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Sample records for cerevisiae mag1 gene

  1. Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase

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    Adhikary, Suraj; Eichman, Brandt F. (Vanderbilt)

    2014-10-02

    DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.

  2. The Reacquisition of Biotin Prototrophy in Saccharomyces cerevisiae Involved Horizontal Gene Transfer, Gene Duplication and Gene Clustering

    OpenAIRE

    Hall, Charles; Dietrich, Fred S

    2007-01-01

    The synthesis of biotin, a vitamin required for many carboxylation reactions, is a variable trait in Saccharomyces cerevisiae. Many S. cerevisiae strains, including common laboratory strains, contain only a partial biotin synthesis pathway. We here report the identification of the first step necessary for the biotin synthesis pathway in S. cerevisiae. The biotin auxotroph strain S288c was able to grow on media lacking biotin when BIO1 and the known biotin synthesis gene BIO6 were introduced t...

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

    Directory of Open Access Journals (Sweden)

    James Ronald

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

  4. Identification and characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Vuralhan, Zeynep; Morais, Marcos A; Tai, Siew-Leng; Piper, Matthew D W; Pronk, Jack T

    2003-08-01

    Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding alpha-keto acids, followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may be oxidized to an acid. This pathway is functional in Saccharomyces cerevisiae, since during growth in glucose-limited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific step in the Ehrlich pathway. S. cerevisiae possesses five candidate genes with sequence similarity to genes encoding thiamine diphosphate-dependent decarboxylases that could encode this activity: YDR380w/ARO10, YDL080C/THI3, PDC1, PDC5, and PDC6. Phenylpyruvate decarboxylase activity was present in cultures grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures. Furthermore, the transcript level of one candidate gene (ARO10) increased 30-fold when phenylalanine replaced ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate decarboxylase enzyme assays indicated that ARO10 was sufficient to encode phenylpyruvate decarboxylase activity in the absence of the four other candidate genes. There was also an alternative activity with a higher capacity but lower affinity for phenylpyruvate. The candidate gene THI3 did not itself encode an active phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (PDC1, PDC5, and PDC6) for the alternative activity. The K(m) and V(max) values of the two activities differed, showing that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to this gene could therefore be important for metabolic engineering

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

    OpenAIRE

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

    1983-01-01

    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...

  6. Behavioral Abnormalities in a Mouse Model of Chronic Toxoplasmosis Are Associated with MAG1 Antibody Levels and Cyst Burden.

    Science.gov (United States)

    Xiao, Jianchun; Li, Ye; Prandovszky, Emese; Kannan, Geetha; Viscidi, Raphael P; Pletnikov, Mikhail V; Yolken, Robert H

    2016-04-01

    There is marked variation in the human response to Toxoplasma gondii infection. Epidemiological studies indicate associations between strain virulence and severity of toxoplasmosis. Animal studies on the pathogenic effect of chronic infection focused on relatively avirulent strains (e.g. type II) because they can easily establish latent infections in mice, defined by the presence of bradyzoite-containing cysts. To provide insight into virulent strain-related severity of human toxoplasmosis, we established a chronic model of the virulent type I strain using outbred mice. We found that type I-exposed mice displayed variable outcomes ranging from aborted to severe infections. According to antibody profiles, we found that most of mice generated antibodies against T. gondii organism but varied greatly in the production of antibodies against matrix antigen MAG1. There was a strong correlation between MAG1 antibody level and brain cyst burden in chronically infected mice (r = 0.82, p = 0.0021). We found that mice with high MAG1 antibody level displayed lower weight, behavioral changes, altered levels of gene expression and immune activation. The most striking change in behavior we discovered was a blunted response to amphetamine-trigged locomotor activity. The extent of most changes was directly correlated with levels of MAG1 antibody. These changes were not found in mice with less cyst burden or mice that were acutely but not chronically infected. Our finding highlights the critical role of cyst burden in a range of disease severity during chronic infection, the predictive value of MAG1 antibody level to brain cyst burden and to changes in behavior or other pathology in chronically infected mice. Our finding may have important implications for understanding the heterogeneous effects of T. gondii infections in human. PMID:27124472

  7. Identification and Characterization of a Novel Biotin Biosynthesis Gene in Saccharomyces cerevisiae

    OpenAIRE

    Wu, Hong; Ito, Kiyoshi; Shimoi, Hitoshi

    2005-01-01

    Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake ye...

  8. Expression of the major heat shock gene of Drosophila melanogaster in Saccharomyces cerevisiae.

    OpenAIRE

    de Banzie, J S; Sinclair, L; Lis, J T

    1986-01-01

    A copy of the gene which encodes the major heat shock protein (hsp70) of D. melanogaster was integrated in both orientations into the genome of S. cerevisiae at the leu2 locus. The level of transcript from the D. melanogaster gene was measured under both normal conditions and conditions which are known to give rise to the heat shock response in S. cerevisiae. In both orientations the D. melanogaster gene gave rise to an abundant transcript in uninduced cells. The level of this transcript was ...

  9. CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Maury, Jerome; Jakociunas, Tadas;

    2015-01-01

    % for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native beta-carotene pathway was reconstructed in S. cerevisiae by simultaneous......Background: One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from...

  10. Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion

    Science.gov (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...

  11. Large-scale evaluation of in silico gene deletions in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Förster, Jochen; Famili, Iman; Palsson, Bernhard Ø;

    2003-01-01

    A large-scale in silico evaluation of gene deletions in Saccharomyces cerevisiae was conducted using a genome-scale reconstructed metabolic model. The effect of 599 single gene deletions on cell viability was simulated in silico and compared to published experimental results. In 526 cases (87...

