering is a powerful strategy for improvement in industrially important, complex properties of S. cerevisiae. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  3. Biogeographical characterization of Saccharomyces cerevisiae wine yeast by molecular methods

    PubMed Central

    Tofalo, Rosanna; Perpetuini, Giorgia; Schirone, Maria; Fasoli, Giuseppe; Aguzzi, Irene; Corsetti, Aldo; Suzzi, Giovanna

    2013-01-01

    Biogeography is the descriptive and explanatory study of spatial patterns and processes involved in the distribution of biodiversity. Without biogeography, it would be difficult to study the diversity of microorganisms because there would be no way to visualize patterns in variation. Saccharomyces cerevisiae, “the wine yeast,” is the most important species involved in alcoholic fermentation, and in vineyard ecosystems, it follows the principle of “everything is everywhere.” Agricultural practices such as farming (organic versus conventional) and floor management systems have selected different populations within this species that are phylogenetically distinct. In fact, recent ecological and geographic studies highlighted that unique strains are associated with particular grape varieties in specific geographical locations. These studies also highlighted that significant diversity and regional character, or ‘terroir,’ have been introduced into the winemaking process via this association. This diversity of wild strains preserves typicity, the high quality, and the unique flavor of wines. Recently, different molecular methods were developed to study population dynamics of S. cerevisiae strains in both vineyards and wineries. In this review, we will provide an update on the current molecular methods used to reveal the geographical distribution of S. cerevisiae wine yeast. PMID:23805132

  4. Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

    PubMed Central

    Feyder, Serge; De Craene, Johan-Owen; Bär, Séverine; Bertazzi, Dimitri L.; Friant, Sylvie

    2015-01-01

    The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes. PMID:25584613

  5. PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae

    PubMed Central

    2010-01-01

    Background In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. Results In the present study we show that overexpression of PGM2 under control of the HXT7'promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. PGM2 overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When PGM2 was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying PGM2 on the integrative plasmid. Conclusion PGM2 overexpression in S. cerevisiae from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity. PMID:20507616

  6. Early manifestations of replicative aging in the yeast Saccharomyces cerevisiae.

    PubMed

    Sorokin, Maksim I; Knorre, Dmitry A; Severin, Fedor F

    2014-01-06

    The yeast Saccharomyces cerevisiae is successfully used as a model organism to find genes responsible for lifespan control of higher organisms. As functional decline of higher eukaryotes can start as early as one quarter of the average lifespan, we asked whether S. cerevisiae can be used to model this manifestation of aging. While the average replicative lifespan of S. cerevisiae mother cells ranges between 15 and 30 division cycles, we found that resistances to certain stresses start to decrease much earlier. Looking into the mechanism, we found that knockouts of genes responsible for mitochondria-to-nucleus (retrograde) signaling, RTG1 or RTG3, significantly decrease the resistance of cells that generated more than four daughters, but not of the younger ones. We also found that even young mother cells frequently contain mitochondria with heterogeneous transmembrane potential and that the percentage of such cells correlates with replicative age. Together, these facts suggest that retrograde signaling starts to malfunction in relatively young cells, leading to accumulation of heterogeneous mitochondria within one cell. The latter may further contribute to a decline in stress resistances.

  7. Role of social wasps in Saccharomyces cerevisiae ecology and evolution.

    PubMed

    Stefanini, Irene; Dapporto, Leonardo; Legras, Jean-Luc; Calabretta, Antonio; Di Paola, Monica; De Filippo, Carlotta; Viola, Roberto; Capretti, Paolo; Polsinelli, Mario; Turillazzi, Stefano; Cavalieri, Duccio

    2012-08-14

    Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vector and natural reservoir of S. cerevisiae during all seasons. We provide experimental evidence that queens of social wasps overwintering as adults (Vespa crabro and Polistes spp.) can harbor yeast cells from autumn to spring and transmit them to their progeny. This result is mirrored by field surveys of the genetic variability of natural strains of yeast. Microsatellites and sequences of a selected set of loci able to recapitulate the yeast strain's evolutionary history were used to compare 17 environmental wasp isolates with a collection of strains from grapes from the same region and more than 230 strains representing worldwide yeast variation. The wasp isolates fall into subclusters representing the overall ecological and industrial yeast diversity of their geographic origin. Our findings indicate that wasps are a key environmental niche for the evolution of natural S. cerevisiae populations, the dispersion of yeast cells in the environment, and the maintenance of their diversity. The close relatedness of several wasp isolates with grape and wine isolates reflects the crucial role of human activities on yeast population structure, through clonal expansion and selection of specific strains during the biotransformation of fermented foods, followed by dispersal mediated by insects and other animals.

  8. Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae.

    PubMed

    Jin, Yong-Su; Jeffries, Thomas W

    2004-07-01

    Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast traditionally used in ethanol production from hexose. However, recombinant S. cerevisiae created in several laboratories have used xylose oxidatively rather than in the fermentative manner that this yeast metabolizes glucose. To understand the differences between glucose and engineered xylose metabolic networks, we performed a flux balance analysis (FBA) and calculated extreme pathways using a stoichiometric model that describes the biochemistry of yeast cell growth. FBA predicted that the ethanol yield from xylose exhibits a maximum under oxygen-limited conditions, and a fermentation experiment confirmed this finding. Fermentation results were largely consistent with in silico phenotypes based on calculated extreme pathways, which displayed several phases of metabolic phenotype with respect to oxygen availability from anaerobic to aerobic conditions. However, in contrast to the model prediction, xylitol production continued even after the optimum aeration level for ethanol production was attained. These results suggest that oxygen (or some other electron accepting system) is required to resolve the redox imbalance caused by cofactor difference between xylose reductase and xylitol dehydrogenase, and that other factors limit glycolytic flux when xylose is the sole carbon source.

  9. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

    PubMed Central

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S.; Flick, Robert; Wolf, Yuri I.; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D.; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M.; Koonin, Eugene V.; Yakunin, Alexander F.

    2015-01-01

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. PMID:26071590

  10. Multilocus sequence typing of oenological Saccharomyces cerevisiae strains.

    PubMed

    Muñoz, Rosario; Gómez, Alicia; Robles, Virginia; Rodríguez, Patricia; Cebollero, Eduardo; Tabera, Laura; Carrascosa, Alfonso V; Gonzalez, Ramon

    2009-12-01

    This study describes the application of a multilocus sequence typing (MLST) analysis for molecular discrimination at the strain level of Spanish wine yeast strains. The discrimination power of MLST is compared to mitochondrial RFLP analysis. Fragments of the ADP1, ACC1, RPN2, GLN4, and ALA1 genes were amplified by PCR from chromosomal DNA of 18 wine Saccharomyces cerevisiae strains. Ten polymorphic sites were found in the five loci analyzed showing 13 different genotypes, with 11 of them represented by only one strain. RFLP analysis of the same 18 wine yeast strains showed seventeen different mitochondrial patterns. Phylogenetic relationships among the strains analyzed, inferred by MLST data, showed wine isolates of S. cerevisiae as a rather homogeneous group. The discrimination potential of mitochondrial RFLP analysis was superior to the MLST scheme used in this work. However, MLST analysis allowed an easy construction of reliable phylogenetic trees. MLST analysis offers the possibility of typing wine S. cerevisiae strains simultaneously to the study of the genetic relationship among them.

  11. Membrane trafficking in the yeast Saccharomyces cerevisiae model.

    PubMed

    Feyder, Serge; De Craene, Johan-Owen; Bär, Séverine; Bertazzi, Dimitri L; Friant, Sylvie

    2015-01-09

    The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes.

  12. Osmo-, Thermo- and Ethanol- Tolerances of Saccharomyces cerevisiae S1

    PubMed Central

    Balakumar, Sandrasegarampillai; Arasaratnam, Vasanthy

    2012-01-01

    Saccharomyces cerevisiae S1, which is a locally isolated and improved strain showed viability at 40, 45 and 50°C and produced ethanol at 40, 43 and 45°C. When the cells were given heat shock at 45°C for 30min and grown at 40°C, 100% viability was observed for 60h, and addition of 200gL−1 ethanol has led to complete cell death at 30h. Heat shock given at 45°C (for 30min) has improved the tolerance to temperature induced ethanol shock leading to 37% viability at 30h. When the cells were subjected to ethanol (200gL−1 for 30 min) and osmotic shock (sorbitol 300gL−1), trehalose contents in the cells were increased. The heat shocked cells showed better viability in presence of added ethanol. Soy flour supplementation has improved the viability of S. cerevisiae S1 to 80% in presence of 100gL−1 added ethanol and to 60% in presence of 300gL−1sorbitol. In presence of sorbitol (200gL−1) and ethanol (50gL−1) at 40°C, 46% viability was retained by S. cerevisiae S1 at 48h and it was improved to 80% by soy flour supplementation. PMID:24031814

  13. Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

    PubMed Central

    Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.

    1998-01-01

    As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. PMID:9464394

  14. Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p

    PubMed Central

    Li, Li; Lipke, Peter N.; Dranginis, Anne M.

    2016-01-01

    ABSTRACT FLO11 encodes a yeast cell wall flocculin that mediates a variety of adhesive phenotypes in Saccharomyces cerevisiae. Flo11p is implicated in many developmental processes, including flocculation, formation of pseudohyphae, agar invasion, and formation of microbial mats and biofilms. However, Flo11p mediates different processes in different yeast strains. To investigate the mechanisms by which FLO11 determines these differences in colony morphology, flocculation, and invasion, we studied gene structure, function, and expression levels. Nonflocculent Saccharomyces cerevisiae Σ1278b cells exhibited significantly higher FLO11 mRNA expression, especially in the stationary phase, than highly flocculent S. cerevisiae var. diastaticus. The two strains varied in cell surface hydrophobicity, and Flo11p contributed significantly to surface hydrophobicity in S. cerevisiae var. diastaticus but not in strain Σ1278b. Sequencing of the FLO11 gene in S. cerevisiae var. diastaticus revealed strain-specific differences, including a 15-amino-acid insertion in the adhesion domain. Flo11p adhesion domains from strain Σ1278b and S. cerevisiae var. diastaticus were expressed and used to coat magnetic beads. The adhesion domain from each strain bound preferentially to homologous cells, and the preferences were independent of the cells in which the adhesion domains were produced. These results are consistent with the idea that strain-specific variations in the amino acid sequences in the adhesion domains cause different Flo11p flocculation activities. The results also imply that strain-specific differences in expression levels, posttranslational modifications, and allelic differences outside the adhesion domains have little effect on flocculation. IMPORTANCE As a nonmotile organism, Saccharomyces cerevisiae employs the cell surface flocculin Flo11/Muc1 as an important means of adapting to environmental change. However, there is a great deal of strain variation in the

  15. Serum Anti-Saccharomyces Cerevisiae Antibodies in Greek Patients with Behcet's Disease

    PubMed Central

    Vaiopoulos, George; Lakatos, Peter Laszlo; Papp, Maria; Kaklamanis, Faedon; Economou, Efrosyni; Zevgolis, Vassilis; Sourdis, John

    2011-01-01

    We tested 59 Greek patients with Behcet's Disease (BD) for serum anti-Saccharomyces cerevisiae antibodies. No increase of these antibodies was detected in the cases compared to 55 healthy unrelated blood donors from the same population. This finding is in contrast with the correlation between Saccharomyces cerevisiae antibodies and BD as reported in other populations. It seems that environmental factors may contribute to disease expression in different populations, producing different effects according to the individual's genetic predisposition. Saccharomyces cerevisiae antibodies do not seem to be of any significance in the Greek population. PMID:21319357

  16. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

    PubMed Central

    Hawkins, Kristy M; Smolke, Christina D

    2010-01-01

    The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches. PMID:18690217

  17. Comparative proteomic analysis of Saccharomyces cerevisiae under different nitrogen sources.

    PubMed

    Zhao, Shaohui; Zhao, Xinrui; Zou, Huijun; Fu, Jianwei; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2014-04-14

    In cultures containing multiple sources of nitrogen, Saccharomyces cerevisiae exhibits a sequential use of nitrogen sources through a mechanism known as nitrogen catabolite repression (NCR). To identify proteins differentially expressed due to NCR, proteomic analysis of S. cerevisiae S288C under different nitrogen source conditions was performed using two-dimensional gel electrophoresis (2-DE), revealing 169 candidate protein spots. Among these 169 protein spots, 121 were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF). The identified proteins were closely associated with four main biological processes through Gene Ontology (GO) categorical analysis. The identification of the potential proteins and cellular processes related to NCR offer a global overview of changes elicited by different nitrogen sources, providing clues into how yeast adapt to different nutritional conditions. Moreover, by comparing our proteomic data with corresponding mRNA data, proteins regulated at the transcriptional and post-transcriptional level could be distinguished. Biological significance In S. cerevisiae, different nitrogen sources provide different growth characteristics and generate different metabolites. The nitrogen catabolite repression (NCR) process plays an important role for S. cerevisiae in the ordinal utilization of different nitrogen sources. NCR process can result in significant shift of global metabolic networks. Previous works on NCR primarily focused on transcriptomic level. The results obtained in this study provided a global atlas of the proteome changes triggered by different nitrogen sources and would facilitate the understanding of mechanisms for how yeast could adapt to different nutritional conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Proteomic analysis of protein methylation in the yeast Saccharomyces cerevisiae.

    PubMed

    Wang, Keyun; Zhou, Yongjin J; Liu, Hongwei; Cheng, Kai; Mao, Jiawei; Wang, Fangjun; Liu, Wujun; Ye, Mingliang; Zhao, Zongbao K; Zou, Hanfa

    2015-01-30

    Protein methylation catalyzed by SAM-dependent methyltransferase represents a major PTM involved in many important biological processes. Because methylation can occur on nitrogen, oxygen and sulfur centers and multiple methylation states exist on the nitrogen centers, methylproteome remains poorly documented. Here we present the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the yeast Saccharomyces cerevisiae based on the online multidimensional μHPLC/MS/MS technology. We identified 43 methylated proteins, containing 68 methylation events associated with 64 methylation sites. More than 90% of these methylation events were previously unannotated in Uniprot database. Our results indicated, 1) over 2.6% of identified S. cerevisiae proteins are methylated, 2) the amino acid residue preference of protein methylation follows the order Lys≫Arg>Asp>Asn≈Gln≈His>Glu>Cys, and 3) the methylation state on nitrogen center is largely exclusive. As our dataset covers various types of methylation centers, it provides rich information about yeast methylproteome and should significantly contribute to the field of protein methylation. In this paper, we presented the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the yeast S. cerevisiae and collected a comprehensive list of proteins methylated on a set of distinct residues (K, R, N, E, D, Q, H, C). Our study provided useful information about the amino acid residue preference and methylation state distributions on nitrogen centers of protein methylation in S. cerevisiae. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

    PubMed Central

    2012-01-01

    Background Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. Methods We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. Results We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. Conclusions In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most

  20. Isoprene hydrocarbons production upon heterologous transformation of Saccharomyces cerevisiae.

    PubMed

    Hong, S-Y; Zurbriggen, A S; Melis, A

    2012-07-01

      Isoprene (2-methyl-1,3-butadiene; C(5) H(8) ) is naturally produced by photosynthesis and emitted in the atmosphere by the leaves of many herbaceous, deciduous and woody plants. Fermentative yeast and fungi (Ascomycota) are not genetically endowed with the isoprene production process. The work investigated whether Ascomycota can be genetically modified and endowed with the property of constitutive isoprene production.   Two different strategies for expression of the IspS gene in Saccharomyces cerevisiae were employed: (i) optimization of codon usage of the IspS gene for specific expression in S. cerevisiae and (ii) multiple independent integrations of the IspS gene in the rDNA loci of the yeast genome. Copy number analysis showed that IspS transgenes were on the average incorporated within about 25% of the endogenous rDNA. Codon use optimization of the Pueraria montana (kudzu vine) IspS gene (SckIspS) for S. cerevisiae showed fivefold greater expression of the IspS protein compared with that of nonoptimized IspS (kIspS). With the strategies mentioned earlier, heterologous expression of the kudzu isoprene synthase gene (kIspS) in S. cerevisiae was tested for stability and as a potential platform of fermentative isoprene production. The multi-copy IspS transgenes were stably integrated and expressed for over 100 generations of yeast cell growth and constitutively produced volatile isoprene hydrocarbons. Secondary chemical modification of isoprene to a number of hydroxylated isoprene derivatives in the sealed reactor was also observed.   Transformation of S. cerevisiae with the Pueraria montana var. lobata (kudzu vine) isoprene synthase gene (IspS) conferred to the yeast cells constitutive isoprene hydrocarbons production in the absence of adverse or toxic effects.   First-time demonstration of constitutive isoprene hydrocarbons production in a fermentative eukaryote operated through the mevalonic acid pathway. The work provides concept validation for the

  1. Effects of Fusariotoxin T-2 on Saccharomyces cerevisiae and Saccharomyces carlsbergensis

    PubMed Central

    Schappert, Keith T.; Khachatourians, George G.

    1983-01-01

    A Fusarium metabolite, T-2 toxin, inhibits the growth of Saccharomyces carlsbergensis and Saccharomyces cerevisiae. The growth inhibitory concentrations of T-2 toxin were 40 and 100 μg/ml, respectively, for exponentially growing cultures of the two yeasts. S. carlsbergensis was more sensitive to the toxin and exhibited a biphasic dose-response curve. Addition of the toxin at 10 μg/ml of S. carlsbergensis culture resulted in a retardation of growth as measured turbidimetrically, after only 30 to 40 min. This action was reversible upon washing the cells free of the toxin. The sensitivity of the yeasts to the toxin was dependent upon the types and concentrations of carbohydrates used in the growth media. The sensitivity of the cells to the toxin decreased in glucose-repressed cultures. These results suggest that T-2 toxin interferes with mitochondrial functions of these yeasts. Images PMID:16346249

  2. Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae.

    PubMed

    Chai, Fenghua; Wang, Ying; Mei, Xueang; Yao, Mingdong; Chen, Yan; Liu, Hong; Xiao, Wenhai; Yuan, Yingjin

    2017-03-29

    Due to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially. By introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell. Saccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating

  3. Exposure to benzene metabolites causes oxidative damage in Saccharomyces cerevisiae.

    PubMed

    Raj, Abhishek; Nachiappan, Vasanthi

    2016-06-01

    Hydroquinone (HQ) and benzoquinone (BQ) are known benzene metabolites that form reactive intermediates such as reactive oxygen species (ROS). This study attempts to understand the effect of benzene metabolites (HQ and BQ) on the antioxidant status, cell morphology, ROS levels and lipid alterations in the yeast Saccharomyces cerevisiae. There was a reduction in the growth pattern of wild-type cells exposed to HQ/BQ. Exposure of yeast cells to benzene metabolites increased the activity of the anti-oxidant enzymes catalase, superoxide dismutase and glutathione peroxidase but lead to a decrease in ascorbic acid and reduced glutathione. Increased triglyceride level and decreased phospholipid levels were observed with exposure to HQ and BQ. These results suggest that the enzymatic antioxidants were increased and are involved in the protection against macromolecular damage during oxidative stress; presumptively, these enzymes are essential for scavenging the pro-oxidant effects of benzene metabolites.

  4. Influence of killer strains of Saccharomyces cerevisiae on wine fermentation.

    PubMed

    Pérez, F; Ramírez, M; Regodón, J A

    2001-09-01

    The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.

  5. Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration.

    PubMed

    Lin, Su-Ju; Kaeberlein, Matt; Andalis, Alex A; Sturtz, Lori A; Defossez, Pierre-Antoine; Culotta, Valeria C; Fink, Gerald R; Guarente, Leonard

    2002-07-18

    Calorie restriction (CR) extends lifespan in a wide spectrum of organisms and is the only regimen known to lengthen the lifespan of mammals. We established a model of CR in budding yeast Saccharomyces cerevisiae. In this system, lifespan can be extended by limiting glucose or by reducing the activity of the glucose-sensing cyclic-AMP-dependent kinase (PKA). Lifespan extension in a mutant with reduced PKA activity requires Sir2 and NAD (nicotinamide adenine dinucleotide). In this study we explore how CR activates Sir2 to extend lifespan. Here we show that the shunting of carbon metabolism toward the mitochondrial tricarboxylic acid cycle and the concomitant increase in respiration play a central part in this process. We discuss how this metabolic strategy may apply to CR in animals.

  6. Regulation of Phospholipid Synthesis in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Carman, George M.; Han, Gil-Soo

    2013-01-01

    The yeast Saccharomyces cerevisiae, with its full complement of organelles, synthesizes membrane phospholipids by pathways that are generally common to those found in higher eukaryotes. Phospholipid synthesis in yeast is regulated in response to a variety of growth conditions (e.g., inositol supplementation, zinc depletion, and growth stage) by a coordination of genetic (e.g., transcriptional activation and repression) and biochemical (e.g., activity modulation and localization) mechanisms. Phosphatidate (PA), whose cellular levels are controlled by the activities of key phospholipid synthesis enzymes, plays a central role in the transcriptional regulation of phospholipid synthesis genes. In addition to the regulation of gene expression, phosphorylation of key phospholipid synthesis catalytic and regulatory proteins controls the metabolism of phospholipid precursors and products. PMID:21275641

  7. Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae

    SciTech Connect

    Hayashi, Naoyuki Kobayashi, Masahiko; Shimizu, Hiroko; Yamamoto, Ken-ichi; Murakami, Seishi; Nishimoto, Takeharu

    2007-11-23

    The Ran GTPase system regulates the direction and timing of several cellular events, such as nuclear-cytosolic transport, centrosome formation, and nuclear envelope assembly in telophase. To gain insight into the Ran system's involvement in chromatin formation, we investigated gene silencing at the telomere in several mutants of the budding yeast Saccharomyces cerevisiae, which had defects in genes involved in the Ran system. A mutation of the RanGAP gene, rna1-1, caused reduced silencing at the telomere, and partial disruption of the nuclear Ran binding factor, yrb2-{delta}2, increased this silencing. The reduced telomere silencing in rna1-1 cells was suppressed by a high dosage of the SIR3 gene or the SIT4 gene. Furthermore, hyperphosphorylated Sir3 protein accumulated in the rna1-1 mutant. These results suggest that RanGAP is required for the heterochromatin structure at the telomere in budding yeast.

  8. Genetic dissection of acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Geng, Peng; Xiao, Yin; Hu, Yun; Sun, Haiye; Xue, Wei; Zhang, Liang; Shi, Gui-Yang

    2016-09-01

    Dissection of the hereditary architecture underlying Saccharomyces cerevisiae tolerance to acetic acid is essential for ethanol fermentation. In this work, a genomics approach was used to dissect hereditary variations in acetic acid tolerance between two phenotypically different strains. A total of 160 segregants derived from these two strains were obtained. Phenotypic analysis indicated that the acetic acid tolerance displayed a normal distribution in these segregants, and suggested that the acetic acid tolerant traits were controlled by multiple quantitative trait loci (QTLs). Thus, 220 SSR markers covering the whole genome were used to detect QTLs of acetic acid tolerant traits. As a result, three QTLs were located on chromosomes 9, 12, and 16, respectively, which explained 38.8-65.9 % of the range of phenotypic variation. Furthermore, twelve genes of the candidates fell into the three QTL regions by integrating the QTL analysis with candidates of acetic acid tolerant genes. These results provided a novel avenue to obtain more robust strains.

  9. Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

    PubMed Central

    Dever, Thomas E.; Kinzy, Terri Goss; Pavitt, Graham D.

    2016-01-01

    In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs. PMID:27183566

  10. Bioethanol production from cellulosic hydrolysates by engineered industrial Saccharomyces cerevisiae.

    PubMed

    Lee, Ye-Gi; Jin, Yong-Su; Cha, Young-Lok; Seo, Jin-Ho

    2017-03-01

    Even though industrial yeast strains exhibit numerous advantageous traits for the production of bioethanol, their genetic manipulation has been limited. This study demonstrates that an industrial polyploidy Saccharomyces cerevisiae JHS200 can be engineered through Cas9 (CRISPR associated protein 9)-based genome editing. Specifically, we generated auxotrophic mutants and introduced a xylose metabolic pathway into the auxotrophic mutants. As expected, the engineered strain (JX123) enhanced ethanol production from cellulosic hydrolysates as compared to other engineered haploid strains. However, the JX123 strain produced substantial amounts of xylitol as a by-product during xylose fermentation. Hypothesizing that the xylitol accumulation might be caused by intracellular redox imbalance from cofactor difference, the NADH oxidase from Lactococcus lactis was introduced into the JX123 strain. The resulting strain (JX123_noxE) not only produced more ethanol, but also produced xylitol less than the JX123 strain. These results suggest that industrial polyploidy yeast can be modified for producing biofuels and chemicals.

