Sample records for saccharomyces cerevisiae encodes

  1. Saccharomyces cerevisiae

    Bojsen, Rasmus K; Andersen, Kaj Scherz; Regenberg, Birgitte


    Microbial biofilms can be defined as multi-cellular aggregates adhering to a surface and embedded in an extracellular matrix (ECM). The nonpathogenic yeast, Saccharomyces cerevisiae, follows the common traits of microbial biofilms with cell-cell and cell-surface adhesion. S. cerevisiae is shown...... pathways including the protein kinase A and a mitogen-activated protein kinase pathway. Advanced genetic tools and resources have been developed for S. cerevisiae including a deletion mutant-strain collection in a biofilm-forming strain background and GFP-fusion protein collections. Furthermore, S....... cerevisiae biofilm is well applied for confocal laser scanning microscopy and fluorophore tagging of proteins, DNA and RNA. These techniques can be used to uncover the molecular mechanisms for biofilm development, drug resistance and for the study of molecular interactions, cell response to environmental...

  2. Regulation of Expression of GLT1, the Gene Encoding Glutamate Synthase in Saccharomyces cerevisiae

    Valenzuela, Lourdes; Ballario, Paola; Aranda, Cristina; Filetici, Patrizia; González, Alicia


    Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whethe...

  3. GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae.

    Avendaño, A; DeLuna, A.; Olivera, H; Valenzuela, L.; A. Gonzalez


    It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP+-glutamate dehydrogenase (NADP+-GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP+-GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to ...

  4. The Saccharomyces cerevisiae ICL2 Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism

    Luttik, Marijke A.H.; Kötter, Peter; Salomons, Florian A.; Klei, Ida J. van der; Dijken, Johannes P. van; Pronk, Jack T.


    The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologous ICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies

  5. PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

    Carter, Andrew T.; Beiche, Flora; Hove-Jensen, Bjarne;


    In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS......) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has...... a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed...

  6. AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae.

    Aouida, Mustapha; Leduc, Anick; Poulin, Richard; Ramotar, Dindial


    Polyamines play essential functions in many aspects of cell biology. Plasma membrane transport systems for the specific uptake of polyamines exist in most eukaryotic cells but have been very recently identified at the molecular level only in the parasite Leishmania. We now report that the high affinity polyamine permease in Saccharomyces cerevisiae is identical to Agp2p, a member of the yeast amino acid transporter family that was previously identified as a carnitine transporter. Deletion of AGP2 dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines, and transformation with an AGP2 expression vector restored polyamine transport in agp2delta mutants. Yeast mutants deficient in polyamine biosynthesis required >10-fold higher concentrations of exogenous putrescine to restore cell proliferation upon deletion of the AGP2 gene. Disruption of END3, a gene required for an early step of endocytosis, increased the abundance of Agp2p, an effect that was paralleled by a marked up-regulation of spermidine transport velocity. Thus, AGP2 encodes the first eukaryotic permease that preferentially uses spermidine over putrescine as a high affinity substrate and plays a central role in the uptake of polyamines in yeast.

  7. The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae.

    Noskov, V; Maki, S.; Kawasaki, Y.; Leem, S H; Ono, B; Araki, H; Pavlov, Y; Sugino, A


    Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a def...

  8. Co-expression of a Saccharomyces diastaticus glucoamylase-encoding gene and a Bacillus amyloliquefaciens alpha-amylase-encoding gene in Saccharomyces cerevisiae.

    Steyn, A J; Pretorius, I S


    A glucoamylase-encoding gene (STA2) from Saccharomyces diastaticus and an alpha-amylase-encoding gene (AMY) from Bacillus amyloliquefaciens were cloned separately into a yeast-integrating shuttle vector (YIp5), generating recombinant plasmids pSP1 and pSP2, respectively. The STA2 and AMY genes were jointly cloned into YIp5, generating plasmid pSP3. Subsequently, the dominant selectable marker APH1, encoding resistance to Geneticin G418 (GtR), was cloned into pSP3, resulting in pSP4. For enhanced expression of GtR, the APH1 gene was fused to the GAL10 promoter and terminated by the URA3 terminator, resulting in pSP5. Plasmid pSP5 was converted to a circular minichromosome (pSP6) by the addition of the ARS1 and CEN4 sequences. Laboratory strains of Saccharomyces cerevisiae transformed with plasmids pSP1 through pSP6, stably produced and secreted glucoamylase and/or alpha-amylase. Brewers' and distillers' yeast transformed with pSP6 were also capable of secreting amylolytic enzymes. Yeast transformants containing pSP1, pSP2 and pSP3 assimilated soluble starch with an efficiency of 69%, 84% and 93%, respectively. The major starch hydrolysis products produced by crude amylolytic enzymes found in the culture broths of the pSP1-, pSP2- and pSP3-containing transformants, were glucose, glucose and maltose (1:1), and glucose and maltose (3:1), respectively. These results confirmed that co-expression of the STA2 and AMY genes synergistically enhanced starch degradation.

  9. Regulation of expression of GLT1, the gene encoding glutamate synthase in Saccharomyces cerevisiae.

    Valenzuela, L; Ballario, P; Aranda, C; Filetici, P; González, A


    Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression. A collection of increasingly larger 5' deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation. The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5' deletion derivatives. Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.

  10. Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1.

    Tishkoff, D X; Johnson, A W; Kolodner, R D


    Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is

  11. Deletion of the Saccharomyces cerevisiae ARO8 gene, encoding an aromatic amino acid transaminase, enhances phenylethanol production from glucose.

    Romagnoli, Gabriele; Knijnenburg, Theo A; Liti, Gianni; Louis, Edward J; Pronk, Jack T; Daran, Jean-Marc


    Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products.

  12. GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae.

    Avendaño, A; Deluna, A; Olivera, H; Valenzuela, L; Gonzalez, A


    It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP+-glutamate dehydrogenase (NADP+-GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP+-GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.

  13. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo


    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

  14. In silicio search for genes encoding peroxisomal proteins in Saccharomyces cerevisiae.

    Kal, A J; Hettema, E H; van den Berg, M; Koerkamp, M G; van Ijlst, L; Distel, B; Tabak, H F


    The biogenesis of peroxisomes involves the synthesis of new proteins that after, completion of translation, are targeted to the organelle by virtue of peroxisomal targeting signals (PTS). Two types of PTSs have been well characterized for import of matrix proteins (PTS1 and PTS2). Induction of the genes encoding these matrix proteins takes place in oleate-containing medium and is mediated via an oleate response element (ORE) present in the region preceding these genes. The authors have searched the yeast genome for OREs preceding open reading frames (ORFs), and for ORFs that contain either a PTS1 or PTS2. Of the ORFs containing an ORE, as well as either a PTS1 or a PTS2, many were known to encode bona fide peroxisomal matrix proteins. In addition, candidate genes were identified as encoding putative new peroxisomal proteins. For one case, subcellular location studies validated the in silicio prediction. This gene encodes a new peroxisomal thioesterase.

  15. The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.

    Teste, M A; Enjalbert, B; Parrou, J L; François, J M


    The YPR184w gene encodes a 1536-amino acid protein that is 34-39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme. The N-terminal part of the protein possesses the four conserved sequences of the alpha-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes. Reliable measurement of alpha-1,4-glucanotransferase and alpha-1, 6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w. The alpha-1, 4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl. Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown. In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

  16. Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

    Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles


    NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.

  17. levadura Saccharomyces Cerevisiae

    B. Aguilar Uscanga


    Full Text Available La pared celular de levaduras representa entre 20 a 30 % de la célula en peso seco. Está compuesta de polisacáridos complejos de β-glucanos, manoproteínas y quitina. Se estudió la composición de los polisacáridos contenidos en la pared celular de la Saccharomyces cerevisiae CEN.PK 113 y se observó el efecto de la variación de la fuente carbono (glucosa, sacarosa, galactosa, maltosa, manosa, etanol y pH (3, 4, 5, 6 en un medio mineral “cell factory”. Las células fueron recolectadas en fase exponencial y se extrajo la pared celular. Los extractos de pared se hidrolizaron con H2SO4 al 72% y las muestras fueron analizadas por cromatografía HPLC. Se realizó una prueba de resistencia al rompimiento celular con una β(1,3-glucanasa, y las células cultivadas a diferentes fuentes carbono y pH. Los resultados del análisis por HPLC, mostraron que la composición de los polisacáridos en la pared celular, varía considerablemente con las modificaciones del medio de cultivo. Se observó que las levaduras cultivadas en sacarosa tienen mayor porcentaje de pared celular (25% y mayor cantidad de glucanos (115µg/mg peso seco y mananos (131µg/mg peso seco, que aquellas levaduras cultivadas en etanol (13% en peso seco. Las levaduras cultivadas a pH 5 presentaron 19% de pared celular en peso seco, mientras que a pH 6 el porcentaje fue menor (14%. El análisis de resistencia al rompimiento celular, mostró que las células cultivadas en etanol y galactosa fueron resistentes al rompimiento enzimático. Se comparó este resultado con el contenido de polisacáridos en la pared celular y concluimos que la resistencia de la célula al rompimiento, no está ligada con la cantidad de β-glucanos contenidos en la pared celular, sino que va a depender del número de enlaces β(1,3 y β(1,6-glucanos, los cuales juegan un rol importante durante el ensamblaje de la pared

  18. Proteomics of Saccharomyces cerevisiae Organelles

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


    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 wi

  19. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Samuelsson, T; Olsson, M


    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  20. Fungal genomics beyond Saccharomyces cerevisiae?

    Hofmann, Gerald; Mcintyre, Mhairi; Nielsen, Jens


    Fungi are used extensively in both fundamental research and industrial applications. Saccharomyces cerevisiae has been the model organism for fungal research for many years, particularly in functional genomics. However, considering the diversity within the fungal kingdom, it is obvious that the a......Fungi are used extensively in both fundamental research and industrial applications. Saccharomyces cerevisiae has been the model organism for fungal research for many years, particularly in functional genomics. However, considering the diversity within the fungal kingdom, it is obvious...... that the application of the existing methods of genome, transcriptome, proteome and metabolome analysis to other fungi has enormous potential, especially for the production of food and food ingredients. The developments in the past year demonstrate that we have only just started to exploit this potential....

  1. Glucose repression in Saccharomyces cerevisiae

    Kayikci, Omur; Nielsen, Jens


    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 gluc......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...

  2. GUP1 of Saccharomyces cerevisiae encodes an O-acyltransferase involved in remodeling of the GPI anchor.

    Bosson, Régine; Jaquenoud, Malika; Conzelmann, Andreas


    The anchors of mature glycosylphosphatidylinositol (GPI)-anchored proteins of Saccharomyces cerevisiae contain either ceramide or diacylglycerol with a C26:0 fatty acid in the sn2 position. The primary GPI lipid added to newly synthesized proteins in the ER consists of diacylglycerol with conventional C16 and C18 fatty acids. Here we show that GUP1 is essential for the synthesis of the C26:0-containing diacylglycerol anchors. Gup1p is an ER membrane protein with multiple membrane-spanning domains harboring a motif that is characteristic of membrane-bound O-acyl-transferases (MBOAT). Gup1Delta cells make normal amounts of GPI proteins but most mature GPI anchors contain lyso-phosphatidylinositol, and others possess phosphatidylinositol with conventional C16 and C18 fatty acids. The incorporation of the normal ceramides into the anchors is also disturbed. As a consequence, the ER-to-Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. Gup1Delta cells have fragile cell walls and a defect in bipolar bud site selection. GUP1 function depends on the active site histidine of the MBOAT motif. GUP1 is highly conserved among fungi and protozoa and the gup1Delta phenotype is partially corrected by GUP1 homologues of Aspergillus fumigatus and Trypanosoma cruzi.

  3. New insights into trehalose metabolism by Saccharomyces cerevisiae: NTH2 encodes a functional cytosolic trehalase, and deletion of TPS1 reveals Ath1p-dependent trehalose mobilization.

    Jules, Matthieu; Beltran, Gemma; François, Jean; Parrou, Jean Luc


    In the yeast Saccharomyces cerevisiae, the synthesis of endogenous trehalose is catalyzed by a trehalose synthase complex, TPS, and its hydrolysis relies on a cytosolic/neutral trehalase encoded by NTH1. In this work, we showed that NTH2, a paralog of NTH1, encodes a functional trehalase that is implicated in trehalose mobilization. Yeast is also endowed with an acid trehalase encoded by ATH1 and an H+/trehalose transporter encoded by AGT1, which can together sustain assimilation of exogenous trehalose. We showed that a tps1 mutant defective in the TPS catalytic subunit cultivated on trehalose, or on a dual source of carbon made of galactose and trehalose, accumulated high levels of intracellular trehalose by its Agt1p-mediated transport. The accumulated disaccharide was mobilized as soon as cells entered the stationary phase by a process requiring a coupling between its export and immediate extracellular hydrolysis by Ath1p. Compared to what is seen for classical growth conditions on glucose, this mobilization was rather unique, since it took place prior to that of glycogen, which was postponed until the late stationary phase. However, when the Ath1p-dependent mobilization of trehalose identified in this study was impaired, glycogen was mobilized earlier and faster, indicating a fine-tuning control in carbon storage management during periods of carbon and energy restriction.

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


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 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...

  5. Viruses and prions of Saccharomyces cerevisiae.

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


    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.

  6. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek;


    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...

  7. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    Nevoigt, Elke


    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...

  8. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    Hove-Jensen, Bjarne


    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  9. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

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


    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.

  10. Effects of pentamidine isethionate on Saccharomyces cerevisiae.

    Ludewig, G.; Williams, J M; Li, Y.; Staben, C


    We used Saccharomyces cerevisiae as a model system in which to examine the mechanism of action of the anti-Pneumocystis drug pentamidine. Pentamidine at low concentrations inhibited S. cerevisiae growth on nonfermentable carbon sources (50% inhibitory concentration [IC50] of 1.25 micrograms/ml in glycerol). Pentamidine inhibited growth on fermentable energy sources only at much higher concentrations (IC50 of 250 micrograms/ml in glucose). Inhibition at low pentamidine concentrations in glycer...

  11. Flocculation of Saccharomyces cerevisiae tup1 mutants.


    Strains of Saccharomyces cerevisiae carrying a mutation in the TUP1 locus exhibited calcium-dependent flocculation. The flocculation had none of the characteristics of sexual agglutination. The flocculation differed from that exhibited by a FLO1 strain in the effect of pH on cation dependence and sensitivity to chemical inactivation.

  12. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

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


    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. Nitrogen Catabolite Repression in Saccharomyces cerevisiae

    Hofman-Bang, H Jacob Peider


    In Saccharomyces cerevisiae the expression of all known nitrogen catabolite pathways are regulated by four regulators known as Gln3, Gat1, Da180, and Deh1. This is known as nitrogen catabolite repression (NCR). They bind to motifs in the promoter region to the consensus sequence S' GATAA 3'. Gln3...

  14. Deletion of PHO13, encoding haloacid dehalogenase type IIA phosphatase, results in upregulation of the pentose phosphate pathway in Saccharomyces cerevisiae.

    Kim, Soo Rin; Xu, Haiqing; Lesmana, Anastashia; Kuzmanovic, Uros; Au, Matthew; Florencia, Clarissa; Oh, Eun Joong; Zhang, Guochang; Kim, Kyoung Heon; Jin, Yong-Su


    The haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes in Saccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identified PHO13 to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion of PHO13 (pho13Δ), we investigated the response of S. cerevisiae to pho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed that pho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced by pho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus, pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation by pho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function of S. cerevisiae Pho13 and the HAD superfamily enzymes and for developing S. cerevisiae strains with industrially attractive phenotypes.

  15. Metabolic Engineering of Ammonium Assimilation in Xylose-Fermenting Saccharomyces cerevisiae Improves Ethanol Production

    Roca, Christophe; Nielsen, Jens; Olsson, Lisbeth


    Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from...

  16. Characteristics of sterol uptake in Saccharomyces cerevisiae.

    Lorenz, R T; Rodriguez, R J; Lewis, T A; Parks, L W


    A Saccharomyces cerevisiae sterol auxotroph, FY3 (alpha hem1 erg7 ura), was used to probe the characteristics of sterol uptake in S. cerevisiae. The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol. When cultured on 1 microgram of sterol ml-1, the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were te...

  17. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen


    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed.

  18. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

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


    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.

  19. Origin of Endogenous DNA Abasic Sites in Saccharomyces cerevisiae


    Abasic (AP) sites are among the most frequent endogenous lesions in DNA and present a strong block to replication. In Saccharomyces cerevisiae, an apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3′-blocked single-strand breaks (M. Guillet and S. Boiteux, EMBO J. 21:2833, 2002). Here, we investigated the origin of endogenous AP sites in yeast. Our results show that the deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the...

  20. Investigation of autonomous cell cycle oscillation in Saccharomyces cerevisiae

    Hansen, Morten Skov


    Autonome Oscillationer i kontinuert kultivering af Saccharomyces cerevisiae Udgangspunktet for dette Ph.d. projekt var at søge at forstå, hvad der gør det muligt at opnå multiple statiske tilstande ved kontinuert kultivering af Saccharomyces cerevisiae med glukose som begrænsende substrat...

  1. Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae

    Hansen, M.K.; Tams, J.W.; Fahrenkrug, Jan;


    G protein-coupled receptor; heterologous expression; membrane protein; Saccharomyces cerevisiae, vasoactive intestinal polypeptide; yeast mating factor-pre-pro *Ga-leader peptide......G protein-coupled receptor; heterologous expression; membrane protein; Saccharomyces cerevisiae, vasoactive intestinal polypeptide; yeast mating factor-pre-pro *Ga-leader peptide...

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

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


    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.

  3. Isocitrate lyase localisation in Saccharomyces cerevisiae cells.

    Chaves, R S; Herrero, P; Ordiz, I; Angeles del Brio, M; Moreno, F


    The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm. This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation. This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted.

  4. [Expression of inulinase genes in the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus].

    Sokolenko, G G; Karpechenko, N A


    Expression of the genes encoding the enzymes involved in inulin, sucrose, and glucose metabolism in the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus was studied. The exon-intron structure of the relevant genes was identified and the primers for quantitative PCR were optimized. Expression of the genes was found to depend on the carbon source. Glucose was shown to exhibit a repressive effect on inulinase synthesis by K. marxianus, while in S. cerevisiae glucose and sucrose were inulinase inducer and repressor, respectively.

  5. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.

    Elena Fossati

    Full Text Available 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.

  6. Saccharomyces cerevisiae metabolism in ecological context

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


    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

  7. Expression of the rgMT gene, encoding for a rice metallothionein-like protein in Saccharomyces cerevisiae and Arabidopsis thaliana

    Shumei Jin; Dan Sun; Ji Wang; Ying Li; Xinwang Wang; Shenkui Liu


    Metallothioneins (MTs) are cysteine-rich proteins of low molecular weight with many attributed functions, such as providing protection against metal toxicity, being involved in regulation of metal ions uptake that can impact plant physiology and providing protection against oxidative stress. However, the precise function of the metallothionein-like proteins such as the one coded for rgMT gene isolated from rice (Oryza sativa L.) is not completely understood. The whole genome analysis of rice (O. sativa) showed that the rgMT gene is homologue to the Os11g47809 on chromosome 11 of O. sativa sp. japonica genome. This study used the rgMT coding sequence to create transgenic lines to investigate the subcellular localization of the protein, as well as the impact of gene expression in yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana under heavy metal ion, salt and oxidative stresses. The results indicate that the rgMT gene was expressed in the cytoplasm of transgenic cells. Yeast cells transgenic for rgMT showed vigorous growth compared to the nontransgenic controls when exposed to 7mM CuCl2, 10 mM FeCl2, 1 M NaCl, 24 mM NaHCO3 and 3.2 mM H2O2, but there was no significant difference for other stresses tested. Similarly, Arabidopsis transgenic for rgMT displayed significantly improved seed germination rates over that of the control when the seeds were stressed with 100 M CuCl2 or 1 mM H2O2. Increased biomass was observed in the presence of 100 M CuCl2, 220 M FeCl2, 3 mM Na2CO3, 5 mM NaHCO3 or 1 mM H2O2. These results indicate that the expression of the rice rgMT gene in transgenic yeast and Arabidopsis is implicated in improving their tolerance for certain salt and peroxide stressors.

  8. The origin recognition complex links replication, sister chromatid cohesion and transcriptional silencing in Saccharomyces cerevisiae

    Suter, Bernhard; Tong, Amy; Chang, Michael; Yu, Lisa; Brown, Grant W; Boone, Charles; Rine, Jasper


    Mutations in genes encoding the origin recognition complex (ORC) of Saccharomyces cerevisiae affect initiation of DNA replication and transcriptional repression at the silent mating-type loci. To explore the function of ORC in more detail, a screen for genetic interactions was undertaken using large

  9. Genome-wide transcriptional response of a Saccharomyces cerevisiae strain with an altered redox metabolism

    Bro, Christoffer; Regenberg, Birgitte; Nielsen, Jens


    The genome-wide transcriptional response of a Saccharomyces cerevisiae strain deleted in GDH1 that encodes a NADP(+)-dependent glutamate dehydrogenase was compared to a wild-type strain under anaerobic steady-state conditions. The GDH1-deleted strain has a significantly reduced NADPH requirement...


    We have sequenced the structural gene and flanking regions for lanosterol 14 alpha-demethylase (14DM) from Saccharomyces cerevisiae. An open reading frame of 530 codons encodes a 60.7-kDa protein. When this gene is disrupted by integrative transformation, the resulting strain req...

  11. Molecular Basis for Saccharomyces cerevisiae Biofilm Development

    Andersen, Kaj Scherz

    of translation of FLO11. In conclusion, I have conducted the first global study of the genetic program for yeast biofilm formation on polystyrene. This work provide several target genes as good basis for further research of biofilm, that I believe can contribute to fields such as cell biology, genetics, system......In this study, I sought to identify genes regulating the global molecular program for development of sessile multicellular communities, also known as biofilm, of the eukaryotic microorganism, Saccharomyces cerevisiae (yeast). Yeast biofilm has a clinical interest, as biofilms can cause chronic......, but only a small subset is previously described as regulators of FLO11. These results reveal that the regulation of biofilm formation and FLO11 is even more complex than what has previously been described. I find that the molecular program for biofilm formation shares many essential components with two...

  12. Synchronization of the Budding Yeast Saccharomyces cerevisiae.

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


    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.

  13. Adaption of Saccharomyces cerevisiae expressing a heterologous protein

    Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars;


    Production of the heterologous protein, bovine aprotinin, in Saccharomyces cerevisiae was shown to affect the metabolism of the host cell to various extent depending on the strain genotype. Strains with different genotypes, industrial and laboroatory, respectively, were investigated. The maximal...

  14. Response of Saccharomyces cerevisiae to cadmium stress

    Moreira, Luciana Mara Costa; Ribeiro, Frederico Haddad; Neves, Maria Jose [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia], e-mail:; Porto, Barbara Abranches Araujo; Amaral, Angela M.; Menezes, Maria Angela B.C. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Lab. de Ativacao Neutronica], e-mail:; Rosa, Carlos Augusto [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Microbiologia], e-mail: carlrosa@icb.ufmg


    The intensification of industrial activity has been greatly contributing with the increase of heavy metals in the environment. Among these heavy metals, cadmium becomes a serious pervasive environmental pollutant. The cadmium is a heavy metal with no biological function, very toxic and carcinogenic at low concentrations. The toxicity of cadmium and several other metals can be mainly attributed to the multiplicity of coordination complexes and clusters that they can form. Some aspects of the cellular response to cadmium were extensively investigated in the yeast Saccharomyces cerevisiae. The primary site of interaction between many toxic metals and microbial cells is the plasma membrane. Plasma-membrane permeabilisation has been reported in a variety of microorganisms following cadmium exposure, and is considered one mechanism of cadmium toxicity in the yeast. In this work, using the yeast strain S. cerevisiae W303-WT, we have investigated the relationships between Cd uptake and release of cellular metal ions (K{sup +} and Na{sup +}) using neutron activation technique. The neutron activation was an easy, rapid and suitable technique for doing these metal determinations on yeast cells; was observed the change in morphology of the strains during the process of Cd accumulation, these alterations were observed by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) during incorporation of cadmium. (author)

  15. Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

    Serge Feyder


    Full Text Available 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.

  16. Membrane trafficking in the yeast Saccharomyces cerevisiae model.

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


    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.

  17. Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network

    Förster, Jochen; Famili, I.; Fu, P.;


    The metabolic network in the yeast Saccharomyces cerevisiae was reconstructed using currently available genomic, biochemical, and physiological information. The metabolic reactions were compartmentalized between the cytosol and the mitochondria, and transport steps between the compartments...... containing 1175 metabolic reactions and 584 metabolites. The number of gene functions included in the reconstructed network corresponds to similar to16% of all characterized ORFs in S. cerevisiae. Using the reconstructed network, the metabolic capabilities of S. cerevisiae were calculated and compared...

  18. Expression of the Saccharomyces cerevisiae DNA repair gene RAD6 that encodes a ubiquitin conjugating enzyme, increases in response to DNA damage and in meiosis but remains constant during the mitotic cell cycle.

    Madura, K; Prakash, S; Prakash, L


    The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating (E2) enzyme and is required for the repair of damaged DNA, mutagenesis, and sporulation. Here, we report our studies on the regulation of RAD6 gene expression after UV damage, during the mitotic cell cycle, in meiosis, and following heat shock and starvation. RAD6 mRNA levels became elevated in cells exposed to UV light, and at all UV doses the increase in mRNA levels was rapid and occurred within 30 min after exposure to UV. RAD6 mRNA levels also increased in sporulating MATa/MAT alpha cells, and the period of maximal accumulation of RAD6 mRNA during meiosis is coincident with the time during which recombination occurs. However, RAD6 mRNA levels showed no periodic fluctuation in the mitotic cell cycle, were not elevated upon heat shock, and fell in cells in the stationary phase of growth. These observations suggest that RAD6 activity is required throughout the cell cycle rather than being restricted to a specific stage, and that during meiosis, high levels of RAD6 activity may be needed at a stage coincident with genetic recombination. The observation that RAD6 transcription is not induced by heat and starvation, treatments that activate stress responses, suggests that the primary role of RAD6 is in the repair of damaged DNA rather than in adapting cells to stress situations.

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

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


    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.

  20. Metabolic engineering of Saccharomyces cerevisiae to improve succinic acid production based on metabolic profiling.

    Ito, Yuma; Hirasawa, Takashi; Shimizu, Hiroshi


    We performed metabolic engineering on the budding yeast Saccharomyces cerevisiae for enhanced production of succinic acid. Aerobic succinic acid production in S. cerevisiae was achieved by disrupting the SDH1 and SDH2 genes, which encode the catalytic subunits of succinic acid dehydrogenase. Increased succinic acid production was achieved by eliminating the ethanol biosynthesis pathways. Metabolic profiling analysis revealed that succinic acid accumulated intracellularly following disruption of the SDH1 and SDH2 genes, which suggests that enhancing the export of intracellular succinic acid outside of cells increases succinic acid production in S. cerevisiae. The mae1 gene encoding the Schizosaccharomyces pombe malic acid transporter was introduced into S. cerevisiae, and as a result, succinic acid production was successfully improved. Metabolic profiling analysis is useful in producing chemicals for metabolic engineering of microorganisms.

  1. Functional profiling of the Saccharomyces cerevisiae genome.

    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


    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.

  2. Arsenate and phosphate interaction in Saccharomyces cerevisiae

    GENG Chun-nu; ZHU Yong-guan


    In the present study, arsenate(As(Ⅴ)) and phosphate(P(Ⅴ)) interactions were investigated in growth, uptake and RNA content in yeast(Saccharomyces cerevisiae). Yeast grew slowly with As(Ⅴ) concentrations increasing in the medium. However, the maximal population density was almost the same among different As(Ⅴ) treatments. It was in the late log phase that yeast growth was augmented by low As(Ⅴ), which was maybe due to the fact that methionine metabolism was stressed by vitamin B6 deprivation, so As(Ⅴ)treatments did not affect maximal population density. However, with P (Ⅴ) concentrations increasing, the maximal population density increased. Therefore, the maximal population density was determined by P (Ⅴ) concentrations in the medium but not by As (Ⅴ)concentrations in the medium. Ycf1p(a tonoplast transpor) transports As(GS)3 into the vacuole, but arsenic(As) remaining in the thalli was 1.27% with As(Ⅴ) exposure for 60 h, from which it can be speculated that the percentage of As transported into vacuole should be lower than 1.27%. However, the percentage of As pumped out of cell was 71.49% with As (Ⅴ) exposure for 68 h. Although two pathways (extrusion and sequestration) were involved in As detoxification in yeast, the extrusion pathway played a major role in As detoxification. RNA content was the highest in the early-log phase and was reduced by As(Ⅴ).

  3. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae.

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


    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.

  4. Redox balancing in recombinant strains of Saccharomyces cerevisiae

    Anderlund, M.


    In metabolically engineered Saccharomyces cerevisiae expressing Pichia stipitis XYL1 and XYL2 genes, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, xylitol is excreted as the major product during anaerobic xylose fermentation and only low yields of ethanol are produced. This has been interpreted as a result of the dual cofactor dependence of XR and the exclusive use of NAD{sup +} by XDH. The excretion of xylitol was completely stopped and the formation of glycerol and acetic acid were reduced in xylose utilising S. cerevisiae strains cultivated in oxygen-limited conditions by expressing lower levels of XR than of XDH. The expression level of XYL1 and XYL2 were controlled by changing the promoters and transcription directions of the genes. A new functional metabolic pathway was established when Thermus thermophilus xylA gene was expressed in S. cerevisiae. The recombinant strain was able to ferment xylose to ethanol when cultivated on a minimal medium containing xylose as only carbon source. In order to create a channeled metabolic transfer in the two first steps of the xylose metabolism, XYL1 and XYL2 were fused in-frame and expressed in S. cerevisiae. When the fusion protein, containing a linker of three amino acids, was co expressed together with native XR and XDH monomers, enzyme complexes consisting of chimeric and native subunits were formed. The total activity of these complexes exhibited 10 and 9 times higher XR and XDH activity, respectively, than the original conjugates, consisting of only chimeric subunits. This strain produced less xylitol and the xylitol yield was lower than with strains only expressing native XR and XDH monomers. In addition, more ethanol and less acetic acid were formed. A new gene encoding the cytoplasmic transhydrogenase from Azotobacter vinelandii was cloned. The enzyme showed high similarity to the family of pyridine nucleotide-disulphide oxidoreductase. To analyse the physiological effect of

  5. Removing cadmium from electroplating wastewater by waste saccharomyces cerevisiae

    DAI Shu-juan; WEI De-zhou; ZHOU Dong-qin; JIA Chun-yun; WANG Yu-juan; LIU Wen-gang


    The appropriate condition and scheme of removing cadmium from electroplating wastewater were investigated by adsorption-precipitation method using waste saccharomyces cerevisiae(WSC) as sorbent. Effect factors on biosorption of cadmium in cadmium-containing electroplating wastewater by waste saccharomyces cerevisiae and precipitation process of waste saccharomyces cerevisiae after adsorbing cadmium were studied. The results show that removal rate of cadmium is over 88% after 30 min adsorbing under the condition of cadmium concentration 26 mg/L, the dosage of waste saccharomyces cerevisiae 16.25 g/L, temperature 18 ℃, pH 6.0 and precipitation time 4 h. Biosorption-precipitation method is effective to remove cadmium in cadmium-containing electroplating wastewater by waste saccharomyces cerevisiae. The SEM, infrared spectroscopy and Zeta-potential of the cells show that chemical chelating is the main adsorption form; electrostatic attraction, hydrogen bonding and van der Waals force all function in adsorption process; and ―NH2―,―C=O―,―C=O―NH―,―CH3, ―OH are the main adsorption groups.

  6. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    Villadsen, Ingrid


    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... many genes and gene products from higher eukaryotes in Saccharomyces cerevisiae because many genes and biological processes are homologous or similar in lower and in higher eukaryotes. The highly developed methods of genetics and molecular biology greatly facilitates studies of higher eukaryotic...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...

  7. Switching the mode of sucrose utilization by Saccharomyces cerevisiae

    Miletti Luiz C


    Full Text Available Abstract 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

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

    Rødkaer, Steven V; Færgeman, Nils J.


    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....

  9. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.


    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a ...

  10. Structure and functional analysis of the multistress response gene DDR2 from Saccharomyces cerevisiae.

    Kobayashi, N; McClanahan, T K; Simon, J R; Treger, J M; McEntee, K


    The DDR2 gene is a multistress response gene in Saccharomyces cerevisiae that is transcriptionally activated by more than thirteen xenobiotic agents and environmental or physiological stresses. The DDR2 gene encodes a small hydrophobic 61 amino acid polypeptide located on chromosome XV adjacent to the SPE2 locus. Disruption alleles of the DDR2 gene have been constructed and these ddr2 delta mutants show no defect in heat shock recovery or thermotolerance and appear normal for DNA damage sensitivity and mutagenesis.

  11. Metabolically engineered Saccharomyces cerevisiae for branched-chain ester productions.

    Yuan, Jifeng; Mishra, Pranjul; Ching, Chi Bun


    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.

  12. Phytochelatins are synthesized by two vacuolar serine carboxypeptidases in Saccharomyces cerevisiae.

    Wünschmann, Jana; Beck, Andreas; Meyer, Laurent; Letzel, Thomas; Grill, Erwin; Lendzian, Klaus J


    Phytochelatins (PCs) are cysteine-rich peptides that chelate heavy metal ions, thereby mediating heavy metal tolerance in plants, fission yeast, and Caenorhabditis elegans. They are synthesized from glutathione by PC synthase, a specific dipeptidyltransferase. While Saccharomyces cerevisiae synthesizes PCs upon exposure to heavy metal ions, the S. cerevisiae genome does not encode a PC synthase homologue. How PCs are synthesized in yeast is unclear. This study shows that the vacuolar serine carboxypeptidases CPY and CPC are responsible for PC synthesis in yeast. The finding of a PCS-like activity of these enzymes in vivo discloses another route for PC biosynthesis in eukaryotes.

  13. Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

    Ramos, W; Tappe, N; Talamantez, J; Friedberg, E C; Tomkinson, A E


    Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joi...

  14. Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

    Penttilä Merja


    Full Text Available Abstract 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

  15. Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

    Vemuri, Goutham; Eiteman, M.A; McEwen, J.E;


    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...... 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...... NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria....

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

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


    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.

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

    Deutch, Charles E.; Marshall, Pamela A.


    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…

  18. The enantioselective b-keto ester reductions by Saccharomyces cerevisiae



    The enantioselective yeast reduction of aromatic b-keto esters, by use of potassium dihydrogen phosphate, calcium phosphate (monobasic), magnesium sulfate and ammonium tartrate (diammonium salt) (10:1:1:50) in water at pH 7 as a buffer for 72–120 h with 45–90 % conversion to the corresponding aromatic -hydroxy esters was achieved by means of Saccharomyces cerevisiae.

  19. Adsorption and Interfacial Electron Transfer of Saccharomyces Cerevisiae

    Hansen, Allan Glargaard; Boisen, Anja; Nielsen, Jens Ulrik;


    We have studied the adsorption and electron-transfer dynamics of Saccharomyces cerevisiae (yeast) iso-l-cytochrome c adsorbed on Au(lll) electrodes in aqueous phosphate buffer media. This cytochrome possesses a thiol group dos e to the protein surface (Cysl02) suitable for linking the protein...

  20. Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae

    Vernis, L.; Piskur, Jure; Diffley, J.F.X.


    The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well...

  1. The Plasma Membrane of Saccharomyces cerevisiae : Structure, Function, and Biogenesis



    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 extens

  2. Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae

    Solis Escalante, D.


    Glycolysis, a biochemical pathway that oxidizes glucose to pyruvate, is at the core of sugar metabolism in Saccharomyces cerevisiae (bakers’ yeast). Glycolysis is not only a catabolic route involved in energy conservation, but also provides building blocks for anabolism. From an applied perspective,

  3. Potential immobilized Saccharomyces cerevisiae as heavy metal removal

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


    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.

  4. Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae.

    DeLuna, Alexander; Quezada, Héctor; Gómez-Puyou, Armando; González, Alicia


    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.

  5. Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production.

    Roca, Christophe; Nielsen, Jens; Olsson, Lisbeth


    Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol(-1), mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.

  6. Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption

    Scalcinati, Gionata; Otero, José Manuel; Van Vleet, Jennifer R. H.;


    Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose...... sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon......-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S. cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling...