  12. Sensitivity to Lovastatin of Saccharomyces cerevisiae Strains Deleted for Pleiotropic Drug Resistance (PDR) Genes

    DEFF Research Database (Denmark)

    Formenti, Luca Riccardo; Kielland-Brandt, Morten

    2011-01-01

    The use of statins is well established in human therapy, and model organisms such as Saccharomyces cerevisiae are commonly used in studies of drug action at molecular and cellular levels. The investigation of the resistance mechanisms towards statins may suggest new approaches to improve therapy...... based on the use of statins. We investigated the susceptibility to lovastatin of S. cerevisiae strains deleted for PDR genes, responsible for exporting hydrophobic and amphi-philic drugs, such as lovastatin. Strains deleted for the genes tested, PDR1, PDR3, PDR5 and SNQ2, exhibited remarkably different...

  13. Classifying genes to the correct Gene Ontology Slim term in Saccharomyces cerevisiae using neighbouring genes with classification learning

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    Tsatsoulis Costas

    2010-05-01

    Full Text Available Abstract Background There is increasing evidence that gene location and surrounding genes influence the functionality of genes in the eukaryotic genome. Knowing the Gene Ontology Slim terms associated with a gene gives us insight into a gene's functionality by informing us how its gene product behaves in a cellular context using three different ontologies: molecular function, biological process, and cellular component. In this study, we analyzed if we could classify a gene in Saccharomyces cerevisiae to its correct Gene Ontology Slim term using information about its location in the genome and information from its nearest-neighbouring genes using classification learning. Results We performed experiments to establish that the MultiBoostAB algorithm using the J48 classifier could correctly classify Gene Ontology Slim terms of a gene given information regarding the gene's location and information from its nearest-neighbouring genes for training. Different neighbourhood sizes were examined to determine how many nearest neighbours should be included around each gene to provide better classification rules. Our results show that by just incorporating neighbour information from each gene's two-nearest neighbours, the percentage of correctly classified genes to their correct Gene Ontology Slim term for each ontology reaches over 80% with high accuracy (reflected in F-measures over 0.80 of the classification rules produced. Conclusions We confirmed that in classifying genes to their correct Gene Ontology Slim term, the inclusion of neighbour information from those genes is beneficial. Knowing the location of a gene and the Gene Ontology Slim information from neighbouring genes gives us insight into that gene's functionality. This benefit is seen by just including information from a gene's two-nearest neighbouring genes.

  14. Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase.

    OpenAIRE

    Walfridsson, M; Hallborn, J; Penttilä, M.; Keränen, S; Hahn-Hägerdal, B

    1995-01-01

    Saccharomyces cerevisiae was metabolically engineered for xylose utilization. The Pichia stipitis CBS 6054 genes XYL1 and XYL2 encoding xylose reductase and xylitol dehydrogenase were cloned into S. cerevisiae. The gene products catalyze the two initial steps in xylose utilization which S. cerevisiae lacks. In order to increase the flux through the pentose phosphate pathway, the S. cerevisiae TKL1 and TAL1 genes encoding transketolase and transaldolase were overexpressed. A XYL1- and XYL2-con...

  15. Physiological studies in aerobic batch cultivations of Saccharomyces cerevisiae strains harboring the MEL1 gene

    DEFF Research Database (Denmark)

    Østergaard, Simon; Roca, Christophe Francois Aime; Ronnow, B.;

    2000-01-01

    Physiological studies of Saccharomyces cerevisiae strains harboring the MEL1 gene were carried out in aerobic batch cultivations on glucose-galactose mixtures and on the disaccharide melibiose, which is hydrolyzed by the enzyme melibiase (Mel1, EC 3.2.1.22) into a glucose and a galactose moiety. ...

  16. Physical evidence for a Saccharomyces cerevisiae transposable element which carries the his4C gene.

    OpenAIRE

    de Bruijn, F; Greer, H

    1981-01-01

    A Saccharomyces cerevisiae transposable element which carries the his4C structural gene and which is capable of transposition, excision, and mutator activity is described. Physical evidence is presented for transposition of the his4C deoxyribonucleic acid sequences to a new location in the genome and for precise excision of these transposed deoxyribonucleic acid sequences in spontaneous his4C- segregants.

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

    Science.gov (United States)

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

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development. PMID:27470141

  18. Quantitative Gene Expression of ERG9 in Model Saccharomyces cerevisiae: Chamomile Extract For Human Cancer Treatment

    Science.gov (United States)

    Hosseinpour, Maryam; Mobini-Dehkordi, Mohsen

    2016-01-01

    Introduction Over expression of squalene synthase gene causes induction of growth tumour and reduction of apoptosis. This gene which is conserved between Saccharomyces cerevisiae yeast and humans, is named (ERG9). Aim In this work, we studied the effect of Matricaria recutita extract on ERG9 gene (squalene synthase) expression in S.cerevisiae which was used as organism model in cancer therapy. Materials and Methods S. cerevisiae was cultured in YPD medium plus 0,250, 1000 and 3000 μg/ml of Matricaria recutita extract and we evaluated the (ERG9) gene expression by Real-time RT-PCR method after 24 hours. Statistical analysis used At least 3 independent experiments were done. Data were analyzed using One-way ANOVA and Dunnett’s test. A p-value of less than 0.01 was considered as significant. Results We found that 250, 1000 and 3000 μg/ml of Matricaria recutita extract could reduce expression of ERG9 gene significantly (p<0.01). Interestingly, the expression of this gene was completely inhibited in 1000 and 3000 μg/ml concentrations. Conclusion This study predicted that Matricaria recutita extract produced anti-cancer effects in humans, because it could inhibit the expression of an analogue key gene in this malignant disease. Further investigations should be made, to study its molecular mechanism of action at the mammal cell level.

