Small Toxic Protein Encoded on Chromosome VII of Saccharomyces cerevisiae (United States)

In a previous study, we found an unknown element that caused growth inhibition after its copy number increased in the 3? region of DIE2 in Saccharomyces cerevisiae. In this study, we further identified this element and observed that overexpression of a small protein (sORF2) of 57 amino acids encoded in this region caused growth inhibition. The transcriptional response and multicopy suppression of the growth inhibition caused by sORF2 overexpression suggest that sORF2 overexpression inhibits the ergosterol biosynthetic pathway. sORF2 was not required in the normal growth of S. cerevisiae, and not conserved in related yeast species including S. paradoxus. Thus, sORF2 (designated as OTO1) is an orphan ORF that determines the specificity of this species. PMID:25781884

Makanae, Koji; Kintaka, Reiko; Ishikawa, Koji; Moriya, Hisao



Cloning and expression of the MEP1 gene encoding an ammonium transporter in Saccharomyces cerevisiae.  


In Saccharomyces cerevisiae, the transport of ammonium across the plasma membrane for use as a nitrogen source is mediated by at least two functionally distinct transport systems whose respective encoding genes are called MEP1 and MEP2. Mutations in the MEP2 gene affect high affinity, low capacity ammonium transport while mutations in the MEP1 gene disrupt a lower affinity, higher capacity system. In this work, the MEP1 gene has been cloned and sequenced and its expression analyzed. The predi...

Marini, A. M.; Vissers, S.; Urrestarazu, A.; Andre?, B.



Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins.  


Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences....

Schatz, P. J.; Pillus, L.; Grisafi, P.; Solomon, F.; Botstein, D.



Mitochondrial and nonmitochondrial citrate synthases in Saccharomyces cerevisiae are encoded by distinct homologous genes.  


Saccharomyces cerevisiae contains two genes, CIT1 and CIT2, encoding functional citrate synthase (K.-S. Kim, M. S. Rosenkrantz, and L. Guarente, Mol. Cell. Biol. 6:1936-1942, 1986). We show here that CIT2 encodes a nonmitochondrial form of citrate synthase. The DNA sequence of CIT2 presented provides a possible explanation for why the CIT2 product, unlike the CIT1 product, fails to be imported into mitochondria. While the products of these two genes are highly homologous, they diverge strikin...

Rosenkrantz, M.; Alam, T.; Kim, K. S.; Clark, B. J.; Srere, P. A.; Guarente, L. P.



The Saccharomyces cerevisiae ADR1 gene is a positive regulator of transcription of genes encoding peroxisomal proteins.  


Expression of the CTA1 gene of Saccharomyces cerevisiae, encoding catalase A, the peroxisomal catalase of this yeast, is sensitive to glucose repression. A DNA fragment cloned as a multicopy plasmid suppressing the glucose repression of CTA1 transcription was demonstrated to contain the ADR1 gene. Multiple copies of ADR1 increased catalase A formation not only on 10% glucose, but also on ethanol medium and in the presence of oleic acid, an inducer of peroxisome proliferation. Compared with wi...

Simon, M.; Adam, G.; Rapatz, W.; Spevak, W.; Ruis, H.



Proline biosynthesis in Saccharomyces cerevisiae: analysis of the PRO3 gene, which encodes delta 1-pyrroline-5-carboxylate reductase.  


The PRO3 gene of Saccharomyces cerevisiae encodes the 286-amino-acid protein delta 1-pyrroline-5-carboxylate reductase [L-proline:NAD(P+) 5-oxidoreductase; EC], which catalyzes the final step in proline biosynthesis. The protein has substantial similarity to the pyrroline carboxylate reductases of diverse bacterial species, soybean, and humans. Using RNA hybridization and measurements of enzyme activity, we have determined that the expression of the PRO3 gene appears to be constitutiv...

Brandriss, M. C.; Falvey, D. A.



Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae. (United States)

The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant. PMID:1409638

Liljelund, P; Mariotte, S; Buhler, J M; Sentenac, A



Deletion of FPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae  


Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this s...

Wei, Na; Xu, Haiqing; Kim, Soo Rin; Jin, Yong-su



The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis. (United States)

In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter. PMID:12923174

Jensen, Laran T; Ajua-Alemanji, Mispa; Culotta, Valeria Cizewski



PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis  


Saccharomyces cerevisiae pas3-mutants are described which conform the pas-phenotype recently reported for the peroxisomal assembly mutants pas1-1 and pas2 (Erdmann, R., M. Veenhuis, D. Mertens, and W.-H Kunau, 1989, Proc. Natl. Acad. Sci. USA. 86:5419-5423). The isolation of pas3- mutants enabled us to clone the PAS3 gene by functional complementation. DNA sequence analysis revealed a 50.6-kD protein with at least one domain of sufficient length and hydrophobicity to span a lipid bilayer. To ...



Saccharomyces cerevisiae SMT4 encodes an evolutionarily conserved protease with a role in chromosome condensation regulation.  


In a search for regulatory genes affecting the targeting of the condensin complex to chromatin in Saccharomyces cerevisiae, we identified a member of the adenovirus protease family, SMT4. SMT4 overexpression suppresses the temperature-sensitive conditional lethal phenotype of smc2-6, but not smc2-8 or smc4-1. A disruption allele of SMT4 has a prominent chromosome phenotype: impaired targeting of Smc4p-GFP to rDNA chromatin. Site-specific mutagenesis of the predicted protease active site cyste...

Strunnikov, A. V.; Aravind, L.; Koonin, E. V.



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

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. PMID:25514203

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



Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase. (United States)

Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3. TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant. The nucleotide sequence of TRP2 as well as that of TRP3 were determined. The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE. On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E. coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein. Based on comparisons of TRP3 amino acid sequence with homologous sequences from E. coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain. These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved. Connector function is thus not dependent on a specific sequence. Nuclease S1 mapping was used to identify mRNA 5' termini. Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA. TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S. cerevisiae general amino acid control system. The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R. (1983) J. Biol. Chem. 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3. PMID:6323449

Zalkin, H; Paluh, J L; van Cleemput, M; Moye, W S; Yanofsky, C



GUP1 and its close homologue GUP2, encoding multi-membrane-spanning proteins involved in active glycerol uptake in Saccharomyces cerevisiae  


Many yeast species can utilise glycerol, both as sole carbon source and as an osmolyte. In Saccharomyces cerevisiae, physiological studies have previously shown the presence of an active uptake system driven by electrogenic proton symport. We have used transposon mutagenesis to isolate mutants affected in the transport of glycerol into the cell. Here we present the identification of YGL084c, encoding a multi-membrane-spanning protein, as being essential for proton symport of glycerol into Sac...

Holst, Bjørn; Lunde, Christina; Lages, Fernanda; Oliveira, Rui Pedro Soares; Lucas, Ca?ndida; Kielland-brandt, Morten



Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling.  


Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpa1 mutations suggests th...

Jahng, K. Y.; Ferguson, J.; Reed, S. I.



Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C.  


The MIF2 gene of Saccharomyces cerevisiae has been implicated in mitosis. Here we provide genetic evidence that MIF2 encodes a centromere protein. Specifically, we found that mutations in MIF2 stabilize dicentric minichromosomes and confer high instability (i.e., a synthetic acentric phenotype) to chromosomes that bear a cis-acting mutation in element I of the yeast centromeric DNA (CDEI). Similarly, we observed synthetic phenotypes between mutations in MIF2 and trans-acting mutations in thre...

Meluh, P. B.; Koshland, D.



Characterization of the Saccharomyces cerevisiae nuclear gene CYB3 encoding a cytochrome b polypeptide of respiratory complex II. (United States)

Computer-assisted structural analysis of the predicted product of the previously described open reading frame (ORF) YKL4 located on the left arm of chromosome XI of Saccharomyces cerevisiae revealed a high degree of similarity (> 50%) to bovine cytochrome b560, the sdhC polypeptide of the Escherichia coli succinate dehydrogenase (SDH) complex and the protein specified by ORF137 located on the chloroplast DNA of Marchantia polymorpha. Disruption of the yeast gene severely impaired mitochondrial function, while Northern analysis showed it to be subject to catabolite repression. Deletion analysis of the CYB3 promoter identified a single HAP2/3/4-binding element that is necessary and sufficient for carbon source-dependent transcriptional regulation. These experiments also suggested the presence of additional, as yet unidentified, transcriptional control elements, both negative and positive. Taken together, these data lead us to conclude that the CYB3 gene encodes the yeast homolog of the bovine cytochrome b560 component of complex II of the mitochondrial electron transport chain. PMID:8152421

Abraham, P R; Mulder, A; van 't Riet, J; Raué, H A



The Saccharomyces Cerevisiae Spt8 Gene Encodes a Very Acidic Protein That Is Functionally Related to Spt3 and Tata-Binding Protein  


Mutations in the Saccharomyces cerevisiae SPT8 gene were previously isolated as suppressors of retrotransposon insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Mutations in SPT8 confer phenotypes similar to those caused by particular mutations in SPT15, which encodes the TATA-binding protein (TBP). These phenotypes are also similar to those caused by mutations in the SPT3 gene, which encodes a protein that directly interacts with TBP. We have now cloned and sequenced the SPT8...

Eisenmann, D. M.; Chapon, C.; Roberts, S. M.; Dollard, C.; Winston, F.



Saccharomyces cerevisiae contains two functional genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase.  


We have isolated two genes from yeast encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase [hydroxymethylglutaryl-coenzyme A reductase (NADPH); HMG-CoA reductase; EC], the rate-limiting enzyme of sterol biosynthesis. These genes, HMG1 and HMG2, were identified by hybridization to a cDNA clone encoding hamster HMG-CoA reductase. DNA sequence analysis reveals homology between the amino acid sequence of the proteins encoded by the two yeast genes and the carboxyl-terminal half of th...

Basson, M. E.; Thorsness, M.; Rine, J.



In silicio search for genes encoding peroxisomal proteins in Saccharomyces cerevisiae. (United States)

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. PMID:11330035

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



The FKB2 gene of Saccharomyces cerevisiae, encoding the immunosuppressant-binding protein FKBP-13, is regulated in response to accumulation of unfolded proteins in the endoplasmic reticulum.  


The FKB2 gene of Saccharomyces cerevisiae encodes a homolog of mammalian FKBP-13, an FK506/rapamycin-binding protein that localizes to the lumen of the endoplasmic reticulum (ER). We have found that FKB2 mRNA levels increase in response to the accumulation of unfolded precursor proteins in the ER. FKB2 mRNA levels are elevated in cells blocked in N-glycosylation--i.e., in wild-type cells treated with tunicamycin and in the sec53-6 mutant grown at the nonpermissive temperature. Mutations that ...

Partaledis, J. A.; Berlin, V.



GUP1 of Saccharomyces cerevisiae Encodes an O-Acyltransferase Involved in Remodeling of the GPI Anchor  


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 dom...

Bosson, Re?gine; Jaquenoud, Malika; Conzelmann, Andreas



Choline transport in Saccharomyces cerevisiae.  


Choline transport of Saccharomyces cerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N,N-dimethylethanolamine, and beta-methylcholine were competitive inhibitors of choline transport, with Ki values of 40.1, 3.1, and 6.9 microM, respectively. Ethanolamine, phosphorylcholine, and var...

Hosaka, K.; Yamashita, S.



FTIR spectroscopic discrimination of Saccharomyces cerevisiae and Saccharomyces bayanus strains. (United States)

In this study, we tested the potential of Fourier-transform infrared absorption spectroscopy to screen, on the one hand, Saccharomyces cerevisiae and non-S. cerevisiae strains and, on the other hand, to discriminate between S. cerevisiae and Saccharomyces bayanus strains. Principal components analysis (PCA), used to compare 20 S. cerevisiae and 21 non-Saccharomyces strains, showed only 2 misclassifications. The PCA model was then used to classify spectra from 14 Samos strains. All 14 Samos strains clustered together with the S. cerevisiae group. This result was confirmed by a routinely used electrophoretic pattern obtained by pulsed-field gel electrophoresis. The method was then tested to compare S. cerevisiae and S. bayanus strains. Our results indicate that identification at the strain level is possible. This first result shows that yeast classification and S. bayanus identification can be feasible in a single measurement. PMID:20921989

Adt, Isabelle; Kohler, Achim; Gognies, Sabine; Budin, Julien; Sandt, Christophe; Belarbi, Abdelkader; Manfait, Michel; Sockalingum, Ganesh D



Mutations in a gene encoding the. cap alpha. subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling  

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Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identically to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to gene encoding the ..cap alpha.. subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpal mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G/sub 1/, deposition of mating-specific cell surface aggultinins, and induction of pheromone-specific mRNa, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.

Jahng, K.Y.; Ferguson, J.; Reed, S.I.



Resistance and Adaptation to Quinidine in Saccharomyces cerevisiae: Role of QDR1 (YIL120w), Encoding a Plasma Membrane Transporter of the Major Facilitator Superfamily Required for Multidrug Resistance  


As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frame YIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden ...

Nunes, Patri?cia A.; Tenreiro, Sandra; Sa?-correia, Isabel



SCUD: Saccharomyces Cerevisiae Ubiquitination Database  

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Full Text Available Abstract Background Ubiquitination is an important post-translational modification involved in diverse biological processes. Therefore, genomewide representation of the ubiquitination system for a species is important. Description SCUD is a web-based database for the ubiquitination system in Saccharomyces cerevisiae (Baker's yeast. We first searched for all the known enzymes involved in the ubiquitination process in yeast, including E1, E2, E3, and deubiquitination enzymes. Then, ubiquitinated substrates were collected by literature search. Especially, E3 and deubiquitination enzymes are classified into classes and subclasses by their shared domains and unique functions. As a result, 42 different E3 enzymes were grouped into corresponding classes and subclasses, and 940 ubiquitinated substrates including mutant substrates were identified. All the enzyme and substrate information are interconnected by hyperlinks, which makes it easy to view the enzyme-specific ubiquitination information. Conclusion This database aims to represent a comprehensive yeast ubiquitination system, and is easily expandable with the further experimental data. We expect that this database will be useful for the research on the ubiquitination systems of other higher organisms. SCUD is accessible at

Jung Jin Woo



The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.  


In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounce...

Douglas, C. M.; Foor, F.; Marrinan, J. A.; Morin, N.; Nielsen, J. B.; Dahl, A. M.; Mazur, P.; Baginsky, W.; Li, W.; El-sherbeini, M.



Processing pathway for protease B of Saccharomyces cerevisiae  


The vacuolar protease B of Saccharomyces cerevisiae is a subtilisin- like protease encoded by the PRB1 gene. Antibodies raised against a synthetic peptide and an Escherichia coli-derived PRB1 open reading frame (ORF) protein cross-react with authentic protease B from yeast. By using these antibodies, the posttranslational biosynthetic pathway of protease B has been elucidated. Preproprotease B is a 76-kD unglycosylated precursor that enters the endoplasmic reticulum (ER), where it receives on...



Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae?  


(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could...

Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu



A Late Form of Nucleophagy in Saccharomyces cerevisiae  


Autophagy encompasses several processes by which cytosol and organelles can be delivered to the vacuole/lysosome for breakdown and recycling. We sought to investigate autophagy of the nucleus (nucleophagy) in the yeast Saccharomyces cerevisiae by employing genetically encoded fluorescent reporters. The use of such a nuclear reporter, n-Rosella, proved the basis of robust assays based on either following its accumulation (by confocal microscopy), or degradation (by immunoblotting), within the ...

Mijaljica, Dalibor; Prescott, Mark; Devenish, Rodney J.



Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae (United States)

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


Ferrofluid modified Saccharomyces cerevisiae cells for biocatalysis.  

Czech Academy of Sciences Publication Activity Database

Ro?. 42, - (2009), s. 521-524. ISSN 0963-9969 R&D Projects: GA MPO 2A-1TP1/094; GA MŠk(CZ) OC 157 Institutional research plan: CEZ:AV0Z60870520 Keywords : Saccharomyces cerevisiae * magnetic fluid * hydrogen peroxide Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.414, year: 2009

Šafa?íková, Miroslava; Mad?rová, Zde?ka; Šafa?ík, Ivo



A family of genes encode the multiple forms of the Saccharomyces cerevisiae ribosomal proteins equivalent to the Escherichia coli L12 protein and a single form of the L10-equivalent ribosomal protein.  


The budding yeast Saccharomyces cerevisiae contains a family of genes that encodes four different but related small acidic ribosomal proteins designated L12eIA, L12eIB, L12eIIA, and L12eIIB and a single larger protein designated L10e. These proteins are equivalent (e) to the L12 and L10 proteins of Escherichia coli that assemble as a 4:1 complex onto the large ribosomal subunit. The five yeast genes (or their cDNAs) have been cloned and sequenced (M. Remacha, M. T. Saenz-Robles, M. D. Vilella...

Newton, C. H.; Shimmin, L. C.; Yee, J.; Dennis, P. P.



The Schizosaccharomyces pombe mam2 gene encodes a putative pheromone receptor which has a significant homology with the Saccharomyces cerevisiae Ste2 protein.  


The fission yeast Schizosaccharomyces pombe has two mating-types, h+ (P) and h- (M). The mam2 mutant exhibits an h(-)-specific sterile phenotype. Nucleotide sequencing of the mam2 gene isolated from an S. pombe genomic library revealed an open reading frame composed of 348 amino acids. The deduced mam2 product is a hydrophobic protein of 39 kDa that has significant sequence similarity (26.3% for identical amino acids) with the transmembrane domains of the Saccharomyces cerevisiae STE2 product...

Kitamura, K.; Shimoda, C.



Manganese biosorption sites of Saccharomyces cerevisiae. (United States)

Experiments conducted by pre-treating the fermentation industrial waste biomass of Saccharomyces cerevisiae with laboratory grade chemicals like formaldehyde-formic acid, ethanol, triethyl phosphite-nitromethane, dithiopyridine and benzene helped in studying the roles played by amines, carboxylic acids, phosphates, sulfhydryl group and lipids present on the cell wall of the biomass in manganese biosorption. Potentiometric titration of S. cerevisiae revealed the presence of carboxyl, phosphate, amine groups. The extent of the contribution of the functional groups and lipids to manganese biosorption was in the order: carboxylic acids > amines > lipids > phosphates. Blocking of sulfhydryl group did not have any significant effect on manganese uptake. PMID:17674651

Parvathi, K; Nareshkumar, R; Nagendran, R



Cell Wall Assembly in Saccharomyces cerevisiae  


An extracellular matrix composed of a layered meshwork of ?-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and i...

Lesage, Guillaume; Bussey, Howard



Lipid nutrition of Saccharomyces cerevisiae in winemaking  


Biosynthesis of cell membrane lipids is a crucial metabolic pathway for the growth and viability of eucaryotic microorganisms. In Saccharomyces cerevisiae, unsaturated fatty acids and ergosterol synthesis needs molecular oxygen. Stuck and sluggish fermentations are related to this aspect of metabolism and constitute a major problem in the wine industry. Anaerobiosis, when lipids are not available in the growth medium, highly stresses cells. They release lipid biosynthesis metabolites and soon...

Marzona, Mario; Belviso, Simona



Piecemeal Microautophagy of Nucleus in Saccharomyces cerevisiae  


Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promote piecemeal microautophagy of the nucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen s...

Roberts, Paul; Moshitch-moshkovitz, Sharon; Kvam, Erik; O Toole, Eileen; Winey, Mark; Goldfarb, David S.



Phosphate transport and sensing in Saccharomyces cerevisiae.  


Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvat...

Wykoff, D. D.; O Shea, E. K.



Checkpoint-control in Yeast Saccharomyces cerevisiae  

International Nuclear Information System (INIS)

Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues. The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway. DNA damage activates a set of proteins whose job is to delay the cell cycle until the damage is repaired. This process was thought to involve the detection of damage by sensor proteins, which transmit a signal to a key protein kinase and thence to downstream targets. Checkpoint mechanisms are interesting because of their link with cancer and we have the eternal hope that checkpoints may somehow present a therapeutic opportunity. (author)


Analysis of silencing in Saccharomyces cerevisiae. (United States)

Silencing assays have proven to be powerful tools not only for understanding how epigenetic processes function and defining the structural components of silent chromatin, but also for a useful readout for characterizing the functions of proteins involved in chromatin biology that influence epigenetic processes directly or indirectly. This chapter describes a collection of assays for monitoring silencing in Saccharomyces cerevisiae, including qualitative and quantitative methods as well as protocols that provide either indirect or direct measurements of the transcriptional state of loci regulated by silent chromatin. PMID:25213251

Miller, Andrew; Kirchmaier, Ann L



Catalase enzyme in mitochondria of Saccharomyces cerevisiae  

Scientific Electronic Library Online (English)

Full Text Available SciELO Chile | Language: English Abstract in english Catalase and superoxide dismutase activities have been explored in the yeast Saccharomyces cerevisiae during batchwise growth experiment. During the diauxic growth in YPD medium high Ys values were obtained (0.415 - 0.423) and correlation between the total activities of both enzymes has been found. [...] A mitochondrial fraction from three type strains of Saccharomyces cerevisiae has been isolated. The purity of this fraction was proved through different enzyme assays: hexokinase, glucose-6-phosphate dehydrogenase, D-amino acid oxidase, isocitric lyase, succinate dehydrogenase. Then the catalase, peroxidase, Mn and Cu/Zn superoxide dismutase activities were evaluated in the mitochondrial fraction. Polyacrylamide gel electrophoresis separations allowed to identify a mitochondrial catalase as a band of 0.239 Rm value. It differed from the two catalase specific bands with Rm values 0.218 and 0.257 obtained from the crude extract. It was proved that the three catalase proteins are charge isomers. A positive correlation between the activity of mitochondrial catalase and Mn superoxide dismutase also takes place. Molecular weight of mitochonrial catalase protein has been determined as 240 kD.

Ventsislava Yankova, Petrova; Tanya Vassileva, Rasheva; Anna V., Kujumdzieva.



Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted  


Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yield...

Diderich, Jasper A.; Raamsdonk, Le?onie M.; Kruckeberg, Arthur L.; Berden, Jan A.; Dam, Karel



Expression of VHHs in Saccharomyces cerevisiae. (United States)

The production of VHHs in microorganisms is relatively straightforward, however the amount of VHH produced per volume unit can vary substantially from hardly detectable to hundreds of milligrams per liter. Expression in Escherichia coli is more commonly used at initial research phase, since production of VHHs for large-scale application in E. coli is for a number of reasons not preferred. Otherwise VHH production in GRAS organisms such as Saccharomyces cerevisiae fits very well with industrial fermentation processes, and in fact the only commercially available VHHs are produced in S. cerevisiae. Immediately after the discovery of heavy chain only antibodies, which are per definition devoid of light chains, it was investigated whether many problems encountered with the production of conventional antibodies in lower eukaryotes were absent during the production of VHHs. Here we provide a protocol for the expression of VHH genes in S. cerevisiae in a fed-batch fermentation process. This protocol is also suitable for the production of multivalent VHHs. PMID:22886258

Gorlani, Andrea; de Haard, Hans; Verrips, Theo



Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.  


A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide....

Stern, D. F.; Zheng, P.; Beidler, D. R.; Zerillo, C.



Adaption of Saccharomyces cerevisiae expressing a heterologous protein  

DEFF Research Database (Denmark)

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 specific growth rate of the strains was reduced by 54% and 33%, respectively, upon the introduction of the gene encoding aprotinin. Growing the strains in sequential shake flask cultivations for 250 generations led to an increased maximal specific growth rate and a decrease in the yield of aprotinin as a result of the adaptation. Determination of the level of mRNA encoding aprotinin and the plasmid copy number pointed to different mechanisms responsible for the decline in aprotinin yield in the different strains. (C) 2008 Elsevier B.V. All rights reserved.

Krogh, Astrid MØrkeberg; Beck, Vibe



A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae. (United States)

The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT alpha, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae. PMID:2673556

Hisatomi, T; Tsuboi, M



Modification of mutation frequency in Saccharomyces Cerevisiae  

International Nuclear Information System (INIS)

In a reverse mutation system, using haploid, histidine-requirinq strain of Saccharomyces cerevisiae, the frequency of uv-induced prototrophs increased if the post-irradiation minimal medium was supplemented with limited amounts of histidine. Addition of natural amino acids or RNA bases in the post-irradiation minimal medium, with or without histidine, also increased the uv-induced mutation frequency. Thus, post-irradiation conditions favouring protein and RNA synthesis, are effective in increasing uv-induced mutations in yeast. As compared to uv light, nitrous acid was more effective in inducing reversions in this strain and the frequency increased if the treated cells were plated on minimal medium supplemented with limited amounts of histidine. However, the addition of amino acids or RNA bases decreased the number of revertants. An additional inclusion of histidine reversed the suppressive effect of these metabolites. The mutation induction processes are thus different or differently modifiable in uv and nitrous acid. (author)


On Cycles in the Transcription Network of Saccharomyces cerevisiae  


Abstract Background We investigate the cycles in the transcription network of Saccharomyces cerevisiae. Unlike a similar network of Escherichia coli, it contains many cycles. We characterize properties of these cycles and their place in the regulatory mechanism of the cell. Results Almost all cycles in the transcription network of Saccharomyces cerevisiae are contained in a single strongly connected component, which we call LSCC (L for "largest"), except for a single cycle of two transcriptio...

Berman Piotr; Jeong Jieun



Proliferation of microbodies in Saccharomyces cerevisiae. (United States)

The development of microbodies in the yeast Saccharomyces cerevisiae was studied in response to different conditions of growth. Various strains of S. cerevisiae were investigated, using cells from the exponential growth phase on glucose as an inoculum in all transfer experiments. Electron microscopy, including serial sectioning, revealed that these cells generally contained one to four small microbodies which were localized in the vicinity of the cell wall and characterized by the presence of catalase. Transfer of these glucose-grown cells into media supplemented with various compounds known to induce microbody proliferation in other yeasts--i.e. uric acid, alkylated amines, amino acids, C2-compounds such as ethanol or acetate, in the presence or absence of compounds that induce oxygen radical formation--did not result in a significant change in the number of microbody profiles observed. Marked microbody proliferation was, however, observed after a shift of cells into media containing oleic acid and was associated with the induction of activities of beta-oxidation enzymes. In addition, catalase and isocitrate lyase were present in enhanced levels. Kinetic experiments suggested that these microbodies developed from those originally present in the inoculum cells. In thin sections up to 14 microbody profiles were occasionally observed, often present in small clusters. Their ultimate volume fraction amounted to 8-10% of the cytoplasmic volume. PMID:3332968

Veenhuis, M; Mateblowski, M; Kunau, W H; Harder, W



A Saccharomyces cerevisiae mutant with increased virulence. (United States)

Saccharomyces cerevisiae, bakers' yeast, is not a pathogen in healthy individuals, but is increasingly isolated from immunocompromised patients. The more frequent isolation of S. cerevisiae clinically raises a number of questions concerning the origin, survival, and virulence of this organism in human hosts. Here we compare the virulence of a human isolate, a strain isolated from decaying fruit, and a common laboratory strain in a mouse infection model. We find that the plant isolate is lethal in mice, whereas the laboratory strain is avirulent. A knockout of the SSD1 gene, which alters the composition and cell wall architecture of the yeast cell surface, causes both the clinical and plant isolates to be more virulent in the mouse model of infection. The hypervirulent ssd1 Delta/ssd1 Delta yeast strain is a more potent elicitor of proinflammatory cytokines from macrophages in vitro. Our data suggest that the increased virulence of the mutant strains is a consequence of unique surface characteristics that overstimulate the proinflammatory response. PMID:12589024

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



Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model  

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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.

Serge Feyder



Kinetics of phosphomevalonate kinase from Saccharomyces cerevisiae. (United States)

The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni²? column. The KM of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K(M) of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V(max) was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²? dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH?=?7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP. PMID:24475236

Garcia, David E; Keasling, Jay D



Saccharomyces cerevisiae Heat Shock Transcription Factor Regulates Cell Wall Remodeling in Response to Heat Shock  


The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates expression of genes encoding heat shock proteins and a variety of other proteins as well. To better understand the cellular roles of Hsf1, we screened multicopy suppressor genes of a temperature-sensitive hsf1 mutation. The RIM15 gene, encoding a protein kinase that is negatively regulated by the cyclic AMP-dependent protein kinase, was identified as a suppressor, but Rim15-regulated stress-responsive tra...

Imazu, Hiromi; Sakurai, Hiroshi



Specific distribution of the Saccharomyces cerevisiae linker histone homolog HHO1p in the chromatin  


In virtually all eukaryotic organisms, linker DNA between nucleosomes is associated with a histone termed linker histone or histone H1. In Saccharomyces cerevisiae, HHO1 encodes a putative linker histone with very significant homology to histone H1. The encoded protein is expressed in the nucleus, but has not been shown to affect global chromatin structure, nor has its deletion shown any detectable phenotype. In vitro chromatin assembly experiments with recombinant HHO1p have shown that it is...

Freidkin, Ilya; Katcoff, Don J.



Snf1-Dependent and Snf1-Independent Pathways of Constitutive ADH2 Expression in Saccharomyces cerevisiae  


The transcription factor Adr1 directly activates the expression of genes encoding enzymes in numerous pathways that are upregulated after the exhaustion of glucose in the yeast Saccharomyces cerevisiae. ADH2, encoding the alcohol dehydrogenase isozyme required for ethanol oxidation, is a highly glucose-repressed, Adr1-dependent gene. Using a genetic screen we isolated >100 mutants in 12 complementation groups that exhibit ADR1-dependent constitutive ADH2 expression on glucose. Temperature-sen...

Voronkova, Valentina; Kacherovsky, Nataly; Tachibana, Christine; Yu, Diana; Young, Elton T.



Identification of a Cryptococcus neoformans gene that directs expression of the cryptic Saccharomyces cerevisiae mannitol dehydrogenase gene.  


The Mtl gene from Cryptococcus neoformans, which confers the ability of Saccharomyces cerevisiae Sc4l YJO to grow on mannitol with substantial NAD-dependent mannitol dehydrogenase activity, was identified. Purifications and characterizations of this enzyme show that it is found in polyploid strain BB1, and the peptide sequence of the enzyme helped identify the saccharomyces gene encoding this mannitol dehydrogenase activity. On the other hand, the Mtl gene of C. neoformans encodes a 346-amino...

Perfect, J. R.; Rude, T. H.; Wong, B.; Flynn, T.; Chaturvedi, V.; Niehaus, W.



Redox balancing in recombinant strains of Saccharomyces cerevisiae  

Energy Technology Data Exchange (ETDEWEB)

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 transhydrogenation between the two coenzyme systems NADP(H) and NAD(H) during anaerobic growth, S. cerevisiae was transformed with a plasma membrane bound AB-transhydrogenase from E. coli and with a cytoplasmic BB-transhydrogenase from A. vinelandii. Expression of both types changed the intracellular nucleotide levels. The NADPH/NADP{sup +} ratio was reduced while the NADH/NAD{sup +} ratio was almost constant. An increased formation of 2-oxoglutarate, glycerol and acetate was observed during anaerobic glucose fermentation 206 refs, 8 figs, 3 tabs

Anderlund, M.



Local Regulatory Variation in Saccharomyces cerevisiae.  

Directory of Open Access Journals (Sweden)

Full Text Available Naturally occurring sequence variation that affects gene expression is an important source of phenotypic differences among individuals within a species. We and others have previously shown that such regulatory variation can occur both at the same locus as the gene whose expression it affects (local regulatory variation and elsewhere in the genome at trans-acting factors. Here we present a detailed analysis of genome-wide local regulatory variation in Saccharomyces cerevisiae. We used genetic linkage analysis to show that nearly a quarter of all yeast genes contain local regulatory variation between two divergent strains. We measured allele-specific expression in a diploid hybrid of the two strains for 77 genes showing strong self-linkage and found that in 52%-78% of these genes, local regulatory variation acts directly in cis. We also experimentally confirmed one example in which local regulatory variation in the gene AMN1 acts in trans through a feedback loop. Genome-wide sequence analysis revealed that genes subject to local regulatory variation show increased polymorphism in the promoter regions, and that some but not all of this increase is due to polymorphisms in predicted transcription factor binding sites. Increased polymorphism was also found in the 3' untranslated regions of these genes. These findings point to the importance of cis-acting variation, but also suggest that there is a diverse set of mechanisms through which local variation can affect gene expression levels.



Purification and characterization of Put1p from Saccharomyces cerevisiae. (United States)

In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH) enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A K(m) value of 36 mM proline and a k(cat)=27 s(-1) were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ(1)) as an electron acceptor with a k(cat)=9.6 s(-1) and a K(m) of 33 microM for CoQ(1). In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast. PMID:20450881

Wanduragala, Srimevan; Sanyal, Nikhilesh; Liang, Xinwen; Becker, Donald F



Identification of a Saccharomyces cerevisiae Glucosidase That Hydrolyzes Flavonoid Glucosides? †  


Baker's yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-?-glucoside revealed considerable ?-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the S. cerevisiae genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic ?- or...

Schmidt, Sabine; Rainieri, Sandra; Witte, Simone; Matern, Ulrich; Martens, Stefan



Switching the mode of metabolism in the yeast Saccharomyces cerevisiae  


The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain ...

Otterstedt, Karin; Larsson, Christer; Bill, Roslyn M.; Sta?hlberg, Anders; Boles, Eckhard; Hohmann, Stefan; Gustafsson, Lena



Metabolic engineering of Saccharomyces cerevisiae for lactose/whey fermentation  


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 metabolisation system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisia...

Domingues, Luci?lia; Guimara?es, Pedro M. R.; Oliveira, Carla Cristina Marques



Isolation of yeast Saccharomyces cerevisiae from unusual natural habitats  


Baker yeast Saccharomyces cerevisiae has been an eukarontic experimental organism since 1960s, becoming even more significant with the determination of its complete nucleotide genome sequence in 1996. Even though its biochemical function in the fermentation process had long remained unclear, its metabolism and products (eg. bread, beer, wine) have been used for millennia. S. cerevisiae yeast represents an important organism for production of recombinant proteins (gene manipulation). Moreover,...

Finz?gar, Bernarda



A Saccharomyces cerevisiae bio-databank for winemaking strain selection  


The winemaking industry faces currently an increased demand for novel Saccharomyces cerevisiae strains that are well adapted to different wine styles and that contribute to improved aromatic characteristics. In this reasoning, the Centro de Biologia Molecular e Ambiental (CBMA) at the University of Minho gathered one of the largest bio-databanks of S. cerevisiae, obtained from winemaking environments in Portugal and France. During the harvest time of 2001 to 2009, 604 grape samples were co...

Neves, J. Drumonde; Vieira, E.; Gambon, Brigitte; Valero, Eva; Gomes, Ana Catarina; Sousa, Susana; Lima, Maria Teresa; Arau?jo, Isabel M.; Santos, Manuel A. S.; Dequin, Sylvie; Casal, Margarida; Schuller, Dorit



The phenotypic heterogeneity of saccharomyces cerevisiae strains from natural environments  


Saccharomyces cerevisiae is the model organism par excellence and stands today at the forefront of molecular biology, genetics and genomics. However, as for many other laboratory model organisms, understanding of the ecological, evolutionary and population genetic features that shaped the biology of S. cerevisiae is underscored by a wealth of knowledge on molecular and cellular biology, mainly obtained from a very limited number of reference laboratory strains. In the last few years, yeast r...