  11. Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae

    PubMed Central

    Walters, Robert W.; Matheny, Tyler; Mizoue, Laura S.; Rao, Bhalchandra S.; Muhlrad, Denise; Parker, Roy

    2017-01-01

    RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5′ nicotinamide-adenine dinucleotide (NAD+) RNA in bacteria. Whether 5′ NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5′ NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae. NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5′ end processed. These results define an additional 5′ RNA cap structure in eukaryotes and raise the possibility that this 5′ NAD+ cap could modulate RNA stability and translation on specific subclasses of mRNAs. PMID:28031484

  12. The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae.

    PubMed

    ter Schure, E G; Silljé, H H; Verkleij, A J; Boonstra, J; Verrips, C T

    1995-11-01

    Saccharomyces cerevisiae was grown in a continuous culture at a single dilution rate with input ammonia concentrations whose effects ranged from nitrogen limitation to nitrogen excess and glucose limitation. The rate of ammonia assimilation (in millimoles per gram of cells per hour) was approximately constant. Increased extracellular ammonia concentrations are correlated with increased intracellular glutamate and glutamine concentrations, increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2), and decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1), as well as decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4. The governing factor of nitrogen metabolism might be the concentration of ammonia rather than its flux.

  13. Conservative Duplication of Spindle Poles during Meiosis in Saccharomyces cerevisiae

    PubMed Central

    Wesp, Andreas; Prinz, Susanne; Fink, Gerald R.

    2001-01-01

    During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p. PMID:11244080

  14. Conservative duplication of spindle poles during meiosis in Saccharomyces cerevisiae.

    PubMed

    Wesp, A; Prinz, S; Fink, G R

    2001-04-01

    During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.

  15. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast

  16. Bioaccumulation of cadmium by growing Zygosaccharomyces rouxii and Saccharomyces cerevisiae.

    PubMed

    Li, Chunsheng; Jiang, Wei; Ma, Ning; Zhu, Yinglian; Dong, Xiaoyan; Wang, Dongfeng; Meng, Xianghong; Xu, Ying

    2014-03-01

    Bioaccumulation via growing cells is a potential technique for heavy metal removal from food materials. The cadmium bioaccumulation characteristics by growing Zygosaccharomyces rouxii and Saccharomyces cerevisiae were investigated. Z. rouxii displayed powerful cadmium removal ability at low cadmium concentrations, which mainly depended on the intracellular cadmium bioaccumulation. The percentage of intracellular cadmium bioaccumulation of both yeasts obviously decreased with the increase of initial biomass and cadmium concentrations. Low pH and elevated concentrations of zinc and copper significantly decreased the intracellular cadmium bioaccumulation of both yeasts but improved the cadmium tolerance and the cell-surface cadmium bioaccumulation of Z. rouxii. Cadmium removal of Z. rouxii was improved by zinc and copper conditionally. Z. rouxii that possessed more powerful cadmium tolerance and removal ability at low pH and high concentration of competing ions can be developed into a potential cadmium removal agent using in complex food environment in future. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Effect of different glucose concentrations on proteome of Saccharomyces cerevisiae.

    PubMed

    Guidi, Francesca; Francesca, Guidi; Magherini, Francesca; Francesca, Magherini; Gamberi, Tania; Tania, Gamberi; Borro, Marina; Marina, Borro; Simmaco, Maurizio; Maurizio, Simmaco; Modesti, Alessandra; Alessandra, Modesti

    2010-07-01

    We performed a proteomic study to understand how Saccharomyces cerevisiae adapts its metabolism during the exponential growth on three different concentrations of glucose; this information will be necessary to understand yeast carbon metabolism in different environments. We induced a natural diauxic shift by growing yeast cells in glucose restriction thus having a fast and complete glucose exhaustion. We noticed differential expressions of groups of proteins. Cells in high glucose have a decreased growth rate during the initial phase of fermentation; in glucose restriction and in high glucose we found an over-expression of a protein (Peroxiredoxin) involved in protection against oxidative stress insult. The information obtained in our study validates the application of a proteomic approach for the identification of the molecular bases of environmental variations such as fermentation in high glucose and during a naturally induced diauxic shift.

  18. The Influence of Microgravity on Invasive Growth in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Van Mulders, Sebastiaan E.; Stassen, Catherine; Daenen, Luk; Devreese, Bart; Siewers, Verena; van Eijsden, Rudy G. E.; Nielsen, Jens; Delvaux, Freddy R.; Willaert, Ronnie

    2011-01-01

    This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Σ1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Σ1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Σ1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium.

  19. Hydrogen peroxide removal with magnetically responsive Saccharomyces cerevisiae cells.

    PubMed

    Safarik, Ivo; Sabatkova, Zdenka; Safarikova, Mirka

    2008-09-10

    Hydrogen peroxide (HP) is a promising chemical sanitizer for use in the food industry. Its residues have to be decomposed, usually using an enzyme process employing catalase. In order to offer an inexpensive biocatalyst and to simplify subsequent manipulation, we have prepared magnetically responsive alginate beads containing entrapped Saccharomyces cerevisiae cells and magnetite microparticles. Larger beads (2-3 mm in diameter) were prepared by dropping the mixture into calcium chloride solution, while microbeads (the diameter of majority of particles ranged between 50 and 100 microm) were prepared using the water in oil emulsification process. In general, microbeads enabled more efficient HP decomposition. The prepared microparticulate biocatalyst caused efficient decomposition of HP in water solutions (up to 2% concentration), leaving very low residual HP concentration after treatment (below 0.001% under appropriate conditions). The biocatalyst was stable; the same catalytic activity was observed after one month storage at 4 degrees C, and the microbeads could be used at least five times.

  20. MPS3 mediates meiotic bouquet formation in Saccharomyces cerevisiae

    PubMed Central

    Conrad, Michael N.; Lee, Chih-Ying; Wilkerson, Joseph L.; Dresser, Michael E.

    2007-01-01

    In meiotic prophase, telomeres associate with the nuclear envelope and accumulate adjacent to the centrosome/spindle pole to form the chromosome bouquet, a well conserved event that in Saccharomyces cerevisiae requires the meiotic telomere protein Ndj1p. Ndj1p interacts with Mps3p, a nuclear envelope SUN domain protein that is required for spindle pole body duplication and for sister chromatid cohesion. Removal of the Ndj1p-interaction domain from MPS3 creates an ndj1Δ-like separation-of-function allele, and Ndj1p and Mps3p are codependent for stable association with the telomeres. SUN domain proteins are found in the nuclear envelope across phyla and are implicated in mediating interactions between the interior of the nucleus and the cytoskeleton. Our observations indicate a general mechanism for meiotic telomere movements. PMID:17495028

  1. Technology development for natural product biosynthesis in Saccharomyces cerevisiae.

    PubMed

    Billingsley, John M; DeNicola, Anthony B; Tang, Yi

    2016-12-01

    The explosion of genomic sequence data and the significant advancements in synthetic biology have led to the development of new technologies for natural products discovery and production. Using powerful genetic tools, the yeast Saccharomyces cerevisiae has been engineered as a production host for natural product pathways from bacterial, fungal, and plant species. With an expanding library of characterized genetic parts, biosynthetic pathways can be refactored for optimized expression in yeast. New engineering strategies have enabled the increased production of valuable secondary metabolites by tuning metabolic pathways. Improvements in high-throughput screening methods have facilitated the rapid identification of variants with improved biosynthetic capabilities. In this review, we focus on the molecular tools and engineering strategies that have recently empowered heterologous natural product biosynthesis.

  2. Regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae.

    PubMed

    Carman, George M; Han, Gil-Soo

    2011-01-01

    The yeast Saccharomyces cerevisiae, with its full complement of organelles, synthesizes membrane phospholipids by pathways that are generally common to those found in higher eukaryotes. Phospholipid synthesis in yeast is regulated in response to a variety of growth conditions (e.g., inositol supplementation, zinc depletion, and growth stage) by a coordination of genetic (e.g., transcriptional activation and repression) and biochemical (e.g., activity modulation and localization) mechanisms. Phosphatidate (PA), whose cellular levels are controlled by the activities of key phospholipid synthesis enzymes, plays a central role in the transcriptional regulation of phospholipid synthesis genes. In addition to the regulation of gene expression, phosphorylation of key phospholipid synthesis catalytic and regulatory proteins controls the metabolism of phospholipid precursors and products.

  3. Parallel Identification of New Genes in Saccharomyces cerevisiae

    PubMed Central

    Oshiro, Guy; Wodicka, Lisa M.; Washburn, Michael P.; Yates, John R.; Lockhart, David J.; Winzeler, Elizabeth A.

    2002-01-01

    Short open reading frames (ORFs) occur frequently in primary genome sequence. Distinguishing bona fide small genes from the tens of thousands of short ORFs is one of the most challenging aspects of genome annotation. Direct experimental evidence is often required. Here we use a combination of expression profiling and mass spectrometry to verify the independent transcription of 138 and the translation of 50 previously nonannotated genes in the Saccharomyces cerevisiae genome. Through combined evidence, we propose the addition of 62 new genes to the genome and provide experimental support for the inclusion of 10 previously identified genes. [The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: V. Velculescu. Supplementary material is available online at http://www.genome.org.] PMID:12176929

  4. Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae.

    PubMed

    Dever, Thomas E; Kinzy, Terri Goss; Pavitt, Graham D

    2016-05-01

    In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs.

  5. Molecular architecture of the Saccharomyces cerevisiae activated spliceosome.

    PubMed

    Rauhut, Reinhard; Fabrizio, Patrizia; Dybkov, Olexandr; Hartmuth, Klaus; Pena, Vladimir; Chari, Ashwin; Kumar, Vinay; Lee, Chung-Tien; Urlaub, Henning; Kastner, Berthold; Stark, Holger; Lührmann, Reinhard

    2016-09-23

    The activated spliceosome (B(act)) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae B(act) complex at 5.8-angstrom resolution. Our model reveals that in B(act), the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5' splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile-the branchsite adenosine-is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5'ss. Our structure suggests that Prp2 adenosine triphosphatase-mediated remodeling leads to conformational changes in Hsh155's HEAT domain that liberate the first-step reactants for catalysis.

  6. Overexpressed ribosomal proteins suppress defective chaperonins in Saccharomyces cerevisiae.

    PubMed

    Kabir, M Anaul; Sherman, Fred

    2008-12-01

    The chaperonin Cct complex of the yeast Saccharomyces cerevisiae is composed of eight different subunits encoded by eight essential genes, CCT1-CCT8. This Cct complex is responsible for the folding of a number of proteins including actin and tubulin. We have isolated and characterized 22 multicopy suppressors of the temperature-sensitive allele, cct4-1, which encodes an altered protein with a G345D replacement that diminishes ATP hydrolysis. Fourteen of the suppressors encode ribosomal proteins, four have roles in ribosome biogenesis, two have phosphatase activities, one is involved in protein synthesis and one of the suppressors corresponded to Cct4p. Some of the suppressors also acted on certain cct1, cct2, cct3 and cct6 mutations. We suggest that certain overexpressed ribosomal and other proteins can act as weak chaperones, phenotypically alleviating the partial defects of mutationally altered Cct subunits.

  7. Saccharomyces cerevisiae and Neurospora crassa contain heavy metal sequestering phytochelatin.

    PubMed

    Kneer, R; Kutchan, T M; Hochberger, A; Zenk, M H

    1992-01-01

    In fungi, cellular resistance to heavy metal cytotoxicity is mediated either by binding of metal ions to proteins of the metallothionein type or by chelation to phytochelatin-peptides of the general formula (gamma-Glu-Cys)n-Gly. Hitherto, only one fungus, Candida glabrata has been shown to contain both metal inactivating systems. Here we show by unambiguous FAB-MS analysis that both a metallothionein-free mutant of Saccharomyces cerevisiae as well as a wildtype strain synthesize phytochelatin (PC2) upon exposure to 250 microM Cd2+ ions. The presence of Zn and/or Cu ions in the nutrient broth also induces PC2 synthesis in this organism. By 109Cd exchange and subsequent monobromobimane fluorescence HPLC, it could be shown that the presence of Cd2+ in the growth medium also induces phytochelatin synthesis in Neurospora crassa, which contains metallothioneins.

  8. Patterns in Saccharomyces cerevisiae yeast colonies via magnetic resonance imaging.

    PubMed

    Tenório, Rômulo P; Barros, Wilson

    2017-01-23

    We report the use of high-resolution magnetic resonance imaging methods to observe pattern formation in colonies of Saccharomyces cerevisiae. Our results indicate substantial signal loss localized in specific regions of the colony rendering useful imaging contrast. This imaging contrast is recognizable as being due to discontinuities in magnetic susceptibility (χ) between different spatial regions. At the microscopic pixel level, the local variations in the magnetic susceptibility (Δχ) induce a loss in the NMR signal, which was quantified via T2 and T2* maps, permitting estimation of Δχ values for different regions of the colony. Interestingly the typical petal/wrinkling patterns present in the colony have a high degree of correlation with the estimated susceptibility distribution. We conclude that the presence of magnetic susceptibility inclusions, together with their spatial arrangement within the colony, may be a potential cause of the susceptibility distribution and therefore the contrast observed on the images.

  9. Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae.

    PubMed

    Nielsen, Jens; Jewett, Michael C

    2008-02-01

    Industrial biotechnology is a rapidly growing field. With the increasing shift towards a bio-based economy, there is rising demand for developing efficient cell factories that can produce fuels, chemicals, pharmaceuticals, materials, nutraceuticals, and even food ingredients. The yeast Saccharomyces cerevisiae is extremely well suited for this objective. As one of the most intensely studied eukaryotic model organisms, a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance can be capitalized upon to enable a substantial increase in the industrial application of this yeast. Developments in genomics and high-throughput systems biology tools are enhancing one's ability to rapidly characterize cellular behaviour, which is valuable in the field of metabolic engineering where strain characterization is often the bottleneck in strain development programmes. Here, the impact of systems biology on metabolic engineering is reviewed and perspectives on the role of systems biology in the design of cell factories are given.

  10. Saccharomyces cerevisiae Yta7 Regulates Histone Gene Expression

    PubMed Central

    Gradolatto, Angeline; Rogers, Richard S.; Lavender, Heather; Taverna, Sean D.; Allis, C. David; Aitchison, John D.; Tackett, Alan J.

    2008-01-01

    The Saccharomyces cerevisiae Yta7 protein is a component of a nucleosome bound protein complex that maintains distinct transcriptional zones of chromatin. We previously found that one protein copurifying with Yta7 is the yFACT member Spt16. Epistasis analyses revealed a link between Yta7, Spt16, and other previously identified members of the histone regulatory pathway. In concurrence, Yta7 was found to regulate histone gene transcription in a cell-cycle-dependent manner. Association at the histone gene loci appeared to occur through binding of the bromodomain-like region of Yta7 with the N-terminal tail of histone H3. Our work suggests a mechanism in which Yta7 is localized to chromatin to establish regions of transcriptional silencing, and that one facet of this cellular mechanism is to modulate transcription of histone genes. PMID:18493054

  11. Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

    PubMed Central

    Symington, Lorraine S.; Rothstein, Rodney; Lisby, Michael

    2014-01-01

    Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell. PMID:25381364

  12. Tolerance of budding yeast Saccharomyces cerevisiae to ultra high pressure

    NASA Astrophysics Data System (ADS)

    Shibata, M.; Torigoe, M.; Matsumoto, Y.; Yamamoto, M.; Takizawa, N.; Hada, Y.; Mori, Y.; Takarabe, K.; Ono, F.

    2014-05-01

    Our studies on the tolerance of plants and animals against very high pressure of several GPa have been extended to a smaller sized fungus, the budding yeast Saccharomyces cerevisiae. Several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate, and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar. It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for longer than 12 h were found dead. The high pressure tolerance of budding yeast is a little weaker than that of tardigrades.

  13. Production of natural products through metabolic engineering of Saccharomyces cerevisiae.

    PubMed

    Krivoruchko, Anastasia; Nielsen, Jens

    2015-12-01

    Many high-value metabolites are produced in nature by organisms that are not ideal for large-scale production. Therefore, interest exists in expressing the biosynthetic pathways of these compounds in organisms that are more suitable for industrial production. Recent years have seen developments in both the discovery of various biosynthetic pathways, as well as development of metabolic engineering tools that allow reconstruction of complex pathways in microorganisms. In the present review we discuss recent advances in reconstruction of the biosynthetic pathways of various high-value products in the yeast Saccharomyces cerevisiae, a commonly used industrial microorganism. Key achievements in the production of different isoprenoids, aromatics and polyketides are presented and the metabolic engineering strategies underlying these accomplishments are discussed.

  14. Preferentially quantized linker DNA lengths in Saccharomyces cerevisiae.

    PubMed

    Wang, Ji-Ping; Fondufe-Mittendorf, Yvonne; Xi, Liqun; Tsai, Guei-Feng; Segal, Eran; Widom, Jonathan

    2008-09-12

    The exact lengths of linker DNAs connecting adjacent nucleosomes specify the intrinsic three-dimensional structures of eukaryotic chromatin fibers. Some studies suggest that linker DNA lengths preferentially occur at certain quantized values, differing one from another by integral multiples of the DNA helical repeat, approximately 10 bp; however, studies in the literature are inconsistent. Here, we investigate linker DNA length distributions in the yeast Saccharomyces cerevisiae genome, using two novel methods: a Fourier analysis of genomic dinucleotide periodicities adjacent to experimentally mapped nucleosomes and a duration hidden Markov model applied to experimentally defined dinucleosomes. Both methods reveal that linker DNA lengths in yeast are preferentially periodic at the DNA helical repeat ( approximately 10 bp), obeying the forms 10n+5 bp (integer n). This 10 bp periodicity implies an ordered superhelical intrinsic structure for the average chromatin fiber in yeast.

  15. On the Mechanism of Gene Silencing in Saccharomyces cerevisiae

    PubMed Central

    Steakley, David Lee; Rine, Jasper

    2015-01-01

    Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation. This study provided strong support for the steric occlusion mechanism by the discovery that RNA polymerase of bacteriophage T7 could be substantially blocked from transcribing from its cognate promoter when embedded in silenced chromatin. Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin. The effectiveness of the Sir protein–based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing. PMID:26082137

  16. Mutants of Saccharomyces cerevisiae with defective vacuolar function

    SciTech Connect

    Kitamoto, K.; Yoshizawa, K.; Ohsumi, Y.; Anraku, Y.

    1988-06-01

    Mutants of the yeast Saccharomyces cerevisiae that have a small vacuolar lysine pool were isolated and characterized. Mutant KL97 (lys1 slp1-1) and strain KL197-1A (slp1-1), a prototrophic derivative of KL97, did not grow well in synthetic medium supplemented with 10 mM lysine. Genetic studies indicated that the slp1-1mutation (for small lysine pool) is recessive and is due to a single chromosomal mutation. Mutant KL97 shows the following pleiotropic defects in vacuolar functions. (i) It has small vacuolar pools for lysine, arginine, and histidine. (ii) Its growth is sensitive to lysine, histidine, Ca/sup 2 +/, heavy metal ions, and antibiotics. (iii) It has many small vesicles but no large central vacuole. (iv) It has a normal amount of the vacuolar membrane marker ..cap alpha..-mannosidase but shows reduced activities of the vacuole sap markers proteinase A, proteinase B, and carboxypeptidase Y.

  17. Draft Genome Sequence of the Yeast Saccharomyces cerevisiae GUJ105 From Gujarat, India

    PubMed Central

    Detroja, Rajesh; Rathore, Ankita

    2016-01-01

    Here, we report the draft genome sequence of Saccharomyces cerevisiae strain GUJ105, isolated clinically. The size of the genome is approximately 11.5 Mb and contains 5,447 protein-coding genes. PMID:27908989

  18. Nitrogen and carbon assimilation by Saccharomyces cerevisiae during Sauvignon blanc juice fermentation.

    PubMed

    Pinu, Farhana R; Edwards, Patrick J B; Gardner, Richard C; Villas-Boas, Silas G

    2014-12-01

    To investigate the assimilation and production of juice metabolites by Saccharomyces cerevisiae during winemaking, we compared the metabolite profiles of 63 Sauvignon blanc (SB) grape juices collected over five harvesting seasons from different locations of New Zealand before and after fermentation by the commercial wine yeast strain EC1118 at 15 °C. Metabolite profiles were obtained using gas chromatography-mass spectrometry and nuclear magnetic resonance and the oenological parameters were determined by Fourier transform infrared spectroscopy. Our results revealed that the amino acids threonine and serine were the most consumed organic nitrogen sources, while proline and gamma-aminobutyric acid were the least consumed amino acids during SB juice fermentation. Saccharomyces cerevisiae metabolised some uncommon nitrogen sources (e.g. norleucine, norvaline and pyroglutamic acid) and several organic acids, including some fatty acids, most likely after fermenting the main juice sugars (glucose, fructose and mannose). However, consumption showed large variation between juices and in some cases between seasons. Our study clearly shows that preferred nitrogen and carbon sources were consumed by S. cerevisiae EC1118 independent of the juice fine composition, whilst the consumption of other nutrient sources mainly depended on the concentration of other juice metabolites, which explains the uniqueness of each barrel of wine. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  19. Micromechanical and surface adhesive properties of single saccharomyces cerevisiae cells

    NASA Astrophysics Data System (ADS)

    Farzi, Bahman; Cetinkaya, Cetin

    2017-09-01

    The adhesion and mechanical properties of a biological cell (e.g. cell membrane elasticity and adhesiveness) are often strong indicators for the state of its health. Many existing techniques for determining mechanical properties of cells require direct physical contact with a single cell or a group of cells. Physical contact with the cell can trigger complex mechanotransduction mechanisms, leading to cellular responses, and consequently interfering with measurement accuracy. In the current work, based on ultrasonic excitation and interferometric (optical) motion detection, a non-contact method for characterizing the adhesion and mechanical properties of single cells is presented. It is experimentally demonstrated that the rocking (rigid body) motion and internal vibrational resonance frequencies of a single saccharomyces cerevisiae (SC) (baker’s yeast) cell can be acquired with the current approach, and the Young’s modulus and surface tension of the cell membrane as well as surface adhesion energy can be extracted from the values of these acquired resonance frequencies. The detected resonance frequency ranges for single SC cells include a rocking (rigid body) frequency of 330  ±  70 kHz and two breathing resonance frequencies of 1.53  ±  0.12 and 2.02  ±  0.31 MHz. Based on these values, the average work-of-adhesion of SC cells on a silicon substrate in aqueous medium is extracted, for the first time, as WASC-Si=16.2+/- 3.8 mJ {{m}-2} . Similarly, the surface tension and the Young’s modulus of the SC cell wall are predicted as {{σ }SC}=0.16+/- 0.02 N {{m}-1} and {{E}SC}= 9.20  ±  2.80 MPa, respectively. These results are compared to those reported in the literature by utilizing various methods, and good agreements are found. The current approach eliminates the measurement inaccuracies associated with the physical contact. Exciting and detecting cell dynamics at micro-second time-scales is significantly faster than the

  20. "Ant" and "grasshopper" life-history strategies in Saccharomyces cerevisiae.

    PubMed

    Spor, Aymé; Wang, Shaoxiao; Dillmann, Christine; de Vienne, Dominique; Sicard, Delphine

    2008-02-13

    From the evolutionary and ecological points of view, it is essential to distinguish between the genetic and environmental components of the variability of life-history traits and of their trade-offs. Among the factors affecting this variability, the resource uptake rate deserves particular attention, because it depends on both the environment and the genetic background of the individuals. In order to unravel the bases of the life-history strategies in yeast, we grew a collection of twelve strains of Saccharomyces cerevisiae from different industrial and geographical origins in three culture media differing for their glucose content. Using a population dynamics model to fit the change of population size over time, we estimated the intrinsic growth rate (r), the carrying capacity (K), the mean cell size and the glucose consumption rate per cell. The life-history traits, as well as the glucose consumption rate, displayed large genetic and plastic variability and genetic-by-environment interactions. Within each medium, growth rate and carrying capacity were not correlated, but a marked trade-off between these traits was observed over the media, with high K and low r in the glucose rich medium and low K and high r in the other media. The cell size was tightly negatively correlated to carrying capacity in all conditions. The resource consumption rate appeared to be a clear-cut determinant of both the carrying capacity and the cell size in all media, since it accounted for 37% to 84% of the variation of those traits. In a given medium, the strains that consume glucose at high rate have large cell size and low carrying capacity, while the strains that consume glucose at low rate have small cell size but high carrying capacity. These two contrasted behaviors may be metaphorically defined as "ant" and "grasshopper" strategies of resource utilization. Interestingly, a strain may be "ant" in one medium and "grasshopper" in another. These life-history strategies are discussed

  1. Production of Dengue 2 Envelope Protein in the Yeast Saccharomyces Cerevisiae. Phase 1

    DTIC Science & Technology

    1990-02-15

    PRODUCTION OF DENGUE 2 ENVELOPE PROTEIN IN THE YEAST SACCHAROMYCES CEREVISIAE FINAL, PHASE I REPORT JOHN M. IVY KATHY HOUTCHENS FEBRUARY 15, 1990...SUBTITLE Production of Dengue 2 Envelope Protein in the Yeast Saccharomyces cerevisiae ( 6. AUTHOR(S) John M. Ivy Kathy Houtchens 7 PERFORMING...DISTRIBUTION CODE 13. ABSTRACT (Mammum 200 words) The four serotypes of dengue viruses are a leading cause of morbidity throughout the tropics and subtropics

  2. Water treatment process and system for metals removal using Saccharomyces cerevisiae

    DOEpatents

    Krauter, Paula A. W.; Krauter, Gordon W.