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

    NARCIS (Netherlands)

    Verwaal, R.; Wang, J.; Meijnen, J.P.; Visser, H.; Sandmann, G.; Berg, van den J.A.; Ooyen, van A.J.J.

    2007-01-01

    To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially ß-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these g

  20. Saccharomyces cerevisiae Signature Genes for Predicting Nitrogen Deficiency during Alcoholic Fermentation▿ †

    OpenAIRE

    Mendes Ferreira, A.; Olmo Muñoz, Marcel·lí del; García Martínez, José; Jiménez Martí, Elena; Leão, C.; Mendes Faia, A.; Pérez Ortín, José Enrique

    2007-01-01

    Genome-wide analysis of the wine yeast strain Saccharomyces cerevisiae PYCC4072 identified 36 genes highly expressed under conditions of low or absent nitrogen in comparison with a nitrogen-replete condition. Reverse transcription-PCR analysis for four of these transcripts with this strain and its validation with another wine yeast strain underlines the usefulness of these signature genes for predicting nitrogen deficiency and therefore the diagnosis of wine stuck/sluggish fermentations.

  1. Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Slater, M R; Craig, E A

    1987-01-01

    The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the ...

  2. Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein.

    OpenAIRE

    Knight, S A; Tamai, K T; Kosman, D J; Thiele, D J

    1994-01-01

    Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome...

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

    OpenAIRE

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

    1991-01-01

    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...

  4. A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Cuperus, Josh T; Lo, Russell S; Shumaker, Lucia; Proctor, Julia; Fields, Stanley

    2015-07-17

    Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We identify variants of the Escherichia coli tet operator (tetO) sequence that bind a TetR-VP16 activator with differential affinity and therefore result in different TetR-VP16 activator-driven expression. By recombining these variants upstream of the genes of a pathway, we generate unique combinations of expression levels. Here, we built a tetO toolkit, which includes the I-OnuI homing endonuclease to create double-strand breaks, which increases homologous recombination by 10(5); a plasmid carrying six variant tetO sequences flanked by I-OnuI sites, uncoupling transformation and recombination steps; an S. cerevisiae-optimized TetR-VP16 activator; and a vector to integrate constructs into the yeast genome. We introduce into the S. cerevisiae genome the three crt genes from Erwinia herbicola required for yeast to synthesize lycopene and carry out the recombination process to produce a population of cells with permutations of tetO variants regulating the three genes. We identify 0.7% of this population as making detectable lycopene, of which the vast majority have undergone recombination at all three crt genes. We estimate a rate of ∼20% recombination per targeted site, much higher than that obtained in other studies. Application of this toolkit to medically or industrially important end products could reduce the time and labor required to optimize the expression of a set of metabolic genes. PMID:25742460

  5. Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: A three factor design

    DEFF Research Database (Denmark)

    Fazio, Alessandro; Jewett, Michael Christopher; Daran-Lapujade, Pascale;

    2008-01-01

    Background: Characterization of cellular growth is central to understanding living systems. Here, we applied a three-factor design to study the relationship between specific growth rate and genome-wide gene expression in 36 steady-state chemostat cultures of Saccharomyces cerevisiae. The three...... factors we considered were specific growth rate, nutrient limitation, and oxygen availability. Results: We identified 268 growth rate dependent genes, independent of nutrient limitation and oxygen availability. The transcriptional response was used to identify key areas in metabolism around which m...

  6. Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bai, Dongmei; Siewers, Verena; Huang, Le;

    2009-01-01

    The construction of mitotically stable yeast strains for heterologous gene or pathway expression often requires chromosomal integration. However, transcription levels vary between different chromosome regions. We therefore characterized 20 different integration sites of the Sacchromyces cerevisiae...... genome by inserting lacZ as a reporter gene under the control of two different promoters and determining expression levels through enzyme activity measurement. An up to 8.7-fold difference was detected between the sites conferring lowest and highest expression, respectively. This opens the opportunity...

  7. Identification of SAS4 and SAS5, two genes that regulate silencing in Saccharomyces cerevisiae.

    OpenAIRE

    Xu, E Y; S. Kim; Replogle, K; Rine, J; Rivier, D H

    1999-01-01

    In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the alpha-mating phenotype to MATalpha HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that correspo...

  8. Ectopic Expression of Arabidopsis Phospholipase A Genes Elucidates Role of Phospholipase Bs in S. cerevisiae Cells

    OpenAIRE

    Zhang, Meng; Zhang, Yan; Giblin, E Michael; Taylor, David C.

    2009-01-01

    In S. cerevisiae neither disruption of the phospholipase B triple knockout mutant (plb1plb2plb3; plb123) nor over-expression of phospholipase Bs (PLBs) result in a phenotype different from wild type. In performing experiments to characterize candidate plant phospholipase (PLA) genes, we found, surprisingly, that ectopic expression of either of two different A. thaliana PLA2 or PLA1 genes in the yeast plb123 mutant completely inhibited cell growth. We proposed that while PLBs might not be esse...

  9. EasyClone 2.0:expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains

    OpenAIRE

    Stovicek, Vratislav; Borja Zamfir, Gheorghe Manuel; Förster, Jochen; Borodina, Irina

    2015-01-01

    Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vector...