Duarte, Ricardo Franco; Casal, Margarida; Schuller, Dorit



Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae.  


How eukaryotic cells sense availability of glucose, their preferred carbon and energy source, is an important, unsolved problem. Bakers' yeast (Saccharomyces cerevisiae) uses two glucose transporter homologs, Snf3 and Rgt2, as glucose sensors that generate a signal for induction of expression of genes encoding hexose transporters (HXT genes). We present evidence that these proteins generate an intracellular glucose signal without transporting glucose. The Snf3 and Rgt2 glucose sensors contain...

Ozcan, S.; Dover, J.; Johnston, M.



The Mitochondrial Alcohol Dehydrogenase Adh3p Is Involved in a Redox Shuttle in Saccharomyces cerevisiae:  


NDI1 is the unique gene encoding the internal mitochondrial NADH dehydrogenase of Saccharomyces cerevisiae. The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone. Surprisingly, NDI1 is not essential for respiratory growth. Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol. Cytosolic NADH can be oxidized by the external NADH dehydrogena...

Bakker, B. M.; Bro, C.; Kotter, P.; Luttik, M. A.; Dijken, J. P.; Pronk, J. T.



Saccharomyces cerevisiae Possesses a Stress-Inducible Glycyl-tRNA Synthetase Gene  


Aminoacyl-tRNA synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes both cytoplasmic and mitochondrial activities, while GRS2 is essentially silent and dispensable under normal conditions. We herein present evidence that expression of GRS2 was drastically induced upon heat shock, ethanol or hydro...

Chen, Shun-jia; Wu, Yi-hua; Huang, Hsiao-yun; Wang, Chien-chia



The STF2p Hydrophilin from Saccharomyces cerevisiae Is Required for Dehydration Stress Tolerance  


The yeast Saccharomyces cerevisiae is able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. The yeast genes encoding hydrophilin proteins were characterised to determine their roles in the dehydration-resistant phenotype, and STF2p was found to be a hydrophilin that is essential for survival after the desiccation-rehydration process. Deletion of STF2 promotes the production of reactive oxygen species and apoptotic cell death ...

Lo?pez-marti?nez, Gema; Rodri?guez-porrata, Boris; Margalef-catala?, Mar; Cordero-otero, Ricardo



Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance?  


The red carotenoid astaxanthin possesses higher antioxidant activity than other carotenoids and has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. In this study, we produced astaxanthin in the budding yeast Saccharomyces cerevisiae by introducing the genes involved in astaxanthin biosynthesis of carotenogenic microorganisms. In particular, expression of genes of the red yeast Xanthophyllomyces dendrorhous encoding phytoene desaturase (crtI product)...

Ukibe, Ken; Hashida, Keisuke; Yoshida, Nobuyuki; Takagi, Hiroshi



Pga1 Is an Essential Component of Glycosylphosphatidylinositol-Mannosyltransferase II of Saccharomyces cerevisiae  


The Saccharomyces cerevisiae essential gene YNL158w/PGA1 encodes an endoplasmic reticulum (ER)-localized membrane protein. We constructed temperature-sensitive alleles of PGA1 by error-prone polymerase chain reaction mutagenesis to explore its biological role. Pulse-chase experiments revealed that the pga1ts mutants accumulated the ER-form precursor of Gas1 protein at the restrictive temperature. Transport of invertase and carboxypeptidase Y were not affected. Triton X-114 phase separation an...

Sato, Keisuke; Noda, Yoichi; Yoda, Koji



Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae ?-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance?  


In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast Saccharomyces cerevisiae induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of PRO1 which encodes the Asp154Asn muta...

Sekine, Tomoko; Kawaguchi, Akari; Hamano, Yoshimitsu; Takagi, Hiroshi



Control of Adaptation to Mating Pheromone by G Protein ? Subunits of Saccharomyces Cerevisiae  


The STE4 gene of the yeast Saccharomyces cerevisiae encodes the ? subunit of a heterotrimeric G protein that mediates response to mating pheromones and influences recovery from pheromone-induced growth arrest. To explore how G(?) subunits regulate response and recovery (adaptation), we isolated and characterized signaling-defective STE4 alleles (STE4(sd)). STE4(sd) mutations resulted in amino acid substitutions in the N-terminal region of Ste4p, proximal to the first of seven repeat units c...

Grishin, A. V.; Weiner, J. L.; Blumer, K. J.



Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.  


The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Ara...

Zanchin, N. I.; Roberts, P.; Desilva, A.; Sherman, F.; Goldfarb, D. S.



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


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-scale synthetic lethal analysis. Combination of orc2-1 and orc5-1 alleles with the complete set of haploid deletion mutants revealed synthetic lethal/sick phenotypes with genes involved in DNA rep...

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



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


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. ...

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



A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA-binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination.  


In the yeast Saccharomyces cerevisiae, recombination between direct repeats is synergistically reduced in rad1 rad52 double mutants, suggesting that the two genes define alternate recombination pathways. Using a classical genetic approach, we searched for suppressors of the recombination defect in the double mutant. One mutation that restores wild-type levels of recombination was isolated. Cloning by complementation and subsequent physical and genetic analysis revealed that it maps to RAF1. T...

Smith, J.; Rothstein, R.



AKR1 encodes a candidate effector of the G beta gamma complex in the Saccharomyces cerevisiae pheromone response pathway and contributes to control of both cell shape and signal transduction.  


Mating pheromones of Saccharomyces cerevisiae control both signal transduction events and changes in cell shape. The G beta gamma complex of the pheromone receptor-coupled G protein activates the signal transduction pathway, leading to transcriptional induction and cell cycle arrest, but how pheromone-dependent signalling leads to cell shape changes is unclear. We used a two-hybrid system to search for proteins that interact with the G beta gamma complex and that might be involved in cell sha...

Pryciak, P. M.; Hartwell, L. H.



Effect of l-Proline on Sake Brewing and Ethanol Stress in Saccharomyces cerevisiae  


During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. l-Proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular l-proline in cells of S. cerevisiae grown under ethanol stress. An l-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1D154N, which encodes the Asp154Asn mutant...

Takagi, Hiroshi; Takaoka, Miki; Kawaguchi, Akari; Kubo, Yoshito



Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae. (United States)

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast. PMID:16161138

Dejong, JingHong M; Liu, Yule; Bollon, Arthur P; Long, Robert M; Jennewein, Stefan; Williams, David; Croteau, Rodney B



Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae  

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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 recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.

Penttilä Merja



Genetiese manipulering van die gis Saccharomyces cerevisiae betreffende polisakkariedbenutting Genetiese manipulering van die gis Saccharomyces cerevisiae betreffende polisakkariedbenutting  

Directory of Open Access Journals (Sweden)

Full Text Available Die gis Saccharomyces cerevisiae word wêreldwyd as die belangrikste kommersiële mikro-organisme bestempel en geniet sogenaamde ABAV-status (Algemeen Beskou As Veilig weens dié gis se eeue lange verbintenis met voedselproduksie (bv. brood, wyn, bier, proteienaanvulling en geurstowwe.Die gis Saccharomyces cerevisiae word wêreldwyd as die belangrikste kommersiële mikro-organisme bestempel en geniet sogenaamde ABAV-status (Algemeen Beskou As Veilig weens dié gis se eeue lange verbintenis met voedselproduksie (bv. brood, wyn, bier, proteienaanvulling en geurstowwe.

I. S. Pretoruis



Construction of strains of Saccharomyces cerevisiae that grow on lactose.  


We have constructed strains of Saccharomyces cerevisiae that grow on lactose (Lac+). S. cerevisiae strain YNN27, which, like all S. cerevisiae, is unable to grow on lactose, was transformed with pKR1B-LAC4-1. This plasmid has a selectable marker gene conferring resistance to the antibiotic G418 and carries a 13-kilobase region of the Kluyveromyces lactis genome including LAC4, a beta-galactosidase gene. Transformants were selected first for G418 resistance and then for growth on lactose. Sout...

Sreekrishna, K.; Dickson, R. C.



Exploring the genetic control of glycolytic oscillations in Saccharomyces Cerevisiae  

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Full Text Available Abstract Background A well known example of oscillatory phenomena is the transient oscillations of glycolytic intermediates in Saccharomyces cerevisiae, their regulation being predominantly investigated by mathematical modeling. To our knowledge there has not been a genetic approach to elucidate the regulatory role of the different enzymes of the glycolytic pathway. Results We report that the laboratory strain BY4743 could also be used to investigate this oscillatory phenomenon, which traditionally has been studied using S. cerevisiae X2180. This has enabled us to employ existing isogenic deletion mutants and dissect the roles of isoforms, or subunits of key glycolytic enzymes in glycolytic oscillations. We demonstrate that deletion of TDH3 but not TDH2 and TDH1 (encoding glyceraldehyde-3-phosphate dehydrogenase: GAPDH abolishes NADH oscillations. While deletion of each of the hexokinase (HK encoding genes (HXK1 and HXK2 leads to oscillations that are longer lasting with lower amplitude, the effect of HXK2 deletion on the duration of the oscillations is stronger than that of HXK1. Most importantly our results show that the presence of beta (Pfk2 but not that of alpha subunits (Pfk1 of the hetero-octameric enzyme phosphofructokinase (PFK is necessary to achieve these oscillations. Furthermore, we report that the cAMP-mediated PKA pathway (via some of its components responsible for feedback down-regulation modulates the activity of glycoytic enzymes thus affecting oscillations. Deletion of both PDE2 (encoding a high affinity cAMP-phosphodiesterase and IRA2 (encoding a GTPase activating protein- Ras-GAP, responsible for inactivating Ras-GTP abolished glycolytic oscillations. Conclusions The genetic approach to characterising the glycolytic oscillations in yeast has demonstrated differential roles of the two types of subunits of PFK, and the isoforms of GAPDH and HK. Furthermore, it has shown that PDE2 and IRA2, encoding components of the cAMP pathway responsible for negative feedback regulation of PKA, are required for glycolytic oscillations, suggesting an enticing link between these cAMP pathway components and the glycolysis pathway enzymes shown to have the greatest role in glycolytic oscillation. This study suggests that a systematic genetic approach combined with mathematical modelling can advance the study of oscillatory phenomena.

Williamson Thomas



[Tolerance of Saccharomyces cerevisiae to monoterpenes--a review]. (United States)

Tolerance of Saccharomyces cerevisiae to monoterpenes is important in both metabolic engineering of the yeast to produce these chemicals de novo and efficient use of biomass containing these chemicals. Understanding the mechanisms in the tolerance of S. cerevisiae to monoterpenes could facilitate the construction of yeast strains with enhanced monoterpenes resistance, and therefore improve related bioprocesses. Monoterpenes could disturb the redox balance in S. cerevisiae, therefore increase the accumulation of reactive oxygen species (ROS) and result in cell death. S. cerevisiae has to systematically improve its antioxidative ability to deal with the ROS induced damage. The current review summarized the recent developments in demonstration of the tolerance of S. cerevisiae to different typical monoterpenes mainly from the aspect of the antioxidative mechanisms. Based on the analysis of the previous works, further attempts to demonstrate the mechanisms were proposed. PMID:24028054

Liu, Jidong; Zhou, Jingwen; Chen, Jian



Phosphoglycerate mutase knock-out mutant Saccharomyces cerevisiae: Physiological investigation and transcriptome analysis  


Abstract The yeast Saccharomyces cerevisiae is able to adapt its metabolism to grow on different carbon sources and shift to non-fermentative growth on C2 or C3 carbon sources (ethanol, acetate or glycerol) requires activation of gluconeogenesis. Here we studied the response to deletion of the glycolytic and gluconeogenic gene GPM1, encoding for phosphoglycerate mutase. It was previously shown that a S. cerevisiae strain with non-functional copies of GPM1 only grow when glycerol an...

Papini, Marta; Nookaew, Intawat; Scalcinati, Gionata; Siewers, Verena; Nielsen, Jens



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.




Hydrogen peroxide removal with magnetically responsive Saccharomyces cerevisiae cells.  

Czech Academy of Sciences Publication Activity Database

Ro?. 56, - (2008), s. 7925-7928. ISSN 0021-8561 R&D Projects: GA MPO 2A-1TP1/094; GA MŠk OC 157 Institutional research plan: CEZ:AV0Z60870520 Keywords : magnetic alginate beads * catalase * magnetic separation * Saccharomyces cerevisiae cells * hydrogen peroxide Subject RIV: GM - Food Processing Impact factor: 2.562, year: 2008

Šafa?ík, Ivo; Mad?rová, Zde?ka; Šafa?íková, Miroslava



Pathways of ultraviolet mutability in Saccharomyces cerevisiae  

International Nuclear Information System (INIS)

Seven umr mutants of Saccharomyces cerevisiae which had reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were tested for sensitivity to L-canavanine relative to one wild-type UMR strain and one slightly UV-sensitive but phenotypically umr+ strain (mutant 306). Relative UV mutation resistance was estimated by dividing the UV fluence needed to yield a particular induced mutation frequency by that needed to reach the same frequency in the genotypic wild-type strain. The umr5 and umr6 strains were especially sensitive to canavanine growth inhibition, while umr1 was no more sensitive than either wild type; umr2, umr3, umr4, a umr7 and ? umr7 were equally sensitive to an intermediate degree. Incubation at 30degC of wild-type cells plated on canavanine-selective agar for increasingly longer times before UV irradiation resulted in decreasing UV mutation frequencies (reduced to 50% in 1.6 h). All umr strains tested in this way lost UV mutability faster than wild type, including mutant 306, umr1 (not sensitive to growth inhibition), and umr6 (very sensitive to growth inhibition). Cells were grown to stationary phase in YEPD growth medium and assayed for arginine and tryptophan transport into the cell. The umr6 strain, which had weak UV mutation resistance but high sensitivity to canavanine growth inhibition, transported arginine and tryptophan at essentially wild-type levels. The umr1 strain, however, which has moderate UV mutatain, however, which has moderate UV mutation resistance and normal canavanine toxicity, transported both amino acids at rates tenfold higher than wild type. The data suggest that increased canavanine toxicity does not necessarily lead to defective mutability at CAN1, and that mutational deficiency cannot result solely from increased canavanine toxicity. Although exposure to canavanine was shown to block mutation fixation and/or expression, it is suggested that the degree of growth inhibition is not strictly correlated with the degree of mutation resistance


Prezygotic reproductive isolation between Saccharomyces cerevisiae and Saccharomyces paradoxus  


Background: Matings between different Saccharomyces sensu stricto yeast species produce sexually sterile hybrids, so individuals should avoid mating with other species. Any mechanism that reduces the frequency of interspecific matings will confer a selective advantage. Here we test the ability of two closely-related Saccharomyces sensu stricto species to select their own species as mates and avoid hybridisation.Results: We set up mate choice tests, using five independently isolated pairs of s...

Maclean, C. J.; Greig, D.



Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae. (United States)

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. It is encoded by the HEM15 gene in the yeast Saccharomyces cerevisiae. Five hem15 mutants causing defective heme synthesis and protoporphyrin accumulation were investigated. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. The substitution L62F caused a five-fold increase in Vmax and 32-fold and four-fold increases in the KM's for protoporphyrin and metal. Replacements of the conserved G47 by S and S102 by F increased the KM for protoporphyrin 10-fold without affecting the affinity for metal or enzyme activity. Two amino acid changes, L205P and P221L, produced a thermosensitive phenotype. In vivo heme synthesis, the amount of immunodetected protein, and ferrochelatase activity measured in vitro were more affected in cells grown at 37 degrees C than at 30 degrees C. The effects of these mutations on the enzyme function are discussed with respects to ferrochelatase structure and mechanism of action. PMID:8818224

Góra, M; Chaciñska, A; Rytka, J; Labbe-Bois, R



Electrical stimulation of saccharomyces cerevisiae cultures / Estimulação elétrica de células de Saccharomyces cerevisiae  

Scientific Electronic Library Online (English)

Full Text Available Modulação do potencial de membrana celular endógeno por um campo elétrico externo influencia a estrutura e função dos compartimentos da membrana, de suas proteínas e da bi-camada lipídica. Neste trabalho, os efeitos da aplicação de potencial no crescimento de Saccharomyces cerevisiae foram caracteri [...] zados por experimentos simples, mas conclusivos. O perfil temporal de crescimento celular e a divisão celular foram investigados como respostas macroscópicas ao estímulo elétrico. Experimentos controle foram conduzidos em condições idênticas, exceto pela ausência de potencial aplicado. Através de análise comparativa, verificou-se que o estímulo elétrico alterou o ciclo celular como foi possível observar através da medida da dispersão de tamanho celular de cada população, sugerindo um possível sincronismo na divisão celular. Análise do espectro de potência foi empregada para sustentar o aumento no sincronismo, e uma modelagem matemática foi conduzida para determinar mudanças na cinética de crescimento celular. Parâmetros cinéticos do modelo tipo Monod para crescimento foram determinados por regressão não-linear. A constante de afinidade (a saber, K S) apresentou uma dependência com o potencial aplicado, sugerindo mudanças no transporte através da membrana celular. Verificou-se, também, que o estresse promovido eletroquimicamente inibiu o crescimento e induziu mudanças na viabilidade celular. Abstract in english 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 conclu [...] sive experiments. Cell growth time profile and cell division were investigated as macroscopic response to the electrical stimulation. Control experiments were conducted under identical conditions except for the absence of applied potential. Through comparative analysis, electrical stimulation was verified to alter cell cycle as smaller sized population was observed, suggesting that a synchrony in cell division was promoted. Power spectral analysis was employed to sustain synchrony enhancement, and mathematical modeling was conducted for determining kinetic growth changes. Monod type kinetic parameters for growth were determined by non-linear regression. The affinity constant (namely kS) presented a dependence on applied potential suggesting changes on transport across cell membrane. Electrochemically promoted stress was also verified to inhibit growth as well as to induce changes on cell viability.

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



KIN28 encodes a C-terminal domain kinase that controls mRNA transcription in Saccharomyces cerevisiae but lacks cyclin-dependent kinase-activating kinase (CAK) activity.  


The Saccharomyces cerevisiae gene KIN28 is a member of the cyclin-dependent kinase (CDK) family. The Kin28 protein shares extensive sequence identity with the vertebrate CDK-activating kinase MO15 (Cdk7), which phosphorylates CDKs in vitro on a critical threonine residue. Kin28 and MO15 have recently been found to copurify with the transcription factor IIH (TFIIH) holoenzyme of yeast and human cells, respectively. Although TFIIH is capable of phosphorylating the C-terminal domain (CTD) of RNA...

Cismowski, M. J.; Laff, G. M.; Solomon, M. J.; Reed, S. I.



Evidence for Domesticated and Wild Populations of Saccharomyces cerevisiae.  

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Full Text Available Saccharomyces cerevisiae is predominantly found in association with human activities, particularly the production of alcoholic beverages. S. paradoxus, the closest known relative of S. cerevisiae, is commonly found on exudates and bark of deciduous trees and in associated soils. This has lead to the idea that S. cerevisiae is a domesticated species, specialized for the fermentation of alcoholic beverages, and isolates of S. cerevisiae from other sources simply represent migrants from these fermentations. We have surveyed DNA sequence diversity at five loci in 81 strains of S. cerevisiae that were isolated from a variety of human and natural fermentations as well as sources unrelated to alcoholic beverage production, such as tree exudates and immunocompromised patients. Diversity within vineyard strains and within saké strains is low, consistent with their status as domesticated stocks. The oldest lineages and the majority of variation are found in strains from sources unrelated to wine production. We propose a model whereby two specialized breeds of S. cerevisiae have been created, one for the production of grape wine and one for the production of saké wine. We estimate that these two breeds have remained isolated from one another for thousands of years, consistent with the earliest archeological evidence for winemaking. We conclude that although there are clearly strains of S. cerevisiae specialized for the production of alcoholic beverages, these have been derived from natural populations unassociated with alcoholic beverage production, rather than the opposite.



Genes from Debaryomyces hansenii increase salt tolerance in Saccharomyces cerevisiae W303. (United States)

The yeast Debaryomyces hansenii has been chosen as a model for molecular studies of tolerance to NaCl. A gene library was built and transformants of Saccharomyces cerevisiae W303 containing genes from D. hansenii were selected for their ability to grow in the presence of high concentrations of NaCl and/or low concentrations of KCl. In three of these transformants 500 mM NaCl improved growth at pH 7.6 like in D. hansenii but not in S. cerevisiae. One of the plasmids restored growth at 50 microM KCl and K(+) uptake in a mutant of S. cerevisiae lacking genes that encode K(+) transporters. PMID:12702302

Prista, Catarina; Soeiro, Ana; Vesely, Paul; Almagro, Anabel; Ramos, José; Loureiro-Dias, Maria C



Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae. (United States)

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter(-1)) rather than GGOH (0.2 mg liter(-1)) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter(-1) GGOH and 6.5 mg liter(-1) squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter(-1) GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering. PMID:19592534

Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu



Primary structure of the RAD52 gene in Saccharomyces cerevisiae.  


The RAD52 gene of Saccharomyces cerevisiae, which is involved in genetic recombination and DNA repair, was cloned by transformation of rad52-1 mutant cells to methyl methanesulfonate resistance with BamHI fragments of Rad+ genomic DNA inserted into the Escherichia coli-S. cerevisiae shuttle vector YRp7. A plasmid carrying a 2.0-kilobase BamHI fragment was found to partially complement methyl methanesulfonate sensitivity of the rad52-1 mutant. By using this fragment as a hybridization probe, a...

Adzuma, K.; Ogawa, T.; Ogawa, H.



Cloning of the ASN1 and ASN2 genes encoding asparagine synthetases in Saccharomyces cerevisiae: differential regulation by the CCAAT-box-binding factor. (United States)

Two new yeast genes, named ASN1 and ASN2, were isolated by complementation of the growth defect of an asparagine auxotrophic mutant. Genetical analysis indicates that these two genes are allelic to the asnA and asnB loci described previously. Simultaneous disruption of both genes leads to a total asparagine auxotrophy, while disruption of asn1 or asn2 alone has no effect on growth under tested conditions. Nucleotide sequences of ASN1 and ASN2 revealed striking similarities with genes encoding asparagine synthetase (AS) from other organisms. Regulation of ASN1 and ASN2 expression was studied using lacZ fusions and both genes were found to be several times less expressed in the absence of the transcription activator Gcn4p. The HAP complex, another transcription factor that binds to CCAAT-box sequences, was shown to specifically affect ASN1 expression. Hap2p and Hap3p subunits of the HAP complex are required for optimal expression of ASN1, while the Hap4p regulatory subunit, which is required for regulation by the carbon source, plays a minor role in this process. Consistent with the weak effect of Hap4p, the carbon source does not significantly affect expression of ASN1. Our results show that the role of the HAP complex is not limited to activation of genes required for respiratory metabolism. PMID:8951815

Dang, V D; Valens, M; Bolotin-Fukuhara, M; Daignan-Fornier, B



The Acyl-CoA synthetases encoded within FAA1 and FAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import, activation, and intracellular Utilization  

DEFF Research Database (Denmark)

Exogenous long-chain fatty acids are activated to coenzyme A derivatives prior to metabolic utilization. In the yeast Saccharomyces cerevisiae, the activation of these compounds prior to metabolic utilization proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p. Faa1p or Faa4p are essential for long-chain fatty acid import, suggesting that one or both of these enzymes are components of the fatty acid transport system, which also includes Fat1p. By monitoring the intracellular accumulation of the fluorescent long-chain fatty acid analogue 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, long-chain fatty acid transport was shown to be severely restricted in a faa1 Delta faa4 Delta strain. These data established for the first time a mechanistic linkage between the import and activation of exogenous fatty acids in yeast. To investigate this linkage further, oleoyl CoA levels were defined following incubation of wild type and mutant cells with limiting concentrations of exogenous oleate. These studies demonstrated oleoyl CoA levels were reduced to less than 10% wild-type levels in faa1 Delta and faa1 Delta faa4 Delta strains. Defects in metabolic utilization and intracellular trafficking were also found in the fatty acyl-CoA synthetase-deficient strains. The faa1 Delta faa4 Delta strain had a marked reduction in endogenous acyl-CoA pools, suggesting these enzymes play a role in maintenance of endogenous acyl-CoA pools, metabolism and trafficking. In addition, this strain had levels of in vivo beta-oxidation of exogenous oleate reduced 3-fold when compared with the isogenic parent. Northern analyses demonstrated an additional defect in fatty acid trafficking as FAA1 or FAA4 were required for the transcriptional regulation of the genes encoding the peroxisomal enzymes acyl-CoA oxidase (POX1) and medium-chain acyl-CoA synthetase (FAA2). These data support the hypothesis that fatty acyl-CoA synthetase (Faa1p or Faa4p) functions as a component of the fatty acid import system by linking import and activation of exogenous fatty acids to intracellular utilization and signaling.

Færgeman, Nils J.; Black, P N



A Member of the Sugar Transporter Family, Stl1p Is the Glycerol/H+ Symporter in Saccharomyces cerevisiae  


Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol tra...

Ferreira, Ce?lia; Voorst, Frank; Martins, Anto?nio; Neves, Luisa; Oliveira, Rui; Kielland-brandt, Morten C.; Lucas, Ca?ndida; Brandt, Anders



Genome adaptation to chemical stress: clues from comparative transcriptomics in Saccharomyces cerevisiae and Candida glabrata  


Comparative transcriptomics of Saccharomyces cerevisiae and Candida glabrata revealed a remarkable conservation of response to drug-induced stress, despite underlying differences in the regulatory networks.

Lelandais, Gae?lle; Tanty, Ve?ronique; Geneix, Colette; Etchebest, Catherine; Jacq, Claude; Devaux, Fre?de?ric



Adenine deaminase and adenine utilization in Saccharomyces cerevisiae.  


Compared with other purine salvage and nitrogen catabolism enzymatic activities, adenine deaminase (adenine aminohydrolase [AAH]; EC activity in Saccharomyces cerevisiae is uniquely regulated. AAH specific activity is not induced by adenine and is reduced sevenfold when cells are cultivated in medium containing proline in place of ammonium as the sole nitrogen source. Exogenous adenine enters metabolic pathways primarily via the function of either AAH or adenine phosphoribosyltransfe...

Deeley, M. C.



Early meiotic transcripts are highly unstable in Saccharomyces cerevisiae.  


Meiosis in Saccharomyces cerevisiae requires the induction of a large number of genes whose mRNAs accumulate at specific times during meiotic development. This study addresses the role of mRNA stability in the regulation of meiosis-specific gene expression. Evidence is provided below demonstrating that the levels of meiotic mRNAs are exquisitely regulated by both transcriptional control and RNA turnover. The data show that (i) early meiotic transcripts are extremely unstable when expressed du...

Surosky, R. T.; Esposito, R. E.



Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae.  


In fungi and many other organisms, a thick outer cell wall is responsible for determining the shape of the cell and for maintaining its integrity. The budding yeast Saccharomyces cerevisiae has been a useful model organism for the study of cell wall synthesis, and over the past few decades, many aspects of the composition, structure, and enzymology of the cell wall have been elucidated. The cell wall of budding yeasts is a complex and dynamic structure; its arrangement alters as the cell grow...

Cid, V. J.; Dura?n, A.; Del Rey, F.; Snyder, M. P.; Nombela, C.; Sa?nchez, M.



A mechanism of palindromic gene amplification in Saccharomyces cerevisiae  


Selective gene amplification is associated with normal development, neoplasia, and drug resistance. One class of amplification events results in large arrays of inverted repeats that are often complex in structure, thus providing little information about their genesis. We made a recombination substrate in Saccharomyces cerevisiae that frequently generates palindromic duplications to repair a site-specific double-strand break in strains deleted for the SAE2 gene. The resulting palindromes are ...

Rattray, Alison J.; Shafer, Brenda K.; Neelam, Beena; Strathern, Jeffrey N.



Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae  


Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending al...

Dornfeld, K. J.; Livingston, D. M.



Genes Required for Vacuolar Acidity in Saccharomyces Cerevisiae  


Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein. Thirty nine vacuolar pH (Vph(-)) mutants were identified. Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes. The remaining mutants defined eight complementation groups of vph mutations. No alleles of the VAT2 or TFP1 genes (know...

Preston, R. A.; Reinagel, P. S.; Jones, E. W.



Meiotic Recombination at the Ends of Chromosomes in Saccharomyces cerevisiae  


Meiotic reciprocal recombination (crossing over) was examined in the outermost 60–80 kb of almost all Saccharomyces cerevisiae chromosomes. These sequences included both repetitive gene-poor subtelomeric heterochromatin-like regions and their adjacent unique gene-rich euchromatin-like regions. Subtelomeric sequences underwent very little crossing over, exhibiting approximately two- to threefold fewer crossovers per kilobase of DNA than the genomic average. Surprisingly, the adjacent euchrom...

Barton, Arnold B.; Pekosz, Michael R.; Kurvathi, Rohini S.; Kaback, David B.



Global response of Saccharomyces cerevisiae to an alkylating agent  


DNA chip technology enables simultaneous examination of how ?6,200 Saccharomyces cerevisiae gene transcript levels, representing the entire genome, respond to environmental change. By using chips bearing oligonucleotide arrays, we show that, after exposure to the alkylating agent methyl methanesulfonate, ?325 gene transcript levels are increased and ?76 are decreased. Of the 21 genes that already were known to be induced by a DNA-damaging agent, 18 can be sco...

Jelinsky, Scott A.; Samson, Leona D.



Information propagation within the Genetic Network of Saccharomyces cerevisiae  


Abstract Background A gene network's capacity to process information, so as to bind past events to future actions, depends on its structure and logic. From previous and new microarray measurements in Saccharomyces cerevisiae following gene deletions and overexpressions, we identify a core gene regulatory network (GRN) of functional interactions between 328 genes and the transfer functions of each gene. Inferred connections are verified by gene enrichment. Results We find that this core networ...

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



Construction of a flocculent saccharomyces cerevisiae fermenting lactose  


A flocculent Saccharomyces cerevisiae strain with the ability to express both the LAC4 (coding for b-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus was constructed. This recombinant strain is not only able to grow on lactose, but it can also ferment this substrate. To our knowledge this is the first time that a recombinant S. cervisiae has been found to ferment lactose in a way comparable to that of the existing lactose-fermenting ...

Domingues, Luci?lia; Teixeira, J. A.; Lima, Nelson



The Snf1 Protein Kinase in the Yeast Saccharomyces cerevisiae  


In yeast, Saccharomyces cerevisiae, the Snf1 protein kinase is primarily known as a key component of the glucose repression regulatory cascade. The Snf1 kinase is highly conserved among eukaryotes and its mammalian homolog AMPK is responsible for energy homeostasis in cells, organs and whole bodies. Failure in the AMPK regulatory cascade leads to metabolic disorders, such as obesity or type 2 diabetes. The knowledge about the Snf1 protein kinase remains to be of much interest in s...

Usaite, Renata; Olsson, Lisbeth; Nielsen, Jens



Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems  


Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity a...

Dicarlo, James; Norville, Julie; Mali, Prashant; Rios Villanueva, Xavier; Aach, John Dennis; Church, George Mcdonald



Dynamics of the Saccharomyces cerevisiae Transcriptome during Bread Dough Fermentation  


The behavior of yeast cells during industrial processes such as the production of beer, wine, and bioethanol has been extensively studied. In contrast, our knowledge about yeast physiology during solid-state processes, such as bread dough, cheese, or cocoa fermentation, remains limited. We investigated changes in the transcriptomes of three genetically distinct Saccharomyces cerevisiae strains during bread dough fermentation. Our results show that regardless of the genetic background, all thr...

Aslankoohi, Elham; Zhu, Bo; Rezaei, Mohammad Naser; Voordeckers, Karin; Maeyer, Dries; Marchal, Kathleen; Dornez, Emmie; Courtin, Christophe M.; Verstrepen, Kevin J.



Discontinuity Induced Bifurcations in a Model of Saccharomyces cerevisiae  


We perform a bifurcation analysis of the mathematical model of Jones and Kompala [K.D. Jones and D.S. Kompala, Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures, J. Biotech., 71:105-131, 1999]. Stable oscillations arise via Andronov-Hopf bifurcations and exist for intermediate values of the dilution rate as has been noted from experiments previously. A variety of discontinuity induced bifurcations arise from a lack of global...

Simpson, D. J. W.; Kompala, D. S.; Meiss, J. D.



Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells.  


To obtain a broad perspective of the events leading to spontaneous loss of heterozygosity (LOH), we have characterized the genetic alterations that functionally inactivated the URA3 marker hemizygously or heterozygously situated either on chromosome III or chromosome V in diploid Saccharomyces cerevisiae cells. Analysis of chromosome structure in a large number of LOH clones by pulsed-field gel electrophoresis and PCR showed that chromosome loss, allelic recombination, and chromosome aberrati...

Hiraoka, M.; Watanabe, K.; Umezu, K.; Maki, H.



Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts  


Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnol...

Barbosa, Catarina; Lage, Patri?cia; Vilela, Alice; Mendes-faia, Arlete; Mendes-ferreira, Ana



Biogeographical characterisation of Saccharomyces cerevisiae wine yeast by molecular methods  


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”. Agricultur...




Biogeographical characterization of Saccharomyces cerevisiae wine yeast by molecular methods  


Biogeography is the descriptive and explanatory study of spatial patterns and processes involved in the distribution of biodiversity. Without biogeography, it would be difficult to study the diversity of microorganisms because there would be no way to visualize patterns in variation. Saccharomyces cerevisiae, “the wine yeast,” is the most important species involved in alcoholic fermentation, and in vineyard ecosystems, it follows the principle of “everything is everywhere.” Agricultur...

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



Isolation and Partial Purification of the Saccharomyces cerevisiae Cytokinetic Apparatus  


Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin...

Young, Brian A.; Buser, Christopher; Drubin, David G.



Rearrangements of highly polymorphic regions near telomeres of Saccharomyces cerevisiae.  


We have examined the mitotic and meiotic properties of telomeric regions in various laboratory strains of yeast. Using a sequence (Y probe) derived from a cloned yeast telomere (J. Szostak and E. Blackburn, Cell 29:245-255, 1982), we found that various strains of Saccharomyces cerevisiae show extensive polymorphisms of restriction endonuclease fragment length. Some of the variation in the lengths of telomeric fragments appears to be under the control of a small number of genes. When DNA from ...

Horowitz, H.; Thorburn, P.; Haber, J. E.



Comparative Genomics of Saccharomyces cerevisiae Natural Isolates for Bioenergy Production  


Lignocellulosic plant material is a viable source of biomass to produce alternative energy including ethanol and other biofuels. However, several factors—including toxic byproducts from biomass pretreatment and poor fermentation of xylose and other pentose sugars—currently limit the efficiency of microbial biofuel production. To begin to understand the genetic basis of desirable traits, we characterized three strains of Saccharomyces cerevisiae with robust growth in a pretreated lignocell...

Wohlbach, Dana J.; Rovinskiy, Nikolay; Lewis, Jeffrey A.; Sardi, Maria; Schackwitz, Wendy S.; Martin, Joel A.; Deshpande, Shweta; Daum, Christopher G.; Lipzen, Anna; Sato, Trey K.; Gasch, Audrey P.