  7. Development of a system for multicopy gene integration in Saccharomyces cerevisiae.

    Semkiv, Marta V; Dmytruk, Kostyantyn V; Sibirny, Andriy A


    In this study we describe construction and evaluation of a vector for multicopy integration in yeast Saccharomyces cerevisiae. In this vector a modified selective marker and a reporter gene PHO8 (encoding alkaline phosphatase) were flanked with delta sequences of the Ty1 transposon. Modified by error-prone PCR version of selection marker kanMX4 was obtained from Escherichia coli clone with impaired geneticin (G418) resistance. The attenuation of kanMX4 gene provides an opportunity to select for explicitly multicopy integration of the module in S. cerevisiae using moderate (200 mg L(-1)) antibiotic concentrations. The developed system provided integration of 3-10 copies of the module in the genome of S. cerevisiae. High copy integration events were confirmed by qRT-PCR, Southern hybridization and reporter enzyme activity measurements.

  8. Understanding the 3-hydroxypropionic acid tolerance mechanism in Saccharomyces cerevisiae

    Kildegaard, Kanchana Rueksomtawin; Juncker, Agnieszka; Hallstrom, Bjorn;


    a sustainable alternative for production of acrylic acid from renewable feedstocks. We are establishing Saccharomyces cerevisiae as an alternative host for 3HP production. However, 3HP also inhibits yeast grow th at level well below what is desired for commercial applications. Therefore, we are aiming...... to improve 3HP tolerance in S. cerevisiae by applying adaptive evolution approach. We have generated yeast strains with sign ificantly improved capacity for tolerating 3HP when compared to the wild-type. We will present physiolo gical characterization, genome re-sequencing, and transcriptome analysis...

  9. Expression and secretion of Aspergillus niger glucoamylase in Saccharomyces cerevisiae

    李文清; 何鸣; 罗进贤


    Aspergillus niger glucoamylase GA 1 cDNA was inserted in between the yeast PGK promoter and terminator on plasmid pMA91. The resultant plasmid pMAG69 was introduced into Saccharomyces cerevisiae GRF18 by protoplast transformation. The A niger GA I cDNA was expressed efficiently under the contiol of PGK promoter and 99% of the gene products were secreted into the culture medium using its own signal sequence The recombmant yeast can digest 87% of starch in 2 d in the medium containing 10% starch. The recombinant plasmid pMAG69 can exist stably in 5. cerevisiae.

  10. Genetic mapping of Ty elements in Saccharomyces cerevisiae.

    Klein, H L; Petes, T. D.


    We used transformation to insert a selectable marker at various sites in the Saccharomyces cerevisiae genome occupied by the transposable element Ty. The vector CV9 contains the LEU2+ gene and a portion of the repeated element Ty1-17. Transformation with this plasmid resulted in integration of the vector via a reciprocal exchange using homology at the LEU2 locus or at the various Ty elements that are dispersed throughout the S. cerevisiae genome. These transformants were used to map genetical...

  11. Saccharomyces cerevisiae: a versatile eukaryotic system in virology

    Breinig Tanja


    Full Text Available Abstract The yeast Saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. Less known is its value for virus research, an area in which Saccharomyces cerevisiae has proven to be very fruitful as well. The present review will discuss the main achievements of yeast-based studies in basic and applied virus research. These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production.

  12. Investigation of nutrient sensing in the yeast Saccharomyces cerevisiae

    Eckert-Boulet, Nadine


    Gæren Saccharomyces cerevisiae har udviklet komplekse regulatoriske systemer til at kontrollere ekspression af de proteiner, der importerer næringsstoffer, således at disse kun bliver produceret, når der er brug for dem. Dette er tilfældet for hexose-transportører samt aminosyre-transportører (di......Gæren Saccharomyces cerevisiae har udviklet komplekse regulatoriske systemer til at kontrollere ekspression af de proteiner, der importerer næringsstoffer, således at disse kun bliver produceret, når der er brug for dem. Dette er tilfældet for hexose-transportører samt aminosyre...

  13. Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

    Verwaal, R.; Jiang, Y.; Wang, J.; Daran, J.M.; Sandmann, G.; Berg, van den J.A.; Ooyen, van A.J.J.


    To obtain insight into the genome-wide transcriptional response of heterologous carotenoid production in Saccharomyces cerevisiae, the transcriptome of two different S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous grown in carbon-limited chemosta

  14. YPA: an integrated repository of promoter features in Saccharomyces cerevisiae


    This study presents the Yeast Promoter Atlas (YPA, or database, which aims to collect comprehensive promoter features in Saccharomyces cerevisiae. YPA integrates nine kinds of promoter features including promoter sequences, genes’ transcription boundaries—transcription start sites (TSSs), five prime untranslated regions (5′-UTRs) and three prime untranslated regions (3′UTRs), TATA boxes, transcription factor binding sites (TFBSs), nucleo...

  15. Cellular Memory of Acquired Stress Resistance in Saccharomyces cerevisiae

    Guan, Qiaoning; Haroon, Suraiya; Bravo, Diego González; Will, Jessica L.; Gasch, Audrey P.


    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 H2O2. We set out to characterize ...

  16. Sucrose And Saccharomyces Cerevisiae: A Relationship Most Sweet.

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


    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 f...

  17. Interaction of Saccharomyces cerevisiae with gold: toxicity and accumulation.

    Karamushka, V I; Gadd, G M


    This paper examines the effects of ionic gold on Saccharomyces cerevisiae, as determined by long-term (growth in gold-containing media) and short-term interactions (H+ efflux activity). An increasing gold concentration inhibited growth and at gold concentration used. Both Ca and Mg enhanced the inhibitory effect of gold on the yeast cells with Ca showing a stronger inhibitory effect than Mg.

  18. The enantioselective b-keto ester reductions by Saccharomyces cerevisiae



    Full Text Available The enantioselective yeast reduction of aromatic b-keto esters, by use of potassium dihydrogen phosphate, calcium phosphate (monobasic, magnesium sulfate and ammonium tartrate (diammonium salt (10:1:1:50 in water at pH 7 as a buffer for 72–120 h with 45–90 % conversion to the corresponding aromatic -hydroxy esters was achieved by means of Saccharomyces cerevisiae.

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

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


    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 dehydro...

  20. Calcium dependence of Eugenol tolerance and toxicity in Saccharomyces cerevisiae

    Stephen K Roberts; Martin McAinsh; Hanna Cantopher; Sean Sandison


    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. ...

  1. Antiproliferative effects of Matricaria chamomilla on Saccharomyces cerevisiae

    Hosseinpour Maryam; Mobini-Dehkordi Mohsen; Saffar Behnaz; Teimori Hossein


    Introduction: The Matricaria chamomilla plant is one of the most important plants used for the therapeutic purposes. More than 120 chemical constituents have been identified in Matricaria chamomile plant including 28 terpenoids and 36 flavonoids. This plant has a variety of therapeutic applications including the treatment of diabetes, eczema, wounds and gastrointestinal diseases. The Saccharomyces cerevisiae yeast is a non-pathogenic organism that is used as a model for pathogenic yeasts in o...

  2. Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

    D'Amore, T; C.J. Panchal; Stewart, G G


    An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar af...

  3. Influence of dough freezing on Saccharomyces cerevisiae metabolism

    Pejin Dušanka J.; Došanović Irena S.; Popov Stevan D.; Suturović Zvonimir J.; Ranković Jovana A.; Dodić Siniša N.; Dodić Jelena M.; Vučurović Vesna M.


    The need to freeze dough is increasing in bakery production. Frozen dough can be stored for a long time without quality change. The capacity of bakery production can be increased in this way, and in the same time, the night shifts can be decreased. Yeast cells can be damaged by freezing process resulting in poor technological quality of dough after defrostation (longer fermentation of dough). The influence of frozen storage time of dough on survival percentage of Saccharomyces cerevisiae was ...

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

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


    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.

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

    Chunxiao eWang


    Full Text Available 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.

  6. Influence of organic acids and organochlorinated insecticides on metabolism of Saccharomyces cerevisiae

    Pejin Dušanka J.


    Full Text Available Saccharomyces cerevisiae is exposed to different stress factors during the production: osmotic, temperature, oxidative. The response to these stresses is the adaptive mechanism of cells. The raw materials Saccharomyces cerevisiae is produced from, contain metabolism products of present microorganisms and protective agents used during the growth of sugar beet for example the influence of acetic and butyric acid and organochlorinated insecticides, lindan and heptachlor, on the metabolism of Saccharomyces cerevisiae was investigated and presented in this work. The mentioned compounds affect negatively the specific growth rate, yield, content of proteins, phosphorus, total ribonucleic acids. These compounds influence the increase of trechalose and glycogen content in the Saccharomyces cerevisiae cells.

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

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


    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.

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

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina


    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

  9. Evidence of Natural Hybridization in Brazilian Wild Lineages of Saccharomyces cerevisiae.

    Barbosa, Raquel; Almeida, Pedro; Safar, Silvana V B; Santos, Renata Oliveira; Morais, Paula B; Nielly-Thibault, Lou; Leducq, Jean-Baptiste; Landry, Christian R; Gonçalves, Paula; Rosa, Carlos A; Sampaio, José Paulo


    The natural biology of Saccharomyces cerevisiae, the best known unicellular model eukaryote, remains poorly documented and understood although recent progress has started to change this situation. Studies carried out recently in the Northern Hemisphere revealed the existence of wild populations associated with oak trees in North America, Asia, and in the Mediterranean region. However, in spite of these advances, the global distribution of natural populations of S. cerevisiae, especially in regions were oaks and other members of the Fagaceae are absent, is not well understood. Here we investigate the occurrence of S. cerevisiae in Brazil, a tropical region where oaks and other Fagaceae are absent. We report a candidate natural habitat of S. cerevisiae in South America and, using whole-genome data, we uncover new lineages that appear to have as closest relatives the wild populations found in North America and Japan. A population structure analysis revealed the penetration of the wine genotype into the wild Brazilian population, a first observation of the impact of domesticated microbe lineages on the genetic structure of wild populations. Unexpectedly, the Brazilian population shows conspicuous evidence of hybridization with an American population of Saccharomyces paradoxus. Introgressions from S. paradoxus were significantly enriched in genes encoding secondary active transmembrane transporters. We hypothesize that hybridization in tropical wild lineages may have facilitated the habitat transition accompanying the colonization of the tropical ecosystem.

  10. Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae

    Bergman, Alexandra; Siewers, Verena; Nielsen, Jens;


    Phosphoketolases catalyze an energy-and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does...... C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some...... demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could...

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

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


    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.

  12. Transcriptome-Based Characterization of Interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in Lactose-Grown Chemostat Cocultures

    Mendes, F.; Sieuwerts, S.; De Hulster, E.; Almering, M.J.; Luttik, M.A.; Pronk, J.T.; Smid, E.J.; Bron, P.A.; Daran-Lapujade, P.


    Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaric

  13. Transcriptome-based characterization of interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in lactose-grown chemostat cocultures

    Mendes, F.; Sieuwerts, S.; Hulster, de E.; Almering, M.J.; Luttik, M.A.H.; Pronk, J.T.; Smid, E.J.; Baron, P.A.; Daran-Lapujade, P.


    Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaric

  14. Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

    Milne, N; Luttik, M A H; Cueto Rojas, H F; Wahl, A; van Maris, A J A; Pronk, J T; Daran, J M


    In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae.

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

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


    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.

  16. Saccharomyces cerevisiae as a starter culture in Mycella.

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


    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.

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

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


    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.

  18. Integration of Transcriptional and Posttranslational Regulation in a Glucose Signal Transduction Pathway in Saccharomyces cerevisiae

    Kim, Jeong-Ho; Brachet, Valérie; Moriya, Hisao; Johnston, Mark


    Expression of the HXT genes encoding glucose transporters in the budding yeast Saccharomyces cerevisiae is regulated by two interconnected glucose-signaling pathways: the Snf3/Rgt2-Rgt1 glucose induction pathway and the Snf1-Mig1 glucose repression pathway. The Snf3 and Rgt2 glucose sensors in the membrane generate a signal in the presence of glucose that inhibits the functions of Std1 and Mth1, paralogous proteins that regulate the function of the Rgt1 transcription factor, which binds to th...

  19. Cloning and Characterization of a Sulfonate/α-Ketoglutarate Dioxygenase from Saccharomyces cerevisiae

    Hogan, Deborah A; Auchtung, Thomas A.; Hausinger, Robert P.


    The Saccharomyces cerevisiae open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to Escherichia coli taurine/α-ketoglutarate dioxygenase and 27% identity to Ralstonia eutropha TfdA, a herbicide-degrading enzyme. Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/α-ketoglutarate dioxygenase. Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates being is...

  20. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T


    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification.

  1. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...


    ... Saccharomyces cerevisiae: exemption from the requirement of a tolerance. 180.1246 Section 180.1246 Protection of... Saccharomyces cerevisiae: exemption from the requirement of a tolerance. This regulation establishes an... Hydrolysate from Saccharomyces cerevisiae on all food commodities when applied/used for the management...

  2. Overproduction of fatty acids in engineered Saccharomyces cerevisiae.

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


    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.

  3. Antiproliferative effects of Matricaria chamomilla on Saccharomyces cerevisiae

    Hosseinpour Maryam


    Full Text Available Introduction: The Matricaria chamomilla plant is one of the most important plants used for the therapeutic purposes. More than 120 chemical constituents have been identified in Matricaria chamomile plant including 28 terpenoids and 36 flavonoids. This plant has a variety of therapeutic applications including the treatment of diabetes, eczema, wounds and gastrointestinal diseases. The Saccharomyces cerevisiae yeast is a non-pathogenic organism that is used as a model for pathogenic yeasts in order to identify compounds with antifungal properties and also to identify functional mechanism of these compounds. The aim of this study is to investigate the antifungal effect of Matricaria chamomilla hydroalcoholic extract on S. cerevisiae yeast. Methods: In this study Matricaria chamomilla extract was prepared by maceration method. In order to study the extract effect on growth and survival rate of the yeast cell, the spectrophotometry and methylene blue staining methods were used. Excel and SPSS 11 softwares were used to determine amounts and to infer the difference between control and treatment samples. Results: Results obtained from spectrophotometry and analyses of methylene blue staining showed that the Matricaria chamomilla extract at the concentration of 3000 μg/ml caused a significant decrease in the yeast growth and reduced the cells survival rate up to 48% (p< 0.05. Conclusion: Results of this research confirm that the hydroalcoholic extract of Matricaria chamomilla has antiproliferative effect on Saccharomyces cerevisiae.

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

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


    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).

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

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


    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.

  6. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

    Hou, Jin; Österlund, Tobias; Liu, Zihe


    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

  7. Purification of fluorescently labeled Saccharomyces cerevisiae Spindle Pole Bodies

    Davis, Trisha N.


    Centrosomes are components of the mitotic spindle responsible for organizing microtubules and establishing a bipolar spindle for accurate chromosome segregation. In budding yeast, Saccharomyces cerevisiae, the centrosome is called the spindle pole body, a highly organized tri-laminar structure embedded in the nuclear envelope. Here we describe a detailed protocol for the purification of fluorescently labeled spindle pole bodes from S. cerevisiae. Spindle pole bodies are purified from yeast using a TAP-tag purification followed by velocity sedimentation. This highly reproducible TAP-tag purification method improves upon previous techniques and expands the scope of in vitro characterization of yeast spindle pole bodies. The genetic flexibility of this technique allows for the study of spindle pole body mutants as well as the study of spindle pole bodies during different stages of the cell cycle. The ease and reproducibility of the technique makes it possible to study spindle pole bodies using a variety of biochemical, biophysical, and microscopic techniques. PMID:27193850

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

    Fernandez, N; Puente, P; Leal, F


    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.

  9. Magnetically altered ethanol fermentation capacity of Saccharomyces cerevisiae

    Galonja-Corghill Tamara


    Full Text Available We studied the effect of static magnetic fields on ethanol production by yeast Saccharomyces cerevisiae 424A (LNH-ST using sugar cane molasses during the fermentation in an enclosed bioreactor. Two static NdFeB magnets were attached to a cylindrical tube reactor with their opposite poles (north to south, creating 150 mT magnetic field inside the reactor. Comparable differences emerged between the results of these two experimental conditions. We found ethanol productivity to be 15% higher in the samples exposed to 150 mT magnetic field.

  10. Nitrogen catabolite repression of asparaginase II in Saccharomyces cerevisiae.

    Dunlop, P C; Meyer, G M; Roon, R J


    The biosynthesis of asparaginase II in Saccharomyces cerevisiae is subject to strong catabolite repression by a variety of nitrogen compounds. In the present study, asparaginase II synthesis was examined in a wild-type yeast strain and in strains carrying gdhA, gdhCR, or gdhCS mutations. The following effects were observed: (i) In the wild-type strain, the biosynthesis of asparaginase II was strongly repressed when either 10 mM ammonium sulfate or various amino acids (10 mM) served as the sou...

  11. Adsorption and Interfacial Electron Transfer of Saccharomyces Cerevisiae

    Andersen, Jens Enevold Thanulov


    We have studied the adsorption and electron-transfer dynamics of Saccharomyces cerevisiae (yeast) iso-l-cytochrome c adsorbed on Au(lll) electrodes in aqueous phosphate buffer media. This cytochrome possesses a thiol group dos e to the protein surface (Cysl02) suitable for linking the protein...... negative ofthe equilibrium potential of YCC, where the protein is electrochemically functional. The MCS data show tensile differential stress signals when YCC is adsorbed on a gold-coate d MCS, with distinguishable adsorption phases in the time range from

  12. Differential repair of UV damage in Saccharomyces cerevisiae.

    Terleth, C; van Sluis, C A; van de Putte, P


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

  13. Metabolic engineering of Saccharomyces cerevisiae for optimizing 3HP production

    Jensen, Niels Bjerg; Maury, Jerome; Oberg, Fredrik;


    and the market for acrylate products exceeds USD 100 billion. As an alternative to oil and gas derived acrylic acid, 3-hydroxypropionic (3HP) acid produced from renewable sources is highly desired, because 3HP can easily be converted into acrylic acid. We are setting out to produce 3HP in yeast Saccharomyces...... cerevisiae. One main reason for selecting Baker's yeast as host organism is that yeast has a high tolerance towards low pH in comparison to bacteria, e.g. E. coli. Hence, it lowers the consumption of base for neutralization of growth media when compared to bacteria. The preferred engineered pathway towards 3...

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

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


    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.

  15. Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in Saccharomyces cerevisiae.

    Baek, Seung-Ho; Kwon, Eunice Y; Kim, Yong Hwan; Hahn, Ji-Sook


    There is an increasing demand for microbial production of lactic acid (LA) as a monomer of biodegradable poly lactic acid (PLA). Both optical isomers, D-LA and L-LA, are required to produce stereocomplex PLA with improved properties. In this study, we developed Saccharomyces cerevisiae strains for efficient production of D-LA. D-LA production was achieved by expressing highly stereospecific D-lactate dehydrogenase gene (ldhA, LEUM_1756) from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 in S. cerevisiae lacking natural LA production activity. D-LA consumption after glucose depletion was inhibited by deleting DLD1 encoding D-lactate dehydrogenase and JEN1 encoding monocarboxylate transporter. In addition, ethanol production was reduced by deleting PDC1 and ADH1 genes encoding major pyruvate decarboxylase and alcohol dehydrogenase, respectively, and glycerol production was eliminated by deleting GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase. LA tolerance of the engineered D-LA-producing strain was enhanced by adaptive evolution and overexpression of HAA1 encoding a transcriptional activator involved in weak acid stress response, resulting in effective D-LA production up to 48.9 g/L without neutralization. In a flask fed-batch fermentation under neutralizing condition, our evolved strain produced 112.0 g/L D-LA with a yield of 0.80 g/g glucose and a productivity of 2.2 g/(L · h).

  16. Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae.

    Coschigano, P W; Miller, S. M; Magasanik, B


    We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This U...

  17. Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains

    Míriam Cristina Sakuragui Matuo


    Full Text Available Saccharomyces cerevisiae has been widely used in mutagenicity tests due to the presence of a cytochrome P-450 system, capable of metabolizing promutagens to active mutagens. There are a large number of S. cerevisiae strains with varying abilities to produce cytochrome P-450. However, strain selection and ideal cultivation conditions are not well defined. We compared cytochrome P-450 levels in four different S. cerevisiae strains and evaluated the cultivation conditions necessary to obtain the highest levels. The amount of cytochrome P-450 produced by each strain varied, as did the incubation time needed to reach the maximum level. The highest cytochrome P-450 concentrations were found in media containing fermentable sugars. The NCYC 240 strain produced the highest level of cytochrome P-450 when grown in the presence of 20 % (w/v glucose. The addition of ethanol to the media also increased cytochrome P-450 synthesis in this strain. These results indicate cultivation conditions must be specific and well-established for the strain selected in order to assure high cytochrome P-450 levels and reliable mutagenicity results.Linhagens de Saccharomyces cerevisiae tem sido amplamente empregadas em testes de mutagenicidade devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias pró-mutagênicas à sua forma ativa. Devido à grande variedade de linhagens de S. cerevisiae com diferentes capacidades de produção de citocromo P-450, torna-se necessária a seleção de cepas, bem como a definição das condições ideais de cultivo. Neste trabalho, foram comparados os níveis de citocromo P-450 em quatro diferentes linhagens de S. cerevisiae e avaliadas as condições de cultivo necessárias para obtenção de altas concentrações deste sistema enzimático. O maior nível enzimático foi encontrado na linhagem NCYC 240 em presença de 20 % de glicose (p/v. A adição de etanol ao meio de cultura também produziu um aumento na s

  18. Fermentation of xylose to produce ethanol by recombinant Saccharomyces cerevisiae strain containing XYLA and XKS1

    LIU Xiaolin; JIANG Ning; HE Peng; LU Dajun; SHEN An


    Fermentation of the pentose sugar xylose to produce ethanol using lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyce cerevisise, an efficient ethanol producer, cannot utilize xylose because it lacks the ability to convert xylose to its isomer xylulose. In this study, XYLA gene encoding xylose isomerase (XI) from Thermoanaerobacter tengcongensis MB4T and XKS1 gene encoding xylulokinase (XK) from Pichia stipitis were cloned and functionally coexpressed in Saccharomyces cerevisiae EF-326 to construct a recombinant xylose-utilizing strain. The resulting strain S. cerevisiae EF 1014 not only grew on xylose as sole carbon source, but also produced ethanol under anaerobic conditions. Fermentations performed with different xylose concentrations at different temperatures demonstrated that the highest ethanol productivity was 0.11 g/g xylose when xylose concentration was provided at 50 g/L. Under this condition, 28.4% of xylose was consumed and 1.54 g/L xylitol was formed. An increasing fermentation temperature from 30℃ to 37℃ did not improve ethanol yield.

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

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


    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.

  20. Saccharomyces cerevisiae as a model organism: a comparative study.

    Hiren Karathia

    Full Text Available BACKGROUND: Model organisms are used for research because they provide a framework on which to develop and optimize methods that facilitate and standardize analysis. Such organisms should be representative of the living beings for which they are to serve as proxy. However, in practice, a model organism is often selected ad hoc, and without considering its representativeness, because a systematic and rational method to include this consideration in the selection process is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this work we propose such a method and apply it in a pilot study of strengths and limitations of Saccharomyces cerevisiae as a model organism. The method relies on the functional classification of proteins into different biological pathways and processes and on full proteome comparisons between the putative model organism and other organisms for which we would like to extrapolate results. Here we compare S. cerevisiae to 704 other organisms from various phyla. For each organism, our results identify the pathways and processes for which S. cerevisiae is predicted to be a good model to extrapolate from. We find that animals in general and Homo sapiens in particular are some of the non-fungal organisms for which S. cerevisiae is likely to be a good model in which to study a significant fraction of common biological processes. We validate our approach by correctly predicting which organisms are phenotypically more distant from S. cerevisiae with respect to several different biological processes. CONCLUSIONS/SIGNIFICANCE: The method we propose could be used to choose appropriate substitute model organisms for the study of biological processes in other species that are harder to study. For example, one could identify appropriate models to study either pathologies in humans or specific biological processes in species with a long development time, such as plants.

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

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


    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.

  2. Characteristics of Zn2+ Biosorption by Saccharomyces cerevisiae


    Objective To investigate the characteristics of Zn2+ biosorption and the release of cations during the process of Zn2+biosorption by intact cells of Saccharomyces cerevisiae. Methods The batch adsorption test was used to study the biosorption equilibrium and isotherm. Zn2+ concentration was measured with atomic adsorption spectrophotometer (AAS) AAS 6.Vario. Results When the initial concentration of Zn2+ ranged between 0.08 and 0.8 mmol/L, the initial pH was natural (about 5.65), the sorbent concentration was about 1 g/L and the capacity of Zn2+ biosorption was from 74.8 to 654.8 μmol/g. The pH value increased by 0.55-1.28 and the intracellular cations (K+, Mg2+, Na+, Ca2+) of the cells were released during the process of Zn2+ biosorption. Conclusion Ion exchange was one of the mechanisms for Zn2+ biosorption. The biomass of Saccharomyces cerevisiae is a potential biosorbent for the removal of Zn2+ from aqueous solution. More work needs to be done before putting it into practical application.

  3. Investigation of the Best Saccharomyces cerevisiae Growth Condition

    Salari, Roshanak; Salari, Rosita


    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

  4. Plasma membrane electron transport in Saccharomyces cerevisiae depends on the presence of mitochondrial respiratory subunits.

    Herst, Patries M; Perrone, Gabriel G; Dawes, Ian W; Bircham, Peter W; Berridge, Michael V


    Most investigations into plasma membrane electron transport (PMET) in Saccharomyces cerevisiae have focused on the inducible ferric reductase responsible for iron uptake under iron/copper-limiting conditions. In this paper, we describe a PMET system, distinct from ferric reductase, which reduces the cell-impermeable water-soluble tetrazolium dye, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-1), under normal iron/copper conditions. WST-1/1-methoxy-phenazine methosulphate reduction was unaffected by anoxia and relatively insensitive to diphenyleneiodonium. Dye reduction was increased when intracellular NADH levels were high, which, in S. cerevisiae, required deletion of numerous genes associated with NADH recycling. Genome-wide screening of all viable nuclear gene-deletion mutants of S. cerevisiae revealed that, although mitochondrial electron transport per se was not required, the presence of several nuclear and mitochondrially encoded subunits of respiratory complexes III and IV was mandatory for PMET. This suggests some form of interaction between components of mitochondrial and plasma membrane electron transport. In support of this, mitochondrial tubular networks in S. cerevisiae were shown to be located in close proximity to the plasma membrane using confocal microscopy.

  5. Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation.

    Dong, Shi-Jun; Lin, Xiang-Hua; Li, Hao


    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.

  6. Saccharomyces cerevisiae Is Permissive for Replication of Bovine Papillomavirus Type 1

    Zhao, Kong-Nan; Frazer, Ian H


    We recently demonstrated that Saccharomyces cerevisiae protoplasts can take up bovine papillomavirus type 1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPV virus-like particles are assembled in infected S. cerevisiae cultures from newly synthesized capsid proteins and also package newly synthesized DNA, including full-length and truncated viral DNA and S. cerevisiae-derived DNA. Virus particles prepared in S. cerevisiae are able to convey pa...

  7. Engineering cellular redox balance in Saccharomyces cerevisiae for improved production of L-lactic acid.

    Lee, Ju Young; Kang, Chang Duk; Lee, Seung Hyun; Park, Young Kyoung; Cho, Kwang Myung


    Owing to the growing market for the biodegradable and renewable polymer, polylactic acid, world demand for lactic acid is rapidly increasing. However, the very high concentrations desired for industrial production of the free lactic acid create toxicity and low pH concerns for manufacturers. Saccharomyces cerevisiae is the most well characterized eukaryote, a preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust, commercially compatible workhorse to be exploited for the production of diverse chemicals. S. cerevisiae has also been explored as a host for lactic acid production because of its high acid tolerance. Here, we constructed an L-lactic acid-overproducing S. cerevisiae by redirecting cellular metabolic fluxes to the production of L-lactic acid. To this end, we deleted the S. cerevisiae genes encoding pyruvate decarboxylase 1 (PDC1), L-lactate cytochrome-c oxidoreductase (CYB2), and glycerol-3-phosphate dehydrogenase (GPD1), replacing them with a heterologous L-lactate dehydrogenase (LDH) gene. Two new target genes encoding isoenzymes of the external NADH dehydrogenase (NDE1 and NDE2), were also deleted from the genome to re-engineer the intracellular redox balance. The resulting strain was found to produce L-lactic acid more efficiently (32.6% increase in final L-lactic acid titer). When tested in a bioreactor in fed-batch mode, this engineered strain produced 117 g/L of L-lactic acid under low pH conditions. This result demonstrates that the redox balance engineering should be coupled with the metabolic engineering in the construction of L-lactic acid-overproducing S. cerevisiae.

  8. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J


    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  9. Biogeographical characterisation of Saccharomyces cerevisiae wine yeast by molecular methods

    Rosanna eTofalo


    Full Text Available 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 visualise 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 flavour 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.

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

    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


    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.

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

    Maksim I. Sorokin


    Full Text Available 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 mitochondriato-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.

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

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


    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.

  13. The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability

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


    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.

  14. Osmo-, thermo- and ethanol- tolerances of Saccharomyces cerevisiae S1

    Sandrasegarampillai Balakumar


    Full Text Available Saccharomyces cerevisiae S1, which is a locally isolated and improved strain showed viability at 40, 45 and 50ºC and produced ethanol at 40, 43 and 45ºC. When the cells were given heat shock at 45ºC for 30min and grown at 40ºC, 100% viability was observed for 60h, and addition of 200gl-1 ethanol has led to complete cell death at 30h. Heat shock given at 45ºC (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-1 sorbitol. In presence of sorbitol (200gl-1 and ethanol (50gl-1 at 40ºC, 46% viability was retained by S. cerevisiae S1 at 48h and it was improved to 80% by soy flour supplementation.

  15. Saccharomyces cerevisiae in the Production of Whisk(ey

    Graeme M. Walker


    Full Text Available Whisk(ey is a major global distilled spirit beverage. Whiskies are produced from cereal starches that are saccharified, fermented and distilled prior to spirit maturation. The strain of Saccharomyces cerevisiae employed in whisky fermentations is crucially important not only in terms of ethanol yields, but also for production of minor yeast metabolites which collectively contribute to development of spirit flavour and aroma characteristics. Distillers must therefore pay very careful attention to the strain of yeast exploited to ensure consistency of fermentation performance and spirit congener profiles. In the Scotch whisky industry, initiatives to address sustainability issues facing the industry (for example, reduced energy and water usage have resulted in a growing awareness regarding criteria for selecting new distilling yeasts with improved efficiency. For example, there is now a desire for Scotch whisky distilling yeasts to perform under more challenging conditions such as high gravity wort fermentations. This article highlights the important roles of S. cerevisiae strains in whisky production (with particular emphasis on Scotch and describes key fermentation performance attributes sought in distiller’s yeast, such as high alcohol yields, stress tolerance and desirable congener profiles. We hope that the information herein will be useful for whisky producers and yeast suppliers in selecting new distilling strains of S. cerevisiae, and for the scientific community to stimulate further research in this area.

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

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


    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.

  17. NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, kinetic properties, and physiological roles.

    DeLuna, A; Avendano, A; Riego, L; Gonzalez, A


    In the yeast Saccharomyces cerevisiae, two NADP(+)-dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and alpha-ketoglutarate. The GDH2-encoded NAD(+)-dependent glutamate dehydrogenase degrades glutamate producing ammonium and alpha-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in alpha-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1- and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of alpha-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of alpha-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.

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

    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.


    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

  19. Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae.

    Murakami, Suguru; Shimamoto, Toshi; Nagano, Hideaki; Tsuruno, Masahiro; Okuhara, Hiroaki; Hatanaka, Haruyo; Tojo, Hiromasa; Kodama, Yukiko; Funato, Kouichi


    Ceramide is one of the most important intercellular components responsible for the barrier and moisture retention functions of the skin. Because of the risks involved with using products of animal origin and the low productivity of plants, the availability of ceramides is currently limited. In this study, we successfully developed a system that produces sphingosine-containing human ceramide-NS in the yeast Saccharomyces cerevisiae by eliminating the genes for yeast sphingolipid hydroxylases (encoded by SUR2 and SCS7) and introducing the gene for a human sphingolipid desaturase (encoded by DES1). The inactivation of the ceramidase gene YDC1, overexpression of the inositol phosphosphingolipid phospholipase C gene ISC1, and endoplasmic reticulum localization of the DES1 gene product resulted in enhanced production of ceramide-NS. The engineered yeast strains can serve as hosts not only for providing a sustainable source of ceramide-NS but also for developing further systems to produce sphingosine-containing sphingolipids.

  20. De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae

    Li, Mingji; Kildegaard, Kanchana Rueksomtawin; Chen, Yun;


    Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae...... to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.......73±0.05 mg L−1 resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85±0.31 mg L−1 resveratrol from glucose as the sole carbon source. Next we improved...

  1. Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae

    Hahn-Hägerdal Bärbel


    Full Text Available Abstract Background Baker's yeast (Saccharomyces cerevisiae has been engineered for xylose utilization to enable production of fuel ethanol from lignocellulose raw material. One unresolved challenge is that S. cerevisiae lacks a dedicated transport system for pentose sugars, which means that xylose is transported by non-specific Hxt transporters with comparatively low transport rate and affinity for xylose. Results In this study, we compared three heterologous xylose transporters that have recently been shown to improve xylose uptake under different experimental conditions. The transporters Gxf1, Sut1 and At5g59250 from Candida intermedia, Pichia stipitis and Arabidopsis thaliana, respectively, were expressed in isogenic strains of S. cerevisiae and the transport kinetics and utilization of xylose was evaluated. Expression of the Gxf1 and Sut1 transporters led to significantly increased affinity and transport rates of xylose. In batch cultivation at 4 g/L xylose concentration, improved transport kinetics led to a corresponding increase in xylose utilization, whereas no correlation could be demonstrated at xylose concentrations greater than 15 g/L. The relative contribution of native sugar transporters to the overall xylose transport capacity was also estimated during growth on glucose and xylose. Conclusions Kinetic characterization and aerobic batch cultivation of strains expressing the Gxf1, Sut1 and At5g59250 transporters showed a direct relationship between transport kinetics and xylose growth. The Gxf1 transporter had the highest transport capacity and the highest xylose growth rate, followed by the Sut1 transporter. The range in which transport controlled the growth rate was determined to between 0 and 15 g/L xylose. The role of catabolite repression in regulation of native transporters was also confirmed by the observation that xylose transport by native S. cerevisiae transporters increased significantly during cultivation in xylose and

  2. Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in Saccharomyces cerevisiae

    Hernando, Yolanda; Carter, Andrew T.; Parr, Adrian;


    The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(α)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible co...

  3. Studies of Saccharomyces cerevisiae and Non-Saccharomyces Yeasts during Alcoholic Fermentation

    Kemsawasd, Varongsiri

    to the early death of L. thermotolerans during mixed-culture alcoholic fermentation with S. cerevisiae. These data may be valuable for development of mixed- and sequential starter cultures for tailoring of wine quality and enhanced flavour profiles. As well, we studied the role of nitrogenous sources...... in completion of anaerobic alcoholic fermentation. For both S. cerevisiae and non-Saccharomyces yeasts, some 22 different nitrogenous sources were evaluated for effects on growth and fermentation ability during anaerobic alcoholic fermentation. The data revealed that nitrogen preference is a trait...... that is species-specific; as well, growth enhancement is also dependent upon the type of nitrogen supplementation for each yeast species. This work provides the first detailed analysis of appropriate nitrogen supplementation to improve yeast growth and alcoholic fermentative activity (i.e., glucose consumption...

  4. Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae.

    Siewers, Verena; Chen, Xiao; Huang, Le; Zhang, Jie; Nielsen, Jens


    Non-ribosomal peptides (NRPs) are a diverse family of secondary metabolites with a broad range of biological activities. We started to develop an eukaryotic microbial platform based on the yeast Saccharomyces cerevisiae for heterologous production of NRPs using delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 5' end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 degrees C. When ACVS and PPTase encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast.

  5. Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers.

    Generoso, Wesley Cardoso; Schadeweg, Virginia; Oreb, Mislav; Boles, Eckhard


    Saccharomyces cerevisiae has decisive advantages in industrial processes due to its tolerance to alcohols and fermentation conditions. Butanol isomers are considered as suitable fuel substitutes and valuable biomass-derived chemical building blocks. Whereas high production was achieved with bacterial systems, metabolic engineering of yeast for butanol production is in the beginning. For isobutanol synthesis, combination of valine biosynthesis and degradation, and complete pathway re-localisation into cytosol or mitochondria gave promising results. However, competing pathways, co-factor imbalances and FeS cluster assembly are still major issues. 1-Butanol production via the Clostridium pathway seems to be limited by cytosolic acetyl-CoA, its central precursor. Endogenous 1-butanol pathways have been discovered via threonine or glycine catabolism. 2-Butanol production was established but was limited by B12-dependence.