  10. In vivo robustness analysis of cell division cycle genes in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Hisao Moriya

    2006-07-01

    Full Text Available Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named "genetic tug-of-war" (gTOW that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes. The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.

  11. The CRO-1 gene of Saccharomyces cerevisiae controls mitotic crossing over, chromosomal stability and sporulation

    International Nuclear Information System (INIS)

    The properties of a novel temperature-sensitive recombination-defective mutant of Saccharomyces cerevisiae, cro1-1 is described. The cro1-1 mutant is the first instance of a rec mutation that reduces drastically the rates of spontaneous mitotic crossing-over events but not those of gene conversional events. The cro1-1 mutation thus provides evidence that mitotic crossing-over is dependent upon gene products that are not essential for gene conversional events. The cro1-1 mutation also results in enhanced mitotic-chromosomal instability and MATa/MATα cro1-1/cro1-1 mutants are sporulation deficient. These phenotypes indicate that the CRO1 gene modulates mitotic chromosomal integrity and is essential for normal meiosis. The cro1-1 mutant possesses Holliday junction resolvase activity, hence its recombinational defect does not involve failure to execute this putative final recombinational step. 7 refs., 1 fig., 5 tabs

  12. Genes regulation encoding ADP/ATP carrier in yeasts Saccharomyces cerevisiae and Candida parapsilosis

    International Nuclear Information System (INIS)

    Genes encoding a mitochondrial ADP/ATP carrier (AAC) in yeast Saccharomyces cerevisiae and Candida parapsilosis were investigated. AAC2 is coding for the major AAC isoform in S. cerevisiae. We suggest that AAC2 is a member of a syn-expression group of genes encoding oxidative phosphorylation proteins. Within our previous studies on the regulation of the AAC2 transcription an UAS (-393/-268) was identified that is essential for the expression of this gene. Two functional regulatory cis-elements are located within this UAS -binding sites for an ABFl factor and for HAP2/3/4/5 heteromeric complex. We examined relative contributions and mutual interactions of the ABFl and HAP2/3/4/5 factors in the activation of transcription from the UAS of the AAC2 gene. The whole UAS was dissected into smaller sub-fragments and tested for (i) the ability to form DNA-protein complexes with cellular proteins in vitro, (ii) the ability to confer heterologous expression using AAC3 gene lacking its own promoter, and (iii) the expression of AAC3-lacZ fusion instead of intact AAC3 gene. The obtained results demonstrated that: a) The whole UAS as well as sub-fragment containing only ABF1-binding site are able to form DNA-protein complexes with cellular proteins in oxygen- and heme- dependent manner. The experiments with antibody against the ABF1 showed that the ABF1 factor is one of the proteins binding to AAC2 promoter. We have been unsuccessful to prove the binding of cellular proteins to the HAP2/3/4/5-binding site. However, the presence of HAP2/3/4/5-binding site is necessary to drive a binding of cellular proteins to the ABF1-binding site in carbon source-dependent manner. b) The presence of both ABF1- and HAP2/3/4/5-binding sites and original spacing between them is necessary to confer the growth of Aaac2 mutant strain on non- fermentable carbon source when put in front of AAC3 gene introduced on centromeric vector to Aaac2 mutant strain. c) For the activation of AAC3-lacZ expression on

  13. Population genetic variation in gene expression is associated withphenotypic variation in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Fay, Justin C.; McCullough, Heather L.; Sniegowski, Paul D.; Eisen, Michael B.

    2004-02-25

    The relationship between genetic variation in gene expression and phenotypic variation observable in nature is not well understood. Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understanding the molecular basis and evolution of complex traits. Results: We compared levels of gene expression among nine natural isolates of Saccharomyces cerevisiae grown either in the presence or absence of copper sulfate. Of the nine strains, two show a reduced growth rate and two others are rust colored in the presence of copper sulfate. We identified 633 genes that show significant differences in expression among strains. Of these genes,20 were correlated with resistance to copper sulfate and 24 were correlated with rust coloration. The function of these genes in combination with their expression pattern suggests the presence of both correlative and causative expression differences. But the majority of differentially expressed genes were not correlated with either phenotype and showed the same expression pattern both in the presence and absence of copper sulfate. To determine whether these expression differences may contribute to phenotypic variation under other environmental conditions, we examined one phenotype, freeze tolerance, predicted by the differential expression of the aquaporin gene AQY2. We found freeze tolerance is associated with the expression of AQY2. Conclusions: Gene expression differences provide substantial insight into the molecular basis of naturally occurring traits and can be used to predict environment dependent phenotypic variation.

  14. Cloning and Expression of a Schwanniomyces occidentalis α-Amylase Gene in Saccharomyces cerevisiae

    OpenAIRE

    Wang, Tsung Tsan; Lin, Long Liu; Hsu, Wen Hwei

    1989-01-01

    An α-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 α-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 α-amylase synthesis. The concentration of α-amylase in culture broth produced ...

  15. Model-guided identification of gene deletion targets for metabolic engineering in Saccharomyces cerevisiae.

    Science.gov (United States)

    Brochado, Ana Rita; Patil, Kiran Raosaheb

    2014-01-01

    Identification of metabolic engineering strategies for rerouting intracellular fluxes towards a desired product is often a challenging task owing to the topological and regulatory complexity of metabolic networks. Genome-scale metabolic models help tackling this complexity through systematic consideration of mass balance and reaction directionality constraints over the entire network. Here, we describe how genome-scale metabolic models can be used for identifying gene deletion targets leading to increased production of the desired product. Vanillin production in Saccharomyces cerevisiae is used as a case study throughout this chapter. PMID:24744040

  16. Gene-interleaving patterns of synteny in the Saccharomyces cerevisiae genome: are they proof of an ancient genome duplication event?