Genome-Wide Analysis of Nascent Transcription in Saccharomyces cerevisiae  


The assessment of transcriptional regulation requires a genome-wide survey of active RNA polymerases. Thus, we combined the nuclear run-on assay, which labels and captures nascent transcripts, with high-throughput DNA sequencing to examine transcriptional activity in exponentially growing Saccharomyces cerevisiae. Sequence read data from these nuclear run-on libraries revealed that transcriptional regulation in yeast occurs not only at the level of RNA polymerase recruitment to promoters but ...

Mckinlay, Anastasia; Araya, Carlos L.; Fields, Stanley



Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics.  


We describe mutants of Saccharomyces cerevisiae that are more sensitive than the wild type to the aminoglycoside antibiotics G418, hygromycin B, destomycin A, and gentamicin X2. In addition, the mutants are sensitive to apramycin, kanamycin B, lividomycin A, neamine, neomycin, paromomycin, and tobramycin--antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is caused by mutations in at least three genes, denoted AGS1, AGS2, and AGS3 (for aminoglyco...

Ernst, J. F.; Chan, R. K.



Cloning and heterologous expression of glycosidase genes from Saccharomyces cerevisiae.  


Genomic clones were isolated that code for three glycosidases proposed to be involved in the catabolism of cell wall components in Saccharomyces cerevisiae. alpha-Mannosidase (AMS1), exoglucanase (BGL1), and endochitinase (CTS1) genes were isolated with the aid of filter assays based on the hydrolysis of 4-methylumbelliferyl glycosides, which permitted the in situ monitoring of these glycosidase activities in yeast colonies. Uracil prototrophs resulting from transformation with a multicopy YE...

Kuranda, M. J.; Robbins, P. W.



The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier.  


The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% prot...

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



Transcriptional regulation by ergosterol in the yeast Saccharomyces cerevisiae.  


Sterol biosynthesis in the yeast Saccharomyces cerevisiae is an energy-expensive, aerobic process, requiring heme and molecular oxygen. Heme, also synthesized exclusively during aerobic growth, not only acts as an enzymatic cofactor but also is directly and indirectly responsible for the transcriptional control of several yeast genes. Because of their biosynthetic similarities, we hypothesized that ergosterol, like heme, may have a regulatory function. Sterols are known to play a structural r...

Smith, S. J.; Crowley, J. H.; Parks, L. W.



Regulatory mutations affecting ornithine decarboxylase activity in Saccharomyces cerevisiae.  


We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionin...

Cohn, M. S.; Tabor, C. W.; Tabor, H.



Arsenic oxide-induced thermotolerance in Saccharomyces cerevisiae.  


The growth response of Saccharomyces cerevisiae to arsenite and arsenate and the relationship between the enhancement of heat shock protein (hsp) synthesis caused by these arsenic oxides and thermotolerance are reported. Arsenite and arsenate transiently inhibited cell growth and overall protein synthesis; arsenate enhanced the synthesis of the 42-, 74-, 84-, and 100-kilodalton hsps, whereas arsenite enhanced synthesis of only the 74-kilodalton hsp. The induction of these hsps reached a maxim...

Chang, E. C.; Kosman, D. J.; Willsky, G. R.



Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.  


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...

D Amore, T.; Panchal, C. J.; Stewart, G. G.



An apoptotic cell cycle mutant in Saccharomyces cerevisiae  

DEFF Research Database (Denmark)

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 in which apoptosis can be studied using the novel, temperature sensitive mutant, cdc77. The cdc77 cells are defective in a G1 process, and die show the characteristc signs of apoptosis: condensation of the chromatin, degradation of the inner nuclear membrane, dilation of the space between the nuclear membranes, condensation of the cytoplasm and degradation of DNA to 50kb fragmensts. It should be noted that in yeast, in contrast to higher eukaryotes, the nuclear membrane remain intact and the chromosomes remain uncondensed and invisible during mitosis. The cdc77 mutant exhibit a defect in initiation of DNA synthesis and a much prolonged DNA synthesis under semirestrictive conditions.

Villadsen, Ingrid



Antimutagenic and antioxidant activity of Lisosan G in Saccharomyces cerevisiae. (United States)

In the present study the antimutagenic and antioxidant effects of a powder of grain (Lisosan G) in yeast Saccharomyces cerevisiae were studied. Results showed that Lisosan G treatment decreased significantly the intracellular ROS concentration and mutagenesis induced by hydrogen peroxide in S. cerevisiae D7 strain. The effect of Lisosan G was then evaluated by using superoxide dismutase (SOD) proficient and deficient strains of S. cerevisiae. Lisosan G showed protective activity in sod1? and sod2? mutant strains, indicating an in vivo antioxidant effect. A high radical scavenging activity of Lisosan G was also demonstrated in vitro using the oxygen radical absorbance capacity (ORAC) assay. The obtained results showed a protective effect of Lisosan G in yeast cells, indicating that its antioxidant capacity contributes to its antimutagenic action. PMID:22953954

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



Saccharomyces cerevisiae en la fabricación del licor Cocuy. / Saccharomyces cerevisiae in the manufacturing of Cocuy liquor  

Scientific Electronic Library Online (English)

Full Text Available SciELO Venezuela | Language: Spanish Abstract in spanish El licor "cocuy" es una bebida artesanal, producida por las comunidades rurales en el occidente de Venezuela mediante un proceso de fermentación y destilación del mosto extraído del Agave cocui. Este estudio fue enmarcado en el "Programa Agave" con el propósito de contribuir a rescatar esta activida [...] d productiva tradicional. En vista de la falta de información en relación al proceso autóctono se hicieron estudios de las levaduras fermentadoras, la optimización de la producción de etanol y la utilización del residuo de la destilación (vinaza) como medio de cultivo. Los aislados con mayor capacidad fermentativa fueron seleccionados e identificados mediante parámetros morfológicos y metabólicos. Se compararon los niveles de consumo de azúcar de las levaduras con mayor capacidad fermentativa. Se estudió el efecto de la adición del azúcar blanca comercial y/o del fosfato de amonio y en la producción de alcohol en el proceso artesanal. Las concentraciones de azúcares en el mosto se evaluaron por refractometría, y el contenido de alcohol del licor por hidrometría. La utilización de la vinaza para la producción de biomasa como un componente del medio de cultivo fue comparada con un medio sintético mediante medidas del peso seco de la biomasa. Se confirma el papel de Saccharomyces cerevisiae en el proceso fermentativo espontáneo. Los resultados in situ evidenciaron un efecto favorable de la elevación del contenido de azúcar (11 a 18°Brix) y de la adición de fosfato de amonio dibásico (0,2 g/l). En estas condiciones, el tiempo de fermentación del mosto se acortó y la producción de licor aumentó hasta un 92%. Se demostró la posibilidad de utilizar la vinaza como un componente para un medio de cultivo de esta levadura, para iniciar la fermentación y para la producción de biomasa como una fuente de nutrientes de alto valor nutritivo para aves de corral o caprinos. Se recomienda apoyar los esfuerzos para desarrollar de esta importante fuente de ingresos para los campesinos que habitan las zonas semi-áridas de los estados Falcón y Lara. Abstract in english Liquor cocuy is an alcoholic beverage produced by fermentation and a subsequent distillation process of Agave cocui juice by the communities northwest Venezuela. This study was included in the "Agave cocui Program", which purpose was to rescue this traditional productive activity. Due to the lack of [...] information about this native product fermentative yeasts, alcohol production optimization, and use of distillation residue (nasty wine) as culture medium was investigated. Isolates with the best fermentative capacity were selected and identified by morphological and metabolic studies. Sugars consumption of the yeast with highest fermentative capacity were compared. Effect of white commercial sugar and/or ammonium phosphate dibasic addition to juice was evaluated during the process. Sugars concentration was estimated with a refractometer, measurement of alcohol content of liquor with an hidrometer. The production of yeasts biomass grown in a broth with nasty wine was compared to that in a synthetic one by dry biomass weight determinations. The Saccharomyces cerevisiae role in the spontaneous fermentative process was confirmed. The results in situ proved the favorable effect of extra sugar (11 to 18°Brix) and ammonium phosphate (0,2 g/l). Fermentative period was reduced and liquor production was enhanced to 92%. Use of nasty wine was proposed to obtain yeasts biomass, as fermentation starter or as a source of high nutritive value for poultry and goat feeding. We recommend to support efforts to improve this local activity which represent an important source of income for the farmer at the semi-arid zone at the states of Falcón and Lara.

F, Yegres; G, Fernández-Zeppenfeldt; CG, Padin; L, Rovero; N, Richard-Yegres.



Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases.  


We previously characterized a methionine aminopeptidase (EC; Met-AP1; also called peptidase M) in Saccharomyces cerevisiae, which differs from its prokaryotic homologues in that it (i) contains an N-terminal zinc-finger domain and (ii) does not produce lethality when disrupted, although it does slow growth dramatically; it is encoded by a gene called MAP1. Here we describe a second methionine aminopeptidase (Met-AP2) in S. cerevisiae, encoded by MAP2, which was cloned as a suppresso...

Li, X.; Chang, Y. H.



GPI lipid remodeling in Saccharomyces cerevisiae  


The lipid moieties of mature GPI anchors of S. cerevisiae usually do not contain the diacylglycerol present on CP2, the mature GPI lipid transferred by the GPI transamidase to proteins in the ER. CP2 probably contains the C16 and C18:1 fatty acids found in regular yeast PI, whereas most mature GPI proteins of yeast contain a ceramide moiety, and a minor fraction contains a modified diacylglycerol containing C26:0 in sn2. Recent studies have identified Per1p and Gup1p as be...

Conzelmann, Andreas; Pagac, Martin; Jaquenoud, Malika; Bosson, Re?gine; Ghugtyal, Vikram; Roubaty, Carole; Vionnet, Christine



Metabolic alterations during ascosporogenesis of Saccharomyces cerevisiae  

International Nuclear Information System (INIS)

Sporulation of S. cerevisiae has been shown to alter the profiles of enzymes involved in gluconeogenesis and glycolysis. The enhancement in the levels of total cellular carbohydrates could be correlated with the enhancement in fructose 1,6-diphosphatase and trehalose-phosphate synthetase. The latter activity could account for the 15-fold increase in trehalose levels in sporulating cells. Glucose-6-phosphatase, pyruvate kinase and phosphofructokinase showed continuous decline during ascosporogenesis. The relative incorporation of radioactivity from possible precursors of gluconeogenesis indicated that acetate-2-14C alone could contribute to carbohydrate synthesis. (author)


Saccharomyces cerevisiae: a nomadic yeast with no niche? (United States)

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

Goddard, Matthew R; Greig, Duncan



AFT1: a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae.  


Using a scheme for selecting mutants of Saccharomyces cerevisiae with abnormalities of iron metabolism, we have identified a gene, AFT1, that mediates the control of iron uptake. AFT1 encodes a 78 kDa protein with a highly basic amino terminal domain and a glutamine-rich C-terminal domain, reminiscent of transcriptional activators. The protein also contains an amino terminal and a C-terminal region with 10% His residues. A dominant mutant allele of this gene, termed AFT1-1up, results in high ...

Yamaguchi-iwai, Y.; Dancis, A.; Klausner, R. D.



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


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...

Kim, Jeong-ho; Brachet, Vale?rie; Moriya, Hisao; Johnston, Mark



Ty Element-Induced Temperature-Sensitive Mutations of Saccharomyces Cerevisiae  


Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-...

Kawakami, K.; Shafer, B. K.; Garfinkel, D. J.; Strathern, J. N.; Nakamura, Y.



Mid2 Is a Putative Sensor for Cell Integrity Signaling in Saccharomyces cerevisiae  


Hcs77 is a putative cell surface sensor for cell integrity signaling in Saccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39°C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated the MID2 gene as a dosage suppressor of the cell lysis defect of an hcs77 null mutant. MID2 encodes a putative membrane protein whose function is required for survival of pheromone treatment....

Rajavel, Mathumathi; Philip, Bevin; Buehrer, Benjamin M.; Errede, Beverly; Levin, David E.



Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae.  


The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed f...

Whiteway, M.; Hougan, L.; Thomas, D. Y.



Cha4p of Saccharomyces Cerevisiae Activates Transcription via Serine/Threonine Response Elements  


The CHA1 gene of Saccharomyces cerevisiae encodes the catabolic L-serine (L-threonine) deaminase responsible for the utilization of serine/threonine as nitrogen sources. Previously, we identified two serine/threonine response elements in the CHA1 promoter, UAS(CHA). We report isolation of a mutation, cha4-1, that impairs serine/threonine induction of CHA1 transcription. The cha4-1 allele causes noninducibility of a CHA1p-lacZ translational gene fusion, indicating that Cha4p exerts its action ...

Holmberg, S.; Schjerling, P.



Daughter-specific repression of Saccharomyces cerevisiae HO: Ash1 is the commander  


The GATA-1-like factor Ash1 is a repressor of the HO gene, which encodes an endonuclease that is responsible for mating-type switching in the yeast Saccharomyces cerevisiae. A multi-step programme, which involves a macromolecular protein complex, the secondary structure of ASH1 mRNA and the cell cytoskeleton, enables Ash1 to asymmetrically localize to the daughter cell nucleus in late anaphase and to repress HO transcription. The resulting Ash1 activity prevents the daughter cell from switchi...

Cosma, Maria Pia



Engineering the Saccharomyces cerevisiae ?-Oxidation Pathway to Increase Medium Chain Fatty Acid Production as Potential Biofuel  


Fatty acid-derived biofuels and biochemicals can be produced in microbes using ?-oxidation pathway engineering. In this study, the ?-oxidation pathway of Saccharomyces cerevisiae was engineered to accumulate a higher ratio of medium chain fatty acids (MCFAs) when cells were grown on fatty acid-rich feedstock. For this purpose, the haploid deletion strain ?pox1 was obtained, in which the sole acyl-CoA oxidase encoded by POX1 was deleted. Next, the POX2 gene from Yarrowia lipolytica, which e...

Chen, Liwei; Zhang, Jianhua; Chen, Wei Ning



Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae.  


A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2Val19, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation. It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase. We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases. These result...

Sass, P.; Field, J.; Nikawa, J.; Toda, T.; Wigler, M.



Accuracy of thymine–thymine dimer bypass by Saccharomyces cerevisiae DNA polymerase ?  


The Saccharomyces cerevisiae RAD30 gene functions in error-free replication of UV-damaged DNA. RAD30 encodes a DNA polymerase, Pol ?, which inserts two adenines opposite the two thymines of a cis-syn thymine–thymine (T–T) dimer. Here we use steady-state kinetics to determine the accuracy of DNA synthesis opposite the T–T dimer. Surprisingly, the accuracy of DNA synthesis opposite the damaged DNA is nearly indistinguishable from that opposite nondamaged DNA, with frequencies of misincor...

Washington, M. Todd; Johnson, Robert E.; Prakash, Satya; Prakash, Louise



Construction of killer industrial yeast Saccharomyces cerevisiae HAU-1 and its fermentation performance  

Scientific Electronic Library Online (English)

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

Bijender K., Bajaj; S., Sharma.



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  

DEFF Research Database (Denmark)

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work 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 transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD(+).

Anderlund, M.; Nissen, Torben Lauesgaard



Cloning and DNA sequence analysis of the glucose transporter gene2 from Iranian Saccharomyces cerevisiae  

Directory of Open Access Journals (Sweden)

Full Text Available Introduction: Saccharomyces cerevisiae has 20 genes that encode hexose transporter proteins including HXT1 to HXT17, GAL2, SNF3 and RGT2. Among these gene families, seven genes (HXT1-HXT7 have important role in alcohol production. The aim of this study was the identification and isolation of HXT2 gene from Saccharomyces cerevisiae genome by PCR technique and cloning into vector containing suitable expression promoter in order to design expression vector as a basis to produce recombinant yeast by transformation.Materials and Methods: After designing specific oligonucleotides primers, fragment gene amplified by PCR. Gene HXT2 inserted into pTZ57R vector by restriction enzymes EcoRI and HindIII and T4 ligase. After transformation of pTZ57R/THXT2 into E.coli, plasmid recombinant analysis considered. The final further analysis by restriction enzymes digestion and software were evaluated.Results: HXT2 gene isolated from pTZ57R/THXT2 has correct size in agarose gel electrophoresis. Electrophoresis analysis showed that this gene has correct size on agarose gel. Software study showed that this gene encode proteins with 59.84 KDa molecular weight having 541 amino acids with isoelectric point 8.3.Conclusion: HXT2 gene by PCR optimization from saccharomyces cerevisiae was isolated and cloned into prokaryotic host. This is the first report of isolation and cloning of this gene by using genetic engineering technique in IRAN that can be used for cloning into suitable expression vector to improve alcohol fermentation yield.

Saleh Amiri



Deletion of the GRE3 Aldose Reductase Gene and Its Influence on Xylose Metabolism in Recombinant Strains of Saccharomyces cerevisiae Expressing the xylA and XKS1 Genes  


Saccharomyces cerevisiae ferments hexoses efficiently but is unable to ferment xylose. When the bacterial enzyme xylose isomerase (XI) from Thermus thermophilus was produced in S. cerevisiae, xylose utilization and ethanol formation were demonstrated. In addition, xylitol and acetate were formed. An unspecific aldose reductase (AR) capable of reducing xylose to xylitol has been identified in S. cerevisiae. The GRE3 gene, encoding the AR enzyme, was deleted in S. cerevisiae CEN.PK2-1C, yieldin...

Tra?ff, K. L.; Otero Cordero, R. R.; Zyl, W. H.; Hahn-ha?gerdal, B.



Genome Snapshot: a new resource at the Saccharomyces Genome Database (SGD) presenting an overview of the Saccharomyces cerevisiae genome  


Sequencing and annotation of the entire Saccharomyces cerevisiae genome has made it possible to gain a genome-wide perspective on yeast genes and gene products. To make this information available on an ongoing basis, the Saccharomyces Genome Database (SGD) () has created the Genome Snapshot (). The Genome Snapshot summarizes the current state of knowledge about the genes and chromosomal features of S.cerevisiae. The information is organized into two categories: (i) number of each type of chro...

Hirschman, Jodi E.; Balakrishnan, Rama; Christie, Karen R.; Costanzo, Maria C.; Dwight, Selina S.; Engel, Stacia R.; Fisk, Dianna G.; Hong, Eurie L.; Livstone, Michael S.; Nash, Robert; Park, Julie; Oughtred, Rose; Skrzypek, Marek; Starr, Barry; Theesfeld, Chandra L.



Improving 2-Phenylethanol Production via Ehrlich Pathway Using Genetic Engineered Saccharomyces cerevisiae Strains. (United States)

2-phenylethanol (2-PE) is an important aromatic compound with a rose-like fragrance widely used in food industry and cosmetic manufacture. In order to obtain "natural" 2-PE, the genetically modified budding yeasts were developed and applied for the 2-PE production. The gene ARO8 encoding transaminase and the gene ARO10 encoding decarboxylase in the Ehrlich pathway were expressed in Saccharomyces cerevisiae S288c. The activities of transaminase and decarboxylase were both enhanced in the corresponding recombinant strains. Consequently, the 2-PE yield in the recombinant strains with ARO8 and ARO10 were increased by 9.3 and 16.3 %, respectively, than that in the wild strain. A co-expression vector harboring ARO8 and ARO10 was then introduced into S. cerevisiae S288c, generating the recombinant strain SPO810. The fed-batch fermentation results indicated that the 2-PE yield in SPO810 reached 2.61 g L(-1) after 60 h of cultivation, which was 36.8 % higher than that in the wild strain. These results demonstrated that the 2-PE production was significantly improved by enhanced expression of the two key enzymes encoded by ARO8 and ARO10 in the Ehrlich pathway, providing new perspectives for enhancing "natural" 2-PE production in S. cerevisiae. PMID:25681107

Yin, Sheng; Zhou, Hui; Xiao, Xiao; Lang, Tiandan; Liang, Jingru; Wang, Chengtao



Magnetically altered ethanol fermentation capacity of Saccharomyces cerevisiae  

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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.

Galonja-Corghill Tamara



Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin.  


A Ca2+- and calmodulin (CaM)-dependent protein kinase of Saccharomyces cerevisiae was partially purified by CaM affinity chromatography of the soluble fraction, and the properties of the enzyme were investigated. The protein kinase activity of the affinity-purified preparation was stimulated at least eightfold by the simultaneous presence of Ca2+ and CaM. The enzyme stimulation was strongly inhibited by trifluoperazine (TFP), a CaM antagonist. When the kinase was incubated in the presence of ...

Miyakawa, T.; Oka, Y.; Tsuchiya, E.; Fukui, S.



SAD mutation of Saccharomyces cerevisiae is an extra a cassette.  


Sporulation of Saccharomyces cerevisiae ordinarily requires the a1 function of the a mating type locus. SAD is a dominant mutation that allows strains lacking a1 (MAT alpha/MAT alpha and mata1/MAT alpha diploids) to sporulate. We provide functional and physical evidence that SAD is an extra cassette in the yeast genome, distinct from those at HML, MAT, and HMR. The properties of SAD strains indicate that the a cassette at SAD produces a limited amount of a1 product, sufficient for promoting s...

Kassir, Y.; Hicks, J. B.; Herskowitz, I.



Competing Crossover Pathways Act During Meiosis in Saccharomyces cerevisiae  


In Saccharomyces cerevisiae the MSH4-MSH5, MLH1-MLH3, and MUS81-MMS4 complexes act to promote crossing over during meiosis. MSH4-MSH5, but not MUS81-MMS4, promotes crossovers that display interference. A role for MLH1-MLH3 in crossover control is less clear partly because mlh1? mutants retain crossover interference yet display a decrease in crossing over that is only slightly less severe than that seen in msh4? and msh5? mutants. We analyzed the effects of msh5?, mlh1?, and mms4? single...

Argueso, Juan Lucas; Wanat, Jennifer; Gemici, Zekeriyya; Alani, Eric



Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and partial characterization.  


A plasmid (pNF2000) containing a 9.7-kilobase pair DNA insert that complements the UV sensitivity of rad2-1, rad2-2, and rad2-4 mutants of Saccharomyces cerevisiae has been isolated from a yeast genomic library. Genetic analysis of strains derived by transformation of rad2 mutants with an integrating plasmid containing a 9.3-kilobase pair fragment from pNF2000 shows that the fragment integrates exclusively at the chromosomal rad2 gene. We therefore conclude that this plasmid contains the RAD2...

Naumovski, L.; Friedberg, E. C.



d-Xylulose Fermentation to Ethanol by Saccharomyces cerevisiae  


We used commercial bakers' yeast (Saccharomyces cerevisiae) to study the conversion of d-xylulose to ethanol in the presence of d-xylose. The rate of ethanol production increased with an increase in yeast cell density. The optimal temperature for d-xylulose fermentation was 35°C, and the optimal pH range was 4 to 6. The fermentation of d-xylulose by yeast resulted in the production of ethanol as the major product; small amounts of xylitol and glycerol were also produced. The production of xy...

Chiang, Lin-chang; Gong, Cheng-shung; Chen, Li-fu; Tsao, George T.



mRNA decapping enzyme from ribosomes of Saccharomyces cerevisiae  

International Nuclear Information System (INIS)

By use of [3H]methyl-5'-capped [14C]mRNA from yeast as a substrate, a decapping enzyme activity has been detected in enzyme fractions derived from a high salt wash of ribosomes of Saccharomyces cerevisiae. The product of the decapping reaction is [3H]m7GDP. That the enzyme is not a non-specific pyrophosphatase is suggested by the finding that the diphosphate product, m7GpppA(G), and UDP-glucose are not hydrolyzed


Molecular Genetics of Serine and Threonine Catabolism in Saccharomyces Cerevisiae  


The catabolic L-serine (L-threonine) deaminase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. A mutant, cha1 (catabolism of hydroxyamino acids), lacking this enzyme activity has been isolated. We have cloned the CHA1 gene by complementation of a cha1 mutation. Northern analysis showed that CHA1 mRNA has a size of about 1200 ribonucleotides. CHA1 is probably the structural gene for the enzyme; it is an abundant RNA in cells g...

Petersen, Jgl; Kielland-brandt, M. C.; Nilsson-tillgren, T.; Bornaes, C.; Holmberg, S.



Expression of Heteropolymeric Ferritin Improves Iron Storage in Saccharomyces cerevisiae  


Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp). In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2? and ura3? for YIplac128 and YEp352, respectively. The heterologous expression of phytase was used to determine the expression capability of the host strain. Expression in the...

Kim, Hye-jin; Kim, Hyang-mi; Kim, Ji-hye; Ryu, Kyeong-seon; Park, Seung-moon; Jahng, Kwang-yeup; Yang, Moon-sik; Kim, Dae-hyuk



Isolation of the ARO1 cluster gene of Saccharomyces cerevisiae.  


The AROl cluster gene was isolated by complementation in Saccharomyces cerevisiae after transformation with a comprehensive yeast DNA library of BamHI restriction fragments inserted into the shuttle vector YEp13. Most of the transformants exhibited the expected episomal inheritance of the ARO+ phenotype; however, one stable transformant has been shown to be an integration of the AROl fragment and the vector YEp13 at the arol locus. The insert containing AROl is a 17.2-kilobase pair (kbp) BamH...

Larimer, F. W.; Morse, C. C.; Beck, A. K.; Cole, K. W.; Gaertner, F. H.



[Cycloheximide-dependent mutants of the yeast Saccharomyces cerevisiae]. (United States)

Selection of sup1 and sup2 mutants in the yeast Saccharomyces cerevisiae on cycloheximide containing media revealed classes of mutants that either are completely unable to grow on YAPD without cycloheximide or need this drug under high temperature incubation (30 or 36 degrees C). Some of these mutants also exhibit the growth dependence on another antibiotic--trichodermin, and, at the same time, the osmotic dependence. A hypothesis claiming that sup1 and sup2 mutations cause conformational lability fo yeast cytoplasmic ribosomes has been put forward. It is also proposed that binding of cycloheximide and trichodermin to the mutant ribosomes cause their conformational shift, which compensates the functional defects. PMID:6363204

Mironova, L N; Ter-Avanesian, M D



The Saccharomyces cerevisiae Msh2 and Msh6 proteins form a complex that specifically binds to duplex oligonucleotides containing mismatched DNA base pairs.  


The yeast Saccharomyces cerevisiae encodes six proteins, Msh1p to Msh6p, that show strong amino acid sequence similarity to MutS, a central component of the bacterial mutHLS mismatch repair system. Recent studies with humans and S. cerevisiae suggest that in eukaryotes, specific MutS homolog complexes that display unique DNA mismatch specificities exist. In this study, the S. cerevisiae 109-kDa Msh2 and 140-kDa Msh6 proteins were cooverexpressed in S. cerevisiae and shown to interact in an im...

Alani, E.



A Defect in Mismatch Repair in Saccharomyces Cerevisiae Stimulates Ectopic Recombination between Homeologous Genes by an Excision Repair Dependent Process  


Null mutations in three recombination and DNA repair genes were studied to determine their effects on mitotic recombination between the duplicate AdoMet (S-adenosylmethionine) synthetase genes (SAM1 and SAM2) in Saccharomyces cerevisiae. SAM1 and SAM2, located on chromosomes XII and IV, respectively, encode functionally equivalent although differentially regulated AdoMet synthetases. These similar but not identical (homeologous) genes are 83% homologous at the nucleotide level and this identi...

Bailis, A. M.; Rothstein, R.



Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae  


The cytoplasmic heritable determinant [PSI+] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI+] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [...

Eaglestone, Simon S.; Ruddock, Lloyd W.; Cox, Brian S.; Tuite, Mick F.



Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains  

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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 síntese de citocromo P-450. Estes resultados indicam que as condições de cultivo devem ser específicas e bem definidas para a linhagem selecionada, garantindo assim elevados níveis de citocromo P-450 e, conseqüentemente, a confiabilidade nos testes de mutagenicidade.

Míriam Cristina Sakuragui Matuo



Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese 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 p [...] rodução 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 síntese de citocromo P-450. Estes resultados indicam que as condições de cultivo devem ser específicas e bem definidas para a linhagem selecionada, garantindo assim elevados níveis de citocromo P-450 e, conseqüentemente, a confiabilidade nos testes de mutagenicidade. Abstract in english 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 selectio [...] n 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.

Míriam Cristina Sakuragui, Matuo; Irene Satiko, Kikuchi; Terezinha de Jesus Andreoli, Pinto.



Identification of the constitutive ultradian oscillator of the circadian clock (ENOX1 in Saccharomyces cerevisiae  

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Full Text Available A yeast (Saccharomyces cerevisiae deletion library was screened based on NADH fluorescence using a 384 well plate assay and robotics to identify a yeast isolate lacking the 24 min periodic cell surface oxidase. The oxidase was shown previously to be a candidate ultradian oscillator of the yeast’s biological clock. The cDNA was cloned from a yeast overexpression library and the encoded protein was expressed in bacteria and characterized. Glyceraldehyde-3-phosphate dehydrogenase activity was used as the cellular circadian indicator. The identified gene was YML117W which encodes a ca 126 kDa putative RNA-binding protein. The candidate ENOX1 activity from yeast had functional characteristics similar to those of other constitutive ENOX1 proteins of eukaryotes exhibiting oscillating activities with a temperature independent period length of 24 min phased by melatonin and low frequency electromagnetic fields and susceptible to inhibition by the ENOX1 inhibitor, simalikalactone D. The YML117W deletion mutant cells lacked the ENOX1 clock output present in wild type yeast. The findings identify YML117W as the ENOX1 of Saccharomyces cerevisiae and support its proposed function as an ultradian oscillator of the yeast biological clock.

Sara S. Dick



Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae  


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 fermentatio...

Bellon, Jennifer R.; Schmid, Frank; Capone, Dimitra L.; Dunn, Barbara L.; Chambers, Paul J.



Beta-glucana from Saccharomyces cerevisiae: constitution, bioactivity and obtaining / Beta-glucana de Saccharomyces cerevisiae: constituição, bioatividade e obtenção  

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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.

Raul Jorge Hernan Castro-Gómez



Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae  

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Full Text Available Abstract Background Living cells constantly sense and adapt to redox shifts by the induction of genes whose products act to maintain the cellular redox environment. In the eukaryote Saccharomyces cerevisiae, while stationary cells possess a degree of constitutive resistance towards oxidants, treatment of exponential phase cultures with sub-lethal stresses can lead to the transient induction of protection against subsequent lethal oxidant conditions. The sensors of oxidative stress and the corresponding transcription factors that activate gene expression under these conditions have not yet been completely identified. Results We report the role of SOD1, SOD2 and TPS1 genes (which encode the cytoplasmic Cu/Zn-superoxide dismutase, the mitochondrial Mn-isoform and trehalose-6-phosphate synthase, respectively in the development of resistance to oxidative stress. In all experimental conditions, the cultures were divided into two parts, one was immediately submitted to severe stress (namely: exposure to H2O2, heat shock or ethanol stress while the other was initially adapted to 40°C for 60 min. The deficiency in trehalose synthesis did not impair the acquisition of tolerance to H2O2, but this disaccharide played an essential role in tolerance against heat and ethanol stresses. We also verified that the presence of only one Sodp isoform was sufficient to improve cellular resistance to 5 mM H2O2. On the other hand, while the lack of Sod2p caused high cell sensitivity to ethanol and heat shock, the absence of Sod1p seemed to be beneficial to the process of acquisition of tolerance to these adverse conditions. The increase in oxidation-dependent fluorescence of crude extracts of sod1 mutant cells upon incubation at 40°C was approximately 2-fold higher than in sod2 and control strain extracts. Furthermore, in Western blots, we observed that sod mutants showed a different pattern of Hsp104p and Hsp26p expression also different from that in their control strain. Conclusions Trehalose seemed not to be essential in the acquisition of tolerance to H2O2 stress, but its absence was strongly felt under water stress conditions such as heat and alcoholic stresses. On the other hand, Sod1p could be involved in the control of ROS production; these reactive molecules could signal the induction of genes implicated within cell tolerance to heat and ethanol. The effects of this deletion needs further investigation.

Eleutherio Elis CA



[Anti-Saccharomyces cerevisiae antibodies in patients with Crohn's disease]. (United States)

Inflammatory Bowel Diseases are a group of diseases with chronic inflammation of the gastrointestinal tract, but without proven etiology. Immunologic, environmental, infective and genetic factors equally can play role in their development. Antibodies to an oligomannose epitope of the Saccharomyces cerevisiae demonstrated in 60-70% of the patients with Crohn's disease. The origin and the clinicopathological role are not clarified. It is important that there are no surveys with patients suffering in gluten sensitive enteropathy in the literature. As there are no ASCA survey in Hungary, the aim of this study was to determine the prevalence of the ASCA. The authors examined at their patients the ASCA's occurrence and compared with the clinical picture of the Crohn's disease. The results supported the theory that ASCA positivity correlates with small intestines' Crohn's disease and in these cases both the IgG and IgA type antibodies proved. The antibodies in the sera at the analyzed ASCA positive cases prove a systemic immune response against Saccharomyces cerevisiae and the authors suggest the end of the oral tolerance against the yeast's antigens. The diet restriction (elemental diet, total parenteral nutrition, and fecal diversion) may ameliorate the status of the patients with Crohn's disease. It is speculated that the yeast-free diet as a part of the therapy for the ASCA positive patients can be reasonable: moreover the permanent "forbidding" of the yeast can be an acceptable alternative in case of getting well. PMID:11760647

Barta, Z; Csípö, I; Antal-Szalmás, P; Sipka, S; Szabó, G; Szegedi, G



Polycistronic expression of a ?-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ?-ionone production. (United States)

The flavour and fragrance compound ?-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for ?-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three ?-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of ?-carotene. To enable ?-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the ?-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, ?-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound. PMID:24486029

Beekwilder, Jules; van Rossum, Harmen M; Koopman, Frank; Sonntag, Frank; Buchhaupt, Markus; Schrader, Jens; Hall, Robert D; Bosch, Dirk; Pronk, Jack T; van Maris, Antonius J A; Daran, Jean-Marc



Ciclohexadespipeptide beauvericin degradation by different strains of Saccharomyces cerevisiae. (United States)

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. PMID:23791659

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



Protein Enrichment of Cassava Pulp Fermentation by Saccharomyces cerevisiae  

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Full Text Available The purpose of this study was to determine intestinal digestibility of residual components of cassava pulp solid state fermentation by Saccharomyces cerevisiae for animal feed. Three ruminally cannulated animal were used to measure in situ rumen Dry Matter (DM and Crude Protein (CP degradability characteristics of cassava pulp solid state fermentation by S. cerevisiae. Nylon bags containing 3 g (as fed basis of each feed was immersed in duplicate at each time point in the ventral rumen of each goat for 2, 4, 8, 12, 24, 48 and 72 h. Rumen feed residues from bags of 16 h incubation were used for estimation of lower gut digestibility by the technique of in vitro pepsin-pancreatin digestion. The results of the chemical analysis indicated that fermentation was slightly improved Ruminal Undegradable Protein (RUP of cassava pulp. The highest value of RUP was significantly differ (pS. cerevisiae in cassava pulp. The present results indicate that fermented cassava pulp can improve protein content and ruminal undegradable protein content.