  6. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

    Hou, Jin; Lages, Nuno; Oldiges, M.


    Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation during metabolism. There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output. Therefore, cofactors are emerging to be attractive targets...... to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH...... production, while decreasing mitochondrial NADH lowered ethanol production. However, when these reactions were coupled with NADPH production, the metabolic changes were more moderated. The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations...

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

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


    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.

  8. Interaction among Saccharomyces cerevisiae pheromone receptors during endocytosis

    Chien-I Chang


    Full Text Available This study investigates endocytosis of Saccharomyces cerevisiae α-factor receptor and the role that receptor oligomerization plays in this process. α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, we found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors. Both in vitro and in vivo assays suggested that ligand-induced conformational changes in one Ste2 subunit do not affect neighboring subunits. Therefore, recognition of the endocytosis signal sequence and recognition of the ligand-induced conformational change are likely to be two independent events.

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

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


    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.

  10. Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts.

    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


    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.

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

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


    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.

  12. Preferentially quantized linker DNA lengths in Saccharomyces cerevisiae.

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


    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.

  13. Preferentially quantized linker DNA lengths in Saccharomyces cerevisiae.

    Ji-Ping Wang

    Full Text Available 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.


    Blažena Lavová


    Full Text Available Reactive oxygen species (ROS with reactive nitrogen species (RNS are known to play dual role in biological systems, they can be harmful or beneficial to living systems. ROS can be important mediators of damage to cell structures, including proteins, lipids and nucleic acids termed as oxidative stress. The antioxidant enzymes protect the organism against the oxidative damage caused by active oxygen forms. The role of superoxide dismutase (SOD is to accelerate the dismutation of the toxic superoxide radical, produced during oxidative energy processes, to hydrogen peroxide and molecular oxygen. In this study, SOD activity of three yeast strains Saccharomyces cerevisiae was determined. It was found that SOD activity was the highest (23.7 protein in strain 612 after 28 hours of cultivation. The lowest SOD activity from all tested strains was found after 56 hours of cultivation of strain Gyöng (0.7 protein.

  15. Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states

    Boender, L.G.M.; Almering, M.J.H.; Dijk, M.; Van Maris, A.J.A.; De Winde, J.H.; Pronk, J.T.; Daran-Lapujade, P.


    Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuousl

  16. Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

    Milne, N.; Luttik, M.A.H.; Cueto Rojas, H.F.; Wahl, A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.G.


    In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential pl

  17. Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes

    Albergaria, Helena; Arneborg, Nils


    Winemaking, brewing and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the main biotransformation and Saccharomyces cerevisiae the primary microorganism. Although a wide variety of microbial species may participate in alcoholic fermentation and...

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

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


    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. Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes.

    Wei, Yongjun; Gossing, Michael; Bergenholm, David; Siewers, Verena; Nielsen, Jens


    Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0-C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0), but CB supply is limited. Therefore, CB-like lipids (CBL, which are composed of POP, POS and SOS) are in great demand. Saccharomyces cerevisiae produces TAGs as storage lipids, which are also mainly composed of C16 and C18 fatty acids. However, POP, POS and SOS are not among the major TAG forms in yeast. TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast strains harboring cocoa genes increased 190, 230 and 196% over the control strain, respectively; especially, the potential SOS content of the three yeast strains increased 254, 476 and 354% over the control strain. Moreover, one of the three yeast strains had a 2.25-fold increased TAG content and 6.7-fold higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis.

  20. Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

    Shirley, Derek; Oppert, Cris; Reynolds, Todd B; Miracle, Bethany; Oppert, Brenda; Klingeman, William E; Jurat-Fuentes, Juan Luis


    Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.

  1. Biodiversity of Saccharomyces cerevisiae isolated from a survey of pito production sites in various parts of Ghana.

    Glover, Richard L K; Abaidoo, Robert C; Jakobsen, Mogens; Jespersen, Lene


    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.


    G. P. Shuvaeva


    Full Text Available Summary. Experimental data on an acid and thermal inactivation of a high refined inulinase (2,1-β-D- fructanfructanohydrolase, KF 3.2.17, produced by the race of Saccharomyces cerevisiae VGSh-2 yeast are presented. The strain of S. cerevisiae VGSh-2 was produced by the method of the induced mutagenesis and deposited to the collection of pure cultures of the chair of biochemistry and biotechnology of Voronezh state university of engineering technologies. The cells of source culture (S. cerevisiae XII were affected step-by-step by the ultra-violet radiation (UFR and UFR in a complex with a chemical mutagen (etilenimine. The culture was grown up by the method of liquid-phase deep cultivation on a constant nutrient medium. Refining conditions for inulinase are sorted out. Activity of enzyme dependence on physical and chemical factors (рН and temperature is obtained and numerical values of the main kinetic constants – Km and Vmax are determined. The structure of enzyme molecule is studied by an infrared-spectroscopy method: the type and relative quantity of elements of secondary structure of protein are defined. Substrate binding groups of the active center of an inulinase are found. The comparative analysis of the ability to hydrolysis of inulin in several enzyme preparations from Jerusalem artichoke and to the subsequent their fermentation by the VGSh-2 and XI S. cerevisiae yeasts is carried out. Optimum conditions of enzyme hydrolysis of inulin are selected. Research of the fermentation process of starchcontaining raw materials by yeasts of VGSh-2 and XI races is done. It is established that the using of VGSh-2 S. cerevisiae yeast for a grain wort and the Jerusalem artichoke fermentation, allows to increase an extraction of ethyl alcohol comparing to control race, to improve its quality characteristics, and also allows to predict the using of new race in the food industry for production ethanol from grain raw materials and a fermentation of

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

    Lezzi, Chiara; Bleve, Gianluca; Spagnolo, Stefano; Perrotta, Carla; Grieco, Francesco


    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.

  4. Structure of the Glycosyltransferase Ktr4p from Saccharomyces cerevisiae.

    Dominik D D Possner

    Full Text Available In the yeast Saccharomyces cerevisiae, members of the Kre2/Mnt1 protein family have been shown to be α-1,2-mannosyltransferases or α-1,2-mannosylphosphate transferases, utilising an Mn2+-coordinated GDP-mannose as the sugar donor and a variety of mannose derivatives as acceptors. Enzymes in this family are localised to the Golgi apparatus, and have been shown to be involved in both N- and O-linked glycosylation of newly-synthesised proteins, including cell wall glycoproteins. Our knowledge of the nine proteins in this family is however very incomplete at present. Only one family member, Kre2p/Mnt1p, has been studied by structural methods, and three (Ktr4p, Ktr5p, Ktr7p are completely uncharacterised and remain classified only as putative glycosyltransferases. Here we use in vitro enzyme activity assays to provide experimental confirmation of the predicted glycosyltransferase activity of Ktr4p. Using GDP-mannose as the donor, we observe activity towards the acceptor methyl-α-mannoside, but little or no activity towards mannose or α-1,2-mannobiose. We also present the structure of the lumenal catalytic domain of S. cerevisiae Ktr4p, determined by X-ray crystallography to a resolution of 2.2 Å, and the complex of the enzyme with GDP to 1.9 Å resolution.

  5. Long-chain alkane production by the yeast Saccharomyces cerevisiae.

    Buijs, Nicolaas A; Zhou, Yongjin J; Siewers, Verena; Nielsen, Jens


    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.

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

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


    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.

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

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


    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.

  8. Phosphite disrupts the acclimation of Saccharomyces cerevisiae to phosphate starvation.

    McDonald, A E; Niere, J O; Plaxton, W C


    The influence of phosphite (H2PO3-) on the response of Saccharomyces cerevisiae to orthophosphate (HPO4(2-); Pi) starvation was assessed. Phosphate-repressible acid phosphatase (rAPase) derepression and cell development were abolished when phosphate-sufficient (+Pi) yeast were subcultured into phosphate-deficient (-Pi) media containing 0.1 mM phosphite. By contrast, treatment with 0.1 mM phosphite exerted no influence on rAPase activity or growth of +Pi cells. 31P NMR spectroscopy revealed that phosphite is assimilated and concentrated by yeast cultured with 0.1 mM phosphite, and that the levels of sugar phosphates, pyrophosphate, and particularly polyphosphate were significantly reduced in the phosphite-treated -Pi cells. Examination of phosphite's effects on two PHO regulon mutants that constitutively express rAPase indicated that (i) a potential target for phosphite's action in -Pi yeast is Pho84 (plasmalemma high-affinity Pi transporter and component of a putative phosphate sensor-complex), and that (ii) an additional mechanism exists to control rAPase expression that is independent of Pho85 (cyclin-dependent protein kinase). Marked accumulation of polyphosphate in the delta pho85 mutant suggested that Pho85 contributes to the control of polyphosphate metabolism. Results are consistent with the hypothesis that phosphite obstructs the signaling pathway by which S. cerevisiae perceives and responds to phosphate deprivation at the molecular level.

  9. The evolution of gene expression QTL in Saccharomyces cerevisiae.

    James Ronald

    Full Text Available Understanding the evolutionary forces that influence patterns of gene expression variation will provide insights into the mechanisms of evolutionary change and the molecular basis of phenotypic diversity. To date, studies of gene expression evolution have primarily been made by analyzing how gene expression levels vary within and between species. However, the fundamental unit of heritable variation in transcript abundance is the underlying regulatory allele, and as a result it is necessary to understand gene expression evolution at the level of DNA sequence variation. Here we describe the evolutionary forces shaping patterns of genetic variation for 1206 cis-regulatory QTL identified in a cross between two divergent strains of Saccharomyces cerevisiae. We demonstrate that purifying selection against mildly deleterious alleles is the dominant force governing cis-regulatory evolution in S. cerevisiae and estimate the strength of selection. We also find that essential genes and genes with larger codon bias are subject to slightly stronger cis-regulatory constraint and that positive selection has played a role in the evolution of major trans-acting QTL.

  10. CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

    Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D


    This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory.

  11. Effects of proteinase A on cultivation and viability characteristics of industrial Saccharomyces cerevisiae WZ65

    Hong-bo ZHANG; Hai-feng ZHANG; Qi-he CHEN; Hui RUAN; Ming-liang FU; Guo-qing HE


    Proteinase A (PrA), encoded by PEP4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. It was observed that the lag phase of cell growth of partial PEP4 gene deletion mutant (36 h) and PrA-negative mutant (48 h) was significantly ex-tended, compared with the wild type strain (24 h) (P<0.05), but PrA had no effect on glucose metabolism either under shaking or steady state cultivations. The logistic model was chosen to evaluate the effect of PrA on S. cerevisiae cell growth, and PrA was found to promote cell growth against insufficient oxygen condition in steady state cultivation, but had no effect in shaking culti-vation. The effects of glucose starvation on cell growth of partial PEP4 gene deletion strain and PrA-negative mutant were also evaluated. The results show that PrA partial deficiency increased the adaption ofS. cerevisiae to unfavorable nutrient environment, but had no effect on glucose metabolism under the stress of low glucose. During heat shock test, at 60 ℃ the reduced cell viability rate (RCVR) was 10% for the wild type S. cerevisiae and 90% for both mutant strains (P<0.01), suggesting that PrA was a negative factor for S. cerevisiae cells to survive under heat shock. As temperatures rose from 60 ℃ to 70 ℃, the wild type S. cerevisiae had significantly lower relative glucose consumption rate (RGCR) (61.0% and 80.0%) than the partial mutant (78.0% and 98.5%) and the complete mutant (80.0% and 98.0%) (P<0.05), suggesting that, in coping with heat shock, cells of the PrA mutants increased their glucose consumption to survive. The present study may provide meaningful information for brewing industry; however, the role of PrA in industrial S. cerevisiae physiology is complex and needs to be further investigated.

  12. Major sulfonate transporter Soa1 in Saccharomyces cerevisiae and considerable substrate diversity in its fungal family

    Holt, Sylvester; Kankipati, Harish; De Graeve, Stijn; Van Zeebroeck, Griet; Foulquié-Moreno, Maria R.; Lindgreen, Stinus; Thevelein, Johan M.


    Sulfate is a well-established sulfur source for fungi; however, in soils sulfonates and sulfate esters, especially choline sulfate, are often much more prominent. Here we show that Saccharomyces cerevisiae YIL166C(SOA1) encodes an inorganic sulfur (sulfate, sulfite and thiosulfate) transporter that also catalyses sulfonate and choline sulfate uptake. Phylogenetic analysis of fungal SOA1 orthologues and expression of 20 members in the sul1Δ sul2Δ soa1Δ strain, which is deficient in inorganic and organic sulfur compound uptake, reveals that these transporters have diverse substrate preferences for sulfur compounds. We further show that SOA2, a S. cerevisiae SOA1 paralogue found in S. uvarum, S. eubayanus and S. arboricola is likely to be an evolutionary remnant of the uncharacterized open reading frames YOL163W and YOL162W. Our work highlights the importance of sulfonates and choline sulfate as sulfur sources in the natural environment of S. cerevisiae and other fungi by identifying fungal transporters for these compounds. PMID:28165463

  13. Ecological interactions among Saccharomyces cerevisiae strains: insight into the dominance phenomenon

    Pérez-Torrado, Roberto; Rantsiou, Kalliopi; Perrone, Benedeta; Navarro-Tapia, Elisabeth; Querol, Amparo; Cocolin, Luca


    This study investigates the behaviour of Saccharomyces cerevisiae strains, in order to obtain insight into the intraspecies competition taking place in mixed populations of this species. Two strains of S. cerevisiae, one dominant and one non-dominant, were labelled and mixed, and individual fermentations were set up to study the transcriptomes of the strains by means of RNA-seq. The results obtained suggest that cell-to-cell contact and aggregation, which are driven by the expression of genes that are associated with the cell surface, are indispensable conditions for the achievement of dominance. Observations on mixed aggregates, made up of cells of both strains, which were detected by means of flow cytometry, have confirmed the transcriptomic data. Furthermore, overexpression of the SSU1 gene, which encodes for a transporter that confers resistance to sulphites, provides an ecological advantage to the dominant strain. A mechanistic model is proposed that sheds light on the dominance phenomenon between different strains of the S. cerevisiae species. The collected data suggest that cell-to-cell contact, together with differential sulphite production and resistance is important in determining the dominance of one strain over another. PMID:28266552

  14. Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

    Steen, EricJ.; Chan, Rossana; Prasad, Nilu; Myers, Samuel; Petzold, Christopher; Redding, Alyssa; Ouellet, Mario; Keasling, JayD.


    BackgroundIncreasing energy costs and environmental concerns have motivated engineering microbes for the production of ?second generation? biofuels that have better properties than ethanol.Results& ConclusionsSaccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

  15. Comparative proteomics analysis of engineered Saccharomyces cerevisiae with enhanced biofuel precursor production.

    Xiaoling Tang

    Full Text Available The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production.

  16. Increased ethanol production by deletion of HAP4 in recombinant xylose-assimilating Saccharomyces cerevisiae.

    Matsushika, Akinori; Hoshino, Tamotsu


    The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.

  17. Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol

    Myers Samuel


    Full Text Available Abstract Background Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol. Results and conclusion Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

  18. An improved method of xylose utilization by recombinant Saccharomyces cerevisiae.

    Ma, Tien-Yang; Lin, Ting-Hsiang; Hsu, Teng-Chieh; Huang, Chiung-Fang; Guo, Gia-Luen; Hwang, Wen-Song


    The aim of this study was to develop a method to optimize expression levels of xylose-metabolizing enzymes to improve xylose utilization capacity of Saccharomyces cerevisiae. A xylose-utilizing recombinant S. cerevisiae strain YY2KL, able to express nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-dependent xylose reductase (XR), nicotinamide adenine dinucleotide (NAD(+))-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK), showed a low ethanol yield and sugar consumption rate. To optimize xylose utilization by YY2KL, a recombinant expression plasmid containing the XR gene was transformed and integrated into the aur1 site of YY2KL. Two recombinant expression plasmids containing an nicotinamide adenine dinucleotide phosphate (NADP(+))-dependent XDH mutant and XK genes were dually transformed and integrated into the 5S ribosomal DNA (rDNA) sites of YY2KL. This procedure allowed systematic construction of an S. cerevisiae library with different ratios of genes for xylose-metabolizing enzymes, and well-grown colonies with different xylose fermentation capacities could be further selected in yeast protein extract (YPX) medium (1 % yeast extract, 2 % peptone, and 2 % xylose). We successfully isolated a recombinant strain with a superior xylose fermentation capacity and designated it as strain YY5A. The xylose consumption rate for strain YY5A was estimated to be 2.32 g/gDCW/h (g xylose/g dry cell weight/h), which was 2.34 times higher than that for the parent strain YY2KL (0.99 g/gDCW/h). The ethanol yield was also enhanced 1.83 times by this novel method. Optimal ratio and expression levels of xylose-metabolizing enzymes are important for efficient conversion of xylose to ethanol. This study provides a novel method that allows rapid and effective selection of ratio-optimized xylose-utilizing yeast strains. This method may be applicable to other multienzyme systems in yeast.

  19. High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

    Ahlgren Simon


    Full Text Available Abstract Background The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP. Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. Results We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™ to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. Conclusions Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.

  20. Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae.

    Yu, Dayu; Xu, Fuchao; Zi, Jiachen; Wang, Siyuan; Gage, David; Zeng, Jia; Zhan, Jixun


    Two fungal cyclooligomer depsipeptide synthetases(CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, respectively. The titers of beauvericins (33.8 ± 1.4 mg/l) and bassianolide (21.7± 0.1 mg/l) in the engineered S. cerevisiae BJ5464-NpgA strains were comparable to those in the native producer B. bassiana. Feeding D-hydroxyisovaleric acid (D-Hiv) and the corresponding L-amino acid precursors improved the production of beauvericins and bassianolide. However, the high price of D-Hiv limits its application in large-scale production of these cyclooligomer depsipeptides. Alternatively, we engineered another enzyme, ketoisovalerate reductase (KIVR) from B. bassiana, into S. cerevisiae BJ5464-NpgA for enhanced in situ synthesis of this expensive substrate. Co-expression of BbBEAS and KIVR in the yeast led to significant improvement of the production of beauvericins.The total titer of beauvericin and its congeners (beauvericins A-C) was increased to 61.7 ± 3.0 mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC7159. Supplement of L-Val at 10 mM improved the supply of ketoisovalerate, the substrate of KIVR, which consequently further increased the total titer of beauvericins to 105.8 ± 2.1 mg/l. Using this yeast system,we functionally characterized an unknown CODS from Fusarium venenatum NRRL 26139 as a beauvericin synthetase, which was named as FvBEAS. Our work thus provides a useful approach for functional reconstitution and engineering of fungal CODSs for efficient production of this family of anticancer molecules.

  1. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

    Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber


    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.

  2. Non-coding RNA prediction and verification in Saccharomyces cerevisiae.

    Laura A Kavanaugh


    Full Text Available Non-coding RNA (ncRNA play an important and varied role in cellular function. A significant amount of research has been devoted to computational prediction of these genes from genomic sequence, but the ability to do so has remained elusive due to a lack of apparent genomic features. In this work, thermodynamic stability of ncRNA structural elements, as summarized in a Z-score, is used to predict ncRNA in the yeast Saccharomyces cerevisiae. This analysis was coupled with comparative genomics to search for ncRNA genes on chromosome six of S. cerevisiae and S. bayanus. Sets of positive and negative control genes were evaluated to determine the efficacy of thermodynamic stability for discriminating ncRNA from background sequence. The effect of window sizes and step sizes on the sensitivity of ncRNA identification was also explored. Non-coding RNA gene candidates, common to both S. cerevisiae and S. bayanus, were verified using northern blot analysis, rapid amplification of cDNA ends (RACE, and publicly available cDNA library data. Four ncRNA transcripts are well supported by experimental data (RUF10, RUF11, RUF12, RUF13, while one additional putative ncRNA transcript is well supported but the data are not entirely conclusive. Six candidates appear to be structural elements in 5' or 3' untranslated regions of annotated protein-coding genes. This work shows that thermodynamic stability, coupled with comparative genomics, can be used to predict ncRNA with significant structural elements.

  3. PRODUKSI ETANOL DARI TETES TEBU OLEH Saccharomyces cerevisiae PEMBENTUK FLOK (NRRL – Y 265 (Ethanol Production from Cane Molasses by Flocculant Saccharomyces cerevisiae (NRRL – Y 265

    Agustin Krisna Wardani


    Full Text Available The potential use of sugar cane molasses by flocculant Saccharomyces cerevisiae in ethanol production was investigated. In order to minimize the negative effect of calcium on yeast growth, pretreated sugar cane molasses with dilute acid was performed. The influence of process parameters such as sugar concentration and inoculum concentration were evaluated for enhancing bioethanol production. Result showed that maximum ethanol concentration of 8,792% (b/v was obtained at the best condition of inoculum concentration 10% (v/v and sugar concentration 15% (b/v. Based on the experimental data, maximum yield of ethanol production of 65% was obtained. This result demonstrated the potential of molasses as promising biomass resources for ethanol production. Keywords: Ethanol, preteated cane molasses, flocculant Saccharomyces cerevisiae, fermentation   ABSTRAK Efisiensi produksi bioetanol diperoleh melalui ketepatan pemilihan jenis mikroorganisme, bahan baku, dan kontrol proses fermentasi. Alternatif proses untuk meminimalisasi biaya produksi etanol adalah dengan mengeliminasi tahap pemisahan sentrifugasi sel dari produk karena memerlukan biaya instalasi dan biaya perawatan yang tinggi. Proses sentrifugasi merupakan tahapan penting untuk memisahkan sel mikroba dari medium fermentasi pada produksi bioetanol. Untuk meminimalisir biaya produksi akibat proses tersebut digunakan inokulum Saccharomyces cerevisiae pembentuk flok dan tetes tebu sebagai sumber gula. Penelitian ini bertujuan untuk mendapatkan konsentrasi penambahan inokulum Saccharomyces cerevisiae pembentuk flok dan konsentrasi sumber gula dalam tetes tebu yang tepat dalam produksi etanol yang maksimum. Saccharomyces cerevisiae sebanyak 5%, 10%, dan 15% (v/v diinokulasikan pada medium tetes tebu hasil pretreatment dengan kandungan gula 15%, 20%, dan 25% (b/v pada pH 5. Fermentasi dilakukan pada suhu 30°C dan agitasi 100 rpm selama 72 jam. Etanol tertinggi didapat pada kondisi konsentrasi inokulum

  4. Exploring the northern limit of the distribution of Saccharomyces cerevisiae and Saccharomyces paradoxus in North America.

    Charron, Guillaume; Leducq, Jean-Baptiste; Bertin, Chloé; Dubé, Alexandre K; Landry, Christian R


    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.

  5. Invertase SUC2 Is the Key Hydrolase for Inulin Degradation in Saccharomyces cerevisiae

    Wang, Shi-An; Li, Fu-li


    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.

  6. Physiological impact and context dependency of transcriptional responses: a chemostat study in Saccharomyces cerevisiae

    Tai, S.L.


    This thesis is a compilation of a four-year PhD project on bakers' yeast (Saccharomyces cerevisiae). Since the entire S. cerevisiae genome sequence became available in 1996, DNA-microarray analysis has become a popular high-information-density tool for analyzing gene expression in this important ind

  7. Biopharmaceutical protein production bySaccharomyces cerevisiae: current state and future prospects

    Huang, Mingtao; Bao, Jichen; Nielsen, Jens


    tasks with low cost, high productivity and proper post-translational modifications. The yeast Saccharomyces cerevisiae is one of these preferred cell factories as it meets many of the requirements. There are several reports on improvement of recombinant protein production by S. cerevisiae through...


    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...

  9. Creation of a synthetic xylose-inducible promoter for Saccharomyces cerevisiae

    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...

  10. Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation.

    Nissen, T L; Kielland-Brandt, M C; Nielsen, J; Villadsen, J


    Ethanol is still one of the most important products originating from the biotechnological industry with respect to both value and amount. In addition to ethanol, a number of byproducts are formed during an anaerobic fermentation of Saccharomyces cerevisiae. One of the most important of these compounds, glycerol, is produced by yeast to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD+. The purpose of this study was to evaluate whether a reduced formation of surplus NADH and an increased consumption of ATP in biosynthesis would result in a decreased glycerol yield and an increased ethanol yield in anaerobic cultivations of S. cerevisiae. A yeast strain was constructed in which GLN1, encoding glutamine synthetase, and GLT1, encoding glutamate synthase, were overexpressed, and GDH1, encoding the NADPH-dependent glutamate dehydrogenase, was deleted. Hereby the normal NADPH-consuming synthesis of glutamate from ammonium and 2-oxoglutarate was substituted by a new pathway in which ATP and NADH were consumed. The resulting strain TN19 (gdh1-A1 PGK1p-GLT1 PGK1p-GLN1) had a 10% higher ethanol yield and a 38% lower glycerol yield compared to the wild type in anaerobic batch fermentations. The maximum specific growth rate of strain TN19 was slightly lower than the wild-type value, but earlier results suggest that this can be circumvented by increasing the specific activities of Gln1p and Glt1p even more. Thus, the results verify the proposed concept of increasing the ethanol yield in S. cerevisiae by metabolic engineering of pathways involved in biomass synthesis.

  11. Genome-wide transcriptional response of a Saccharomyces cerevisiae strain with an altered redox metabolism.

    Bro, Christoffer; Regenberg, Birgitte; Nielsen, Jens


    The genome-wide transcriptional response of a Saccharomyces cerevisiae strain deleted in GDH1 that encodes a NADP(+)-dependent glutamate dehydrogenase was compared to a wild-type strain under anaerobic steady-state conditions. The GDH1-deleted strain has a significantly reduced NADPH requirement, and therefore, an altered redox metabolism. Identification of genes with significantly changed expression using a t-test and a Bonferroni correction yielded only 16 transcripts when accepting two false-positives, and 7 of these were Open Reading Frames (ORFs) with unknown function. Among the 16 transcripts the only one with a direct link to redox metabolism was GND1, encoding phosphogluconate dehydrogenase. To extract additional information we analyzed the transcription data for a gene subset consisting of all known genes encoding metabolic enzymes that use NAD(+) or NADP(+). The subset was analyzed for genes with significantly changed expression again with a t-test and correction for multiple testing. This approach was found to enrich the analysis since GND1, ZWF1 and ALD6, encoding the most important enzymes for regeneration of NADPH under anaerobic conditions, were down-regulated together with eight other genes encoding NADP(H)-dependent enzymes. This indicates a possible common redox-dependent regulation of these genes. Furthermore, we showed that it might be necessary to analyze the expression of a subset of genes to extract all available information from global transcription analysis.

  12. [Saccharomyces cerevisiae fungemia in an elderly patient following probiotic treatment].

    Eren, Zehra; Gurol, Yeşim; Sonmezoglu, Meral; Eren, Hatice Seyma; Celik, Gülden; Kantarci, Gülçin


    Saccharomyces cerevisiae, known as baker's yeast, is also used as a probiotic agent to treat gastroenteritis by modulating the endogenous flora and immune system. However, since there have been increasing reports of fungemia due to S.cerevisiae and its subspecies S.boulardii, it is recommended that probiotics should be cautiously used in immunosuppressed patients, people with underlying diseases and low-birth weight babies. To emphasize this phenomenon, in this report, a case of S.cerevisiae fungemia developed in a patient given probiotic treatment for antibiotic-associated diarrhea, was presented. An 88-year-old female patient was admitted to our hospital with left hip pain, hypotension, and confusion. Her medical history included hypertension, chronic renal failure, left knee replacement surgery, and recurrent urinary tract infections due to neurogenic bladder. She was transferred to the intensive care unit with the diagnosis of urosepsis. After obtaining blood and urine samples for culture, empirical meropenem (2 x 500 mg) and linezolid (1 x 600 mg) treatment were administered. A central venous catheter (CVC) was inserted and after one day of inotropic support, her hemodynamic parameters were stabilized. The urine culture obtained on admission yielded extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. Urine culture was repeated after three days and no bacteria were isolated. On the 4th day of admission she developed diarrhea. Toxin A/B tests for Clostridium difficile were negative. To relieve diarrhea, S.boulardii (Reflor 250 mg capsules, Sanofi Aventis, Turkey) was administered twice a day, without opening capsules. Two days later, her C-reactive protein (CRP) level increased from 23.2 mg/L to 100 mg/L without fever. Her blood culture taken from the CVC yielded S.cerevisiae. Linezolid and meropenem therapies were stopped on the 13th and 14th days, respectively, while prophylactic fluconazole therapy was replaced with

  13. Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae

    Henriksen, Peter; Wagner, Sebastian Alexander; Weinert, Brian Tate;


    Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysin...

  14. Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.

    Spevak, W; Fessl, F; Rytka, J; Traczyk, A; Skoneczny, M; Ruis, H


    The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl). Catalase T-deficient mutants were obtained by UV mutagenesis of an S. cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression. Since the second catalase protein of S. cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected u...

  15. Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering.

    Asadollahi, Mohammad A; Maury, Jérôme; Patil, Kiran Raosaheb; Schalk, Michel; Clark, Anthony; Nielsen, Jens


    A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function. Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase encoded by GDH2 was also considered in this study. Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain.

  16. Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose.

    Divate, Nileema R; Chen, Gen-Hung; Wang, Pei-Ming; Ou, Bor-Rung; Chung, Yun-Chin


    A genetic recombinant Saccharomyces cerevisiae starter with high ethanol tolerance capacities was constructed. In this study, the gene of trehalose-6-phosphate synthase (encoded by tps1), which catalyzes the first step in trehalose synthesis, was cloned and overexpressed in S. cerevisiae. Moreover, the gene of neutral trehalase (encoded by nth1, trehalose degrading enzyme) was deleted by using a disruption cassette, which contained long flanking homology regions of nth1 gene (the upstream 0.26 kb and downstream 0.4 kb). The engineered strain increased its tolerance against ethanol and glucose stress. The growth of the wild strain was inhibited when the medium contained 6 % or more ethanol, whereas growth of the engineered strain was affected when the medium contained 10 % or more ethanol. There was no significant difference in the ethanol yield between the wild strain and the engineered strain when the fermentation broth contained 10 % glucose (p > 0.05). The engineered strain showed greater ethanol yield than the wild type strain when the medium contained more than 15 % glucose (p < 0.05). Higher intracellular trehalose accumulation by overexpression of tps1 and deletion of nth1 might provide the ability for yeast to protect against environmental stress.

  17. Transcript-specific translational regulation in the unfolded protein response of Saccharomyces cerevisiae.

    Payne, Tom; Hanfrey, Colin; Bishop, Amy L; Michael, Anthony J; Avery, Simon V; Archer, David B


    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes stress and induces the unfolded protein response (UPR). Genome-wide analysis of translational regulation in response to the UPR-inducing agent dithiothreitol in Saccharomyces cerevisiae is reported. Microarray analysis, confirmed using qRT-PCR, identified transcript-specific translational regulation. Transcripts with functions in ribosomal biogenesis and assembly were translationally repressed. In contrast, mRNAs from known UPR genes, encoding the UPR transcription factor Hac1p, the ER-oxidoreductase Ero1p and the ER-associated protein degradation (ERAD) protein Der1p, were enriched in polysomal fractions, indicating translational up-regulation. Splicing of HAC1 mRNA is shown to be required for efficient ribosomal loading.

  18. Killer toxin of Saccharomyces cerevisiae Y500-4L active against Fleischmann and Itaiquara commercial brands of yeast

    Soares Giselle A.M.


    Full Text Available The strain Saccharomyces cerevisiae Y500-4L, previously selected from the must of alcohol producing plants and showing high fermentative and killer capacities, was characterized according to the interactions between the yeasts and examined for curing and detection of dsRNA plasmids, which code for the killer character. The killer yeast S. cerevisiae Y500-4L showed considerable killer activity against the Fleischmann and Itaiquara commercial brands of yeast and also against the standard killer yeasts K2 (S. diastaticus NCYC 713, K4 (Candida glabrata NCYC 388 and K11 (Torulopsis glabrata ATCC 15126. However S. cerevisiae Y500-4L showed sensitivity to the killer toxin produced by the standard killer yeasts K8 (Hansenula anomala NCYC 435, K9 (Hansenula mrakii NCYC 500, K10 (Kluyveromyces drosophilarum NCYC 575 and K11 (Torulopsis glabrata ATCC 15126. No M-dsRNA plasmid was detected in the S. cerevisiae Y500-4L strain and these results suggest that the genetic basis for toxin production is encoded by chromosomal DNA. The strain S. cerevisiae Y500-4L was more resistant to the loss of the phenotype killer with cycloheximide and incubation at elevated temperatures (40oC than the standard killer yeast S. cerevisiae K1.

  19. Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae

    Vázquez de Aldana Carlos R


    Full Text Available Abstract Background In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. Results Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11 from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. Conclusion Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.

  20. Introducing a new breed of wine yeast: interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae.

    Jennifer R Bellon

    Full Text Available 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.

  1. Genotoxicity assessment of amaranth and allura red using Saccharomyces cerevisiae.

    Jabeen, Hafiza Sumara; ur Rahman, Sajjad; Mahmood, Shahid; Anwer, Sadaf


    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.

  2. Protein disorder reduced in Saccharomyces cerevisiae to survive heat shock

    Vicedo, Esmeralda; Gasik, Zofia; Dong, Yu-An; Goldberg, Tatyana; Rost, Burkhard


    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

  3. Saccharomyces cerevisiae Fermentation Effects on Pollen: Archaeological Implications

    Crystal A. Dozier


    Full Text Available Pollen is the reproductive agent of flowering plants; palynology is utilized by archaeologists because sporopollenin, a major component in the exine of pollen grains, is resistant to decay and morphologically distinctive. Wine, beer, and mead have been identified in the archaeological record by palynological assessment due to indicator species or due to a pollen profile similar to that recovered from honey, a common source of sugar in a variety of fermented beverages. While most palynologists have assumed that pollen grains are resistant to alcoholic fermentation, a recent study in food science implies that pollen is a yeast nutrient because pollen-enriched meads produce more alcohol. The experiment presented here explores the potential distortion of the pollen record through fermentation by brewing a traditional, pollen-rich mead with Saccharomyces cerevisiae. In this experiment, the pollen grains did not undergo any discernible morphological changes nor were distorted in the pollen profile. Any nutrition that the yeast garners from the pollen therefore leaves sporopollenin intact. These results support palynological research on residues of alcoholic beverages and confirms that the fermentation process does not distort the pollen profile of the original substance. The paper concludes with the potential and limits of palynological study to assess fermentation within the archaeological record.

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

    Visser, W.; Scheffers, W.A.; Batenburg-Van Der Vegte, W.H.; Van Dijken, J.P. (Delft Univ. of Technology (Netherlands))


    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.

  5. Biotransformation of malachite green by Saccharomyces cerevisiae MTCC 463.

    Jadhav, J P; Govindwar, S P


    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.

  6. YPA: an integrated repository of promoter features in Saccharomyces cerevisiae.

    Chang, Darby Tien-Hao; Huang, Cheng-Yi; Wu, Chi-Yeh; Wu, Wei-Sheng


    This study presents the Yeast Promoter Atlas (YPA, or database, which aims to collect comprehensive promoter features in Saccharomyces cerevisiae. YPA integrates nine kinds of promoter features including promoter sequences, genes' transcription boundaries-transcription start sites (TSSs), five prime untranslated regions (5'-UTRs) and three prime untranslated regions (3'UTRs), TATA boxes, transcription factor binding sites (TFBSs), nucleosome occupancy, DNA bendability, transcription factor (TF) binding, TF knockout expression and TF-TF physical interaction. YPA is designed to present data in a unified manner as many important observations are revealed only when these promoter features are considered altogether. For example, DNA rigidity can prevent nucleosome packaging, thereby making TFBSs in the rigid DNA regions more accessible to TFs. Integrating nucleosome occupancy, DNA bendability, TF binding, TF knockout expression and TFBS data helps to identify which TFBS is actually functional. In YPA, various promoter features can be accessed in a centralized and organized platform. Researchers can easily view if the TFBSs in an interested promoter are occupied by nucleosomes or located in a rigid DNA segment and know if the expression of the downstream gene responds to the knockout of the corresponding TFs. Compared to other established yeast promoter databases, YPA collects not only TFBSs but also many other promoter features to help biologists study transcriptional regulation.