    Directory of Open Access Journals (Sweden)

    Sun Feng-Jie

    2007-09-01

    Full Text Available Abstract Background Recent comparative genomic studies claim local syntenic gene-interleaving relationships in Ashbya gossypii and Kluyveromyces waltii are compelling evidence for an ancient whole-genome duplication event in Saccharomyces cerevisiae. We here test, using Hannenhalli-Pevzner rearrangement algorithms that address the multiple genome rearrangement problem, whether syntenic patterns are proof of paleopolyploidization. Results We focus on (1 pairwise comparison of gene arrangement sequences in A. gossypii and S. cerevisiae, (2 reconstruction of gene arrangements ancestral to A. gossypii, S. cerevisiae, and K. waltii, (3 synteny patterns arising within and between lineages, and (4 expected gene orientation of duplicate gene sets. The existence of syntenic patterns between ancestral gene sets and A. gossypii, S. cerevisiae, and K. waltii, and other evidence, suggests that gene-interleaving relationships are the natural consequence of topological rearrangements in chromosomes and that a more gradual scenario of genome evolution involving segmental duplication and recombination constitutes a more parsimonious explanation. Furthermore, phylogenetic trees reconstructed under alternative hypotheses placed the putative whole-genome duplication event after the divergence of the S. cerevisiae and K. waltii lineages, but in the lineage leading to K. waltii. This is clearly incompatible with an ancient genome duplication event in S. cerevisiae. Conclusion Because the presence of syntenic patterns appears to be a condition that is necessary, but not sufficient, to support the existence of the whole-genome duplication event, our results prompt careful re-evaluation of paleopolyploidization in the yeast lineage and the evolutionary meaning of syntenic patterns. Reviewers This article was reviewed by Kenneth H. Wolfe (nominated by Nicolas Galtier, Austin L. Hughes (nominated by Eugene Koonin, Mikhail S. Gelfand, and Mark Gerstein.

  17. Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wang, Meng; Li, Sijin; Zhao, Huimin

    2016-01-01

    The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. PMID:26059511

  18. [Involvement of PHO80 and PHO85 genes in Saccharomyces cerevisiae ion tolerance].

    Science.gov (United States)

    Mao, Xi-Cheng; Xia, Yu-Lei; Hu, Ya-Fang; Lu, Chang-De

    2003-01-01

    PHO85 is a versatile gene in Saccharomyces cerevisiae, which is involved in metabolism of inorganic phosphate and usage of carbon source, accumulation of glycogen, regulation of protein stability and cell cycle control. The viability of wild type budding yeast strain YPH499 and its derivative pho85Delta mutant, pho80 mutant, and pap1(pcl-7)Delta mutant in different cations were investigated and their tolerance to the cations(LC(50)) was measured. The results showed that the deletion of PHO85 or PHO80 gene both increased sensibility of Sacchromyces cerevisiae to ions K(+), Mg(2+), Zn(2+), Ca(2+) and Mn(2+), while the deletion of pap1(pcl-7) gene did not lead to such phenotype. The difference between the patterns of relative growth curve of the mutants and wild type strain in the above ions also implied that PHO80 was the unique PCLs in complex with PHO85 CDK, that were contributed to K(+) and Mg(2+) ion homeostasis control and there were some other PCLs besides PHO80 that were involved in Zn(2+), Ca(2+) and Mn(2+) tolerance regulation as cyclin of PHO85 CDK. Furthermore, the amount of the total cellular calcium of pho85Delta mutant, pho80Delta mutant and YPH499 indicated that the ability of calcium accumulation of pho85 mutant and pho80Delta mutant was impaired. PMID:12518234

  19. Determination of the core promoter regions of the Saccharomyces cerevisiae RPS3 gene.

    Science.gov (United States)

    Joo, Yoo Jin; Kim, Jin-Ha; Baek, Joung Hee; Seong, Ki Moon; Lee, Jae Yung; Kim, Joon

    2009-01-01

    Ribosomal protein genes (RPG), which are scattered throughout the genomes of all eukaryotes, are subjected to coordinated expression. In yeast, the expression of RPGs is highly regulated, mainly at the transcriptional level. Recent research has found that many ribosomal proteins (RPs) function in multiple processes in addition to protein synthesis. Therefore, detailed knowledge of promoter architecture as well as gene regulation is important in understanding the multiple cellular processes mediated by RPGs. In this study, we investigated the functional architecture of the yeast RPS3 promoter and identified many putative cis-elements. Using beta-galactosidase reporter analysis and EMSA, the core promoter of RPS3 containing UASrpg and T-rich regions was corroborated. Moreover, the promoter occupancy of RPS3 by three transcription factors was confirmed. Taken together, our results further the current understanding of the promoter architecture and trans-elements of the Saccharomyces cerevisiae RPS3 gene. PMID:19853675

  20. Barosensitivity in Saccharomyces cerevisiae is Closely Associated with a Deletion of the COX1 Gene.

    Science.gov (United States)