C. Yuangklang



Capacidade fermentativa de Saccharomyces cerevisiae enriquecida com ácidos graxos / Fermentative capacity of (Saccharomyces cerevisiae) enriched with fatty acids  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese Levedura de panificaçao (Saccharomyces cerevisiae) foi obtida anaerobicamente (sem e com adição dos ácidos graxos palmítico, oleico e linoleico) e aerobicamente e utilizada em ensaios de fermentação com 14% e 16% de sacarose a 32°C. Não houve diferenças significativas, quanto a viabilidade celular, [...] entre os tratamentos das leveduras com ácido oleico, ácido linoleico e aerobicamente (as quais foram ricas em palmitoleico e oleico). As leveduras enriquecidas com ácido palmítico e anaeróbicas apresentaram maior redução na viabilidade do que com ácidos graxos insaturados. Foi observado um aumento na produção de ácido pirúvico e uma redução nos álcoois superiores com a redução da viabilidade celular. Abstract in english Baker's yeast (Saccharomyces cerevisiae) was obtained anaerobically (with and without the addition of following fatty acids: palmitic, oleic and linoleic) aerobically and utilised infermentation trials with 14 and 16% of sucrose in the medium at 32°C. There were no significant differences among olei [...] c acid, linoleic acid and aerobic treatments (which were rich in palmitoleic and oleic acids) in relation to cellular viability. Yeasts enriched with palmitic acid and under anaerobic conditions showed a higher reduction on viability than those treated with unsaturated fatty acids. An increased production of pyruvic acid and a reduction in higher alcohols with a reduction on cellular viability were observed.

L.E., Gutierrez; A.V.K.O., Annicchino; L., Lucatti.


Histone H1 of Saccharomyces cerevisiae Inhibits Transcriptional Silencing  


Eukaryotic genomes contain euchromatic regions, which are transcriptionally active, and heterochromatic regions, which are repressed. These domains are separated by “barrier elements”: DNA sequences that protect euchromatic regions from encroachment by neighboring heterochromatin. To identify proteins that play a role in the function of barrier elements we have carried out a screen in S. cerevisiae. We recovered the gene HHO1, which encodes the yeast ortholog of histone H1, as a high-copy...

Veron, Marie; Zou, Yanfei; Yu, Qun; Bi, Xin; Selmi, Abdelkader; Gilson, Eric; Defossez, Pierre-antoine



Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae  

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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 at low glucose concentration.

Hahn-Hägerdal Bärbel



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

DEFF Research Database (Denmark)

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 ?-(l-?-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 °C. When ACVS and PPTase encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicatingthat gene copy number was one of the rate-limiting factors for ACV production in yeast.

Siewers, Verena; Chen, Xiao



“A comparison between sugar consumption and ethanol production in wort by immobilized Saccharomyces Cerevisiae, Saccharomyces Ludwigii and Saccharomyces Rouxii on Brewer’S Spent Grain”  


The immobilization of Saccharomyces cerevisiae DSM 70424, Saccharomyces ludwigii DSM 3447 and Saccharomyces rouxii DSM 2531 on brewer's spent grain and then ethanol production and sugar consumption of these immobilized yeasts were investigated. The aim of this study was to investigate the abilities of these three immobilized yeasts for producing alcohol for brewing at two temperatures (7 and 12 °C) using two different sugar levels (one at original level supplied in the brewery and one with 2...

Aniseh Mohammadi; Seyyed Hadi Razavi; Seyyed Mohammad Mousavi; Karamatollah Rezaei



Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae  

International Nuclear Information System (INIS)

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


Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae  

DEFF Research Database (Denmark)

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

Nielsen, Jens; Jewett, Michael Christopher



Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers. (United States)

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. PMID:25286420

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



Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae  

DEFF Research Database (Denmark)

We have investigated glycolytic oscillations under semi-anaerobic conditions in Saccharomyces cerevisiae by means of NADH fluorescence, measurements of intracellular glucose concentration, and mitochondrial membrane potential. The glucose concentration was measured using an optical nanosensor, while mitochondrial membrane potential was measured using the fluorescent dye DiOC(2)(3). The results show that, as opposed to NADH and other intermediates in glycolysis, intracellular glucose is not oscillating. Furthermore, oscillations in NADH and membrane potential are inhibited by the ATP/ADP antiporter inhibitor atractyloside and high concentrations of the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, suggesting that there is a strong coupling between oscillations in mitochondrial membrane potential and oscillations in NADH mediated by the ATP/ADP antiporter and possibly also other respiratory components.

Poulsen, Allan K.; Andersen, Ann Zahle



Inducibility of gene conversion in Saccharomyces cerevisiae treated with MMS. (United States)

At high survival levels (85%), point mutation and gene conversion frequencies were determined in strain D7 of Saccharomyces cerevisiae after treatment with methyl methanesulfonate (MMS) either after cells were incubated in complete medium before plating or following a split-dose protocol. It is shown that induction of gene conversion by MMS post-incubation leads to an additional enhancement in frequency. This increase is not observed for point mutation. By fractionation of the MMS dose (1 mM + 1 mM) with incubation in complete medium between the 2 doses the frequency of gene conversion is twice as high as with a single equal total dose (2 mM). This treatment does not modify the frequencies of point mutation. These data support the notion that an inducible recombinogenic function exists in wild-type yeast. PMID:3526142

Cundari, E; Vellosi, R; Galli, A; Bronzetti, G



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

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 ?-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. PMID:24318365

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



Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae  


During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with div...

Henderson, Clark M.; Zeno, Wade F.; Lerno, Larry A.; Longo, Marjorie L.; Block, David E.



Optimization of feeding strategy for the ergosterol production by yeasts Saccharomyces cerevisiae  


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...

Mojmir Rychtera; Josef Cermak; Jaroslav Votruba; Jan Nahlik; Karel Melzoch; Kent, Christopher A.; Estela Escalante, Waldir D.



Genomic evolution of Saccharomyces cerevisiae under Chinese rice wine fermentation. (United States)

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

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



Cybernetic model for the growth of Saccharomyces cerevisiae on melibiose. (United States)

Cybernetic modeling has traditionally been used in the modeling of microbial growth on multiple substrates. In this paper, cybernetic modeling has been applied to serve as a model for growth on substrates such as melibiose, which are disaccharides and enzymatically degrade to a monosaccharide mixture in the fermentation broth. The enzyme alpha-galactosidase has been shown to be strongly induced in the presence of galactose and severely repressed by glucose. In the present model, the relative concentration of alpha-galactosidase has been linked to that of the key enzyme for galactose metabolism. The enzymatic degradation process is placed under the control of the cybernetic variables. The maximum rate of melibiose degradation vm and the Monod parameters for growth of Saccharomyces cerevisiae on pure glucose and galactose were estimated by batch growth experiments. S. cerevisiae growth on melibiose and a mixture of melibiose and glucose under a variety of preculturing conditions was simulated. Depending on the rate of enzymatic degradation (i.e., the value of vm), the cell mass profile for microbial growth on a disaccharide can resemble profiles for growth on a single substrate (melibiose) or can resemble diauxie growth. Experiments indicate that the model is able to accurately predict the cell mass profiles for yeast growth. PMID:8983203

Gadgil, C J; Bhat, P J; Venkatesh, K V



Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells. (United States)

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. PMID:22996308

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



Metabolic control of transcription: paradigms and lessons from Saccharomyces cerevisiae. (United States)

The comparatively simple eukaryote Saccharomyces cerevisiae is composed of some 6000 individual genes. Specific sets of these genes can be transcribed co-ordinately in response to particular metabolic signals. The resultant integrated response to nutrient challenge allows the organism to survive and flourish in a variety of environmental conditions while minimal energy is expended upon the production of unnecessary proteins. The Zn(II)2Cys6 family of transcriptional regulators is composed of some 46 members in S. cerevisiae and many of these have been implicated in mediating transcriptional responses to specific nutrients. Gal4p, the archetypical member of this family, is responsible for the expression of the GAL genes when galactose is utilized as a carbon source. The regulation of Gal4p activity has been studied for many years, but we are still uncovering both nuances and fundamental control mechanisms that impinge on its function. In the present review, we describe the latest developments in the regulation of GAL gene expression and compare the mechanisms employed here with the molecular control of other Zn(II)2Cys6 transcriptional regulators. This reveals a wide array of protein-protein, protein-DNA and protein-nutrient interactions that are employed by this family of regulators. PMID:18687061

Campbell, Robert N; Leverentz, Michael K; Ryan, Louise A; Reece, Richard J



A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4. (United States)

To investigate the role of the prevacuolar secretion pathway in the trafficking of vacuolar proteins in Candida albicans, the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS4 was cloned and analyzed. Candida albicans VPS4 encodes a deduced AAA-type ATPase that is 75.6% similar to S. cerevisiae Vps4p, and plasmids bearing C. albicans VPS4 complemented the abnormal vacuolar morphology and carboxypeptidase missorting in S. cerevisiae vps4 null mutants. Candida albicans vps4Delta null mutants displayed a characteristic class E vacuolar morphology and multilamellar structures consistent with an aberrant prevacuolar compartment. The C. albicans vps4Delta mutant degraded more extracellular bovine serum albumin than did wild-type strains, which implied that this mutant secreted more extracellular protease activity. These phenotypes were complemented when a wild-type copy of VPS4 was reintroduced into its proper locus. Using a series of protease inhibitors, the origin of this extracellular protease activity was identified as a serine protease, and genetic analyses using a C. albicans vps4Deltaprc1Delta mutant identified this missorted vacuolar protease as carboxypeptidase Y. Unexpectedly, C. albicans Sap2p was not detected in culture supernatants of the vps4Delta mutants. These results indicate that C. albicans VPS4 is required for vacuolar biogenesis and proper sorting of vacuolar proteins. PMID:17506830

Lee, Samuel A; Jones, Jason; Khalique, Zachary; Kot, John; Alba, Mercedes; Bernardo, Stella; Seghal, Alfica; Wong, Brian



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

Energy Technology Data Exchange (ETDEWEB)

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.

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



Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae. (United States)

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. PMID:23608474

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



High level secretion of cellobiohydrolases by Saccharomyces cerevisiae  

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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.

Ahlgren Simon



Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae  

DEFF Research Database (Denmark)

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 level over-expression of HSF1-R206S increased heterologous ?-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.

Hou, Jin; Österlund, Tobias



Efficient direct ethanol production from cellulose by cellulase- and cellodextrin transporter-co-expressing Saccharomyces cerevisiae  


Efficient degradation of cellulosic biomass requires the synergistic action of the cellulolytic enzymes endoglucanase, cellobiohydrolase, and ?-glucosidase. Although there are many reports describing consolidation of hydrolysis and fermentation steps using recombinant Saccharomyces cerevisiae that express cellulolytic enzymes, the efficiency of cellulose degradation has not been sufficiently improved. Although the yeast S. cerevisiae cannot take up cellooligosaccharide, some fungi can take u...

Yamada, Ryosuke; Nakatani, Yuki; Ogino, Chiaki; Kondo, Akihiko



Reconstruction of the biosynthetic pathway for the core fungal polyketide scaffold rubrofusarin in Saccharomyces cerevisiae  

DEFF Research Database (Denmark)

ABSTRACT: 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 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 a biosynthetic pathway for rubrofusarin in S. cerevisiae. First, the Fusarium graminearum gene encoding polyketide synthase 12 (PKS12) was heterologously co-expressed with the Aspergillus fumigatus gene encoding phosphopantetheinyl transferase (npgA) resulting in production of YWA1. This aromatic heptaketide intermediate was converted into nor-rubrofusarin upon expression of the dehydratase gene aurZ from the aurofusarin gene cluster of F. graminearum. Final conversion into rubrofusarin was achieved by expression of the O-methyltransferase encoding gene aurJ, also obtained from the aurofusarin gene cluster, resulting in a titer of 1.1 mg/L. Reduced levels of rubrofusarin were detected when expressing PKS12, npgA, and aurJ alone, presumably due to spontaneous conversion of YWA1 to nor-rubrofusarin. However, the co-expression of aurZ resulted in an approx. six-fold increase in rubrofusarin production. 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 verifies the suggested pathway, and as such, it is the first example of utilizing a synthetic biological “bottom up” approach for the validation of a complex fungal polyketide pathway.

Rugbjerg, Peter; Naesby, Michael



Ecological Success of a Group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii Hybrids in the Northern European Wine-Making Environment  


The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24 Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) pro...

Erny, C.; Raoult, P.; Alais, A.; Butterlin, G.; Delobel, P.; Matei-radoi, F.; Casaregola, S.; Legras, J. L.



Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae*  


Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport...

Castegna, Alessandra; Scarcia, Pasquale; Agrimi, Gennaro; Palmieri, Luigi; Rottensteiner, Hanspeter; Spera, Iolanda; Germinario, Lucrezia; Palmieri, Ferdinando



Analysis of Key Factors Affecting Ethanol Production by Saccharomyces cerevisiae IFST-072011  


Ethanol production by Saccharomyces cerevisiae is affected not only by fermentation conditions (temperature, pH and sugar concentration) but also by the intrinsic factors e.g., culture medium, dissolved O2, immobilization and other micronutrients. In order to investigate the influence of key factors on ethanol production by S. cerevisiae, a laboratory strain S. cerevisiae IFST-072011 was used in this study. Several fermentation runs were carried out varying temperature, pH, sugar concen...

Monzur Morshed Ahmed; Md. Abdul Quayum; Md. Fakruddin; Naiyyum Choudhury



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


Caspofungin inhibits the synthesis of 1, 3-?-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 pl...

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



Utilization by Saccharomyces cerevisiae of 5'-methylthioadenosine as a source of both purine and methionine.  


Cells of the yeast Saccharomyces cerevisiae are normally impermeable to the purine nucleosides adenosine and 5'-deoxy-5'-methylthioadenosine (MTA), a product of polyamine biosynthesis. cordycepin-sensitive, adenosine-utilizing strains of S. cerevisiae were able to use MTA to fulfill an auxotrophic requirement for purine. Cordycepin-sensitive strains carrying a met5 mutation were also able to use MTA as a source of methionine. These MTA-utilizing strains of S. cerevisiae should be useful for m...

Cone, M. C.; Marchitto, K.; Zehfus, B.; Ferro, A. J.



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


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...

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



Metabolic engineering of Saccharomyces cerevisiae for bioconversion of D-xylose to D-xylonate. (United States)

An NAD(+)-dependent D-xylose dehydrogenase, XylB, from Caulobacter crescentus was expressed in Saccharomyces cerevisiae, resulting in production of 17 ± 2 g D-xylonate l(-1) at 0.23 gl(-1)h(-1) from 23 g D-xylose l(-1) (with glucose and ethanol as co-substrates). D-Xylonate titre and production rate were increased and xylitol production decreased, compared to strains expressing genes encoding T. reesei or pig liver NADP(+)-dependent D-xylose dehydrogenases. D-Xylonate accumulated intracellularly to ?70 mgg(-1); xylitol to ?18 mgg(-1). The aldose reductase encoding gene GRE3 was deleted to reduce xylitol production. Cells expressing D-xylonolactone lactonase xylC from C. crescentus with xylB initially produced more extracellular D-xylonate than cells lacking xylC at both pH 5.5 and pH 3, and sustained higher production at pH 3. Cell vitality and viability decreased during D-xylonate production at pH 3.0. An industrial S. cerevisiae strain expressing xylB efficiently produced 43 g D-xylonate l(-1) from 49 g D-xylose l(-1). PMID:22709678

Toivari, Mervi; Nygård, Yvonne; Kumpula, Esa-Pekka; Vehkomäki, Maija-Leena; Ben?ina, Mojca; Valkonen, Mari; Maaheimo, Hannu; Andberg, Martina; Koivula, Anu; Ruohonen, Laura; Penttilä, Merja; Wiebe, Marilyn G



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

DEFF Research Database (Denmark)

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.

Asadollahi, Mohammadali; Maury, Jerome



Improvement of Galactose Uptake in Saccharomyces cerevisiae through Overexpression of Phosphoglucomutase: Example of Transcript Analysis as a Tool in Inverse Metabolic Engineering  


Through genome-wide transcript analysis of a reference strain and two recombinant Saccharomyces cerevisiae strains with different rates of galactose uptake, we obtained information about the global transcriptional response to metabolic engineering of the GAL gene regulatory network. One of the recombinant strains overexpressed the gene encoding the transcriptional activator Gal4, and in the other strain the genes encoding Gal80, Gal6, and Mig1, which are negative regulators of the GAL system,...

Bro, Christoffer; Knudsen, Steen; Regenberg, Birgitte; Olsson, Lisbeth; Nielsen, Jens



A relationship between gene expression and protein interactions on the proteome scale: analysis of the bacteriophage T7 and the yeast Saccharomyces cerevisiae  


The relationship between the similarity of expression patterns for a pair of genes and interaction of the proteins they encode is demonstrated both for the simple genome of the bacteriophage T7 and the considerably more complex genome of the yeast Saccharomyces cerevisiae. Statistical analysis of large-scale gene expression and protein interaction data shows that protein pairs encoded by co-expressed genes interact with each other more frequently than with random proteins. Furthermore, the me...

Grigoriev, Andrei



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

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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.

Soares Giselle A.M.



Characterization and gene expression profiles of thermotolerant Saccharomyces cerevisiae isolates from Thai fruits. (United States)

For industrial applications, fermentation of ethanol at high temperature offers advantages such as reduction in cooling costs, reduced risk of microbial contamination and higher efficiency of fermentation processes including saccharification and continuous ethanol stripping. Three thermotolerant Saccharomyces cerevisiae isolates (C3723, C3751 and C3867) from Thai fruits were capable of growing and producing 38 g/L ethanol up to 41°C. Based on genetic analyses, these isolates were prototrophic and homothallic, with dominant homothallic and thermotolerant phenotypes. After short-term (30 min) and long-term (12 h) exposure at 37°C, expression levels increased for the heat stress-response genes HSP26, SSA4, HSP82, and HSP104 encoding the heat shock proteins small HSP, HSP70, HSP90 and the HSP100 family, respectively. In isolates C3723 and C3867, expression was significantly higher than that in reference isolates W303 and TISTR5606 for TPS1 encoding trehalose-6-phosphate synthase, NTH1 encoding neutral trehalase and GSY1 encoding glycogen synthase. The results suggested that continuous high expression of heat stress-response genes was important for the long-term, heat stress tolerance of these thermotolerant isolates. PMID:22579450

Auesukaree, Choowong; Koedrith, Preeyaporn; Saenpayavai, Pornpon; Asvarak, Thipa; Benjaphokee, Suthee; Sugiyama, Minetaka; Kaneko, Yoshinobu; Harashima, Satoshi; Boonchird, Chuenchit




Scientific Electronic Library Online (English)

Full Text Available O presente trabalho visou o estudo do acúmulo de cádmio (Cd) por Saccharomyces cerevisiae, fermentando mosto de melaço com contaminações controladas em níveis sub-tóxicos do citado metal. As condições de fermentação foram similares às reinantes na produção industrial de etanol. O mosto, não esterili [...] zado, continha 12% de açúcares redutores totais (ART) e pH 4,5. Para a contaminação controlada empregou-se dois sais de cádmio, cloreto e acetato e, quatro níveis de contaminação 0,5; 1,0; 2,0 e 5,0 mg mosto. A inoculação do mosto foi executada com fermento de panificação (10% p/p). Após a fermentação (4 horas) foram determinados, porcentagem de fermento no vinho centrifugado e teor alcoólico. Na levedura separada foram determinados peso úmido, matéria seca, proteína bruta e teores de cádmio por espectrofotometria de absorção atômica. Em todos os níveis de contaminação estudados houve acúmulo de Cd pela levedura e diminuição do rendimento em etanol. Abstract in english The aim of this paper was to study the cadmium (Cd) accumulation by Saccharomyces cerevisiae fermenting wort of molasses, under sub-toxic levels of controlled cadmium contamination. Fermentation conditions were similar to industrial alcohol production. Non-sterelized wort had 12% of total reducing s [...] ugars (w/w) and pH 4.5. For the controlled contamination, two cadmium salts were used (chloride and acetate), at four levels of contamination: 0.5; 1.0; 2.0 and 5.0 mg wort. The inoculation of the wort was carried out with commercial bread yeast (10% w/w). After fermentation (4 hours), samples were evaluated for cellular viability, alcohol content and yeast percentage in the centrifuged wine. The centrifuged yeast cells were evaluated for total fresh and dry weight, total protein, and cadmium concentration by atomic absortion spectroscopy. In all Cd levels, there was cadmium accumulation by yeast and a decrease in ethanol yield.




Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae. (United States)

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain. PMID:21642010

Parachin, Nádia Skorupa; Bergdahl, Basti; van Niel, Ed W J; Gorwa-Grauslund, Marie F



Introducing a new breed of wine yeast: interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae. (United States)

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment. PMID:23614011

Bellon, Jennifer R; Schmid, Frank; Capone, Dimitra L; Dunn, Barbara L; Chambers, Paul J



Functional interactions between potassium and phosphate homeostasis in Saccharomyces cerevisiae. (United States)

Maintenance of ion homeostatic mechanisms is essential for living cells, including the budding yeast Saccharomyces?cerevisiae. Whereas the impact of changes in phosphate metabolism on metal ion homeostasis has been recently examined, the inverse effect is still largely unexplored. We show here that depletion of potassium from the medium or alteration of diverse regulatory pathways controlling potassium uptake, such as the Trk potassium transporters or the Pma1 H(+) -ATPase, triggers a response that mimics that of phosphate (Pi) deprivation, exemplified by accumulation of the high-affinity Pi transporter Pho84. This response is mediated by and requires the integrity of the PHO signaling pathway. Removal of potassium from the medium does not alter the amount of total or free intracellular Pi, but is accompanied by decreased ATP and ADP levels and rapid depletion of cellular polyphosphates. Therefore, our data do not support the notion of Pi being the major signaling molecule triggering phosphate-starvation responses. We also observe that cells with compromised potassium uptake cannot grow under limiting Pi conditions. The link between potassium and phosphate homeostasis reported here could explain the invasive phenotype, characteristic of nutrient deprivation, observed in potassium-deficient yeast cells. PMID:25425491

Canadell, David; González, Asier; Casado, Carlos; Ariño, Joaquín



Metabolic engineering of Saccharomyces cerevisiae for caffeine and theobromine production. (United States)

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. PMID:25133732

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



Structure of the RACK1 dimer from Saccharomyces cerevisiae  

DEFF Research Database (Denmark)

Receptor for activated C-kinase 1 (RACK1) serves as a scaffolding protein in numerous signaling pathways involving kinases and membrane-bound receptors from different cellular compartments. It exists simultaneously as a cytosolic free form and as a ribosome-bound protein. As part of the 40S ribosomal subunit, it triggers translational regulation by establishing a direct link between protein kinase C and the protein synthesis machinery. It has been suggested that RACK1 could recruit other signaling molecules onto the ribosome, providing a signal-specific modulation of the translational process. RACK1 is able to dimerize both in vitro and in vivo. This homodimer formation has been observed in several processes including the regulation of the N-methyl-d-aspartate receptor by the Fyn kinase in the brain and the oxygen-independent degradation of hypoxia-inducible factor 1. The functional relevance of this dimerization is, however, still unclear and the question of a possible dimerization of the ribosome-bound protein is still pending. Here, we report the first structure of a RACK1 homodimer, as determined from two independent crystal forms of the Saccharomyces cerevisiae RACK1 protein (also known as Asc1p) at 2.9 and 3.9 Å resolution. The structure reveals an atypical mode of dimerization where monomers intertwine on blade 4, thus exposing a novel surface of the protein to potential interacting partners. We discuss the significance of the dimer structure for RACK1 function.

Yatime, Laure; Hein, Kim Langemach



Lipid droplet autophagy in the yeast Saccharomyces cerevisiae (United States)

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast. PMID:24258026

van Zutphen, Tim; Todde, Virginia; de Boer, Rinse; Kreim, Martin; Hofbauer, Harald F.; Wolinski, Heimo; Veenhuis, Marten; van der Klei, Ida J.; Kohlwein, Sepp D.



An overview of membrane transport proteins in Saccharomyces cerevisiae. (United States)

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. PMID:8720066

Andre, B



Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae. (United States)

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S-S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C. PMID:23974371

Voutilainen, Sanni P; Nurmi-Rantala, Susanna; Penttilä, Merja; Koivula, Anu



Dynamics of the Saccharomyces cerevisiae transcriptome during bread dough fermentation. (United States)

The behavior of yeast cells during industrial processes such as the production of beer, wine, and bioethanol has been extensively studied. In contrast, our knowledge about yeast physiology during solid-state processes, such as bread dough, cheese, or cocoa fermentation, remains limited. We investigated changes in the transcriptomes of three genetically distinct Saccharomyces cerevisiae strains during bread dough fermentation. Our results show that regardless of the genetic background, all three strains exhibit similar changes in expression patterns. At the onset of fermentation, expression of glucose-regulated genes changes dramatically, and the osmotic stress response is activated. The middle fermentation phase is characterized by the induction of genes involved in amino acid metabolism. Finally, at the latest time point, cells suffer from nutrient depletion and activate pathways associated with starvation and stress responses. Further analysis shows that genes regulated by the high-osmolarity glycerol (HOG) pathway, the major pathway involved in the response to osmotic stress and glycerol homeostasis, are among the most differentially expressed genes at the onset of fermentation. More importantly, deletion of HOG1 and other genes of this pathway significantly reduces the fermentation capacity. Together, our results demonstrate that cells embedded in a solid matrix such as bread dough suffer severe osmotic stress and that a proper induction of the HOG pathway is critical for optimal fermentation. PMID:24056467

Aslankoohi, Elham; Zhu, Bo; Rezaei, Mohammad Naser; Voordeckers, Karin; De Maeyer, Dries; Marchal, Kathleen; Dornez, Emmie; Courtin, Christophe M; Verstrepen, Kevin J



Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae. (United States)

As reported previously, Saccharomyces cerevisiae cells deficient in all four known genes coding for alcohol dehydrogenases (ADH1 through ADH4) produce considerable amounts of ethanol during aerobic growth on glucose. It has been suggested that ethanol production in such adh0 cells is a corollary of acetaldehyde dismutation in mitochondria. This could be substantiated further by showing that mitochondrial ethanol formation requires functional electron transport, while the proton gradient or oxidative phosphorylation does not interfere with reduction of acetaldehyde in isolated mitochondria. This acetaldehyde-reducing activity is different from classical alcohol dehydrogenases in that it is associated with the inner mitochondrial membrane and also is unable to carry out ethanol oxidation. The putative cofactor is NADH + H+ generated by a soluble, matrix-located aldehyde dehydrogenase upon acetaldehyde oxidation to acetate. This enzyme has been purified from mitochondria of glucose-grown cells. It is clearly different from the known mitochondrial aldehyde dehydrogenase, which is absent in glucose-grown cells. Both acetaldehyde-reducing and acetaldehyde-oxidizing activities are also present in the mitochondrial fraction of fermentation-proficient (ADH+) cells. Mitochondrial acetaldehyde dismutation may have some significance in the removal of surplus acetaldehyde and in the formation of acetate in mitochondria during aerobic glucose fermentation. Images FIG. 4 PMID:1938903

Thielen, J; Ciriacy, M




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Full Text Available Research efforts are needed to design and improve the process, which would produce sustainable and economically feasible transportation fuel. The present investigation was undertaken to determine the availability of carbohydrates in hydrolysates derived from different substrates Acacia arabica, Delbergia sisso, Peltophorum Pterocarpur and Perkia biglobosa pods, in the production of ethanol. The enzymatic hydrolysis of the substrates has yielded the significant amount of reducing sugar from the substrates by comparing the effect of enzymes on hydrolysis. The Acacia arabica pods has showed the higher production of reducing sugars when treated with 4% a-amylase whereas Peltophorum pterocarpum has produced lowest yield of reducing sugar at 4% a-amylase enzyme. The optimum temperature required for the activity of a-amylase enzyme in the production of reducing sugars using different substrates were revealed that at 30oC the Acacia arabica has yielded maximum sugars whereas the Perkia biglobosa has showed the minimum yield of reducing sugar. The optimum period of enzyme activity in the production of reducing sugars using different substrates was indicted that the Acacia arabica has showed the maximum yield of reducing sugars during the incubation period of 24 hours whereas minimum yield was observed in Perkia biglobosa. The optimum incubation period of Saccharomyces cerevisiae in the production of ethanol has showed that the seven days of incubation has yielded maximum amount of ethanol using the substrate Acacia arabica.

Vijaya S. Gulalkayi



Lipid droplet autophagy in the yeast Saccharomyces cerevisiae. (United States)

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum-autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast. PMID:24258026

van Zutphen, Tim; Todde, Virginia; de Boer, Rinse; Kreim, Martin; Hofbauer, Harald F; Wolinski, Heimo; Veenhuis, Marten; van der Klei, Ida J; Kohlwein, Sepp D



Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae. (United States)

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

Gonzalez-Perez, David; Alcalde, Miguel



Biosynthesis of diphthamide in the yeast Saccharomyces cerevisiae  

International Nuclear Information System (INIS)

Inactivation of EF-2 by diphtheria toxin requires the presence of a posttranslationally synthesized amino acid residue, diphthamide. The present work was undertaken to study the biosynthetic mechanism of diphthamide synthesis in the yeast Saccharomyces cerevisiae in order to gain better understanding of the biological roles of this unique amino acid residue. Thirty-one haploid ADP-ribosylation-negative mutants, comprising 5 complementation groups, were obtained. One of these mutants contains a toxin-resistant form of EF-2 which can be converted to a toxin-sensitive form through the methylation reaction catalyzed by a S-AdoMet:EF-2 methyltransferase enzyme which is present in other yeast strains. The [3He]methylated residue in the EF-2 modified by the methyltransferase in the presence of S-Ado-L-[3H-methyl]-Met has been analyzed chromatographically following both acid and enzymatic hydrolysis. At the conclusion of the reaction, all of the radiolabel was recovered as diphthine (the unamidated form of diphthamide). The authors conclude that the S-AdoMet:EF-2-methyltransferase is specific for the addition of at least the last two of the three methyl groups present in diphthine


Growth and metabolism of inositol-starved Saccharomyces cerevisiae. (United States)

Upon starvation for inositol, a phospholipid precursor, an inositol-requiring mutant of Saccharomyces cerevisiae has been shown to die if all other conditions are growth supporting. The growth and metabolism of inositol-starved cells has been investigated in order to determine the physiological state leading to "inositolless death". The synthesis of the major inositol-containing phospholipid ceases within 30 min after the removal of inositol from the growth medium. The cells, however, continue in an apparently normal fashion for one generation (2 h under the growth conditions used in this study). The cessation of cell division is not preceded or accompanied by any detectable change in the rate of macromolecular synthesis. When cell division ceases, the cells remain constant in volume, whereas macromolecular synthesis continues at first at an unchanged rate and eventually at a decreasing rate. Macromolecular synthesis terminates after about 4 h of inositol starvation, at approximately the time when the cells begin to die. Cell death is also accompanied by a decline in cellular potassium and adenosine triphosphate levels. The cells can be protected from inositolless death by several treatments that block cellular metabolism. It is concluded that inositol starvation results in a imbalance between the expansion of cell volume and the accumulation of cytoplasmic constituents. This imbalance is very likely the cause of inositolless death. PMID:323239

Henry, S A; Atkinson, K D; Kolat, A I; Culbertson, M R



Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae  

DEFF Research Database (Denmark)

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 oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol 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 of the cofactors. The changes in product profile and intracellular metabolite levels were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation, as estimated from a simple stoichiometric model. The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications.

Hou, Jin; Lages, Nuno



Genetic effects of chlorinated ethylenes in the yeast Saccharomyces cerevisiae. (United States)

The chlorinated ethylenes 1,1-dichloroethylene (vinylidene chloride), trans-1,2-dichloroethylene, trichloroethylene, and tetrachloroethylene (perchloroethylene) were assayed for their ability to induce mitotic gene conversion and point mutation as well as mitotic aneuploidy in diploid strains of the yeast Saccharomyces cerevisiae. From strain D7 late logarithmic-phase cells grown in 20% glucose liquid medium, containing a high level of cytochrome P-450, as well as stationary-phase cells combined with an exogenous metabolic activating system (S9) were used, in order to activate the chlorinated compounds and to produce electrophilic mutagenic intermediates. Only 1,1-dichloroethylene exhibited a dose-dependent genetic activity, while the other ethylenes did not. The 2 ways of metabolic activation were compared and were found to cause approximately the same effect. In contrast to the findings with strain D7, vinylidene chloride, trans-1,2-dichloroethylene, and trichloroethylene induced, without metabolic activation, mitotic chromosomal malsegregation in strain D61.M. The presence of liver homogenate as an activating system did not enhance the respective frequencies of chromosome loss. In the case of tetrachloroethylene, sufficient data have not become available, since this compound showed a highly toxic effect towards yeast cells, decreasing the rate of surviving cells to less than 30% at a concentration of 9.8 mM. PMID:3050501

Koch, R; Schlegelmilch, R; Wolf, H U



D-xylulose fermentation to ethanol by Saccharomyces cerevisiae  

Energy Technology Data Exchange (ETDEWEB)

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).

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



The genes for fifteen ribosomal proteins of Saccharomyces cerevisiae. (United States)

We have isolated recombinant lambda phage carrying the genes for 14 of the ribosomal proteins of the yeast Saccharomyces cerevisiae. Analysis of these and of the plasmid carrying the gene tcm1, which codes for the ribosomal protein responsible for resistance to trichodermin, demonstrates that in general the genes for ribosomal proteins are unlinked. One exceptional recombinant carries the genes for two ribosomal proteins within a 2-kilobase region. DNA fragments bearing individual ribosomal protein genes were used to probe restriction digests of the yeast genome to determine whether any of the genes were duplicated. Only 3 of 12 of the genes are present unequivocally as a single copy. Similar fragments were used to probe blots of mRNA separated on denaturing agarose gels to determine the size of the mRNA for each protein. In each case, the mRNA is near the minimum size necessary to code for its protein. In certain temperature-sensitive mutants which fail to synthesize functional mRNA for ribosomal protein, Rosbash et al. (Rosbash, M., Harris, P. K. W., Woolford, J., and Teem, J. L. (1981) Cell, 24, 679-686) have demonstrated the accumulation of a larger RNA molecule, homologous to a ribosomal protein gene, that appears to be a transcript which retains an intervening sequence. We find that for 8 of the 11 ribosomal protein genes examined, a larger molecule accumulates in such a mutant strain, suggesting that in general transcripts of ribosomal protein genes may have introns. PMID:6268628

Fried, H M; Pearson, N J; Kim, C H; Warner, J R



Improved ethanol fermentation by heterologous endoinulinase and inherent invertase from inulin by Saccharomyces cerevisiae. (United States)

It is hypothesized that introduction of an endoinulinase gene into Saccharomyces cerevisiae will improve its inulin utilization and ethanol fermentation through collaboration between the heterologous endoinulinase and the inherent invertase SUC2. The aim of this work was to test the hypothesis by introducing the endoinulinase gene inuA from Aspergillus niger into S. cerevisiae. The results showed that heterologous inuA expressed in S. cerevisiae selectively digested long chains of inulin into short fructooligosaccharides and parts of these fructooligosaccharides could be efficiently utilized by the yeast. This study demonstrated that collaboration between heterologous endoinulinase and inherent invertase improved inulin degradation and ethanol fermentation in S. cerevisiae. PMID:23683966

Yuan, Bo; Wang, Shi-An; Li, Fu-Li



The Effects of Saccharomyces cerevisiae and Flavomycin on Broiler Growth Performance  

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Full Text Available Ninety days old commercial broilers (Ross PM-3 were used in a completely randomized design to study the effect of Saccharomyces cerevisiae and flavomycin on growth performance of broiler chicks. Three treatments (30 replicates utilized were (1 control, (2 Saccharomyces cerevisiae and (3 flavomycin. A significant increase in gain of birds was observed in birds fed Saccharomyces cerevisiae group in 5 th and flavomycin group on 28 th and 35 th days (P<0.05. Feed consumption of broilers during the 14 days of experiment was not different among treatments. A considerable increase in feed consumption in the treated chicks was recorded in 21st and 37th days of the experiment in flavomycin group. A similar result was found on 28 th day of the experiment in control group. Birds receiving 0.2 % Saccharomyces cerevisiae consumed significantly much more feed during 5th week of experiment. Body weight of broilers at 37 days age were better with flavomycin, Saccharomyces cerevisiae and control groups respectively (P<0.05.