  7. Cellular memory of acquired stress resistance in Saccharomyces cerevisiae.

    Guan, Qiaoning; Haroon, Suraiya; Bravo, Diego González; Will, Jessica L; Gasch, Audrey P


    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.

  8. Sulfate assimilation mediates tellurite reduction and toxicity in Saccharomyces cerevisiae.

    Ottosson, Lars-Göran; Logg, Katarina; Ibstedt, Sebastian; Sunnerhagen, Per; Käll, Mikael; Blomberg, Anders; Warringer, Jonas


    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.

  9. Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae.

    Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian


    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.

  10. Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

    Danuza Nogueira Moysés


    Full Text Available 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.

  11. An overview of membrane transport proteins in Saccharomyces cerevisiae.

    Andre, B


    All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport. It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms. This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae. This analysis was conducted at a time when about 65% of all yeast genes were available in public databases. In addition to approximately 60 transport proteins whose function was at least partially known, approximately 100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms. While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization. The proteins revealed by this in silicio analysis also include a putative K+ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na+-solute symporters, a protein very similar to electroneural cation-chloride cotransporters, and a putative Na+-H+ antiporter. A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing.

  12. Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae.

    Bogonez, E; Satrústegui, J; Machado, A


    The activity of glutamate dehydrogenase (NADP+) (EC; 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.

  13. Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

    Dilworth, David; Nelson, Christopher J.


    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

  14. Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.

    Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra


    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.

  15. D-xylulose fermentation to ethanol by Saccharomyces cerevisiae

    Chiang, L.C.; Gong, C.S.; Chen, L.F.; Tsao, G.T.


    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).

  16. mRNA quality control pathways in Saccharomyces cerevisiae

    Satarupa Das; Biswadip Das


    Efficient production of translation-competent mRNAs involves processing and modification events both in the nucleus and cytoplasm which require a number of complex machineries at both co-transcriptional and post-transcriptional levels. Mutations in the genomic sequence sometimes result in the formation of mutant non-functional defective messages. In addition, the enormous amounts of complexities involved in the biogenesis of mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional counterpart. Subsequent translation of these mutant and defective populations of messenger RNAs could possibly result in the unfaithful transmission of genetic information and thus is considered a threat to the survival of the cell. To prevent this possibility, mRNA quality control systems have evolved both in the nucleus and cytoplasm in eukaryotes to scrutinize various stages of mRNP biogenesis and translation. In this review, we will focus on the physiological role of some of these mRNA quality control systems in the simplest model eukaryote Saccharomyces cerevisiae.

  17. Direct mating between diploid sake strains of Saccharomyces cerevisiae.

    Hashimoto, Shinji; Aritomi, Kazuo; Minohara, Takafumi; Nishizawa, Yoshinori; Hoshida, Hisashi; Kashiwagi, Susumu; Akada, Rinji


    Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATalpha mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.

  18. Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16.

    Bang, D D; Timmermans, V; Verhage, R; Zeeman, A M; van de Putte, P; Brouwer, J


    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.

  19. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)


    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.

  20. Calcium dependence of eugenol tolerance and toxicity in Saccharomyces cerevisiae.

    Roberts, Stephen K; McAinsh, Martin; Cantopher, Hanna; Sandison, Sean


    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.

  1. Ecological and Genetic Barriers Differentiate Natural Populations of Saccharomyces cerevisiae.

    Clowers, Katie J; Heilberger, Justin; Piotrowski, Jeff S; Will, Jessica L; Gasch, Audrey P


    How populations that inhabit the same geographical area become genetically differentiated is not clear. To investigate this, we characterized phenotypic and genetic differences between two populations of Saccharomyces cerevisiae that in some cases inhabit the same environment but show relatively little gene flow. We profiled stress sensitivity in a group of vineyard isolates and a group of oak-soil strains and found several niche-related phenotypes that distinguish the populations. We performed bulk-segregant mapping on two of the distinguishing traits: The vineyard-specific ability to grow in grape juice and oak-specific tolerance to the cell wall damaging drug Congo red. To implicate causal genes, we also performed a chemical genomic screen in the lab-strain deletion collection and identified many important genes that fell under quantitative trait loci peaks. One gene important for growth in grape juice and identified by both the mapping and the screen was SSU1, a sulfite-nitrite pump implicated in wine fermentations. The beneficial allele is generated by a known translocation that we reasoned may also serve as a genetic barrier. We found that the translocation is prevalent in vineyard strains, but absent in oak strains, and presents a postzygotic barrier to spore viability. Furthermore, the translocation was associated with a fitness cost to the rapid growth rate seen in oak-soil strains. Our results reveal the translocation as a dual-function locus that enforces ecological differentiation while producing a genetic barrier to gene flow in these sympatric populations.

  2. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides.

    Dai, Zhubo; Liu, Yi; Zhang, Xianan; Shi, Mingyu; Wang, Beibei; Wang, Dong; Huang, Luqi; Zhang, Xueli


    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.

  3. Calcium dependence of eugenol tolerance and toxicity in Saccharomyces cerevisiae.

    Stephen K Roberts

    Full Text Available 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.

  4. Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine production.

    Jin, Lu; Bhuiya, Mohammad Wadud; Li, Mengmeng; Liu, XiangQi; Han, Jixiang; Deng, WeiWei; Wang, Min; Yu, Oliver; Zhang, Zhengzhu


    Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g., tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.

  5. Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine production.

    Lu Jin

    Full Text Available Caffeine (1, 3, 7-trimethylxanthine and theobromine (3, 7-dimethylxanthine are the major purine alkaloids in plants, e.g., tea (Camellia sinensis and coffee (Coffea arabica. Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT and Camellia sinensis caffeine synthase (TCS in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.

  6. The network architecture of the Saccharomyces cerevisiae genome.

    Stephen A Hoang

    Full Text Available We propose a network-based approach for surmising the spatial organization of genomes from high-throughput interaction data. Our strategy is based on methods for inferring architectural features of networks. Specifically, we employ a community detection algorithm to partition networks of genomic interactions. These community partitions represent an intuitive interpretation of genomic organization from interaction data. Furthermore, they are able to recapitulate known aspects of the spatial organization of the Saccharomyces cerevisiae genome, such as the rosette conformation of the genome, the clustering of centromeres, as well as tRNAs, and telomeres. We also demonstrate that simple architectural features of genomic interaction networks, such as cliques, can give meaningful insight into the functional role of the spatial organization of the genome. We show that there is a correlation between inter-chromosomal clique size and replication timing, as well as cohesin enrichment. Together, our network-based approach represents an effective and intuitive framework for interpreting high-throughput genomic interaction data. Importantly, there is a great potential for this strategy, given the rich literature and extensive set of existing tools in the field of network analysis.


    V. N. Bayraktar


    Full Text Available The biochemical constituents in wine stocks that influence the flavor and quality of wine are investigated in the paper. The tested parameters consist of volume fraction of ethanol, residual sugar, phenolic compounds, tartaric, malic, citric, lactic, acetic acids, titratable acidity and volatile acids. The wine stocks that were received from white and red grape varieties Tairov`s selection were tested. There was a correlation between titratable acidity and volatile acids in the wine stocks from white and red grape varieties. High correlation was also found between lactic and acetic acids, between volatile acids, acetic acid and sugar. It was determined that wine stocks with a high concentration of ethanol originated from those yeast strains of Saccharomyces cerevisiae, in a fermented grape must of high speed of enzyme activity. The taste of wine stocks correlated with the ratio of tartaric to malic acid. Analysis showed significant differences between the varieties of white and red wine stocks in concentrations of organic acids, phenolic compounds, residual sugar, and volume fraction of ethanol. Positive correlation was indicated for both studied groups for volatile acids and acetic acid, tartaric, malic, lactic acids and total sugar. Prospective yeast cultures with high productivity of alcohol (ethanol were selected for winemaking biotechnology.

  8. Data on dynamic study of cytoophidia in Saccharomyces cerevisiae

    Hui Li


    Full Text Available The data in this paper are related to the research article entitled “Filamentation of metabolic enzymes in Saccharomyces cerevisiae” Q.J. Shen et al. (2016 [1]. Cytoophidia are filamentous structures discovered in fruit flies (doi:10.1016/S1673-8527(0960046-1 J.L. Liu (2010 [2], bacteria (doi:10.1038/ncb2087 M. Ingerson-Mahar et al. (2010 [3], yeast (doi:10.1083/jcb.201003001; doi:10.1242/bio.20149613 C. Noree et al. (2010 and J. Zhang, L. Hulme, J.L. Liu (2014 [4,5] and human cells (doi:10.1371/journal.pone.0029690; doi:10.1016/j.jgg.2011.08.004 K. Chen et al. (2011 and W.C. Carcamo et al. (2011 ( [6,7]. However, there is little research on the motility of the cytoophidia. Here we selected cytoophidia formed by 6 filament-forming proteins in the budding yeast S. cerevisiae, and performed living-cell imaging of cells expressing the proteins fused with GFP. The dynamic features of the six types of cytoophidia were analyzed. In the data, both raw movies and analysed results of the dynamics of cytoophidia are presented.

  9. Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    Gonzalez-Perez, David; Alcalde, Miguel


    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

  10. Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history.

    Legras, Jean-Luc; Merdinoglu, Didier; Cornuet, Jean-Marie; Karst, Francis


    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.

  11. Genetic Basis for Saccharomyces cerevisiae Biofilm in Liquid Medium

    Andersen, Kaj Scherz; Bojsen, Rasmus Kenneth; Gro Rejkjær Sørensen, Laura


    Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics...... than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the S1278b background and found 71 genes that were essential for biofilm development. Quantitative...... northern blots further revealed that AIM1, ASG1, AVT1, DRN1, ELP4, FLO8, FMP10, HMT1, KAR5, MIT1, MRPL32, MSS11, NCP1, NPR1, PEP5, PEX25, RIM8, RIM101, RGT1, SNF8, SPC2, STB6, STP22, TEC1, VID24, VPS20, VTC3, YBL029W, YBL029C-A, YFL054C, YGR161W-C, YIL014C-A, YIR024C, YKL151C, YNL200C, YOR034C-A, and YOR...

  12. The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

    Hu, Y.; Cooper, T. G.; Kohlhaw, G B


    The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopro...

  13. Responsiveness to Exogenous Camp of a Saccharomyces Cerevisiae Strain Conferred by Naturally Occurring Alleles of Pde1 and Pde2

    Mitsuzawa, H.


    The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity...

  14. Molecular cloning and expression of the Saccharomyces cerevisiae RFC3 gene, an essential component of replication factor C.

    Li, X.; Burgers, P M


    Yeast replication factor C (RF-C) is a multi-polypeptide complex required for processive DNA replication by DNA polymerases delta and epsilon. The gene encoding the 40-kDa subunit of the Saccharomyces cerevisiae RF-C (RFC3) has been cloned. The RFC3 gene is required for yeast cell growth and has been mapped to the left arm of chromosome XIV. The deduced amino acid sequence of the RFC3 gene shows a high homology to the 36-, 37-, and 40-kDa subunits of human RF-C (also called activator 1), with...

  15. Quantitative comparison of transient growth of Saccharomyces cerevisiae, Saccharomyces kluyveri, and Kluyveromyces lactis.

    Herwig, Christoph; Von Stockar, Urs


    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

  16. Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae.

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


    Variations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes. S. cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source. With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold. The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased. Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription. At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations. It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.

  17. A novel approach for the improvement of ethanol fermentation by Saccharomyces cerevisiae

    Hou, L.; Cao, X.; Wang, C. [Tianjin Univ. of Science and Technology, Tianjin (China). Key Laboratory of Food Nutrition and Safety


    The partial substitution of fossil fuels with bioethanol has become an important strategy for the use of renewable energy. Ethanol production is generally achieved through fermentation of starch or sugar-based feedstock by Saccharomyces cerevisiae. In order to meet the growing demand for ethanol, there is a need for new yeast strains that can produce ethanol more efficiently and cost effectively. This paper presented a new genome engineering approach that was developed to improve ethanol production by S. cerevisiae. In this study, the aneuploid strain constructed on the base of tetraploid cells was shown to have favourable metabolic traits in very high gravity (VHG) fermentation with 300 g/L glucose as the carbon source. The tetraploid strain was constructed using the plasmid YCplac33-GHK, which comprised the HO gene encoding the site-specific HO endonucleases. The aneuploid strain, WT4-M, was chosen and screened once the tetraploid cells were treated with methyl benzimidazole-2-yl-carbamate to induce loss of mitotic chromosomes. The aneuploid strain WT4-M increased ethanol production as well as osmotic and thermal tolerance. The sugar to ethanol conversion rate also improved. It was concluded that this new approach is valuable for creating yeast strains with better fermentation characteristics. 25 refs., 3 figs.

  18. Development of N- and O-linked oligosaccharide engineered Saccharomyces cerevisiae strain.

    Abe, Hiroko; Tomimoto, Kazuya; Fujita, Yasuko; Iwaki, Tomoko; Chiba, Yasunori; Nakayama, Ken-Ichi; Nakajima, Yoshihiro


    Yeast cells have been engineered for the production of glycoproteins as biopharmaceuticals with humanized N-linked oligosaccharides. The suppression of yeast-specific O-mannosylation is important to reduce immune response and to improve heterologous protein productivity in the production of biopharmaceuticals. However, so far, there are few reports of the engineering of both N-linked and O-linked oligosaccharides in yeast cells. In the present study, we describe the generation of a Saccharomyces cerevisiae strain capable of producing a glycoprotein with humanized Man5GlcNAc2 N-linked oligosaccharides, an intermediate of mammalian hybrid- and complex-type oligosaccharides, while suppressing O-mannosylation. First, a yeast strain that produces a glycoprotein with Man5GlcNAc2 was isolated by introducing msdS encoding α-1,2-mannosidase into a strain synthesizing Man8GlcNAc2 N-linked oligosaccharides. Next, to suppress O-mannosylation, an O-mannosyltransferase-deficient strain was generated by disrupting PMT1 and PMT2 Although the relative amount of O-linked oligosaccharides in the disruptant was reduced to approximately 40% of that in wild type cells, this strain exhibited growth defects and decreased protein productivity. To overcome the growth defects, we applied a mutagenesis technique that is based on the disparity theory of evolution. Finally, to improve protein productivity of the growth-recovered strain, vacuolar proteases PEP4 and PRB1 were further disrupted. Thus, by combining genetic engineering and disparity mutagenesis, we generated an Saccharomyces cerevisiae strain whose N- and O-linked oligosaccharide synthetic pathways were engineered to effectively produce the heterologous protein.

  19. Overexpression of Erg11p by the Regulatable GAL1 Promoter Confers Fluconazole Resistance in Saccharomyces cerevisiae

    Kontoyiannis, Dimitrios P.; Sagar, Namita; Hirschi, Kendal D.


    The contribution of the dosage of target enzyme P-450 14α-demethylase (14αDM) to fluconazole resistance in both Candida albicans and Saccharomyces cerevisiae remains unclear. Here, we show that overexpression of Saccharomyces P-450 14αDM in S. cerevisiae, under the control of the regulatable promoter GAL1, results in azole resistance.

  20. Low oxygen levels as a trigger for enhancement of respiratory metabolism in Saccharomyces cerevisiae

    Wiebe Marilyn G


    Full Text Available Abstract Background The industrially important yeast Saccharomyces cerevisiae is able to grow both in the presence and absence of oxygen. However, the regulation of its metabolism in conditions of intermediate oxygen availability is not well characterised. We assessed the effect of oxygen provision on the transcriptome and proteome of S. cerevisiae in glucose-limited chemostat cultivations in anaerobic and aerobic conditions, and with three intermediate (0.5, 1.0 and 2.8% oxygen levels of oxygen in the feed gas. Results The main differences in the transcriptome were observed in the comparison of fully aerobic, intermediate oxygen and anaerobic conditions, while the transcriptome was generally unchanged in conditions receiving different intermediate levels (0.5, 1.0 or 2.8% O2 of oxygen in the feed gas. Comparison of the transcriptome and proteome data suggested post-transcriptional regulation was important, especially in 0.5% oxygen. In the conditions of intermediate oxygen, the genes encoding enzymes of the respiratory pathway were more highly expressed than in either aerobic or anaerobic conditions. A similar trend was also seen in the proteome and in enzyme activities of the TCA cycle. Further, genes encoding proteins of the mitochondrial translation machinery were present at higher levels in all oxygen-limited and anaerobic conditions, compared to fully aerobic conditions. Conclusion Global upregulation of genes encoding components of the respiratory pathway under conditions of intermediate oxygen suggested a regulatory mechanism to control these genes as a response to the need of more efficient energy production. Further, cells grown in three different intermediate oxygen levels were highly similar at the level of transcription, while they differed at the proteome level, suggesting post-transcriptional mechanisms leading to distinct physiological modes of respiro-fermentative metabolism.

  1. Impact of oxygenation on the performance of three non-Saccharomyces yeasts in co-fermentation with Saccharomyces cerevisiae.

    Shekhawat, Kirti; Bauer, Florian F; Setati, Mathabatha E


    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.

  2. Biological Treatment of Textile Effluent Using Candida zeylanoides and Saccharomyces cerevisiae Isolated from Soil

    O. P. Abioye


    Full Text Available This study evaluates the efficacy of yeasts isolated from soil in the treatment of textile wastewater. Two yeast species were isolated from soil; they were identified as Candida zeylanoides and Saccharomyces cerevisiae. The yeasts were inoculated into flask containing effluent and incubated for 15 days. Saccharomyces cerevisiae showed the most significant treatment capacity with a 66% reduction in BOD; this was followed closely by Candida zeylanoides with 57.3% reduction in BOD and a consortium of the two species showed the least remediation potential of 36.9%. The use of Saccharomyces cerevisiae and Candida zeylanoides in treatment of textile wastewater will help to limit the adverse environmental and health implications associated with disposal of untreated effluent into water bodies.

  3. Prevalence reduction of pathogens in poultry fed with Saccharomyces cerevisiae

    Fanelli, A.


    Full Text Available Description of the subject. The growth of new antibiotic-resistant strains of pathogens represents a huge problem in poultry rearing. There is evidence that dietary yeast could be effective in the protection against a variety of pathogens that can affect poultry health and cause foodborne diseases in humans. Since still few or contradictory information are available for this topic. Objectives. The objective of this study was to investigate the effects of live yeast supplementation in broiler chickens on Salmonella enteritidis and Campylobacter jejuni content in feces, cecum, and skin. Method. Supplemented yeast consisted of Saccharomyces cerevisiae (Levucell® SB20, type boulardii I-1079, Lallemand, France and was administered at a rate of 1 x 106 CFU·g-1 of feed. On day ten of life, birds were orally challenged with S. enteritidis (1 x 105 CFU/bird and C. jejuni (3 x 105 CFU/bird. Growth performance, and coliforms, yeasts and lactobacilli enumeration were evaluated on day 0, 10, 20 and 38. Ten and eighteen days post infection (PI, 10 animals per replicate were slaughtered and pooled ceca content were analyzed for yeast enumeration and Salmonella and Campylobacter frequency and enumeration. The presence and the enumeration of Salmonella and Campylobacter in neck and breast skin were performed on one subject per replicate. Results. Dietary S. cerevisiae increased yeast and lactobacilli (p = 0.01 count, while Salmonella enumeration and frequency significantly decreased in neck (p = 0.03 and tended to decrease in cecum (p = 0.06, feces (p = 0.06, and breast (p = 0.08. On 10d PI Campylobacter presence was decreased in cecum (p = 0.01, feces (p < 0.01, breast skin (p = 0.04 and neck skin (p < 0.01, while the enumeration was found to be lower in feces (p < 0.01 and neck skin (p = 0.05. At the end of the trial the frequency of this pathogen was decreased in feces (p < 0.01, and breast skin (p = 0.02, while the enumeration was diminished in cecum (p

  4. Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

    Quarterman, Josh; Skerker, Jeffrey M; Feng, Xueyang; Liu, Ian Y; Zhao, Huimin; Arkin, Adam P; Jin, Yong-Su


    In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without

  5. Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae.

    Najafpour, Ghasem; Younesi, Habibollah; Syahidah Ku Ismail, Ku


    Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase

  6. Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley

    Camila M.P.B.S. de Ponzzes-Gomes


    Full Text Available The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 x 10(5 cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.

  7. Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley

    de Ponzzes-Gomes, Camila M.P.B.S.; de Mélo, Dângelly L.F.M.; Santana, Caroline A.; Pereira, Giuliano E.; Mendonça, Michelle O.C.; Gomes, Fátima C.O.; Oliveira, Evelyn S.; Barbosa, Antonio M.; Trindade, Rita C.; Rosa, Carlos A.


    The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 × 105 cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production. PMID:25242923

  8. Nucleotide sequence of the GDH gene coding for the NADP-specific glutamate dehydrogenase of Saccharomyces cerevisiae.

    Nagasu, T; Hall, B D


    The isolation of the Saccharomyces cerevisiae gene for NADP-dependent glutamate dehydrogenase (NADP-GDH) by cross hybridization to the Neurospora crassa am gene, known to encode for NADP-GDH is described. Two DNA fragments selected from a yeast genomic library in phage lambda gt11 were shown by restriction analysis to share 2.5 kb of common sequence. A yeast shuttle vector (CV13) carrying either to the cloned fragments complements the gdh- strain of S. cerevisiae and directs substantial overproduction of NADP-GDH. One of the cloned fragments was sequenced, and the deduced amino acid (aa) sequence of the yeast NADP-GDH is 64% homologous to N. crassa, 51% to Escherichia coli and 24% to bovine NADP-GDHs.

  9. Compositions and methods for modeling Saccharomyces cerevisiae metabolism


    The invention provides an in silica model for determining a S. cerevisiae physiological function. The model includes a data structure relating a plurality of S. cerevisiae reactants to a plurality of S. cerevisiae reactions, a constraint set for the plurality of S. cerevisiae reactions......, and commands for determining a distribution of flux through the reactions that is predictive of a S. cerevisiae physiological function. A model of the invention can further include a gene database containing information characterizing the associated gene or genes. The invention further provides methods...... for making an in silica S. cerevisiae model and methods for determining a S. cerevisiae physiological function using a model of the invention. The invention provides an in silica model for determining a S. cerevisiae physiological function. The model includes a data structure relating a plurality of S...

  10. High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.

    Verwaal, René; Wang, Jing; Meijnen, Jean-Paul; Visser, Hans; Sandmann, Gerhard; van den Berg, Johan A; van Ooyen, Albert J J


    To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.

  11. Nanofiltration concentration of extracellular glutathione produced by engineered Saccharomyces cerevisiae.

    Sasaki, Kengo; Hara, Kiyotaka Y; Kawaguchi, Hideo; Sazuka, Takashi; Ogino, Chiaki; Kondo, Akihiko


    This study aimed to optimize extracellular glutathione production by a Saccharomyces cerevisiae engineered strain and to concentrate the extracellular glutathione by membrane separation processes, including ultrafiltration (UF) and nanofiltration (NF). Synthetic defined (SD) medium containing 20 g L(-1) glucose was fermented for 48 h; the fermentation liquid was passed through an UF membrane to remove macromolecules. Glutathione in this permeate was concentrated for 48 h to 545.1 ± 33.6 mg L(-1) using the NF membrane; this was a significantly higher concentration than that obtained with yeast extract peptone dextrose (YPD) medium following 96 h NF concentration (217.9 ± 57.4 mg L(-1)). This higher glutathione concentration results from lower cellular growth in SD medium (final OD600 = 6.9 ± 0.1) than in YPD medium (final OD600 = 11.0 ± 0.6) and thus higher production of extracellular glutathione (16.0 ± 1.3 compared to 9.2 ± 2.1 mg L(-1) in YPD medium, respectively). Similar fermentation and membrane processing of sweet sorghum juice containing 20 g L(-1) total sugars provided 240.3 ± 60.6 mg L(-1) glutathione. Increased extracellular production of glutathione by this engineered strain in SD medium and subsequent UF permeation and NF concentration in shortend time may help realize industrial recovery of extracellular glutathione.

  12. Interaction between lanthanide ions and Saccharomyces cerevisiae cells.

    Ene, Cristian D; Ruta, Lavinia L; Nicolau, Ioana; Popa, Claudia V; Iordache, Virgil; Neagoe, Aurora D; Farcasanu, Ileana C


    Lanthanides are a group of non-essential elements with important imaging and therapeutic applications. Although trivalent lanthanide ions (Ln³⁺) are used as potent blockers of Ca²⁺ channels, the systematic studies correlating Ln³⁺ accumulation and toxicity to Ca²⁺ channel blocking activity are scarce. In this study, we made use of the eukaryotic model Saccharomyces cerevisiae to investigate the correlation between Ln³⁺ accumulation, their toxicity and their capacity to block the exogenous stress-induced Ca²⁺ influx into the cytosol. It was found that the Ln³⁺ blocked the Ca²⁺ entry into the yeast cells only when present at concentration high enough to allow rapid binding to cell surface. At lower concentrations, Ln³⁺ were taken up by the cell, but Ca²⁺ blockage was no longer achieved. At 1 mM concentration, all ions from the Ln³⁺ series could block Ca²⁺ entry into cytosol with the exception of La³⁺, and to a lesser extent, Pr³⁺ and Nd³⁺. The plasma membrane Ca²⁺-channel Cch1/Mid1 contributed to La³⁺ and Gd³⁺ entry into the cells, with a significant preference for La³⁺. The results open the possibility to obtain cells loaded with controlled amounts and ratios of Ln³⁺.

  13. Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae.

    Jongedijk, Esmer; Cankar, Katarina; Ranzijn, Jorn; van der Krol, Sander; Bouwmeester, Harro; Beekwilder, Jules


    Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028 mg/l (+)-limonene and 0.060 mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12 mg/l (+)-limonene and 0.49 mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems.

  14. New Genes Involved in Osmotic Stress Tolerance in Saccharomyces cerevisiae

    Gonzalez, Ramon; Morales, Pilar; Tronchoni, Jordi; Cordero-Bueso, Gustavo; Vaudano, Enrico; Quirós, Manuel; Novo, Maite; Torres-Pérez, Rafael; Valero, Eva


    Adaptation to changes in osmolarity is fundamental for the survival of living cells, and has implications in food and industrial biotechnology. It has been extensively studied in the yeast Saccharomyces cerevisiae, where the Hog1 stress activated protein kinase was discovered about 20 years ago. Hog1 is the core of the intracellular signaling pathway that governs the adaptive response to osmotic stress in this species. The main endpoint of this program is synthesis and intracellular retention of glycerol, as a compatible osmolyte. Despite many details of the signaling pathways and yeast responses to osmotic challenges have already been described, genome-wide approaches are contributing to refine our knowledge of yeast adaptation to hypertonic media. In this work, we used a quantitative fitness analysis approach in order to deepen our understanding of the interplay between yeast cells and the osmotic environment. Genetic requirements for proper growth under osmotic stress showed both common and specific features when hypertonic conditions were induced by either glucose or sorbitol. Tolerance to high-glucose content requires mitochondrial function, while defective protein targeting to peroxisome, GID-complex function (involved in negative regulation of gluconeogenesis), or chromatin dynamics, result in poor survival to sorbitol-induced osmotic stress. On the other side, the competitive disadvantage of yeast strains defective in the endomembrane system is relieved by hypertonic conditions. This finding points to the Golgi-endosome system as one of the main cell components negatively affected by hyperosmolarity. Most of the biological processes highlighted in this analysis had not been previously related to osmotic stress but are probably relevant in an ecological and evolutionary context. PMID:27733850

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

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


    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.

  16. [Construction and fermentation control of reductive TCA pathway for malic acid production in Saccharomyces cerevisiae].

    Yan, Daojiang; Wang, Caixia; Zhou, Jiemin; Liu, Yilan; Yang, Maohua; Xing, Jianmin


    Malic acid is widely used in food, and chemical industries. Through overexpressing pyruvate carboxylase and malate dehydrogenase in pdc1-deficient Saccharomyces cerevisiae, malic acid was successfully produced through the reductive TCA pathway. No malic acid was detected in wild type Saccharomyces cerevisiae, however, 45 mmol/L malic acid was produced in engineered strain, and the concentration of byproduct ethanol also reduced by 18%. The production of malic acid enhanced 6% by increasing the concentration of Ca2+. In addition, the final concentration reached 52.5 mmol/L malic acid by addition of biotin. The increasing is almost 16% higher than that of the original strain.

  17. Screening of Non- Saccharomyces cerevisiae Strains for Tolerance to Formic Acid in Bioethanol Fermentation.

    Oshoma, Cyprian E; Greetham, Darren; Louis, Edward J; Smart, Katherine A; Phister, Trevor G; Powell, Chris; Du, Chenyu


    Formic acid is one of the major inhibitory compounds present in hydrolysates derived from lignocellulosic materials, the presence of which can significantly hamper the efficiency of converting available sugars into bioethanol. This study investigated the potential for screening formic acid tolerance in non-Saccharomyces cerevisiae yeast strains, which could be used for the development of advanced generation bioethanol processes. Spot plate and phenotypic microarray methods were used to screen the formic acid tolerance of 7 non-Saccharomyces cerevisiae yeasts. S. kudriavzeii IFO1802 and S. arboricolus 2.3319 displayed a higher formic acid tolerance when compared to other strains in the study. Strain S. arboricolus 2.3319 was selected for further investigation due to its genetic variability among the Saccharomyces species as related to Saccharomyces cerevisiae and availability of two sibling strains: S. arboricolus 2.3317 and 2.3318 in the lab. The tolerance of S. arboricolus strains (2.3317, 2.3318 and 2.3319) to formic acid was further investigated by lab-scale fermentation analysis, and compared with S. cerevisiae NCYC2592. S. arboricolus 2.3319 demonstrated improved formic acid tolerance and a similar bioethanol synthesis capacity to S. cerevisiae NCYC2592, while S. arboricolus 2.3317 and 2.3318 exhibited an overall inferior performance. Metabolite analysis indicated that S. arboricolus strain 2.3319 accumulated comparatively high concentrations of glycerol and glycogen, which may have contributed to its ability to tolerate high levels of formic acid.

  18. Lycopene overproduction in Saccharomyces cerevisiae through combining pathway engineering with host engineering

    Chen, Yan; Xiao, Wenhai; Wang, Ying; Liu, Hong; Li, Xia; Yuan, Yingjin


    Background Microbial production of lycopene, a commercially and medically important compound, has received increasing concern in recent years. Saccharomyces cerevisiae is regarded as a safer host for lycopene production than Escherichia coli. However, to date, the lycopene yield (mg/g DCW) in S. cerevisiae was lower than that in E. coli and did not facilitate downstream extraction process, which might be attributed to the incompatibility between host cell and heterologous pathway. Therefore, ...

  19. Industrial Systems Biology of Saccharomyces cerevisiae Enables Novel Succinic Acid Cell Factory

    Otero, José Manuel; Cimini, Donatella; Patil, Kiran Raosaheb


    Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought......-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals....

  20. Selection of Indigenous Saccharomyces cerevisiae Strains from Kutjevo Wine Growing Area at the Laboratoy Scale

    Sandi Orlić; Nenad Očić; Ana Jeromel; Katarina Huić; Sulejman Redžepović


    The use of selected yeasts for winemaking has clear advantages over traditional spontaneous fermentation. Selection of wine yeasts is usually carried out within the Saccharomyces cerevisiae species. Yeast strains produce different amount of secondary compounds that impart specific characteristics to the wines. This suggests that it is necessary to isolate naturally occuring autochthone strains, which exhibit a metabolic profile that corresponds to each wine. Twenty two strains of S.cerevisiae...

  1. Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae.

    Lewis, T A; Taylor, F R; Parks, L W


    Wild-type Saccharomyces cerevisiae do not accumulate exogenous sterols under aerobic conditions, and a mutant allele conferring sterol auxotrophy (erg7) could be isolated only in strains with a heme deficiency. delta-Aminolevulinic acid (ALA) fed to a hem1 (ALA synthetase-) erg7 (2,3-oxidosqualene cyclase-) sterol-auxotrophic strain of S. cerevisiae inhibited sterol uptake, and growth was negatively affected when intracellular sterol was depleted. The inhibition of sterol uptake (and growth o...

  2. Accumulation and chemical states of radiocesium by fungus Saccharomyces cerevisiae

    Ohnuki, Toshihiko; Sakamoto, Fuminori; Kozai, Naofumi; Yamasaki, Shinya; Yu, Qianqian


    After accident of Fukushima Daiichi Nuclear Power Plant, the fall-out radiocesium was deposited on the ground. Filamentous fungus is known to accumulate radiocesium in environment, even though many minerals are involved in soil. These facts suggest that fungus affect the migration behavior of radiocesium in the environment. However, accumulation mechanism of radiocesium by fungus is not understood. In the present study, accumulation and chemical states change of Cs by unicellular fungus of Saccharomyces cerevisiae have been studied to elucidate the role of microorganisms in the migration of radiocesium in the environment. Two different experimental conditions were employed; one is the accumulation experiments of radiocesium by S. cerevisiae from the agar medium containing 137Cs and a mineral of zeolite, vermiculite, smectite, mica, or illite. The other is the experiments using stable cesium to examine the chemical states change of Cs. In the former experiment, the cells were grown on membrane filter of 0.45 μm installed on the agar medium. After the grown cells were weighed, radioactivity in the cells was measured by an autoradiography technique. The mineral weight contents were changed from 0.1% to 1% of the medium. In the latter experiment, the cells were grown in the medium containing stable Cs between 1 mM and 10mM. The Cs accumulated cells were analyzed by SEM-EDS and EXAFS. The adsorption experiments of cesium by the cells under resting condition were also conducted to test the effect of cells metabolic activity. Without mineral in the medium, cells of S. cerevisiae accumulated 1.5x103 Bq/g from the medium containing 137Cs of 2.6x102 Bq/g. When mineral was added in the medium, concentration of 137Cs in the cells decreased. The concentration of 137Cs in the cells from the medium containing different minerals were in the following order; smectite, illite, mica > vermiculite > zeolite. This order was nearly the same as the inverse of distribution coefficient of

  3. Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

    Rossouw, D.; Heyns, E. H.; Setati, M. E.; Bosch, S.


    The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ and tdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines. PMID:23793638

  4. Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

    Zelle, Rintze M; de Hulster, Erik; van Winden, Wouter A; de Waard, Pieter; Dijkema, Cor; Winkler, Aaron A; Geertman, Jan-Maarten A; van Dijken, Johannes P; Pronk, Jack T; van Maris, Antonius J A


    Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

  5. Prioritized Expression of BDH2 under Bulk Translational Repression and Its Contribution to Tolerance to Severe Vanillin Stress in Saccharomyces cerevisiae

    Ishida, Yoko; Nguyen, Trinh T. M.; Kitajima, Sakihito; Izawa, Shingo


    Vanillin is a potent fermentation inhibitor derived from the lignocellulosic biomass in biofuel production, and high concentrations of vanillin result in the pronounced repression of bulk translation in Saccharomyces cerevisiae. Studies on genes that are efficiently translated even in the presence of high concentrations of vanillin will be useful for improving yeast vanillin tolerance and fermentation efficiency. The BDH1 and BDH2 genes encode putative medium-chain alcohol dehydrogenase/reduc...

  6. Involvement of Sac1 phosphoinositide phosphatase in the metabolism of phosphatidylserine in the yeast Saccharomyces cerevisiae.

    Tani, Motohiro; Kuge, Osamu


    Sac1 is a phosphoinositide phosphatase that preferentially dephosphorylates phosphatidylinositol 4-phosphate. Mutation of SAC1 causes not only the accumulation of phosphoinositides but also reduction of the phosphatidylserine (PS) level in the yeast Saccharomyces cerevisiae. In this study, we characterized the mechanism underlying the PS reduction in SAC1-deleted cells. Incorporation of (32) P into PS was significantly delayed in sac1∆ cells. Such a delay was also observed in SAC1- and PS decarboxylase gene-deleted cells, suggesting that the reduction in the PS level is caused by a reduction in the rate of biosynthesis of PS. A reduction in the PS level was also observed with repression of STT4 encoding phosphatidylinositol 4-kinase or deletion of VPS34 encoding phophatidylinositol 3-kinase. However, the combination of mutations of SAC1 and STT4 or VPS34 did not restore the reduced PS level, suggesting that both the synthesis and degradation of phosphoinositides are important for maintenance of the PS level. Finally, we observed an abnormal PS distribution in sac1∆ cells when a specific probe for PS was expressed. Collectively, these results suggested that Sac1 is involved in the maintenance of a normal rate of biosynthesis and distribution of PS.