    Nomura, Kazuki; Iwahashi, Hitoshi; Iguchi, Akinori; Shigematsu, Toru

    2015-05-01

    High hydrostatic pressure causes physical stress to microorganisms; therefore, this technology may be applied to food pasteurization without introducing the unfavorable effects of thermal denaturation. However, its application is limited to high-value foods because the treatment requires a robust steel vessel and expensive pressurization equipment. To reduce these costs, we studied the pasteurization of Saccharomyces cerevisiae using relatively moderate high-pressure levels. A mutant strain isolated by ultraviolet mutagenesis showed significant loss of viability under high-pressure conditions. Gene expression analysis of the mutant strain revealed that it incurred a deletion of the COX1 gene. Our results suggest that the pressure-sensitivity can readily be introduced into industrial/food microorganisms by complementing a COX1 deleted mitochondria. PMID:25881710

  1. Effects of the rad52 gene on recombination in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, γ-ray-induced, uv-induced, and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Intra- and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the hisl-1/hisl-315 and trp5-2/trp5-48 heteroalleles. Gene-centromere recombination was also not observed in rad52/rad52 diploids. No γ-ray-induced intragenic mitotic recombination is seen in rad52/rad52 diploids and uv-induced intragenic recombination is greatly reduced. However, spontaneous mitotic recombination is not similarly affected. The RAD52 gene thus functions in recombination in meiosis and in γ-ray and uv-induced mitotic recombination but not in spontaneous mitotic recombination

  2. POS5 Gene of Saccharomyces cerevisiae Encodes a Mitochondrial NADH Kinase Required for Stability of Mitochondrial DNA

    OpenAIRE

    Strand, Micheline K.; Stuart, Gregory R.; Longley, Matthew J.; Graziewicz, Maria A.; Dominick, Olivia C.; Copeland, William C.

    2003-01-01

    In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD+, with a twofold preference for NADH. Disruption of POS5 inc...

  3. Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins▿

    OpenAIRE

    Payne, T.; Finnis, C.; Evans, L R; Mead, D. J.; Avery, S V; Archer, D. B.; Sleep, D.

    2008-01-01

    The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regul...

  4. Abundant Gene-by-Environment Interactions in Gene Expression Reaction Norms to Copper within Saccharomyces cerevisiae

    OpenAIRE

    Hodgins-Davis, Andrea; Adomas, Aleksandra B; Warringer, Jonas; Townsend, Jeffrey P.

    2012-01-01

    Genetic variation for plastic phenotypes potentially contributes phenotypic variation to populations that can be selected during adaptation to novel ecological contexts. However, the basis and extent of plastic variation that manifests in diverse environments remains elusive. Here, we characterize copper reaction norms for mRNA abundance among five Saccharomyces cerevisiae strains to 1) describe population variation across the full range of ecologically relevant copper concentrations, from st...

  5. The rhp6+ gene of Schizosaccharomyces pombe: a structural and functional homolog of the RAD6 gene from the distantly related yeast Saccharomyces cerevisiae.

    NARCIS (Netherlands)

    P. Reynolds (Paul); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); S. Prakash; L. Prakash

    1990-01-01

    textabstractThe RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin conjugating enzyme and is required for DNA repair, DNA-damage-induced mutagenesis and sporulation. Here, we show that RAD6 and the rhp6+ gene from the distantly related yeast Schizosaccharomyces pombe share a high degree of st

  6. Repression of nitrogen catabolic genes by ammonia and glutamine in nitrogen-limited continuous cultures of Saccharomyces cerevisiae

    NARCIS (Netherlands)

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

    1998-01-01

    Growth of Saccharomyces cerevisiae on ammonia and glutamine decreases the expression of many nitrogen catabolic genes to low levels. To discriminate between ammonia- and glutamine-driven repression of GAP1, PUT4, GDH1 and GLN1, a gln1-37 mutant was used. This mutant is not able to convert ammonia in

  7. Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene.

    OpenAIRE

    Xiao, W; Derfler, B; J. Chen; Samson, L

    1991-01-01

    We previously identified and characterized biochemically an O6-methylguanine (O6MeG) DNA repair methyltransferase (MTase) in the yeast Saccharomyces cerevisiae and showed that it recognizes both O6MeG and O4-methylthymine (O4MeT) in vitro. Here we characterize the cloned S. cerevisiae O6MeG DNA MTase gene (MGT1) and determine its in vivo role in protecting yeast from DNA alkylation damage. We isolated a yeast DNA fragment that suppressed alkylation-induced killing and mutation in Escherichia ...

  8. [Cloning and expression of bacteriophage FMV lysocyme gene in cells of yeasts Saccharomyces cerevisiae and Pichia pastoris].

    Science.gov (United States)

    Kozlov, D G; Cheperigin, S E; Chestkov, A V; Krylov, V N; Tsygankov, Iu D

    2010-03-01

    Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form. PMID:20391778

  9. Local synteny and codon usage contribute to asymmetric sequence divergence of Saccharomyces cerevisiae gene duplicates

    Directory of Open Access Journals (Sweden)

    Bergthorsson Ulfar

    2011-09-01

    Full Text Available Abstract Background Duplicated genes frequently experience asymmetric rates of sequence evolution. Relaxed selective constraints and positive selection have both been invoked to explain the observation that one paralog within a gene-duplicate pair exhibits an accelerated rate of sequence evolution. In the majority of studies where asymmetric divergence has been established, there is no indication as to which gene copy, ancestral or derived, is evolving more rapidly. In this study we investigated the effect of local synteny (gene-neighborhood conservation and codon usage on the sequence evolution of gene duplicates in the S. cerevisiae genome. We further distinguish the gene duplicates into those that originated from a whole-genome duplication (WGD event (ohnologs versus small-scale duplications (SSD to determine if there exist any differences in their patterns of sequence evolution. Results For SSD pairs, the derived copy evolves faster than the ancestral copy. However, there is no relationship between rate asymmetry and synteny conservation (ancestral-like versus derived-like in ohnologs. mRNA abundance and optimal codon usage as measured by the CAI is lower in the derived SSD copies relative to ancestral paralogs. Moreover, in the case of ohnologs, the faster-evolving copy has lower CAI and lowered expression. Conclusions Together, these results suggest that relaxation of selection for codon usage and gene expression contribute to rate asymmetry in the evolution of duplicated genes and that in SSD pairs, the relaxation of selection stems from the loss of ancestral regulatory information in the derived copy.