    2002-01-01

    A process and a system for removal of metals from ground water or from soil by bioreducing or bioaccumulating the metals using metal tolerant microorganisms Saccharomyces cerevisiae. Saccharomyces cerevisiae is tolerant to the metals, able to bioreduce the metals to the less toxic state and to accumulate them. The process and the system is useful for removal or substantial reduction of levels of chromium, molybdenum, cobalt, zinc, nickel, calcium, strontium, mercury and copper in water.

  3. Switching the mode of sucrose utilization by Saccharomyces cerevisiae

    PubMed Central

    Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

    2008-01-01

    Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose

  4. Transcriptional changes associated with ethanol tolerance in Saccharomyces cerevisiae.

    PubMed

    Stanley, Dragana; Chambers, Paul J; Stanley, Grant A; Borneman, Anthony; Fraser, Sarah

    2010-09-01

    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.

  5. The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier.

    PubMed

    Cameron, D R; Cooper, D G; Neufeld, R J

    1988-06-01

    The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier.

  6. The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier.

    PubMed Central

    Cameron, D R; Cooper, D G; Neufeld, R J

    1988-01-01

    The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier. PMID:3046488

  7. Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae.

    PubMed

    Mitsumori, Risa; Ohashi, Tomoe; Kugou, Kazuto; Ichino, Ayako; Taniguchi, Kei; Ohta, Kunihiro; Uchida, Hiroyuki; Oki, Masaya

    2016-07-01

    In Saccharomyces cerevisiae, the HMR, HML, telomere and rDNA regions are silenced. Silencing at the rDNA region requires Sir2, and silencing at the HMR, HML and telomere regions requires binding of a protein complex, consisting of Sir2, Sir3 and Sir4, that mediates repression of gene expression. Here, several novel Sir3 binding domains, termed CN domains (Chromosomal Novel Sir3 binding region), were identified using chromatin immunoprecipitation (ChIP) on chip analysis of S. cerevisiae chromosomes. Furthermore, analysis of G1-arrested cells demonstrated that Sir3 binding was elevated in G1-arrested cells compared with logarithmically growing asynchronous cells, and that Sir3 binding varied with the cell cycle. In addition to 14 CN regions identified from analysis of logarithmically growing asynchronous cells (CN1-14), 11 CN regions were identified from G1-arrested cells (CN15-25). Gene expression at some CN regions did not differ between WT and sir3Δ strains. Sir3 at conventional heterochromatic regions is thought to be recruited to chromosomes by Sir2 and Sir4; however, in this study, Sir3 binding occurred at some CN regions even in sir2Δ and sir4Δ backgrounds. Taken together, our results suggest that Sir3 exhibits novel binding parameters and gene regulatory functions at the CN binding domains.

  8. Mead production: selection and characterization assays of Saccharomyces cerevisiae strains.

    PubMed

    Pereira, Ana Paula; Dias, Teresa; Andrade, João; Ramalhosa, Elsa; Estevinho, Letícia M

    2009-08-01

    Mead is a traditional drink, which results from the alcoholic fermentation of diluted honey carried out by yeasts. However, when it is produced in a homemade way, mead producers find several problems, namely, the lack of uniformity in the final product, delayed and arrested fermentations, and the production of "off-flavours" by the yeasts. These problems are usually associated with the inability of yeast strains to respond and adapt to unfavourable and stressful growth conditions. The main objectives of this work were to evaluate the capacity of Saccharomyces cerevisiae strains, isolated from honey of the Trás-os-Montes (Northeast Portugal), to produce mead. Five strains from honey, as well as one laboratory strain and one commercial wine strain, were evaluated in terms of their fermentation performance under ethanol, sulphur dioxide and osmotic stress. All the strains showed similar behaviour in these conditions. Two yeasts strains isolated from honey and the commercial wine strain were further tested for mead production, using two different honey (a dark and a light honey), enriched with two supplements (one commercial and one developed by the research team), as fermentation media. The results obtained in this work show that S. cerevisiae strains isolated from honey, are appropriate for mead production. However it is of extreme importance to take into account the characteristics of the honey, and supplements used in the fermentation medium formulation, in order to achieve the best results in mead production.

  9. Saccharomyces cerevisiae contains two functional citrate synthase genes.

    PubMed Central

    Kim, K S; Rosenkrantz, M S; Guarente, L

    1986-01-01

    The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed. Images PMID:3023912

  10. Anaerobic glycerol production by Saccharomyces cerevisiae strains under hyperosmotic stress.

    PubMed

    Modig, Tobias; Granath, Katarina; Adler, Lennart; Lidén, Gunnar

    2007-05-01

    Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80-90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.

  11. Proteome analysis of aerobically and anaerobically grown Saccharomyces cerevisiae cells.

    PubMed

    Bruckmann, Astrid; Hensbergen, Paul J; Balog, Crina I A; Deelder, André M; Brandt, Raymond; Snoek, I S Ishtar; Steensma, H Yde; van Heusden, G Paul H

    2009-01-30

    The yeast Saccharomyces cerevisiae is able to grow under aerobic as well as anaerobic conditions. We and others previously found that transcription levels of approximately 500 genes differed more than two-fold when cells from anaerobic and aerobic conditions were compared. Here, we addressed the effect of anaerobic growth at the post-transcriptional level by comparing the proteomes of cells isolated from steady-state glucose-limited anaerobic and aerobic cultures. Following two-dimensional gel electrophoresis and mass spectrometry we identified 110 protein spots, corresponding to 75 unique proteins, of which the levels differed more than two-fold between aerobically and anaerobically-grown cells. For 21 of the 110 spots, the intensities decreased more than two-fold whereas the corresponding mRNA levels increased or did not change significantly under anaerobic conditions. The intensities of the other 89 spots changed in the same direction as the mRNA levels of the corresponding genes, although to different extents. For some genes of glycolysis a small increase in mRNA levels, 1.5-2 fold, corresponded to a 5-10 fold increase in protein levels. Extrapolation of our results suggests that transcriptional regulation is the major but not exclusive mechanism for adaptation of S. cerevisiae to anaerobic growth conditions.

  12. Lactose fermentation by engineered Saccharomyces cerevisiae capable of fermenting cellobiose.

    PubMed

    Liu, Jing-Jing; Zhang, Guo-Chang; Oh, Eun Joong; Pathanibul, Panchalee; Turner, Timothy L; Jin, Yong-Su

    2016-09-20

    Lactose is an inevitable byproduct of the dairy industry. In addition to cheese manufacturing, the growing Greek yogurt industry generates excess acid whey, which contains lactose. Therefore, rapid and efficient conversion of lactose to fuels and chemicals would be useful for recycling the otherwise harmful acid whey. Saccharomyces cerevisiae, a popular metabolic engineering host, cannot natively utilize lactose. However, we discovered that an engineered S. cerevisiae strain (EJ2) capable of fermenting cellobiose can also ferment lactose. This finding suggests that a cellobiose transporter (CDT-1) can transport lactose and a β-glucosidase (GH1-1) can hydrolyze lactose by acting as a β-galactosidase. While the lactose fermentation by the EJ2 strain was much slower than the cellobiose fermentation, a faster lactose-fermenting strain (EJ2e8) was obtained through serial subcultures on lactose. The EJ2e8 strain fermented lactose with a consumption rate of 2.16g/Lh. The improved lactose fermentation by the EJ2e8 strain was due to the increased copy number of cdt-1 and gh1-1 genes. Looking ahead, the EJ2e8 strain could be exploited for the production of other non-ethanol fuels and chemicals from lactose through further metabolic engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Energy coupling in Saccharomyces cerevisiae: selected opportunities for metabolic engineering.

    PubMed

    de Kok, Stefan; Kozak, Barbara U; Pronk, Jack T; van Maris, Antonius J A

    2012-06-01

    Free-energy (ATP) conservation during product formation is crucial for the maximum product yield that can be obtained, but often overlooked in metabolic engineering strategies. Product pathways that do not yield ATP or even demand input of free energy (ATP) require an additional pathway to supply the ATP needed for product formation, cellular maintenance, and/or growth. On the other hand, product pathways with a high ATP yield may result in excess biomass formation at the expense of the product yield. This mini-review discusses the importance of the ATP yield for product formation and presents several opportunities for engineering free-energy (ATP) conservation, with a focus on sugar-based product formation by Saccharomyces cerevisiae. These engineering opportunities are not limited to the metabolic flexibility within S. cerevisiae itself, but also expression of heterologous reactions will be taken into account. As such, the diversity in microbial sugar uptake and phosphorylation mechanisms, carboxylation reactions, product export, and the flexibility of oxidative phosphorylation via the respiratory chain and H(+) -ATP synthase can be used to increase or decrease free-energy (ATP) conservation. For product pathways with a negative, zero or too high ATP yield, analysis and metabolic engineering of the ATP yield of product formation will provide a promising strategy to increase the product yield and simplify process conditions. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  14. Sweet wine production by two osmotolerant Saccharomyces cerevisiae strains.

    PubMed

    García-Martínez, Teresa; de Lerma, Nieves López; Moreno, Juan; Peinado, Rafael A; Millán, M Carmen; Mauricio, Juan C

    2013-06-01

    The use of Saccharomyces cerevisiae to produce sweet wine is difficult because yeast is affected by a hyperosmotic stress due to the high sugar concentrations in the fermenting must. One possible alternative could be the coimmobilization of the osmotolerant yeast strains S. cerevisiae X4 and X5 on Penicillium chrysogenum strain H3 (GRAS) for the partial fermentation of raisin musts. This immobilized has been, namely, as yeast biocapsules. Traditional sweet wine (that is, without fermentation of the must) and must partially fermented by free yeast cells were also used for comparison. Partially fermented sweet wines showed higher concentration of the volatile compounds than traditionally produced wines. The wines obtained by immobilized yeast cells reached minor concentrations of major alcohols than wines by free cells. The consumption of specific nitrogen compounds was dependent on yeast strain and the cellular immobilization. A principal component analysis shows that the compounds related to the response to osmotic stress (glycerol, acetaldehyde, acetoin, and butanediol) clearly differentiate the wines obtained with free yeasts but not the wines obtained with immobilized yeasts. © 2013 Institute of Food Technologists®

  15. MPR1 as a novel selection marker in Saccharomyces cerevisiae.

    PubMed

    Ogawa-Mitsuhashi, Kaoru; Sagane, Koji; Kuromitsu, Junro; Takagi, Hiroshi; Tsukahara, Kappei

    2009-11-01

    L-Azetidine-2-carboxylic acid (AZC) is a toxic four-membered ring analogue of L-proline that is transported into cells by proline transporters. AZC and L-proline in the cells are competitively incorporated into nascent proteins. When AZC is present in a minimum medium, misfolded proteins are synthesized in the cells, thereby inhibiting cell growth. The MPR1 gene has been isolated from the budding yeast Saccharomyces cerevisiae Sigma1278b as a multicopy suppressor of AZC-induced growth inhibition. MPR1 encodes a novel acetyltransferase that detoxifies AZC via N-acetylation. Since MPR1 is absent in the laboratory strain of S. cerevisiae S288C, it could be a positive selection marker that confers AZC resistance in the S288C background strains. To examine the usefulness of MPR1, we constructed some plasmid vectors that harboured MPR1 under the control of various promoters and introduced them into the S288C-derived strains. The expression of MPR1 conferred AZC resistance that was largely dependent on the expression level of MPR1. In an additional experiment, the galactose-inducible MPR1 and ppr1(+), the fission yeast Schizosaccharomyces pombe homologue of MPR1, were used for gene disruption by homologous recombination, and here AZC-resistant colonies were also successfully selected. We concluded that our MPR1-AZC system provides a powerful tool for yeast transformation. Copyright (c) 2009 John Wiley & Sons, Ltd.

  16. Modulation of efficiency of translation termination in Saccharomyces cerevisiae.

    PubMed

    Nizhnikov, Anton A; Antonets, Kirill S; Inge-Vechtomov, Sergey G; Derkatch, Irina L

    2014-01-01

    Nonsense suppression is a readthrough of premature termination codons. It typically occurs either due to the recognition of stop codons by tRNAs with mutant anticodons, or due to a decrease in the fidelity of translation termination. In the latter case, suppressors usually promote the readthrough of different types of nonsense codons and are thus called omnipotent nonsense suppressors. Omnipotent nonsense suppressors were identified in yeast Saccharomyces cerevisiae in 1960s, and most of subsequent studies were performed in this model organism. Initially, omnipotent suppressors were localized by genetic analysis to different protein- and RNA-encoding genes, mostly the components of translational machinery. Later, nonsense suppression was found to be caused not only by genomic mutations, but also by epigenetic elements, prions. Prions are self-perpetuating protein conformations usually manifested by infectious protein aggregates. Modulation of translational accuracy by prions reflects changes in the activity of their structural proteins involved in different aspects of protein synthesis. Overall, nonsense suppression can be seen as a "phenotypic mirror" of events affecting the accuracy of the translational machine. However, the range of proteins participating in the modulation of translation termination fidelity is not fully elucidated. Recently, the list has been expanded significantly by findings that revealed a number of weak genetic and epigenetic nonsense suppressors, the effect of which can be detected only in specific genetic backgrounds. This review summarizes the data on the nonsense suppressors decreasing the fidelity of translation termination in S. cerevisiae, and discusses the functional significance of the modulation of translational accuracy.

  17. Proteomic analysis of Saccharomyces cerevisiae under high gravity fermentation conditions.

    PubMed

    Pham, Trong Khoa; Chong, Poh Kuan; Gan, Chee Sian; Wright, Phillip C

    2006-12-01

    Saccharomyces cerevisiae KAY446 was utilized for ethanol production, with glucose concentrations ranging from 120 g/L (normal) to 300 g/L (high). Although grown in a high glucose environment, S. cerevisiae still retained the ability to produce ethanol with a high degree of glucose utilization. iTRAQ-mediated shotgun proteomics was applied to identify relative expression change of proteins under the different glucose conditions. A total of 413 proteins were identified from three replicate, independent LC-MS/MS runs. Unsurprisingly, many proteins in the glycolysis/gluconeogenesis pathway showed significant changes in expression level. Twenty five proteins involved in amino acid metabolism decreased their expression, while the expressions of 12 heat-shock related proteins were also identified. Under high glucose conditions, ethanol was produced as a major product. However, the assimilation of glucose as well as a number of byproducts was also enhanced. Therefore, to optimize the ethanol production under very high gravity conditions, a number of pathways will need to be deactivated, while still maintaining the correct cellular redox or osmotic state. Proteomics is demonstrated here as a tool to aid in this forward metabolic engineering.

  18. Brefeldin A causes a defect in secretion in Saccharomyces cerevisiae.

    PubMed

    Vogel, J P; Lee, J N; Kirsch, D R; Rose, M D; Sztul, E S

    1993-02-15

    Brefeldin A (BFA) blocks secretion in mammalian cells and causes the redistribution of Golgi resident membrane proteins to the endoplasmic reticulum (Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071-1080). The target(s) of BFA and its mechanism of action remain unknown. The yeast Saccharomyces cerevisiae represents an ideal organism in which to identify the BFA targets, since many molecules essential for vesicular traffic have been already identified taking advantage of the powerful genetics of this system. Unfortunately, wild type S. cerevisiae strains are largely insensitive to BFA (Hayashi, T., Takatsuki, A., and Tamura, G. (1982) Agric. Biol. Chem. 46, 2241-2248). Here we demonstrate that an erg6 mutant (Gaber, R., Copple, D., Kennedy, B., Vidal, M., and Bard, M. (1989) Mol. Cell. Biol. 9, 3447-3456) defective in the biosynthesis of ergosterol is sensitive to BFA. Treatment of erg6 cells with BFA results in an arrest in growth and causes a block in secretion similar to that seen in mammalian cells treated with BFA. Our data suggest that the changes in the erg6 strain allows BFA entry and that this strain can be used to examine the molecular mechanism of BFA action.

  19. Metabolomic approach for improving ethanol stress tolerance in Saccharomyces cerevisiae.

    PubMed

    Ohta, Erika; Nakayama, Yasumune; Mukai, Yukio; Bamba, Takeshi; Fukusaki, Eiichiro

    2016-04-01

    The budding yeast Saccharomyces cerevisiae is widely used for brewing and ethanol production. The ethanol sensitivity of yeast cells is still a serious problem during ethanol fermentation, and a variety of genetic approaches (e.g., random mutant screening under selective pressure of ethanol) have been developed to improve ethanol tolerance. In this study, we developed a strategy for improving ethanol tolerance of yeast cells based on metabolomics as a high-resolution quantitative phenotypic analysis. We performed gas chromatography-mass spectrometry analysis to identify and quantify 36 compounds on 14 mutant strains including knockout strains for transcription factor and metabolic enzyme genes. A strong relation between metabolome of these mutants and their ethanol tolerance was observed. Data mining of the metabolomic analysis showed that several compounds (such as trehalose, valine, inositol and proline) contributed highly to ethanol tolerance. Our approach successfully detected well-known ethanol stress related metabolites such as trehalose and proline thus, to further prove our strategy, we focused on valine and inositol as the most promising target metabolites in our study. Our results show that simultaneous deletion of LEU4 and LEU9 (leading to accumulation of valine) or INM1 and INM2 (leading to reduction of inositol) significantly enhanced ethanol tolerance. This study shows the potential of the metabolomic approach to identify target genes for strain improvement of S. cerevisiae with higher ethanol tolerance. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Ethanol production using immobilized Saccharomyces cerevisiae in lyophilized cellulose gel.

    PubMed

    Winkelhausen, Eleonora; Velickova, Elena; Amartey, Samuel A; Kuzmanova, Slobodanka

    2010-12-01

    A new lyophilization technique was used for immobilization of Saccharomyces cerevisiae cells in hydroxyethylcellulose (HEC) gels. The suitability of the lyophilized HEC gels to serve as immobilization matrices for the yeast cells was assessed by calculating the immobilization efficiency and the cell retention in three consecutive batches, each in duration of 72 h. Throughout the repeated batch fermentation, the immobilization efficiency was almost constant with an average value of 0.92 (12-216 h). The maximum value of cell retention was 0.24 g immobilized cells/g gel. Both parameters indicated that lyophilized gels are stable and capable of retaining the immobilized yeast cells. Showing the yeast cells propagation within the polymeric matrix, the scanning electron microscope images also confirmed that the lyophilization technique for immobilization of S. cerevisiae cells in the HEC gels was successful. The activity of the immobilized yeast cells was demonstrated by their capacity to convert glucose to ethanol. Ethanol yield of 0.40, 0.43 and 0.30 g ethanol/g glucose corresponding to 79%, 84% and 60% of the theoretical yield was attained in the first, second and third batches, respectively. The cell leakage was less than 10% of the average concentration of the immobilized cells.

  1. Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells.

    PubMed

    Lezzi, Chiara; Bleve, Gianluca; Spagnolo, Stefano; Perrotta, Carla; Grieco, Francesco

    2012-12-01

    It has been demonstrated that Agaricus bisporus tyrosinase is able to oxidize various phenolic compounds, thus being an enzyme of great importance for a number of biotechnological applications. The tyrosinase-coding PPO2 gene was isolated by reverse-transcription polymerase chain reaction (RT-PCR) using total RNA extracted from the mushroom fruit bodies as template. The gene was sequenced and cloned into pYES2 plasmid, and the resulting pY-PPO2 recombinant vector was then used to transform Saccharomyces cerevisiae cells. Native polyacrylamide gel electrophoresis followed by enzymatic activity staining with L-3,4-dihydroxyphenylalanine (L-DOPA) indicated that the recombinant tyrosinase is biologically active. The recombinant enzyme was overexpressed and biochemically characterized, showing that the catalytic constants of the recombinant tyrosinase were higher than those obtained when a commercial tyrosinase was used, for all the tested substrates. The present study describes the recombinant production of A. bisporus tyrosinase in active form. The produced enzyme has similar properties to the one produced in the native A. bisporus host, and its expression in S. cerevisiae provides good potential for protein engineering and functional studies of this important enzyme.

  2. Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae

    PubMed Central

    Aimanianda, Vishukumar; Clavaud, Cécile; Simenel, Catherine; Fontaine, Thierry; Delepierre, Muriel; Latgé, Jean-Paul

    2009-01-01

    Despite its essential role in the yeast cell wall, the exact composition of the β-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant β-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ∼38 kDa and could be hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR (1H and 13C) analyses confirmed it to be a β-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two β-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[14C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for β-(1,6)-glucan synthetic activity were defined. PMID:19279004

  3. Long-chain alkane production by the yeast Saccharomyces cerevisiae.

    PubMed

    Buijs, Nicolaas A; Zhou, Yongjin J; Siewers, Verena; Nielsen, Jens

    2015-06-01

    In the past decade industrial-scale production of renewable transportation biofuels has been developed as an alternative to fossil fuels, with ethanol as the most prominent biofuel and yeast as the production organism of choice. However, ethanol is a less efficient substitute fuel for heavy-duty and maritime transportation as well as aviation due to its low energy density. Therefore, new types of biofuels, such as alkanes, are being developed that can be used as drop-in fuels and can substitute gasoline, diesel, and kerosene. Here, we describe for the first time the heterologous biosynthesis of long-chain alkanes by the yeast Saccharomyces cerevisiae. We show that elimination of the hexadecenal dehydrogenase Hfd1 and expression of a redox system are essential for alkane biosynthesis in yeast. Deletion of HFD1 together with expression of an alkane biosynthesis pathway resulted in the production of the alkanes tridecane, pentadecane, and heptadecane. Our study provides a proof of principle for producing long-chain alkanes in the industrial workhorse S. cerevisiae, which was so far limited to bacteria. We anticipate that these findings will be a key factor for further yeast engineering to enable industrial production of alkane based drop-in biofuels, which can allow the biofuel industry to diversify beyond bioethanol.

  4. Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Tyo, Keith E J; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2012-08-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S. cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.

  5. Ras proteins control mitochondrial biogenesis and function in Saccharomyces cerevisiae.

    PubMed

    Hlavatá, L; Nyström, T

    2003-01-01

    The evolutionarily conserved Ras proteins function as a point of convergence for different signaling pathways in eukaryotes and have been implicated in both aging and cancer development. In Saccharomyces cerevisiae the plasma membrane proteins Ras1 and Ras2 are sensing the nutritional status of the environments, e.g., the abundance and quality of available carbon sources. The cAMP-protein kinase A pathway is the most explored signaling pathway controlled by Ras proteins; it affects a large number of genes, some of which are important to defend the cell against oxidative stress. In addition, recent analysis has shown that the Ras system of yeast is involved in the development of mitochondria and in regulating their activity. As a sensor of environmental status and an effector of mitochondrial activity, Ras serves as a Rosetta stone of cellular energy transduction. This review summarizes the physical and functional involvement of Ras proteins and Ras-dependent signaling pathways in mitochondrial function in S. cerevisiae. Since mitochondria produce harmful reactive oxygen species as an inevitable byproduct and are partly under control of Ras, illuminating these regulatory interactions may improve our understanding of both cancer and aging.

  6. Role of social wasps in Saccharomyces cerevisiae ecology and evolution

    PubMed Central

    Stefanini, Irene; Dapporto, Leonardo; Legras, Jean-Luc; Calabretta, Antonio; Di Paola, Monica; De Filippo, Carlotta; Viola, Roberto; Capretti, Paolo; Polsinelli, Mario; Turillazzi, Stefano; Cavalieri, Duccio

    2012-01-01

    Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vector and natural reservoir of S. cerevisiae during all seasons. We provide experimental evidence that queens of social wasps overwintering as adults (Vespa crabro and Polistes spp.) can harbor yeast cells from autumn to spring and transmit them to their progeny. This result is mirrored by field surveys of the genetic variability of natural strains of yeast. Microsatellites and sequences of a selected set of loci able to recapitulate the yeast strain’s evolutionary history were used to compare 17 environmental wasp isolates with a collection of strains from grapes from the same region and more than 230 strains representing worldwide yeast variation. The wasp isolates fall into subclusters representing the overall ecological and industrial yeast diversity of their geographic origin. Our findings indicate that wasps are a key environmental niche for the evolution of natural S. cerevisiae populations, the dispersion of yeast cells in the environment, and the maintenance of their diversity. The close relatedness of several wasp isolates with grape and wine isolates reflects the crucial role of human activities on yeast population structure, through clonal expansion and selection of specific strains during the biotransformation of fermented foods, followed by dispersal mediated by insects and other animals. PMID:22847440

  7. Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae.

    PubMed

    Bakker, B M; Overkamp, K M; van Maris AJ; Kötter, P; Luttik, M A; van Dijken JP; Pronk, J T

    2001-01-01

    In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.

  8. Exploring the Saccharomyces cerevisiae Volatile Metabolome: Indigenous versus Commercial Strains

    PubMed Central

    Alves, Zélia; Melo, André; Figueiredo, Ana Raquel; Coimbra, Manuel A.; Gomes, Ana C.; Rocha, Sílvia M.