  7. The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

    Leem, S H; Ropp, P A; Sugino, A


    We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

  8. [Construction of high sulphite-producing industrial strain of Saccharomyces cerevisiae].

    Qu, Na; He, Xiu-ping; Guo, Xue-na; Liu, Nan; Zhang, Bo-run


    In the process of beer storage and transportation, off-flavor can be produced for oxidation of beer. Sulphite is important for stabilizing the beer flavor because of its antioxidant activity. However, the low level of sulphite synthesized by the brewing yeast is not enough to stabilize beer flavor. Three enzymes involve sulphite biosynthesis in yeast. One of them, APS kinase (encoded by MET14) plays important role in the process of sulphite formation. In order to construct high sulphite-producing brewing yeast strain for beer production, MET14 gene was cloned and overexpressed in industrial strain of Saccharomyces cerevisiae. Primer 1 (5'-TGTGAATTCCTGTACACCAATGGCTACT-3', EcoR I) and primer 2 (5'-TATAAGCTTGATGA GGTGGATGAAGACG-3', HindIII) were designed according to the MET14 sequence in GenBank. A 1.1kb DNA fragment containing the open reading frame and terminator of MET14 gene was amplified from Saccharomyces cerevisiae YSF-5 by PCR, and inserted into YEp352 to generate recombinant plasmid pMET14. To express MET14 gene properly in S. cerevisiae, the recombinant expression plasmids pPM with URA3 gene as the selection marker and pCPM with URA3 gene and copper resistance gene as the selection marker for yeast transformation were constructed. In plasmid pPM, the PGK1 promoter from plasmid pVC727 was fused with the MET14 gene from pMET14, and the expression cassette was inserted into the plasmid YEp352. The dominant selection marker, copper-resistance gene expression cassette CUP1-MTI was inserted in plasmid pPM to result in pCPM. Restriction enzyme analysis showed that plasmids pPM and pCPM were constructed correctly. The laboratory strain of S. cerevisiae YS58 with ura3, trp1, leu2, his4 auxotroph was transformed with plasmid pPM. Yeast transformants were screened on synthetic minimal medium (SD) containing leucine, histidine and tryptophan. The sulphite production of the transformants carrying pPM was 2 fold of that in the control strain YS58, which showed that the

  9. Electrical stimulation of saccharomyces cerevisiae cultures Estimulação elétrica de células de Saccharomyces cerevisiae

    Araújo,Ofelia Q.F.; Coelho, Maria Alice Z.; Margarit,Isabel C.P.; Vaz-Junior,Carlos A.; Maria Helena M. Rocha-Leão


    Modulation of cell endogenous membrane potential by an external electrical field influences the structure and function of membrane compartments, proteins and lipid bi-layer. In this work, the effects of applied potential on Saccharomyces cerevisiae growth were characterized through simple yet conclusive experiments. Cell growth time profile and cell division were investigated as macroscopic response to the electrical stimulation. Control experiments were conducted under identical conditions e...

  10. Overexpression of Sbe2p, a Golgi Protein, Results in Resistance to Caspofungin in Saccharomyces cerevisiae

    Osherov, Nir; May, Gregory S.; Albert, Nathaniel D.; Kontoyiannis, D. P.


    Caspofungin inhibits the synthesis of 1, 3-β-d-glucan, an essential cell wall target in fungi. Genetic studies in the model yeast Saccharomyces cerevisiae have shown that mutations in FKS1 and FKS2 genes result in caspofungin resistance. However, direct demonstration of the role of gene overexpression in caspofungin resistance has been lacking. We transformed wild-type S. cerevisiae with an S. cerevisiae URA3-based GAL1 cDNA library and selected transformants in glucose synthetic complete pla...

  11. Expression of the E.coli pntA and pntB genes encoding nicotinamide nucleotide transhydrogenase in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation

    Anderlund, M.; Nissen, Torben Lauesgaard; Nielsen, Jens Bredal;


    in reoxidation of NADH to NAD(+) in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD(+) by NADPH and by NADH in the presence of NADP(+), which demonstrated that an active enzyme...... was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase...... in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD(+) NADPH, and NADP(+) were measured in cells expressing...

  12. Chromosome VIII disomy influences the nonsense suppression efficiency and transition metal tolerance of the yeast Saccharomyces cerevisiae.

    Zadorsky, S P; Sopova, Y V; Andreichuk, D Y; Startsev, V A; Medvedeva, V P; Inge-Vechtomov, S G


    The SUP35 gene of the yeast Saccharomyces cerevisiae encodes the translation termination factor eRF3. Mutations in this gene lead to the suppression of nonsense mutations and a number of other pleiotropic phenotypes, one of which is impaired chromosome segregation during cell division. Similar effects result from replacing the S. cerevisiae SUP35 gene with its orthologues. A number of genetic and epigenetic changes that occur in the sup35 background result in partial compensation for this suppressor effect. In this study we showed that in S. cerevisiae strains in which the SUP35 orthologue from the yeast Pichia methanolica replaces the S. cerevisiae SUP35 gene, chromosome VIII disomy results in decreased efficiency of nonsense suppression. This antisuppressor effect is not associated with decreased stop codon read-through. We identified SBP1, a gene that localizes to chromosome VIII, as a dosage-dependent antisuppressor that strongly contributes to the overall antisuppressor effect of chromosome VIII disomy. Disomy of chromosome VIII also leads to a change in the yeast strains' tolerance of a number of transition metal salts.

  13. Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering.

    Chen, Yingying; Stabryla, Lisa; Wei, Na


    Development of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

  14. Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism

    Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi


    Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis. PMID:28241048

  15. Growth and fermentation characteristics of Saccharomyces cerevisiae NK28 isolated from kiwi fruit.

    Lee, Jong-Sub; Park, Eun-Hee; Kim, Jung-Wan; Yeo, Soo-Hwan; Kim, Myoung-Dong


    The influences of glucose concentration, initial medium acidity (pH), and temperature on the growth and ethanol production of Saccharomyces cerevisiae NK28, which was isolated from kiwi fruit, were examined in shake flask cultures. The optimal glucose concentration, initial medium pH, and temperature for ethanol production were 200 g/l, pH 6.0, and 35oC, respectively. Under this growth condition, S. cerevisiae NK28 produced 98.9 ± 5.67 g/l ethanol in 24 h with a volumetric ethanol production rate of 4.12 ± 0.24 g/l·h. S. cerevisiae NK28 was more tolerant to heat and ethanol than laboratory strain S. cerevisiae BY4742, and its tolerance to ethanol and fermentation inhibitors was comparable to that of an ethanologen, S. cerevisiae D5A.

  16. Oral administration of myostatin-specific recombinant Saccharomyces cerevisiae vaccine in rabbit.

    Liu, Zhongtian; Zhou, Gang; Ren, Chonghua; Xu, Kun; Yan, Qiang; Li, Xinyi; Zhang, Tingting; Zhang, Zhiying


    Yeast is considered as a simple and cost-effective host for protein expression, and our previous studies have proved that Saccharomyces cerevisiae can deliver recombinant protein and DNA into mouse dendritic cells and can further induce immune responses as novel vaccines. In order to know whether similar immune responses can be induced in rabbit by oral administration of such recombinant S. cerevisiae vaccine, we orally fed the rabbits with heat-inactivated myostatin-recombinant S. cerevisiae for 5 weeks, and then myostatin-specific antibody in serum was detected successfully by western blotting and ELISA assay. The rabbits treated with myostatin-recombinant S. cerevisiae vaccine grew faster and their muscles were much heavier than that of the control group. As a common experimental animal and a meat livestock with great economic value, rabbit was proved to be the second animal species that have been successfully orally immunized by recombinant S. cerevisiae vaccine after mice.

  17. Clinical Saccharomyces cerevisiae isolates cannot cross the epithelial barrier in vitro.

    Pérez-Torrado, Roberto; Llopis, Silvia; Jespersen, Lene; Fernández-Espinar, Teresa; Querol, Amparo


    Saccharomyces cerevisiae is generally considered to be a safe organism and is essential to produce many different kinds of foods as well as being widely used as a dietary supplement. However, several isolates, which are genetically related to brewing and baking yeasts, have shown virulent traits, being able to produce human infections in immunodeficient patients. Previously it has been shown that the administration of S. cerevisiae clinical isolates can lead to systemic infections, reaching several organs in murine systems. In this work, we studied S. cerevisiae clinical isolates in an in vitro intestinal epithelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. The results showed that, in contrast to C. glabrata and C. albicans, S. cerevisiae was not able to cross the intestinal barrier. We concluded that S. cerevisiae can only perform opportunistic or passive crossings when epithelial barrier integrity is previously compromised.

  18. Elucidating TOR Signaling and Rapamycin Action: Lessons from Saccharomyces cerevisiae

    Crespo, José L; Hall, Michael N.


    TOR (target of rapamycin) is a phosphatidylinositol kinase-related protein kinase that controls cell growth in response to nutrients. Rapamycin is an immunosuppressive and anticancer drug that acts by inhibiting TOR. The modes of action of TOR and rapamycin are remarkably conserved from S. cerevisiae to humans. The current understanding of TOR and rapamycin is derived largely from studies with S. cerevisiae. In this review, we discuss the contributions made by S. cerevisiae to understanding r...

  19. Physiology of Saccharomyces cerevisiae during cell cycle oscillations.

    Duboc, P; Marison, I; von Stockar, U


    Synchronized populations of Saccharomyces cerevisiae CBS 426 are characterized by autonomous oscillations of process variables. CO2 evolution rate, O2 uptake rate and heat production rate varied by a factor of 2 for a continuous culture grown at a dilution rate of 0.10 h-1. Elemental analysis showed that the carbon mass fraction of biomass did not change. Since the reactor is not at steady state, the elemental and energy balances were calculated on cumulated quantities, i.e. the integral of the reaction rates. It was possible to show that carbon, degree of reduction and energy balances matched. Application of simple mass balance principles for non-steady state systems indicated that oscillations were basically characterized by changes in biomass production rate. In addition, the amount of intermediates, e.g. ethanol or acetate, produced or consumed was negligible. Growth rate was low during the S-phase (0.075 h-1) and high during the G2, M and G1 phases (0.125 h-1) for a constant dilution rate of 0.10 h-1. However, nitrogen, ash, sulfur and potassium content showed systematic increases during the S-phase (bud initiation). Cell component analyses showed that changes in cellular fractions during oscillations (storage carbohydrate content decreased during the S-phase) were due to changes in production rates, particularly for protein and carbohydrates. Nevertheless, using the data evaluation techniques for dynamic systems presented here, it was shown that storage carbohydrates are not consumed during the S-phase. Only the synthesis rate of the different cell components changed depending on position in cell cycle. The growth process may be divided into two phenomena: the formation of new cells during mitosis with a low yield, and size increase of new born cells with high yield. Both kinetic and stoichiometric coefficients varied with the position in the oscillation: the results showed that biomass structure changed and that specific growth rate, as well as biomass yield

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

    Aymé Spor

    Full Text Available 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

  1. Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1

    Zhou Cong-Zhao


    Full Text Available Abstract Background As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG. Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications. Results The crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1 from Saccharomyces cerevisiae has been determined at 2.37 Å resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-α helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 ± 9 μM for 6-phosphogluconate and of 35 ± 6 μM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution. Conclusion The overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not

  2. Engineering of Saccharomyces cerevisiae for production of resveratrol and its derivatives

    Li, Mingji

    Resveratrol is a natural potent antioxidant with multiple beneficial effects on human health and is therefore used in medical, food, and cosmetic areas. In my PhD thesis I describe how I engineered yeast cell factory Saccharomyces cerevisiae for production of resveratrol by fermentation of cheap ...

  3. Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of L-Arabinose

    Wisselink, H.W.; Toirkens, M.J.; Del Rosario Franco Berriel, M.; Winkler, A.A.; Van Dijken, J.P.; Pronk, J.T.; Van Maris, A.J.A.


    For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as L-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organ

  4. Separate and Simultaneous enzymatic hydrolysis and fermentation of wheat hemicellulose with recombinant xylose utilizing Saccharomyces cerevisiae

    Olsson, Lisbeth; Sørensen, H. R.; Dam, B. P;


    Fermentations with three different xylose-utilizing recombinant Saccharomyces cerevisiae strains (F12, CR4, and CB4) were performed using two different wheat hemicellulose substrates, unfermented starch free fibers, and an industrial ethanol fermentation residue, vinasse. With CR4 and F12...

  5. amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae

    Solis-Escalante, D.; Kuijpers, N.G.A.; Bongaerts, N.; Bolat, I.; Bosman, L.; Pronk, J.T.; Daran, J.M.; Daran-Lapujade, P.A.S.


    Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, ar

  6. Growth-rate regulated genes have profound impact on interpretation of transcriptome profiling in Saccharomyces cerevisiae

    Regenberg, Birgitte; Grotkjaer, Thomas; Winther, Ole


    Growth rate is central to the development of cells in all organisms. However, little is known about the impact of changing growth rates. We used continuous cultures to control growth rate and studied the transcriptional program of the model eukaryote Saccharomyces cerevisiae, with generation time...

  7. Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains

    Baleiras Couto, M.M.; Eijsma, B.; Hofstra, H.; Huis in 't Veld, J.H.J.; Vossen, J.M.B.M. van der


    Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligon

  8. Implementation of communication-mediating domains for non-ribosomal peptide production in Saccharomyces cerevisiae

    Siewers, Verena; San-Bento, Rita; Nielsen, Jens


    Saccharomyces cerevisiae has in several cases been proven to be a suitable host for the production of natural products and was recently exploited for the production of non-ribosomal peptides. Synthesis of non-ribosomal peptides (NRPs) is mediated by NRP synthetases (NRPSs), modular enzymes, which...

  9. Natural and modified promoters for tailored metabolic engineering of the yeast Saccharomyces cerevisiae

    Hubmann, Georg; Thevelein, Johan M; Nevoigt, Elke


    The ease of highly sophisticated genetic manipulations in the yeast Saccharomyces cerevisiae has initiated numerous initiatives towards development of metabolically engineered strains for novel applications beyond its traditional use in brewing, baking, and wine making. In fact, baker's yeast has be

  10. Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae

    Silljé, H H; Paalman, J W; ter Schure, E G; Olsthoorn, S Q; Verkleij, A J; Boonstra, Johannes; Verrips, C T


    Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. B

  11. The role of the nutrients in G1 phase progression of Saccharomyces cerevisiae

    Paalman, Johannes Wilhelmus Gerardus


    Organisms that have optimally integrated fast reproduction with high survival have a strategic advantage over less adapted species during evolution. This thesis focusses on the main decision point in the life cycle of the yeast Saccharomyces cerevisiae, at which the cell decides to rapidly grow and

  12. Ctk1 function is necessary for full translation initiation activity in Saccharomyces cerevisiae

    Coordes, Britta; Brünger, Katharina M; Burger, Kaspar;


    Translation is a fundamental and highly regulated cellular process. Previously, we reported that the kinase and transcription elongation factor Ctk1 increases fidelity during translation elongation in Saccharomyces cerevisiae. Here, we show that loss of Ctk1 function also affects the initiation s...

  13. A mathematical model of the mating signal transduction pathway in the yeast Saccharomyces cerevisiae. Final report

    Thomas Ivan Milac


    Outline of two major goals in my proposal for this fellowship. First goal having no previous training in biology, was to become knowledgeable of the paradigms, experimental techniques, and current research interests of molecular biology. Second goal was to construct a mathematical model of the mating signal transduction pathway in the yeast Saccharomyces cerevisiae.

  14. Glucose and maltose metabolism in MIG1-disrupted and MAL-constitutive strains of Saccharomyces cerevisiae

    Klein, Christopher; Olsson, Lisbeth; Rønnow, B;


    The alleviation of glucose control of maltose metabolism brought about by MIG1 disruption was compared to that by MAL overexpression in a haploid Saccharomyces cerevisiae strain. The sugar consumption profiles during cultivation of the wild type, single transformants and a double transformant in ...

  15. DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

    Pedersen, Jakob Madsen; Fredsøe, Jacob Christian; Rødgaard, Morten Terpager;


    To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-re...... transcriptional activation of genes with a repressible/inducible mode of regulation....

  16. Conditions With High Intracellular Glucose Inhibit Sensing Through Glucose Sensor Snf3 in Saccharomyces cerevisiae

    Karhumaa, Kaisa; Wu, B.Q.; Kielland-Brandt, Morten


    Gene expression in micro-organisms is regulated according to extracellular conditions and nutrient concentrations. In Saccharomyces cerevisiae, non-transporting sensors with high sequence similarity to transporters, that is, transporter-like sensors, have been identified for sugars as well...

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


    developing subunit dengue vaccines or recombinant live viral vaccines. Subunit vaccines may eventually include synthetic dengue peptides or recombinant... dengue proteins expressed in microorganisms, and live viral vectors such as vaccinia may express in vivo immunogenic dengue peptides . Durin...PRODUCTION OF DENGUE 2 ENVELOPE PROTEIN IN THE YEAST SACCHAROMYCES CEREVISIAE FINAL, PHASE I REPORT JOHN M. IVY KATHY HOUTCHENS FEBRUARY 15, 1990

  18. Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carbosylation, oxaloacetate reduction and malate export

    Zelle, R.M.; Hulster, de E.; Winden, van W.A.; Waard, de P.; Dijkema, C.; Winkler, A.A.; Geertman, J.M.A.


    Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production

  19. Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export

    Zelle, R.M.; De Hulster, E.; Van Winden, W.A.; De Waard, P.; Dijkema, C.; Winkler, A.A.; Geertman, J.M.; Van Dijken, J.P.; Pronk, J.T.; Van Maris, A.J.A.


    Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production

  20. The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix

    Li, M; Phylip, L H; Lees, W E;


    Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2...

  1. An oxalyl-CoA synthetase is important for oxalate metabolism in Saccharomyces cerevisiae

    Although oxalic acid is common in nature, our understanding of the mechanism(s) regulating its turnover remains incomplete. In this study we identify Saccharomyces cerevisiae acyl-activating enzyme 3 (ScAAE3) as an enzyme capable of catalyzing the conversion of oxalate to oxalyl-CoA. Based on our fi...

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

    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...

  3. Investigating genotype-phenotype relationships in Saccharomyces cerevisiae metabolic network through stoichiometric modeling

    Brochado, Ana Rita

    . This chapter aims at providing the reader with relevant state-of-the-art information concerning Systems Biology, Genome-Scale Metabolic Modeling and Metabolic Engineering. Particular attention is given to the yeast Saccharomyces cerevisiae, the eukaryotic model organism used thought the thesis....

  4. Dissection of transcriptional regulation networks and prediction of gene functions in Saccharomyces cerevisiae

    Boorsma, A.


    Molecular biology aims to unravel the functions of cells by studying cellular processes at the molecular level. Amodel organism that is well established in molecular biology is bakers yeast (Saccharomyces cerevisiae). Bakers yeast cells are remarkably similar to human cells, but much easier to grow

  5. The significance of peroxisome function in chronological aging of Saccharomyces cerevisiae

    Lefevre, Sophie D.; van Roermund, Carlo W.; Wanders, Ronald J. A.; Veenhuis, Marten; van der Klei, Ida J.


    Summary We studied the chronological lifespan of glucose-grown Saccharomyces cerevisiae in relation to the function of intact peroxisomes. We analyzed four different peroxisome-deficient (pex) phenotypes. These included Delta pex3 cells that lack peroxisomal membranes and in which all peroxisomal pr

  6. Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae.

    Hill, A.; Bloom, K.


    The introduction of a conditional centromere into chromosome III of Saccharomyces cerevisiae provided an opportunity to evaluate phenotypic and karyotypic consequences in cells harboring dicentric chromosomes upon entry into mitosis. A mitotic pause ensued, and monocentric derivatives of chromosome III were generated at a high frequency.

  7. Genome-wide transcription survey on flavour production in Saccharomyces cerevisiae

    Schoondermark-Stolk, S.A.; Jansen, M.D.; Verkleij, A.J.; Verrips, C.T.; Euverink, G.J.W.; Dijkhuizen, L.; Boonstra, J.


    The yeast Saccharomyces cerevisiae is widely used as aroma producer in the preparation of fermented foods and beverages. During food fermentations, secondary metabolites like 3-methyl-1-butanol, 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutanoate and 3-methylbutyrate emerge. These four compounds have

  8. One-hybrid screens at the Saccharomyces cerevisiae HMR locus identify novel transcriptional silencing factors.

    Andrulis, Erik D; Zappulla, David C; Alexieva-Botcheva, Krassimira; Evangelista, Carlos; Sternglanz, Rolf


    In Saccharomyces cerevisiae, genes located at the telomeres and the HM loci are subject to transcriptional silencing. Here, we report results of screening a Gal4 DNA-binding domain hybrid library for proteins that cause silencing when targeted to a silencer-defective HMR locus. PMID:15020450

  9. Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

    Romagnoli, G.; Luttik, M.A.H.; Kötter, P.; Pronk, J.T.; Daran, J.M.


    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share seque

  10. Metabolism of phosphatidylcholine and its implications for lipid acyl chain composition in Saccharomyces cerevisiae

    de Kroon, A.I.P.M.


    Phosphatidylcholine (PC) is a very abundant membrane lipid in most eukaryotes including the model organism Saccharomyces cerevisiae. Consequently, the molecular species profile of PC, i.e. the ensemble of PC molecules with acyl chains differing in number of carbon atoms and double bonds, is importan

  11. PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae.

    Xu, Haiqing; Kim, Sooah; Sorek, Hagit; Lee, Youngsuk; Jeong, Deokyeol; Kim, Jungyeon; Oh, Eun Joong; Yun, Eun Ju; Wemmer, David E; Kim, Kyoung Heon; Kim, Soo Rin; Jin, Yong-Su


    The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

  12. Mixing of vineyard and oak-tree ecotypes of Saccharomyces cerevisiae in North American vineyards.

    Hyma, Katie E; Fay, Justin C


    Humans have had a significant impact on the distribution and abundance of Saccharomyces cerevisiae through its widespread use in beer, bread and wine production. Yet, similar to other Saccharomyces species, S. cerevisiae has also been isolated from habitats unrelated to fermentations. Strains of S. cerevisiae isolated from grapes, wine must and vineyards worldwide are genetically differentiated from strains isolated from oak-tree bark, exudate and associated soil in North America. However, the causes and consequences of this differentiation have not yet been resolved. Historical differentiation of these two groups may have been influenced by geographic, ecological or human-associated barriers to gene flow. Here, we make use of the relatively recent establishment of vineyards across North America to identify and characterize any active barriers to gene flow between these two groups. We examined S. cerevisiae strains isolated from grapes and oak trees within three North American vineyards and compared them to those isolated from oak trees outside of vineyards. Within vineyards, we found evidence of migration between grapes and oak trees and potential gene flow between the divergent oak-tree and vineyard groups. Yet, we found no vineyard genotypes on oak trees outside of vineyards. In contrast, Saccharomyces paradoxus isolated from the same sources showed population structure characterized by isolation by distance. The apparent absence of ecological or genetic barriers between sympatric vineyard and oak-tree populations of S. cerevisiae implies that vineyards play an important role in the mixing between these two groups.

  13. Growth temperature exerts differential physiological and transcriptional responses in laboratory and wine strains of Saccharomyces cerevisiae

    Pizarra, Francisco J.; Jewett, Michael Christopher; Nielsen, Jens;


    Laboratory strains of Saccharomyces cerevisiae have been widely used as a model for studying eukaryotic cells and mapping the molecular mechanisms of many different human diseases. Industrial wine yeasts, on the other hand, have been selected on the basis of their adaptation to stringent environm......Laboratory strains of Saccharomyces cerevisiae have been widely used as a model for studying eukaryotic cells and mapping the molecular mechanisms of many different human diseases. Industrial wine yeasts, on the other hand, have been selected on the basis of their adaptation to stringent......-limited, anaerobic, steady-state chemostat cultures. Physiological characterization revealed that the growth temperature strongly impacted the biomass yield of both strains. Moreover, we found that the wine yeast was better adapted to mobilizing resources for biomass production and that the laboratory yeast...... global insight into how growth temperature affects differential physiological and transcriptional responses in laboratory and wine strains of S. cerevisiae....

  14. Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

    Carlsen, Simon; Ajikumar, Parayil Kumaran; Formenti, Luca Riccardo;


    the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered...... yeast showed that the pathway is active until the synthesis of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe–4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two...... steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U–13C6 glucose and growth experiments, we found that the ISC assembly...

  15. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Brian H. Carrick


    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  16. Improvement of oxidized glutathione fermentation by thiol redox metabolism engineering in Saccharomyces cerevisiae.

    Hara, Kiyotaka Y; Aoki, Naoko; Kobayashi, Jyumpei; Kiriyama, Kentaro; Nishida, Keiji; Araki, Michihiro; Kondo, Akihiko


    Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase ( genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

  17. Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols.

    Sheng, Jiayuan; Stevens, Joseph; Feng, Xueyang


    Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6-C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 &C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.

  18. Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield.

    Basso, Thiago O; de Kok, Stefan; Dario, Marcelo; do Espirito-Santo, Júlio Cézar A; Müller, Gabriela; Schlölg, Paulo S; Silva, Carlos P; Tonso, Aldo; Daran, Jean-Marc; Gombert, Andreas K; van Maris, Antonius J A; Pronk, Jack T; Stambuk, Boris U


    Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

  19. Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress.

    Rodríguez-Vargas, Sonia; Sánchez-García, Alicia; Martínez-Rivas, Jose Manuel; Prieto, Jose Antonio; Randez-Gil, Francisca


    Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Delta(9) position. We expressed two sunflower (Helianthus annuus) oleate Delta(12) desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Delta(9,12), the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15 degrees C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp(+) or Trp(-) strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30 degrees C or 15 degrees C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.

  20. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

    Carrick, Brian H; Hao, Linxuan; Smaldino, Philip J; Engelke, David R


    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  1. Transcription of hexose transporters of Saccharomyces cerevisiae is affected by change in oxygen provision

    Ruohonen Laura


    Full Text Available Abstract Background The gene family of hexose transporters in Saccharomyces cerevisiae consists of 20 members; 18 genes encoding transporters (HXT1-HXT17, GAL2 and two genes encoding sensors (SNF3, RGT2. The effect of oxygen provision on the expression of these genes was studied in glucose-limited chemostat cultivations (D = 0.10 h-1, pH 5, 30°C. Transcript levels were measured from cells grown in five steady state oxygen levels (0, 0.5, 1, 2.8 and 20.9% O2, and from cells under conditions in which oxygen was introduced to anaerobic cultures or removed from cultures receiving oxygen. Results The expression pattern of the HXT gene family was distinct in cells grown under aerobic, hypoxic and anaerobic conditions. The transcription of HXT2, HXT4 and HXT5 was low when the oxygen concentration in the cultures was low, both under steady state and non-steady state conditions, whereas the expression of HXT6, HXT13 and HXT15/16 was higher in hypoxic than in fully aerobic or anaerobic conditions. None of the HXT genes showed higher transcript levels in strictly anaerobic conditions. Expression of HXT9, HXT14 and GAL2 was not detected under the culture conditions studied. Conclusion When oxygen becomes limiting in a glucose-limited chemostat cultivation, the glucose uptake rate per cell increases. However, the expression of none of the hexose transporter encoding genes was increased in anaerobic conditions. It thus seems that the decrease in the moderately low affinity uptake and consequently the relative increase of high affinity uptake may itself allow the higher specific glucose consumption rate to occur in anaerobic compared to aerobic conditions.

  2. A synthetic suicide riboswitch for the high-throughput screening of metabolite production in Saccharomyces cerevisiae.

    Lee, Sang-Woo; Oh, Min-Kyu


    Artificial devices such as the synthetic riboswitch have shown potential to introduce unnatural phenotypic perturbation because its synthetic traits are distinct from that of innate metabolism. In this study, a riboswitch, a small regulatory element found in RNAs, was employed to reprogram microorganisms to produce valuable metabolites. A self-cleaving ribozyme glmS, found in gram-positive bacteria, cleaves its own transcript in response to the intracellular glucosamine 6-phosphate (GlcN6P) concentration. The glmS ribozyme was integrated into the 3'-untranslated region of FCY1, which encodes cytosine deaminase in Saccharomyces cerevisiae to construct a suicide riboswitch for evolutionary engineering. Growth of the strain harboring the suicide riboswitch was hampered by the addition of fluorocytosine, and was recovered as metabolite level increased. By using this riboswitch, we isolated a N-acetyl glucosamine (GlcNAc) producer strain by screening an efficient glutamine-fructose-6-phosphate transaminase (Gfa1p) and haloacid dehalogenase-like phosphatases (HAD phosphatases) originated from Escherichia coli. The suicide riboswitch was also applied to different metabolite by using artificial allosteric ribozyme. Since the mechanisms used in this work are universal in microorganisms, our synthetic suicide riboswitch can be applied to a wide range of organisms and can be exploited to the efficient and high-throughput screening of inconspicuous phenotypes.

  3. Mutants of Saccharomyces cerevisiae with defects in acetate metabolism: isolation and characterization of Acn- mutants.

    McCammon, M T


    The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn- ("ACetate Nonutilizing") mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism.

  4. Quantitative transcriptome, proteome, and sulfur metabolite profiling of the Saccharomyces cerevisiae response to arsenite.

    Thorsen, Michael; Lagniel, Gilles; Kristiansson, Erik; Junot, Christophe; Nerman, Olle; Labarre, Jean; Tamás, Markus J


    Arsenic is ubiquitously present in nature, and various mechanisms have evolved enabling cells to evade toxicity and acquire tolerance. Herein, we explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent arsenic (arsenite) by quantitative transcriptome, proteome, and sulfur metabolite profiling. Arsenite exposure affected transcription of genes encoding functions related to protein biosynthesis, arsenic detoxification, oxidative stress defense, redox maintenance, and proteolytic activity. Importantly, we observed that nearly all components of the sulfate assimilation and glutathione biosynthesis pathways were induced at both gene and protein levels. Kinetic metabolic profiling evidenced a significant increase in the pools of sulfur metabolites as well as elevated cellular glutathione levels. Moreover, the flux in the sulfur assimilation pathway as well as the glutathione synthesis rate strongly increased with a concomitant reduction of sulfur incorporation into proteins. By combining comparative genomics and molecular analyses, we pinpointed transcription factors that mediate the core of the transcriptional response to arsenite. Taken together, our data reveal that arsenite-exposed cells channel a large part of assimilated sulfur into glutathione biosynthesis, and we provide evidence that the transcriptional regulators Yap1p and Met4p control this response in concert.

  5. Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains.

    Kato, Hiroko; Suyama, Hiroaki; Yamada, Ryosuke; Hasunuma, Tomohisa; Kondo, Akihiko


    To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.

  6. Glucose-based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae.

    Germann, Susanne M; Baallal Jacobsen, Simo A; Schneider, Konstantin; Harrison, Scott J; Jensen, Niels B; Chen, Xiao; Stahlhut, Steen G; Borodina, Irina; Luo, Hao; Zhu, Jiangfeng; Maury, Jérôme; Forster, Jochen


    Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N-acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L-tryptophan hydroxylase, a 5-hydroxy-L-tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O-methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co-factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L(-1) in a 76h fermentation using simulated fed-batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin.

  7. Change in activity of serine palmitoyltransferase affects sensitivity to syringomycin E in yeast Saccharomyces cerevisiae.

    Toume, Moeko; Tani, Motohiro


    Syringomycin E is a cyclic lipodepsipeptide produced by strains of the plant bacterium Pseudomonas syringae pv. syringae. Genetic studies involving the yeast Saccharomyces cerevisiae have revealed that complex sphingolipids play important roles in the action of syringomycin E. Here, we found a novel mutation that confers resistance to syringomycin E on yeast; that is, a deletion mutant of ORM1 and ORM2, which encode negative regulators of serine palmitoyltransferase catalyzing the initial step of sphingolipid biosynthesis, exhibited resistance to syringomycin E. On the contrary, overexpression of Orm2 resulted in high sensitivity to the toxin. Moreover, overexpression of Lcb1 and Lcb2, catalytic subunits of serine palmitoyltransferase, causes resistance to the toxin, whereas partial repression of expression of Lcb1 had the opposite effect. Partial reduction of complex sphingolipids by repression of expression of Aur1, an inositol phosphorylceramide synthase, also resulted in high sensitivity to the toxin. These results suggested that an increase in sphingolipid biosynthesis caused by a change in the activity of serine palmitoyltransferase causes resistance to syringomycin E.

  8. IMP2, a gene involved in the expression of glucose-repressible genes in Saccharomyces cerevisiae.

    Lodi, T; Goffrini, P; Ferrero, I; Donnini, C


    Two mutants carrying different deletions of the IMP2 coding sequence of Saccharomyces cerevisiae, delta T1, which encodes a protein lacking the last 26 C-terminal amino acids, and delta T2, which completely lacks the coding region, were analysed for derepression of glucose-repressible maltose, galactose, raffinose and ethanol utilization pathways in response to glucose limitation. The role of the IMP2 gene product in the regulation of carbon catabolite repressible enzymes maltase, invertase, alcohol dehydrogenase, NAD-dependent glutamate dehydrogenase (NAD-GDH) and L-lactate:ferricytochrome-c oxidoreductase (L-LCR) was also analysed. The IMP2 gene product is required for the rapid glucose derepression of all above-mentioned carbon source utilization pathways and of all the enzymes except for L-LCR. NAD-GDH is regulated by IMP2 in the opposite way and, in fact, this enzyme was released at higher levels in both imp2 mutants than in the wild-type strain. Therefore, the product of IMP2 appears to be involved in positive and negative regulation. Both deletions result in growth and catalytic defects; in some cases partial modification of the gene product yielded more dramatic effects than its complete absence. Moreover, evidence is provided that the IMP2 gene product regulates galactose- and maltose-inducible genes at the transcriptional level and is a positive regulator of maltase, maltose permease and galactose permease gene expression.

  9. Requirements for E1A dependent transcription in the yeast Saccharomyces cerevisiae

    Mymryk Joe S


    Full Text Available Abstract Background The human adenovirus type 5 early region 1A (E1A gene encodes proteins that are potent regulators of transcription. E1A does not bind DNA directly, but is recruited to target promoters by the interaction with sequence specific DNA binding proteins. In mammalian systems, E1A has been shown to contain two regions that can independently induce transcription when fused to a heterologous DNA binding domain. When expressed in Saccharomyces cerevisiae, each of these regions of E1A also acts as a strong transcriptional activator. This allows yeast to be used as a model system to study mechanisms by which E1A stimulates transcription. Results Using 81 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, CSR, INO80, ISW1, NuA3, NuA4, Mediator, PAF, RSC, SAGA, SAS, SLIK, SWI/SNF and SWR1 transcriptional regulatory complexes on E1A dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on transcriptional activation. Conclusion Our analysis indicates that the two activation domains of E1A function via distinct mechanisms, identify new factors regulating E1A dependent transcription and suggest that yeast can serve as a valid model system for at least some aspects of E1A function.

  10. Heterologous Expression of Amylase Gene from Saccharomycopsis fibuligera in an Industrial Strain of Saccharomyces cerevisiae

    LIU Zeng-ran; ZHANG Guang-yi; LONG Zhang-fu; LIU Shi-gui


    An α-amylase encoding gene was amplified by polymerase chain reaction from Saccharomycopsis fibuligera and inserted into a shuttle vector YEp352,together with the yeast phosphoglycerate kinase 1 promoter and α-factor signal gene. The recombinant expression plasmid pLA8α was transformed into an industrial strain of Saccharomyces cerevisiae Sc-11. The activity of the α-amylase produced by the transformant Sc-11-pLA8α was 6.3 U/mL and the starch utilization rate in YPS medium was 42 %. The purified amylase was analyzed by SDS-PAGE,showing a molecular weight of 55×103 protein band. Furthermore, the residual sugar, ethanol and some volatile compounds in the fermented worts under simulating brewing conditions were determined by chromatographic analyses. The fermentation characteristics of Sc-11-pLA8α were similar to that of Sc-11 and only minor changes in the concentration of flavor compounds could be observed.