  10. Genomewide screening for genes associated with gliotoxin resistance and sensitivity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chamilos, Georgios; Lewis, Russell E; Lamaris, Gregory A; Albert, Nathaniel D; Kontoyiannis, Dimitrios P

    2008-04-01

    Gliotoxin (GT) is a secondary fungal metabolite with pleiotropic immunosuppressive properties that have been implicated in Aspergillus virulence. However, the mechanisms of GT cytotoxicity and its molecular targets in eukaryotic cells have not been fully characterized. We screened a haploid library of Saccharomyces cerevisiae single-gene deletion mutants (4,787 strains in EUROSCARF) to identify nonessential genes associated with GT increased resistance (GT-IR) and increased sensitivity (GT-IS). The susceptibility of the wild-type parental strain BY4741 to GT was initially assessed by broth microdilution methods using different media. GT-IR and GT-IS were defined as a fourfold increase and decrease, respectively, in MIC, and this was additionally confirmed by susceptibility testing on agar yeast extract-peptone-glucose plates. The specificity of GT-IR and GT-IS mutants exhibiting normal growth compared with the wild-type strain was further tested in studies of their susceptibility to conventional antifungal agents, cycloheximide, and H2O2. GT-IR was associated with the disruption of genes acting in general metabolism (OPI1, SNF1, IFA38), mitochondrial function (RTG2), DNA damage repair (RAD18), and vesicular transport (APL2) and genes of unknown function (YGL235W, YOR345C, YLR456W, YGL072C). The disruption of three genes encoding transsulfuration (CYS3), mitochondrial function (MEF2), and an unknown function (YKL037W) led to GT-IS. Specificity for GT-IR and GT-IS was observed in all mutants. Importantly, the majority (69%) of genes implicated in GT-IR (6/10) and GT-IS (2/3) have human homologs. We identified novel Saccharomyces genes specifically implicated in GT-IR or GT-IS. Because most of these genes are evolutionarily conserved, further characterization of their function could improve our understanding of GT cytotoxicity mechanisms in humans. PMID:18212113

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

    Directory of Open Access Journals (Sweden)

    Karhumaa Kaisa

    2011-07-01

    Full Text Available Abstract 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

  12. Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brahmachari Samir K

    2001-10-01

    Full Text Available Abstract Background Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes. These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes. Results A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed. The sequence was inserted within a nucleosome positioned upstream of the β-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10Upstream Activating Sequences (UASg. Upon derepression with galactose, β-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences. This reduction in expression is correlated with reduced transcription. Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells. Conclusion We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription. Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells.

  13. Quantitative transcription dynamic analysis reveals candidate genes and key regulators for ethanol tolerance in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ma Menggen

    2010-06-01

    Full Text Available Abstract Background Derived from our lignocellulosic conversion inhibitor-tolerant yeast, we generated an ethanol-tolerant strain Saccharomyces cerevisiae NRRL Y-50316 by enforced evolutionary adaptation. Using a newly developed robust mRNA reference and a master equation unifying gene expression data analyses, we investigated comparative quantitative transcription dynamics of 175 genes selected from previous studies for an ethanol-tolerant yeast and its closely related parental strain. Results A highly fitted master equation was established and applied for quantitative gene expression analyses using pathway-based qRT-PCR array assays. The ethanol-tolerant Y-50316 displayed significantly enriched background of mRNA abundance for at least 35 genes without ethanol challenge compared with its parental strain Y-50049. Under the ethanol challenge, the tolerant Y-50316 responded in consistent expressions over time for numerous genes belonging to groups of heat shock proteins, trehalose metabolism, glycolysis, pentose phosphate pathway, fatty acid metabolism, amino acid biosynthesis, pleiotropic drug resistance gene family and transcription factors. The parental strain showed repressed expressions for many genes and was unable to withstand the ethanol stress and establish a viable culture and fermentation. The distinct expression dynamics between the two strains and their close association with cell growth, viability and ethanol fermentation profiles distinguished the tolerance-response from the stress-response in yeast under the ethanol challenge. At least 82 genes were identified as candidate and key genes for ethanol-tolerance and subsequent fermentation under the stress. Among which, 36 genes were newly recognized by the present study. Most of the ethanol-tolerance candidate genes were found to share protein binding motifs of transcription factors Msn4p/Msn2p, Yap1p, Hsf1p and Pdr1p/Pdr3p. Conclusion Enriched background of transcription abundance

  14. The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth.

    OpenAIRE

    Yoko-o, T; Matsui, Y; Yagisawa, H; Nojima, H; Uno, I; Toh-E, A

    1993-01-01

    Using the polymerase chain reaction technique, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) in the yeast Saccharomyces cerevisiae. The nucleotide sequence indicates that the gene encodes a polypeptide of 869 amino acid residues with a calculated molecular mass of 101 kDa. This polypeptide has both the X and Y regions conserved among mammalian PLC-beta, -gamma, and -delta, and the structure is most similar to that of mammalian PLC-delta. This ...