Control of the Saccharomyces cerevisiae regulatory gene PET494: transcriptional repression by glucose and translational induction by oxygen.  


The product of the Saccharomyces cerevisiae nuclear gene PET494 is required to promote the translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII). The level of cytochrome c oxidase activity is affected by several different environmental conditions, which also influence coxIII expression. We have studied the regulation of PET494 to test whether the level of its expression might modulate coxIII translation in response to these conditions. A pet494::lacZ fusion ...

Marykwas, D. L.; Fox, T. D.



Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae  


Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very ...

Souza, A. C.; Mesquita, J. F.; Panek, A. D.; Silva, J. T.; Paschoalin, V. M. F.



Rtr1 Is the Saccharomyces cerevisiae Homolog of a Novel Family of RNA Polymerase II-Binding Proteins?  


Cells must rapidly sense and respond to a wide variety of potentially cytotoxic external stressors to survive in a constantly changing environment. In a search for novel genes required for stress tolerance in Saccharomyces cerevisiae, we identified the uncharacterized open reading frame YER139C as a gene required for growth at 37°C in the presence of the heat shock mimetic formamide. YER139C encodes the closest yeast homolog of the human RPAP2 protein, recently identified as a novel RNA poly...

Gibney, Patrick A.; Fries, Thomas; Bailer, Susanne M.; Morano, Kevin A.



Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures.  


Expression of two Saccharomyces cerevisiae genes (YG101 and YG103) that are related to the gene encoding inducible 70K protein (hsp70) is repressed upon heat shock. Mutations of the two genes were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles. No phenotypic effect of single mutations of either gene was detected. However, cells containing both YG101 and YG103 mutations showed altered growth properties; double-mutation cells possess an optimal grow...

Craig, E. A.; Jacobsen, K.



Exo1 Roles for Repair of DNA Double-Strand Breaks and Meiotic Crossing Over in Saccharomyces cerevisiae  


The MRE11, RAD50, and XRS2 genes of Saccharomyces cerevisiae are involved in the repair of DNA double-strand breaks (DSBs) produced by ionizing radiation and by radiomimetic chemicals such as methyl methanesulfonate (MMS). In these mutants, single-strand DNA degradation in a 5? to 3? direction from DSB ends is reduced. Multiple copies of the EXO1 gene, encoding a 5? to 3? double-strand DNA exonuclease, were found to suppress the high MMS sensitivity of these mutants. The exo1 single m...

Tsubouchi, Hideo; Ogawa, Hideyuki



Polymorphisms in DNA polymerase ? affect the mtDNA stability and the NRTI-induced mitochondrial toxicity in Saccharomyces cerevisiae. (United States)

Several pathological mutations have been identified in human POLG gene, encoding for the catalytic subunit of Pol ?, the solely mitochondrial replicase in animals and fungi. However, little is known regarding non-pathological polymorphisms found in this gene. Here we studied, in the yeast model Saccharomyces cerevisiae, eight human polymorphisms. We found that most of them are not neutral but enhanced both mtDNA extended mutability and the accumulation of mtDNA point mutations, either alone or in combination with a pathological mutation. In addition, we found that the presence of some SNPs increased the stavudine and/or zalcitabine-induced mtDNA mutability and instability. PMID:25462018

Baruffini, Enrico; Ferrari, Jessica; Dallabona, Cristina; Donnini, Claudia; Lodi, Tiziana



Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae.  


Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two...

Schnier, J.; Schwelberger, H. G.; Smit-mcbride, Z.; Kang, H. A.; Hershey, J. W.



GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces?cerevisiae  


We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the ? subunit of the eIF-2 complex and contains a mutation in the G2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein...

Huang, Han-kuei; Yoon, Heejeong; Hannig, Ernest M.; Donahue, Thomas F.



Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair.  


Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of th...

Qiu, J.; Guan, M. X.; Bailis, A. M.; Shen, B.



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

Energy Technology Data Exchange (ETDEWEB)

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.

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



EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. (United States)

Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out. PMID:24151867

Jensen, Niels B; Strucko, Tomas; Kildegaard, Kanchana R; David, Florian; Maury, Jérôme; Mortensen, Uffe H; Forster, Jochen; Nielsen, Jens; Borodina, Irina



Development of a cellulolytic Saccharomyces cerevisiae strain with enhanced cellobiohydrolase activity. (United States)

Consolidated bioprocessing (CBP) is a promising technology for lignocellulosic ethanol production, and the key is the engineering of a microorganism that can efficiently utilize cellulose. Development of Saccharomyces cerevisiae for CBP requires high level expression of cellulases, particularly cellobiohydrolases (CBH). In this study, to construct a CBP-enabling yeast with enhanced CBH activity, three cassettes containing constitutively expressed CBH-encoding genes (cbh1 from Aspergillus aculeatus, cbh1 and cbh2 from Trichoderma reesei) were constructed. T. reesei eg2, A. aculeatus bgl1, and the three CBH-encoding genes were then sequentially integrated into the S. cerevisiae W303-1A chromosome via ?-sequence-mediated integration. The resultant strains W1, W2, and W3, expressing uni-, bi-, and trifunctional cellulases, respectively, exhibited corresponding cellulase activities. Furthermore, both the activities and glucose producing activity ascended. The growth test on cellulose containing plates indicated that CBH was a necessary component for successful utilization of crystalline cellulose. The three recombinant strains and the control strains W303-1A and AADY were evaluated in acid- and alkali-pretreated corncob containing media with 5 FPU exogenous cellulase/g biomass loading. The highest ethanol titer (g/l) within 7 days was 5.92 ± 0.51, 18.60 ± 0.81, 28.20 ± 0.84, 1.40 ± 0.12, and 2.12 ± 0.35, respectively. Compared with the control strains, W3 efficiently fermented pretreated corncob to ethanol. To our knowledge, this is the first study aimed at creating cellulolytic yeast with enhanced CBH activity by integrating three types of CBH-encoding gene with a strong constitutive promoter Ptpi. PMID:25164958

Hong, Jiefang; Yang, Huajun; Zhang, Kun; Liu, Cheng; Zou, Shaolan; Zhang, Minhua



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

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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.

Wiebe Marilyn G



Compositions and methods for modeling Saccharomyces cerevisiae metabolism  

DEFF Research Database (Denmark)

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. 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.

Palsson, Bernard unknown


Cellular responses of Saccharomyces cerevisiae to DNA damage  

International Nuclear Information System (INIS)

Full text. Several experimental strategies have been used to study responses of S. cerevisiae cells to DNA damage. One approach was based on the isolation of novel genes, the expression of which is induced by lesions in DNA. One of these genes, DIN7, was cloned and partially characterized previously. The product of DIN7 belongs to a large family of proteins involved in DNA repair and mutagenesis. This family includes Rad2, Rad27 and ExoI proteins of S. cerevisiae and their respective human homologues, all of which are endowed with DNA nuclease activity. To study cellular function of Din7 we constructed the pPK3 plasmid carrying DIN7 fused to the GAL1 promoter. Effects of DIN7 overproduction on the phenotypes of wild-type cells and of rad27 and exoI mutants were examined. Overproduction of Din7 does not seem to affect the proficiency of wild-type S. cerevisiae cells in recombination and mutagenesis. Also, overexpression of DIN7 does not suppress the deficiency of the EXOI gene product, the closest homologue of Din7, both in recombination and in controlling the fidelity of DNA replication. Unexpectedly, we found that elevated levels of Din7 result in a very high frequency of mitochondrial rho- mutants. A high frequency of production of rho- mutants wa s also observed in strains defective in the functioning of the Dun1 protein kinase involved in signal transmission in cells exposed to DNA damaging agents. Interestingly, deficiency of Dun1 results also in a significant derepression of the DIN7 gene. Experiments are under way to distinguish whether a high cellular level of Din7 specifically decreases stability of mitochondrial DNA or affects stability of chromosomal DNA as well. Analysis of previously constructed S. cerevisiae strains carrying random geno mic fusions with reporter lacZ gene, allowed us to identify the reading frame YBR173c, on chromosome II as a novel damage inducible gene - DIN8. We have shown that DIN8-lacZ fusion is induced in yeast cells treated with MMS or exposed to UV light. Northern RNA analysis indicates that DIN8 is induced in response to DNA damage at the transcriptional level. DIN8 was cloned and the phenotype of cells with disruption of the gene is under study. POL2-MEC1-RAD53-DUN1-signal transducing pathway has recently been postulated to be involved in the regulation of response of S. cerevisiae cells to DNA-damaging agents. We analyzed the expression of a known damage inducible DNA-repair gene, MAG1, encoding 3-methyladenine glycosylase, in S. cerevisiae strains carrying MAG1 ::lacZ fusion and deficient in either POL2, MEC1, RAD53 or DUN1 function. ?-galactosidase activity was assayed in cycling cells exposed to MMS or UV light. It was found that, in contrast to model DNA damage inducible RNR genes, neither mutation in t he sensory C-terminal part of polymerase ? (pol2-11) nor the in the Mec1, Sad1/Rad53 or Dun1 cellular kinases blocks the induction of MAG1 in response to MMS or UV light in cycling yeasts. (author)


Expresión heteróloga de un péptido multiepitópico de células B de M. tuberculosis en Saccharomyces cerevisiae  

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Full Text Available Saccharomyces cerevisiae ha sido ampliamente utilizada como sistema de expresión de proteínas heterólogas. El presente trabajo se encaminó hacia la expresión en Saccharomyces cerevisiae de un péptido de epitopes múltiples de M. tuberculosis. Con dicho propósito el péptido quimérico denominado B2 fue clonado en dos vectores de expresión de esta levadura con promotores regulables por galactosa y sulfato cúprico, respectivamente. Luego de los experimentos de inducción, la expresión del péptido B2 se analizó mediante SDS/PAGE y Western blot. El análisis por Western blot confirmó la expresión del péptido B2, al hacerse la inducción con 100 ?M de CuSO4 durante toda la noche. No ocurrió así en los experimentos donde se utilizó la galactosa como inductor con todas las condiciones ensayadas. Estos resultados mostraron que la levadura Saccharomyces cerevisiae pudiera ser un buen hospedero alternativo para la expresión de péptidos multiepitópicos de M. tuberculosis.

Norazmi Mohd Nor



Prevalence reduction of pathogens in poultry fed with Saccharomyces cerevisiae  

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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 < 0.05 and feces (p < 0.05. Conclusions. The present study shows that the inclusion of Levucell® SB20 can significantly control Campylobacter carriage in chickens with some positive effects also on Salmonella presence, thus reducing the contamination of carcasses at slaughtering and preventing human foodborne diseases.

Fanelli, A.



Analysis of HIV-1 Vpr determinants responsible for cell growth arrest in Saccharomyces cerevisiae  

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Full Text Available Abstract Background The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clément, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816–48826, 2002. In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae. Results Our data clearly shows that integrity of N-terminal ?-helix I (17–33 and ?-helix III (53–83 is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides. Conclusions Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes ?-helix I (17–33 and ?-helix III (53–83. Furthermore, the mechanism(s underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells.

Yao Xiao-Jian



Predicting functional upstream open reading frames in Saccharomyces cerevisiae  

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Full Text Available Abstract Background Some upstream open reading frames (uORFs regulate gene expression (i.e., they are functional and can play key roles in keeping organisms healthy. However, how uORFs are involved in gene regulation is not yet fully understood. In order to get a complete view of how uORFs are involved in gene regulation, it is expected that a large number of experimentally verified functional uORFs are needed. Unfortunately, wet-experiments to verify that uORFs are functional are expensive. Results In this paper, a new computational approach to predicting functional uORFs in the yeast Saccharomyces cerevisiae is presented. Our approach is based on inductive logic programming and makes use of a novel combination of knowledge about biological conservation, Gene Ontology annotations and genes' responses to different conditions. Our method results in a set of simple and informative hypotheses with an estimated sensitivity of 76%. The hypotheses predict 301 further genes to have 398 novel functional uORFs. Three (RPC11, TPK1, and FOL1 of these 301 genes have been hypothesised, following wet-experiments, by a related study to have functional uORFs. A comparison with another related study suggests that eleven of the predicted functional uORFs from genes LDB17, HEM3, CIN8, BCK2, PMC1, FAS1, APP1, ACC1, CKA2, SUR1, and ATH1 are strong candidates for wet-lab experimental studies. Conclusions Learning based prediction of functional uORFs can be done with a high sensitivity. The predictions made in this study can serve as a list of candidates for subsequent wet-lab verification and might help to elucidate the regulatory roles of uORFs.

Kristiansson Erik



Metabolic engineering of Saccharomyces cerevisiae for optimizing 3HP production  

DEFF Research Database (Denmark)

The finite nature of fossil resources and the negative influence of CO2 emissions on the global climate are key drivers in development of new biological processes. These are based on renewable resources such as sugar, starch, and biomass and aim at replacing chemical production from fossil fuels. Polyacrylates are a substantial part of the different plastic varieties found on the market. This kind of plastic is derived from acrylic acid, which is currently produced from propylene, a by-product of ethylene and gasoline production. Annually, more than one billion kilograms of acrylic acid is produced 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 3HP has a substantial need for NADPH equivalents. Consequently, a yeast host with elevated NADPH availability is preferred. We will redirect several of the glycolysis steps in order to increase the NADPH generation per glucose molecule and thereby increase 3HP production. We believe this strain will be of high interest for other NADPH demanding biosynthetic routes.

Jensen, Niels Bjerg; Maury, Jerome



Capturing of the monoterpene olefin limonene produced in Saccharomyces cerevisiae. (United States)

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. PMID:25164098

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



[Construction of Saccharomyces cerevisiae cell factories for lycopene production]. (United States)

For microbial production of lycopene, the lycopene synthetic genes from Pantoea agglomerans were integrated into Saccharomyces cerevisiae strain BY4742, to obtain strain ZD-L-000 for production of 0.17 mg · L(-1) lycopene. Improving supplies of isoprenoid precursors was then investigated for increasing lycopene production. Four key genes were chosen to be overexpressed, inclu- ding truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMG1), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, a mutated global regulatory factor gene (upc2.1), a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1), which is a key enzyme in the diterpenoid synthetic pathway, and GGPP synthase gene (SaGGPS) from Sulfolobus acidocaldarius. Over-expression of upc2.1 could not improve lycopene production, while over-expression of tHMGI , BTS1-ERG20 and SaGGPS genes led to 2-, 16. 9- and20. 5-fold increase of lycopene production, respectively. In addition, three effective genes, tHMG1, BTS1-ERG20 and SaGGPS, were integrated into rDNA sites of ZD-L-000, resulting in strain ZD-L-201 for production of 13.23 mg · L(-1) lycopene, which was 77-fold higher than that of the parent strain. Finally, two-phase extractive fermentation was performed. The titer of lycopene increased 10-fold to 135.21 mg · L(-1). The engineered yeast strains obtained in this work provided the basis for fermentative production of lycopene. PMID:25751950

Shi, Ming-Yu; Liu Yi; Wang, Dong; Lu, Fu-Ping; Huang, Lu-Qi; Dai, Zhu-Bo; Zhang, Xue-Li



Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae. (United States)

A diffusion-based microreactor system operated with a reaction volume of 8 ?L is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV-depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle-image-velocimetry (?PIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient K(L)a as well as mechanical stability of the device were accomplished for a membrane thickness of 300 ?m. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm. PMID:20945484

Edlich, Astrid; Magdanz, Veronika; Rasch, Detlev; Demming, Stefanie; Aliasghar Zadeh, Shobeir; Segura, Rodrigo; Kähler, Christian; Radespiel, Rolf; Büttgenbach, Stephanus; Franco-Lara, Ezequiel; Krull, Rainer



Membrane stress caused by octanoic acid in Saccharomyces cerevisiae. (United States)

In order to compete with petroleum-based fuel and chemicals, engineering a robust biocatalyst that can convert renewable feedstocks into biorenewable chemicals, such as carboxylic acids, is increasingly important. However, product toxicity is often problematic. In this study, the toxicity of the carboxylic acids hexanoic, octanoic, and decanoic acid on Saccharomyces cerevisiae was investigated, with a focus on octanoic acid. These compounds are completely inhibitory at concentrations of magnitude 1 mM, and the toxicity increases as chain length increases and as media pH decreases. Transciptome analysis, reconstruction of gene regulatory network, and network component analysis suggested decreased membrane integrity during challenge with octanoic acid. This was confirmed by quantification of dose-dependent and chain length-dependent induction of membrane leakage, though membrane fluidity was not affected. This induction of membrane leakage could be significantly decreased by a period of pre-adaptation, and this pre-adaptation was accompanied by increased oleic acid content in the membrane, significantly increased production of saturated lipids relative to unsaturated lipids, and a significant increase in the average lipid chain length in the membrane. However, during adaptation cell surface hydrophobicity was not altered. The supplementation of oleic acid to the medium not only elevated the tolerance of yeast cells to octanoic acid but also attenuated the membrane leakiness. However, while attempts to mimic the oleic acid supplementation effects through expression of the Trichoplusia ni acyl-CoA ?9 desaturase OLE1(TniNPVE desaturase) were able to increase the oleic acid content, the magnitude of the increase was not sufficient to reproduce the supplementation effect and increase octanoic acid tolerance. Similarly, introduction of cyclopropanated fatty acids through expression of the Escherichia coli cfa gene was not helpful for tolerance. Thus, we have provided quantitative evidence that carboxylic acids damage the yeast membrane and that manipulation of the lipid content of the membrane can increase tolerance, and possibly production, of these valuable products. PMID:23435986

Liu, Ping; Chernyshov, Andriy; Najdi, Tarek; Fu, Yao; Dickerson, Julie; Sandmeyer, Suzanne; Jarboe, Laura



Genetic basis for Saccharomyces cerevisiae biofilm in liquid medium. (United States)

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 ?1278b 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 YOR223W controlled biofilm through FLO11 induction. Almost all deletion mutants that were unable to form biofilms in liquid medium also lost the ability to form surface-spreading biofilm colonies (mats) on agar and 69% also lost the ability to grow invasively. The protein kinase A isoform Tpk3p functioned specifically in biofilm and mat formation. In a tpk3 mutant, transcription of FLO11 was induced three-fold compared with wild-type, but biofilm development and cell-cell adhesion was absent, suggesting that Tpk3p regulates FLO11 positive posttranscriptionally and negative transcriptionally.The study provides a resource of biofilm-influencing genes for additional research on biofilm development and suggests that the regulation of FLO11 is more complex than previously anticipated. PMID:25009170

Scherz, Kaj; Andersen; Bojsen, Rasmus; Gro, Laura; Rejkjær; Sørensen; Weiss, Martin; Nielsen; Lisby, Michael; Folkesson, Anders; Regenberg, Birgitte



Structural and Functional Insights into Saccharomyces cerevisiae Riboflavin Biosynthesis Reductase RIB7  


Saccharomyces cerevisiae RIB7 (ScRIB7) is a potent target for anti-fungal agents because of its involvement in the riboflavin biosynthesis pathway as a NADPH-dependent reductase. However, the catalytic mechanism of riboflavin biosynthesis reductase (RBSRs) is controversial, and enzyme structure information is still lacking in eukaryotes. Here we report the crystal structure of Saccharomyces cerevisiae RIB7 at 2.10 Å resolution and its complex with NADPH at 2.35 Å resolution. ScRIB7 exists a...

Lv, Zongyang; Sun, Jian; Liu, Yingfang



Computational identification of non-coding RNAs in Saccharomyces cerevisiae by comparative genomics  


We screened for new structural non-coding RNAs (ncRNAs) in the genome sequence of the yeast Saccharomyces cerevisiae using computational comparative analysis of genome sequences from five related species of Saccharomyces. The screen identified 92 candidate ncRNA genes. Thirteen showed discrete transcripts when assayed by northern blot. Of these, eight appear to be novel ncRNAs ranging in size from 268 to 775 nt, including three new H/ACA box small nucleolar RNAs.

Mccutcheon, John P.; Eddy, Sean R.



Saccharomyces cerevisiae KNU5377 Stress Response during High-Temperature Ethanol Fermentation  


Fuel ethanol production is far more costly to produce than fossil fuels. There are a number of approaches to cost-effective fuel ethanol production from biomass. We characterized stress response of thermotolerant Saccharomyces cerevisiae KNU5377 during glucose-based batch fermentation at high temperature (40°C). S. cerevisiae KNU5377 (KNU5377) transcription factors (Hsf1, Msn2/4, and Yap1), metabolic enzymes (hexokinase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydroge...

Kim, Il-sup; Kim, Young-saeng; Kim, Hyun; Jin, Ingnyol; Yoon, Ho-sung



Hxt-Carrier-Mediated Glucose Efflux upon Exposure of Saccharomyces cerevisiae to Excess Maltose  


When wild-type Saccharomyces cerevisiae strains pregrown in maltose-limited chemostat cultures were exposed to excess maltose, release of glucose into the external medium was observed. Control experiments confirmed that glucose release was not caused by cell lysis or extracellular maltose hydrolysis. To test the hypothesis that glucose efflux involved plasma membrane glucose transporters, experiments were performed with an S. cerevisiae strain in which all members of the hexose transporter (H...

Jansen, M. L. A.; Winde, J. H.; Pronk, J. T.



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


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 chemostat cultures was analysed. The strains exhibited different absolute carotenoid levels as well as different intermediate profiles. These discrepancies were further sustained by the difference of the t...

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



Role of Nitrogen and Carbon Transport, Regulation, and Metabolism Genes for Saccharomyces cerevisiae Survival In Vivo†  


Saccharomyces cerevisiae is both an emerging opportunistic pathogen and a close relative of pathogenic Candida species. To better understand the ecology of fungal infection, we investigated the importance of pathways involved in uptake, metabolism, and biosynthesis of nitrogen and carbon compounds for survival of a clinical S. cerevisiae strain in a murine host. Potential nitrogen sources in vivo include ammonium, urea, and amino acids, while potential carbon sources include glucose, lactate,...

Kingsbury, Joanne M.; Goldstein, Alan L.; Mccusker, John H.



Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae.  


We have cloned a functional centromeric DNA sequence from Saccharomyces cerevisiae. Using the 2 mu chromosome-loss mapping technique and meiotic tetrad analysis, we have identified this DNA sequence as the centromere of chromosome V (CEN5). The CEN5 sequence has been localized on an 1,100-base-pair BamHI-BglII restriction fragment. Plasmids containing CEN5 and an autonomously replicating sequence are mitotically stable in S. cerevisiae and segregate in a Mendelian fashion during meiosis.

Maine, G. T.; Surosky, R. T.; Tye, B. K.



Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose.  


By using a modified technique to measure glucose uptake in Saccharomyces cerevisiae, potential uncertainties have been identified in previous determinations. These previous determinations had led to the proposal that S. cerevisiae contained a constitutive low-affinity glucose transporter and a glucose-repressible high-affinity transporter. We show that, upon transition from glucose-repressed to -derepressed conditions, the maximum rate of glucose transport is constant and only the affinity fo...

Walsh, M. C.; Smits, H. P.; Scholte, M. E.; Dam, K.



Analysis of the Metabolic Response of Saccharomyces Cerevisiae to DNA Damaging Agents  


Saccharomyces cerevisiae, commonly known as Baker?s yeast, is a eukaryotic model organism widely used in biotechnology research. Its genome has a high degree of similarity to humans, and research done on S. cerevisiae can give us a better understanding of the mechanisms involved and the cellular responses to anti-cancer drugs. Yeast is therefore usefool in increasing the effectiveness of anti-cancer drugs.The main goal for this master thesis was to investigate the metabolic response of S...

Rey, Simon Scheel



Continuous ethanol production from sugar beet thick juice by Saccharomyces cerevisiae immobilized onto sugar beet pulp  


The immobilization of Saccharomyces cerevisiae onto sugar beet pulp (SBP) by natural adhesion is an efficient and low-cost method for retaining high biocatalyst density in the ethanol fermentation system. In the present study, cells of S. cerevisiae 163, were immobilized by natural adhesion onto SBP. The retention of immobilized cells attained the level of about 1.7×1011 cells/gram of dry SBP. Continuous ethanol production from sugar beet thick juice (TJ) ...

Vu?urovi? Vesna M.; Razmovski Radojka N.; Milji? Uroš D.; Puškaš Vladimir S.



Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR  


Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that ...

Martorell, P.; Querol, A.; Ferna?ndez-espinar, M. T.



The use of genetically modified Saccharomyces cerevisiae strains in the wine industry  


During the last decades, science and food technology have contributed at an accelerated rate to the introduction of new products to satisfy nutritional, socio-economic and quality requirements. With the emergence of modern molecular genetics, the industrial importance of Saccharomyces cerevisiae, continuously extended. The demand for suitable genetically modified (GM) S. cerevisiae strains for the biofuel, bakery and beverage industries or for the production of biotechnological products (e.g....

Schuller, Dorit; Casal, Margarida



A collection of indigenous saccharomyces cerevisiae strains from appellations of origin in Portugal and France  


The model organism Saccharomyces cerevisiae stands today at the forefront of molecular biology and functional analysis in genetics and genomics. However, understanding of the ecological, evolutionary and population genetic features that shaped the biology of this species is underscored by a wealth of knowledge on molecular and cellular biology obtained from a very limited number of laboratory strains. In this reasoning, we constituted one of the largest bio-databanks of S. cerevisiae that wer...

Vieira, E.; Neves, J. Drumonde; Gambon, Brigitte; Valero, Eva; Gomes, Ana Catarina; Sousa, Susana; Lima, Maria Teresa; Arau?jo, Isabel M.; Santos, Manuel A. S.; Dequin, Sylvie; Casal, Margarida; Schuller, Dorit



L-Carnosine Affects the Growth of Saccharomyces cerevisiae in a Metabolism-Dependent Manner  


The dipeptide L-carnosine (?-alanyl-L-histidine) has been described as enigmatic: it inhibits growth of cancer cells but delays senescence in cultured human fibroblasts and extends the lifespan of male fruit flies. In an attempt to understand these observations, the effects of L-carnosine on the model eukaryote, Saccharomyces cerevisiae, were examined on account of its unique metabolic properties; S. cerevisiae can respire aerobically, but like some tumor cells, it can also exhibit a metabol...

Cartwright, Stephanie P.; Bill, Roslyn M.; Hipkiss, Alan R.



Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.  


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 GO...

Folch, J. L.; Antaramia?n, A.; Rodri?guez, L.; Bravo, A.; Brunner, A.; Gonza?lez, A.



Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors  


We determined the genome-wide environmental stress response (ESR) expression profile of Candida glabrata, a human pathogen related to Saccharomyces cerevisiae. Despite different habitats, C. glabrata, S. cerevisiae, Schizosaccharomyces pombe and Candida albicans have a qualitatively similar ESR. We investigate the function of the C. glabrata syntenic orthologues to the ESR transcription factor Msn2. The C. glabrata orthologues CgMsn2 and CgMsn4 contain a motif previously referred to as HD1 (h...

Roetzer, Andreas; Gregori, Christa; Jennings, Ann Marie; Quintin, Jessica; Ferrandon, Dominique; Butler, Geraldine; Kuchler, Karl; Ammerer, Gustav; Schu?ller, Christoph



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

Energy Technology Data Exchange (ETDEWEB)

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

Uribe, S.; Rangel, P.; Espinola, G.; Aguirre, G. (Universidad Nacional Autonoma de Mexico, Mexico City (Mexico))



Galacturonic Acid Inhibits the Growth of Saccharomyces cerevisiae on Galactose, Xylose, and Arabinose  


The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch...

Huisjes, Eline H.; Hulster, Erik; Dam, Jan C.; Pronk, Jack T.; Maris, Antonius J. A.



Genotypic and pheno-metabolomic characterization of a Saccharomyces cerevisiae strain collection  


O objectivo do presente trabalho consistiu em explorar a diversidade fenotípica de isolados naturais de Saccharomyces cerevisiae, através de métodos de alto débito combinados com dados bioanalíticos. Uma colecção de 187 estirpes de S. cerevisiae foi constituída com isolados de origens geográficas e aplicações tecnológicas diferentes. Alguns alelos de microssatélites e alguns fenótipos foram identificados como responsáveis pela grande variabilidade entre estirpes. Todas as estir...

Duarte, Ricardo Franco; Mendes, Ine?s; Castro, C. C.; Silva, J. S.; Xavier, A. L.; Neves, J. Drumonde; Oliveira, C.; Martins, Rui C.; Oliveira, J. M.; Ferreira, Anto?nio C.; Schuller, Dorit



Integrated analysis of regulatory and metabolic networks reveals novel regulatory mechanisms in Saccharomyces cerevisiae  


We describe the use of model-driven analysis of multiple data types relevant to transcriptional regulation of metabolism to discover novel regulatory mechanisms in Saccharomyces cerevisiae. We have reconstructed the nutrient-controlled transcriptional regulatory network controlling metabolism in S. cerevisiae consisting of 55 transcription factors regulating 750 metabolic genes, based on information in the primary literature. This reconstructed regulatory network coupled with an existing geno...

Herrga?rd, Markus J.; Lee, Baek-seok; Portnoy, Vasiliy; Palsson, Bernhard Ø.



Adjustment of trehalose metabolism in wine Saccharomyces cerevisiae strains to modify ethanol yields. (United States)

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 (tps1? and tdh3?), 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

Rossouw, D; Heyns, E H; Setati, M E; Bosch, S; Bauer, F F



Links between replication and recombination in Saccharomyces cerevisiae: A hypersensitive requirement for homologous recombination in the absence of Rad27 activity  


The RAD27 gene of Saccharomyces cerevisiae encodes a 5?-3? flap exo/endonuclease, which plays an important role during DNA replication for Okazaki fragment maturation. Genetic studies have shown that RAD27 is not essential for growth, although rad27? mutants are temperature sensitive. Moreover, they exhibit increased sensitivity to alkylating agents, enhanced spontaneous recombination, and repetitive DNA instability. The conditional lethality conferred by the rad27? mutation indicates t...

Debrauwe?re, He?le?ne; Loeillet, Sophie; Lin, Waka; Lopes, Judith; Nicolas, Alain



REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis  

Energy Technology Data Exchange (ETDEWEB)

The REV1 gene of Saccharomyces cerevisiae is required for normal induction of mutations by physical and chemical agents. We have determined the sequence of a 3,485-base-pair segment of DNA that complements the rev1-1 mutant. Gene disruption was used to confirm that this DNA contained the REV1 gene. The sequenced segment contains a single long open reading frame, which can encode a polypeptide of 985 amino acid residues. The REV1 transcript is 3.1 kilobase pairs in length. Frameshift mutations introduced into the open reading frame yielded a Rev-phenotype. A base substitution, encoding Gly-193 to Arg-193, was found in this open reading frame in rev1-1. Deletion mutants, lacking segments of the 5' region of REV1, had intermediate mutability relative to REV1 and rev1-1; a complete deletion exhibited lower mutability than rev1-1. REV1 is not an essential gene. An in-frame fusion of the 5' end of the REV1 open reading frame to the lacZ gene produced beta-galactosidase activity constitutively. The predicted REV1 protein is hydrophilic, with a predicted pI of 9.82. No homologies to RAD1, RAD2, RAD3, RAD7, or RAD10 proteins were noted. A 152-residue internal segment displayed 25% identity with UMUC protein.

Larimer, F.W.; Perry, J.R.; Hardigree, A.A.



Suppressor analysis of the mpt5/htr1/uth4/puf5 deletion in Saccharomyces cerevisiae. (United States)

The MPT5/HTR1/UTH4/PUF5 gene encodes an RNA-binding Puf-family protein in Saccharomyces cerevisiae. The Deltampt5 cells exhibit pleiotropic phenotypes, including the G2/M arrest of the cell cycle and weakened cell wall at high temperatures. The Deltampt5 disruptant was also hydroxyurea (HU) sensitive. In this study we screened deletion suppressors to rescue the temperature sensitivity of Deltampt5, and identified dsf1 (YEL070W), dsf2 (YBR007C), sir2, sir3, sir4 and swe1. Multicopy suppressors identified were PKC1 and its upstream genes, but not the downstream MAPK cascade genes. The overexpression of PKC1, however, did not suppress the HU sensitivity of Deltampt5. In contrast, both the HU- and temperature-sensitivities of a-type Deltampt5 cells were suppressed by each sir deletion or a multicopy of MATalpha2, suggesting that a diploid-type expression is involved. We found that a diploid-specific IME4 gene encoding an RNA-modifying protein was responsible for the suppression of the temperature sensitivity, but not of the HU sensitivity. Furthermore, the suppression of the HU sensitivity depended on PUF4, another Puf-family gene, and overexpression of PUF4 suppressed only the HU sensitivity of Deltampt5. The protein level of Puf4 was not affected by the sir mutation. Thus, these Ime4 and Puf4 proteins play complementary roles to rescue the defects in Deltampt5 Deltasir cells. PMID:16328373

Ohkuni, Kentaro; Kikuchi, Yoshiko; Hara, Kazuhiro; Taneda, Tsuya; Hayashi, Naoyuki; Kikuchi, Akihiko



Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics. (United States)

In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H?-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H?-ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism. PMID:23763276

Walther, Thomas; Mtimet, Narjes; Alkim, Ceren; Vax, Amélie; Loret, Marie-Odile; Ullah, Azmat; Gancedo, Carlos; Smits, Gertien J; François, Jean Marie



Estudo do equilíbrio e cinética da biossorção do pb2+ por saccharomyces cerevisiae Equilibrium and kinetic study of pb2+ biosorption by saccharomyces cerevisiae  


The biosorption, based on the use of biomass for removal of ions is distinguished as an innovative and promising technology when compared with the traditional methods. In this context, the aim of the present work is to use Saccharomyces cerevisiae as biosorbent for the retention of Pb2+ metal ions. Factorial design was used for evaluation of the process. The observed equilibrium data were well described by Langmuir and Freundlich adsorption isotherms. The maximum adsorption capacity was 1486....