  11. Overexpression of ESBP6 improves lactic acid resistance and production in Saccharomyces cerevisiae.

    Sugiyama, Minetaka; Akase, Shin-Pei; Nakanishi, Ryota; Kaneko, Yoshinobu; Harashima, Satoshi


    Polylactic acid plastics are receiving increasing attention for the control of atmospheric CO2 emissions. Lactic acid, the building block for polylactic acid, is produced by fermentation technology from renewable carbon sources. The yeast Saccharomyces cerevisiae, harboring the lactate dehydrogenases gene (LDH), produces lactic acid at a large scale due to its strong acid resistance, to its simple nutritional requirements and to its ease of genetic engineering. Since improvement of lactic acid resistance is correlated with an increase of lactic acid production under non-neutralizing condition, we isolated a novel gene that enhances lactic acid resistance using a multi-copy yeast genomic DNA library. In this study, we identified the ESBP6 gene, which increases lactic acid resistance when overexpressed and which encodes a protein with similarity to monocarboxylate permeases. Although ESBP6 was not induced in response to lactic acid stress, it caused weak but reproducible sensitivity to lactic acid when disrupted. Furthermore, intracellular pH in the ESBP6 overexpressing strain was higher than that in the wild-type strain under lactic acid stressed condition, suggesting that Esbp6 plays some roles in lactic acid adaptation response. The ESBP6 overexpressing strain carrying the LDH gene induced 20% increase in lactic acid production compared with the wild-type strain carrying the LDH gene under non-neutralizing conditions. These results indicate that overexpression of ESBP6 provides a novel and useful tool to improve lactic acid resistance and lactic acid production in yeast.

  12. Identification of Genes in Saccharomyces cerevisiae that Are Haploinsufficient for Overcoming Amino Acid Starvation

    Nancy S. Bae


    Full Text Available The yeast Saccharomyces cerevisiae responds to amino acid deprivation by activating a pathway conserved in eukaryotes to overcome the starvation stress. We have screened the entire yeast heterozygous deletion collection to identify strains haploinsufficient for growth in the presence of sulfometuron methyl, which causes starvation for isoleucine and valine. We have discovered that cells devoid of MET15 are sensitive to sulfometuron methyl, and loss of heterozygosity at the MET15 locus can complicate screening the heterozygous deletion collection. We identified 138 cases of loss of heterozygosity in this screen. After eliminating the issues of the MET15 loss of heterozygosity, strains isolated from the collection were retested on sulfometuron methyl. To determine the general effect of the mutations for a starvation response, SMM-sensitive strains were tested for the ability to grow in the presence of canavanine, which induces arginine starvation, and strains that were MET15 were also tested for growth in the presence of ethionine, which causes methionine starvation. Many of the genes identified in our study were not previously identified as starvation-responsive genes, including a number of essential genes that are not easily screened in a systematic way. The genes identified span a broad range of biological functions, including many involved in some level of gene expression. Several unnamed proteins have also been identified, giving a clue as to possible functions of the encoded proteins.

  13. Identification of the fifth subunit of Saccharomyces cerevisiae replication factor C.

    Gary, S L; Burgers, M J


    Yeast replication factor C (RF-C) is a multipolypeptide complex required for chromosomal DNA replication. Previously this complex was known to consist of at least four subunits. We here report the identification of a fifth RF-C subunit from Saccharomyces cerevisiae, encoded by the RFC5 (YBR0810) gene. This subunit exhibits highest homology to the 38 kDa subunit (38%) of human RF-C (activator 1). Like the other four RFC genes, the RFC5 gene is essential for yeast viability, indicating an essential function for each subunit. RFC5 mRNA is expressed at steady-state levels throughout the mitotic cell cycle. Upon overexpression in Escherichia coli Rfc5p has an apparent molecular mass of 41 kDa. Overproduction of RF-C activity in yeast is dependent on overexpression of the RFC5 gene together with overexpression of the RFC1-4 genes, indicating that the RFC5 gene product forms an integral subunit of this replication factor. Images PMID:8559655

  14. A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae.

    Bae, Jung-Hoon; Sung, Bong Hyun; Seo, Jeong-Woo; Kim, Chul Ho; Sohn, Jung-Hoon


    Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.

  15. In vitro screening of probiotic properties of Saccharomyces cerevisiae var. boulardii and food-borne Saccharomyces cerevisiae strains

    van der Aa Kuhle, Alis; Skovgaard, Kerstin; Jespersen, Lene


    strain when the cells were pre- and coincubated with S. cerevisiae var. boulardii even though this yeast strain was low adhesive (5.4%), suggesting that adhesion is not a mandatory prerequisite for such a probiotic effect. A strain of S. cerevisiae isolated from West African sorghum beer exerted similar...

  16. Systems Biology of Saccharomyces cerevisiae Physiology and its DNA Damage Response

    Fazio, Alessandro

    damage response (Chapter 5). When DNA damage is repaired, cells restart the cell cycle and resume growth. This process is called damage recovery. In S. cerevisiae, the molecular mechanism of recovery relies on dephosphorylation of Rad53 by protein phosphatases (PPs), that, in case of recovery from MMS......The yeast Saccharomyces cerevisiae is a model organism in biology, being widely used in fundamental research, the first eukaryotic organism to be fully sequenced and the platform for the development of many genomics techniques. Therefore, it is not surprising that S. cerevisiae has also been widely...... used in the field of systems biology during the last decade. This thesis investigates S. cerevisiae growth physiology and DNA damage response by using a systems biology approach. Elucidation of the relationship between growth rate and gene expression is important to understand the mechanisms regulating...

  17. Opportunistic strains of Saccharomyces cerevisiae: a potential risk sold in food products

    Roberto ePérez-Torrado


    Full Text Available In recent decades, fungal infections have emerged as an important health problem associated with more people who present deficiencies in the immune system, such as HIV or transplanted patients. Saccharomyces cerevisiae is one of the emerging fungal pathogens with a unique characteristic: its presence in many food products. S. cerevisiae has an impeccably good food safety record compared to other microorganisms like virus, bacteria and some filamentous fungi. However, humans unknowingly and inadvertently ingest large viable populations of S. cerevisiae (home-brewed beer or dietary supplements that contain yeast. In the last few years, researchers have studied the nature of S. cerevisiae strains and the molecular mechanisms related to infections. Here we review the last advance made in this emerging pathogen and we discuss the implication of using this species in food products.

  18. Removal of Strontium Ions by Immobilized Saccharomyces Cerevisiae in Magnetic Chitosan Microspheres

    Yanan Yin


    Full Text Available A novel biosorbent, immobilized Saccharomyces cerevisiae in magnetic chitosan microspheres was prepared, characterized, and used for the removal of Sr2+ from aqueous solution. The structure and morphology of immobilized S. cerevisiae before and after Sr2+adsorption were observed using scanning electron microscopy with energy dispersive X-ray spectroscopy. The experimental results showed that the Langmuir and Freundlich isotherm models could be used to describe the Sr2+ adsorption onto immobilized S. cerevisiae microspheres. The maximal adsorption capacity (qm was calculated to be 81.96 mg/g by the Langmuir model. Immobilized S. cerevisiae was an effective adsorbent for the Sr2+ removal from aqueous solution.

  19. Stress Tolerance Variations in Saccharomyces cerevisiae Strains from Diverse Ecological Sources and Geographical Locations.

    Yan-Lin Zheng

    Full Text Available The budding yeast Saccharomyces cerevisiae is a platform organism for bioethanol production from various feedstocks and robust strains are desirable for efficient fermentation because yeast cells inevitably encounter stressors during the process. Recently, diverse S. cerevisiae lineages were identified, which provided novel resources for understanding stress tolerance variations and related shaping factors in the yeast. This study characterized the tolerance of diverse S. cerevisiae strains to the stressors of high ethanol concentrations, temperature shocks, and osmotic stress. The results showed that the isolates from human-associated environments overall presented a higher level of stress tolerance compared with those from forests spared anthropogenic influences. Statistical analyses indicated that the variations of stress tolerance were significantly correlated with both ecological sources and geographical locations of the strains. This study provides guidelines for selection of robust S. cerevisiae strains for bioethanol production from nature.

  20. A dynamic flux balance model and bottleneck identification of glucose, xylose, xylulose co-fermentation in Saccharomyces cerevisiae

    Economically viable production of lignocellulosic ethanol requires efficient conversion of feedstock sugars to ethanol. Saccharomyces cerevisiae cannot ferment xylose, the main five-carbon sugars in biomass, but can ferment xylulose, an enzymatically derived isomer. Xylulose fermentation is slow rel...

  1. Study on the Growth-promoting Effect of Pseudosciaena crocea with Saccharomyces cerevisiae P13%植物乳杆菌Saccharomyces cerevisiae P13对大黄鱼的促生长作用研究

    廖志勇; 缪雄伟; 林利


    [目的]研究植物乳杆菌Saccharomyces cerevisiae P13对大黄鱼生长和抗病能力的影响.[方法]用合0、104、106、108、1010cfu/kg植物乳杆菌Saccharomyces cerevisiae P13含量的饲料喂养大黄鱼4周后,测定大黄鱼的体重增长率、饲料转化率和感染病菌后的存活率.[结果]大黄鱼后肠中的Saccharomyces cerevisiae P13菌数量在喂食过程中显著增加,含106~1010 cfu/kg Saccharomyces cerevisiae P13的配方饲料明显促进了大黄鱼的体重增长率和摄食率.喂食含106~ 1010cfu/kg Saccharomyces cerevisiae P13的配方饲料后大黄鱼对Streptococcus sp.的抵抗力明显增强.[结论]植物乳杆菌Saccharomyces cerevisiae P13能促进大黄鱼生长,并增强大黄鱼的抗病力.

  2. Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation.

    Amaya-Delgado, L; Herrera-López, E J; Arrizon, Javier; Arellano-Plaza, M; Gschaedler, A


    Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.

  3. Transcriptional responses to glucose in Saccharomyces cerevisiae strains lacking a functional protein kinase A

    Livas Daniela


    Full Text Available Abstract Background The pattern of gene transcripts in the yeast Saccharomyces cerevisiae is strongly affected by the presence of glucose. An increased activity of protein kinase A (PKA, triggered by a rise in the intracellular concentration of cAMP, can account for many of the effects of glucose on transcription. In S. cerevisiae three genes, TPK1, TPK2, and TPK3, encode catalytic subunits of PKA. The lack of viability of tpk1 tpk2 tpk3 triple mutants may be suppressed by mutations such as yak1 or msn2/msn4. To investigate the requirement for PKA in glucose control of gene expression, we have compared the effects of glucose on global transcription in a wild-type strain and in two strains devoid of PKA activity, tpk1 tpk2 tpk3 yak1 and tpk1 tpk2 tpk3 msn2 msn4. Results We have identified different classes of genes that can be induced -or repressed- by glucose in the absence of PKA. Representative examples are genes required for glucose utilization and genes involved in the metabolism of other carbon sources, respectively. Among the genes responding to glucose in strains devoid of PKA some are also controlled by a redundant signalling pathway involving PKA activation, while others are not affected when PKA is activated through an increase in cAMP concentration. On the other hand, among genes that do not respond to glucose in the absence of PKA, some give a full response to increased cAMP levels, even in the absence of glucose, while others appear to require the cooperation of different signalling pathways. We show also that, for a number of genes controlled by glucose through a PKA-dependent pathway, the changes in mRNA levels are transient. We found that, in cells grown in gluconeogenic conditions, expression of a small number of genes, mainly connected with the response to stress, is reduced in the strains lacking PKA. Conclusions In S. cerevisiae, the transcriptional responses to glucose are triggered by a variety of pathways, alone or in

  4. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

    Aouida, Mustapha


    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

  5. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

    Shuhei Noda

    Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

  6. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae.

    Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka; Mahfouz, Magdy M


    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S. cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S. cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production.

  7. Bioethanol production from Gracilaria verrucosa using Saccharomyces cerevisiae adapted to NaCl or galactose.

    Nguyen, Trung Hau; Ra, Chae Hun; Sunwoo, InYung; Jeong, Gwi-Taek; Kim, Sung-Koo


    This study examined the pretreatment, enzymatic saccharification, and fermentation of the red macroalgae Gracilaria verrucosa using adapted saccharomyces cerevisiae to galactose or NaCl for the increase of bioethanol yield. Pretreatment with thermal acid hydrolysis to obtain galactose was carried out with 11.7% (w/v) seaweed slurry and 373 mM H2SO4 at 121 °C for 59 min. Glucose was obtained from enzymatic hydrolysis. Enzymatic saccharification was performed with a mixture of 16 U/mL Celluclast 1.5L and Viscozyme L at 45 °C for 48 h. Ethanol fermentation in 11.7% (w/v) seaweed hydrolysate was carried out using Saccharomyces cerevisiae KCTC 1126 adapted or non-adapted to high concentrations of galactose or NaCl. When non-adapted S. cerevisiae KCTC 1126 was used, the ethanol productivity was 0.09 g/(Lh) with an ethanol yield of 0.25. Ethanol productivity of 0.16 and 0.19 g/(Lh) with ethanol yields of 0.43 and 0.48 was obtained using S. cerevisiae KCTC 1126 adapted to high concentrations of galactose and NaCl, respectively. Adaptation of S. cerevisiae KCTC 1126 to galactose or NaCl increased the ethanol yield via adaptive evolution of the yeast.

  8. Local isolate of Saccharomyces cerevisiae as biocompetitive agent of Aspergillus flavus

    Eni Kusumaningtyas


    Full Text Available Aspergillus flavus is a toxigenic fungus that contaminates feed and influences the animal health. Saccharomyces cerevisiae can be used as a biocompetitive agent to control the contamination. The ability of local isolate of S. cerevisiae as a biocompetitive agent for A. flavus was evaluated. A. flavus (30ml was swept on Sabouraud dextrose agar (SDA, while S. cerevisiae was swept on its left and right. Plates were incubated at 28oC for nine days. Lytic activity of S. cerevisiae was detected by pouring its suspension on the centre of the cross streaks of A. flavus. Plates were incubated at 28oC for five days. Growth inhibition of A. flavus by S. cerevisiae was determined by mixing the two fungi on Potato dextrose broth and incubated at 28oC for 24 hours. Total colony of A. flavus were then observed at incubation time of 2, 4, 6 and 24 hours by pour plates method on the SDA plates and incubated on 28oC for two days. Growth of hyphae of A. flavus sweep were inhibited with the swept of S. cerevisiae. The width of A. flavus colony treated with S. cerevisiae is narrower (3,02 cm than that of control ( 4,60 cm. The growth of A. flavus was also inhibited on the centre of cross streak where the S. cerevisiae poured. S. cerevisiae gradually reduced the colony number of A. flavus in the mixed culture of broth fungi ie. 14 x 103 CFU/ml while colony number of control is 80 x 103 CFU/ml. Results showed that S. cerevisiae could be used as biocompetitive agent of A. flavus.

  9. Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

    De Craene, Johan-Owen; Courte, Fanny; Rinaldi, Bruno; Fitterer, Chantal; Herranz, Mari Carmen; Schmitt-Keichinger, Corinne; Ritzenthaler, Christophe; Friant, Sylvie


    The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

  10. A co-activator of nitrogen-regulated transcription in Saccharomyces cerevisiae.

    Soussi-Boudekou, S; André, B


    In Saccharomyces cerevisiae, the transcription factors Gln3p and Nil1p of the GATA family play a determinant role in expression of genes that are subject to nitrogen catabolite repression. Here we report the isolation of a new yeast mutant, gan1-1, exhibiting dramatically decreased NAD-linked glutamate dehydrogenase (NAD-GDH) and glutamine synthetase (GS) activities. The GAN1 gene was cloned and found to encode a 488-amino-acid polypeptide bearing no typical DNA binding domain. Gan1p is required for full expression of GLN1, GDH2 and also other nitrogen utilization genes, including GAP1, PUT4, MEP2 and GDH1. The extent to which Gan1p is required, however, varies according to the gene and to the nitrogen source available. We show that Gan1p is in fact involved in Gln3p- and Nil1p-dependent transcription. In the case of Gln3p-dependent transcription, the degree to which Gan1p is required appears to be gene specific. The contribution of Gan1p to gene expression is also influenced by the nitrogen status of the cell. We found that GAN1 is identical to ADA1, which encodes a component of the ADA/GCN5 co-activator complex. Ada1/Gan1p thus represents the first reported case of an accessory protein (a co-activator) linking the GATA-binding proteins Gln3p and Nil1p, mediating nitrogen-regulated transcription, to the basal transcription machinery.

  11. Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

    Katarzyna Jarmoszewicz

    Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

  12. Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae

    Xu Deng


    Full Text Available The yeast Saccharomyces cerevisiae IMA multigene family encodes four isomaltases sharing high sequence identity from 65% to 99%. Here, we explore their functional diversity, with exhaustive in-vitro characterization of their enzymological and biochemical properties. The four isoenzymes exhibited a preference for the α-(1,6 disaccharides isomaltose and palatinose, with Michaëlis–Menten kinetics and inhibition at high substrates concentration. They were also able to hydrolyze trisaccharides bearing an α-(1,6 linkage, but also α-(1,2, α-(1,3 and α-(1,5 disaccharides including sucrose, highlighting their substrate ambiguity. While Ima1p and Ima2p presented almost identical characteristics, our results nevertheless showed many singularities within this protein family. In particular, Ima3p presented lower activities and thermostability than Ima2p despite only three different amino acids between the sequences of these two isoforms. The Ima3p_R279Q variant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240 significantly increased the stability of Ima3p and supported the role of prolines in thermostability. The most distant protein, Ima5p, presented the lowest optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specific amino acids for determining the specificity for α-(1,6 substrates. We finally found a mixed inhibition by maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitive at very low isomaltose concentrations and uncompetitive as the substrate concentration increased. Altogether, this work illustrates that a gene family encoding proteins with strong sequence similarities can lead to enzyme with notable differences in biochemical and enzymological properties.

  13. Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. (Univ. di Milano (Italy))


    Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli [beta]-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. The authors showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

  14. Expression of an Aspergillus niger Phytase Gene (phyA) in Saccharomyces cerevisiae

    Han, Yanming; Wilson, David B.; Lei, Xin Gen


    Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase’s activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an act...

  15. Glutatation Transferasas de clase Omega en Saccharomyces cerevisiae: Estudio Bioquímico y Funcional

    Barreto Parra, Lina Patricia


    Saccharomyces cerevisiae posseeix dues glutatió transferases (GST) anomenades Gtt1i Gtt2, amb capacitat de conjugar una molècula de glutatió amb el substrat estàndarCDNB. Aquests dos enzims no són clasificables dins de les classes convencionalsdescrites en base a l'estructura de les GST d'eucariotes superiors, encara que guardencerta similitud estructural amb els membres de la classe Zeta. En aquesta memòria esdescriu la caracterització de tres GST de classe Omega en S. cerevisiae anomenadesG...

  16. Improved ethanol production from whey Saccharomyces cerevisiae using permeabilized cells of Kluyveromyces marxianus

    Rosenberg, M. [Slovak Technical Univ., Bratislava (Slovakia). Dept. of Biochemical Technology; Tomaska, M. [Slovak Technical Univ., Bratislava (Slovakia). Dept. of Biochemical Technology; Kanuch, J. [Slovak Technical Univ., Bratislava (Slovakia). Dept. of Biochemical Technology; Sturdik, E. [Slovak Technical Univ., Bratislava (Slovakia). Dept. of Biochemical Technology


    Permeabilized cells of Kluyveromyces marxianus CCY eSY2 were tested as the source of lactase in the ethanol fermentation of concentrated deproteinized whey (65-70 g/l lactose) by Saccharomyces cerevisiae CCY 10-13-14. Rapid lactose hydrolysis by small amounts of permeabilized cells following the fermentation of released glucose and galactose by S. cerevisiae resulted in a twofold enhancement of the overall volumetric productivity (1.03 g/lxh), compared to the fermentation in which the lactose was directly fermented by K. marxianus. (orig.)

  17. Potential extra-ribosomal functions of ribosomal proteins in Saccharomyces cerevisiae.

    Lu, Hui; Zhu, Yi-Fei; Xiong, Juan; Wang, Rong; Jia, Zhengping


    Ribosomal proteins (RPs), are essential components of the ribosomes, the molecular machines that turn mRNA blueprints into proteins, as they serve to stabilize the structure of the rRNA, thus improving protein biosynthesis. In addition, growing evidence suggests that RPs can function in other cellular roles. In the present review, we summarize several potential extra-ribosomal functions of RPs in ribosomal biogenesis, transcription activity, translation process, DNA repair, replicative life span, adhesive growth, and morphological transformation in Saccharomyces cerevisiae. However, the future in-depth studies are needed to identify these novel secondary functions of RPs in S. cerevisiae.

  18. Incorporating Protein Biosynthesis into the Saccharomyces cerevisiae Genome-scale Metabolic Model

    Olivares Hernandez, Roberto

    Based on stoichiometric biochemical equations that occur into the cell, the genome-scale metabolic models can quantify the metabolic fluxes, which are regarded as the final representation of the physiological state of the cell. For Saccharomyces Cerevisiae the genome scale model has been......, translation initiation, translation elongation, translation termination, translation elongation, and mRNA decay. Considering these information from the mechanisms of transcription and translation, we will include this stoichiometric reactions into the genome scale model for S. Cerevisiae to obtain the first...

  19. Rad52 multimerization is important for its nuclear localization in Saccharomyces cerevisiae

    Plate, Iben; Albertsen, Line; Lisby, Michael


    Rad52 is essential for all homologous recombination and DNA double strand break repair events in Saccharomyces cerevisiae. This protein is multifunctional and contains several domains that allow it to interact with DNA as well as with different repair proteins. However, it has been unclear how Rad......52 enters the nucleus. In the present study, we have used a combination of mutagenesis and sequence analysis to show that Rad52 from S. cerevisiae contains a single functional pat7 type NLS essential for its nuclear localization. The region containing the NLS seems only to be involved in nuclear...

  20. Engineering of carbon catabolite repression in recombinant xylose fermenting Saccharomyces cerevisiae

    Roca, Christophe Francois Aime; Haack, Martin Brian; Olsson, Lisbeth


    Two xylose-fermenting glucose-derepressed Saccharomyces cerevisiae strains were constructed in order to investigate the influence of carbon catabolite repression on xylose metabolism. S. cerevisiae CPB.CR2 (Deltamig1, XYL1, XYL2, XKS1) and CPB.MBH2 (Deltamig1, Deltamig2, XYL1, XYL2, XKS1) were...... of CPB.CR2, where the cells are assumed to grow under non-repressive conditions as they sense almost no glucose, invertase activity was lower during growth on xylose and glucose than on glucose only. The 3-fold reduction in invertase activity could only be attributed to the presence of xylose, suggesting...

  1. Growth-rate dependency of de novo resveratrol production in chemostat cultures of an engineered Saccharomyces cerevisiae strain

    Vos, T.; De la Torre Cortes, P.; Van Gulik, W.M.; Pronk, J.T.; Daran-Lapujade, P.A.S.


    Introduction: Saccharomyces cerevisiae has become a popular host for production of non-native compounds. The metabolic pathways involved generally require a net input of energy. To maximize the ATP yield on sugar in S. cerevisiae, industrial cultivation is typically performed in aerobic, sugar-limit

  2. Integrated phospholipidomics and transcriptomics analysis of Saccharomyces cerevisiae with enhanced tolerance to a mixture of acetic acid, furfural, and phenol

    A mixture of acetic acid, furfural and phenol (AFP), three representative lignocellulose derived inhibitors, significantly inhibited the growth and bioethanol production of Saccharomyces cerevisiae. In order to uncover mechanisms behind the enhanced tolerance of an inhibitor-tolerant S.cerevisiae s...

  3. High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous

    Verwaal, R.; Wang, J.; Meijnen, J.P.; Visser, H.; Sandmann, G.; Berg, van den J.A.; Ooyen, van A.J.J.


    To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially ß-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these g

  4. Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyes cerevisiae improves ethanol production

    Roca, Christophe Francois Aime; Nielsen, Jens; Olsson, Lisbeth


    Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase...

  5. ISOTERMAS DE ADSORÇÃO DE CÁDMIO POR Saccharomyces cerevisiae ISOTHERMS OF CADMIUM ADSORPTION BY Saccharomyces cerevisae

    Silvana ALBERTINI


    Full Text Available Com o objetivo de determinar as isotermas de adsorção de cádmio por Saccharomyces cerevisiae, foram utilizados os sais cloreto e nitrato de cádmio nas concentrações de 5, 10, 20, 40, 60, 80 e 100mg L-1. A biomassa foi produzida a partir de uma cultura "starter"de Saccharomyces cerevisiae IZ 1904. Após o contato de 16h entre o microrganismo e as soluções em estudo, a biomassa foi separada por centrifugação e o teor de cádmio residual foi determinado no sobrenadante por espectrofotometria de absorção atômica. Para os dois sais empregados foi observado um acúmulo crescente de cádmio nas concentrações de 5, 10, 20 e 40mg L-1. Nas concentrações de 60, 80 e 100mg L-1 foi observado que a levedura acumulou teores menores do metal, evidenciando danos na parede celular, nem sempre acompanhados de iguais danos da membrana citoplasmática, tais alterações da parede visualizadas por microscopia eletrônica de varredura.With the objective of determining the isotherms of cadmium the adsorption by Saccharomyces cerevisiae, the chloride and nitrate salts were used in the concentrations of 5, 10, 20, 40, 60, 80, and 100mg L-1. The biomass was produced from a starter culture of Saccharomyces cerevisiae IZ 1904. After a 16h contact between the microrganism and solutions of study the biomass was separated by a centrifuge and the cadmium residue content was determined at the supernatant by atomic adsorption spectrophotometry. For the two salts used a growing accumulation of cadmium was observed at concentrations of 5, 10, 20, and 40mg L-1. In the concentrations of 60, 80 and 100mg L-1 a decreasing of the accumulation of the metal was observed, evidencing damages of the cellular wall, which they're not accompanied always by damages of the citoplasmatic membrane, visualized by scanning electron microscopy.

  6. Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering

    Sanchez, R.G.; Karhumaa, Kaisa; Fonseca, C.;


    to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate......Background: Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced. Results: Evolutionary engineering was used...... of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during...

  7. Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes

    Albertsen, Line; Chen, Yun; Bach, Lars Stougaard


    The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast ma...... demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product. ©American Society for Microbiology. All rights reserved.......The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may...... increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology...

  8. Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine

    Qin, Jiufu; Zhou, Yongjin J.; Krivoruchko, Anastasia;


    Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolite...... the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals.......Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites...... of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway...

  9. Production of volatile and sulfur compounds by ten Saccharomyces cerevisiae strains inoculated in Trebbiano must

    Francesca ePatrignani


    Full Text Available In wines, the presence of sulphur compounds is the resulting of several contributions among which yeast metabolism. The characterization of the starter Saccharomyces cerevisiae needs to be performed also taking into account this ability even if evaluated together with the overall metabolic profile. In this perspective, principal aim of this experimental research was the evaluation of the volatile profiles, throughout GC/MS technique coupled with solid phase micro extraction, of wines obtained throughout the fermentation of 10 strains of Saccharomyces cerevisiae. In addition, the production of sulphur compounds was further evaluated by using a gas-chromatograph coupled with a Flame Photometric Detector. Specifically, the ten strains were inoculated in Trebbiano musts and the fermentations were monitored for 19 days. In the produced wines, volatile and sulphur compounds as well as amino acid concentrations were investigated. Also the physico-chemical characteristics of the wines and their electronic nose profiles were evaluated.

  10. Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae

    Siewers, Verena; Chen, Xiao; Huang, Le


    -(l-α-aminoadipyl)–l-cysteinyl–d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases...

  11. EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

    Jérôme Maury

    Full Text Available Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP. The best 3HP producing clone, with 5.45 g.L(-1 of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

  12. EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

    Maury, Jérôme; Germann, Susanne M; Baallal Jacobsen, Simo Abdessamad; Jensen, Niels B; Kildegaard, Kanchana R; Herrgård, Markus J; Schneider, Konstantin; Koza, Anna; Forster, Jochen; Nielsen, Jens; Borodina, Irina


    Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L(-1) of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

  13. Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae.

    Zelicof, A; Gatica, J; Gerst, J E


    We have isolated a rat cDNA whose expression suppresses the physiological consequences of the chromosomal disruption of CAP, the gene encoding the adenylyl cyclase-associated protein of Saccharomyces cerevisiae. Yeast CAP is a bifunctional protein: the NH2 terminus is necessary and sufficient for cellular responsiveness to activated RAS proteins, while the COOH terminus is required for normal cellular morphology and growth control. The rat MCH1 cDNA encodes a protein of 474 amino acids that is 36% identical to S. cerevisiae CAP and is capable of suppressing the loss of the COOH-terminal functions of CAP when expressed in yeast. The MCH1 protein therefore appears to be a structural and functional homolog of the yeast cyclase-associated proteins. Northern analysis of MCH1 gene expression shows it to be constitutively expressed in all cell and tissue types examined. The cloning of a rat homolog of CAP, in addition to the cloning of a human CAP homolog by Matviw et al. (Matviw, H., Yu, G., and Young, D. (1992) Mol. Cell. Biol. 12, 5033-5040), demonstrates that both cyclase-associated proteins and their functions may have evolved with mammalian cells.

  14. A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae.

    Pineda-Lucena, Antonio; Liao, Jack C C; Cort, John R; Yee, Adelinda; Kennedy, Michael A; Edwards, Aled M; Arrowsmith, Cheryl H


    As part of the Northeast Structural Genomics Consortium pilot project focused on small eukaryotic proteins and protein domains, we have determined the NMR structure of the protein encoded by ORF YML108W from Saccharomyces cerevisiae. YML108W belongs to one of the numerous structural proteomics targets whose biological function is unknown. Moreover, this protein does not have sequence similarity to any other protein. The NMR structure of YML108W consists of a four-stranded beta-sheet with strand order 2143 and two alpha-helices, with an overall topology of betabetaalphabetabetaalpha. Strand beta1 runs parallel to beta4, and beta2:beta1 and beta4:beta3 pairs are arranged in an antiparallel fashion. Although this fold belongs to the split betaalphabeta family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds.

  15. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan


    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase.

  16. Crystallization and preliminary X-ray diffraction analysis of the mitochondrial transcription factor sc-mtTFB from Saccharomyces cerevisiae.

    Schubot, F D; Chen, C J; Rose, J P; Wang, B C


    Eukaryotic mitochondria contain a distinct mini-chromosome. In yeast, transcription of the mitochondrial genome is mediated by a nuclear-encoded RNA polymerase consisting of a single polypeptide core enzyme and a specificity factor termed sc-mtTFB which bears some similarity to bacterial sigma-factors. sc-mtTFB from Saccharomyces cerevisiae has been cloned, expressed, purified and crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 89.7, b = 44.6, c = 98.9 A, beta = 110 degrees. Based on one molecule per asymmetric unit, the solvent content is estimated to be 48%. Small crystals of dimensions 0.01 x 0.05 x 0.13 mm diffract to at least 2.7 A resolution on a rotating-anode X-ray source.

  17. Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

    In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

  18. Preparation of a Saccharomyces cerevisiae cell-free extract for in vitro translation.

    Wu, Cheng; Sachs, Matthew S


    Eukaryotic cell-free in vitro translation systems have been in use since the 1970s. These systems can faithfully synthesize polypeptides when programmed with mRNA, enabling the production of polypeptides for analysis as well as permitting analyses of the cis- and trans-acting factors that regulate translation. Here we describe the preparation and use of cell-free translation systems from the yeast Saccharomyces cerevisiae.

  19. α-Synuclein-induced Aggregation of Cytoplasmic Vesicles in Saccharomyces cerevisiae

    Soper, James H.; Roy, Subhojit; Stieber, Anna; Lee, Eliza; Wilson, Robert B.; Trojanowski, John Q.; Burd, Christopher G.; Lee, Virginia M.-Y.


    Aggregated α-synuclein (α-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, α-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human α-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of ye...

  20. The effect of millimeter waves at the yeast Saccharomyces cerevisiae during heliogeophysical disturbances

    Rogacheva, Svetlana M.; Babaeva, Milena I.


    The isolated and combined effect of heliogeophysical factors and low intensive electromagnetic radiation of millimeter diapason at the metachromasia reaction of the yeast Saccharomyces cerevisiae was studied. It was established that longterm influence of EMR 65 GHz induced changes in the response of cells towards heliogeomagnetic disturbance. On our opinion millimeter waves may reduce the effect of heliogeophysical factors on living organisms because of destabilization of the intracellular water structure.


    Suresh P. Kamble; Jyotsna C. Borate


    Invertase from Saccharomyces cerevisiae is high cost enzyme and primarily used in the confectionary industry. For large scale production of the enzyme, feasible synthetic medium with appropriate supplemented nutrients are required. The effect of carbon source on invertase production is well known, but little is known about the effect of different nitrogen source. The aim of the present study is to see the effect of different nitrogen sources on the production of invertase in submerged ferment...

  2. Multi-Capillary Column-Ion Mobility Spectrometry of Volatile Metabolites Emitted by Saccharomyces Cerevisiae

    Christoph Halbfeld; Ebert, Birgitta E.; Blank, Lars M.


    Volatile organic compounds (VOCs) produced during microbial fermentations determine the flavor of fermented food and are of interest for the production of fragrances or food additives. However, the microbial synthesis of these compounds from simple carbon sources has not been well investigated so far. Here, we analyzed the headspace over glucose minimal salt medium cultures of Saccharomyces cerevisiae using multi-capillary column-ion mobility spectrometry (MCC-IMS). The high sensitivity and f...

  3. Production of β-galactosidase from recombinant Saccharomyces cerevisiae grown on lactose


    Improved productivity and costs reduction in fermentation processes may be attained by using flocculating cell cultures. The production of extracellular heterologous β-galactosidase by recombinant flocculating Saccharomyces cerevisiae cells, expressing the lacA gene (coding for β-galactosidase) of Aspergillus niger under the ADHI promotor and terminator in a bioreactor was studied. The effects of lactose concentration and yeast extract concentration on β-galactosidase production i...

  4. Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network

    Østergaard, Simon; Olsson, Lisbeth; Johnston, M.


    in the pathway, and ultimately, increasing metabolic flux through the pathway of interest, By manipulating the GAL gene regulatory network of Saccharomyces cerevisiae, which is a tightly regulated system, we produced prototroph mutant strains, which increased the flux through the galactose utilization pathway...... media. The improved galactose consumption of the gal mutants did not favor biomass formation, but rather caused excessive respiro-fermentative metabolism, with the ethanol production rate increasing linearly with glycolytic flux....

  5. The Saccharomyces cerevisiae SCRaMbLE system and genome minimization

    Dymond, Jessica; Boeke, Jef


    We have recently reported the first partially synthetic eukaryotic genome. Saccharomyces cerevisiae chromosomes synIXR and semi-synVIL are fully synthetic versions of the right arm of chromosome IX and the telomeric segment of the left arm of chromosome VI, respectively, and represent the beginning of the synthetic yeast genome project, Sc2.0, that progressively replaces native yeast DNA with synthetic sequences. We have designed synthetic chromosome sequences according to principles specifyi...

  6. Heterologous expression and purification of wheat storage proteins in the yeast Saccharomyces cerevisiae


    Im Rahmen des Teilprojektes “Expression und Produktion von Weizenspeicherproteinen in der Hefe Saccharomyces cerevisiae“ des BMBF-Leitprojektes „Entwicklung von Weizen-, Roggen- und Gerstenproteinen ohne Zöliakietoxizität und deren Verwendung zur Herstellung von Lebensmitteln“ (Förderkennzeichen 0312246C) sollten die Ausbeute der heterolog in S. cerevisiae exprimierten Weizenproteine gesteigert werden, um sie für Zöliakietoxizitätsteste einzusetzen. Durch Optimierungsstrategien des Substrates...

  7. L’utilizzo di ceppi modificati di saccharomyces cerevisiae in enologia

    Schuller, Dorit; Casal, Margarida


    Trata-se de uma tradução do seguinte artigo D. Schuller e M. Casal. 2005. The use of genetically modified yeast in the wine industry. Applied Microbiology and Biotechnology 68, 292-304. Negli ultimi decenni, la scienza e la tecnologia degli alimenti hanno contribuito all’introduzione di nuovi prodotti per soddisfare le richieste nutrizionali, socio-economiche e di qualità. Con la moderna genetica molecolare, l’importanza industriale di Saccharomyces Cerevisiae è accresciuta ulteriormen...

  8. Intensification of alcoholic fermentation upon dehydration-rehydration of the yeast Saccharomyces cerevisiae

    Zikmanis, P.B.; Kruce, R.V.; Auzina, L.P.; Margevica, M.V.; Beker, M.J.


    In comparison with intact yeast, dehydrated-rehydrated cells of Saccharomyces cerevisiae show significantly higher ethanol production from exogenous substrate under both anaerobic and aerobic conditions, particularly when low concentration (0.1%) of glucose are used. For populations with a higher percentage of viable rehydrated cells (above 70%) a more notable decrease in the Pasteur effect (the difference between the quantity of ethanol formed under anaerobic and aerobic conditions) is observed. (orig.)