    2015-01-01

    Winemaking is a highly industrialized process and a number of commercial Saccharomyces cerevisiae strains are used around the world, neglecting the diversity of native yeast strains that are responsible for the production of wines peculiar flavours. The aim of this study was to in-depth establish the S. cerevisiae volatile metabolome and to assess inter-strains variability. To fulfill this objective, two indigenous strains (BT2652 and BT2453 isolated from spontaneous fermentation of grapes collected in Bairrada Appellation, Portugal) and two commercial strains (CSc1 and CSc2) S. cerevisiae were analysed using a methodology based on advanced multidimensional gas chromatography (HS-SPME/GC×GC-ToFMS) tandem with multivariate analysis. A total of 257 volatile metabolites were identified, distributed over the chemical families of acetals, acids, alcohols, aldehydes, ketones, terpenic compounds, esters, ethers, furan-type compounds, hydrocarbons, pyrans, pyrazines and S-compounds. Some of these families are related with metabolic pathways of amino acid, carbohydrate and fatty acid metabolism as well as mono and sesquiterpenic biosynthesis. Principal Component Analysis (PCA) was used with a dataset comprising all variables (257 volatile components), and a distinction was observed between commercial and indigenous strains, which suggests inter-strains variability. In a second step, a subset containing esters and terpenic compounds (C10 and C15), metabolites of particular relevance to wine aroma, was also analysed using PCA. The terpenic and ester profiles express the strains variability and their potential contribution to the wine aromas, specially the BT2453, which produced the higher terpenic content. This research contributes to understand the metabolic diversity of indigenous wine microflora versus commercial strains and achieved knowledge that may be further exploited to produce wines with peculiar aroma properties. PMID:26600152

  9. Human G protein-coupled receptor studies in Saccharomyces cerevisiae.

    PubMed

    Liu, Rongfang; Wong, Winsy; IJzerman, Adriaan P

    2016-08-15

    G protein-coupled receptors (GPCRs) are one of the largest families of membrane proteins, with approximately 800 different GPCRs in the human genome. Signaling via GPCRs regulates many biological processes, such as cell proliferation, differentiation, and development. In addition, many receptors have a pivotal role in immunophysiology. Many hormones and neurotransmitters are ligands for these receptors, and hence it is not surprising that many drugs, either mimicking or blocking the action of the bodily substances, have been developed. It is estimated that 30-40% of current drugs on the market target GPCRs. Further identifying and elucidating the functions of GPCRs will provide opportunities for novel drug discovery, including for immunotherapy. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a very important and useful platform in this respect. There are many advantages of using a yeast assay system, as it is cheap, safe and stable; it is also convenient for rapid feasibility and optimization studies. Moreover, it offers a "null" background when studying human GPCRs. New developments regarding human GPCRs expressed in a yeast platform are providing insight into GPCR activation and signaling, and facilitate agonist and antagonist identification. In this review we summarize the latest findings regarding human G-protein-coupled receptors in studies using S. cerevisiae, ever since the year 2005 when we last published a review on this topic. We describe 11 families of GPCRs in detail, while including the principles and developments of each yeast system applied to these different GPCRs and highlight and generalize the experimental findings of GPCR function in these systems. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

    PubMed

    Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development.

  11. Oxygen requirements for formation and activity of the squalene expoxidase in Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Jahnke, L.; Klein, H. P.

    1983-01-01

    The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the 'mitochondrial' fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the 'microsomal' fraction. At 0.03 percent (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent K sub m for oxygen of 0.38 percent (vol/vol) was determined from a crude particulate preparation for the epoxidase.

  12. The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains.

    PubMed Central

    Duncan, K; Edwards, R M; Coggins, J R

    1987-01-01

    The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined. The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555. Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme. The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes. PMID:2825635

  13. Oxygen requirements for formation and activity of the squalene expoxidase in Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Jahnke, L.; Klein, H. P.

    1983-01-01

    The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the 'mitochondrial' fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the 'microsomal' fraction. At 0.03 percent (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent K sub m for oxygen of 0.38 percent (vol/vol) was determined from a crude particulate preparation for the epoxidase.

  14. Exploring the northern limit of the distribution of Saccharomyces cerevisiae and Saccharomyces paradoxus in North America.

    PubMed

    Charron, Guillaume; Leducq, Jean-Baptiste; Bertin, Chloé; Dubé, Alexandre K; Landry, Christian R

    2014-03-01

    We examined the northern limit of Saccharomyces cerevisiae and Saccharomyces paradoxus in northeast America. We collected 876 natural samples at 29 sites and applied enrichment methods for the isolation of mesophilic yeasts. We uncovered a large diversity of yeasts, in some cases, associated with specific substrates. Sequencing of the ITS1, 5.8S and ITS2 loci allowed to assign 226 yeast strains at the species level, including 41 S. paradoxus strains. Our intensive sampling suggests that if present, S. cerevisiae is rare at these northern latitudes. Our sampling efforts spread across several months of the year revealed that successful sampling increases throughout the summer and diminishes significantly at the beginning of the fall. The data obtained on the ecological context of yeasts corroborate what was previously reported on Pichiaceae, Saccharomycodaceae, Debaryomycetaceae and Phaffomycetaceae yeast families. We identified 24 yeast isolates that could not be assigned to any known species and that may be of taxonomic, medical, or biotechnological importance. Our study reports new data on the taxonomic diversity of yeasts and new resources for studying the evolution and ecology of S. paradoxus.

  15. Influence of temperature and nutrient strength on the susceptibility of Saccharomyces cerevisiae to heavy metals

    SciTech Connect

    Hsu, T.; Lee, L.W.; Chang, T.H. )

    1992-09-01

    Saccharomyces cerevisiae is not only a key microorganism in brewing or fermentation processes, it has also been employed for monitoring aquatic pollutants. The major advantage of using Saccharomyces cerevisiae as a bioassay system is that this yeast can be easily obtained as dry pellets from commercial sources at low cost. In addition to its economical aspect, Saccharomyces cerevisiae, like other microorganisms, is easy to handle, grows rapidly, and provides a large number of homogeneous individuals for utilization in toxicity tests. Although cell growth, cell viability, electron transport and mitochondrial respiration of Saccharomyces cerevisiaes have all been selected as parameters for toxicity assessment, measuring cell growth by absorbance is by farm the most convenient and rapid method when large amounts of water samples are to be tested. Mochida et al. (1988), however, reported that Saccharomyces cerevisiae was five to ten times less sensitive than cell culture systems to cadmium, mercury and nickel, when cell growth of both systems was monitored. This relative insensitivity to heavy metals might handicap the practical use of this yeast strain for bioassays. Since previous studies indicated that the susceptibility of microorganisms to environmental toxicants can be influenced by incubation temperature and nutrient strength, we attempted to examine the effect of incubation temperature and nutrient strength on the susceptibility of Saccharomyces cerevisiae to heavy metals in order to obtain the optimum bioassay sensitivity. In this study, we used cadmium and mercury as model toxicants. 9 refs., 2 figs., 1 tab.

  16. The effect of Saccharomyces cerevisiae on the stability of the herbicide glyphosate during bread leavening.

    PubMed

    Low, F L; Shaw, I C; Gerrard, J A

    2005-01-01

    To investigate the ability of baker's yeast (Saccharomyces cerevisiae) to degrade the herbicide glyphosate during the fermentation cycle of the breadmaking process. Aqueous glyphosate was added to bread ingredients and kneaded by commercially available breadmaking equipment into dough cultures. Cultures were incubated in the breadmaker throughout the fermentation cycle. The recovery of glyphosate levels following fermentation was determined, thus allowing an estimation of glyphosate degradation by yeast. It was shown, for the first time, that S. cerevisiae plays a role in metabolizing glyphosate during the fermentation stages of breadmaking. Approximately 21% was degraded within 1 h. As a result of projected increases in the glyphosate use on wheat and the role of bread as a dietary staple, this may contribute to more informed decisions being made relating to the use of glyphosate on glyphosate-resistant wheat, from a public health/regulatory perspective.

  17. ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

  18. Invertase SUC2 Is the Key Hydrolase for Inulin Degradation in Saccharomyces cerevisiae

    PubMed Central

    Wang, Shi-An

    2013-01-01

    Specific Saccharomyces cerevisiae strains were recently found to be capable of efficiently utilizing inulin, but genetic mechanisms of inulin hydrolysis in yeast remain unknown. Here we report functional characteristics of invertase SUC2 from strain JZ1C and demonstrate that SUC2 is the key enzyme responsible for inulin metabolism in S. cerevisiae. PMID:23104410

  19. Construction of an artificial pathway for isobutanol biosynthesis in the cytosol of Saccharomyces cerevisiae.

    PubMed

    Matsuda, Fumio; Kondo, Takashi; Ida, Kengo; Tezuka, Hironori; Ishii, Jun; Kondo, Akihiko

    2012-01-01

    To increase isobutanol production in Saccharomyces cerevisiae, the valine biosynthetic pathway was activated by overexpression of the relevant enzymes in the mitochondria and the cytosol. Native mitochondrial enzymes were overepxressed in the cytosol by deleting the mitochondrial transit peptides. The metabolically engineered S. cerevisiae possessing the cytosolic pathway showed increased isobutanol production (63 ± 4 mg/L).

  20. ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

  1. [Invertase Overproduction May Provide for Inulin Fermentation by Selection Strains of Saccharomyces cerevisiae].

    PubMed

    Naumov, G I; Naumova, E S

    2015-01-01

    In some recent publications, the ability of selection strains of Saccharomyces cerevisiae to ferment inulin was attributed to inulinase activity. The review summarizes the literature data indicating that overproduction of invertase, an enzyme common to S. cerevisiae, may be responsible for this phenomenon.

  2. Invertase SUC2 Is the key hydrolase for inulin degradation in Saccharomyces cerevisiae.

    PubMed

    Wang, Shi-An; Li, Fu-Li

    2013-01-01

    Specific Saccharomyces cerevisiae strains were recently found to be capable of efficiently utilizing inulin, but genetic mechanisms of inulin hydrolysis in yeast remain unknown. Here we report functional characteristics of invertase SUC2 from strain JZ1C and demonstrate that SUC2 is the key enzyme responsible for inulin metabolism in S. cerevisiae.

  3. Creation of a synthetic xylose-inducible promoter for Saccharomyces cerevisiae

    USDA-ARS?s Scientific Manuscript database

    Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. S. cerevisiae strains engineered for xylose fermentation have been made using constitutive promoters to express the req...

  4. Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains.

    PubMed

    Yu, S; Jeppsson, H; Hahn-Hägerdal, B

    1995-12-01

    Xylulose fermentation by four strains of Saccharomyces cerevisiae and two strains of xylose-fermenting yeasts, Pichia stipitis CBS 6054 and Candida shehatae NJ 23, was compared using a mineral medium at a cell concentration of 10 g (dry weight)/l. When xylulose was the sole carbon source and fermentation was anaerobic, S. cerevisiae ATCC 24860 and CBS 8066 showed a substrate consumption rate of 0.035 g g cells-1 h-1 compared with 0.833 gg cells-1 h-1 for glucose. Bakers' yeast and S. cerevisiae isolate 3 consumed xylulose at a much lower rate although they fermented glucose as rapidly as the ATCC and the CBS strains. While P. stipitis CBS 6054 consumed both xylulose and glucose very slowly under anaerobic conditions, C. shehatae NJ 23 fermented xylulose at a rate of 0.345 gg cells-1 h-1, compared with 0.575 gg cells-1 h-1 for glucose. For all six strains, the addition of glucose to the xylulose medium did not enhance the consumption of xylulose, but increased the cell biomass concentrations. When fermentation was performed under oxygen-limited conditions, less xylulose was consumed by S. cerevisiae ATCC 24860 and C. shehatae NJ 23, and 50%- 65% of the assimilated carbon could not be accounted for in the products determined.

  5. Metabolism of sulfur amino acids in Saccharomyces cerevisiae.

    PubMed Central

    Thomas, D; Surdin-Kerjan, Y

    1997-01-01

    Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases

  6. Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering.

    PubMed

    Lee, Ki-Sung; Hong, Min-Eui; Jung, Suk-Chae; Ha, Suk-Jin; Yu, Byung Jo; Koo, Hyun Min; Park, Sung Min; Seo, Jin-Ho; Kweon, Dae-Hyuk; Park, Jae Chan; Jin, Yong-Su

    2011-03-01

    Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate

  7. Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases

    PubMed Central

    2013-01-01

    Background Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol. Results The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l-1 (0.038 and 0.028 g l-1 h-1), respectively, after 10 days. With a substrate load of 200 g l-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l-1 ethanol (0.58 g l-1 h-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l-1 ethanol (0.36 g l-1 h-1). Conclusions In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases. PMID:24286270

  8. Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation.

    PubMed

    Dong, Shi-Jun; Lin, Xiang-Hua; Li, Hao

    2015-11-01

    During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation.

  9. Swi/SNF-GCN5-dependent chromatin remodelling determines induced expression of GDH3, one of the paralogous genes responsible for ammonium assimilation and glutamate biosynthesis in Saccharomyces cerevisiae.

    PubMed

    Avendaño, Amaranta; Riego, Lina; DeLuna, Alexander; Aranda, Cristina; Romero, Guillermo; Ishida, Cecilia; Vázquez-Acevedo, Miriam; Rodarte, Beatriz; Recillas-Targa, Félix; Valenzuela, Lourdes; Zonszein, Sergio; González, Alicia

    2005-07-01

    It is accepted that Saccharomyces cerevisiae genome arose from complete duplication of eight ancestral chromosomes; functionally normal ploidy was recovered because of the massive loss of 90% of duplicated genes. There is evidence that indicates that part of this selective conservation of gene pairs is compelling to yeast facultative metabolism. As an example, the duplicated NADP-glutamate dehydrogenase pathway has been maintained because of the differential expression of the paralogous GDH1 and GDH3 genes, and the biochemical specialization of the enzymes they encode. The present work has been aimed to the understanding of the regulatory mechanisms that modulate GDH3 transcriptional activation. Our results show that GDH3 expression is repressed in glucose-grown cultures, as opposed to what has been observed for GDH1, and induced under respiratory conditions, or under stationary phase. Although GDH3 pertains to the nitrogen metabolic network, and its expression is Gln3p-regulated, complete derepression is ultimately determined by the carbon source through the action of the SAGA and SWI/SNF chromatin remodelling complexes. GDH3 carbon-mediated regulation is over-imposed to that exerted by the nitrogen source, highlighting the fact that operation of facultative metabolism requires strict control of enzymes, like Gdh3p, involved in biosynthetic pathways that use tricarboxylic acid cycle intermediates.

  10. Exclusion of Saccharomyces kudriavzevii from a wine model system mediated by Saccharomyces cerevisiae.

    PubMed

    Arroyo-López, F Noé; Pérez-Través, Laura; Querol, Amparo; Barrio, Eladio

    2011-06-01

    This study investigated the competition and potential hybrid generation between the species Saccharomyces cerevisiae and S. kudriavzevii in a wine-model environment. Our main goal was to understand why S. kudriavzevii has not been found in wine fermentations whilst their hybrids are present. Auxotrophic mutants (Ura(-) and Lys(-)) were used to favour the selection of hybrids and to specifically differentiate the two species in mixed fermentations carried out at different temperatures (17 °C, 24 °C and 31 °C). Both yeasts showed a reduction in their maximum specific growth rates in mixed fermentations, indicating a clear antagonistic effect between the two microorganisms. Temperature played an important role in this competition. In this way, S. kudriavzevii was less affected at 17 °C, but S. cerevisiae was clearly the best competitor at 31 °C, preventing the growth of S. kudriavzevii. Population levels of S. kudriavzevii always significantly decreased in the presence of S. cerevisiae. Ethanol was measured throughout the fermentations and in all cases S. kudriavzevii growth was arrested when ethanol levels were < 5 g/l, indicating that this compound did not influence the competitive exclusion of S. kudriavzevii. Killer factors were also discarded due to the K(-) R(-) phenotype of both strains. Finally, no prototrophic interspecific hybrids were isolated in small-scale fermentations at any temperature assayed. Our results show that the lack of competitiveness exhibited by S. kudriavzevii, especially at high temperatures, explains the absence of this species in wine fermentations, suggesting that natural S. cerevisiae × S. kudriavzevii hybrids most likely originated in wild environments rather than in industrial fermentations.

  11. Adaptive Evolution of a Lactose-Consuming Saccharomyces cerevisiae Recombinant▿

    PubMed Central

    Guimarães, Pedro M. R.; François, Jean; Parrou, Jean Luc; Teixeira, José A.; Domingues, Lucília

    2008-01-01

    The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (β-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media. PMID:18245248

  12. Mutagenic effect of freezing on mitochondrial DNA of Saccharomyces cerevisiae.

    PubMed

    Stoycheva, T; Venkov, P; Tsvetkov, Ts

    2007-06-01

    Although suggested in some studies, the mutagenic effect of freezing has not been proved by induction and isolation of mutants. Using a well-defined genetic model, we supply in this communication evidence for the mutagenic effect of freezing on mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae. The cooling for 2 h at +4 degrees C, followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C resulted in induction of respiratory mutations. The immediate freezing in liquid nitrogen was without mutagenic effect. The study of the stepwise procedure showed that the induction of respiratory mutants takes place during the freezing at -10 and -20 degrees C of cells pre-cooled at +4 degrees C. The genetic crosses of freeze-induced mutants evidenced their mitochondrial rho- origin. The freeze-induced rho- mutants are most likely free of simultaneous nuclear mutations. The extracellular presence of cryoprotectants did not prevent the mutagenic effect of freezing while accumulation of cryoprotectors inside cells completely escaped mtDNA from cryodamage. Although the results obtained favor the notion that the mutagenic effect of freezing on yeast mtDNA is due to formation and growth of intracellular ice crystals, other reasons, such as impairment of mtDNA replication or elevated levels of ROS production are discussed as possible explanations of the mutagenic effect of freezing. It is concluded that: (i) freezing can be used as a method for isolation of mitochondrial mutants in S. cerevisiae and (ii) given the substantial development in cryopreservation of cells and tissues, special precautions should be made to avoid mtDNA damage during the cryopreservation procedures.

  13. Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae.

    PubMed

    Farquhar, R; Honey, N; Murant, S J; Bossier, P; Schultz, L; Montgomery, D; Ellis, R W; Freedman, R B; Tuite, M F

    1991-12-01

    Protein disulfide isomerase (PDI) is an enzyme involved in the catalysis of disulfide bond formation in secretory and cell-surface proteins. Using an oligodeoxyribonucleotide designed to detect the conserved 'thioredoxin-like' active site of vertebrate PDIs, we have isolated a gene encoding PDI from the lower eukaryote, Saccharomyces cerevisiae. The nucleotide sequence and deduced open reading frame of the cloned gene predict a 530-amino-acid (aa) protein of Mr 59,082 and a pI of 4.1, physical properties characteristic of mammalian PDIs. Furthermore, the aa sequence shows 30-32% identity with mammalian and avian PDI sequences and has a very similar overall organisation, namely the presence of two approx. 100-aa segments, each of which is repeated, with the most significant homologies to mammalian and avian PDIs being in the regions (a, a') that contain the conserved 'thioredoxin-like' active site. The N-terminal region has the characteristics of a cleavable secretory signal sequence and the C-terminal four aa (-His-Asp-Glu-Leu) are consistent with the protein being a component of the S. cerevisiae endoplasmic reticulum. Transformants carrying multiple copies of this gene (designated PDI1) have tenfold higher levels of PDI activity and overproduce a protein of the predicted Mr. The PDI1 gene is unique in the yeast genome and encodes a single 1.8-kb transcript that is not found in stationary phase cells. Disruption of the PDI1 gene is haplo-lethal indicating that the product of this gene is essential for viability.

  14. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    PubMed Central

    van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes

  15. Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.

    PubMed

    Guimarães, Pedro M R; François, Jean; Parrou, Jean Luc; Teixeira, José A; Domingues, Lucília

    2008-03-01

    The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (beta-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media.

  16. Histone Deacetylases with Antagonistic Roles in Saccharomyces cerevisiae Heterochromatin Formation.

    PubMed

    Thurtle-Schmidt, Deborah M; Dodson, Anne E; Rine, Jasper

    2016-09-01

    As the only catalytic member of the Sir-protein gene-silencing complex, Sir2's catalytic activity is necessary for silencing. The only known role for Sir2's catalytic activity in Saccharomyces cerevisiae silencing is to deacetylate N-terminal tails of histones H3 and H4, creating high-affinity binding sites for the Sir-protein complex, resulting in association of Sir proteins across the silenced domain. This histone deacetylation model makes the simple prediction that preemptively removing Sir2's H3 and H4 acetyl substrates, by mutating these lysines to unacetylatable arginines, or removing the acetyl transferase responsible for their acetylation, should restore silencing in the Sir2 catalytic mutant. However, this was not the case. We conducted a genetic screen to explore what aspect of Sir2's catalytic activity has not been accounted for in silencing. Mutation of a nonsirtuin histone deacetylase, Rpd3, restored Sir-protein-based silencing in the absence of Sir2's catalytic activity. Moreover, this antagonism could be mediated by either the large or the small Rpd3-containing complex. Interestingly, this restoration of silencing appeared independent of any known histone H3 or H4 substrates of Rpd3 Investigation of Sir-protein association in the Rpd3 mutant revealed that the restoration of silencing was correlated with an increased association of Sir proteins at the silencers, suggesting that Rpd3 was an antagonist of Sir2's function in nucleation of Sir proteins to the silencer. Additionally, restoration of silencing by Rpd3 was dependent on another sirtuin family member, Hst3, indicating multiple antagonistic roles for deacetylases in S. cerevisiae silencing. Copyright © 2016 by the Genetics Society of America.

  17. The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae.

    PubMed

    García-Ríos, Estéfani; Morard, Miguel; Parts, Leopold; Liti, Gianni; Guillamón, José M

    2017-02-14

    Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci. In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions' adaptive nature. The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.

  18. Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

    PubMed Central

    Salusjärvi, Laura; Kankainen, Matti; Soliymani, Rabah; Pitkänen, Juha-Pekka; Penttilä, Merja; Ruohonen, Laura

    2008-01-01

    Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains

  19. Direct conversion of starch to ethanol using recombınant Saccharomyces cerevisiae containing glucoamylase gene

    NASA Astrophysics Data System (ADS)

    Purkan, P.; Baktir, A.; Puspaningsih, N. N. T.; Ni'mah, M.

    2017-09-01

    Saccharomyces cerevisiae is known for its high fermentative capacity, high ethanol yield and its high ethanol tolerance. The yeast is inability converting starch (relatively inexpensive substrate) into biofuel ethanol. Insertion of glucoamylase gene in yeast cell of Saccharomyces cerevisiae had been done to increase the yeast function in ethanol fermentation from starch. Transformation of yeast of S. cerevisiae with recombinant plasmid yEP-GLO1 carrying gene encoding glucoamylase (GLO1) produced the recombinant yeast which enable to degrade starch. Optimizing of bioconversion process of starch into ethanol by the yeast of recombinant Saccharomyces cerevisiae [yEP-GLO1] had been also done. Starch concentration which could be digested by recombinant yeast of S. cerevisiae [yEP-GLO1] was 10% (w/v). Bioconversion of starch having concentration 10% (b/v) using recombinant yeast of S. cerevisiae BY5207 [yEP-GLO1] could result ethanol as 20% (v/v) to alcoholmeter and 19,5% (v/v) to gas of chromatography. Otherwise, using recombinant yeast S. cerevisiae S. cerevisiae AS3324 [yEP-GLO1] resulted ethanol as 17% (v/v) to alcoholmeter and 17,5% (v/v) to gas of chromatography. The highest ethanol in starch bioconversion using both recombinant yeasts BY5207 and AS3324 could be resulted on 144 hours of fermentation time as well as in pH 5.

  20. Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

    PubMed Central

    Bellon, Jennifer R.; Schmid, Frank; Capone, Dimitra L.; Dunn, Barbara L.; Chambers, Paul J.

    2013-01-01

    Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment. PMID:23614011

  1. Introducing a new breed of wine yeast: interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae.

    PubMed

    Bellon, Jennifer R; Schmid, Frank; Capone, Dimitra L; Dunn, Barbara L; Chambers, Paul J

    2013-01-01

    Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.

  2. Regulation of lysine transport by feedback inhibition in Saccharomyces cerevisiae.

    PubMed Central

    Morrison, C E; Lichstein, H C

    1976-01-01

    A steady-state level of about 240 nmol/mg (dry wt) occurs during lysine transport in Saccharomyces cerevisiae. No subsequent efflux of the accumulated amino acid was detected. Two transport systems mediate lysine transport, a high-affinity, lysine-specific system and an arginine-lysine system for which lysine exhibits a lower affinity. Preloading with lysine, arginine, glutamic acid, or aspartic acid inhibited lysine transport activity; preloading with glutamine, glycine, methionine, phenylalanine, or valine had little effect; however, preloading with histidine stimulated lysine transport activity. These preloading effects correlated with fluctuations in the intracellular lysine and/or arginine pool: lysine transport activity was inhibited when increases in the lysine and/or arginine pool occurred and was stimulated when decreases in the lysine and/or arginine pool occurred. After addition of lysine to a growing culture, lysine transport activity was inhibited more than threefold in one-third of the doubling time of the culture. These results indicate that the lysine-specific and arginine-lysine transport systems are regulated by feedback inhibition that may be mediated by intracellular lysine and arginine. PMID:767329

  3. Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae

    SciTech Connect

    Dar, R. D.; Karig, D. K.; Cooke, J. F.; Cox, C. D.; Simpson, M. L.