Joelma Morais Ferreira; Flávio Luiz Honorato da Silva; Odelsia Leonor Sanchez de Alsina; Líbia de Sousa Conrado Oliveira; Eliane Bezerra Cavalcanti; Wolia Costa Gomes



Viabilidade celular de Saccharomyces cerevisiae cultivada em associação com bactérias contaminantes da fermentação alcoólica / Cellular viability of Saccharomyces cerevisiae cultivated in association with contaminant bacteria of alcoholic fermentation  

Scientific Electronic Library Online (English)

Full Text Available O objetivo deste trabalho foi estudar a influência de bactérias dos gêneros Bacillus e Lactobacillus, bem como de seus produtos metabólicos, na redução da viabilidade celular de leveduras Saccharomyces cerevisiae. As bactérias Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lacto [...] bacillus fermentum e Lactobacillus plantarum foram cultivadas em associação com a levedura S. cerevisiae (cepa Y-904) por 72 horas a 32 °C, sob agitação. A viabilidade celular, a taxa de brotamento e a população de células de S. cerevisiae e a acidez total, a acidez volátil e o pH dos meios de cultivos foram determinados às 0, 24, 48 e 72 horas do cultivo misto. As culturas de bactérias foram tratadas através do calor, de agente antimicrobiano e de irradiação. Os resultados mostraram que apenas os meios de cultivo mais acidificados, contaminados com as bactérias ativas L. fermentum e B. subtilis, provocaram redução na viabilidade celular de S. cerevisiae. Excetuando a bactéria B. subtilis tratada com radiação gama, as demais bactérias tratadas pelos diferentes processos (calor, irradiação e antimicrobiano) não causaram diminuição da viabilidade celular e da população de S. cerevisiae, indicando que a presença isolada dos metabólitos celulares dessas bactérias não foi suficiente para reduzir a porcentagem de células vivas de S. cerevisiae. Abstract in english The aim of this project was to study the influence of the bacteria Bacillus and Lactobacillus, as well as their metabolic products to decrease the cellular viability of the yeast Saccharomyces cerevisiae. The bacteria Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus [...] fermentum and Lactobacillus plantarum were cultivated in association with yeast S. cerevisiae (strain Y-904) for 72 hours at 32 ºC under agitation. The cellular viability, budding rate and population of S. Cerevisiae and the total acidity, volatile acidity and pH of culture medium were determined at 0, 24, 48 and 72 hours of incubation of the mixed culture. The bacteria cultures were treated by heat sterilization, antibacterial agent and irradiation. The results showed that only the more acidified culture medium, contaminated with active bacteria L. fermentum and B. subtilis, caused a reduction in the yeast cellular viability. Except for the bacteria B. subtilis treated for radiation, the other bacteria treated by the different procedures (heat, radiation and antibacterial) did not cause a reduction in the cellular viability of S. cerevisiae, indicating that the isolated presence of the cellular metabolic of these bacteria was not enough to reduce the percentage of the living yeast cells.

Thais de Paula, Nobre; Jorge, Horii; André Ricardo, Alcarde.



Effect of carbon source on lysine-mediated inhibition of postexponential growth of Saccharomyces cerevisiae.  


Lysine-mediated inhibition of postexponential growth in Saccharomyces cerevisiae occurred when glucose, fructose, or maltose, but not lactate, pyruvate, or ethanol, was used as the carbon source. Arginine starvation is not responsible for the inhibitory effect, since neither the intracellular pool of glucose-grown (inhibited) cells nor that of lactate-grown (noninhibited) cells contained arginine.

Watson, T. G.



Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol (United States)

The effects of limonene concentration, enzyme loading, and pH on ethanol production from simultaneous saccharification and fermentation (SSF) of citrus peel waste by Saccharomyces cerevisiae were studied at 37 C. Prior to SSF, citrus peel waste underwent a steam explosion process combined with fla...


Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion (United States)

Recombinant constructs of barley '-amylase and Lentinula edodes glucoamylase genes were integrated into the chromosomes of Saccharomyces cerevisiae. The insertion was confirmed by PCR amplification of the gene sequence in the chromosomes. The expression was analyzed by SDS-PAGE of the enzymes puri...


Microbial Cells as Biosorbents for Heavy Metals: Accumulation of Uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa  


Uranium accumulated extracellularly on the surfaces of Saccharomyces cerevisiae cells. The rate and extent of accumulation were subject to environmental parameters, such as pH, temperature, and interference by certain anions and cations. Uranium accumulation by Pseudomonas aeruginosa occurred intracellularly and was extremely rapid (

Strandberg, Gerald W.; Shumate, Starling E.; Parrott, John R.



Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass (United States)

The Aspergillus niger ferulic acid esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interactio...


Saccharomyces cerevisiae Mixed Culture of Blackberry (Rubus ulmifolius L.) Juice: Synergism in the Aroma Compounds Production  


Blackberry (Rubus sp.) juice was fermented using four different strains of Saccharomyces cerevisiae (Vitilevure-CM4457, Enoferm-T306, ICV-K1, and Greroche Rhona-L3574) recognized because of their use in the wine industry. A medium alcoholic graduation spirit (

Bautista-rosales, Pedro Ulises; Ragazzo-sa?nchez, Juan Arturo; Ruiz-montan?ez, Gabriela; Ortiz-basurto, Rosa Isela; Luna-solano, Guadalupe; Caldero?n-santoyo, Montserrat



Rpg1p/eIF3a of Saccharomyces cerevisiae mediates formation of stress granules.  

Czech Academy of Sciences Publication Activity Database

2004, L145. [Annual meeting /44./. Washington (US), 04.12.2004-08.12.2004] R&D Projects: GA ?R GA204/02/1424 Grant ostatní: GA MŠk1(CZ) KONTAKT 2004/09 Institutional research plan: CEZ:AV0Z5020903 Keywords : rpg1p/eif3a * saccharomyces cerevisiae Subject RIV: EE - Microbiology, Virology

Špryngar, Martin; Janatová, Ivana; Wolinski, H.; Kohlwein, S. D.; Hašek, Ji?í


Invert sugar formation with Saccharomyces cerevisiae cells encapsulated in magnetically responsive alginate microparticles.  

Czech Academy of Sciences Publication Activity Database

Ro?. 321, - (2009), s. 1478-1481. ISSN 0304-8853 R&D Projects: GA MPO 2A-1TP1/094; GA MŠk(CZ) OC 157 Institutional research plan: CEZ:AV0Z60870520 Keywords : magnetic alginate microbeads * Saccharomyces cerevisiae * invertase Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.204, year: 2009

Šafa?ík, Ivo; Mad?rová, Zde?ka; Šafa?íková, Miroslava



Multivariate analyses of cellular carbohydrates and fatty acids of Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae.  


Quantitative data of major cellular carbohydrates distinguished Candida albicans or Torulopsis glabrata from Saccharomyces cerevisiae but not C. albicans from T. glabrata. Multivariate analyses of both carbohydrate and fatty acid variables (I. Brondz, I. Olsen, and M. Sjöström, J. Clin. Microbiol. 27:2815-2819, 1989), however, differentiated all three species.

Brondz, I.; Olsen, I.



Ethanol-Independent Biofilm Formation by a Flor Wine Yeast Strain of Saccharomyces cerevisiae?  


Flor strains of Saccharomyces cerevisiae form a biofilm on the surface of wine at the end of fermentation, when sugar is depleted and growth on ethanol becomes dependent on oxygen. Here, we report greater biofilm formation on glycerol and ethyl acetate and inconsistent formation on succinic, lactic, and acetic acids.

Zara, Severino; Gross, Michael K.; Zara, Giacomo; Budroni, Marilena; Bakalinsky, Alan T.



Protective Role of Mitochondrial Superoxide Dismutase against High Osmolarity, Heat and Metalloid Stress in Saccharomyces cerevisiae.  

Czech Academy of Sciences Publication Activity Database

Ro?. 52, ?. 2 (2007), s. 120-126. ISSN 0015-5632 R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z50200510 Keywords : saccharomyces cerevisiae * mitochondrial mn-sod * protein Subject RIV: EE - Microbiology, Virology Impact factor: 0.989, year: 2007

Dziadkowiec, D.; Krasowska, A.; Liebner, A.; Sigler, Karel



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

DEFF Research Database (Denmark)

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 times varying between 2 and 35 hours.

Regenberg, Birgitte; Grotkjaer, Thomas



Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae. (United States)

Metabolic engineering of microorganisms is an alternative and attractive route for production of valuable terpenoids that are usually extracted from plant sources. Tanshinones are the bioactive components of Salvia miltiorrhizha Bunge, which is a well-known traditional Chinese medicine widely used for treatment of many cardiovascular diseases. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase-like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2?mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over-expression of a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1) gene did improve supply of farnesyl diphosphate (FPP), production of miltiradiene was not increased while large amounts of squalene (78?mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8?mg/L by improving supply of GGPP through over-expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over-expressing ERG20-BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2?mg/L. Combinatorial over-expression of tHMGR-upc2.1 and ERG20-BTS1-SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8?mg/L. Finally, fed-batch fermentation was performed, and 488?mg/L miltiradiene was produced. The yeast strains engineered in this work provide a basis for creating an alternative way for production of tanshinones in place of extraction from plant sources. PMID:22566191

Dai, Zhubo; Liu, Yi; Huang, Luqi; Zhang, Xueli



Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae. (United States)

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value. PMID:17002315

Xu, Hengyu; West, Ann H; Cook, Paul F



Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1  

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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-? 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 ?M for 6-phosphogluconate and of 35 ± 6 ?M 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 necessary for the dimerization. This domain also works as a lid on the substrate binding pocket to control the binding of substrate and the release of product, so it is indispensable for the 6PGDH activity. Moreover, the co-crystallized citrate molecules, which mimic the binding mode of the substrate 6-phosphogluconate, provided us a novel strategy to design the 6PDGH inhibitors.

Zhou Cong-Zhao



Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism  

DEFF Research Database (Denmark)

BACKGROUND: Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. RESULTS: The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively. CONCLUSIONS: Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial improvement in isobutanol production for the referencestrain, but not for the ILV2 ILV3 ILV5 overexpression strain. This result implies that other constraints besides the enzyme activities for the supply of 2-ketoisovalerate may become bottlenecks for isobutanol production after ILV2, ILV3, and ILV5 have been overexpressed, which most probably includes the valine inhibition to Ilv2.

Chen, Xiao; Nielsen, Kristian Fog



Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield. (United States)

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. PMID:21963484

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



Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress. (United States)

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. PMID:17071783

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



Radiosensitivity of Saccharomyces cerevisiae W303-1A and BY4741 Strains  

International Nuclear Information System (INIS)

Saccharomyces cerevisiae, a simple eukaryotic cell, has been widely used as a model for all eukaryotes including humans for the study of fundamental cellular processes such as DNA replication, DNA recombination, cell cycle, cell division and metabolism. Numerous laboratory strains are used in yeast research. Most of the mutants have been derived from the two widely used laboratory strains W303-1A and BY4741. While BY4741 is a derivative of S288C, used in the systematic sequencing of the S. cerevisiae genome, strains with a W303 background serve in many physiological and biochemical studies. It was found in a recent study that W303-1A contains a mutant allele of YBP1, ybp1-1, encoding four amino acid substitutions, that results in increased peroxide sensitivity. Mutation of ybp1-1 is not a complete loss of function allele as it is more resistant to peroxides than the knock-out mutant. Ybp1 is required for oxidation of specific cysteine residues of the transcription factor Yap1p resulting in the nuclear localization of Yap1p in response to stress. Ionizing radiation (IR) can produce highly reactive hydroxyl radicals through the decomposition of cellular water, such as superoxide anion radical, hydrogen peroxide, hydroxyl radical. These reactive oxygen species (ROS) can cause wide-ranging cellular damage, including DNA double-strand breaks (DSBs), lipid peroxidation, and protein modification. Also, ROS produced by IR cause oxidative stress. Detoxification enzymes are active stress. Detoxification enzymes are activated for ROS scavenging against oxidative stress. Also, antioxidants are used for detoxification of ROS and reduction of oxidative damage. NAC, one of the antioxidants, is a precursor for glutathione (GSH). The aim of the present study was to compare the differences in radiosensitivity associated cell viability between the two strains. Also, effect of NAC against IR on cell protection was investigated


Radiosensitivity of Saccharomyces cerevisiae W303-1A and BY4741 Strains  

Energy Technology Data Exchange (ETDEWEB)

Saccharomyces cerevisiae, a simple eukaryotic cell, has been widely used as a model for all eukaryotes including humans for the study of fundamental cellular processes such as DNA replication, DNA recombination, cell cycle, cell division and metabolism. Numerous laboratory strains are used in yeast research. Most of the mutants have been derived from the two widely used laboratory strains W303-1A and BY4741. While BY4741 is a derivative of S288C, used in the systematic sequencing of the S. cerevisiae genome, strains with a W303 background serve in many physiological and biochemical studies. It was found in a recent study that W303-1A contains a mutant allele of YBP1, ybp1-1, encoding four amino acid substitutions, that results in increased peroxide sensitivity. Mutation of ybp1-1 is not a complete loss of function allele as it is more resistant to peroxides than the knock-out mutant. Ybp1 is required for oxidation of specific cysteine residues of the transcription factor Yap1p resulting in the nuclear localization of Yap1p in response to stress. Ionizing radiation (IR) can produce highly reactive hydroxyl radicals through the decomposition of cellular water, such as superoxide anion radical, hydrogen peroxide, hydroxyl radical. These reactive oxygen species (ROS) can cause wide-ranging cellular damage, including DNA double-strand breaks (DSBs), lipid peroxidation, and protein modification. Also, ROS produced by IR cause oxidative stress. Detoxification enzymes are activated for ROS scavenging against oxidative stress. Also, antioxidants are used for detoxification of ROS and reduction of oxidative damage. NAC, one of the antioxidants, is a precursor for glutathione (GSH). The aim of the present study was to compare the differences in radiosensitivity associated cell viability between the two strains. Also, effect of NAC against IR on cell protection was investigated

Park, Ji Young; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)



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

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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.

Ruohonen Laura



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

...that consists of the reagents used to measure, by immunochemical techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S. cerevisiae antibodies may aid in the diagnosis of...



Quantitative modeling of the Saccharomyces cerevisiae FLR1 regulatory network using an S-system formalism. (United States)

In this study we address the problem of finding a quantitative mathematical model for the genetic network regulating the stress response of the yeast Saccharomyces cerevisiae to the agricultural fungicide mancozeb. An S-system formalism was used to model the interactions of a five-gene network encoding four transcription factors (Yap1, Yrr1, Rpn4 and Pdr3) regulating the transcriptional activation of the FLR1 gene. Parameter estimation was accomplished by decoupling the resulting system of nonlinear ordinary differential equations into a larger nonlinear algebraic system, and using the Levenberg-Marquardt algorithm to fit the models predictions to experimental data. The introduction of constraints in the model, related to the putative topology of the network, was explored. The results show that forcing the network connectivity to adhere to this topology did not lead to better results than the ones obtained using an unrestricted network topology. Overall, the modeling approach obtained partial success when trained on the nonmutant datasets, although further work is required if one wishes to obtain more accurate prediction of the time courses. PMID:21976379

Calçada, Dulce; Vinga, Susana; Freitas, Ana T; Oliveira, Arlindo L



ATP16 Genes and Neighboring ORFs Are Duplicated on Chromosome IV in Saccharomyces cerevisiae  

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Full Text Available We present evidence that there were two closely linked copies of the ATP16 (YDL004w gene encoding the delta subunit of F1F0-ATPase complex on chromosome IV in laboratory strains, W303-1A, W303-1B, DC5, LL20, SEY2102, YPH499, and S288C of Saccharomyces cerevisiae. We previously reported that there were 2-3 copies of ATP1 (alpha, ATP2 (beta and ATP3 (gamma on chromosomes II, X, and II, respectively. Homologous recombination of the ATP16 with HIS3 (YOR202w, gene walking, and long-PCR analyses showed that two ATP16 were present on the same chromosome as above genes. The gene walking estimated that the two ATP16 were separated by approximately 8.4 kb using ATP16 and its neighboring DNAs as probes, designated the proximal ATP16 to the telomere as ATP16a and to the distal as ATP16b. Although the nucleotide sequences of ATP16a and ATP16b were identical including 833 bases upstream- and 937 bases downstream of ATP16, they might be expressed differently.

Tadashi Mabuchi



A synthetic suicide riboswitch for the high-throughput screening of metabolite production in Saccharomyces cerevisiae. (United States)

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. PMID:25596509

Lee, Sang-Woo; Oh, Min-Kyu



Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae  

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Combinatorial genetic libraries are powerful tools for diversifying and optimizing biomolecules. The process of library assembly is a major limiting factor for library complexity and quality. Gap repair by homologous recombination in Saccharomyces cerevisiae can facilitate in vivo assembly of DNA fragments sharing short patches of sequence homology, thereby supporting generation of high-complexity libraries without compromising fidelity. In this study, we have optimized the ordered assembly of three DNA fragments into a gapped vector by in vivo homologous recombination. Assembly is achieved by co-transformation of the DNA fragments and the gapped vector, using a modified lithium acetate protocol. The optimal gap-repair efficiency is found at a 1:80 molar ratio of gapped vector to each of the three fragments. We measured gap-repair efficiency in different genetic backgrounds and observed increased efficiency in mutants carrying a deletion of the SGS1 helicase-encoding gene. Using our experimental conditions, agap-repair efficiency of > 10(6) plasmid-harbouring colonies/µg gapped vector DNA is obtained in a single transformation, with a recombination fidelity > 90%.

Eckert-Boulet, Nadine Valerie; Pedersen, Mette Louise



Exocytosis and Endocytosis of Small Vesicles across the Plasma Membrane in Saccharomyces cerevisiae  

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Full Text Available When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase, phosphoenolpyruvate carboxykinase, isocitrate lyase, and malate dehydrogenase, as well as the non-gluconeogenic enzymes glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A, are secreted into the periplasm. In the extracellular fraction, these secreted proteins are associated with small vesicles that account for more than 90% of the total number of extracellular structures observed. When glucose is added to glucose-starved cells, FBPase is internalized and associated with clusters of small vesicles in the cytoplasm. Specifically, the internalization of FBPase results in the decline of FBPase and vesicles in the extracellular fraction and their appearance in the cytoplasm. The clearance of extracellular vesicles and vesicle-associated proteins from the extracellular fraction is dependent on the endocytosis gene END3. This internalization is regulated when cells are transferred from low to high glucose. It is rapidly occurring and is a high capacity process, as clusters of vesicles occupy 10%–20% of the total volume in the cytoplasm in glucose re-fed cells. FBPase internalization also requires the VPS34 gene encoding PI3K. Following internalization, FBPase is delivered to the vacuole for degradation, whereas proteins that are not degraded may be recycled.

Kathryn Stein



Comprehensive X-Ray Structural Studies of the Quinolinate Phosphoribosyl Transferase (BNA6) From Saccharomyces Cerevisiae  

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Quinolinic acid phosphoribosyl transferase (QAPRTase, EC is a 32 kDa enzyme encoded by the BNA6 gene in yeast and catalyzes the formation of nicotinate mononucleotide from quinolinate and 5-phosphoribosyl-1-pyrophosphate (PRPP). QAPRTase plays a key role in the tryptophan degradation pathway via kynurenine, leading to the de novo biosynthesis of NAD{sup +} and clearing the neurotoxin quinolinate. To improve our understanding of the specificity of the eukaryotic enzyme and the course of events associated with catalysis, we have determined the crystal structures of the apo and singly bound forms with the substrates quinolinate and PRPP. This reveals that the enzyme folds in a manner similar to that of various prokaryotic forms which are {approx}30% identical in sequence. In addition, the structure of the Michaelis complex is approximated by PRPP and the quinolinate analogue phthalate bound to the active site. These results allow insight into the kinetic mechanism of QAPRTase and provide an understanding of structural diversity in the active site of the Saccharomyces cerevisiae enzyme when compared to prokaryotic homologues.

di Luccio, E.; Wilson, D.K.



Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103  

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Full Text Available Abstract Background The carbonic anhydrases (CAs are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: ?-, ?-, ?-, ?-, ?-, ?- CAs, with ?-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of ?-CA Nce103/YNL036W. Results We determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 Å resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable. Conclusion The quaternary structure of Nce103 resembles the typical plant type ?-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.

Chen Yuxing



Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production. (United States)

2 ?m-based episomal expression vectors are widely used in Saccharomyces cerevisiae for recombinant protein production and synthetic pathway optimization. In this study, we report a new approach to increase the plasmid copy number (PCN) and thus improve the expression of plasmid-encoded proteins. This was achieved by combining destabilization of the marker protein with decreasing the marker gene transcription level. Destabilization of the marker protein alone by fusing a ubiquitin/N-degron tag (ubi-tag) to the N-terminus of the Ura3 marker protein could increase the PCN and activity of LacZ expressed from the same vector. When arginine was exposed at the N-terminus of the marker protein after cleavage of ubiquitin, the PCN and LacZ activity were increased by 70-80%. Replacement of the native URA3 promoter with the HXT1, KEX2 or URA3-d promoter resulted in an increase in the PCN and LacZ activity by about 30-100%. Combining the ubi-tag and promoter modification of the marker gene, increased the PCN and LacZ activity by threefold. We also demonstrated that this new expression vectors can be used to increase enzyme activity by improving patchoulol production by threefold. PMID:22487308

Chen, Yun; Partow, Siavash; Scalcinati, Gionata; Siewers, Verena; Nielsen, Jens



Automated Yeast Mating Protocol Using Open Reading Frames from Saccharomyces cerevisiae Genome to Improve Yeast Strains for Cellulosic Ethanol Production (United States)

Engineering the industrial ethanologen Saccharomyces cerevisiae to utilize pentose sugars from lignocellulosic biomass is critical for commercializing cellulosic fuel ethanol production. Approaches to engineer pentose-fermenting yeasts have required expression of additional genes. We implemented a...


The Snf1 Protein Kinase in the Yeast Saccharomyces cerevisiae  

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In yeast, Saccharomyces cerevisiae, the Snf1 protein kinase is primarily known as a key component of the glucose repression regulatory cascade. The Snf1 kinase is highly conserved among eukaryotes and its mammalian homolog AMPK is responsible for energy homeostasis in cells, organs and whole bodies. Failure in the AMPK regulatory cascade leads to metabolic disorders, such as obesity or type 2 diabetes. The knowledge about the Snf1 protein kinase remains to be of much interest in studying yeast carbon metabolism and human biology. To investigate the effect of Snf1 kinase and its regulatory subunit Snf4 on the regulation of glucose and galactose metabolism, I physiologically characterized ?snf1, ?snf4, and ?snf1?snf4 CEN.PK background yeast strains in glucose and glucose-galactose mixture batch cultivations (chapter 2). The results of this study showed that delayed induction of galactose catabolism was SNF1 or SNF4 gene deletion specific. In comparison to the reference strain, growth delay on galactose was found to last 2.4 times (7 hours) longer for the ?snf4, 3.1 times (10.5 hours) longer for the ?snf1, and 9.6 times (43 hours) longer for the ?snf1?snf4 strains. The maximum specific growth rates on galactose were found to be two to three times lower for the recombinant strains compared to the reference strain (0.13 h-1) and were found to be 0.07 h-1 for the ?snf1, 0.08 h-1 for the ?snf4 and 0.04 h-1 for the ?snf1?snf4 strain. In contrast to what is generally believed, the study showed that the Snf1 kinase was not solely responsible for the derepression of galactose metabolism. To investigate the regulatory role of Snf1 kinase on a global scale, the global scale mRNA, large-scale yeast quantitative proteome and metabolome datasets were generated. One of the largest yeast global quantitative proteome datasets (2388 proteins) to date was generated using Multidimensional Protein Identification Technology followed by quantitation using stable isotope labeling approach (chapter 3). The stable isotope labeling was compared to the spectral counting quantitative approach and the study showed that the stable isotope labeling approach is highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found, the major reason behind the discrepancy was the lack of reproducible sampling for proteins with low spectral counts. To reconstruct a regulatory map of the yeast Snf1 protein kinase, I used the abundances of 5716 mRNAs, 2388 proteins, and 44 metabolites measured for the wild-type, ?snf1, ?snf4, and ?snf1?snf4 strains. By integrating these measurements with global protein-protein-interactions, protein-DNAinteractions and a genome-scale metabolic model, I mapped the complete network of interactions around the protein kinase Snf1 (chapters 4, 5). Through these interactions, I identified how the Snf1 protein kinase regulated cellular metabolism on gene or protein level. The study revealed that the Snf1 protein kinase played a far more extensive role in controlling both carbon and energy metabolism than previously anticipated. Similar to the function of AMPK in humans, my findings showed that Snf1 was a low energy checkpoint. Our results suggested that it was possible to use yeast more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals.

Usaite, Renata



Ecological success of a group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrids in the northern european wine-making environment. (United States)

The hybrid nature of lager-brewing yeast strains has been known for 25 years; however, yeast hybrids have only recently been described in cider and wine fermentations. In this study, we characterized the hybrid genomes and the relatedness of the Eg8 industrial yeast strain and of 24 Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrid yeast strains used for wine making in France (Alsace), Germany, Hungary, and the United States. An array-based comparative genome hybridization (aCGH) profile of the Eg8 genome revealed a typical chimeric profile. Measurement of hybrids DNA content per cell by flow cytometry revealed multiple ploidy levels (2n, 3n, or 4n), and restriction fragment length polymorphism analysis of 22 genes indicated variable amounts of S. kudriavzevii genetic content in three representative strains. We developed microsatellite markers for S. kudriavzevii and used them to analyze the diversity of a population isolated from oaks in Ardèche (France). This analysis revealed new insights into the diversity of this species. We then analyzed the diversity of the wine hybrids for 12 S. cerevisiae and 7 S. kudriavzevii microsatellite loci and found that these strains are the products of multiple hybridization events between several S. cerevisiae wine yeast isolates and various S. kudriavzevii strains. The Eg8 lineage appeared remarkable, since it harbors strains found over a wide geographic area, and the interstrain divergence measured with a (??)(2) genetic distance indicates an ancient origin. These findings reflect the specific adaptations made by S. cerevisiae/S. kudriavzevii cryophilic hybrids to winery environments in cool climates. PMID:22344648

Erny, C; Raoult, P; Alais, A; Butterlin, G; Delobel, P; Matei-Radoi, F; Casaregola, S; Legras, J L



Systems Biology of Saccharomyces cerevisiae Physiology and its DNA Damage Response  

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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 cell growth. In order to study this relationship, we have grown S. cerevisiae cells in chemostat at defined growth rates and measured the transcriptional response. We have applied a complex experimental design, involving three factors: specific growth rate, oxygen availability and nutrient limitation. We have identified 268 growth rate dependent genes. These genes were used to identify key areas of the metabolism around which expression changes were significantly associated and we found nucleotide synthesis and ATP producing and consuming reactions. Moreover, by scoring the significance of overlap between growth rate dependent genes and known transcription factor (TF) target sets, we identified 13 TFs, involved in stress response, cell cycle and ribosome biogenesis, that appeared to coordinate the response at increasing growth rates. Therefore, in this study we have identified a more conservative set of growth dependent genes by using a multi-factorial experimental design. Moreover, new insights into the metabolic response and transcriptional regulation of these genes have been provided by using systems biology tools (Chapter 3). One of the prerequisite of systems biology should be the standardization and reproducibility of experimental and analytical techniques, in order to allow the comparison of data generated in different laboratories. With the aim of addressing this aspect, we have collaborated in a large study involving ten laboratories, constituting the Yeast Systems Biology Network (YSBN). S. cerevisiae cultivations were performed in a single laboratory and samples were sent to the other partners. The experimental design involved two factors: strain (CEN.PK113-7D and YSBN2) and growth condition (batch and chemostat). Transcriptome was measured with four different platforms (Affymetrix, Agilent, qPCR and TRAC), metabolome was analyzed in seven laboratories, using different protocols, and enzyme activities were determined in two different laboratories. The comparison of the analyses showed that reproducibility of the results was affected by the laboratory and the protocol used. Transcription and enzyme activity analyses gave consistent results, while metabolite level measurements showed some variability. Therefore, even though the source of biomass was unique, the reproducibility of data appeared to be a challenging task. Nevertheless, we were able to perform an integrative analysis and discover that the lower biomass yield of CEN.PK113-7D was due to higher protein turnover than YSBN2; this finding would not be achievable using a single omics dataset. Moreover, the generated datasets are a valuable resource for the yeast systems biology community (Chapter 4). Upon DNA damage, S. cerevisiae cells respond activating the so-called cell cycle checkpoints that promote damage repair and viability. The activation of these checkpoints depends on kinase cascades and regulation of transcription is one of the responses elicited by checkpoint activation. Therefore, we have decided to investigate the transcriptional and phenotypic responses to the alkylating agent methyl methanesulfonate (MMS) of mutant strains carrying deletions of genes encoding protein kinases (Mec1, Tel1, Rad53, Dun1, Chk1, Alk1) and protein phosphatases (Ptc3, Pph3, Oca1) involved in DNA damage response (DDR). We have discovered a prominent role for Rad53, Mec1 and Tel1 in transcriptional response. Moreover, we have shown for the first time the important role of O

Fazio, Alessandro



Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation. (United States)

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. PMID:23329062

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




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Full Text Available Fermentation products are indigenous to many civilizations, and they have been produced by industriessince a long time. Saccharomyces cerevisiae S288C (commonly known as baker's yeast is the strain mainly usedin the Glucose based fermentation industries. We have seen the use of same yeast strain at different places withdifferent Phenotypic Constraints. The way to improve the adaptability of considered strain for desired phenotypicconditions, using smart selection of genes through cybernetic modeling is illustrated. Phylogenetic homologues forall S. cerevisiae S288c Glucose Fermentation pathway genes were screened to search evolutionarily relatedfunctional domains in other yeast strains like Saccharomyces cerevisiae YJM789, Candida glabrata CBS138,Kluyveromyces lactis NRRL Y-1140, Ashbya gossypii ATCC10895 etc., which are adapted naturally in different setof environment. We observed that Saccharomyces cerevisiae YJM789, Candida glabrata CBS138, Ashbyagossypii ATCC10895, Kluyveromyces lactis NRRL Y-1140 possess highly conserved functional domains, whichcan be carefully selected based on usage. This study aims at designing an algorithm to select and incorporateevolutionary homologues for genes of a considered strain, which mostly show sub-optimal performance in thedesired set of experimental constraints. Such a consideration of native microenvironment and evolutionarycloseness in the selection of functional homologues of the entire genetic set can thus be significantly fruitful.




Biotransformation of Carmoisine and Reactive Black 5 Dyes Using Saccharomyces cerevisiae  

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Full Text Available Saccharomyces cerevisiae (baker’s yeast is the most important industrial microorganisms. This yeast is commonly used as a leavening agent in baking bread and bakery products, where it produces carbon dioxide from converting of the fermentable sugars present in the dough. Nowadays, industrial and chemical activities led to produce new compounds with new kinds of contamination in the environment. Discharge of untreated or partially treated industrial sewage has created the contamination problems of rivers and lakes such as drugs, oil, heavy metals, paints, pesticides and various chemical compounds in them. Hence, it is necessary to control and reduce the levels of these compounds in wastewater and bring them to permissible values. This study aims to study the bioconversion potential of commonly available Saccharomyces cerevisiae for the two textile dyes of Carmoisine and Reactive Black 5. Reaction mixtures for biotransformation of dyes included 50 mg/l Carmoisine or 25 mg/l Reactive Black 5 and 1% dried harvested cells of S. cerevisiae (bread’s yeast were tested. Harvested dry and wet yeast were studied for this purpose. The results show that harvested cells of Saccharomyces cerevisiae are able to bioconvert Carmoisine and Reactive Black 5. Reactive Black 5, Carmoisine are degraded by biotransformation 85% and 53% within 24 hours in water at the room temperature.

Abbas Sadeghi



Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase, in Saccharomyces cerevisiae.  


In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated ...

Sekiya-kawasaki, Mariko; Abe, Mitsuhiro; Saka, Ayaka; Watanabe, Daisuke; Kono, Keiko; Minemura-asakawa, Masayo; Ishihara, Satoru; Watanabe, Takahide; Ohya, Yoshikazu



"A comparison between sugar consumption and ethanol production in wort by immobilized Saccharomyces Cerevisiae, Saccharomyces Ludwigii and Saccharomyces Rouxii on Brewer's Spent Grain"  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english The immobilization of Saccharomyces cerevisiae DSM 70424, Saccharomyces ludwigii DSM 3447 and Saccharomyces rouxii DSM 2531 on brewer's spent grain and then ethanol production and sugar consumption of these immobilized yeasts were investigated. The aim of this study was to investigate the abilities [...] of these three immobilized yeasts for producing alcohol for brewing at two temperatures (7 and 12 °C) using two different sugar levels (one at original level supplied in the brewery and one with 2.5% (w/v), added glucose to the wort). Increasing both parameters resulted in higher alcohol production by all the yeasts studied. At 7 °C and with original wort density the ethanol content at the end of fermentation was 2.7% (v/v) for S. cerevisiae, 1.7% for S. ludwigii and 2.0% for S. rouxii. After the addition of 2.5% (w/v) glucose at the same temperature (7 °C), the alcohol production was increased to 4.1, 2.8 and 4.1%, respectively. Similar improvements were observed when the fermentation was carried out at 12 °C with/without the addition of glucose to the wort. However, temperature indicated greater influence on S. ludwigii than did on S. rouxii and S. cerevisiae. The immobilization as carried out in this study impacted both S. ludwigii and S. rouxii in a way that they could consume maltose under certain conditions.

Aniseh, Mohammadi; Seyyed Hadi, Razavi; Seyyed Mohammad, Mousavi; Karamatollah, Rezaei.



Manganese toxicity and Saccharomyces cerevisiae Mam3p, a member of the ACDP (ancient conserved domain protein) family. (United States)

Manganese is an essential, but potentially toxic, trace metal in biological systems. Overexposure to manganese is known to cause neurological deficits in humans, but the pathways that lead to manganese toxicity are largely unknown. We have employed the bakers' yeast Saccharomyces cerevisiae as a model system to identify genes that contribute to manganese-related damage. In a genetic screen for yeast manganese-resistance mutants, we identified S. cerevisiae MAM3 as a gene which, when deleted, would increase cellular tolerance to toxic levels of manganese and also increased the cell's resistance towards cobalt and zinc. By sequence analysis, Mam3p shares strong similarity with the mammalian ACDP (ancient conserved domain protein) family of polypeptides. Mutations in human ACDP1 have been associated with urofacial (Ochoa) syndrome. However, the functions of eukaryotic ACDPs remain unknown. We show here that S. cerevisiae MAM3 encodes an integral membrane protein of the yeast vacuole whose expression levels directly correlate with the degree of manganese toxicity. Surprisingly, Mam3p contributes to manganese toxicity without any obvious changes in vacuolar accumulation of metals. Furthermore, through genetic epistasis studies, we demonstrate that MAM3 operates independently of the well-established manganese-trafficking pathways in yeast, involving the manganese transporters Pmr1p, Smf2p and Pho84p. This is the first report of a eukaryotic ACDP family protein involved in metal homoeostasis. PMID:15498024

Yang, Mei; Jensen, Laran T; Gardner, Allison J; Culotta, Valeria C



Tomato QM-like protein protects Saccharomyces cerevisiae cells against oxidative stress by regulating intracellular proline levels. (United States)

Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants. PMID:16751508

Chen, Changbin; Wanduragala, Srimevan; Becker, Donald F; Dickman, Martin B



Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae  

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Full Text Available Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b fourth intron] and aI4 [COX1 (cytochrome c oxidase subunit 1 fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA. bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p and mutations in one of the PPR (pentatricopeptide repeat motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.

J. Ignacio Moreno



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

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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.

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



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


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 a...