  9. Effects of Furfural on the Respiratory Metabolism of Saccharomyces cerevisiae in Glucose-Limited Chemostats

    Sarvari Horvath, I; Franzén, C J; Taherzadeh, M J; Niklasson, C; Lidén, Gunnar


    Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual fur...

  10. The Using of Millimeter Waves for Biosynthetic Processes Stimulation in Saccharomyces Cerevisiae

    Usatîi Agafia; Chiseliţa Natalia; Efremova Nadejda; Borisova Tamara


    The results of influence of three frequencies of electromagnetic radiation of highfrequency range (EMR EHF) on the biosynthesis of carbohydrates, β-glucan, proteins, catalase activity by Saccharomyces cerevisiae CNMN -Y-20 yeast strain were analysed. It was established that frequency of f= 53,33 GHz stimulates the biosynthesis of carbohydrates, including β-glucan and frequency of f= 42,19 GHz promotes the increase of protein content and catalase. The indicated frequencies of EMR EHF are offer...

  11. Efficient ammonium uptake and mobilization of vacuolar arginine by Saccharomyces cerevisiae wine strains during wine fermentation

    Crepin, Lucie; SANCHEZ, Isabelle,; Nidelet, Thibault; Dequin, Sylvie


    Background Under N-limiting conditions, Saccharomyces cerevisiae strains display a substantial variability in their biomass yield from consumed nitrogen -in particular wine yeasts exhibit high growth abilities- that is correlated with their capacity to complete alcoholic fermentation, a trait of interest for fermented beverages industries. The aim of the present work was to assess the contribution of nitrogen availability to the strain-specific differences in the ability to efficiently use N-...

  12. Inactivation of Saccharomyces cerevisiae suspended in orange juice using high-intensity pulsed electric fields.

    Elez-Martínez, Pedro; Escolà-Hernández, Joan; Soliva-Fortuny, Robert C; Martín-Belloso, Olga


    Saccharomyces cerevisiae is often associated with the spoilage of fruit juices. The purpose of this study was to evaluate the effect of high-intensity pulsed electric field (HIPEF) treatment on the survival of S. cerevisiae suspended in orange juice. Commercial heat-sterilized orange juice was inoculated with S. cerevisiae (CECT 1319) (10(8) CFU/ml) and then treated by HIPEFs. The effects of HIPEF parameters (electric field strength, treatment time, pulse polarity, frequency, and pulse width) were evaluated and compared to those of heat pasteurization (90 degrees C/min). In all of the HIPEF experiments, the temperature was kept below 39 degrees C. S. cerevisiae cell damage induced by HIPEF treatment was observed by electron microscopy. HIPEF treatment was effective for the inactivation of S. cerevisiae in orange juice at pasteurization levels. A maximum inactivation of a 5.1-log (CFU per milliliter) reduction was achieved after exposure of S. cerevisiae to HIPEFs for 1,000 micros (4-micros pulse width) at 35 kV/cm and 200 Hz in bipolar mode. Inactivation increased as both the field strength and treatment time increased. For the same electric field strength and treatment time, inactivation decreased when the frequency and pulse width were increased. Electric pulses applied in the bipolar mode were more effective than those in the monopolar mode for destroying S. cerevisiae. HIPEF processing inactivated S. cerevisiae in orange juice, and the extent of inactivation was similar to that obtained during thermal pasteurization. HIPEF treatments caused membrane damage and had a profound effect on the intracellular organization of S. cerevisiae.

  13. Comparison between two selected Saccharomyces cerevisiae strains as fermentation starters in the production of traditional cachaça

    Fátima de Cássia Oliveira Gomes; Roberta Amália de Carvalho Araújo; Patrícia Silva Cisalpino; Elizabeth Spangler Andrade Moreira; Carlos Leomar Zani; Carlos Augusto Rosa


    Two Saccharomyces cerevisiae strains were tested as the starter yeasts in a traditional cachaça distillery. The strains used were S. cerevisiae UFMG-A829, isolated from a cachaça fermentation process, and S. cerevisiae K1-V1116, obtained from the wine industry. The permanence of each strain in the fermentation must was determined by RAPD (Random Amplified Polymorphic DNA)-PCR, with primer M13. Both yeast strains were prevalent in the vats for approximately 30 days. Indigenous non-Saccharomyce...

  14. Removal of lead, mercury and nickel using the yeast Saccharomyces cerevisiae

    Cherlys Infante J.


    Full Text Available Objective. In this study the biomass of the yeast Saccharomyces cerevisiae was used to remove lead, mercury and nickel in the form of ions dissolved in water. Materials and methods. Synthetic solutions were prepared containing the three heavy metals, which were put in contact with viable microorganisms at different conditions of pH, temperature, aeration and agitation. Results. Both individual variables and the interaction effects influenced the biosorption process. Throughout the experimental framework it was observed that the biomass of Saccharomyces cerevisiae removed a higher percentage of lead (86.4% as compared to mercury and nickel (69.7 and 47.8% respectively. When the pH was set at a value of 5 the effect was positive for all three metals. Conclusions. pH was the variable that had a greater influence on the biosorption of lead on the biomass of Saccharomyces cerevisiae. The affinity of the heavy metals for the biomass followed the order Pb>Hg>Ni.

  15. Effect of Yeast : Saccharomyces cerevisiae and Marine Yeast as probiotic supplement on performance of poultry

    I Putu Kompiang


    Full Text Available An experiment had been conducted to evaluate the effect of marine yeast and Saccharomyces cerevisiae (Sc as probiotic supplement on poultry performance. Marine yeast isolated from rotten sea-weed and commercial Saccharomyces cerevisiae were used. Evaluation was conducted by comparing performance of broiler chicken supplemented with marine yeast or Sc, which were given through drinking water (5 ml/l to negative control (feed without antibiotic growth promotor/GPA, positive control (feed with GPA, and reference commercial probiotic. Forty DOC broiler birds were used for each treatment, divided into 4 replicates (10 birds/replicate and raised in wire cages for 5 weeks. Body weight and feed consumption were measured weekly and mortality was recorded during the trial. The results showed that there were no significant difference on the birds performance among marine yeast, Sc, positive control and probiotic reference control treatments. However their effects on bird performance were better (P<0.05 than treatment of negative control. It is concluded that marine yeast or Saccharomyces cerevisiae could replace the function of antibiotic as a growth promotant.

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

    González, Sara S; Barrio, Eladio; Querol, Amparo


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

  17. Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation.

    Lopez, Claudia Lorena Fernandez; Beaufort, Sandra; Brandam, Cédric; Taillandier, Patricia


    Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces.

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

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


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

    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


    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.

  20. Mechanisms of appearance of the Pasteur effect in Saccharomyces cerevisiae: inactivation of sugar transport systems.

    Lagunas, R; Dominguez, C; Busturia, A; Sáez, M J


    Saccharomyces cerevisiae does not show a noticeable Pasteur effect (activation of sugar catabolism by anaerobiosis) when growing with an excess of sugar and nitrogen source, but it does do so after exhaustion of the nitrogen source in the medium (resting state). We have found that this different behavior of growing and resting S. cerevisiae seems due to differences in the contribution of respiration to catabolism under both states. Growing S. cerevisiae respired only 3 to 20% of the catabolized sugar, depending on the sugar present; the remainder was fermented. In contrast, resting S. cerevisiae respired as much as 25 to 100% of the catabolized sugar. These results suggest that a shift to anaerobiosis would have much greater energetic consequences in resting than in growing S. cerevisiae. In resting S. cerevisiae anaerobiosis would strongly decrease the formation of ATP; as a consequence, various regulatory mechanisms would switch on, producing the observed increase of the rate of glycolysis. The greater significance that respiration reached in resting cells was not due to an increase of the respiratory capacity itself, but to a loss of fermentation which turned respiration into the main catabolic pathway. The main mechanism involved in the loss of fermentation observed during nitrogen starvation was a progressive inactivation of the sugar transport systems that reduced the rate of fermentation to less than 10% of the value observed in growing cells. Inactivation of the sugar transports seems a consequence of the turnover of the sugar carriers whose apparent half-lives were 2 to 7 h.

  1. The golden root, Rhodiola rosea, prolongs lifespan but decreases oxidative stress resistance in yeast Saccharomyces cerevisiae.

    Bayliak, Maria M; Lushchak, Volodymyr I


    The effect of aqueous extract from R. rosea root on lifespan and the activity of antioxidant enzymes in budding yeast Saccharomyces cerevisiae have been studied. The supplementation of the growth medium with R. rosea extract decreased survival of exponentially growing S. cerevisiae cells under H(2)O(2)-induced oxidative stress, but increased viability and reproduction success of yeast cells in stationary phase. The extract did not significantly affect catalase activity and decreased SOD activity in chronologically aged yeast population. These results suggest that R. rosea acts as a stressor for S. cerevisiae cells, what sensitizes yeast cells to oxidative stress at exponential phase, but induces adaptation in stationary phase cells demonstrating the positive effect on yeast survival without activation of major antioxidant enzymes.

  2. A new biological test of water toxicity-yeast Saccharomyces cerevisiae conductometric test.

    Dolezalova, Jaroslava; Rumlova, Lubomira


    This new biological test of water toxicity is based on monitoring of specific conductivity changes of yeast Saccharomyces cerevisiae suspension as a result of yeast fermentation activity inhibition in toxic conditions. The test was verified on ten substances with various mechanisms of toxic effect and the results were compared with two standard toxicity tests based on Daphnia magna mobility inhibition (EN ISO 6341) and Vibrio fischeri bioluminescence inhibition (EN ISO 11348-2) and with the results of the S. cerevisiae lethal test (Rumlova and Dolezalova, 2012). The new biological test - S. cerevisiae conductometric test - is an express method developed primarily for field conditions. It is applicable in case of need of immediate information about water toxicity. Fast completion is an advantage of this test (time necessary for test completion is about 60min), the test is simple and the test organism - dried instant yeast - belongs among its biggest advantages because of its long-term storage life and broad availability.

  3. Parameter Optimization for Enhancement of Ethanol Yield by Atmospheric Pressure DBD-Treated Saccharomyces cerevisiae

    Dong, Xiaoyu; Yuan, Yulian; Tang, Qian; Dou, Shaohua; Di, Lanbo; Zhang, Xiuling


    In this study, Saccharomyces cerevisiae (S. cerevisiae) was exposed to dielectric barrier discharge plasma (DBD) to improve its ethanol production capacity during fermentation. Response surface methodology (RSM) was used to optimize the discharge-associated parameters of DBD for the purpose of maximizing the ethanol yield achieved by DBD-treated S. cerevisiae. According to single factor experiments, a mathematical model was established using Box-Behnken central composite experiment design, with plasma exposure time, power supply voltage, and exposed-sample volume as impact factors and ethanol yield as the response. This was followed by response surface analysis. Optimal experimental parameters for plasma discharge-induced enhancement in ethanol yield were plasma exposure time of 1 min, power voltage of 26 V, and an exposed sample volume of 9 mL. Under these conditions, the resulting yield of ethanol was 0.48 g/g, representing an increase of 33% over control.

  4. Toxicity detection using lysosomal enzymes, glycoamylase and thioredoxin fused with fluorescent protein in Saccharomyces cerevisiae.

    Nguyen, Ngoc-Tu; Shin, Hwa-Yoon; Kim, Yang-Hoon; Min, Jiho


    Saccharomyces cerevisiae is the simplest and a favorite eukaryotic system that contains lysosome and thus, is a suitable organism for monitoring some toxic effects in environmental pollution. In this study, S. cerevisiae was transformed with two recombinant plasmids. Sporulation-specific glycoamylase (SGA1), which was upregulated in response to arsenic, was fused with the blue fluorescent protein (BFP) for the construction of an oxidative stress-causing chemicals sensor. Additionally, thioredoxin (TRX2), a protein overexpressed exclusively under tetracycline's influence, fused with the cyan fluorescent protein (CFP) to create a detector for this kind of chemical. In summary, we developed two recombinant S. cerevisiae that facilitate the detection of both kinds of toxic chemicals, specifically visualized by different color indicators.


    N.A. WULFF


    Full Text Available A levedura Saccharomyces cerevisiae estimula o acúmulo de fitoalexinas e tem potencial para ser utilizada como agente de controle alternativo no tratamento de doenças fúngicas em sorgo. São descritos aqui os procedimentos iniciais para a purificação de elicitores de fitoalexinas em sorgo, os quais são extraídos das células da levedura S. cerevisiae por autoclavagem, indicando serem termoestáveis. Após precipitacão com etanol, em concentrações finais de 50 e 80%, as moléculas elicitoras permanecem em solução. O acúmulo de fitoalexinas nos mesocótilos é mais elevado quanto maiores os teores de proteínas das amostras elicitoras.The yeast Saccharomyces cerevisiae stimulates phytoalexin accumulation and is a potential agent for biological control of fungal diseases in sorghum. The present investigation establishes the initial steps to purify elicitor molecules of phytoalexins in sorghum from S. cerevisiae. These molecules are extracted using heat and remain in solution after ethanol precipitation. They are active even after autoclaving, thus showing to be thermostable. A correlation between phytoalexin accumulation in mesocotyls and increasing amounts of protein on elicitor samples was observed.

  6. Saccharomyces cerevisiae Forms D-2-Hydroxyglutarate and Couples Its Degradation to D-Lactate Formation via a Cytosolic Transhydrogenase.

    Becker-Kettern, Julia; Paczia, Nicole; Conrotte, Jean-François; Kay, Daniel P; Guignard, Cédric; Jung, Paul P; Linster, Carole L


    The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to α-ketoglutarate. Depletion of D-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1.

  7. Bioethanol production by a flocculent hybrid, CHFY0321 obtained by protoplast fusion between Saccharomyces cerevisiae and Saccharomyces bayanus

    Choi, Gi-Wook; Kang, Hyun-Woo; Kim, Yule [Changhae Institute of Cassava and Ethanol Research, Changhae Ethanol Co., LTD, Palbok-Dong 829, Dukjin-Gu, Jeonju 561-203 (Korea); Um, Hyun-Ju; Kim, Mina; Kim, Yang-Hoon [Department of Microbiology, Chungbuk National University, 410 Sungbong-Ro, Heungduk-Gu, Cheongju 361-763 (Korea)


    Fusion hybrid yeast, CHFY0321, was obtained by protoplast fusion between non-flocculent-high ethanol fermentative Saccharomyces cerevisiae CHY1011 and flocculent-low ethanol fermentative Saccharomyces bayanus KCCM12633. The hybrid yeast was used together with the parental strains to examine ethanol production in batch fermentation. Under the conditions tested, the fusion hybrid CHFY0321 flocculated to the highest degree and had the capacity to ferment well at pH 4.5 and 32 C. Simultaneous saccharification and fermentation for ethanol production was carried out using a cassava (Manihot esculenta) powder hydrolysate medium containing 19.5% (w v{sup -1}) total sugar in a 5 l lab scale jar fermenter at 32 C for 65 h with an agitation speed of 2 Hz. Under these conditions, CHFY0321 showed the highest flocculating ability and the best fermentation efficiency for ethanol production compared with those of the wild-type parent strains. CHFY0321 gave a final ethanol concentration of 89.8 {+-} 0.13 g l{sup -1}, a volumetric ethanol productivity of 1.38 {+-} 0.13 g l{sup -1} h{sup -1}, and a theoretical yield of 94.2 {+-} 1.58%. These results suggest that CHFY0321 exhibited the fermentation characteristics of S. cerevisiae CHY1011 and the flocculent ability of S. bayanus KCCM12633. Therefore, the strong highly flocculent ethanol fermentative CHFY0321 has potential for improving biotechnological ethanol fermentation processes. (author)

  8. Genomics and Biochemistry of Saccharomyces cerevisiae Wine Yeast Strains.

    Eldarov, M A; Kishkovskaia, S A; Tanaschuk, T N; Mardanov, A V


    Saccharomyces yeasts have been used for millennia for the production of beer, wine, bread, and other fermented products. Long-term "unconscious" selection and domestication led to the selection of hundreds of strains with desired production traits having significant phenotypic and genetic differences from their wild ancestors. This review summarizes the results of recent research in deciphering the genomes of wine Saccharomyces strains, the use of comparative genomics methods to study the mechanisms of yeast genome evolution under conditions of artificial selection, and the use of genomic and postgenomic approaches to identify the molecular nature of the important characteristics of commercial wine strains of Saccharomyces. Succinctly, data concerning metagenomics of microbial communities of grapes and wine and the dynamics of yeast and bacterial flora in the course of winemaking is provided. A separate section is devoted to an overview of the physiological, genetic, and biochemical features of sherry yeast strains used to produce biologically aged wines. The goal of the review is to convince the reader of the efficacy of new genomic and postgenomic technologies as tools for developing strategies for targeted selection and creation of new strains using "classical" and modern techniques for improving winemaking technology.

  9. Partial Rescue of pos5 Mutants by YEF1 and UTR1 Genes in Saccharomyces cerevisiae

    Yong-Fu LI; Feng SHI


    Three NAD kinase homologs, encoded by UTR1, POS5 and YEF1 genes, are found in the yeast Saccharomyces cerevisiae and proven to be important sources of NADPH for the cell. Pos5p, existing in the mitochondrial matrix, is critical for higher temperature endurance and mitochondrial functions, such as glycerol usability and arginine biosynthesis. Through constructing the high-copy expression plasmids of YEF1 and UTR1, which contained the green fluorescent protein reporter tag at their 3' terminus, and introducing them into POS5 gene deletion mutants (i.e. pos5, utr1pos5, yef1pos5 and utr1yef1pos5), the high-copy YEF1 and UTR1 plasmids carrying transformants for pos5 mutants were obtained. Their temperature sensitivity and growth phenotype on media with glycerol as the sole carbon source, or on media without arginine, were checked. Results showed the partial rescue of mitochondrial dysfunctions and temperature sensitivity of pos5 mutants by the high-copy YEF1 gene, and of glycerol growth defect and temperature sensitivity by the high-copy UTR1 gene, which confirmed the potential supplying ability of Yef1p and Utr1p for mitochondrial NADP(H) and implied the weak transport of NADP from cytosol to mitochondria. However, even through the green fluorescent protein reporter label, the subcellular localization of Yef1p and Utr1p in yeast cells could not be observed, which indicated the low expression level of these two NAD kinase homologs.

  10. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    Lee, Byung-Kwon [University of Tennessee, Knoxville (UTK); Jung, Kyung-Sik [University of Tennessee, Knoxville (UTK); Son, Cagdas D [ORNL; Kim, Heejung [University of Tennessee, Knoxville (UTK); Verberkmoes, Nathan C [ORNL; Arshava, Boris [College of Staten Island; Naider, Fred [College of Staten Island; Becker, Jeffrey Marvin [ORNL


    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  11. Unconventional genomic architecture in the budding yeast saccharomyces cerevisiae masks the nested antisense gene NAG1.

    Ma, Jun; Dobry, Craig J; Krysan, Damian J; Kumar, Anuj


    The genomic architecture of the budding yeast Saccharomyces cerevisiae is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene, NAG1 (for nested antisense gene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand. NAG1 encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by beta-galactosidase analysis of a NAG1-lacZ fusion. NAG1 is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (nag1-1) disruptive for NAG1 but silent with respect to YGR031W, defines a role for NAG1 in yeast cell wall biogenesis; microarray profiling of nag1-1 indicates decreased expression of genes contributing to cell wall organization, and the nag1-1 mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus, NAG1 is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.

  12. Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.

    Christopher L Brett

    Full Text Available Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pH(v in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pH(v (5.27±0.13 was resistant to acid stress (5.28±0.14 but shifted significantly in response to alkali stress (5.83±0.13. Of 107 mutants that displayed aberrant pH(v under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pH(v dysregulation in a neo1(ts mutant restored viability whereas cholesterol accumulation in human NPC1(-/- fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation.

  13. Whole genome sequencing of Saccharomyces cerevisiae: from genotype to phenotype for improved metabolic engineering applications

    Asadollahi Mohammad A


    Full Text Available Abstract Background The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. Results In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c. Considering only metabolic genes (782 of 5,596 annotated genes, a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications. Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10 and ergosterol biosynthetic pathway (ERG8, ERG9. Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that

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

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


    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.

  15. Comportamento celular e resposta antioxidante diferenciados de Saccharomyces cerevisiae e de Saccharomyces chevalieri ao metavanadato de amónio Different cellular behaviour and antioxidant response of Saccharomyces cerevisiae and Saccharomyces chevalieri growing in presence of ammonium metavanadate

    R. Ferreira


    Full Text Available A fermentação do vinho é um processo microbiológico complexo que requere a presença de leveduras adaptadas a condições de stresse. No ambiente celular de organismos aeróbios ocorrem naturalmente espécies reactivas de oxigénio (ROS como subprodutos da respiração mitocondrial. A elevada reactividade destas espécies químicas pode gerar danos moleculares que, em alguns casos, levam à morte celular. Em condições fisiológicas normais ou como resposta ao stresse oxidativo, a célula pode desencadear respostas adaptativas que envolvem mecanismos antioxidantes como os enzimas glutationo redutase (GR; EC e catalases T (CAT T; EC e A (CAT A; EC O vanádio, um metal pesado presente em alguns fitofármacos, pode também com portar-se como um gerador de ROS, alterando o estado redox intracelular e exercendo efeitos nocivos em leveduras expostas a quantidade excessiva deste elemento. O principal objectivo deste trabalho foi comparar o efeito do metavanadato de amónio (NH4VO3, um sal pentavalente de vanádio, na viabilidade celular e nas actividades enzimáticas GR, CAT T e CAT A das leveduras vínicas Saccharomyces cerevisiae UE-ME3 e Saccharomyces chevalieri UE-ME1. Os resultados obtidos mostram que S. chevalieri UE-ME1 revelou menor tolerância ao NH4VO3 do que S. cerevisiae UE-ME3, uma vez que culturas de S. chevalieri não sobreviveram para valores de concentração do sal de vanádio superiores a 7,5 mM enquanto que células de S. cerevisiae mantiveram-se viáveis em presença de metavanadato de amónio 75 mM. As actividades enzimáticas estudadas apresentaram em S. chevalieri valores muito inferiores aos que foram determinados em S. cerevisiae embora em ambas as espécies de levedura o NH4VO3 pareça comportarse como um indutor de stresse oxidativo ao provocar um decréscimo significativo da actividade GR (PThe fermentation of wine is a complex microbiological process which requires yeast adaptation to stress

  16. Comparison between two selected Saccharomyces cerevisiae strains as fermentation starters in the production of traditional cachaça

    Fátima de Cássia Oliveira Gomes


    Full Text Available Two Saccharomyces cerevisiae strains were tested as the starter yeasts in a traditional cachaça distillery. The strains used were S. cerevisiae UFMG-A829, isolated from a cachaça fermentation process, and S. cerevisiae K1-V1116, obtained from the wine industry. The permanence of each strain in the fermentation must was determined by RAPD (Random Amplified Polymorphic DNA-PCR, with primer M13. Both yeast strains were prevalent in the vats for approximately 30 days. Indigenous non-Saccharomyces and indigenous S. cerevisiae strains were isolated in lower counts during the fermentation period. Indigenous S. cerevisiae strains were molecularly distinct when compared to the starter yeasts. The two yeasts appeared promising starter yeasts in the fermentation process to produce traditional cachaça.Duas linhagens de Saccharomyces cerevisiae foram testadas como iniciadoras em uma destilaria de cachaça. Foram utilizadas as linhagens de S. cerevisiae UFMG-A829, isolada de fermentação de cachaça, e S. cerevisiae K1-V1116, de origem vinícola. A permanência de cada linhagem durante a fermentação foi determinada por RAPD (Random Amplified Polymorphic DNA-PCR, utilizando o iniciador M13. As duas linhagens predominaram nas dornas de fermentação por aproximadamente 30 dias. Leveduras não-Saccharomyces e S. cerevisiae indígenas foram isoladas em menor proporção durante o experimento. As linhagens de S. cerevisiae indígenas apresentaram perfis moleculares distintos em relação às linhagens iniciadoras. As duas linhagens foram promissoras para serem utilizadas como iniciadoras do processo fermentativo para a produção da cachaça.

  17. Directed Evolution towards Increased Isoprenoid Production in Saccharomyces cerevisiae

    Carlsen, Simon; Nielsen, Michael Lynge; Kielland-Brandt, Morten

    diversity. The most common way of producing these compounds is by organic synthesis. Organic synthesis does however have several disadvantages for production of secondary metabolites such as low yields due to the complex structures, which makes this way of production economically unfeasible. Microbial...... population of S. cerevisiae clones will afterwards be screened using the isoprenoid molecule lycopene as a model compound, hereby enabling the isolation of phenotypes producing higher amounts of isoprenoid. The property making lycopene ideal for screening is its system of 11 conjugated double bonds, which...

  18. Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes: role of physiological fitness and microbial interactions.

    Albergaria, Helena; Arneborg, Nils


    Winemaking, brewing and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the main biotransformation and Saccharomyces cerevisiae the primary microorganism. Although a wide variety of microbial species may participate in alcoholic fermentation and contribute to the sensory properties of end-products, the yeast S. cerevisiae invariably dominates the final stages of fermentation. The ability of S. cerevisiae to outcompete other microbial species during alcoholic fermentation processes, such as winemaking, has traditionally been ascribed to its high fermentative power and capacity to withstand the harsh environmental conditions, i.e. high levels of ethanol and organic acids, low pH values, scarce oxygen availability and depletion of certain nutrients. However, in recent years, several studies have raised evidence that S. cerevisiae, beyond its remarkable fitness for alcoholic fermentation, also uses defensive strategies mediated by different mechanisms, such as cell-to-cell contact and secretion of antimicrobial peptides, to combat other microorganisms. In this paper, we review the main physiological features underlying the special aptitude of S. cerevisiae for alcoholic fermentation and discuss the role of microbial interactions in its dominance during alcoholic fermentation, as well as its relevance for winemaking.

  19. Effects of sequential mixed cultures of Wickerhamomyces anomalus and Saccharomyces cerevisiae on apple cider fermentation.

    Ye, Mengqi; Yue, Tianli; Yuan, Yahong


    The fermentation of cider by mixed cultures of Wickerhamomyces anomalus and Saccharomyces cerevisiae was carried out to study their effect on the cider quality. The results showed that growth of W. anomalus and S. cerevisiae was affected by each other during co-fermentation process. All the mixed cultures produced statistically the same level of ethanol as S. cerevisiae monoculture. The mixed fermentation could produce more variety and higher amounts of acetate esters, ethyl esters, higher alcohols, aldehydes, and ketones. Sensory evaluation demonstrated that ciders obtained from co-fermentation with W. anomalus gained higher scores than ciders fermented by pure S. cerevisiae, especially the co-fermentation cultures WS3, WS4, WS6, and WS8. Only 3 days of fermentation with W. anomalus in sequential mixtures were enough to improve the quality of cider. Wickerhamomyces anomalus could be used in association with S. cerevisiae to improve the quality of cider. The modulation of inoculation time may provide an effective means of manipulating cider aroma for different characteristics.

  20. Effects of Yeast (Saccharomyces Cerevisiae Feed Supplement on Milk Production and its Composition in Tunisian Holstein Friesian Cows

    Maamouri O.


    Full Text Available A 105-day feed trial was conducted to evaluate the effect of probiotic feed supplement containing Saccharomyces cerevisiae on milk yield and its composition in Holstein Friesian cows. The trial was conducted in the region of Sidi Bouzid in the west of Tunisia. Effects of Saccharomyces cerevisiae have been investigated on eight Holstein Friesian cows randomly divided into two groups of four animals on the basis of age, body weight, average milk yield, and lactation number. The first group was supplemented with 2.5 g/cow/day of probiotic yeast Saccharomyces cerevisiae (2.5 1010 CFU/day and the second group (control was without the yeast. The study showed that supplementation with 2.5 g of yeast Saccharomyces cerevisiae per cow per day or 2.5 1010 CFU/day tended (P < 0.06 to increase milk production by 1.1 kg/cow. By cons, there was a significant increase of fat (P < 0.01; 52.8 and 46.9 g/cow/day and protein (P < 0.05; 41.7 and 38.7 g/cow/day content both for treated and control group, respectively. It is concluded that supplementation of Saccharomyces cerevisiae at 2.5 1010 CFU/day in the diet of dairy cows may have positive influence on milk fat and protei n yield (g/cow/day.

  1. The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

    Dang, V D; Bohn, C.; Bolotin-Fukuhara, M; Daignan-Fornier, B.


    In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on a...

  2. Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.

    Miller, S. M; Magasanik, B


    We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to...

  3. The Ski7 Antiviral Protein Is an EF1-α Homolog That Blocks Expression of Non-Poly(A) mRNA in Saccharomyces cerevisiae

    Benard, Lionel; Carroll, Kathleen; Valle, Rosaura C. P.; Masison, Daniel C.; Wickner, Reed B.


    We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-α. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining w...

  4. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

    Bao, Zehua; Xiao, Han; Liang, Jing; Zhang, Lu; Xiong, Xiong; Sun, Ning; Si, Tong; Zhao, Huimin


    One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

  5. Heterologous expression of MlcE in Saccharomyces cerevisiae provides resistance to natural and semi-synthetic statins

    Ana Ley


    Full Text Available Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key enzyme in cholesterol biosynthesis. Their extensive use in treatment and prevention of cardiovascular diseases placed statins among the best selling drugs. Construction of Saccharomyces cerevisiae cell factory for the production of high concentrations of natural statins will require establishment of a non-destructive self-resistance mechanism to overcome the undesirable growth inhibition effects of statins. To establish active export of statins from yeast, and thereby detoxification, we integrated a putative efflux pump-encoding gene mlcE from the mevastatin-producing Penicillium citrinum into the S. cerevisiae genome. The resulting strain showed increased resistance to both natural statins (mevastatin and lovastatin and semi-synthetic statin (simvastatin when compared to the wild type strain. Expression of RFP-tagged mlcE showed that MlcE is localized to the yeast plasma and vacuolar membranes. We provide a possible engineering strategy for improvement of future yeast based production of natural and semi-synthetic statins.

  6. Potential of a Saccharomyces cerevisiae recombinant strain lacking ethanol and glycerol biosynthesis pathways in efficient anaerobic bioproduction

    Hirasawa, Takashi; Ida, Yoshihiro; Furuasawa, Chikara; Shimizu, Hiroshi


    Saccharomyces cerevisiae shows high growth activity under low pH conditions and can be used for producing acidic chemicals such as organic acids as well as fuel ethanol. However, ethanol can also be a problematic by-product in the production of chemicals except for ethanol. We have reported that a stable low-ethanol production phenotype was achieved by disrupting 6 NADH-dependent alcohol dehydrogenase genes of S. cerevisiae. Moreover, the genes encoding the NADH-dependent glycerol biosynthesis enzymes were further disrupted because the ADH-disrupted recombinant strain showed high glycerol production to maintain intracellular redox balance. The recombinant strain incapable producing ethanol and glycerol could have the potential to be a host for producing metabolite(s) whose biosynthesis is coupled with NADH oxidation. Indeed, we successfully achieved almost 100% yield for L-lactate production using this recombinant strain as a host. In addition, the potential of our constructed recombinant strain for efficient bioproduction, particularly under anaerobic conditions, is also discussed. PMID:24247205

  7. Beta-glucana from Saccharomyces cerevisiae: constitution, bioactivity and obtaining / Beta-glucana de Saccharomyces cerevisiae: constituição, bioatividade e obtenção

    Raul Jorge Hernan Castro-Gómez


    Full Text Available b-glucans are polysaccharides that constitute the structure of the cell wall of yeast, fungi and some cereals, which differs each other by the linkages between glucose units. An important source of these polymers is the Saccharomyces cerevisiae cell wall, which is a yeast widely used in industrial processes of fermentation. The b-glucan is considered to be a modifier of biological response due to its immunomodulator potential. When it is recognized by specific cellular receptors, have the ability to enhance the host’s immune response. Other beneficial effects such as anticarcinogenic, antimutagenic, hypocholesterolemic and blood sugar reduction have also been related to the b-glucan. The aim of this literature review was expand scientific knowledge about the constitution and bioactivity of b-glucan, including its recognition by the immune system, as well as its obtaining from S. cerevisiae cell wall.b-glucanas são polissacarídeos constituintes estruturais da parede celular de leveduras, fungos e alguns cereais, que se diferenciam pelo tipo de ligação presente entre as unidades de glicose. Uma importante fonte destes polissacarídeos é a parede celular de Saccharomyces cerevisiae, uma levedura amplamente empregada em processos industriais de fermentação. A b-glucana é considerada um modificador da resposta biológica devido ao seu potencial imunomodulador, pois ao ser reconhecida por receptores celulares específicos tem habilidade de realçar a resposta imune do hospedeiro. Outros efeitos benéficos como anticarcinogênico, antimutagênico, hipocolesterolêmico e hipoglicêmico também têm sido relacionados à b-glucana Esta revisão de literatura teve por objetivo agregar conhecimentos científicos sobre a constituição e bioatividade da b glucana, incluindo seu reconhecimento pelo sistema imune, bem como, a obtenção a partir da parede celular de S. cerevisiae.

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

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


    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.

  9. Enological characterization of natural hybrids from Saccharomyces cerevisiae and S. kudriavzevii.

    González, Sara S; Gallo, Luisa; Climent, M A Dolores; Barrio, Eladio; Querol, Amparo


    The effect of yeasts on wine flavor response is of primary importance. The genus Saccharomyces, and mainly the species Saccharomyces cerevisiae, is responsible for alcoholic fermentation. Recently, several novel yeast isolates from wines have been described as hybrid yeasts between S. cerevisiae x S. kudriavzevii. We have analyzed their influence on two grape musts (Macabeo and Tempranillo) in fermentations conducted at four different temperatures (14, 18, 22 and 32 degrees C) by studying volatile compound production, sugar assimilation and other characteristics influencing the enological properties of wine caused by the impact of yeast. Hybrid yeasts behave particularly well at 14, 18 and 22 degrees C and the commercial strain of S. cerevisiae (T73) is better adapted at higher temperatures. Regarding the production of glycerol, acetic acid and malic acid, the hybrids display moderate behavior and concerning aromatic compound production, they are greater producers of higher alcohols. The behavior displayed by these hybrids in the fermentations studied in this work leads us to conclude that the use of hybrid strains can constitute an advantage in wine making.

  10. Improving the productivity of S-adenosyl-l-methionine by metabolic engineering in an industrial Saccharomyces cerevisiae strain.

    Zhao, Weijun; Hang, Baojian; Zhu, Xiangcheng; Wang, Ri; Shen, Minjie; Huang, Lei; Xu, Zhinan


    S-Adenosyl-l-methionine (SAM) is an important metabolite having prominent roles in treating various diseases. In order to improve the production of SAM, the regulation of three metabolic pathways involved in SAM biosynthesis were investigated in an industrial yeast strain ZJU001. GLC3 encoded glycogen-branching enzyme (GBE), SPE2 encoded SAM decarboxylase, as well as ERG4 and ERG6 encoded key enzymes in ergosterol biosynthesis, were knocked out in ZJU001 accordingly. The results indicated that blocking of either glycogen pathway or SAM decarboxylation pathway could improve the SAM accumulation significantly in ZJU001, while single disruption of either ERG4 or ERG6 gene had no obvious effect on SAM production. Moreover, the double mutant ZJU001-GS with deletion of both GLC3 and SPE2 genes was also constructed, which showed further improvement of SAM accumulation. Finally, SAM2 was overexpressed in ZJU001-GS to give the best SAM-producing recombinant strain ZJU001-GS-SAM2, in which 12.47g/L SAM was produced by following our developed pseudo-exponential fed-batch cultivation strategy, about 81.0% increase comparing to its parent strain ZJU001. The present work laid a solid base for large-scale SAM production with the industrial Saccharomyces cerevisiae strain.

  11. Activation of waste brewer's yeast Saccharomyces cerevisiae for bread production

    Popov Stevan D.


    Full Text Available The waste brewer's yeast S. cerevisiae (activated and non-activated was compared with the commercial baker's yeast regarding the volume of developed gas in dough, volume and freshness stability of produced bread. The activation of waste brewer's yeast resulted in the increased volume of developed gas in dough by 100% compared to non-activated brewer's yeast, and the obtained bread is of more stable freshness compared to bread produced with baker's yeast. The activation of BY affects positively the quality of produced bread regarding bread volume. The volume of developed gas in dough prepared with the use of non-activated BY was not sufficient, therefore, it should not be used as fermentation agent, but only as an additive in bread production process for bread freshness preservation. Intense mixing of dough results in more compressible crumb 48 hrs after baking compared to high-speed mixing.