    2010-09-01

    Stochasticity is an inherent feature of complex systems with nanoscale structure. In such systems information is represented by small collections of elements (e.g. a few electrons on a quantum dot), and small variations in the populations of these elements may lead to big uncertainties in the information. Unfortunately, little is known about how to work within this inherently noisy environment to design robust functionality into complex nanoscale systems. Here, we look to the biological cell as an intriguing model system where evolution has mediated the trade-offs between fluctuations and function, and in particular we look at the relationships and trade-offs between stochastic and deterministic responses in the gene expression of budding yeast (Saccharomyces cerevisiae). We find gene regulatory arrangements that control the stochastic and deterministic components of expression, and show that genes that have evolved to respond to stimuli (stress) in the most strongly deterministic way exhibit the most noise in the absence of the stimuli. We show that this relationship is consistent with a bursty 2-state model of gene expression, and demonstrate that this regulatory motif generates the most uncertainty in gene expression when there is the greatest uncertainty in the optimal level of gene expression.

  4. Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Perez, Adam M.; Finnigan, Gregory C.; Roelants, Françoise M.; Thorner, Jeremy

    2016-01-01

    Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast Saccharomyces cerevisiae is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop. PMID:27847804

  5. Anti-Saccharomyces cerevisiae as unusual antibodies in autoimmune hepatitis.

    PubMed

    Fagoonee, S; De Luca, L; De Angelis, C; Castelli, A; Rizzetto, M; Pellicano, R

    2009-03-01

    Autoantibodies are disease markers of autoimmune hepatitis (AIH). Antinuclear antibodies, smooth muscle antibodies, antibodies to liver/kidney microsome type 1, and perinuclear antibodies to neutrophil cytoplasm constitute the ''conventional'' battery of autoantibodies, while an emerging interest to evaluate new autoantibodies as diagnostic or prognostic markers, such as the anti-Saccharomyces cerevisiae antibodies, is detectable (ASCA). This paper focuses mainly on the findings and the potential role of ASCA in AIH. These antibodies are present in 5-6.3% of blood donors and in the gastrointestinal setting, ASCA have been found most often in Crohn's disease and with lower frequency in the course of ulcerative colitis and celiac disease. Furthermore, they have been described, to a lesser extent, in patients with primary sclerosing cholangitis and primary biliary cirrhosis and in AIH. ASCA occur in 20-30% of patients suffering from AIH with a statistically significant increase observed only for IgG ASCA in type 1 AIH. This probably indicates collateral immune reactivities to the primary pathogenic process. The outcome of hepatitis is not influenced by the presence of ASCA. In conclusion, ASCA positivity does not imply that there exists a distinct subgroup of patients with AIH and these autoantibodies are not involved in the pathogenetic mechanism of AIH.

  6. Lipid droplet autophagy in the yeast Saccharomyces cerevisiae.

    PubMed

    van Zutphen, Tim; Todde, Virginia; de Boer, Rinse; Kreim, Martin; Hofbauer, Harald F; Wolinski, Heimo; Veenhuis, Marten; van der Klei, Ida J; Kohlwein, Sepp D

    2014-01-01

    Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum-autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.

  7. Tor1 regulates protein solubility in Saccharomyces cerevisiae.

    PubMed

    Peters, Theodore W; Rardin, Matthew J; Czerwieniec, Gregg; Evani, Uday S; Reis-Rodrigues, Pedro; Lithgow, Gordon J; Mooney, Sean D; Gibson, Bradford W; Hughes, Robert E

    2012-12-01

    Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase.

  8. Tor1 regulates protein solubility in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Rardin, Matthew J.; Czerwieniec, Gregg; Evani, Uday S.; Reis-Rodrigues, Pedro; Lithgow, Gordon J.; Mooney, Sean D.; Gibson, Bradford W.; Hughes, Robert E.

    2012-01-01

    Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase. PMID:23097491

  9. Fredericamycin A affects mitochondrial inheritance and morphology in Saccharomyces cerevisiae.

    PubMed

    Imamura, Yuko; Yukawa, Masashi; Kimura, Ken-ichi; Takahashi, Hidetoshi; Suzuki, Yoshihiro; Ojika, Makoto; Sakagami, Youji; Tsuchiya, Eiko

    2005-11-01

    Fredericamycin A (FMA) is an antibiotic product of Streptomyces griseus that exhibits modest antitumor activity in vivo and in vitro, but, its functions in vivo are poorly understood. We identified this compound as an inducer of G1 arrest in the yeast, Saccharomyces cerevisiae. FMA exhibits an IC50 of 24 nM towards the growth of a disruptant of multi-drug resistance genes, W303-MLC30, and its cytotoxicity is a function of the time of exposure as well as drug dose. Addition of 0.8 microM of FMA caused aggregation of mitochondria within 10 min of incubation and the drug induced petites at high frequency after 4 h of incubation. Rho(-) cells were about 20 times more resistant to FMA than isogenic rho(+) cells. Overexpression of topoisomerase I, a previously suggested target of the drug, did not alleviate the sensitivity of the cells to FMA or the aggregation of mitochondria. Our results suggest that mitochondria are the primary target site of FMA.

  10. A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae.

    PubMed

    Fisk, H A; Yaffe, M P

    1999-06-14

    The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination.

  11. Response of Saccharomyces cerevisiae to stress-free acidification.

    PubMed

    Chen, Allen Kuan-Liang; Gelling, Cristy; Rogers, Peter L; Dawes, Ian W; Rosche, Bettina

    2009-02-01

    Genome-wide transcriptional analysis of a Saccharomyces cerevisiae batch culture revealed that more than 829 genes were regulated in response to an environmental shift from pH 6 to pH 3 by added sulfuric acid. This shift in pH was not detrimental to the rate of growth compared to a control culture that was maintained at pH 6 and the transcriptional changes most strikingly implicated not up- but down-regulation of stress responses. In addition, the transcriptional changes upon acid addition indicated remodeling of the cell wall and central carbon metabolism. The overall trend of changes was similar for the pH-shift experiment and the pH 6 control. However, the changes in the pH 6 control were much weaker and occurred 2.5 h later than in the pH-shift experiment. Thus, the reaction to the steep pH decrease was an immediate response within the normal repertoire of adaptation shown in later stages of fermentation at pH 6. Artificially preventing the yeast from acidifying the medium may be considered physiologically stressful under the tested conditions.

  12. Molecular analysis of the PHO81 gene of Saccharomyces cerevisiae.

    PubMed Central

    Creasy, C L; Madden, S L; Bergman, L W

    1993-01-01

    The PHO81 gene product is a positive regulatory factor required for the synthesis of the phosphate repressible acid phosphatase (encoded by the PHO5 gene) in Saccharomyces cerevisiae. Genetic analysis has suggested that PHO81 may be the signal acceptor molecule; however, the biochemical function of the PHO81 gene product is not known. We have cloned the PHO81 gene and sequenced the promoter. A PHO81-LacZ fusion was shown to be a valid reporter since its expression is regulated by the level of inorganic phosphate and is controlled by the same regulatory factors that regulate PHO5 expression. To elucidate the mechanism by which PHO81 functions, we have isolated and cloned dominant mutations in the PHO81 gene which confer constitutive synthesis of acid phosphatase. We have demonstrated that overexpression of the negative regulatory factor, PHO80, but not the negative regulatory factor PHO85, partially blocks the constitutive acid phosphatase synthesis in a strain containing a dominant constitutive allele of PHO81. This suggests that PHO81 may function by interacting with PHO80 or that these molecules compete for the same target. Images PMID:8493108

  13. Coordinated regulation of growth genes in Saccharomyces cerevisiae.

    PubMed

    Slattery, Matthew G; Heideman, Warren

    2007-05-15

    It is imperative that quiescent Saccharomyces cerevisiae cells respond rapidly to fresh medium: the cell that initiates growth and division soonest has the most progeny. Several laboratories have used DNA microarrays to identify transcripts that are altered when fresh medium is added to quiescent cells. We combined published data with our own to address several questions: Do these experiments taken together identify a core set of genes that is reproducibly affected when quiescent cells are stimulated by nutrient repletion? Is this gene set coregulated in response to other environmental challenges? Does promoter histone occupancy correlate with the mRNA data? Despite diverse experimental designs, the data were highly correlated, generating a set of nutrient repletion transcripts. Glucose addition accounted for the response. These transcripts were also coregulated in response to diverse stresses. Promoters were associated with increased histone acetylation and decreased histone occupancy when induced, and high histone occupancy with low acetylation when repressed. The presence of RRPE and PAC promoter elements correlated with nutrient responsiveness and a dynamic pattern of histone occupancy and acetylation. Correlative evidence supports the idea that some mRNAs may be upregulated by release from sequestration in RNA-protein complexes.

  14. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

  15. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae

    PubMed Central

    Conrad, Michaela; Schothorst, Joep; Kankipati, Harish Nag; Van Zeebroeck, Griet; Rubio-Texeira, Marta; Thevelein, Johan M

    2014-01-01

    The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms. Many specific nutrient responses have been elucidated in great detail. This has led to important new concepts and insight into nutrient-controlled cellular regulation. Major highlights include the central role of the Snf1 protein kinase in the glucose repression pathway, galactose induction, the discovery of a G-protein-coupled receptor system, and role of Ras in glucose-induced cAMP signaling, the role of the protein synthesis initiation machinery in general control of nitrogen metabolism, the cyclin-controlled protein kinase Pho85 in phosphate regulation, nitrogen catabolite repression and the nitrogen-sensing target of rapamycin pathway, and the discovery of transporter-like proteins acting as nutrient sensors. In addition, a number of cellular targets, like carbohydrate stores, stress tolerance, and ribosomal gene expression, are controlled by the presence of multiple nutrients. The protein kinase A signaling pathway plays a major role in this general nutrient response. It has led to the discovery of nutrient transceptors (transporter receptors) as nutrient sensors. Major shortcomings in our knowledge are the relationship between rapid and steady-state nutrient signaling, the role of metabolic intermediates in intracellular nutrient sensing, and the identity of the nutrient sensors controlling cellular growth. PMID:24483210

  16. Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

    PubMed Central

    Orlean, Peter

    2012-01-01

    The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins. PMID:23135325

  17. Factors involved in anaerobic growth of Saccharomyces cerevisiae.

    PubMed

    Ishtar Snoek, I S; Yde Steensma, H

    2007-01-01

    Life in the absence of molecular oxygen requires several adaptations. Traditionally, the switch from respiratory metabolism to fermentation has attracted much attention in Saccharomyces cerevisiae, as this is the basis for the use of this yeast in the production of alcohol and in baking. It has also been clear that under anaerobic conditions the yeast is not able to synthesize sterols and unsaturated fatty acids and that for anaerobic growth these have to be added to the media. More recently it has been found that many more factors play a role. Several other biosynthetic reactions also require molecular oxygen and the yeast must have alternatives for these. In addition, the composition of the cell wall and cell membrane show major differences when aerobic and anaerobic cells are compared. All these changes are reflected by the observation that the transcription of more than 500 genes changes significantly between aerobically and anaerobically growing cultures. In this review we will give an overview of the factors that play a role in the survival in the absence of molecular oxygen.

  18. Dimethyl sulfoxide induces oxidative stress in the yeast Saccharomyces cerevisiae.

    PubMed

    Sadowska-Bartosz, Izabela; Pączka, Aleksandra; Mołoń, Mateusz; Bartosz, Grzegorz

    2013-12-01

    Dimethyl sulfoxide (DMSO) is used as a cryoprotectant for the preservation of cells, including yeast, and as a solvent for chemical compounds. We report that DMSO induces oxidative stress in the yeast. Saccharomyces cerevisiae wt strain EG-103 and its mutants Δsod1, Δsod2, and Δsod1 Δsod2 were used. Yeast were subjected to the action of 1-14% DMSO for 1 h at 28 °C. DMSO induced a concentration-dependent inhibition of yeast growth, the effect being more pronounced for mutants devoid of SOD (especially Δsod1 Δsod2). Cell viability was compromised. DMSO-concentration-dependent activity loss of succinate dehydrogenase, a FeS enzyme sensitive to oxidative stress, was observed. DMSO enhanced formation of reactive oxygen species, estimated with dihydroethidine in a concentration-dependent manner, the effect being again more pronounced in mutants devoid of superoxide dismutases. The content of cellular glutathione was increased with increasing DMSO concentrations, which may represent a compensatory response. Membrane fluidity, estimated by fluorescence polarization of DPH, was decreased by DMSO. These results demonstrate that DMSO, although generally considered to be antioxidant, induces oxidative stress in yeast cells.

  19. Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae

    DOE PAGES

    Dar, R. D.; Karig, D. K.; Cooke, J. F.; ...

    2010-09-01

    Stochasticity is an inherent feature of complex systems with nanoscale structure. In such systems information is represented by small collections of elements (e.g. a few electrons on a quantum dot), and small variations in the populations of these elements may lead to big uncertainties in the information. Unfortunately, little is known about how to work within this inherently noisy environment to design robust functionality into complex nanoscale systems. Here, we look to the biological cell as an intriguing model system where evolution has mediated the trade-offs between fluctuations and function, and in particular we look at the relationships and trade-offsmore » between stochastic and deterministic responses in the gene expression of budding yeast (Saccharomyces cerevisiae). We find gene regulatory arrangements that control the stochastic and deterministic components of expression, and show that genes that have evolved to respond to stimuli (stress) in the most strongly deterministic way exhibit the most noise in the absence of the stimuli. We show that this relationship is consistent with a bursty 2-state model of gene expression, and demonstrate that this regulatory motif generates the most uncertainty in gene expression when there is the greatest uncertainty in the optimal level of gene expression.« less

  20. Recycling Carbon Dioxide during Xylose Fermentation by Engineered Saccharomyces cerevisiae.

    PubMed

    Xia, Peng-Fei; Zhang, Guo-Chang; Walker, Berkley; Seo, Seung-Oh; Kwak, Suryang; Liu, Jing-Jing; Kim, Heejin; Ort, Donald R; Wang, Shu-Guang; Jin, Yong-Su

    2017-02-17

    Global climate change caused by the emission of anthropogenic greenhouse gases (GHGs) is a grand challenge to humanity. To alleviate the trend, the consumption of fossil fuels needs to be largely reduced and alternative energy technologies capable of controlling GHG emissions are anticipated. In this study, we introduced a synthetic reductive pentose phosphate pathway (rPPP) into a xylose-fermenting Saccharomyces cerevisiae strain SR8 to achieve simultaneous lignocellulosic bioethanol production and carbon dioxide recycling. Specifically, ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and phosphoribulokinase from Spinacia oleracea were introduced into the SR8 strain. The resulting strain with the synthetic rPPP was able to exhibit a higher yield of ethanol and lower yields of byproducts (xylitol and glycerol) than a control strain. In addition, the reduced release of carbon dioxide by the engineered strain was observed during xylose fermentation, suggesting that the carbon dioxide generated by pyruvate decarboxylase was partially reassimilated through the synthetic rPPP. These results demonstrated that recycling of carbon dioxide from the ethanol fermentation pathway in yeast can be achieved during lignocellulosic bioethanol production through a synthetic carbon conservative metabolic pathway. This strategy has a great potential to alleviate GHG emissions during the production of second-generation ethanol.

  1. Tolerance of budding yeast Saccharomyces cerevisiae to ultra high pressure

    NASA Astrophysics Data System (ADS)

    Ono, Fumihisa; Shibata, Michiko; Torigoe, Motoki; Matsumoto, Yuta; Yamamoto, Shinsuke; Takizawa, Noboru; Hada, Yoshio; Mori, Yoshihisa; Takarabe, Kenichi

    2013-06-01

    In our previous studies on the tolerance of small plants and animals to the high hydrostatic pressure of 7.5 GPa, it was shown that all the living samples could be borne at this high pressure, which is more than one order of magnitude higher than the proteinic denaturation pressure. To make this inconsistency clear, we have extended these studies to a smaller sized fungus, budding yeast Saccharomyces cerevisiae. A several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate (PC72, Sumitomo 3M), and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar (PDA). It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for 12 and 24 h were found dead. The high pressure tolerance of budding yeast is weaker than that of tardigrades.

  2. Dynamics of the Saccharomyces cerevisiae Transcriptome during Bread Dough Fermentation

    PubMed Central

    Aslankoohi, Elham; Zhu, Bo; Rezaei, Mohammad Naser; Voordeckers, Karin; De Maeyer, Dries; Marchal, Kathleen; Dornez, Emmie

    2013-01-01

    The behavior of yeast cells during industrial processes such as the production of beer, wine, and bioethanol has been extensively studied. In contrast, our knowledge about yeast physiology during solid-state processes, such as bread dough, cheese, or cocoa fermentation, remains limited. We investigated changes in the transcriptomes of three genetically distinct Saccharomyces cerevisiae strains during bread dough fermentation. Our results show that regardless of the genetic background, all three strains exhibit similar changes in expression patterns. At the onset of fermentation, expression of glucose-regulated genes changes dramatically, and the osmotic stress response is activated. The middle fermentation phase is characterized by the induction of genes involved in amino acid metabolism. Finally, at the latest time point, cells suffer from nutrient depletion and activate pathways associated with starvation and stress responses. Further analysis shows that genes regulated by the high-osmolarity glycerol (HOG) pathway, the major pathway involved in the response to osmotic stress and glycerol homeostasis, are among the most differentially expressed genes at the onset of fermentation. More importantly, deletion of HOG1 and other genes of this pathway significantly reduces the fermentation capacity. Together, our results demonstrate that cells embedded in a solid matrix such as bread dough suffer severe osmotic stress and that a proper induction of the HOG pathway is critical for optimal fermentation. PMID:24056467

  3. Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae.

    PubMed Central

    Thielen, J; Ciriacy, M

    1991-01-01

    As reported previously, Saccharomyces cerevisiae cells deficient in all four known genes coding for alcohol dehydrogenases (ADH1 through ADH4) produce considerable amounts of ethanol during aerobic growth on glucose. It has been suggested that ethanol production in such adh0 cells is a corollary of acetaldehyde dismutation in mitochondria. This could be substantiated further by showing that mitochondrial ethanol formation requires functional electron transport, while the proton gradient or oxidative phosphorylation does not interfere with reduction of acetaldehyde in isolated mitochondria. This acetaldehyde-reducing activity is different from classical alcohol dehydrogenases in that it is associated with the inner mitochondrial membrane and also is unable to carry out ethanol oxidation. The putative cofactor is NADH + H+ generated by a soluble, matrix-located aldehyde dehydrogenase upon acetaldehyde oxidation to acetate. This enzyme has been purified from mitochondria of glucose-grown cells. It is clearly different from the known mitochondrial aldehyde dehydrogenase, which is absent in glucose-grown cells. Both acetaldehyde-reducing and acetaldehyde-oxidizing activities are also present in the mitochondrial fraction of fermentation-proficient (ADH+) cells. Mitochondrial acetaldehyde dismutation may have some significance in the removal of surplus acetaldehyde and in the formation of acetate in mitochondria during aerobic glucose fermentation. Images FIG. 4 PMID:1938903

  4. Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts.

    PubMed

    Gallone, Brigida; Steensels, Jan; Prahl, Troels; Soriaga, Leah; Saels, Veerle; Herrera-Malaver, Beatriz; Merlevede, Adriaan; Roncoroni, Miguel; Voordeckers, Karin; Miraglia, Loren; Teiling, Clotilde; Steffy, Brian; Taylor, Maryann; Schwartz, Ariel; Richardson, Toby; White, Christopher; Baele, Guy; Maere, Steven; Verstrepen, Kevin J

    2016-09-08

    Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  5. Reversal of PCNA Ubiquitylation by Ubp10 in Saccharomyces cerevisiae

    PubMed Central

    Gallego-Sánchez, Alfonso; Andrés, Sonia; Conde, Francisco; San-Segundo, Pedro A.; Bueno, Avelino

    2012-01-01

    Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2BK123 ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in response to the simple presence of ubPCNA. In agreement with this, we show that Ubp10 forms a complex with PCNA in vivo. Interestingly, we also show that deletion of UBP10 alters in different ways the interaction of PCNA with DNA polymerase ζ–associated protein Rev1 and with accessory subunit Rev7. While deletion of UBP10 enhances PCNA–Rev1 interaction, it decreases significantly Rev7 binding to the sliding clamp. Finally, we report that Ubp10 counteracts Rad18 E3-ubiquitin ligase activity on PCNA at lysine 164 in such a manner that deregulation of Ubp10 expression causes tolerance impairment and MMS hypersensitivity. PMID:22829782

  6. Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae.

    PubMed

    Juozapaitis, Mindaugas; Serva, Andrius; Zvirbliene, Aurelija; Slibinskas, Rimantas; Staniulis, Juozas; Sasnauskas, Kestutis; Shiell, Brian J; Wang, Lin-Fa; Michalski, Wojtek P

    2007-03-01

    Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.

  7. Host Factors That Affect Ty3 Retrotransposition in Saccharomyces cerevisiae

    PubMed Central

    Aye, Michael; Irwin, Becky; Beliakova-Bethell, Nadejda; Chen, Eric; Garrus, Jennifer; Sandmeyer, Suzanne

    2004-01-01

    The retrovirus-like element Ty3 of Saccharomyces cerevisiae integrates at the transcription initiation region of RNA polymerase III. To identify host genes that affect transposition, a collection of insertion mutants was screened using a genetic assay in which insertion of Ty3 activates expression of a tRNA suppressor. Fifty-three loci were identified in this screen. Corresponding knockout mutants were tested for the ability to mobilize a galactose-inducible Ty3, marked with the HIS3 gene. Of 42 mutants tested, 22 had phenotypes similar to those displayed in the original assay. The proteins encoded by the defective genes are involved in chromatin dynamics, transcription, RNA processing, protein modification, cell cycle regulation, nuclear import, and unknown functions. These mutants were induced for Ty3 expression and assayed for Gag3p protein, integrase, cDNA, and Ty3 integration upstream of chromosomal tDNAVal(AAC) genes. Most mutants displayed differences from the wild type in one or more intermediates, although these were typically not as severe as the genetic defect. Because a relatively large number of genes affecting retrotransposition can be identified in yeast and because the majority of these genes have mammalian homologs, this approach provides an avenue for the identification of potential antiviral targets. PMID:15579677

  8. Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae.

    PubMed

    DeLuna, Alexander; Quezada, Héctor; Gómez-Puyou, Armando; González, Alicia

    2005-03-25

    The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.

  9. Phylogenetic Portrait of the Saccharomyces cerevisiae Functional Genome

    PubMed Central

    Gibney, Patrick A.; Hickman, Mark J.; Bradley, Patrick H.; Matese, John C.; Botstein, David

    2013-01-01

    The genome of budding yeast (Saccharomyces cerevisiae) contains approximately 5800 protein-encoding genes, the majority of which are associated with some known biological function. Yet the extent of amino acid sequence conservation of these genes over all phyla has only been partially examined. Here we provide a more comprehensive overview and visualization of the conservation of yeast genes and a means for browsing and exploring the data in detail, down to the individual yeast gene, at http://yeast-phylogroups.princeton.edu. We used data from the OrthoMCL database, which has defined orthologs from approximately 150 completely sequenced genomes, including diverse representatives of the archeal, bacterial, and eukaryotic domains. By clustering genes based on similar patterns of conservation, we organized and visualized all the protein-encoding genes in yeast as a single heat map. Most genes fall into one of eight major clusters, called “phylogroups.” Gene ontology analysis of the phylogroups revealed that they were associated with specific, distinct trends in gene function, generalizations likely to be of interest to a wide range of biologists. PMID:23749449

  10. Proteomic Profiling of Autophagosome Cargo in Saccharomyces cerevisiae

    PubMed Central

    Morimoto, Mayumi; Fujii, Kiyonaga; Noda, Nobuo N.; Inagaki, Fuyuhiko; Ohsumi, Yoshinori

    2014-01-01

    Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy. PMID:24626240

  11. Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae.

    PubMed

    Bogonez, E; Satrústegui, J; Machado, A

    1985-06-01

    The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.

  12. RNA–DNA sequence differences in Saccharomyces cerevisiae

    PubMed Central

    Wang, Isabel X.; Grunseich, Christopher; Chung, Youree G.; Kwak, Hojoong; Ramrattan, Girish; Zhu, Zhengwei; Cheung, Vivian G.