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



Toxicity and biosorption of metals by saccharomyces cerevisiae, amorphotheca resinae and azolla filiculoides  


The value of H+ efflux in assessing and understanding metal interactions with Saccharomyces cerevisiae was investigated for its potential use as a rapid means of toxicity assessment for a range of metals Toxicity decreased in the order Cu2+ > Cd2+ > Pb2+ > Co2+ > Sr2+. Toxic effects can be alleviated by external Ca2+. The effect of Cu2+ and Co2+ on S cerevisiae growth, and the intracellular localisation of Cu2+, were studied in order to gain a better understanding of their toxicity. S cer...

Fogarty, Robert V.



Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae.  


The DNA segments containing the ADR1 gene and a mutant allele, ADR1-5c, have been isolated by complementation of function in Saccharomyces cerevisiae. The ADR1 gene is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when S. cerevisiae cells are grown on a nonfermentable carbon source, whereas the ADR1-5c allele allows ADHII synthesis even during glucose repression. A plasmid pool consisting of yeast DNA fragments isolated from a strain carrying the ADR1-5c alle...

Denis, C. L.; Young, E. T.



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

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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.)

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



Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds  


An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second...

Sanseverino, John; Gupta, Rakesh K.; Layton, Alice C.; Patterson, Stacey S.; Ripp, Steven A.; Saidak, Leslie; Simpson, Michael L.; Schultz, T. Wayne; Sayler, Gary S.



Saccharomyces cerevisiae BLYAS, a New Bioluminescent Bioreporter for Detection of Androgenic Compounds?  


A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes requ...

Eldridge, Melanie L.; Sanseverino, John; Layton, Alice C.; Easter, James P.; Schultz, T. Wayne; Sayler, Gary S.



The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.  


The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are...

Racenis, P. V.; Lai, J. L.; Das, A. K.; Mullick, P. C.; Hajra, A. K.; Greenberg, M. L.



A short region from the LEU2 gene of Saccharomyces cerevisiae functions as an ARS in the yeast Saccharomyces exiguus Yp74L-3. (United States)

We examined the autonomously replicating sequence (ARS) activity of some fragments derived from the LEU2 region of Saccharomyces cerevisiae onto Saccharomyces exiguus Yp74L-3. A DNA fragment functioning as an ARS in S. exiguus, but not in S. cerevisiae, was shown to exist. The ARS activity for S. exiguus was reduced by the 2-microm plasmid origin of S. cerevisiae when both elements coexisted on a single circular plasmid. Analysis of ARS activity with the PCR products from the fragment revealed that the ARS-acting sequence was located in the 3'-terminal area of the transcribed region of the LEU2 gene of S. cerevisiae. It is suggested that the ARS recognition system in S. exiguus is significantly different from that of S. cerevisiae. PMID:9806982

Hisatomi, T; Wada, Y; Fujisaki, C; Tsuboi, M



O emprego de fermento de pão, Saccharomyces cerevisiae, na síntese de feromônios / Baker's yeast, Saccharomyces cerevisiae, as a tool for the synthesis of pheromones  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese [...] Abstract in english The use of pheromones in integrated pest management has been increasing in the last years due to environmental concern. This development is accompanied by the search for simple, efficient and less aggressive synthetic methodologies for the preparation of pheromones. One of these methodologies includ [...] es microbiological reactions, more specifically biocatalytic reduction of carbonyl compounds using baker's yeast (Saccharomyces cerevisiae). This review presents the use of baker's yeast as an easy and cheap alternative to obtain enantiomerically enriched compounds employed in the synthesis of pheromones.

Patrícia T., Baraldi; Arlene G., Corrêa.



Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine  


Spermidine and its derivative, hypusinated eIF5A, are essential for the growth of Saccharomyces cerevisiae. Very low concentrations of spermidine (10?8 M) are sufficient for the growth of S. cerevisiae polyamine auxotrophs (spe1?, spe2?, and spe3?). Under these conditions, even though the growth rate is near normal, the internal concentration of spermidine is

Chattopadhyay, Manas K.; Park, Myung Hee; Tabor, Herbert



Integrated phospholipidomics and transcriptomics analysis of Saccharomyces cerevisiae with enhanced tolerance to a mixture of acetic acid, furfural, and phenol (United States)

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...


Digestibility and nutrient intake in Mangalarga Marchador mares supplemented with Saccharomyces cerevisiae during aerobic training  

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Full Text Available The study evaluated the effect of yeast supplementation on the digestibility and intake of nutrients of Mangalarga Marchador horses in training. Fourteen Mangalarga Marchador mares were divided into two groups: Probiotic (horses supplemented with 20 g of Saccharomyces cerevisiae daily and Control. The diet consisted of commercial concentrate and roughage in the ratio of 50:50. The mares were trained for six weeks, Monday to Saturday, and the exercise performed daily alternating work on a treadmill and automatic walker. Nutrient digestibility was assessed using the indicator LIPE® (6 days end fecal collection was performed for five days. Was analyzed DM, NDF, ADF, CP, GE, hemicelluloses and dry matter intake. There was not difference (P>0,05 in any of the variables analyzed. Supplementation with 20 g of Saccharomyces cerevisiae does not affect the digestibility and nutrient intake in mares Mangalarga Marchador submitted six weeks of aerobic training.

Tiago Resende Garcia



Effects of altered 5'-flanking sequences on the in vivo expression of a Saccharomyces cerevisiae tRNATyr gene.  


Deletion mutations ending in the 5'-flanking sequences of the Saccharomyces cerevisiae SUP4-o gene have been analyzed for their effects on gene expression. This ochre-suppressing tRNATyr gene was cloned into a S. cerevisiae centromeric plasmid, and its level of in vivo expression was monitored by observing the suppressor phenotype of the gene after transformation into S. cerevisiae. A deletion mutant that retains only four base pairs of the 5'-flanking sequence is profoundly deficient in expr...

Shaw, K. J.; Olson, M. V.



Stimulation of Strontium Accumulation in Linoleate-Enriched Saccharomyces cerevisiae Is a Result of Reduced Sr2+ Efflux  


The influence of modified plasma membrane fatty acid composition on cellular strontium accumulation in Saccharomyces cerevisiae was investigated. Growth of S. cerevisiae in the presence of 1 mM linoleate (18:2) (which results in 18:2 incorporation to ?70% of total cellular and plasma membrane fatty acids, with no effect on growth rate) yielded cells that accumulated Sr2+ intracellularly at approximately twice the rate of S. cerevisiae grown without a fatty acid supplement. This effect was e...

Avery, Simon V.; Smith, Shareeka L.; Ghazi, A. Mohamad; Hoptroff, Michael J.



Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels  


Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from...

Chen, Changbin; Wanduragala, Srimevan; Becker, Donald F.; Dickman, Martin B.



Activation of signaling pathways related to cell wall integrity and multidrug resistance by organic solvent in Saccharomyces cerevisiae. (United States)

Organic solvents are toxic to living cells. In eukaryotes, cells with organic solvent tolerance have only been found in Saccharomyces cerevisiae. Although several factors contributing to organic solvent tolerance have been identified in previous studies, the mechanism of how yeast cells naturally respond to organic solvent stress is not known. We demonstrated that the pleiotropic drug resistance (PDR) pathway contributed to response to organic solvent stress. Activation of the PDR pathway by mutations in the transcription factors Pdr1p and Pdr3p led to organic solvent tolerance. Exposure to organic solvents also induced transcription levels of PDR5, which encodes a major drug efflux pump. Overproduction of Pdr5p improved organic solvent tolerance, presumably by exporting organic solvents out of the cell. In addition, we showed that the cell wall integrity (CWI) pathway was induced in response to organic solvents to upregulate genes encoding the cell wall-related proteins Wsc3p and Ynl190wp. WSC3 and YNL190W were upregulated independently of the PDR pathway. Among the components of the CWI pathway, the cell surface sensors (Wsc3p and Mid2p) and the transcription factors (Swi4p and Swi6p) appeared to be particularly involved in the response to organic solvents. Our findings indicate that S. cerevisiae activates two different signaling pathways, the PDR pathway and the CWI pathway, to cope with stresses from organic solvents. PMID:24378717

Nishida, Nao; Jing, Dongyu; Kuroda, Kouichi; Ueda, Mitsuyoshi



Gene expression profiles and intracellular contents of stress protectants in Saccharomyces cerevisiae under ethanol and sorbitol stresses. (United States)

In response to osmotic stress, proline is accumulated in many bacterial and plant cells. During various stresses, the yeast Saccharomyces cerevisiae induces glycerol or trehalose synthesis, but the fluctuations in gene expression and intracellular levels of proline in yeast are not yet well understood. We previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. In this study, we examined the relationships between the gene expression profiles and intracellular contents of glycerol, trehalose, and proline under stress conditions. When yeast cells were exposed to 1 M sorbitol stress, the expression of GPD1 encoding glycerol-3-phosphate dehydrogenase is induced, leading to glycerol accumulation. In contrast, in the presence of 9% ethanol, the rapid induction of TPS2 encoding trehalose-6-phosphate phosphatase resulted in trehalose accumulation. We found that intracellular proline levels did not increase immediately after addition of sorbitol or ethanol. However, the expressions of genes involved in proline synthesis and degradation did not change during exposure to these stresses. It appears that the elevated proline levels are due primarily to an increase in proline uptake from a nutrient medium caused by the induction of PUT4. These results suggest that S. cerevisiae cells do not accumulate proline in response to sorbitol or ethanol stress different from other organisms. PMID:18351334

Kaino, Tomohiro; Takagi, Hiroshi



Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose. (United States)

The gene mel1, encoding alpha-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and beta-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanol-producing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular alpha-galactosidase and beta-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC 1 co-expressing 2 genes could achieve 0.29 OD600 h(-1) and a biomass yield up to 7.8 g l(-1) dry cell weight on medium containing 10.0 g l(-1) cellobiose and 10.0 g l(-1) melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG 1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l(-1) ethanol was produced from 100 g of cellulose supplied with 5 g l(-1) melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield. PMID:22586919

Zhang, L; Guo, Z P; Ding, Z Y; Wang, Z X; Shi, G Y



[Regularities of the induction of point mutation in the yeast Saccharomyces cerevisiae after exposure to gamma-radiation]. (United States)

A tester system consisting of six isogenic strains was used to study the regularities of the induction of point mutations in the yeast Saccharomyces cerevisiae after exposure to gamma-radiation. This system allowed the identification of all six possible substitutions in the Cys-22 codon of the CYC1 gene encoding iso-1-cytochrome c. The dose dependence of the frequency of these six base-pair substitutions was shown to be linear-quadratic. The pattern of base substitutions did not depend on the doses of gamma-irradiation used (from 125 to 1000 Gy) and predominantly included GC-->AT transitions and AT-->TA transversions. The possible mechanisms of gamma-ray mutagenesis leading to a nonlinear dose dependence were considered, and the spectra of mutations obtained for different yeast genes and for Escherichia coli were compared. PMID:9879010

Liubimova, K A; Anikin, S A; Koltovaia, N A; Krasavin, E A



A novel member of the split betaalphabeta fold: Solution structure of the hypothetical protein YML108W from Saccharomyces cerevisiae  

International Nuclear Information System (INIS)

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 open reading frame 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 b-sheet with strand order 2143 and two a-helices, with an overall topology of bbabba. Strand b1 runs parallel to b4, and b2:b1 and b4:b3 pairs are arranged in an antiparallel fashion. While this fold belongs to the split bab family, it appears to be unique among this family; it is a novel arrangement of secondary structure, thereby expanding the universe of protein folds


Ethanol fermentation of a diluted molasses medium by Saccharomyces cerevisiae immobilized on chrysotile  


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 wit...

Monte Alegre Ranulfo; Rigo Maurício; Joekes Inés



Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.  


Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabo...

Ma, H.; Botstein, D.



Processing Body and Stress Granule Assembly Occur by Independent and Differentially Regulated Pathways in Saccharomyces cerevisiae  


A variety of ribonucleoprotein (RNP) granules form in eukaryotic cells to regulate the translation, decay, and localization of the encapsulated messenger RNA (mRNAs). The work here examined the assembly and function of two highly conserved RNP structures, the processing body (P body) and the stress granule, in the yeast Saccharomyces cerevisiae. These granules are induced by similar stress conditions and contain translationally repressed mRNAs and a partially overlapping set of protein consti...

Shah, Khyati H.; Zhang, Bo; Ramachandran, Vidhya; Herman, Paul K.



Nutritional regulation of late meiotic events in Saccharomyces cerevisiae through a pathway distinct from initiation.  


The IME1 gene is essential for initiation of meiosis in the yeast Saccharomyces cerevisiae, although it is not required for growth. Here we report that in stationary-phase cultures containing low concentration of glucose, cells overexpressing IME1 undergo the early meiotic events, including DNA replication, commitment to recombination, and synaptonemal complex formation and dissolution. In contrast, later meiotic events, such as chromosome segregation, commitment to meiosis, and spore formati...

Lee, R. H.; Honigberg, S. M.



Selenite-induced cell death in Saccharomyces cerevisiae: protective role of glutaredoxins  


Unlike in higher organisms, selenium is not essential for growth in Saccharomyces cerevisiae. In this species, it causes toxic effects at high concentrations. In the present study, we show that when supplied as selenite to yeast cultures growing under fermentative metabolism, its effects can be dissected into two death phases. From the time of initial treatment, it causes loss of membrane integrity and genotoxicity. Both effects occur at higher levels in mutants lacking Grx1p and Grx2p than i...

Izquierdo A?lvarez, Alicia; Casas Herranz, Celia; Herrero Perpin?a?n, Enrique



Growth-rate-dependent regulation of the expression and inactivation of thymidylate synthase in Saccharomyces cerevisiae.  


Thymidylate synthase activity fluctuated dramatically as cultures of Saccharomyces cerevisiae progressed through the different stages of batch culture growth. During logarithmic growth these yeast cultures each contained about 40 microU (1 microU is 1 pmol of 3H released per min) of thymidylate synthase activity per 10(8) haploid cells, but as cultures entered the stationary phase and during the stationary phase, activity dropped dramatically, eventually reaching undetectable levels. Stimulat...

Greenwood, M. T.; Calmels, E. M.; Storms, R. K.



p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.  


CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the functi...

Sikorski, R. S.; Michaud, W. A.; Hieter, P.



Expression and partial purification of enzymatically active recombinant Ty1 integrase in Saccharomyces cerevisiae.  


Integration of the Saccharomyces cerevisiae retrotransposon Ty1 into the genome requires Ty1 integrase (IN). Apparent functions of Ty1 IN are target-site determination, cleavage, and joining of donor strands. To further study the mechanism of Ty1 integration, an IN expression plasmid has been constructed for use in yeast. The recombinant IN coding sequence differs from mature Ty1 IN associated with Ty1 virus-like particles only in that it has several additional N-terminal am...

Moore, S. P.; Garfinkel, D. J.



Monoubiquitination Is Sufficient To Signal Internalization of the Maltose Transporter in Saccharomyces cerevisiae  


Monoubiquitination of the 12-transmembrane segment (12-TMS) Saccharomyces cerevisiae maltose transporter promoted the maximal internalization rate of this protein. This modification is similar to that of the 7-TMS ?-factor receptor but different from that of the 12-TMS uracil and general amino acid permeases. This result shows that binding of ubiquitin-Lys63 chains is not required for maximal internalization of all 12-TMS-containing proteins.

Lucero, Pilar; Pen?alver, E?lida; Vela, Laura; Lagunas, Rosario



Purification and properties of saccharopine dehydrogenase (glutamate forming) in the Saccharomyces cerevisiae lysine biosynthetic pathway.  


Saccharopine dehydrogenase (glutamate forming) of the biosynthetic pathway of lysine in Saccharomyces cerevisiae was purified 1,122-fold by using acid precipitation, ammonium sulfate precipitation, DEAE-Sepharose, gel filtration, and Reactive Red-120 agarose chromatography. The enzyme exhibited a native molecular size of 69,000 daltons by gel filtration and consisted of a single 50,000-dalton polypeptide based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was read...

Storts, D. R.; Bhattacharjee, J. K.



A glucose transporter chimera confers a dominant negative glucose starvation phenotype in Saccharomyces cerevisiae.  


A family of glucose transporters mediates glucose uptake in Saccharomyces cerevisiae. We show that the dominant mutation GSF4-1, which impairs glucose repression of SUC2, results in a nonfunctional chimera of the transporters Hxt1p and Hxt4p. Hxt1/4p inhibits the function of wild-type glucose transporters. Similar mutations may facilitate analysis of the major facilitator superfamily.

Sherwood, P. W.; Katic, I.; Sanz, P.; Carlson, M.



Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae  


The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here ...

Elbing, Karin; Larsson, Christer; Bill, Roslyn M.; Albers, Eva; Snoep, Jacky L.; Boles, Eckhard; Hohmann, Stefan; Gustafsson, Lena



Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae.  


Functional TATA elements in the 5' untranslated region of the CYC1 gene in the yeast Saccharomyces cerevisiae have been defined by transcriptional analysis of site-directed mutations. Five sites previously suggested to contain functional TATA elements were altered individually and in all possible combinations. The results indicated that only two elements are required for transcription at the normal level and the normal start sites. The two functional TATA elements are located at sites -178 an...

Li, W. Z.; Sherman, F.



Analysis of Plasmid Deletion Induced by Ionizing Radiation in Yeast Saccharomyces cerevisiae  

International Nuclear Information System (INIS)

The article is dedicated to the research of plasmid system YCpL2 with help of quantitative analysis of deletion formation. The cells of yeast Saccharomyces cerevisiae were irradiated by ?-ray with the flux of 0.7 Gy/min and energy of 1.3 MeV as well as heavy ion beam 11B with energy 32 MeV/n. The deletion of plasmid DNA has been analyzed by genetic and restriction analysis


Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate?recombination  


Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination. Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler DNA. We showed previously that T-DNA can also be transferred from A. tumefaciens to Saccharomyces cerevisiae and integrates into the yeast genome via ...

Bundock, Paul; Hooykaas, Paul?J ?J



Pds1p is required for faithful execution of anaphase in the yeast, Saccharomyces cerevisiae  


To identify mutations that cause defects in mitosis, a collection of mutants in Saccharomyces cerevisiae was screened by a rapid visual assay for abnormal chromosome segregation. From this screen we identified one mutation, pds1-1 that was independently identified in an alternative screen for mutants that exhibit inviability after transient exposure to nocodazole and precocious disassociation of sister chromatids (Guacci, V., A. Yamamoto, A. Strunnikov, J. Kingsbury, E. Hogan, P. Meluh, and D...



Multiple Roles of the Cox20 Chaperone in Assembly of Saccharomyces cerevisiae Cytochrome c Oxidase  


The Cox2 subunit of Saccharomyces cerevisiae cytochrome c oxidase is synthesized in the mitochondrial matrix as a precursor whose leader peptide is rapidly processed by the inner membrane protease following translocation to the intermembrane space. Processing is chaperoned by Cox20, an integral inner membrane protein whose hydrophilic domains are located in the intermembrane space, and Cox20 remains associated with mature, unassembled Cox2. The Cox2 C-tail domain is exported post-translationa...

Elliott, Leah E.; Saracco, Scott A.; Fox, Thomas D.



Genes required to maintain telomeres in the absence of telomerase in Saccharomyces cerevisiae  


In the absence of telomerase, Saccharomyces cerevisiae telomeres erode leading to senescence. Rare cells can survive after this stage as they can elongate their telomeres utilizing homologous recombination. Two different types of survivors can be easily distinguished by Southern blot. Type I survivor cells, elongate the telomere by amplifying Y elements and require RAD51, RAD54, RAD55 and RAD57 for establishment. Type II survivors elongate their telomere by amplifying TG1-3 repeats, however, ...

Alotaibi, Mohammad Kdaimes H.



Cloning and Expression of a Schwanniomyces occidentalis ?-Amylase Gene in Saccharomyces cerevisiae  


An ?-amylase gene (AMY) was cloned from Schwanniomyces occidentalis CCRC 21164 into Saccharomyces cerevisiae AH22 by inserting Sau3AI-generated DNA fragments into the BamHI site of YEp16. The 5-kilobase insert was shown to direct the synthesis of ?-amylase. After subclones containing various lengths of restricted fragments were screened, a 3.4-kilobase fragment of the donor strain DNA was found to be sufficient for ?-amylase synthesis. The concentration of ?-amylase in culture broth produ...

Wang, Tsung Tsan; Lin, Long Liu; Hsu, Wen Hwei



In Vivo Analysis of the Mechanisms for Oxidation of Cytosolic NADH by Saccharomyces cerevisiae Mitochondria  


During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In Saccharomyces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance o...

Overkamp, K. M.; Bakker, B. M.; Kotter, P.; Tuijl, A.; Vries, S.; Dijken, J. P.; Pronk, J. T.



Isoproturon induce Saccharomyces cerevisiae UE-ME3 proliferation in glucose starvation conditions.  


Isoproturon (IPU), an herbicide used in winter crops, often persists in soils and aquifers at levels considered toxic by European legislation. Whereas it may be involved in triggering of serious illnesses, it’s urgent to find microorganisms that could contribute to its elimination. The main purpose of this study was to evaluate the effect of IPU on the antioxidant response of the wine-wild Saccharomyces cerevisiae UE-ME3 from Alentejo, Portugal, that present great resistant to vanadium and ...

Candeias, M.; Alves-pereira, I.; Ferreira, Rui



Robust industrial Saccharomyces cerevisiae strains for very high gravity bio-ethanol fermentations  


The application and physiological background of two industrial Saccharomyces cerevisiae strains, isolated from harsh industrial environments, were studied in Very High Gravity (VHG) bio-ethanol fermentations. VHG laboratory fermentations, mimicking industrially relevant conditions, were performed with PE-2 and CA1185 industrial strains and the CEN.PK113-7D laboratory strain. The industrial isolates produced remarkable high ethanol titres (>19%, v/v) and accumulated an increased content of ste...

Pereira, Francisco B.; Guimara?es, Pedro M. R.; Teixeira, J. A.; Domingues, Luci?lia



Different Expression Systems for Production of Recombinant Proteins in Saccharomyces cerevisiae  


Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and ?-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a ...

Liu, Zihe; Tyo, Keith E. J.; Marti?nez, Jose? L.; Petranovic, Dina; Nielsen, Jens



Translational Accuracy during Exponential, Postdiauxic, and Stationary Growth Phases in Saccharomyces cerevisiae  


When the yeast Saccharomyces cerevisiae shifts from rapid growth on glucose to slow growth on ethanol, it undergoes profound changes in cellular metabolism, including the destruction of most of the translational machinery. We have examined the effect of this metabolic change, termed the diauxic shift, on the frequency of translational errors. Recoding sites are mRNA sequences that increase the frequency of translational errors, providing a convenient reporter of translational accuracy. We fou...

Stahl, Guillaume; Salem, Samia N. Ben; Chen, Lifeng; Zhao, Bing; Farabaugh, Philip J.



Glucose uptake and catabolite repression in dominant HTR1 mutants of Saccharomyces cerevisiae.  


Growth and carbon metabolism in triosephosphate isomerase (delta tpi1) mutants of Saccharomyces cerevisiae are severely inhibited by glucose. By using this feature, we selected for secondary site revertants on glucose. We defined five complementation groups, some of which have previously been identified as glucose repression mutants. The predominant mutant type, HTR1 (hexose transport regulation), is dominant and causes various glucose-specific metabolic and regulatory defects in TPI1 wild-ty...

Ozcan, S.; Freidel, K.; Leuker, A.; Ciriacy, M.



The DNA repair capability of cdc9, the saccharomyces cerevisiae mutant defective in DNA ligase  

International Nuclear Information System (INIS)

The cell cycle mutant, cdc9, in the yeast Saccharomyces cerevisiae is defective in DNA ligase with the consequence to be deficient in the repair of DNA damaged by methyl methane sulphonate. On the other hand survival of cdc9 after irradiation by ?-rays is little different from that of the wild-type, even after a period of stress at the restrictive temperature. The mutant cdc9 is not allelic with any known rad or mms mutants. (orig./AJ)


Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae  


Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur. Mol. Biol. Organ.) J. 4:1861-1866). Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed. The internal pH of the intermediate compartment under energy depletion was determined by fl...



Nonhomologous Synapsis and Reduced Crossing over in a Heterozygous Paracentric Inversion in Saccharomyces Cerevisiae  


Homologous chromosome synapsis (``homosynapsis'') and crossing over are well-conserved aspects of meiotic chromosome behavior. The long-standing assumption that these two processes are causally related has been challenged recently by observations in Saccharomyces cerevisiae of significant levels of crossing over (1) between small sequences at nonhomologous locations and (2) in mutants where synapsis is abnormal or absent. In order to avoid problems of local sequence effects and of mutation pl...

Dresser, M. E.; Ewing, D. J.; Harwell, S. N.; Coody, D.; Conrad, M. N.



Activation of cycasin to a mutagen for Saccharomyces cerevisiae by rat intestinal flora.  


Genetic test systems involving microorganisms and liver enzyme preparations may be insufficient to detect compounds that require breakdown by enzymes provided by the microbial flora of the intestinal tract. A method is described for providing such activation and for simultaneously testing the potential genetic activity of breakdown products in an indicator organism. Parabiotic chambers containing Saccharomyces cerevisiae genetic test organisms in one chamber were separated by a membrane filte...

Mayer, V. W.; Goin, C. J.



Improvement of Phytase Activity by a New Saccharomyces cerevisiae Strain Using Statistical Optimization  


Using statistical optimization, we enhanced the activity of phytase by a new Saccharomyces cerevisiae strain cultured in mineral medium. Concentrations of carbon source and inducer of phytase production were optimized using a 22 full factorial CCD and response surface methodology (RSM). Urea was fixed as nitrogen source in culture medium (0.15%, w/v). The culture medium consisting of 2.5% sucrose and 0.5% sodium phytate optimally supported the maximum phytase activity. In addition, we found t...

Ries, Edi Franciele; Alves Macedo, Gabriela



High-cell-density fermentation of Saccharomyces cerevisiae for the optimisation of mead production  


Mead is a traditional drink that contains 8 % and 18 % (v/v) of ethanol, resulting from the alcoholic fermentation of diluted honey by yeasts. Mead fermentation is a time-consuming process and the quality of the final product is highly variable. Therefore, the present investigation had two main objectives: first, to determine the adequate inoculum size of two commercial wine-making strains of Saccharomyces cerevisiae for the optimisation of mead fermentation; and second, to determine if an in...

Pereira, A. P.; Ferreira, A. Mendes; Oliveira, J. M.; Estevinho, L. M.; Faia, A. Mendes



Biosynthesis of phosphoinositol-containing sphingolipids from phosphatidylinositol by a membrane preparation from Saccharomyces cerevisiae.  


Incubation of membranes prepared from Saccharomyces cerevisiae with [32P]phosphatidyl[3H]inositol resulted in the transfer of both labels to two products which were characterized as two species of inositolphosphoceramide, differing in the ceramide portion of the molecule. The products were characterized on the basis of stability in mild alkali, mobility on silica gel-impregnated paper, chromatography on silicic acid columns, and release of inositol phosphate upon base hydrolysis. The reaction...

Becker, G. W.; Lester, R. L.



Continuous ethanol fermentation of lactose by a recombinant flocculating saccharomyces cerevisiae strain  


Alcohol fermentation of lactose was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for b-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus. Data on yeast fermentation and growth on a medium containing lactose as the sole carbon source are presented. In the range of studied lactose concentrations, total lactose consumption was observed with a conversion yield of ethanol close to t...

Domingues, Luci?lia; Dantas, Maria M.; Lima, Nelson; Teixeira, J. A.



Anomalies in the transcriptional regulatory network of the yeast Saccharomyces cerevisiae  


We investigate the structural and dynamical properties of the transcriptional regulatory network of the yeast {\\it Saccharomyces cerevisiae} and compare it with two unbiased ensembles: one obtained by reshuffling the edges and the other generated by mimicking the transcriptional regulation mechanism within the cell. Both ensembles reproduce the degree distributions (the first -by construction- exactly and the second approximately), degree-degree correlations and the $k$-core...

Tugrul, Murat; Kabakcioglu, Alkan



Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains  


Incorporation of strain-specific synthetic DNA tags into yeast Saccharomyces cerevisiae gene-deletion strains has enabled identification of gene functions by massively parallel growth rate analysis. However, it is important to confirm the sequences of these tags, because mutations introduced during construction could lead to significant errors in hybridization performance. To validate this experimental system, we sequenced 11,812 synthetic 20-mer molecular bar codes and adjacent sequences (>1...

Eason, Robert G.; Pourmand, Nader; Tongprasit, Waraporn; Herman, Zelek S.; Anthony, Kevin; Jejelowo, Olufisayo; Davis, Ronald W.; Stolc, Viktor



Hybridization and Polyploidization of Saccharomyces cerevisiae Strains by Transformation-Associated Cell Fusion  


Hybrid or polyploid clones of Saccharomyces cerevisiae produced by protoplast fusion were easily isolated by selecting transformants with the plasmid phenotype because the transformation was directly associated with cell fusion. When haploid cells were used as the original strain, the transformants were mostly diploids with a significant fraction of polyploids (triploids or tetraploids). Repeated transformation after curing the plasmid gave rise to clones with higher ploidy, but the frequency...

Takagi, Atsuko; Harashima, Satoshi; Oshima, Yasuji



Gcn4 Is Required for the Response to Peroxide Stress in the Yeast Saccharomyces cerevisiae  


An oxidative stress occurs when reactive oxygen species overwhelm the cellular antioxidant defenses. We have examined the regulation of protein synthesis in Saccharomyces cerevisiae in response to oxidative stress induced by exposure to hydroperoxides (hydrogen peroxide, and cumene hydroperoxide), a thiol oxidant (diamide), and a heavy metal (cadmium). Examination of translational activity indicates that these oxidants inhibit translation at the initiation and postinitiation phases. Inhibitio...

Mascarenhas, Claire; Edwards-ingram, Laura C.; Zeef, Leo; Shenton, Daniel; Ashe, Mark P.; Grant, Chris M.



Global Analyses of Sumoylated Proteins in Saccharomyces cerevisiae: Induction of Protein Sumoylation by Cellular Stresses*  


We have undertaken a global analysis of sumoylated proteins in Saccharomyces cerevisiae by tandem mass spectrometry. Exposure of cells to oxidative and ethanol stresses caused large increases in protein sumoylation. A large number of new sumoylated proteins were identified in untreated, hydrogen peroxide-treated, and ethanol-treated cells. These proteins are known to be involved in diverse cellular processes, including gene transcription, protein translation, DNA replication, chromosome segre...

Zhou, Weidong; Ryan, Jennifer J.; Zhou, Huilin



SO2 protects the amino nitrogen metabolism of Saccharomyces cerevisiae under thermal stress  


Thermal stress conditions during alcoholic fermentation modify yeasts' plasma membrane since they become more hyperfluid, which results in a loss of bilayer integrity. In this study, the influence of elevated temperatures on nitrogen metabolism of a Saccharomyces cerevisiae strain was studied, as well as the effect of different concentrations of SO2 on nitrogen metabolism under thermal stress conditions. The results obtained revealed that amino nitrogen consumption was lower in the fermentati...

Anci?n?azpilicueta, Carmen; Barriuso?esteban, Blanca; Nieto?rojo, Rodrigo; Aristiza?bal?lo?pez, Nerea



Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae  


Through metabolic engineering microorganisms can be engineered to produce new products and further produce these with higher yield and productivities. Here, we expressed the bacterial polyhydroxybutyrate (PHB) pathway in the yeast Saccharomyces cerevisiae and we further evaluated the effect of engineering the formation of acetyl coenzyme A (acetyl-CoA), an intermediate of the central carbon metabolism and precursor of the PHB pathway, on heterologous PHB production by yeast. We engineered the...

Kocharin, Kanokarn; Chen, Yun; Siewers, Verena; Nielsen, Jens



Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast  


Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (?nth1), acid trehalase mutants (?ath1), and double mutants (?nth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disrupti...

Shima, Jun; Hino, Akihiro; Yamada-iyo, Chie; Suzuki, Yasuo; Nakajima, Ryouichi; Watanabe, Hajime; Mori, Katsumi; Takano, Hiroyuki



Population Size Drives Industrial Saccharomyces cerevisiae Alcoholic Fermentation and Is under Genetic Control?†‡  


Alcoholic fermentation (AF) conducted by Saccharomyces cerevisiae has been exploited for millennia in three important human food processes: beer and wine production and bread leavening. Most of the efforts to understand and improve AF have been made separately for each process, with strains that are supposedly well adapted. In this work, we propose a first comparison of yeast AFs in three synthetic media mimicking the dough/wort/grape must found in baking, brewing, and wine making. The fermen...

Albertin, Warren; Marullo, Philippe; Aigle, Michel; Dillmann, Christine; Vienne, Dominique; Bely, Marina; Sicard, Delphine



Evaluation of stress tolerance and fermentative behavior of indigenous Saccharomyces cerevisiae  


Sixty six indigenous Saccharomyces cerevisiae strains were evaluated in stressful conditions (temperature, osmolarity, sulphite and ethanol tolerance) and also ability to flocculate. Eighteen strains showed tolerant characteristics to these stressful conditions, growing at 42 °C, in 0.04% sulphite, 1 mol L?1 NaCl and 12% ethanol. No flocculent characteristics were observed. These strains were evaluated according to their fermentative performance in sugar cane juice. The conversion factors ...

Ramos, Ci?ntia Lacerda; Duarte, Whasley Ferreira; Freire, Ana Luiza; Dias, Disney Ribeiro; Eleutherio, Elis Cristina Arau?jo; Schwan, Rosane Freitas



Cybernetic modeling of spontaneous oscillations in continuous cultures of Saccharomyces cerevisiae. (United States)

We have developed a 'cybernetic' model to simulate the dynamic competition between all the available metabolic pathways of yeast Saccharomyces cerevisiae. This computer model predicts all the key experimentally observed aspects of the sustained oscillations in all the measured concentrations in continuous cultures, such as the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitation rate are changed. PMID:10483111

Kompala, D S



Regulation of Thermotolerance by Stress-Induced Transcription Factors in Saccharomyces cerevisiae?  


The heat shock transcription factor Hsf1 and the general stress transcription factors Msn2 and Msn4 (Msn2/4) are major regulators of the heat shock response in Saccharomyces cerevisiae. Here, we show that transcriptional activation of their target genes, including HSP104, an antistress chaperone gene, is obligatory for thermotolerance. Although Hsf1 activity might be necessary before the exposure of cells to high temperature, severe heat shock induced the binding of hyperphosphorylated Hsf1 t...

Yamamoto, Noritaka; Maeda, Yuka; Ikeda, Aya; Sakurai, Hiroshi



Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae.  


The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxida...

Davidson, J. F.; Whyte, B.; Bissinger, P. H.; Schiestl, R. H.



Dynamic association of transcriptional activation domains and regulatory regions in Saccharomyces cerevisiae heat shock factor  


In Saccharomyces cerevisiae, the heat shock transcription factor (HSF) is thought to be a homotypic trimer that is bound to the promoters of heat shock protein (HSP) genes at both normal and heat shock temperatures. Exposure to heat shock greatly and rapidly induces HSF transcriptional activity without further increasing DNA-binding affinity. It is believed that HSF is under negative regulation at normal growth temperatures, but the detailed mechanism by which HSF is activated is still not cl...

Chen, Tianxin; Parker, Carl S.



Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae.  