  12. Saccharomyces cerevisiae SCY1发酵kefir的工艺研究%Production of Kefir via Milk Fermentation by Saccharomyces cerevisiae SCY1

    李理; 马栋; 张静


    本文采用Saccharomyces cerevisiae SCY1和乳酸菌混合发酵牛乳制备kefir,分别研究了接种量、灭菌条件、发酵温度和加糖量对kefir风味的影响,最终确定最佳工艺条件为:XPL-1接种量为0.0400 g/L,SCY1接种量为103个/mL;灭菌条件为80~85℃下灭菌10 min;发酵温度为32℃;加糖量为4%.通过此工艺条件制备的kefir,具有独特的风味和较高的营养价值.

  13. Modulation of gluconeogenesis and lipid production in an engineered oleaginous Saccharomyces cerevisiae transformant.

    Kamisaka, Yasushi; Kimura, Kazuyoshi; Uemura, Hiroshi; Ledesma-Amaro, Rodrigo


    We previously created an oleaginous Saccharomyces cerevisiae transformant as a dga1 mutant overexpressing Dga1p lacking 29 amino acids at the N-terminal (Dga1∆Np). Because we have already shown that dga1 disruption decreases the expression of ESA1, which encodes histone acetyltransferase, the present study was aimed at exploring how Esa1p was involved in lipid accumulation. We based our work on the previous observation that Esa1p acetylates and activates phosphoenolpyruvate carboxykinase (PEPCK) encoded by PCK1, a rate-limiting enzyme in gluconeogenesis, and subsequently evaluated the activation of Pck1p by yeast growth with non-fermentable carbon sources, thus dependent on gluconeogenesis. This assay revealed that the ∆dga1 mutant overexpressing Dga1∆Np had much lower growth in a glycerol-lactate (GL) medium than the wild-type strain overexpressing Dga1∆Np. Moreover, overexpression of Esa1p or Pck1p in mutants improved the growth, indicating that the ∆dga1 mutant overexpressing Dga1∆Np had lower activities of Pck1p and gluconeogenesis due to lower expression of ESA1. In vitro PEPCK assay showed the same trend in the culture of the ∆dga1 mutant overexpressing Dga1∆Np with 10 % glucose medium, indicating that Pck1p-mediated gluconeogenesis decreased in this oleaginous transformant under the lipid-accumulating conditions introduced by the glucose medium. The growth of the ∆dga1 mutant overexpressing Dga1∆Np in the GL medium was also improved by overexpression of acetyl-CoA synthetase, Acs1p or Acs2p, indicating that supply of acetyl-CoA was crucial for Pck1p acetylation by Esa1p. In addition, the ∆dga1 mutant without Dga1∆Np also showed better growth in the GL medium, indicating that decreased lipid accumulation was enhancing Pck1p-mediated gluconeogenesis. Finally, we found that overexpression of Ole1p, a fatty acid ∆9-desaturase, in the ∆dga1 mutant overexpressing Dga1∆Np improved its growth in the GL medium. Although the exact

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

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


    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.

  15. [The cloning and expression of the gene for beta-galactosidase from Candida pseudotropicalis yeasts in Saccharomyces cerevisiae cells].

    Tretiak, K A; Zakal'skiĭ, A E; Gudz', S P


    The gene of beta-galactosidase of lactose-assimilating yeast Candida pseudotropicalis was cloned in pG2 and pBG2-3 hybrid shuttle vectors and expressed in Saccharomyces cerevisiae laboratory strains under the control of own promoter. The plasmids were able to replicate autonomously with relative stability in transformants of baker's yeasts. The availability of glucose or lactose in the medium influenced the recombinant plasmid stability and the expression of the cloned gene. A number of experiments have shown that the LAC+ phenotype in pG2-transformed Saccharomyces cerevisiae was due to the expression of the Candida pseudotropicalis lactose permease gene that is probably located in SaIG1/XhoI DNA fragment about 4.3 kb long. Southern hybridization experiments showed that LAC(+)-transformants of Saccharomyces cerevisiae contained both autonomously-replicative, and integrative pG2 plasmid.

  16. Optimization of feeding strategy for the ergosterol production by yeasts Saccharomyces cerevisiae

    Mojmir Rychtera


    Full Text Available Objective of this study was to optimize ergosterol production by yeast strain Saccharomyces cerevisiae with the use of computer controlled feeding of cultivation medium. Baker´s yeasts strain of Saccharomyces cerevisiae originally modified and selected as mutant D7 was further applied in an industrial scale and also in this investigation. Composition of cultivation medium was optimized with the use of a modified Rosenbrock´s method with regard to following components: glucose, yeast extract, ammonium sulphate, potassium dihydrogen phosphate, magnesium sulphate and calcium chloride. Cultivation of yeast culture was performed in 7 L laboratory bioreactor with a working volume of 5 L equipped with a control unit and linked to a computer, with dissolved oxygen tension measurement, oxygen and carbon dioxide analyzers. BIOGENES prototype software was created from the commercial control system Genesis for Windows 3.0 (GFW, from Iconics and CLIPS 6.04 for the PC-Windows platform. From various factors affecting sterol biosynthesis a specific growth rate was chosen. Feed rate was controlled according to mathematical model. In this case it dealt with a design of optimal profile of specific growth rate with consequent calculation of carbon dioxide profile. Sterol concentration in the dry biomass increased from 1.0 % up to 3 %. Key words: Saccharomyces cerevisiae yeasts, ergosterol, fed-batch cultivation control, effect of the specific growth rate. Resumen: El objetivo de este estudio fue optimizar la producción de ergosterol por una cepa de levadura Saccharomyces cerevisiae, controlando la alimentación de medio de cultivo por computadora. La cepa de levadura panadera Saccharomyces cerevisiae originalmente modificada y seleccionada como mutante D7 fue posteriormente utilizada a escala industrial y también para esta investigación. La composición del medio de cultivo fue optimizada usando el método modificado de Rosenbrock respecto a los siguientes

  17. Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae

    Tremaine, Mary; Hebert, Alexander S.; Myers, Kevin S.; Sardi, Maria; Dickinson, Quinn; Reed, Jennifer L.; Zhang, Yaoping; Coon, Joshua J.; Hittinger, Chris Todd; Gasch, Audrey P.; Landick, Robert


    The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism. PMID:27741250

  18. Disruption of the Saccharomyces cerevisiae homologue to the murine fatty acid transport protein impairs uptake and growth on long-chain fatty acids

    Færgeman, Nils J.; DiRusso, C C; Elberger, A;


    described in 3T3-L1 adipocytes (Schaffer and Lodish (1994) Cell 79, 427-436), suggesting a similar function. Disruption of FAT1 results in 1) an impaired growth in YPD medium containing 25 microM cerulenin and 500 microM fatty acid (myristate (C14:0), palmitate (C16:0), or oleate (C18:1)); 2) a marked......The yeast Saccharomyces cerevisiae is able to utilize exogenous fatty acids for a variety of cellular processes including beta-oxidation, phospholipid biosynthesis, and protein modification. The molecular mechanisms that govern the uptake of these compounds in S. cerevisiae have not been described....... We report the characterization of FAT1, a gene that encodes a putative membrane-bound long-chain fatty acid transport protein (Fat1p). Fat1p contains 623 amino acid residues that are 33% identical and 54% with similar chemical properties as compared with the fatty acid transport protein FATP...

  19. Genomic diversity of Saccharomyces cerevisiae yeasts associated with alcoholic fermentation of bacanora produced by artisanal methods.

    Álvarez-Ainza, M L; Zamora-Quiñonez, K A; Moreno-Ibarra, G M; Acedo-Félix, E


    Bacanora is a spirituous beverage elaborated with Agave angustifolia Haw in an artisanal process. Natural fermentation is mostly performed with native yeasts and bacteria. In this study, 228 strains of yeast like Saccharomyces were isolated from the natural alcoholic fermentation on the production of bacanora. Restriction analysis of the amplified region ITS1-5.8S-ITS2 of the ribosomal DNA genes (RFLPr) were used to confirm the genus, and 182 strains were identified as Saccharomyces cerevisiae. These strains displayed high genomic variability in their chromosomes profiles by karyotyping. Electrophoretic profiles of the strains evaluated showed a large number of chromosomes the size of which ranged between 225 and 2200 kpb approximately.

  20. Vanadium pentoxide effects on stress responses in wine Saccharomyces cerevisiae strain UE-ME3.

    Rosado, Tânia; Conim, Ana; Alves-Pereira, Isabel; Ferreira, Rui


    Vanadium pentoxide mainly used as catalyst in sulphuric acid, maleic anhydride and ceramics industry, is a pollutant watering redistributed around the environment. Research on biological influence of vanadium pentoxide has gained major importance because it exerts toxic effects on a wide variety of biological systems. In this work we intent to evaluate the effects of vanadium pentoxide ranging from 0 to 2 mM in culture media on a wine wild-type Saccharomyces cerevisiae from Alentejo region of Portugal. Our results show that 2.0 mM vanadium pentoxide in culture medium induced a significant increase of malonaldehyde level and Glutathione peroxidase activity, a slightly increase of Catalase A activity as well as a decrease of wet weight and mitochondrial NADH cit c reductase of S. cerevisiae UE-ME(3). Also our results show that cycloheximide prevent cell death when cells grows 30 min in presence of 1.5 mM of vanadium pentoxide.

  1. The Bioconversion of Red Ginseng Ethanol Extract into Compound K by Saccharomyces cerevisiae HJ-014.

    Choi, Hak Joo; Kim, Eun A; Kim, Dong Hee; Shin, Kwang-Soo


    A β-glucosidase producing yeast strain was isolated from Korean traditional rice wine. Based on the sequence of the YCL008c gene and analysis of the fatty acid composition, the isolate was identified as Saccharomyces cerevisiae strain HJ-014. S. cerevisiae HJ-014 produced ginsenoside Rd, F2, and compound K from the ethanol extract of red ginseng. The production was increased by shaking culture, where the bioconversion efficiency was increased 2-fold compared to standing culture. The production of ginsenoside F2 and compound K was time-dependent and thought to proceed by the transformation pathway of: red ginseng extract→Rd→F2→compound K. The optimum incubation time and concentration of red ginseng extract for the production of compound K was 96 hr and 4.5% (w/v), respectively.

  2. Biogenic amine accumulation in silver carp sausage inoculated with Lactobacillus plantarum plus Saccharomyces cerevisiae.

    Nie, Xiaohua; Zhang, Qilin; Lin, Shengli


    The effect of an amine-negative mixed starter culture (Lactobacillus plantarum ZY40 plus Saccharomyces cerevisiae JM19) on biogenic amine accumulation in fermented silver carp sausage was studied. Microbial counts, pH, titratable acid and free amino acids were also determined. Putrescine, cadaverine and tyramine were the main amines formed during sausage fermentation. The contents of putrescine and cadaverine were greatly reduced by the addition of L. plantarum ZY40 plus S. cerevisiae JM19, whereas tyramine accumulation was enhanced as compared to the control batch. Histamine and spermidine were not affected by the mixed starter culture, and their levels varied slightly throughout the fermentation. Besides, no positive correction between pH, free amino acid content and biogenic amine accumulation were found.

  3. Microarray analysis of p-anisaldehyde-induced transcriptome of Saccharomyces cerevisiae.

    Yu, Lu; Guo, Na; Yang, Yi; Wu, Xiuping; Meng, Rizeng; Fan, Junwen; Ge, Fa; Wang, Xuelin; Liu, Jingbo; Deng, Xuming


    p-Anisaldehyde (4-methoxybenzaldehyde), an extract from Pimpinella anisum L. seeds, is a potential novel preservative. To reveal the possible action mechanism of p-anisaldehyde against microorganisms, yeast-based commercial oligonucleotide microarrays were used to analyze the genome-wide transcriptional changes in response to p-anisaldehyde. Quantitative real-time RT-PCR was performed for selected genes to verify the microarray results. We interpreted our microarray data with the clustering tool, T-profiler. Analysis of microarray data revealed that p-anisaldehyde induced the expression of genes related to sulphur assimilation, aromatic aldehydes metabolism, and secondary metabolism, which demonstrated that the addition of p-anisaldehyde may influence the normal metabolism of aromatic aldehydes. This genome-wide transcriptomics approach revealed first insights into the response of Saccharomyces cerevisiae (S. cerevisiae) to p-anisaldehyde challenge.

  4. Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid

    Sergio eGiannattasio


    Full Text Available Beyond its classical biotechnological applications such as food and beverage production or as a cell factory, the yeast Saccharomyces cerevisiae is a valuable model organism to study fundamental mechanisms of cell response to stressful environmental changes. Acetic acid is a physiological product of yeast fermentation and it is a well-known food preservative due to its antimicrobial action. Acetic acid has recently been shown to cause yeast cell death and aging. Here we shall focus on the molecular mechanisms of S. cerevisiae stress adaptation and programmed cell death in response to acetic acid. We shall elaborate on the intracellular signaling pathways involved in the cross-talk of pro-survival and pro-death pathways underlying the importance of understanding fundamental aspects of yeast cell homeostasis to improve the performance of a given yeast strain in biotechnological applications.

  5. Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.

    Duina, Andrea A; Miller, Mary E; Keeney, Jill B


    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.

  6. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A


    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

  7. Selection of Indigenous Saccharomyces cerevisiae Strains from Kutjevo Wine Growing Area at the Laboratoy Scale

    Sandi Orlić


    Full Text Available The use of selected yeasts for winemaking has clear advantages over traditional spontaneous fermentation. Selection of wine yeasts is usually carried out within the Saccharomyces cerevisiae species. Yeast strains produce different amount of secondary compounds that impart specific characteristics to the wines. This suggests that it is necessary to isolate naturally occuring autochthone strains, which exhibit a metabolic profile that corresponds to each wine. Twenty two strains of S.cerevisiae, isolated from the Kutjevo region (Gornji and Donji Hrnjevec, Mitrovac, Graševina grapes, were tested for: fermentation vigor, ethanol resistance, volatile acidity, H2S production and β-glucosidase, polygalacturonase, and killer activity. From the results of this investigation we are able to select two yeast strains (RO 1272 and RO 1284 for more detailed fermentation trials and possible use as a starter culture in production of typical wines.

  8. Z curve theory-based analysis of the dynamic nature of nucleosome positioning in Saccharomyces cerevisiae.

    Wu, Xueting; Liu, Hui; Liu, Hongbo; Su, Jianzhong; Lv, Jie; Cui, Ying; Wang, Fang; Zhang, Yan


    Nucleosome is the elementary structural unit of eukaryotic chromatin. Instability of nucleosome positioning plays critical roles in chromatin remodeling in differentiation and disease. In this study, we investigated nucleosome dynamics in the Saccharomyces cerevisiae genome using a geometric model based on Z curve theory. We identified 52,941 stable nucleosomes and 7607 dynamic nucleosomes, compiling them into a genome-wide nucleosome dynamic positioning map and constructing a user-friendly visualization platform ( Our approach achieved a sensitivity of 90.31% and a specificity of 87.76% for S. cerevisiae. Analysis revealed transcription factor binding sites (TFBSs) were enriched in linkers. And among the sparse nucleosomes around TFBSs, dynamic nucleosomes were slightly preferred. Gene Ontology (GO) enrichment analysis indicated that stable and dynamic nucleosomes were enriched on genes involved in different biological processes and functions. This study provides an approach for comprehending chromatin remodeling and transcriptional regulation of genes.

  9. New and efficient method using Saccharomyces cerevisiae mutants for identification of siderophores produced by microorganisms.

    Park, Yong-Sung; Kim, Ji-Hyun; Chang, Hyo-Ihl; Kim, Seung-Wook; Paik, Hyun-Dong; Kang, Chang-Won; Kim, Tae-Hyoung; Sung, Ha-Chin; Yun, Cheol-Won


    The separation and identification of siderophores produced by microorganisms is a time-consuming and an expensive procedure. We have developed a new and efficient method to identify siderophores using well-established Saccharomyces cerevisiae deletion mutants. The Deltafet3,arn strains fail to sustain growth, even when specific siderophores are supplied, and mutants are siderophore-specific: Deltafet3,arn2 for triacetylfusarinine C (TAFC), Deltafet3,arn1,sit1 for ferrichrome (FC), and Deltafet3,sit1 for ferrioxamine B (FOB). The culture broth of Fusarium graminearum was separated by HPLC, and each peak was subjected to a plate assay using S. cerevisiae mutants. We have found that each peak contained specific siderophores produced by F. graminearum, and these coincided with reference siderophores. This method is quite novel because nobody tried this method to identify the siderophores. Furthermore, this method will save time and cost in the identification of siderophores produced by microorganisms.

  10. Fed-batch bioreactor process with recombinant Saccharomyces cerevisiae growing on cheese whey

    R. Rech


    Full Text Available Saccharomyces cerevisiae strain W303 was transformed with two yeast integrative plasmids containing Kluyveromyces lactis LAC4 and LAC12 genes that codify beta-galactosidase and lactose permease respectively. The BLR030 recombinant strain was selected due to its growth and beta-galactosidase production capacity. Different culture media based on deproteinized cheese whey (DCW were tested and the best composition (containing DCW, supplemented with yeast extract 1 %, and peptone 3 % (w/v was chosen for bioreactor experiments. Batch, and fed-batch cultures with linear ascending feeding for 25 (FB25, 35 (FB35, and 50 (FB50 hours, were performed. FB35 and FB50 produced the highest beta-galactosidase specific activities (around 1,800 U/g cells, and also the best productivities (180 U/L.h. Results show the potential use of fed-batch cultures of recombinant S. cerevisiae on industrial applications using supplemented whey as substrate.

  11. Mini-Review: The Role of Saccharomyces cerevisiae in the Production of Gin and Vodka

    Matthew Pauley


    Full Text Available The spirit beverages of vodka and gin are often produced from a neutral spirits base. These neutral spirits are derived from the distillation of fermented carbohydrates of agricultural origin. The fermentations in the production of these beverages are not often reported in great detail and to some extent are shrouded in mystery. The roles of fermentation and the yeast Saccharomyces cerevisiae are essential to the complete process, and without fermentation there would not be alcohol to distil. Nevertheless, it is not the yeast that is perceived to contribute to the distinctive consumer experiences, which are associated with these beverages. However, there are opportunities for the development of new strains of S. cerevisiae for the production of neutral spirits, which have a high ethanol yield, are tolerant of ethanol stress, and produce low levels of congeners.

  12. Reconstruction of the biosynthetic pathway for the core fungal polyketide scaffold rubrofusarin in Saccharomyces cerevisiae

    Rugbjerg, Peter; Naesby, Michael; Mortensen, Uffe Hasbro


    production in easily fermentable and genetically engineerable organisms, such as Saccharomyces cerevisiae and Escherichia coli are desirable. Rubrofusarin is an orange polyketide pigment that is a common intermediate in many different fungal biosynthetic pathways. RESULTS: In this study, we established......BACKGROUND: Fungal polyketides include commercially important pharmaceuticals and food additives, e.g. the cholesterol-lowering statins and the red and orange monascus pigments. Presently, production relies on isolation of the compounds from the natural producers, and systems for heterologous....... CONCLUSIONS: The reconstructed pathway for rubrofusarin in S. cerevisiae allows the production of a core scaffold molecule with a branch-point role in several fungal polyketide pathways, thus paving the way for production of further natural pigments and bioactive molecules. Furthermore, the reconstruction...

  13. Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains

    Sonderegger, M.; Jeppsson, M.; Larsson, C.;


    Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrol......Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components...... in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation...

  14. Saccharomyces cerevisiae biofilm tolerance towards systemic antifungals depends on growth phase

    Bojsen, Rasmus Kenneth; Regenberg, Birgitte; Folkesson, Sven Anders


    used Saccharomyces cerevisiae as a model for drug susceptibility of yeast biofilms. Confocal laser scanning microscopy showed that S. cerevisiae and C. glabrata form similarly structured biofilms and that the viable cell numbers were significantly reduced by treatment of mature biofilms......Background : Biofilm-forming Candida species cause infections that can be difficult to eradicate, possibly because of antifungal drug tolerance mechanisms specific to biofilms. In spite of decades of research, the connection between biofilm and drug tolerance is not fully understood. Results : We...... with amphotericin B but not voriconazole, flucytosine, or caspofungin. We showed that metabolic activity in yeast biofilm cells decreased with time, as visualized by FUN-1 staining, and mature, 48-hour biofilms contained cells with slow metabolism and limited growth. Time-kill studies showed that in exponentially...

  15. Changes and roles of membrane compositions in the adaptation of Saccharomyces cerevisiae to ethanol.

    Wang, Yanfeng; Zhang, Shuxian; Liu, Huaqing; Zhang, Lei; Yi, Chenfeng; Li, Hao


    Bioethanol fermentation by Saccharomyces cerevisiae is often stressed by the accumulation of ethanol. Cell membrane is the first assaulting target of ethanol. Ethanol-adapted S. cerevisiae strains provide opportunity to shed light on membrane functions in the ethanol tolerance. This study aimed at clarifying the roles of cell membrane in the ethanol tolerance of S. cerevisiae through comparing membrane components between S. cerevisiae parental strain and ethanol-adapted strains. A directed evolutionary engineering was performed to obtain the ethanol-adapted S. cerevisiae strains. The parental, ethanol-adapted M5 and M10 strains were selected to be compared the percentage of viable cells after exposing to ethanol stress and cell membrane compositions (i.e., ergosterol, trehalose, and fatty acids). Compared with the parental strain, M5 or M10 strain had higher survival rate in the presence of 10% v/v ethanol. Compared with that in the parental strain, contents of trehalose, ergosterol, and fatty acids increased about 15.7, 12.1, and 29.3%, respectively, in M5 strain, and about 47.5, 107.8, and 61.5%, respectively, in M10 strain. Moreover, expression differences of genes involved in fatty acids metabolisms among the parental, M5 and M10 strains were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and results demonstrated that M5 or M10 strain had higher expression of ACC1 and OLE1 than the parental strain. These results indicated that although being exposed to step-wise increased ethanol, S. cerevisiae cells might remodel membrane components or structure to adapt to the ethanol stress.

  16. Global Transcriptional and Physiological Responses of Saccharomyces cerevisiae to Ammonium, L-Alanine, or L-Glutamine Limitation

    Usaite, Renata; Patil, Kiran Raosaheb; Grotkjær, Thomas;


    The yeast Saccharomyces cerevisiae encounters a range of nitrogen sources at various concentrations in its environment. The impact of these two parameters on transcription and metabolism was studied by growing S. cerevisiae in chemostat cultures with L-glutamine, L-alanine, or L-ammonium in limit......The yeast Saccharomyces cerevisiae encounters a range of nitrogen sources at various concentrations in its environment. The impact of these two parameters on transcription and metabolism was studied by growing S. cerevisiae in chemostat cultures with L-glutamine, L-alanine, or L...... activity in L-alanine-limited cells. The changes in these cells were found to be focused around pyruvate, acetyl coenzyme A, glyoxylate, and alpha-ketoglutarate via increased levels of ALT1, DAL7, PYC1, GDH2, and ADH5 and decreased levels of GDH3, CIT2, and ACS1 transcripts. The transcript profiles were...

  17. Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

    Thomsen, Rune; Libri, Domenico; Boulay, Jocelyne


    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...... 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...

  18. Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

    Jakociunas, Tadas; Bonde, Ida; Herrgard, Markus


    CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces...... cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains...

  19. Ethanol fermentation of a diluted molasses medium by Saccharomyces cerevisiae immobilized on chrysotile

    Monte Alegre Ranulfo


    Full Text Available In this work, the catalytic role of chrysotile support on the acceleration of alcoholic fermentation under non-aseptic conditions by Saccharomyces cerevisiae was investigated. The fermentation medium employed consisted only of diluted sugar-cane molasses. In the batch fermentations process with immobilized yeasts, the initial rate of CO2 production increased roughly 27 % during the first 30 minutes, compared to systems containing no chrysotile. A study of continuous alcoholic fermentation with chrysotile in the reactor bed showed a higher ethanol production rate at the different dilution rates investigated compared to similar fermentations without chrysotile.

  20. Kinetics of volatile metabolites during alcoholic fermentation of cane molasses by Saccharomyces cerevisiae

    Cachot, T.; Mueller, M.; Pons, M.N. (Centre National de la Recherche Scientifique, 54 - Nancy (France). Lab. des Sciences du Genie Chimique)


    The kinetics of ethanol, acetaldehyde, ethyl acetate and fusel alcohols during alcoholic fermentations on cane molasses by Saccharomyces cerevisiae have been obtained via an in-situ gas membrane sensor connected to a gas chromatograph. Various operation parameters have been investigated such as inoculum rate, molasses concentration, operation mode (batch, fed-batch). The modification of fusel alcohols kinetics in response to addition of amino acids has been studied as well as the assimilation of two intermediary aldehydes (isovaleraldehyde and isobutyraldehyde) in the fusel alcohol synthesis pathway. (orig.).

  1. Análisis funcional del complejo p24 en Saccharomyces cerevisiae

    Aguilera Romero, María Auxiliadora


    El propósito general de esta tesis es el análisis molecular de la función del complejo p24 en los procesos selectivos de transporte vesicular entre el RE y el aparato de Golgi en la levadura Saccharomyces cerevisiae. ... y: 'Times New Roman','serif'; font-size: 12pt">Para abordar este objetivo general se han planteado los siguientes objetivos concretos:1. Estudio de la función del complejo p24 en la formación de las vesículas COPI derivadas del Golgi.2. Análisis del requerimiento del compl...

  2. Expression of α-Amylase Gene of Bacillus licheniformis in Saccharomyces cerevisiae

    罗进贤; 黎杨; 李文清; 张添元; 何鸣


    The secretive expression vector has been constructed using the promoter and signal se-quence of yeast MF-α1 factor,and the Bacillus licheniformis α-amylase gene without promoter and signal se-quence has been inserted into the downstream of the signal sequence on the vector.After the readjustment ofthe reading frame,the amylase gene was expressed in Saccharomyces cerevisiae and the product was secretedfrom it.The properties of enzymes secreted from yeast and B.subtilis are compared,and the mechanism ofthe gene expression and product secretion are discussed.

  3. Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis

    Nissen, Torben Lauesgaard; Hamann, Claus Wendelboe; Kielland-Brandt, M. C.;


    Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass....... The modest effect of the GPD1 deletion under anaerobic conditions on the maximum specific growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth. Strain TN6 (gpd1-Delta 1 gpd2-Delta 1) was unable to grow under anaerobic conditions due...

  4. Asparaginase II of Saccharomyces cerevisiae: selection of four mutations that cause derepressed enzyme synthesis.

    Kamerud, J Q; Roon, R J


    A positive selection method was used to isolate four Saccharomyces cerevisiae mutations that cause derepressed synthesis of asparaginase II. The four mutations (and1, and2, and3, and4) were neither closely linked to each other nor linked to previously characterized mutations (asp3, asp6) which cause the complete loss of asparaginase II activity. One of the new mutations (and4) was shown to be allelic to gdh-CR, a pleiotropic mutation which causes derepressed synthesis of a number of enzymes of nitrogen catabolism.

  5. Mutation of the mitochondrial large ribosomal RNA can provide pentamidine resistance to Saccharomyces cerevisiae.

    Örs, Ş Tomris; Akdoğan, Emel; Dunn, Cory D


    Pentamidine is used to treat several trypanosomal diseases, as well as opportunistic infection by pathogenic fungi. However, the relevant targets of this drug are unknown. We isolated dominant mutations providing pentamidine resistance to Saccharomyces cerevisiae, one of which was localized to mitochondrial DNA. Next-generation sequencing revealed alteration of a widely conserved base at the peptidyl transferase center of the mitochondrial 21S ribosomal RNA. Our results provide a potential rationale for the toxicity of this drug to patients, and we discuss whether blockade of mitochondrial translation is the mechanism by which pathogenic fungi or protists are killed by pentamidine.

  6. Isolation of Cytokinetic Actomyosin Rings from Saccharomyces cerevisiae and Schizosaccharomyces pombe

    Palani, Saravanan; Chew, Ting Gang; Balasubramanian, Mohan K.


    Cytokinesis is the final stage of cell division, through which cellular constituents of mother cells are partitioned into two daughter cells resulting in the increase in cell number. In animal and fungal cells cytokinesis is mediated by an actomyosin contractile ring, which is attached to the overlying cell membrane. Contraction of this ring after chromosome segregation physically severs the mother cell into two daughters. Here we describe methods for the isolation and partial purification of the actomyosin ring from the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which can serve as in vitro systems to facilitate biochemical and ultrastructural analysis of cytokinesis in these genetically tractable model systems. PMID:26519310

  7. Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

    Montiel, J F; Norbury, C J; Tuite, M F; Dobson, M J; Mills, J S; Kingsman, A J; Kingsman, S M


    We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence. PMID:6320114

  8. Tight Coupling of Metabolic Oscillations and Intracellular Water Dynamics in Saccharomyces cerevisiae

    Thoke, Henrik Seir; Tobiesen, Asger; Brewer, Jonathan R.


    We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute...... conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D...

  9. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna


    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level.

  10. Crystallization and preliminary X-ray diffraction data of alpha-galactosidase from Saccharomyces cerevisiae.

    Fernández-Leiro, Rafael; Pereira-Rodríguez, Angel; Cerdán, M Esperanza; Becerra, Manuel; Sanz-Aparicio, Juliana


    Saccharomyces cerevisiae alpha-galactosidase is a highly glycosylated extracellular protein that catalyzes the hydrolysis of alpha-galactosidic linkages in various glucids. Its enzymatic activity is of interest in many food-related industries and has biotechnological applications. Glycosylated and in vitro deglycosylated protein samples were both assayed for crystallization, but only the latter gave good-quality crystals that were suitable for X-ray crystallography. The crystals belonged to space group P42(1)2, with unit-cell parameters a = b = 101.24, c = 111.52 A. A complete diffraction data set was collected to 1.95 A resolution using a synchrotron source.

  11. Simultaneous raw starch hydrolysis and ethanol fermentation by glucoamylase from Rhizoctonia solani and Saccharomyces cerevisiae.

    Singh, D; Dahiya, J S; Nigam, P


    Crude glucoamylase preparation from Rhizoctonia solani was used to saccharify raw and cooked starch. Various concentrations of potato starch and wheat flour from 10-40%, w/v were used for mashing but 30% was found to be the optimal and economical. The saccharified mash yielded 5.89%, v/v ethanol in a simultaneous saccharification and fermentation process using a yeast strain Saccharomyces cerevisiae (SC-39) at 35 degrees C for 4 days. Removal of inhibitory substances from the fermenting broth through dialysis caused considerable increase in ethanol production.

  12. On-line cell mass monitoring of Saccharomyces cerevisiae cultivations by multi-wavelength fluorescence

    Haack, Martin Brian; Eliasson, Anna; Olsson, Lisbeth


    The catalyst in bioprocesses, i.e. the cell mass, is one of the most challenging and important variables to monitor in bioprocesses. In the present study, cell mass in cultivations with Saccharomyces cerevisiae was monitored on-line with a non-invasive in situ placed sensor measuring multi......-line monitoring of culture fluorescence can be used for estimation of the cell mass concentration during cultivations....... in a decomposition of the multivariate fluorescent landscape, whereby underlying spectra of the individual intrinsic fluorophors present in the cell mass were estimated. Furthermore, gravimetrically determined cell mass concentration was used together with the fluorescence spectra for calibration and validation...

  13. Sensitivity of Saccharomyces cerevisiae defective in TOR signaling pathway to carbonyl/oxidative stress

    Valishkevych B. V.


    Full Text Available Aim. To investigate the influence of carbonyl/oxidative stress induced by glyoxal, methylglyoxal and hydrogen peroxide on the survival of Saccharomyces cerevisiae, defective for different parts of TOR- signaling pathway, grown on glucose or fructose. Methods. The assessment of number of colony-forming units to determine the yeast reproductive ability. Results. It was shown that at certain concentrations the mentioned above toxicants caused an increase in yeast survival, indicating the hormetic effect. Conclusions. The TOR signaling pathway is involved in the hormetic effect, but it is specific for each strain and depends on the type of carbohydrate in the incubation medium.

  14. Cloning and expression in Saccharomyces cerevisiae of chit2 gene from Beauveria bassiana

    SONG Jin-zhu; YANG Xiao-xue; WANG Yun; YANG Qian


    To study recycled trashes from shrimps and crabs in the sea through chitinase secreted by microor-ganisms, the chitinase gene chit2 was cloned and sequenced from Beauveria bassiana by the polymerase chain reaction (PCR), and was ligated into the yeast expression vector pYES2. The expression vector plasmid was transformed into Saccharomyces cerevisiae H158. Gene expression took place upon induction with 2% galac-tose. The measurement of enzyme activity shows that the expression production can be expressed in active forms and secreted to the medium. The enzyme activity approaches the peak of 0. 63 U/mL when the culture time is 36 h.

  15. Role of phosphate in the regulation of the Pasteur effect in Saccharomyces cerevisiae.

    Lagunas, R; Gancedo, C


    The occurrence of the Pasteur effect in Saccharomyces cerevisiae in several conditions has been examined. In these conditions measurements of a series of metabolites potentially involved in the regulation of the effect were performed. These included, among others, adenine nucleotides, citrate, fructose 2,6-bisphosphate and phosphate. Only phosphate changed in a consistent way, increasing in anaerobiosis when the Pasteur effect occurred. It is concluded that, with the available data, only phosphate may be considered as a regulator of the Pasteur effect in this microorganism.

  16. SAS4 and SAS5 are locus-specific regulators of silencing in Saccharomyces cerevisiae.

    Xu, E. Y; S. Kim; Rivier, D H


    Sir2p, Sir3p, Sir4p, and the core histones form a repressive chromatin structure that silences transcription in the regions near telomeres and at the HML and HMR cryptic mating-type loci in Saccharomyces cerevisiae. Null alleles of SAS4 and SAS5 suppress silencing defects at HMR; therefore, SAS4 and SAS5 are negative regulators of silencing at HMR. This study revealed that SAS4 and SAS5 contribute to silencing at HML and the telomeres, indicating that SAS4 and SAS5 are positive regulators of ...

  17. Increasing ethanol productivity during xylose fermentation by cell recycling of recombinant Saccharomyces cerevisiae

    Roca, Christophe Francois Aime; Olsson, Lisbeth


    The influence of cell recycling of xylose-fermenting Saccharomyces cerevisiae TMB3001 was investigated during continuous cultivation on a xylose-glucose mixture. By using cell recycling at the dilution rate (D) of 0.05 h(-1), the cell-mass concentration could be increased from 2.2 g l(-1) to 22 g l...... ethanol productivity was in the range of 0.23-0.26 g g(-1) h(-1) with or without cell recycling, showing that an increased cell-mass concentration did not influence the efficiency of the yeast....

  18. Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae


    The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S pha...

  19. [Geothermal water as part of culture medium and morpho-physiological properties of Saccharomyces cerevisiae].

    Abramov, Sh A; Kotenko, S Ts; Khalilova, E A; Kisrieva, Iu S


    Morphophysiological changes in Saccharomyces cerevisiae gamma-503 cells cultivated in nutrient media containing geothermal water as a source of mineral substances were studied. The optimal mineralization of the medium was found to be 4.0 g/l, supplemented with 2.6 g/l (NH4)2HPO4. These conditions provided active growth and development of the culture with high yields of the biomass and the maximal enzymatic activity. Differences in cellular structures at certain stages of metabolism were demonstrated.

  20. Behavior of Lactobacillus plantarum and Saccharomyces cerevisiae in fresh and thermally processed orange juice.

    Alwazeer, Duried; Cachon, Remy; Divies, Charles


    Lactobacillus plantarum and Saccharomyces cerevisiae are acid-tolerant microorganisms that are able to spoil citrus juices before and after pasteurization. The growth of these microorganisms in orange juice with and without pasteurization was investigated. Two samples of orange juice were inoculated with ca. 10(5) CFU/ml of each microorganism. Others were inoculated with ca. 10(7) CFU/ml of each microorganism and then thermally treated. L. plantarum populations were reduced by 2.5 and 6 and 2 log10 CFU/ml, respectively. Samples of heated and nonheated juice were incubated at 15 degrees C for 20 days. Injured populations of L. plantarum decreased by ca. 2 log10 CFU/ml during the first 70 h of storage, but those of S. cerevisiae did not decrease. The length of the lag phase after pasteurization increased 6.2-fold for L. plantarum and 1.9-fold for S. cerevisiae, and generation times increased by 41 and 86%, respectively. The results of this study demonstrate the differences in the capabilities of intact and injured cells of spoilage microorganisms to spoil citrus juice and the different thermal resistance levels of cells. While L. plantarum was more resistant to heat treatment than S. cerevisiae was, growth recovery after pasteurization was faster for the latter microorganism.