    2016-01-01

    Alterations of RNA sequences and structures, such as those from editing and alternative splicing, result in two or more RNA transcripts from a DNA template. It was thought that in yeast, RNA editing only occurs in tRNAs. Here, we found that Saccharomyces cerevisiae have all 12 types of RNA–DNA sequence differences (RDDs) in the mRNA. We showed these sequence differences are propagated to proteins, as we identified peptides encoded by the RNA sequences in addition to those by the DNA sequences at RDD sites. RDDs are significantly enriched at regions with R-loops. A screen of yeast mutants showed that RDD formation is affected by mutations in genes regulating R-loops. Loss-of-function mutations in ribonuclease H, senataxin, and topoisomerase I that resolve RNA–DNA hybrids lead to increases in RDD frequency. Our results demonstrate that RDD is a conserved process that diversifies transcriptomes and proteomes and provide a mechanistic link between R-loops and RDDs. PMID:27638543

  13. Kinetic and Morphological Observations on Saccharomyces cerevisiae During Spheroplast Formation

    PubMed Central

    Darling, Sven; Theilade, Jørgen; Birch-Andersen, Aksel

    1969-01-01

    A strain of Saccharomyces cerevisiae which produced elongated cells under our growth conditions was investigated. By digestion of the cell walls with snail enzyme, the cells became spheroplasts after a transient state which we termed “prospheroplast.” The prospheroplast could be lysed like the spheroplast, but it retained the shape of the original yeast cell if osmotically protected. Prospheroplasts and spheroplasts were prepared, and thin sections of samples taken throughout the process of wall removal were studied in the electron microscope, at regular intervals up to the time of complete conversion to spheroplasts. In addition, cell wall remnants recovered from spheroplast preparations were shadow cast for electron microscopy. This material revealed structures resembling bud scars with attached membranous matter. The kinetic studies showed that after a certain period of time all cells were transformed into prospheroplasts, whereas spheroplast formation started later, depending on the enzyme concentration. In sections, the prospheroplasts appeared to be formed by detachment of the cell walls. Both the prospheroplasts and the spheroplasts showed asymmetric cytoplasmic membranes in which the outer leaflets appeared coated with a dense fibrillar layer. The experiments suggest that, after enzyme digestion, the cytoplasmic membrane retains a coating which is rigid in the prospheroplast but which loses rigidity when the cell is transformed into a spheroplast. Images PMID:5784226

  14. Dynamics of the Saccharomyces cerevisiae transcriptome during bread dough fermentation.

    PubMed

    Aslankoohi, Elham; Zhu, Bo; Rezaei, Mohammad Naser; Voordeckers, Karin; De Maeyer, Dries; Marchal, Kathleen; Dornez, Emmie; Courtin, Christophe M; Verstrepen, Kevin J

    2013-12-01

    The behavior of yeast cells during industrial processes such as the production of beer, wine, and bioethanol has been extensively studied. In contrast, our knowledge about yeast physiology during solid-state processes, such as bread dough, cheese, or cocoa fermentation, remains limited. We investigated changes in the transcriptomes of three genetically distinct Saccharomyces cerevisiae strains during bread dough fermentation. Our results show that regardless of the genetic background, all three strains exhibit similar changes in expression patterns. At the onset of fermentation, expression of glucose-regulated genes changes dramatically, and the osmotic stress response is activated. The middle fermentation phase is characterized by the induction of genes involved in amino acid metabolism. Finally, at the latest time point, cells suffer from nutrient depletion and activate pathways associated with starvation and stress responses. Further analysis shows that genes regulated by the high-osmolarity glycerol (HOG) pathway, the major pathway involved in the response to osmotic stress and glycerol homeostasis, are among the most differentially expressed genes at the onset of fermentation. More importantly, deletion of HOG1 and other genes of this pathway significantly reduces the fermentation capacity. Together, our results demonstrate that cells embedded in a solid matrix such as bread dough suffer severe osmotic stress and that a proper induction of the HOG pathway is critical for optimal fermentation.

  15. Effect of calcofluor white on chitin synthases from Saccharomyces cerevisiae.

    PubMed Central

    Roncero, C; Valdivieso, M H; Ribas, J C; Durán, A

    1988-01-01

    The growths of Saccharomyces cerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3), defective in chitin synthase 1 (Chs1) but showing a new chitin synthase activity (Chs2), were affected by Calcofluor. To be effective, the interaction of Calcofluor with growing cells had to occur at around pH 6. Treatment of growing cells from these strains with the fluorochrome led to an increase in the total levels of Chs1 and Chs2 activities measured on permeabilized cells. During treatment, basal levels (activities expressed in the absence of exogenous proteolytic activation) of Chs1 and Chs2 increased nine- and fourfold, respectively, through a mechanism dependent on protein synthesis, since the effect was abolished by cycloheximide. During alpha-factor treatment, both Chs1 and Chs2 levels increased; however, as opposed to what occurred during the mitotic cell cycle, there was no further increase in Chs1 or Chs2 activities by Calcofluor treatment. Images PMID:2965145

  16. Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects.

    PubMed

    Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

    2016-02-25

    Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.

  17. Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

    PubMed Central

    Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

    2016-01-01

    Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review. PMID:26927067

  18. Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history.

    PubMed

    Legras, Jean-Luc; Merdinoglu, Didier; Cornuet, Jean-Marie; Karst, Francis

    2007-05-01

    Fermented beverages and foods have played a significant role in most societies worldwide for millennia. To better understand how the yeast species Saccharomyces cerevisiae, the main fermenting agent, evolved along this historical and expansion process, we analysed the genetic diversity among 651 strains from 56 different geographical origins, worldwide. Their genotyping at 12 microsatellite loci revealed 575 distinct genotypes organized in subgroups of yeast types, i.e. bread, beer, wine, sake. Some of these groups presented unexpected relatedness: Bread strains displayed a combination of alleles intermediate between beer and wine strains, and strains used for rice wine and sake were most closely related to beer and bread strains. However, up to 28% of genetic diversity between these technological groups was associated with geographical differences which suggests local domestications. Focusing on wine yeasts, a group of Lebanese strains were basal in an F(ST) tree, suggesting a Mesopotamia-based origin of most wine strains. In Europe, migration of wine strains occurred through the Danube Valley, and around the Mediterranean Sea. An approximate Bayesian computation approach suggested a postglacial divergence (most probable period 10,000-12,000 bp). As our results suggest intimate association between man and wine yeast across centuries, we hypothesize that yeast followed man and vine migrations as a commensal member of grapevine flora.

  19. Peroxisomal fatty acid uptake mechanism in Saccharomyces cerevisiae.

    PubMed

    van Roermund, Carlo W T; Ijlst, Lodewijk; Majczak, Wiktor; Waterham, Hans R; Folkerts, Hendrik; Wanders, Ronald J A; Hellingwerf, Klaas J

    2012-06-08

    Peroxisomes play a major role in human cellular lipid metabolism, including fatty acid β-oxidation. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy, which is caused by mutations in ABCD1. The biochemical hallmark of X-linked adrenoleukodystrophy is the accumulation of very long chain fatty acids (VLCFAs) due to impaired peroxisomal β-oxidation. Although this suggests a role of ABCD1 in VLCFA import into peroxisomes, no direct experimental evidence is available to substantiate this. To unravel the mechanism of peroxisomal VLCFA transport, we use Saccharomyces cerevisiae as a model organism. Here we provide evidence that in this organism very long chain acyl-CoA esters are hydrolyzed by the Pxa1p-Pxa2p complex prior to the actual transport of their fatty acid moiety into the peroxisomes with the CoA presumably being released into the cytoplasm. The Pxa1p-Pxa2p complex functionally interacts with the acyl-CoA synthetases Faa2p and/or Fat1p on the inner surface of the peroxisomal membrane for subsequent re-esterification of the VLCFAs. Importantly, the Pxa1p-Pxa2p complex shares this molecular mechanism with HsABCD1 and HsABCD2.

  20. Peroxisomal Fatty Acid Uptake Mechanism in Saccharomyces cerevisiae*

    PubMed Central

    van Roermund, Carlo W. T.; IJlst, Lodewijk; Majczak, Wiktor; Waterham, Hans R.; Folkerts, Hendrik; Wanders, Ronald J. A.; Hellingwerf, Klaas J.

    2012-01-01

    Peroxisomes play a major role in human cellular lipid metabolism, including fatty acid β-oxidation. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy, which is caused by mutations in ABCD1. The biochemical hallmark of X-linked adrenoleukodystrophy is the accumulation of very long chain fatty acids (VLCFAs) due to impaired peroxisomal β-oxidation. Although this suggests a role of ABCD1 in VLCFA import into peroxisomes, no direct experimental evidence is available to substantiate this. To unravel the mechanism of peroxisomal VLCFA transport, we use Saccharomyces cerevisiae as a model organism. Here we provide evidence that in this organism very long chain acyl-CoA esters are hydrolyzed by the Pxa1p-Pxa2p complex prior to the actual transport of their fatty acid moiety into the peroxisomes with the CoA presumably being released into the cytoplasm. The Pxa1p-Pxa2p complex functionally interacts with the acyl-CoA synthetases Faa2p and/or Fat1p on the inner surface of the peroxisomal membrane for subsequent re-esterification of the VLCFAs. Importantly, the Pxa1p-Pxa2p complex shares this molecular mechanism with HsABCD1 and HsABCD2. PMID:22493507

  1. Cellular memory of acquired stress resistance in Saccharomyces cerevisiae.

    PubMed

    Guan, Qiaoning; Haroon, Suraiya; Bravo, Diego González; Will, Jessica L; Gasch, Audrey P

    2012-10-01

    Cellular memory of past experiences has been observed in several organisms and across a variety of experiences, including bacteria "remembering" prior nutritional status and amoeba "learning" to anticipate future environmental conditions. Here, we show that Saccharomyces cerevisiae maintains a multifaceted memory of prior stress exposure. We previously demonstrated that yeast cells exposed to a mild dose of salt acquire subsequent tolerance to severe doses of H(2)O(2). We set out to characterize the retention of acquired tolerance and in the process uncovered two distinct aspects of cellular memory. First, we found that H(2)O(2) resistance persisted for four to five generations after cells were removed from the prior salt treatment and was transmitted to daughter cells that never directly experienced the pretreatment. Maintenance of this memory did not require nascent protein synthesis after the initial salt pretreatment, but rather required long-lived cytosolic catalase Ctt1p that was synthesized during salt exposure and then distributed to daughter cells during subsequent cell divisions. In addition to and separable from the memory of H(2)O(2) resistance, these cells also displayed a faster gene-expression response to subsequent stress at >1000 genes, representing transcriptional memory. The faster gene-expression response requires the nuclear pore component Nup42p and serves an important function by facilitating faster reacquisition of H(2)O(2) tolerance after a second cycle of salt exposure. Memory of prior stress exposure likely provides a significant advantage to microbial populations living in ever-changing environments.

  2. Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    PubMed Central

    Gonzalez-Perez, David; Alcalde, Miguel

    2014-01-01

    The ligninolytic enzymatic consortium produced by white-rot fungi is one of the most efficient oxidative systems found in nature, with many potential applications that range from the production of 2nd generation biofuels to chemicals synthesis. In the current study, two high redox potential oxidoreductase fusion genes (laccase -Lac- and versatile peroxidase -Vp-) that had been evolved in the laboratory were re-assembled in Saccharomyces cerevisiae. First, cell viability and secretion were assessed after co-transforming the Lac and Vp genes into yeast. Several expression cassettes were inserted in vivo into episomal bi-directional vectors in order to evaluate inducible promoter and/or terminator pairs of different strengths in an individual and combined manner. The synthetic white-rot yeast model harboring Vp(GAL1/CYC1)-Lac(GAL10/ADH1) displayed up to 1000 and 100 Units per L of peroxidase and laccase activity, respectively, representing a suitable point of departure for future synthetic biology studies. PMID:24830983

  3. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides.

    PubMed

    Dai, Zhubo; Liu, Yi; Zhang, Xianan; Shi, Mingyu; Wang, Beibei; Wang, Dong; Huang, Luqi; Zhang, Xueli

    2013-11-01

    Ginsenosides are the primary bioactive components of ginseng, which is a popular medicinal herb and exhibits diverse pharmacological activities. Protopanaxadiol is the aglycon of several dammarane-type ginsenosides, which also has anticancer activity. For microbial production of protopanaxadiol, dammarenediol-II synthase and protopanaxadiol synthase genes of Panax ginseng, together with a NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana, were introduced into Saccharomyces cerevisiae, resulting in production of 0.05 mg/g DCW protopanaxadiol. Increasing squalene and 2,3-oxidosqualene supplies through overexpressing truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, farnesyl diphosphate synthase, squalene synthase and 2,3-oxidosqualene synthase genes, together with increasing protopanaxadiol synthase activity through codon optimization, led to 262-fold increase of protopanaxadiol production. Finally, using two-phase extractive fermentation resulted in production of 8.40 mg/g DCW protopanaxadiol (1189 mg/L), together with 10.94 mg/g DCW dammarenediol-II (1548 mg/L). The yeast strains engineered in this work can serve as the basis for creating an alternative way for production of ginsenosides in place of extraction from plant sources.

  4. Biotransformation of malachite green by Saccharomyces cerevisiae MTCC 463.

    PubMed

    Jadhav, J P; Govindwar, S P

    2006-03-01

    In recent years, use of microbial biomass for decolourization of textile industry wastewater is becoming a promising alternative in which some bacteria and fungi are used to replace present treatment processes. Saccharomyces cerevisiae MTCC 463 decolourized the triphenylmethane dyes (malachite green, cotton blue, methyl violet and crystal violet) by biosorption, showing different decolourization patterns. However, malachite green decolourized by biosorption at the initial stage and further biodegradation occurred, about 85% in plain distilled water within 7 h, and about 95.5% in 5% glucose medium within 4 h, under aerobic conditions and at room temperature. Decolourization of malachite green depends on various conditions, such as concentration of dye, concentration of cells, composition of medium and agitation. HPLC, UV-VIS, FTIR and TLC analysis of samples extracted with ethyl acetate from decolourized culture flasks confirmed the biodegradation of malachite green into several metabolites. A study of the enzymes responsible for the biodegradation of malachite green in the control and cells obtained after decolourization showed the activities of laccase, lignin peroxidase, NADH-DCIP reductase, malachite green reductase and aminopyrine N-demethylase in control cells. A significant increase in the activities of NADH-DCIP reductase and MG reductase was observed in the cells obtained after decolourization, indicating a major involvement of reductases in malachite green degradation.

  5. Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.

    PubMed

    Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra

    2016-01-01

    This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jen1, Ady2, Fps1 and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jen1 and Ady2 can mediate the uptake of the anionic form of these substrates by a H(+)-symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fps1 channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions.

  6. Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

    PubMed Central

    van Zutphen, Tim; Todde, Virginia; de Boer, Rinse; Kreim, Martin; Hofbauer, Harald F.; Wolinski, Heimo; Veenhuis, Marten; van der Klei, Ida J.; Kohlwein, Sepp D.

    2014-01-01

    Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast. PMID:24258026

  7. Influence of culture conditions on glutathione production by Saccharomyces cerevisiae.

    PubMed

    Santos, Lucielen Oliveira; Gonzales, Tatiane Araujo; Ubeda, Beatriz Torsani; Monte Alegre, Ranulfo

    2007-12-01

    A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20-30 degrees C), agitation rate (100-300 rpm), initial pH (5.0-7.0), inoculum concentration (5-15%), and glucose concentration (30-70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2(5-2)), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2(2)) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20 degrees C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.

  8. A quantitative assay for telomere protection in Saccharomyces cerevisiae.

    PubMed Central

    DuBois, Michelle L; Haimberger, Zara W; McIntosh, Martin W; Gottschling, Daniel E

    2002-01-01

    Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This "chromosomal cap" assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected. PMID:12136006

  9. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

  10. Endomitotic effect of a cell cycle mutation of Saccharomyces cerevisiae

    SciTech Connect

    Schild, D.; Ananthaswamy, H.N.; Mortimer, R.K.

    1981-03-01

    A recessive temperature-sensitive mutation of Saccharomyces cerevisiae has been isolated and shown to cause an increase in ploidy in both haploids and diploids. Genetic analysis revealed that the strain carrying the mutation was an aa diploid, although MNNG mutagenesis had been done on an a haploid strain. When the mutant strain was crossed with an ..cap alpha cap alpha.. diploid and the resultant tetraploid sporulated, some of the meiotic progeny of this tetraploid were themselves tetraploid, as shown by both genetic analysis and DNA measurements, instead of diploid as expected of tetraploid meiosis. The ability of these tetraploids to continue to produce tetraploid meiotic progeny was followed for four generations. It was found that tetraploidization was independent of sporulation temperature, but was dependent on the temperature of germination and the growth of the spores. Increase in ploidy occurred when the spores were germinated and grown at 30/sup 0/, but did not occur at 23/sup 0/. Two cycles of sporulation and growth at 23/sup 0/ resulted in haploids, which were shown to diploidize within 24 hr when grown at 30/sup 0/.

  11. Metabolically engineered Saccharomyces cerevisiae for enhanced isoamyl alcohol production.

    PubMed

    Yuan, Jifeng; Chen, Xue; Mishra, Pranjul; Ching, Chi-Bun

    2017-01-01

    Higher chain alcohols have gained much attention as next generation transport fuels because of their higher energy density and low moisture absorption capacity compared to ethanol. In the present study, we attempted to engineer Saccharomyces cerevisiae for the synthesis of isoamyl alcohol via de novo leucine biosynthetic pathway coupled with Ehrlich degradation pathway. To achieve high-level production of isoamyl alcohol, two strategies are used in the current study: (1) reconstruction of a chromosome-based leucine biosynthetic pathway under the control of galactose-inducible promoters; (2) overexpression of the mitochondrial 2-isopropylmalate (α-IPM) transporter to boost the transportation of α-IPM from mitochondria to the cytosol. We found engineered yeast cells with a combinatorially assembled leucine biosynthetic pathway coupled with the Ehrlich degradation pathway resulted in high-level production of isoamyl alcohol; however, there was still a significant amount of isobutanol co-formed during the fermentation process. Further introducing an α-IPM transporter not only boosted the isoamyl alcohol biosynthetic pathway activity but also reduced isobutanol to a much lower level. Taken together, our work represents the first study to construct a chromosome-based leucine biosynthetic pathway for isoamyl alcohol production. Furthermore, the utilization of the mitochondrial compartment coupled with the transporter engineering serves as an effective approach to minimize the by-product formation and to improve the isoamyl alcohol production.

  12. D-lactic acid production by metabolically engineered Saccharomyces cerevisiae.

    PubMed

    Ishida, Nobuhiro; Suzuki, Tomiko; Tokuhiro, Kenro; Nagamori, Eiji; Onishi, Toru; Saitoh, Satoshi; Kitamoto, Katsuhiko; Takahashi, Haruo

    2006-02-01

    Poly D-lactic acid is an important polymer because it improves the thermostability of poly L-lactic acid by the stereo complex formation. We constructed a metabolically engineered Saccharomyces cerevisiae that produces D-lactic acid efficiently. In this recombinant, the coding region of pyruvate decarboxylase 1 (PDC1) was completely deleted, and two copies of the D-lactate dehydrogenase (D-LDH) gene from Leuconostoc mesenteroides subsp. mesenteroides strain NBRC3426 were introduced into the genome. The D-lactate production reached 61.5 g/l, the amount of glucose being transformed into D-lactic acid being 61.2% under neutralizing conditions. Additionally, the yield of free D-lactic acid was also shown to be 53.0% under non-neutralizing conditions. It was confirmed that D-lactic acid of extremely high optical purity of 99.9% or higher. Our finding obtained the possibility of a new approach for pure d-lactic acid production without a neutralizing process compared with other techniques involving lactic acid bacteria and transgenic Escherichia coli.

  13. Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae.

    PubMed Central

    Murray, A W; Claus, T E; Szostak, J W

    1988-01-01

    We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation. The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules. The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA. The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule. Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA. The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase. The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division. Images PMID:3062364

  14. TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae

    PubMed Central

    Welch, Aaron Z.; Gibney, Patrick A.; Botstein, David; Koshland, Douglas E.

    2013-01-01

    Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance. PMID:23171550

  15. Dissection of Filamentous Growth by Transposon Mutagenesis in Saccharomyces Cerevisiae

    PubMed Central

    Mosch, H. U.; Fink, G. R.

    1997-01-01

    Diploid Saccharomyces cerevisiae strains starved for nitrogen undergo a developmental transition from growth as single yeast form (YF) cells to a multicellular form consisting of filaments of pseudohyphal (PH) cells. Filamentous growth is regulated by an evolutionarily conserved signaling pathway that includes the small GTP-binding proteins Ras2p and Cdc42p, the protein kinases Ste20p, Ste11p and Ste7p, and the transcription factor Ste12p. Here, we designed a genetic screen for mutant strains defective for filamentous growth (dfg) to identify novel targets of the filamentation signaling pathway, and we thereby identified 16 different genes, CDC39, STE12, TEC1, WHI3, NAB1, DBR1, CDC55, SRV2, TPM1, SPA2, BNI1, DFG5, DFG9, DFG10, BUD8 and DFG16, mutations that block filamentous growth. Phenotypic analysis of dfg mutant strains genetically dissects filamentous growth into the cellular processes of signal transduction, bud site selection, cell morphogenesis and invasive growth. Epistasis tests between dfg mutant alleles and dominant activated alleles of the RAS2 and STE11 genes, RAS2(Val19) and STE11-4, respectively, identify putative targets for the filamentation signaling pathway. Several of the genes described here have homologues in filamentous fungi, where they also regulate fungal development. PMID:9055077

  16. Transcriptional response of Saccharomyces cerevisiae to desiccation and rehydration.

    PubMed

    Singh, Jatinder; Kumar, Deept; Ramakrishnan, Naren; Singhal, Vibha; Jervis, Jody; Garst, James F; Slaughter, Stephen M; DeSantis, Andrea M; Potts, Malcolm; Helm, Richard F

    2005-12-01

    A transcriptional analysis of the response of Saccharomyces cerevisiae strain BY4743 to controlled air-drying (desiccation) and subsequent rehydration under minimal glucose conditions was performed. Expression of genes involved in fatty acid oxidation and the glyoxylate cycle was observed to increase during drying and remained in this state during the rehydration phase. When the BY4743 expression profile for the dried sample was compared to that of a commercially prepared dry active yeast, strikingly similar expression changes were observed. The fact that these two samples, dried by different means, possessed very similar transcriptional profiles supports the hypothesis that the response to desiccation is a coordinated event independent of the particular conditions involved in water removal. Similarities between "stationary-phase-essential genes" and those upregulated during desiccation were also noted, suggesting commonalities in different routes to reduced metabolic states. Trends in extracellular and intracellular glucose and trehalose levels suggested that the cells were in a "holding pattern" during the rehydration phase, a concept that was reinforced by cell cycle analyses. Application of a "redescription mining" algorithm suggested that sulfur metabolism is important for cell survival during desiccation and rehydration.

  17. Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae.

    PubMed

    Ohya, Y; Uno, I; Ishikawa, T; Anraku, Y

    1987-10-01

    Calmodulin from the yeast Saccharomyces cerevisiae was purified to complete homogeneity by hydrophobic interaction chromatography and HPLC gel filtration. The biochemical properties of the purified protein as calmodulin were examined under various criteria and its similarity and dissimilarity to other calmodulins have been described. Like other calmodulins, yeast calmodulin activated bovine phosphodiesterase and pea NAD kinase in a Ca2+-dependent manner, but its concentration for half-maximal activation was 8-10 times that of bovine calmodulin. The amino acid composition of yeast calmodulin was different from those of calmodulins from other lower eukaryotes in that it contained no tyrosine, but more leucine and had a high ratio of serine to threonine. Yeast calmodulin did not contain tryptophanyl or tyrosyl residues, so its ultraviolet spectrum reflected the absorbance of phenylalanyl residues, and had a molar absorption coefficient at 259 nm of 1900 M-1 cm-1. Ca2+ ions changed the secondary structure of yeast calmodulin, causing a 3% decrease in the alpha-helical content, unlike its effect on other calmodulins. Antibody against yeast calmodulin did not cross-react with bovine calmodulin, and antibody against bovine calmodulin did not cross-react with yeast calmodulin, presumably due to differences in the amino acid sequences of the antigenic sites. It is concluded that the molecular structure of yeast calmodulin differs from those of calmodulins from other sources, but that its Ca2+-dependent regulatory functions are highly conserved and essentially similar to those of calmodulins of higher eukaryotes.

  18. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D

    2016-03-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.

  19. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae

    PubMed Central

    Kennedy, Erin K.; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C.; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D.

    2016-01-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2ARts1 either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase. PMID:26715668

  20. Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion

    PubMed Central

    Carman, George M.; Han, Gil-Soo

    2007-01-01

    The synthesis of phospholipids in the yeast Saccharomyces cerevisiae is regulated by zinc, an essential mineral required for growth and metabolism. Cells depleted of zinc contain increased levels of phosphatidylinositol and decreased levels of phosphatidylethanolamine. In addition to the major phospholipids, the levels of the minor phospholipids phosphatidate and diacylglycerol pyrophosphate decrease in the vacuole membrane of zinc-depleted cells. Alterations in phosphatidylinositol and phosphatidylethanolamine can be ascribed to an increase in PIS1-encoded phosphatidylinositol synthase activity and to decreases in the activities of CDP-diacylglycerol pathway enzymes including the CHO1-encoded phosphatidylserine synthase, respectively. Alterations in the minor vacuole membrane phospholipids are due to the induction of the DPP1-encoded diacylglycerol pyrophosphate phosphatase. These changes in the activities of phospholipid biosynthetic enzymes result from differential regulation of gene expression at the level of transcription. Under zinc-deplete conditions, the positive transcription factor Zap1p stimulates the expression of the DPP1 and PIS1 genes through the cis-acting element UASZRE. In contrast, the negative regulatory protein Opi1p, which is involved in inositol-mediated regulation of phospholipid synthesis, represses the expression of the CHO1 gene through the cis-acting element UASINO. Regulation of phospholipid synthesis may provide an important mechanism by which cells cope with the stress of zinc depletion, given the roles that phospholipids play in the structure and function of cellular membranes. PMID:16807089

  1. Regulation of profilin localization in Saccharomyces cerevisiae by phosphoinositide metabolism.

    PubMed

    Ostrander, D B; Gorman, J A; Carman, G M

    1995-11-10

    Profilin is an actin- and phosphatidylinositol 4,5-bisphosphate-binding protein that plays a role in the organization of the cytoskeleton and may be involved in growth factor signaling pathways. The subcellular localization of profilin was examined in the yeast Saccharomyces cerevisiae. Immunoblot analysis showed that profilin was localized in both the plasma membrane and cytosolic fractions of the cell. Actin was bound to the profilin localized in the cytosol. The association of profilin with the membrane was peripheral and mediated through interaction with phospholipid. The phospholipid dependence of profilin for membrane binding was examined in vitro using pure profilin and defined unilamellar phospholipid vesicles. The presence of phosphatidylinositol 4,5-bisphosphate in phospholipid vesicles was required for maximum profilin binding. Moreover, the binding of profilin to phospholipid vesicles was dependent on the surface concentration of phosphatidylinositol 4,5-bisphosphate. The subcellular localization of profilin was examined in vivo under growth conditions (i.e. inositol starvation of ino1 cells and glucose starvation of respiratory deficient cells) where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were depleted. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate levels resulted in a translocation of profilin from the plasma membrane to the cytosolic fraction. Profilin translocated back to the membrane fraction from the cytosol under growth conditions where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were replenished. These results suggested that phosphoinositide metabolism played a role in the localization of profilin.