The effects of osmoprotectants (such as glycine betaine and proline) and particulate materials on the fermentation of very high concentrations of glucose by the brewing strain Saccharomyces cerevisiae (uvarum) NCYC 1324 were studied. The yeast growing at 20 degrees C consumed only 15 g of the sugar per 100 ml from a minimal medium which initially contained 35% (wt/vol) glucose. Supplementing the medium with a mixture of glycine betaine, glycine, and proline increased the amount of sugar ferme...

Thomas, K. C.; Hynes, S. H.; Ingledew, W. M.



Effects of Furfural on the Respiratory Metabolism of Saccharomyces cerevisiae in Glucose-Limited Chemostats  


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...

Sarvari Horvath, I.; Franze?n, C. J.; Taherzadeh, M. J.; Niklasson, C.; Lide?n, Gunnar



Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins  


This study demonstrates mismatch repair (MMR) reactions reconstituted in vitro with purified Saccharomyces cerevisiae proteins. Biochemical analysis of MMR in vitro showed that MMR required mispair binding by the MutS homolog 2–MutS homolog 6 complex and corresponded to the Exonuclease 1-dependent subpathway of MMR. The reactions observed involved the formation of long excision tracts whose length was consistent with the length of MMR-dependent gene conversion tracts in vivo. The availabili...

Bowen, Nikki; Smith, Catherine E.; Srivatsan, Anjana; Willcox, Smaranda; Griffith, Jack D.; Kolodner, Richard D.



Histone H3 and H4 gene deletions in Saccharomyces cerevisiae  


The genome of haploid Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 genes. Strains with deletions of each of these loci were constructed by gene replacement techniques. Mutants containing deletions of either gene set were viable, however meiotic segregants lacking both histone H3 and H4 gene loci were inviable. In haploid cells no phenotypic expression of the histone gene deletions was observed; deletion mutants had wild-type growth rates, were not temperature sen...



Carbon Starvation Can Induce Energy Deprivation and Loss of Fermentative Capacity in Saccharomyces cerevisiae  


Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showe...

Thomsson, Elisabeth; Larsson, Christer; Albers, Eva; Nilsson, Annika; Franze?n, Carl Johan; Gustafsson, Lena



High expression of heterologous proteins by Saccharomyces cerevisiae grown on ethanol  


The production of recombinant proteins is of great importance for industrial applications in fields such as pharmaceutical ingredients and industrial enzymes. One of these products are camelid antibody fragments, produced by Saccharomyces cerevisiae in high cell density fed batch fermentation processes, using glucose as sole carbon source. To improve this production, process optimisation was performed on both biological and technical aspects. First the whole production system was analysed for...

Laar, Antonius Martinus Johannes



Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition.  


One major mechanism of copper toxicity towards microorganisms is disruption of plasma membrane integrity. In this study, the influence of plasma membrane fatty acid composition on the susceptibility of Saccharomyces cerevisiae to Cu2+ toxicity was investigated. Microbial fatty acid composition is highly variable, depending on both intrinsic and environmental factors. Manipulation was achieved in this study by growth in fatty acid-supplemented medium. Whereas cells grown under standard conditi...

Avery, S. V.; Howlett, N. G.; Radice, S.



Sequential injection kinetic flow assay for monitoring glycerol in a sugar fermentation process by saccharomyces cerevisiae  


A sequential injection system to monitor glycerol in a Saccharomyces cerevisiae fermentation process was developed. The method relies on the rate of formation of nicotinamide adenine dinucleotide in its reduced form (NADH, measured spectrophotometrically at 340 nm) from the reaction of glycerol with NAD+ cofactor, catalysed by the enzyme glycerol dehydrogenase present in solution. This procedure enables the determination of glycerol between 0.046 and 0.46 g/l, (corresponding to yeast ferment...

Hueso Domi?nguez, Karina B.; To?th, Ildiko? V.; Souto, M. Renata S.; Mendes, Filipa; Garci?a Mari?a, Ca?ndido; Vasconcelos, Isabel; Rangel, Anto?nio O. S. S.



Haploidy, Diploidy and Evolution of Antifungal Drug Resistance in Saccharomyces cerevisiae  


We tested the hypothesis that the time course of the evolution of antifungal drug resistance depends on the ploidy of the fungus. The experiments were designed to measure the initial response to the selection imposed by the antifungal drug fluconazole up to and including the fixation of the first resistance mutation in populations of Saccharomyces cerevisiae. Under conditions of low drug concentration, mutations in the genes PDR1 and PDR3, which regulate the ABC transporters implicated in res...

Anderson, James B.; Sirjusingh, Caroline; Ricker, Nicole



Efficient fermentation of an improved synthetic grape must by enological and laboratory strains of Saccharomyces cerevisiae  


Grape must or freshly pressed grape juice is a complex chemical matrix that impacts the efficiency of yeast fermentation. The composition of natural grape must (NGM) can be variable; thus, to ensure reproducibility, a synthetic grape must (SGM) with defined composition is commonly used. The aim of this work was to create conditions to advance the use of Saccharomyces cerevisiae laboratory strains for wine fermentation studies, considering previous results obtained for enological strains ferme...

Viana, Tiago; Loureiro-dias, Maria C.; Prista, Catarina



Towards fermentation of galacturonic acid-containing feedstocks with Saccharomyces cerevisiae:  


The ambition to reduce our current dependence on fossil transportation fuels has driven renewed interest in bioethanol. Pectin-rich feedstocks like sugar beet pulp and citrus peel, which are currently sold as cattle feed, are promising raw materials for the production of bioethanol. This thesis explores the challenges related to the fermentation of pectin-rich hydrolysates with Saccharomyces cerevisiae. Galacturonic acid is a major constituent of pectin-rich hydrolysates. Achieving efficient ...

Huisjes, E. H.



A Kinetic Study of the Fermentation of Cane Sugar Using Saccharomyces cerevisiae  


The fermentation of cane sugar as substrate by Saccharomyces cerevisiae (enzyme) was critically investigated to obtain certain useful kinetic parameters and to determine the effect of temperature, pH, substrate and yeast (enzyme) concentration on the rate of fermentation. The results indicate that the rate of fermentation (measured as rate of production of CO2) increased in proportion with temperature (optimum 32°C - 36°C), pH (optimum 5.5) substrate (op...

Egharevba Felix; Ogbebor Clara; Akpoveta Oshevwiyo Vincent



Role of Heat Shock Transcription Factor in Saccharomyces cerevisiae Oxidative Stress Response? †  


The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates the transcription of a set of genes that contain heat shock elements (HSEs) in their promoters and function in diverse cellular processes, including protein folding. Here, we show that Hsf1 activates the transcription of various target genes when cells are treated with oxidizing reagents, including the superoxide anion generators menadione and KO2 and the thiol oxidants diamide and 1-chloro-2,4-dinitroben...

Yamamoto, Ayako; Ueda, Junko; Yamamoto, Noritaka; Hashikawa, Naoya; Sakurai, Hiroshi



Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae.  


We isolated two genes for extracellular beta-glucosidase, BGL1 and BGL2, from the genomic library of the yeast Saccharomycopsis fibuligera. Gene products (BGLI and BGLII) were purified from the culture fluids of Saccharomyces cerevisiae transformed with BGL1 and BGL2, respectively. Molecular weights of BGLI and BGLII were estimated to be 220,000 and 200,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two beta-glucosidases showed the same enzymatic char...

Machida, M.; Ohtsuki, I.; Fukui, S.; Yamashita, I.



Impact of xylose and mannose on central metabolism of yeast Saccharomyces cerevisiae  


In this study, understanding of the central metabolism was improved by quantification of metabolite concentrations, enzyme activities, protein abundances, and gene transcript concentrations. Intracellular fluxes were estimated by applying stoichiometric models of metabolism. The methods were applied in the study of yeast Saccharomyces cerevisiae in two separate projects. A xylose project aimed at improved utilization of D-xylose as a substrate for, e.g., producing biomaterial-based fuel ethan...

Pitka?nen, Juha-pekka



Simultaneous yet Independent Regulation of Actin Cytoskeletal Organization and Translation Initiation by Glucose in Saccharomyces cerevisiae  


Acute glucose deprivation rapidly but transiently depolarizes the actin cytoskeleton and inhibits translation initiation in Saccharomyces cerevisiae. Neither rapid actin depolarization nor translation inhibition upon glucose removal occurs in a reg1 disruptant, which is defective in glucose repression, or in the tpk1w mutant, which has weak cAPK activity. In the absence of additional glucose, recovery of either actin polarization or translation initiation relies upon respiration, the Snf1p pr...

Uesono, Yukifumi; Ashe, Mark P.; Toh-e, Akio



Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein  


The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms...

Kagawa, Wataru; Arai, Naoto; Ichikawa, Yuichi; Saito, Kengo; Sugiyama, Shusei; Saotome, Mika; Shibata, Takehiko; Kurumizaka, Hitoshi



Diversion of Flux toward Sesquiterpene Production in Saccharomyces cerevisiae by Fusion of Host and Heterologous Enzymes? †  


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 be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the int...

Albertsen, Line; Chen, Yun; Bach, Lars S.; Rattleff, Stig; Maury, Jerome; Brix, Susanne; Nielsen, Jens; Mortensen, Uffe H.



Topological basis of signal integration in the transcriptional-regulatory network of the yeast, Saccharomyces cerevisiae  


BACKGROUND. Signal recognition and information processing is a fundamental cellular function, which in part involves comprehensive transcriptional regulatory (TR) mechanisms carried out in response to complex environmental signals in the context of the cell's own internal state. However, the network topological basis of developing such integrated responses remains poorly understood. RESULTS. By studying the TR network of the yeast Saccharomyces cerevisiae we show that a...

Farkas, Ille?s J.; Wu, Chuang; Chennubhotla, Chakra; Bahar, Ivet; Oltvai, Zolta?n N.



Fructo-Oligosaccharide Synthesis by Mutant Versions of Saccharomyces cerevisiae Invertase ?†  


Efficient enzymatic synthesis of tailor-made prebiotic fructo-oligosaccharides (FOS) used in functional food formulation is a relevant biotechnological objective. We have engineered the Saccharomyces cerevisiae invertase (Suc2) to improve its transferase activity and to identify the enzymatic determinants for product specificity. Amino acid replacement (W19Y, N21S, N24S) within a conserved motif (?-fructosidase) specifically increased the synthesis of 6-kestose up to 10-fold. Mutants with lo...

Lafraya, A?lvaro; Sanz-aparicio, Julia; Polaina, Julio; Mari?n-navarro, Julia



Heterozygous Screen in Saccharomyces cerevisiae Identifies Dosage-Sensitive Genes That Affect Chromosome Stability  


Current techniques for identifying mutations that convey a small increased cancer risk or those that modify cancer risk in carriers of highly penetrant mutations are limited by the statistical power of epidemiologic studies, which require screening of large populations and candidate genes. To identify dosage-sensitive genes that mediate genomic stability, we performed a genomewide screen in Saccharomyces cerevisiae for heterozygous mutations that increase chromosome instability in a checkpoin...

Strome, Erin D.; Wu, Xiaowei; Kimmel, Marek; Plon, Sharon E.



Optimisation of the production of cathepsin L1 from a recombinant saccharomyces cerevisiae  


Cathepsin L1 is a cysteine protease that has been previously isolated and functionally expressed in Saccharomyces cerevisiae. It has the potential to be employed as a vaccine for liver-fluke disease in cattle and other ruminants. Production of this recombinant enzyme, which is secreted into the media from recombinant yeast, was studied initially in shake flask cultures and subsequently in 5L and 15L fermenters. In early studies, low productivity and especially variations in Cathepsin L1 p...

O Donovan, Eimear C.



POB3 is required for both transcription and replication in the yeast Saccharomyces cerevisiae.  


Spt16 and Pob3 form stable heterodimers in Saccharomyces cerevisiae, and homologous proteins have also been purified as complexes from diverse eukaryotes. This conserved factor has been implicated in both transcription and replication and may affect both by altering the characteristics of chromatin. Here we describe the isolation and properties of a set of pob3 mutants and confirm that they have defects in both replication and transcription. Mutation of POB3 caused the Spt(-) phenotype, spt16...

Schlesinger, M. B.; Formosa, T.



Specific Genetic Interactions Between Spindle Assembly Checkpoint Proteins and B-Type Cyclins in Saccharomyces cerevisiae  


The B-type cyclin Clb5 is involved primarily in control of DNA replication in Saccharomyces cerevisiae. We conducted a synthetic genetic array (SGA) analysis, testing for synthetic lethality between the clb5 deletion and a selected 87 deletions related to diverse aspects of cell cycle control based on GO annotations. Deletion of the spindle checkpoint genes BUB1 and BUB3 caused synthetic lethality with clb5. The spindle checkpoint monitors the attachment of spindles to the kinetochore or spin...

Ikui, Amy E.; Cross, Frederick R.



Fractionation of Phenolic Compounds Extracted from Propolis and Their Activity in the Yeast Saccharomyces cerevisiae  


We have here investigated the activities of Slovenian propolis extracts in the yeast Saccharomyces cerevisiae, and identified the phenolic compounds that appear to contribute to these activities. We correlated changes in intracellular oxidation and cellular metabolic energy in these yeasts with the individual fractions of the propolis extracts obtained following solid-phase extraction. The most effective fraction was further investigated according to its phenolic compounds.

Petelinc, Tanja; Polak, Tomaz?; Dems?ar, Lea; Jamnik, Polona



EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae  


Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. Th...

Jensen, Niels Bjerg; Strucko, Tomas; Kildegaard, Kanchana Rueksomtawin; David, Florian; Maury, Jerome; Mortensen, Uffe Hasbro; Forster, Jochen; Nielsen, Jens; Borodina, Irina



The daughters of Saccharomyces cerevisiaeRAS2val19 mutant are born old.  

Czech Academy of Sciences Publication Activity Database

Bratislava : SAS, 2007, s. 80-80. ISSN 1336-4839. [Annual Conference on Yeasts /35./. Smolenice (SK), 16.05.2007-18.05.2007] R&D Projects: GA ?R GA301/03/0289; GA ?R GA301/07/0339; GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z50200510 Keywords : saccharomyces cerevisiae Subject RIV: EE - Microbiology, Virology

Pichová, Alena; Sigler, Karel


Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production  


Yeast to directly convert cellulose and, especially, the microcrystalline cellulose into bioethanol, was engineered through display of minicellulosomes on the cell surface of Saccharomyces cerevisiae. The construction and cell surface attachment of cellulosomes were accomplished with two individual miniscaffoldins to increase the display level. All of the cellulases including a celCCA (endoglucanase), a celCCE (cellobiohydrolase), and a Ccel_2454 (?-glucosidase) were cloned from Clostridium ...

Fan, Li-hai; Zhang, Zi-jian; Yu, Xiao-yu; Xue, Ya-xu; Tan, Tian-wei



Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae  


Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and...

Zechmann, Bernd; Liou, Liang-chun; Koffler, Barbara E.; Horvat, Lucija; Tomas?ic?, Ana; Fulgosi, Hrvoje; Zhang, Zhaojie



Effects of mill stream flours technological quality on fermentative activity of baker's yeast Saccharomyces cerevisiae  


This work in concerned with the interdependence between technological quality of mill stream flours and fermentative activity of baker's yeast Saccharomyces cerevisiae. Each mill stream flour has its own specific properties, determined by the particle size, technological phase of its formation and part of the wheat kernel it consists of. Biochemical complexity of dough during examination of fermentative activity of baker's yeast confirmed the influence of a number of physical and biochemical ...

Miri? Katarina V.; Pejin Dušanka J.



Saccharomyces cerevisiae Srs2 DNA Helicase Selectively Blocks Expansions of Trinucleotide Repeats  


Trinucleotide repeats (TNRs) undergo frequent mutations in families afflicted with certain neurodegenerative disorders and in model organisms. TNR instability is modulated both by the repeat tract itself and by cellular proteins. Here we identified the Saccharomyces cerevisiae DNA helicase Srs2 as a potent and selective inhibitor of expansions. srs2 mutants had up to 40-fold increased expansion rates of CTG, CAG, and CGG repeats. The expansion phenotype was specific, as mutation rates at dinu...

Bhattacharyya, Saumitri; Lahue, Robert S.



Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae  


Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tole...

Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna



Altered immunochemical reactivity of Saccharomyces cerevisiae a-cells after alpha-factor-induced morphogenesis.  


Antibodies were raised against Saccharomyces cerevisiae a-cells that had been exposed to the sex pheromone, alpha-factor. After adsorption of the antiserum with diploid cells, antibodies remained that reacted specifically with the mannan from haploid cells. The characteristic determinant was observed in mannan from pheromone-treated a-cells, in mannan from untreated alpha-cells, and at a much lower concentration, in mannan from control a-cells. The antigens from these three mannans appeared t...

Lipke, P. N.; Ballou, C. E.



High Osmolarity Extends Life Span in Saccharomyces cerevisiae by a Mechanism Related to Calorie Restriction  


Calorie restriction (CR) extends life span in many different organisms, including mammals. We describe here a novel pathway that extends the life span of Saccharomyces cerevisiae mother cells but does not involve a reduction in caloric content of the media, i.e., there is growth of yeast cells in the presence of a high concentration of external osmolytes. Like CR, this longevity-promoting response to high osmolarity requires SIR2, suggesting a common mechanism of life span regulation. Genetic...

Kaeberlein, Matt; Andalis, Alex A.; Fink, Gerald R.; Guarente, Leonard



A Synthetic Interaction between CDC20 and RAD4 in Saccharomyces cerevisiae upon UV Irradiation  


Regulation of DNA repair can be achieved through ubiquitin-mediated degradation of transiently induced proteins. In Saccharomyces cerevisiae, Rad4 is involved in damage recognition during nucleotide excision repair (NER) and, in conjunction with Rad23, recruits other proteins to the site of damage. We identified a synthetic interaction upon UV exposure between Rad4 and Cdc20, a protein that modulates the activity of the anaphase promoting complex (APC/C), a multisubunit E3 ubiquitin ligase co...

Bernadette Connors; Lauren Rochelle; Asela Roberts; Graham Howard



Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair.  


The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpa...

Verhage, R. A.; Gool, A. J.; Groot, N.; Hoeijmakers, J. H.; Putte, P.; Brouwer, J.



Saccharomyces cerevisiae Produces a Yeast Substance that Exhibits Estrogenic Activity in Mammalian Systems (United States)

Partially purified lipid extracts of Saccharomyces cerevisiae contain a substance that displaces tritiated estradiol from rat uterine cytosol estrogen receptors. The yeast product induces estrogenic bioresponses in mammalian systems as measured by induction of progesterone receptors in cultured MCF-7 human breast cancer cells and by a uterotrophic response and progesterone receptor induction after administration to ovariectomized mice. The findings raise the possibility that bakers' yeast may be a source of environmental estrogens.

Feldman, David; Stathis, Peter A.; Hirst, Margaret A.; Price Stover, E.; Do, Yung S.; Kurz, Walter



Histone H4 Lysine 20 of Saccharomyces cerevisiae is monomethylated and functions in subtelomeric silencing§  


Histones undergo posttranslational modifications that are linked to important biological processes. Previous studies have indicated that lysine methylation correlating with closed or repressive chromatin is absent in the budding yeast Saccharomyces cerevisiae, including at H4 lysine 20 (K20). Here we provide functional evidence for H4 K20 monomethylation (K20me1) in budding yeast. H4 K20me1 is detectable on endogenous H4 by western analysis using methyl-specific antibodies, and the signal is ...

Edwards, Christopher R.; Dang, Weiwei; Berger, Shelley L.



Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae  


We used the yeast MEL1 gene for secreted ?-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi ?-1,4-exoglucanase was inserted into one cartridge to create a fusion of the ?-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. ...

Curry, Claudia; Gilkes, Neil; O Neill, Gary; Miller, Robert C.; Skipper, Nigel



Tripartite structure of the Saccharomyces cerevisiae arginase (CAR1) gene inducer-responsive upstream activation sequence.  


Arginase (CAR1) gene expression in Saccharomyces cerevisiae is induced by arginine. The 5' regulatory region of CAR1 contains four separable regulatory elements--two inducer-independent upstream activation sequences (UASs) (UASC1 and UASC2), an inducer-dependent UAS (UASI), and an upstream repression sequence (URS1) which negatively regulates CAR1 and many other yeast genes. Here we demonstrate that three homologous DNA sequences originally reported to be present in the inducer-responsive UAS...

Viljoen, M.; Kovari, L. Z.; Kovari, I. A.; Park, H. D.; Vuuren, H. J.; Cooper, T. G.



Genome-Wide Mapping of the Cohesin Complex in the Yeast Saccharomyces cerevisiae  


In eukaryotic cells, cohesin holds sister chromatids together until they separate into daughter cells during mitosis. We have used chromatin immunoprecipitation coupled with microarray analysis (ChIP chip) to produce a genome-wide description of cohesin binding to meiotic and mitotic chromosomes of Saccharomyces cerevisiae. A computer program, PeakFinder, enables flexible, automated identification and annotation of cohesin binding peaks in ChIP chip data. Cohesin sites are highly conserved in...

Glynn, Earl F.; Megee, Paul C.; Yu, Hong-guo; Mistrot, Cathy; Unal, Elcin; Koshland, Douglas E.; Derisi, Joseph L.; Gerton, Jennifer L.



Inactivation of Saccharomyces cerevisiae suspended in orange juice using high-intensity pulsed electric fields. (United States)

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. PMID:15553647

Elez-Martínez, Pedro; Escolà-Hernández, Joan; Soliva-Fortuny, Robert C; Martín-Belloso, Olga



Anti-Saccharomyces cerevisiae autoantibodies in autoimmune diseases: from bread baking to autoimmunity. (United States)

Saccharomyces cerevisiae is best known as the baker's and brewer's yeast, but its residual traces are also frequent excipients in some vaccines. Although anti-S. cerevisiae autoantibodies (ASCAs) are considered specific for Crohn's disease, a growing number of studies have detected high levels of ASCAs in patients affected with autoimmune diseases as compared with healthy controls, including antiphospholipid syndrome, systemic lupus erythematosus, type 1 diabetes mellitus, and rheumatoid arthritis. Commensal microorganisms such as Saccharomyces are required for nutrition, proper development of Peyer's aggregated lymphoid tissue, and tissue healing. However, even the commensal nonclassically pathogenic microbiota can trigger autoimmunity when fine regulation of immune tolerance does not work properly. For our purposes, the protein database of the National Center for Biotechnology Information (NCBI) was consulted, comparing Saccharomyces mannan to several molecules with a pathogenetic role in autoimmune diseases. Thanks to the NCBI bioinformation technology tool, several overlaps in molecular structures (50-100 %) were identified when yeast mannan, and the most common autoantigens were compared. The autoantigen U2 snRNP B? was found to conserve a superfamily protein domain that shares 83 % of the S. cerevisiae mannan sequence. Furthermore, ASCAs may be present years before the diagnosis of some associated autoimmune diseases as they were retrospectively found in the preserved blood samples of soldiers who became affected by Crohn's disease years later. Our results strongly suggest that ASCAs' role in clinical practice should be better addressed in order to evaluate their predictive or prognostic relevance. PMID:23292495

Rinaldi, Maurizio; Perricone, Roberto; Blank, Miri; Perricone, Carlo; Shoenfeld, Yehuda



The toxic potential of an industrial effluent determined with the Saccharomyces cerevisiae-based assay. (United States)

Increasing levels of environmental pollution and the continuous monitoring of water quality both request specific and sensitive methods for the detection of detrimental water contents. On a regulatory basis genotoxicity is assessed by the standard umu-test (ISO 13829) that responds to DNA damage induced by chemicals. The focus of this study was the examination of the toxic potential of samples taken from the wastewater treatment plant of a refinery factory to explore the applicability of the Saccharomyces cerevisiae (bakers yeast) test for the detection of bio-available genotoxic activity in complex matrices. The toxic potential of samples without pre-treatment and following centrifugation was determined with the eukaryotic Saccharomyces cerevisiae bioassay based on the transcriptional activation of the green fluorescent protein (gfp) fused to the DNA damage inducible RAD54 promoter and general growth inhibition. Primary effluent samples were taken as qualified sterile spot samples from the final effluent of the purification plant. The Saccharomyces cerevisiae assay yielded geno- and cytotoxic responses in all complex untreated and centrifuged samples with high reproducibility. The obtained results suggest that the yeast assay is suited as a screening tool to monitor genotoxic potential of wastewater. PMID:16002118

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



Evaluation of wet-feeding wheat-based diets containing Saccharomyces cerevisiae to broiler chickens. (United States)

1. This experiment investigated the effects of water and Saccharomyces cerevisiae added to wheat-based diets on gastrointestinal, blood and performance parameters of broiler chickens. 2. A total of 160 one-d-old male broiler chicks were given air-dry or wet diets, with or without S. cerevisiae supplementation (0 and 20 g/kg air-dry feed) ad libitum to 42 d. 3. Feeding broilers with a diet mixed with water in a ratio of 1·2 : 1·0 increased body weight, feed intake, abdominal fat, carcase weight, feed transit time and blood HDL (high density lipoprotein) (without yeast). Supplementation with S. cerevisiae increased DM digestibility but reduced ileal pH, ileal coliform population and abdominal fat content. 4. There was a significant interaction between S. cerevisiae and wet feeding, with S. cerevisiae supplementation inducing a significant increase in body weight and feed intake but a reduction of relative abdominal fat and ileal pH of broilers fed on wet diets. 5. It is concluded that wet feeding improved growth performance by increasing feed intake and that the addition of a culture of S. cerevisiae had a growth stimulating effect, as the inclusion of yeast in wet wheat-based broiler diets generated greater responses than yeast in dry-based diets. PMID:21161784

Afsharmanesh, M; Barani, M; Silversides, F G



Pengaruh Media Tumbuh terhadap Kadar Protein Saccharomyces cerevisiae dalam Pembuatan Protein Sel Tunggal  

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Full Text Available The aim of this research was to examine the influence of difference growth media, i. e. tofu liquid waste, tofu solid waste, and coconut water in various composition and Yeast Extract Peptone Dextrose (YEPD, to protein contents of Saccharomyces cerevisiae in Single Cell Protein (SCP production. The framework of this research was that tofu liquid waste, tofu solid waste, and coconut water were containing a lot of carbons, nitrogens, minerals, and vitamin that could be used as growth medium of S. cerevisiae to produce SCP, which was commonly used. The medium from tofu liquid waste and the coconut water were made by ratio 2:1, 1:1, 1:2 and added with tofu solid waste 1.5 g and 2.5 g. Then, the measurement of pH medium, the amount of cell, cell dried weight, and the protein content in S. cerevisiae was done. The measurement of protein content was done by Lowry method. The result of the research showed that growth media influenced protein content of S. cerevisiae. Protein content of S. cerevisiae cultured in tofu liquid waste- coconut water was lower then in YEPD medium. The protein content of S. cerevisiae cultured in tofu liquid waste and coconut water ratio 1:2, added with 2.5 g tofu solid waste was higher then in other medium composition.




Saccharomyces cerevisiae : a model to uncover molecular mechanisms for yeast biofilm biology.  

DEFF Research Database (Denmark)

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 to produce an ECM and respond to quorum sensing, and multi-cellular aggregates have lowered susceptibility to antifungals. Adhesion is mediated by a family of cell surface proteins of which Flo11 has been shown to be essential for biofilm development. FLO11 expression is regulated via a number of regulatory 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 cues, cell-to-cell variation and niches in S. cerevisiae biofilm. Being closely related to Candida species, S. cerevisiae is a model to investigate biofilms of pathogenic yeast.

Bojsen, Rasmus K; Andersen, Kaj Scherz



Efeito do nitrito sobre a fermentação alcoólica realizada por Saccharomyces cerevisiae / Effect of nitrite on alcoholic fermentation carried out with Saccharomyces cerevisiae  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese O efeito de concentrações de até 80 ppm de nitrito sobre a fermentação alcoólica foi estudado com levedura de panificação (Saccharomyces cerevisiae). Houve aumento no tempo de fermentação com adição de nitrito sem afetar a produção de etanol. Com a adição de 60 e 80 ppm de NO2-, ocorreu redução na v [...] iabilidade celular e brotamento acompanhada por aumento no acúmulo de trealose e glicogênio. Aumentando a concentração de nitrito houve aumento no álcool n-propílico e redução nos teores de álcoois isobutílico e isoamílico. Abstract in english The effect of nitrite up to 80 ppm on alcoholic fermentation was studied with baker's yeast (Saccharomyces cerevisiae). There was an increase in fermentation time but not effect on ethanol yield. With the addition of 60 and 80 ppm NO2- there was a reduction on cell viabilitty and budding with corres [...] pondent increase on trehalose and glycogen accumulation. Increasing nitrite concentration resulted in increase on n-propilic alcohol level and a reduction on isobutilic and isoamilic alcohols content.

L.E., Gutierrez; V.F. de Martin, Orelli.


A new biological test of water toxicity-yeast Saccharomyces cerevisiae conductometric test. (United States)

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. PMID:25461558

Dolezalova, Jaroslava; Rumlova, Lubomira



Utilizing an endogenous pathway for 1-butanol production in Saccharomyces cerevisiae. (United States)

Microbial production of higher alcohols from renewable feedstock has attracted intensive attention thanks to its potential as a source for next-generation gasoline substitutes. Here we report the discovery, characterization and engineering of an endogenous 1-butanol pathway in Saccharomyces cerevisiae. Upon introduction of a single gene deletion adh1?, S. cerevisiae was able to accumulate more than 120 mg/L 1-butanol from glucose in rich medium. Precursor feeding, ¹³C-isotope labeling and gene deletion experiments demonstrated that the endogenous 1-butanol production was dependent on catabolism of threonine in a manner similar to fusel alcohol production by the Ehrlich pathway. Specifically, the leucine biosynthesis pathway was engaged in the conversion of key 2-keto acid intermediates. Overexpression of the pathway enzymes and elimination of competing pathways achieved the highest reported 1-butanol titer in S. cerevisiae (242.8 mg/L). PMID:24412568

Si, Tong; Luo, Yunzi; Xiao, Han; Zhao, Huimin



Ethanol production by Saccharomyces cerevisiae using lignocellulosic hydrolysate from Chrysanthemum waste degradation. (United States)

Ethanol production derived from Saccharomyces cerevisiae fermentation of a hydrolysate from floriculture waste degradation was studied. The hydrolysate was produced from Chrysanthemum (Dendranthema grandiflora) waste degradation by Pleurotus ostreatus and characterized to determine the presence of compounds that may inhibit fermentation. The products of hydrolysis confirmed by HPLC were cellobiose, glucose, xylose and mannose. The hydrolysate was fermented by S. cerevisiae, and concentrations of biomass, ethanol, and glucose were determined as a function of time. Results were compared to YGC modified medium (yeast extract, glucose and chloramphenicol) fermentation. Ethanol yield was 0.45 g g(-1), 88 % of the maximal theoretical value. Crysanthemum waste hydrolysate was suitable for ethanol production, containing glucose and mannose with adequate nutrients for S. cerevisiae fermentation and low fermentation inhibitor levels. PMID:23117675

Quevedo-Hidalgo, Balkys; Monsalve-Marín, Felipe; Narváez-Rincón, Paulo César; Pedroza-Rodríguez, Aura Marina; Velásquez-Lozano, Mario Enrique



Ethanol Production from Sago Waste Using Saccharomyces cerevisiae Vits-M1  

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Full Text Available The present study deals with the biotechnological production of ethanol from sago waste materials. As petroleum has become depleted, renewable energy production has started to gain attention all over the world, including the production of ethanol from sago wastes. In our research we have standardized the production of ethanol from sago wastes using Saccharomyces cerevisiae strain isolated from molasses. The production of ethanol was carried out by means of simultaneous saccharification with acids, followed by fermentation. The yeast strains were isolated from either batter or molasses and the taxonomy was studied by phenotypic characters in comparison with the standard strain Saccharomyces cerevisiae MTCC 173. Among the two isolated strains, S. cerevisiae VITS-M1 isolated from molasses showed better survival rate in different sugars such as glucose, sucrose, maltose and galactose except lactose; it also showed better survival rate at high ethanol concentration and at acidic pH. The saccharification process of sago liquid waste and solid waste was standardized using hydrochloric acid and sulphuric acid under different treatments. The fermented product, ethanol was distilled using laboratory model distillation unit and measured qualitatively using gas chromatography in comparison with the standard analytical grade ethanol. The overall experimental data indicates that the sago liquid waste yielded more ethanol by simultaneous saccharification with 0.3N HCl and 0.3N H2SO4 and fermentation with the S. cerevisiae VITS-M1 isolated from molasses.

D. Subashini




Directory of Open Access Journals (Sweden)

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.




Saccharomyces cerevisiae can secrete Sapp1p proteinase of Candida parapsilosis but cannot use it for efficient nitrogen acquisition. (United States)

Secreted aspartic proteinase Sapp1p of Candida parapsilosis represents one of the factors contributing to the pathogenicity of the fungus. The proteinase is synthesized as an inactive pre-pro-enzyme, but only processed Sapp1p is secreted into extracellular space. We constructed a plasmid containing the SAPP1 coding sequence under control of the ScGAL1 promoter and used it for proteinase expression in a Saccharomyces cerevisiae kex2? mutant. Because Sapp1p maturation depends on cleavage by Kex2p proteinase, the kex2? mutant secreted only the pro-form of Sapp1p. Characterization of this secreted proteinase form revealed that the Sapp1p signal peptide consists of 23 amino acids. Additionally, we prepared a plasmid with the SAPP1 coding sequence under control of its authentic CpSAPP1 promoter, which contains two GATAA motifs. While in C. parapsilosis SAPP1 expression is repressed by good low molecular weight nitrogen sources (e.g., ammonium ions), S. cerevisiae cells harboring this plasmid secreted a low concentration of active proteinase regardless of the type of nitrogen source used. Quantitative real-time PCR analysis of a set of genes related to nitrogen metabolism and uptake (GAT1, GLN3, STP2, GAP1, OPT1, and PTR2) obtained from S. cerevisiae cells transformed with either plasmid encoding SAPP1 under control of its own promoter or empty vector and cultivated in media containing various nitrogen sources also suggested that SAPP1 expression can be connected with the S. cerevisiae regulatory network. However, this regulation occurs in a different manner than in C. parapsilosis. PMID:23812814

Vinterová, Zuzana; Bauerová, Václava; Dostál, Ji?í; Sychrová, Hana; Hrušková-Heidingsfeldová, Olga; Pichová, Iva



Bioethanol production by a flocculent hybrid, CHFY0321 obtained by protoplast fusion between Saccharomyces cerevisiae and Saccharomyces bayanus  

Energy Technology Data Exchange (ETDEWEB)

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)

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)



Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae  


This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis. Following reduction, alkylation, trypsin...

Saleem, Ramsey A.; Aitchison, John D.



Glycerol metabolism and transport activity regulation in Saccharomyces cerevisiae  


With the purpose of studying the correlation between glycerol metabolic pathway and glycerol active transport (1) in S. cerevisiae, an extensive study on glycerol transport was elaborated in all the available mutants from the genes of glycerol metabolic pathway having W303 as common genetic background: gut1?, gut2?, gpp1?, gpp2?, gpd?1 and gpd2? and several double mutants. For this purpose we chose to diagnostic active transport determining uptake kinetic parameters...

Lages, Fernanda; Oliveira, Rui Pedro Soares; Lucas, Ca?ndida