  2. Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc.

    PubMed

    Iwanyshyn, Wendy M; Han, Gil-Soo; Carman, George M

    2004-05-21

    Zinc is an essential nutrient required for the growth and metabolism of eukaryotic cells. In this work, we examined the effects of zinc depletion on the regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae. Zinc depletion resulted in a decrease in the activity levels of the CDP-diacylglycerol pathway enzymes phosphatidylserine synthase, phosphatidylserine decarboxylase, phosphatidylethanolamine methyltransferase, and phospholipid methyltransferase. In contrast, the activity of phosphatidylinositol synthase was elevated in response to zinc depletion. The level of Aut7p, a marker for the induction of autophagy, was also elevated in zinc-depleted cells. For the CHO1-encoded phosphatidylserine synthase, the reduction in activity in response to zinc depletion was controlled at the level of transcription. This regulation was mediated through the UAS(INO) element and by the transcription factors Ino2p, Ino4p, and Opi1p that are responsible for the inositol-mediated regulation of UAS(INO)-containing genes involved in phospholipid synthesis. Analysis of the cellular composition of the major membrane phospholipids showed that zinc depletion resulted in a 66% decrease in phosphatidylethanolamine and a 29% increase in phosphatidylinositol. A zrt1Delta zrt2Delta mutant (defective in the plasma membrane zinc transporters Zrt1p and Zrt2p) grown in the presence of zinc exhibited a phospholipid composition similar to that of wild type cells depleted for zinc. These results indicated that a decrease in the cytoplasmic levels of zinc was responsible for the alterations in phospholipid composition.

  3. Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae.

    PubMed

    Hu, Kejin; Guo, Shuguang; Yan, Gonghong; Yuan, Wenjie; Zheng, Yin; Jiang, Yu

    2016-04-01

    In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth-related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12-rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2(W2041R) ) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non-covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization. © 2016 John Wiley & Sons Ltd.

  4. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae.

    PubMed

    Conrad, Michaela; Schothorst, Joep; Kankipati, Harish Nag; Van Zeebroeck, Griet; Rubio-Texeira, Marta; Thevelein, Johan M

    2014-03-01

    The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms. Many specific nutrient responses have been elucidated in great detail. This has led to important new concepts and insight into nutrient-controlled cellular regulation. Major highlights include the central role of the Snf1 protein kinase in the glucose repression pathway, galactose induction, the discovery of a G-protein-coupled receptor system, and role of Ras in glucose-induced cAMP signaling, the role of the protein synthesis initiation machinery in general control of nitrogen metabolism, the cyclin-controlled protein kinase Pho85 in phosphate regulation, nitrogen catabolite repression and the nitrogen-sensing target of rapamycin pathway, and the discovery of transporter-like proteins acting as nutrient sensors. In addition, a number of cellular targets, like carbohydrate stores, stress tolerance, and ribosomal gene expression, are controlled by the presence of multiple nutrients. The protein kinase A signaling pathway plays a major role in this general nutrient response. It has led to the discovery of nutrient transceptors (transporter receptors) as nutrient sensors. Major shortcomings in our knowledge are the relationship between rapid and steady-state nutrient signaling, the role of metabolic intermediates in intracellular nutrient sensing, and the identity of the nutrient sensors controlling cellular growth. © 2014 The Authors. FEMS Microbiology Reviews published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

  5. Calcium dependence of eugenol tolerance and toxicity in Saccharomyces cerevisiae.

    PubMed

    Roberts, Stephen K; McAinsh, Martin; Cantopher, Hanna; Sandison, Sean

    2014-01-01

    Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections.

  6. Dual effects of plant steroidal alkaloids on Saccharomyces cerevisiae.

    PubMed

    Simons, Veronika; Morrissey, John P; Latijnhouwers, Maita; Csukai, Michael; Cleaver, Adam; Yarrow, Carol; Osbourn, Anne

    2006-08-01

    Many plant species accumulate sterols and triterpenes as antimicrobial glycosides. These secondary metabolites (saponins) provide built-in chemical protection against pest and pathogen attack and can also influence induced defense responses. In addition, they have a variety of important pharmacological properties, including anticancer activity. The biological mechanisms underpinning the varied and diverse effects of saponins on microbes, plants, and animals are only poorly understood despite the ecological and pharmaceutical importance of this major class of plant secondary metabolites. Here we have exploited budding yeast (Saccharomyces cerevisiae) to investigate the effects of saponins on eukaryotic cells. The tomato steroidal glycoalkaloid alpha-tomatine has antifungal activity towards yeast, and this activity is associated with membrane permeabilization. Removal of a single sugar from the tetrasaccharide chain of alpha-tomatine results in a substantial reduction in antimicrobial activity. Surprisingly, the complete loss of sugars leads to enhanced antifungal activity. Experiments with alpha-tomatine and its aglycone tomatidine indicate that the mode of action of tomatidine towards yeast is distinct from that of alpha-tomatine and does not involve membrane permeabilization. Investigation of the effects of tomatidine on yeast by gene expression and sterol analysis indicate that tomatidine inhibits ergosterol biosynthesis. Tomatidine-treated cells accumulate zymosterol rather than ergosterol, which is consistent with inhibition of the sterol C(24) methyltransferase Erg6p. However, erg6 and erg3 mutants (but not erg2 mutants) have enhanced resistance to tomatidine, suggesting a complex interaction of erg mutations, sterol content, and tomatidine resistance.

  7. Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16.

    PubMed Central

    Bang, D D; Timmermans, V; Verhage, R; Zeeman, A M; van de Putte, P; Brouwer, J

    1995-01-01

    The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci [Bang et al. (1992) Nucleic Acids Res. 20, 3925-3931]. More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae [Waters et al. (1993) Mol. Gen. Genet. 239, 28-32]. We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation. Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA. In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced. The basal level of the RAD16 transcript is constant during the mitotic cell cycle. G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation. However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle. Images PMID:7784171

  8. Genes Required for Vacuolar Acidity in Saccharomyces Cerevisiae

    PubMed Central

    Preston, R. A.; Reinagel, P. S.; Jones, E. W.

    1992-01-01

    Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein. Thirty nine vacuolar pH (Vph(-)) mutants were identified. Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes. The remaining mutants defined eight complementation groups of vph mutations. No alleles of the VAT2 or TFP1 genes (known to encode subunits of the vacuolar H(+)-ATPase) were identified in the Vph(-) screen. Strains bearing mutations in any of six of the VPH genes failed to grow on medium buffered at neutral pH; otherwise, none of the vph mutations caused notable growth inhibition on standard yeast media. Expression of the vacuolar protease, carboxypeptidase Y, was defective in strains bearing vph4 mutations but was apparently normal in strains bearing any of the other vph mutations. Defects in vacuolar morphology at the light microscope level were evident in all Vph(-) mutants. Strains that contained representative mutant alleles of the 17 previously described PEP genes were assayed for vacuolar pH; mutations in seven of the PEP genes (including PEP3, PEP5, PEP6 and PEP7) caused loss of vacuolar acidity. PMID:1628805

  9. A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae

    PubMed Central

    Steidle, Elizabeth A.; Chong, Lucy S.; Wu, Mingxuan; Crooke, Elliott; Fiedler, Dorothea; Resnick, Adam C.; Rolfes, Ronda J.

    2016-01-01

    Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, the Saccharomyces cerevisiae homolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. We identified a novel phosphatase of the recently discovered atypical dual specificity phosphatase family as a physiological inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the β-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5 or IP7) in vitro. In vivo, siw14Δ yeast mutants possess increased IP7 levels, whereas heterologous SIW14 overexpression eliminates IP7 from cells. IP7 levels increased proportionately when siw14Δ was combined with ddp1Δ or vip1Δ, indicating independent activity by the enzymes encoded by these genes. We conclude that Siw14 is a physiological phosphatase that modulates inositol pyrophosphate metabolism by dephosphorylating the IP7 isoform 5PP-IP5 to IP6. PMID:26828065

  10. Post-Transcriptional Regulation of Iron Homeostasis in Saccharomyces cerevisiae

    PubMed Central

    Martínez-Pastor, María Teresa; de Llanos, Rosa; Romero, Antonia María; Puig, Sergi

    2013-01-01

    Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in a wide variety of biological processes. Recent studies in Saccharomyces cerevisiae have shown that in response to iron deficiency, an RNA-binding protein denoted Cth2 coordinates a global metabolic rearrangement that aims to optimize iron utilization. The Cth2 protein contains two Cx8Cx5Cx3H tandem zinc fingers (TZFs) that specifically bind to adenosine/uridine-rich elements within the 3′ untranslated region of many mRNAs to promote their degradation. The Cth2 protein shuttles between the nucleus and the cytoplasm. Once inside the nucleus, Cth2 binds target mRNAs and stimulates alternative 3′ end processing. A Cth2/mRNA-containing complex is required for export to the cytoplasm, where the mRNA is degraded by the 5′ to 3′ degradation pathway. This post-transcriptional regulatory mechanism limits iron utilization in nonessential pathways and activates essential iron-dependent enzymes such as ribonucleotide reductase, which is required for DNA synthesis and repair. Recent findings indicate that the TZF-containing tristetraprolin protein also functions in modulating human iron homeostasis. Elevated iron concentrations can also be detrimental for cells. The Rnt1 RNase III exonuclease protects cells from excess iron by promoting the degradation of a subset of the Fe acquisition system when iron levels rise. PMID:23903042

  11. Metabolically engineered Saccharomyces cerevisiae for branched-chain ester productions.

    PubMed

    Yuan, Jifeng; Mishra, Pranjul; Ching, Chi Bun

    2016-12-10

    Medium branched-chain esters can be used not only as a biofuel but are also useful chemicals with various industrial applications. The development of economically feasible and environment friendly bio-based fuels requires efficient cell factories capable of producing desired products in high yield. Herein, we sought to use a number of strategies to engineer Saccharomyces cerevisiae for high-level production of branched-chain esters. Mitochondrion-based expression of ATF1 gene in a base strain with an overexpressed valine biosynthetic pathway together with expression of mitochondrion-relocalized α-ketoacid decarboxylase (encoded by ARO10) and alcohol dehydrogenase (encoded by ADH7) not only produced isobutyl acetate, but also 3-methyl-1-butyl acetate and 2-methyl-1-butyl acetate. Further segmentation of the downstream esterification step into the cytosol to utilize the cytosolic acetyl-CoA pool for acetyltransferase (ATF)-mediated condensation enabled an additional fold improvement of ester productions. The best titre attained in the present study is 260.2mg/L isobutyl acetate, 296.1mg/L 3-methyl-1-butyl acetate and 289.6mg/L 2-methyl-1-butyl acetate. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. A novel selection system for chromosome translocations in Saccharomyces cerevisiae.

    PubMed Central

    Tennyson, Rachel B; Ebran, Nathalie; Herrera, Anissa E; Lindsley, Janet E

    2002-01-01

    Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors. While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements. This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae. A translocation YAC was created that contains the breakpoint cluster region from the human MLL gene, a gene frequently involved in translocations in leukemia patients, flanked by positive and negative selection markers. A translocation between the YAC and a yeast chromosome, whose breakpoint falls within the MLL DNA, physically separates the markers and forms the basis for the selection. When RAD52 is deleted, essentially all of the selected and screened cells contain simple translocations. The detectable translocation rates are the same in haploids and diploids, although the mechanisms involved and true translocation rates may be distinct. A unique double-strand break induced within the MLL sequences increases the number of detectable translocation events 100- to 1000-fold. This novel system provides a tractable assay for answering basic mechanistic questions about the development of chromosomal translocations. PMID:11973293

  13. Kem Mutations Affect Nuclear Fusion in Saccharomyces Cerevisiae

    PubMed Central

    Kim, J.; Ljungdahl, P. O.; Fink, G. R.

    1990-01-01

    We have identified mutations in three genes of Saccharomyces cerevisiae, KEM1, KEM2 and KEM3, that enhance the nuclear fusion defect of kar1-1 yeast during conjugation. The KEM1 and KEM3 genes are located on the left arm of chromosome VII. Kem mutations reduce nuclear fusion whether the kem and the kar1-1 mutations are in the same or in different parents (i.e., in both kem kar1-1 X wild-type and kem X kar1-1 crosses). kem1 X kem1 crosses show a defect in nuclear fusion, but kem1 X wild-type crosses do not. Mutant kem1 strains are hypersensitive to benomyl, lose chromosomes at a rate 10-20-fold higher than KEM(+) strains, and lose viability upon nitrogen starvation. In addition, kem1/kem1 diploids are unable to sporulate. Cells containing a kem1 null allele grow very poorly, have an elongated rod-shape and are defective in spindle pole body duplication and/or separation. The KEM1 gene, which is expressed as a 5.5-kb mRNA transcript, contains a 4.6-kb open reading frame encoding a 175-kD protein. PMID:2076815

  14. Respiratory Development in Saccharomyces cerevisiae Grown at Controlled Oxygen Tension

    PubMed Central

    Rogers, P. J.; Stewart, P. R.

    1973-01-01

    Saccharomyces cerevisiae was grown in batch culture over a wide range of oxygen concentrations, varying from the anaerobic condition to a maximal dissolved oxygen concentration of 3.5 μM. The development of cells was assayed by measuring amounts of the aerobic cytochromes aa3, b, c, and c1, the cellular content of unsaturated fatty acids and ergosterol, and the activity of respiratory enzyme complexes. The half-maximal levels of membrane-bound cytochromes aa3, b, and c1, were reached in cells grown in O2 concentrations around 0.1 μM; this was similar to the oxygen concentration required for half-maximal levels of unsaturated fatty acid and sterol. However, the synthesis of ubiquinone and cytochrome c and the increase in fumarase activity were essentially linear functions of the dissolved oxygen concentration up to 3.5 μM oxygen. The synthesis of the succinate dehydrogenase, succinate cytochrome c reductase, and cytochrome c oxidase complexes showed different responses to changes in O2 concentration in the growth medium. Cyanide-insensitive respiration and P450 cytochrome content were maximal at 0.25 μM oxygen and declined in both more anaerobic and aerobic conditions. Cytochrome c peroxidase and catalase activities in cell-free homogenates were high in all but the most strictly anaerobic cells. PMID:4352179

  15. Genetic Analysis of Default Mating Behavior in Saccharomyces Cerevisiae

    PubMed Central

    Dorer, R.; Boone, C.; Kimbrough, T.; Kim, J.; Hartwell, L. H.

    1997-01-01

    Haploid Saccharomyces cerevisiae cells find each other during conjugation by orienting their growth toward each other along pheromone gradients (chemotropism). However, when their receptors are saturated for pheromone binding, yeast cells must select a mate by executing a default pathway in which they choose a mating partner at random. We previously demonstrated that this default pathway requires the SPA2 gene. In this report we show that the default mating pathway also requires the AXL1, FUS1, FUS2, FUS3, PEA2, RVS161, and BNI1 genes. These genes, including SPA2, are also important for efficient cell fusion during chemotropic mating. Cells containing null mutations in these genes display defects in cell fusion that subtly affect mating efficiency. In addition, we found that the defect in default mating caused by mutations in SPA2 is partially suppressed by multiple copies of two genes, FUS2 and MFA2. These findings uncover a molecular relationship between default mating and cell fusion. Moreover, because axl1 mutants secrete reduced levels of a-factor and are defective at both cell fusion and default mating, these results reveal an important role for a-factor in cell fusion and default mating. We suggest that default mating places a more stringent requirement on some aspects of cell fusion than does chemotropic mating. PMID:9135999

  16. Biotransformation of mogrosides from Siraitia grosvenorii Swingle by Saccharomyces cerevisiae.

    PubMed

    Chiu, Chun-Hui; Wang, Reuben; Lee, Cho-Ching; Lo, Yi-Chen; Lu, Ting-Jang

    2013-07-24

    Mogrosides are a group of triterpenoidal saponins from the fruit of Siraitia grosvenorii Swingle; they are intensely sweet and have consequently been used as a substitute for sugar by the food industry. The lack of efficient methods to produce specific mogrosides has hindered investigation of the relationship between their structure and bioactivity, e.g., down-regulation of blood glucose levels, anti-inflammation, and antiviral infection. Here, we attempt to selectively convert the major saponin mogroside V, a mogrol pentaglucoside, into mogroside III E, a triglucoside, via the β-glucosidases of the budding yeast Saccharomyces cerevisiae. We report that the β-glucopyranosyl and β-glucopyranosyl-(1→2)-β-d-glucopyranosyl attached on C-3 and -24 of mogrol, respectively, were resistant to hydrolysis by yeast β-d-glucosidases. We further screened 16 mutants bearing single defective glucanase or glucosidase genes, thereby demonstrating that Exg1 is a major enzyme of the initiation of mogroside V conversion. Deletion of the KRE6 gene unexpectedly facilitated the production of mogroside III E in yeast culture. This paper demonstrates that yeast knockout mutants are a valuable tool for saponin modification and for studying the specificity of glucosidase function.

  17. Cell cycle regulation of homologous recombination in Saccharomyces cerevisiae.

    PubMed

    Mathiasen, David P; Lisby, Michael

    2014-03-01

    Homologous recombination (HR) contributes to maintaining genome integrity by facilitating error-free repair of DNA double-strand breaks (DSBs) primarily during the S and G2 phases of the mitotic cell cycle, while nonhomologous end joining (NHEJ) is the preferred pathway for DSB repair in G1 phase. The decision to repair a DSB by NHEJ or HR is made primarily at the level of DSB end resection, which is inhibited by the Ku complex in G1 and promoted by the Sae2 and Mre11 nucleases in S/G2 . The cell cycle regulation of HR is accomplished both at the transcription level and at the protein level through post-translational modification, degradation and subcellular localization. Cyclin-dependent kinase Cdc28 plays an established key role in these events, while the role of transcriptional regulation and protein degradation are less well understood. Here, the cell cycle regulatory mechanisms for mitotic HR in Saccharomyces cerevisiae are reviewed, and evolutionarily conserved principles are highlighted. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  18. d-Xylulose Fermentation to Ethanol by Saccharomyces cerevisiae.

    PubMed

    Chiang, L C; Gong, C S; Chen, L F; Tsao, G T

    1981-08-01

    We used commercial bakers' yeast (Saccharomyces cerevisiae) to study the conversion of d-xylulose to ethanol in the presence of d-xylose. The rate of ethanol production increased with an increase in yeast cell density. The optimal temperature for d-xylulose fermentation was 35 degrees C, and the optimal pH range was 4 to 6. The fermentation of d-xylulose by yeast resulted in the production of ethanol as the major product; small amounts of xylitol and glycerol were also produced. The production of xylitol was influenced by pH as well as temperature. High pH values and low temperatures enhanced xylitol production. The rate of d-xylulose fermentation decreased when the production of ethanol yielded concentrations of 4% or more. The slow conversion rate of d-xylulose to ethanol was increased by increasing the yeast cell density. The overall production of ethanol from d-xylulose by yeast cells under optimal conditions was 90% of the theoretical yield.

  19. Protective Effects of Arginine on Saccharomyces cerevisiae Against Ethanol Stress

    PubMed Central

    Cheng, Yanfei; Du, Zhaoli; Zhu, Hui; Guo, Xuena; He, Xiuping

    2016-01-01

    Yeast cells are challenged by various environmental stresses in the process of industrial fermentation. As the currently main organism for bio-ethanol production, Saccharomyces cerevisiae suffers from ethanol stress. Some amino acids have been reported to be related to yeast tolerance to stresses. Here the relationship between arginine and yeast response to ethanol stress was investigated. Marked inhibitions of ethanol on cell growth, expression of genes involved in arginine biosynthesis and intracellular accumulation of arginine were observed. Furthermore, extracellular addition of arginine can abate the ethanol damage largely. To further confirm the protective effects of arginine on yeast cells, yeast strains with different levels of arginine content were constructed by overexpression of ARG4 involved in arginine biosynthesis or CAR1 encoding arginase. Intracellular arginine was increased by 18.9% or 13.1% respectively by overexpression of ARG4 or disruption of CAR1, which enhanced yeast tolerance to ethanol stress. Moreover, a 41.1% decrease of intracellular arginine was observed in CAR1 overexpressing strain, which made yeast cells keenly sensitive to ethanol. Further investigations indicated that arginine protected yeast cells from ethanol damage by maintaining the integrity of cell wall and cytoplasma membrane, stabilizing the morphology and function of organellae due to low ROS generation. PMID:27507154

  20. Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

    PubMed Central

    Kron, S J; Styles, C A; Fink, G R

    1994-01-01

    Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape. Images PMID:7841518

  1. Sulfate assimilation mediates tellurite reduction and toxicity in Saccharomyces cerevisiae.

    PubMed

    Ottosson, Lars-Göran; Logg, Katarina; Ibstedt, Sebastian; Sunnerhagen, Per; Käll, Mikael; Blomberg, Anders; Warringer, Jonas

    2010-10-01

    Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism.

  2. Comparative Genomics of Saccharomyces cerevisiae Natural Isolates for Bioenergy Production

    PubMed Central

    Wohlbach, Dana J.; Rovinskiy, Nikolay; Lewis, Jeffrey A.; Sardi, Maria; Schackwitz, Wendy S.; Martin, Joel A.; Deshpande, Shweta; Daum, Christopher G.; Lipzen, Anna; Sato, Trey K.; Gasch, Audrey P.

    2014-01-01

    Lignocellulosic plant material is a viable source of biomass to produce alternative energy including ethanol and other biofuels. However, several factors—including toxic byproducts from biomass pretreatment and poor fermentation of xylose and other pentose sugars—currently limit the efficiency of microbial biofuel production. To begin to understand the genetic basis of desirable traits, we characterized three strains of Saccharomyces cerevisiae with robust growth in a pretreated lignocellulosic hydrolysate or tolerance to stress conditions relevant to industrial biofuel production, through genome and transcriptome sequencing analysis. All stress resistant strains were highly mosaic, suggesting that genetic admixture may contribute to novel allele combinations underlying these phenotypes. Strain-specific gene sets not found in the lab strain were functionally linked to the tolerances of particular strains. Furthermore, genes with signatures of evolutionary selection were enriched for functional categories important for stress resistance and included stress-responsive signaling factors. Comparison of the strains’ transcriptomic responses to heat and ethanol treatment—two stresses relevant to industrial bioethanol production—pointed to physiological processes that were related to particular stress resistance profiles. Many of the genotype-by-environment expression responses occurred at targets of transcription factors with signatures of positive selection, suggesting that these strains have undergone positive selection for stress tolerance. Our results generate new insights into potential mechanisms of tolerance to stresses relevant to biofuel production, including ethanol and heat, present a backdrop for further engineering, and provide glimpses into the natural variation of stress tolerance in wild yeast strains. PMID:25364804

  3. Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation

    PubMed Central

    Ha, Suk-Jin; Galazka, Jonathan M.; Rin Kim, Soo; Choi, Jin-Ho; Yang, Xiaomin; Seo, Jin-Ho; Louise Glass, N.; Cate, Jamie H. D.; Jin, Yong-Su

    2011-01-01

    The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily glucose and xylose. However, strains of Saccharomyces cerevisiae presently used in bioethanol production ferment glucose but not xylose. Yeasts engineered to ferment xylose do so slowly, and cannot utilize xylose until glucose is completely consumed. To overcome these bottlenecks, we engineered yeasts to coferment mixtures of xylose and cellobiose. In these yeast strains, hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular β-glucosidase following import by a high-affinity cellodextrin transporter. Intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation allowing coconsumption of cellobiose and xylose. The resulting yeast strains, cofermented cellobiose and xylose simultaneously and exhibited improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. We also observed improved yields and productivities from cofermentation experiments performed with simulated cellulosic hydrolyzates, suggesting this is a promising cofermentation strategy for cellulosic biofuel production. The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production. PMID:21187422

  4. Brazilian propolis protects Saccharomyces cerevisiae cells against oxidative stress

    PubMed Central

    de Sá, Rafael A.; de Castro, Frederico A.V.; Eleutherio, Elis C.A.; de Souza, Raquel M.; da Silva, Joaquim F.M.; Pereira, Marcos D.

    2013-01-01

    Propolis is a natural product widely used for humans. Due to its complex composition, a number of applications (antimicrobial, antiinflammatory, anesthetic, cytostatic and antioxidant) have been attributed to this substance. Using Saccharomyces cerevisiae as a eukaryotic model we investigated the mechanisms underlying the antioxidant effect of propolis from Guarapari against oxidative stress. Submitting a wild type (BY4741) and antioxidant deficient strains (ctt1Δ, sod1Δ, gsh1Δ, gtt1Δ and gtt2Δ) either to 15 mM menadione or to 2 mM hydrogen peroxide during 60 min, we observed that all strains, except the mutant sod1Δ, acquired tolerance when previously treated with 25 μg/mL of alcoholic propolis extract. Such a treatment reduced the levels of ROS generation and of lipid peroxidation, after oxidative stress. The increase in Cu/Zn-Sod activity by propolis suggests that the protection might be acting synergistically with Cu/Zn-Sod. PMID:24516431

  5. Protein disorder reduced in Saccharomyces cerevisiae to survive heat shock

    PubMed Central

    Vicedo, Esmeralda; Gasik, Zofia; Dong, Yu-An; Goldberg, Tatyana; Rost, Burkhard

    2015-01-01

    Recent experiments established that a culture of Saccharomyces cerevisiae (baker’s yeast) survives sudden high temperatures by specifically duplicating the entire chromosome III and two chromosomal fragments (from IV and XII). Heat shock proteins (HSPs) are not significantly over-abundant in the duplication. In contrast, we suggest a simple algorithm to “ postdict ” the experimental results: Find a small enough chromosome with minimal protein disorder and duplicate this region. This algorithm largely explains all observed duplications. In particular, all regions duplicated in the experiment reduced the overall content of protein disorder. The differential analysis of the functional makeup of the duplication remained inconclusive. Gene Ontology (GO) enrichment suggested over-representation in processes related to reproduction and nutrient uptake. Analyzing the protein-protein interaction network (PPI) revealed that few network-central proteins were duplicated. The predictive hypothesis hinges upon the concept of reducing proteins with long regions of disorder in order to become less sensitive to heat shock attack. PMID:26673203

  6. Hed1 Promotes Meiotic Crossover Formation in Saccharomyces cerevisiae.

    PubMed

    Kong, Yoon-Ju; Joo, Jeong-Hwan; Kim, Keun Pil; Hong, Soogil

    2017-02-28

    Homologous recombination occurs between homologous chromosomes and is significantly involved in programmed double-strand break (DSB) repair. Activation of two recombinases, Rad51 and Dmc1, is essential for an interhomolog bias during meiosis. Rad51 participates in both mitotic and meiotic recombination, and its strand exchange activity is regulated by an inhibitory factor during meiosis. Thus, activities of Rad51 and Dmc1 are coordinated to promote homolog bias. It has been reported that Hed1, a meiosis-specific protein in budding yeast, regulates Rad51-dependent recombination activity. Here, we investigated the role of Hed1 in meiotic recombination by ectopic expression of the protein after pre-meiotic replication in Saccharomyces cerevisiae. DNA physical analysis revealed that the overexpression of Hed1 delays the DSB-to-joint molecule (JM) transition and promotes interhomolog JM formation. The study indicates a possible role of Hed1 in controlling the strand exchange activity of Rad51 and, eventually, meiotic crossover formation.

  7. Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae

    SciTech Connect

    Dar, Roy D.; Karig, David K; Cooke, John F; Cox, Chris D.; Simpson, Michael L

    2010-01-01

    Stochasticity is an inherent feature of complex systems with nanoscale structure. In such systems information is represented by small collections of elements (e.g. a few electrons on a quantum dot), and small variations in the populations of these elements may lead to big uncertainties in the information. Unfortunately, little is known about how to work within this inherently noisy environment to design robust functionality into complex nanoscale systems. Here, we look to the biological cell as an intriguing model system where evolution has mediated the trade-offs between fluctuations and function, and in particular we look at the relationships and trade-offs between stochastic and deterministic responses in the gene expression of budding yeast (Saccharomyces cerevisiae). We find gene regulatory arrangements that control the stochastic and deterministic components of expression, and show that genes that have evolved to respond to stimuli (stress) in the most strongly deterministic way exhibit the most noise in the absence of the stimuli. We show that this relationship is consistent with a bursty 2-state model of gene expression, and demonstrate that this regulatory motif generates the most uncertainty in gene expression when there is the greatest uncertainty in the optimal level of gene expression.

  8. Calcium signaling and copper toxicity in Saccharomyces cerevisiae cells.

    PubMed

    Ruta, Lavinia L; Popa, Claudia V; Nicolau, Ioana; Farcasanu, Ileana C

    2016-12-01

    To respond to metal surpluses, cells have developed intricate ways of defense against the excessive metallic ions. To understand the ways in which cells sense the presence of toxic concentration in the environment, the role of Ca(2+) in mediating the cell response to high Cu(2+) was investigated in Saccharomyces cerevisiae cells. It was found that the cell exposure to high Cu(2+) was accompanied by elevations in cytosolic Ca(2+) with patterns that were influenced not only by Cu(2+) concentration but also by the oxidative state of the cell. When Ca(2+) channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca(2+) pool under Cu(2+) stress was the vacuolar Ca(2+) channel, Yvc1, also activated by the Cch1-mediated Ca(2+) influx. Using yeast mutants defective in the Cu(2+) transport across the plasma membrane, it was found that the Cu(2+)-dependent Ca(2+) elevation could correlate not only with the accumulated metal, but also with the overall oxidative status. Moreover, it was revealed that Cu(2+) and H2O2 acted in synergy to induce Ca(2+)-mediated responses to external stress.

  9. D-xylulose fermentation to ethanol by Saccharomyces cerevisiae

    SciTech Connect

    Chiang, L.C.; Gong, C.S.; Chen, L.F.; Tsao, G.T.

    1981-08-01

    Commercial bakers' yeast (Saccharomyces cerevisiae) was used to study the conversion of D-xylulose to ethanol in the presence of D-xylose. The rate of ethanol production increased with an increase in yeast cell density. The optimal temperature for D-xylulose fermentation was 35 degrees Celcius, and the optimal pH range was 4 to 6. The fermentation of D-xylulose by yeast resulted in the production of ethanol as the major product; small amounts of xylitol and glycerol were also produced. The production of xylitol was influenced by pH as well as temperature. High pH values and low temperatures enhanced xylitol production. The rate of D-xylulose fermentation decreased when the production of ethanol yielded concentrations of 4% or more. The slow conversion rate of D-xylulose to ethanol was increased by increasing the yeast cell density. The overall production of ethanol from D-xylulose by yeast cells under optimal conditions was 90% of the theoretical yield. (Refs. 21).

  10. Codon recognition during frameshift suppression in Saccharomyces cerevisiae.

    PubMed Central

    Gaber, R F; Culbertson, M R

    1984-01-01

    A genetic approach has been used to establish the molecular basis of 4-base codon recognition by frameshift suppressor tRNA containing an extra nucleotide in the anticodon. We have isolated all possible base substitution mutations at the position 4 (N) in the 3'-CCCN-5' anticodon of a Saccharomyces cerevisiae frameshift suppressor glycine tRNA encoded by the SUF16 gene. Base substitutions at +1 frameshift sites in the his4 gene have also been obtained such that all possible 4-base 5'-GGGN-3' codons have been identified. By testing for suppression in different strains that collectively represent all 16 possible combinations of position 4 nucleotides, we show that frameshift suppression does not require position 4 base pairing. Nonetheless, position 4 interactions influence the efficiency of suppression. Our results suggest a model in which 4-base translocation of mRNA on the ribosome is directed primarily by the number of nucleotides in the anticodon loop, whereas the resulting efficiency of suppression is dependent on the nature of position 4 nucleotides. Images PMID:6390183

  11. Genotoxicity assessment of amaranth and allura red using Saccharomyces cerevisiae.

    PubMed

    Jabeen, Hafiza Sumara; ur Rahman, Sajjad; Mahmood, Shahid; Anwer, Sadaf

    2013-01-01

    Amaranth (E123) and Allura red (E129), very important food azo dyes used in food, drug, paper, cosmetic and textile industries, were assessed for their genotoxic potential through comet assay in yeast cells. Comet assay was standardized by with different concentration of H(2)O(2). Concentrations of Amaranth and Allura red were maintained in sorbitol buffer starting from 9.76 to 5,000 μg/mL and 1 × 10(4) cells were incubated at two different incubation temperatures 28 and 37°C. Amaranth (E123) and Allura red (E129) were found to exhibit their genotoxic effect directly in Saccharomyces cerevisiae. No significant genotoxic activity was observed for Amaranth and Allura red at 28°C but at 37°C direct relation of Amaranth concentration with comet tail was significant and no positive relation was seen with time exposure factor. At 37°C the minimum concentration of Amaranth and Allura red at which significant DNA damage observed through comet assay was 1,250 μg/mL in 2nd h post exposure time. The results indicated that food colors should be carefully used in baking products as heavy concentration of food colors could affect the fermentation process of baking.

  12. Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae.

    PubMed

    Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian

    2014-10-01

    Natural products with anti-aging property have drawn great attention recently but examples of such compounds are exceedingly scarce. By applying a high-throughput assay based on yeast chronological lifespan measurement, we screened the anti-aging activity of 144 botanical materials and found that dried roots of Salvia miltiorrhiza Bunge have significant anti-aging activity. Tanshinones isolated from the plant including cryptotanshione, tanshinone I, and tanshinone IIa, are the active components. Among them, cryptotanshinone can greatly extend the budding yeast Saccharomyces cerevisiae chronological lifespan (up to 2.5 times) in a dose- and the-time-of-addition-dependent manner at nanomolar concentrations without disruption of cell growth. We demonstrate that cryptotanshinone prolong chronological lifespan via a nutrient-dependent regime, especially essential amino acid sensing, and three conserved protein kinases Tor1, Sch9, and Gcn2 are required for cryptotanshinone-induced lifespan extension. In addition, cryptotanshinone significantly increases the lifespan of SOD2-deleted mutants. Altogether, those data suggest that cryptotanshinone might be involved in the regulation of, Tor1, Sch9, Gcn2, and Sod2, these highly conserved longevity proteins modulated by nutrients from yeast to humans.

  13. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

    PubMed

    Krogan, Nevan J; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P; Punna, Thanuja; Peregrín-Alvarez, José M; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D; Paccanaro, Alberto; Bray, James E; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J; Gandi, Kiran; Thompson, Natalie J; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H Y; Butland, Gareth; Altaf-Ul, Amin M; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S; Ingles, C James; Hughes, Timothy R; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J; Emili, Andrew; Greenblatt, Jack F

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

  14. Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae.

    PubMed

    Yedidi, Ravikiran S; Fatehi, Amatullah K; Enenkel, Cordula

    The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence. [Formula: see text].

  15. Water-Transfer Slows Aging in Saccharomyces cerevisiae

    PubMed Central

    Cohen, Aviv; Weindling, Esther; Rabinovich, Efrat; Nachman, Iftach; Fuchs, Shai; Chuartzman, Silvia; Gal, Lihi; Schuldiner, Maya; Bar-Nun, Shoshana

    2016-01-01

    Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process. PMID:26862897

  16. In vivo Reconstitution of Algal Triacylglycerol Production in Saccharomyces cerevisiae

    PubMed Central

    Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

    2016-01-01

    The current fascination with algal biofuel production stems from a high lipid biosynthetic capacity and little conflict with land plant cultivation. However, the mechanisms which enable algae to accumulate massive oil remain elusive. An enzyme for triacylglycerol (TAG) biosynthesis in Chlamydomonas reinhardtii, CrDGTT2, can produce a large amount of TAG when expressed in yeast or higher plants, suggesting a unique ability of CrDGTT2 to enhance oil production in a heterologous system. Here, we performed metabolic engineering in Saccharomyces cerevisiae by taking advantage of CrDGTT2. We suppressed membrane phospholipid biosynthesis at the log phase by mutating OPI3, enhanced TAG biosynthetic pathway at the stationary phase by overexpressing PAH1 and CrDGTT2, and suppressed TAG hydrolysis on growth resumption from the stationary phase by knocking out DGK1. The resulting engineered yeast cells accumulated about 70-fold of TAG compared with wild type cells. Moreover, TAG production was sustainable. Our results demonstrated the enhanced and sustainable TAG production in the yeast synthetic platform. PMID:26913021

  17. Acetaldehyde addition throughout the growth phase alleviates the phenotypic effect of zinc deficiency in Saccharomyces cerevisiae.

    PubMed

    Cheraiti, Naoufel; Sauvage, François-Xavier; Salmon, Jean-Michel

    2008-01-01

    During experiments to determine the effects of exogenously added acetaldehyde on pure cultures of various yeast strains, we discovered that an early acetaldehyde perfusion during the growth phase allowed several yeasts to partially overcome the phenotypic effects of zinc depletion during alcoholic fermentation. We, therefore, performed genome-wide expression and proteomic analysis on an industrial Saccharomyces cerevisiae yeast strain (VL1) growing in zinc-replete or zinc-depleted conditions in the presence of perfused acetaldehyde to identify molecular markers of this effect. Zinc depletion severely affects ethanol production and therefore nicotinamide adenine dinucleotide (NAD) regeneration, although we observed partial compensation by the upregulation of the poorly efficient Fe-dependent Adh4p in our conditions. A coordinate metabolic response was indeed observed in response to the early acetaldehyde perfusion, and particularly of the lower part of glycolysis, leading to the cellular replenishment of NAD cofactor. These various findings suggest that acetaldehyde exchange between strains may inhibit the growth of some yeast strains while encouraging the growth of others. This phenomenon could be particularly important for understanding the ecology of colonization of complex fermentation media by S. cerevisiae after elimination of non-Saccharomyces yeasts.

  18. Functional Survey for Heterologous Sugar Transport Proteins, Using Saccharomyces cerevisiae as a Host▿

    PubMed Central

    Young, Eric; Poucher, Ashley; Comer, Austin; Bailey, Alexandra; Alper, Hal

    2011-01-01

    Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences. PMID:21421781

  19. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    PubMed

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

  20. RNAseq-based transcriptome comparison of Saccharomyces cerevisiae strains isolated from diverse fermentative environments.

    PubMed

    Ibáñez, Clara; Pérez-Torrado, Roberto; Morard, Miguel; Toft, Christina; Barrio, Eladio; Querol, Amparo

    2017-09-18

    Transcriptome analyses play a central role in unraveling the complexity of gene expression regulation in Saccharomyces cerevisiae. This species, one of the most important microorganisms for humans given its industrial applications, shows an astonishing degree of genetic and phenotypic variability among different strains adapted to specific environments. In order to gain novel insights into the Saccharomyces cerevisiae biology of strains adapted to different fermentative environments, we analyzed the whole transcriptome of three strains isolated from wine, flor wine or mezcal fermentations. An RNA-seq transcriptome comparison of the different yeasts in the samples obtained during synthetic must fermentation highlighted the differences observed in the genes that encode mannoproteins, and in those involved in aroma, sugar transport, glycerol and alcohol metabolism, which are important under alcoholic fermentation conditions. These differences were also observed in the physiology of the strains after mannoprotein and aroma determinations. This study offers an essential foundation for understanding how gene expression variations contribute to the fermentation differences of the strains adapted to unequal fermentative environments. Such knowledge is crucial to make improvements in fermentation processes and to define targets for the genetic improvement or selection of wine yeasts. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Cocoa (Theobroma cacao) Polyphenol-rich Extract Increases the Chronological Lifespan of Saccharomyces cerevisiae.

    PubMed

    Baiges, I; Arola, L

    2016-01-01

    Saccharomyces cerevisiae is a model organism with conserved aging pathways. Yeast chronological lifespan experiments mimic the processes involved in human non-dividing tissues, such as the nervous system or skeletal muscle, and can speed up the search for biomolecules with potential anti-aging effects before proceeding to animal studies. To test the effectiveness of a cocoa polyphenol-rich extract (CPE) in expanding the S. cerevisiae chronological lifespan in two conditions: in the stationary phase reached after glucose depletion and under severe caloric restriction. Using a high-throughput method, wild-type S. cerevisiae and its mitochondrial manganese-dependent superoxide dismutase null mutant (sod2Δ) were cultured in synthetic complete dextrose medium. After 2 days, 0, 5 and 20 mg/ml of CPE were added, and viability was measured throughout the stationary phase. The effects of the major components of CPE were also evaluated. To determine yeast lifespan under severe caloric restriction conditions, cultures were washed with water 24 h after the addition of 0 and 20 mg/ml of CPE, and viability was followed over time. CPE increased the chronological lifespan of S. cerevisiae during the stationary phase in a dose-dependent manner. A similar increase was also observed in (sod2Δ). None of the major CPE components (theobromine, caffeine, maltodextrin, (-)-epicatechin, (+)-catechin and procyanidin B2) was able to increase the yeast lifespan. CPE further increased the yeast lifespan under severe caloric restriction. CPE increases the chronological lifespan of S. cerevisiae through a SOD2-independent mechanism. The extract also extends yeast lifespan under severe caloric restriction conditions. The high-throughput assay used makes it possible to simply and rapidly test the efficacy of a large number of compounds on yeast aging, requiring only small amounts, and is thus a convenient screening assay to accelerate the search for biomolecules with potential anti-aging effects.

  2. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture.

  3. Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis.

    PubMed

    Pericolini, Eva; Gabrielli, Elena; Ballet, Nathalie; Sabbatini, Samuele; Roselletti, Elena; Cayzeele Decherf, Amélie; Pélerin, Fanny; Luciano, Eugenio; Perito, Stefano; Jüsten, Peter; Vecchiarelli, Anna

    2017-01-02

    Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection.

  4. Biodiversity of Saccharomyces cerevisiae isolated from a survey of pito production sites in various parts of Ghana.

    PubMed

    Glover, Richard L K; Abaidoo, Robert C; Jakobsen, Mogens; Jespersen, Lene

    2005-10-01

    Biodiversity among Saccharomyces cerevisiae predominating the spontaneous fermentation of Dagarti pito in Ghana was assessed. Two hundred and forty-nine isolates obtained from samples of dried yeast taken from commercial pito production sites in eight geographical regions of Ghana were characterized phenotypically by colony and cell morphology as well as carbohydrate assimilation profiling. Yeast populations ranged between 10(6) and 10(8) cfug(-1). Ninety-nine percent of the isolates (247) investigated showed macro-and micro morphological characteristics typical of S. cerevisiae. Of these, 72% (179) had assimilation profiles similar to S. cerevisiae while 28% (68) had assimilation profiles atypical of S. cerevisiae or any other member of the Saccharomyces sensu stricto complex. Amplification of the region spanning the two intergenic transcribed spacers (ITS) and the 5.8S ribosomal gene (ITS1-5.8S rDNA-ITS2), followed by restriction analysis, as well as determination of chromosome length polymorphism by pulsed field gel electrophoresis (PFGE) of 25 representative isolates strongly indicated that all belonged to S. cerevisiae, notwithstanding the phenotypic differences. Sequencing of the mitochondrial cytochrome-c oxidase II gene (COX 2) and the actin-encoding gene (ACT1) of four isolates, confirmed their close relatedness to S. cerevisiae, particularly to the type strain CBS1171 (98.7%), as well as other members of the Saccharomyces sensu stricto complex. Twenty isolates selected from eight geographical regions of Ghana and investigated for their technological properties, showed different patterns of growth and flocculation but otherwise similar technological characteristica. Most of the isolates produced pito having sensory attributes, which compared favourably with commercially produced pito.

  5. Quantitative comparison of transient growth of Saccharomyces cerevisiae, Saccharomyces kluyveri, and Kluyveromyces lactis.

    PubMed

    Herwig, Christoph; Von Stockar, Urs

    2003-03-30

    A multitude of metabolic regulations occur in yeast, particularly under dynamic process conditions, such as under sudden glucose excess. However, quantification of regulations and classification of yeast strains under these conditions have yet to be elucidated, which requires high-frequency and consistent quantification of the metabolic response. The present study aimed at quantifying the dynamic regulation of the central metabolism of strains Saccharomyces cerevisiae, S. kluyveri, and Kluyveromyces lactis upon sudden glucose excess, accomplished by a shift-up in dilution rate inside of the oxidative region using a small metabolic flux model. It was found that, under transient growth conditions, S. kluyveri behaved like K. lactis, while classification using steady-state conditions would position S. kluyveri close to S. cerevisiae. For transient conditions and based on the observation whether excess glucose is initially used for catabolism (energy) or anabolism (carbon), we propose to classify strains into energy-driven, such as S. cerevisiae, and carbon-driven, such as S. kluyveri and K. lactis, strains. Furthermore, it was found that the delayed onset of fermentative catabolism in carbon-driven strains is a consequence of low catabolic flux and the initial shunt of glucose in non-nitrogen-containing biomass constituents. The MFA model suggests that energy limitation forced the cell to ultimately increase catabolic flux, while the capacity of oxidative catabolism is not sufficient to process this flux oxidatively. The combination of transient experiments and its exploitation with reconciled intrinsic rates using a small metabolic model could corroborate earlier findings of metabolic regulations, such as tight glucose control in carbon-driven strains and transient changes in biomass composition, as well as explore new regulations, such as assimilation of ethanol before glucose. The benefit from using small metabolic flux models is the richness of information and the

  6. Depletion of Saccharomyces cerevisiae in psoriasis patients, restored by Dimethylfumarate therapy (DMF).

    PubMed

    Eppinga, Hester; Thio, H Bing; Schreurs, Marco W J; Blakaj, Blerdi; Tahitu, Ruena I; Konstantinov, Sergey R; Peppelenbosch, Maikel P; Fuhler, Gwenny M

    2017-01-01

    Psoriasis and inflammatory bowel disease (IBD) are chronic inflammatory diseases sharing similar pathogenic pathways. Intestinal microbial changes such as a decrease of bakers' yeast Saccharomyces cerevisiae have been reported in IBD, suggesting the presence of a gut-skin axis. To investigate whether the S. cerevisiae abundance was altered in psoriasis patients versus healthy controls, and whether dimethylfumarate (DMF) interacted with this yeast. Using qPCR, faecal samples were compared between psoriasis patients without DMF (n = 30), psoriasis patients with DMF (n = 28), and healthy controls (n = 32). Faecal S. cerevisiae abundance was decreased in psoriasis compared to healthy controls (p<0.001). Interestingly, DMF use raised S. cerevisiae levels (p<0.001). Gastrointestinal adverse-effects of DMF were correlated with a higher S. cerevisiae abundance (p = 0.010). In vitro, a direct effect of DMF on S. cerevisiae growth was observed. In addition, anti-Saccharomyces cerevisiae antibodies were not elevated in psoriasis. The abundance of baker's yeast S. cerevisiae is decreased in psoriasis patients, but appears to be restored upon DMF use. S. cerevisiae is generally classified as a yeast with beneficial immunomodulatory properties, but may also be involved in the occurrence of DMF's gastrointestinal adverse-effects. Potentially, DMF might be a new therapy for IBD.

  7. Impact of oxygenation on the performance of three non-Saccharomyces yeasts in co-fermentation with Saccharomyces cerevisiae.

    PubMed

    Shekhawat, Kirti; Bauer, Florian F; Setati, Mathabatha E

    2017-03-01

    The sequential or co-inoculation of grape must with non-Saccharomyces yeast species and Saccharomyces cerevisiae wine yeast strains has recently become a common practice in winemaking. The procedure intends to enhance unique aroma and flavor profiles of wine. The extent of the impact of non-Saccharomyces strains depends on their ability to produce biomass and to remain metabolically active for a sufficiently long period. However, mixed-culture wine fermentations tend to become rapidly dominated by S. cerevisiae, reducing or eliminating the non-Saccharomyces yeast contribution. For an efficient application of these yeasts, it is therefore essential to understand the environmental factors that modulate the population dynamics of such ecosystems. Several environmental parameters have been shown to influence population dynamics, but their specific effect remains largely uncharacterized. In this study, the population dynamics in co-fermentations of S. cerevisiae and three non-Saccharomyces yeast species: Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima, was investigated as a function of oxygen availability. In all cases, oxygen availability strongly influenced population dynamics, but clear species-dependent differences were observed. Our data show that L. thermotolerans required the least oxygen, followed by T. delbrueckii and M. pulcherrima. Distinct species-specific chemical volatile profiles correlated in all cases with increased persistence of non-Saccharomyces yeasts, in particular increases in some higher alcohols and medium chain fatty acids. The results highlight the role of oxygen in regulating the succession of yeasts during wine fermentations and suggests that more stringent aeration strategies would be necessary to support the persistence of non-Saccharomyces yeasts in real must fermentations.

  8. Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. Results We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6–7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. Conclusions We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography. PMID:22694812

  9. Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

    SciTech Connect

    Steen, EricJ.; Chan, Rossana; Prasad, Nilu; Myers, Samuel; Petzold, Christopher; Redding, Alyssa; Ouellet, Mario; Keasling, JayD.

    <