  15. MGA2 Is Involved in the Low-Oxygen Response Element-Dependent Hypoxic Induction of Genes in Saccharomyces cerevisiae

    OpenAIRE

    Jiang, Yide; Vasconcelles, Michael J.; Wretzel, Sharon; Light, Anne; Martin, Charles E.; Goldberg, Mark A.

    2001-01-01

    Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example, OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1 expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-induced OLE1 expression, we examined OLE1 expres...

  16. Studio nel sistema modello Saccharomyces cerevisiae di mutazioni patologiche nel gene POLG codificante la DNA polimerasi mitocondriale

    OpenAIRE

    Baruffini, Enrico

    2008-01-01

    Il lavoro riportato in questa tesi concerne lo studio nel sistema modello Saccharomyces cerevisiae di mutazioni patologiche nel gene umano POLG che codifica per la polimerasi mitocondriale gamma associate a patologie mitocondriali. Nello studio sono state introdotte sette mutazioni in POLG nelle posizioni equivalenti del gene ortologo di lievito MIP1. E’ stato studiato l’effetto di queste mutazioni sulla stabilità del DNA mitocondriale, dimostrando che tutte le mutazioni provocano un incremen...

  17. The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity.

    OpenAIRE

    Lin, S J; Culotta, V C

    1995-01-01

    In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as superoxide dismutase (SOD) and catalase. Here we describe the isolation and characterization of another gene in the yeast Saccharomyces cerevisiae that plays a critical role in detoxification of reactive oxygen species. This gene, named ATX1, was originally isolated by its ability to suppress oxygen toxicity in yeast lacking SOD. ATX1 encodes a 8.2-kDa poly...

  18. Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences.

    OpenAIRE

    Ogden, J E; Stanway, C; Kim, S.; Mellor, J; Kingsman, A J; Kingsman, S M

    1986-01-01

    The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell. To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene. Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full trans...

  19. Bulk segregant analysis by high-throughput sequencing reveals a novel xylose utilization gene from Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jared W Wenger

    2010-05-01

    Full Text Available Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing.

  20. EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Stovicek, Vratislav; Borja, Gheorghe M; Forster, Jochen; Borodina, Irina

    2015-11-01

    Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway. PMID:26376869

  1. RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates

    International Nuclear Information System (INIS)

    The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. The authors have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which the authors have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids. 48 references, 6 figures

  2. A comprehensive gene regulatory network for the diauxic shift in Saccharomyces cerevisiae.

    Science.gov (United States)

    Geistlinger, Ludwig; Csaba, Gergely; Dirmeier, Simon; Küffner, Robert; Zimmer, Ralf

    2013-10-01

    Existing machine-readable resources for large-scale gene regulatory networks usually do not provide context information characterizing the activating conditions for a regulation and how targeted genes are affected. Although this information is essentially required for data interpretation, available networks are often restricted to not condition-dependent, non-quantitative, plain binary interactions as derived from high-throughput screens. In this article, we present a comprehensive Petri net based regulatory network that controls the diauxic shift in Saccharomyces cerevisiae. For 100 specific enzymatic genes, we collected regulations from public databases as well as identified and manually curated >400 relevant scientific articles. The resulting network consists of >300 multi-input regulatory interactions providing (i) activating conditions for the regulators; (ii) semi-quantitative effects on their targets; and (iii) classification of the experimental evidence. The diauxic shift network compiles widespread distributed regulatory information and is available in an easy-to-use machine-readable form. Additionally, we developed a browsable system organizing the network into pathway maps, which allows to inspect and trace the evidence for each annotated regulation in the model. PMID:23873954

  3. Identification of dosage-sensitive genes in Saccharomyces cerevisiae using the genetic tug-of-war method

    OpenAIRE

    Makanae, Koji; Kintaka, Reiko; Makino, Takashi; Kitano, Hiroaki; Moriya, Hisao

    2013-01-01

    Gene overexpression beyond a permissible limit causes defects in cellular functions. However, the permissible limits of most genes are unclear. Previously, we developed a genetic method designated genetic tug-of-war (gTOW) to measure the copy number limit of overexpression of a target gene. In the current study, we applied gTOW to the analysis of all protein-coding genes in the budding yeast Saccharomyces cerevisiae. We showed that the yeast cellular system was robust against an increase in t...

  4. Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

    OpenAIRE

    Naumovski, L; Friedberg, E C

    1982-01-01

    We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective ...

  5. Effects of the rad52 gene on recombination in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Effects of the rad 52 mutation in Saccharomyces cerevisiae on meiotic, γ-ray-induced, uv-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the his1-1/his1-315 and trp-5-2/trp5-48 heteroalleles. Gene-centromere recombination also was not observed in rad/52/rad52 diploids. No γ-ray- or uv-induced intragenic mitotic recombination is seen in rad52/rad52 diploids. The rate of spontaneous mitotic recombination is lowered five-fold at the his1-1/his1-315 and leu1-c/leu1-12 heteroalleles. Spontaneous reversion rates of both his1-1 and his1-315 were elevated 10 to 20 fold in rad52/rad52 diploids. The RAD52 gene function is required for spontaneous mitotic recombination, uv- and γ-ray-induced mitotic recombination and mitotic recombination

  6. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae

    Science.gov (United States)

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  8. RAD6/sup +/ gene of Saccharomyces cerevisiae codes for two mutationally separable deoxyribonucleic acid repair functions

    International Nuclear Information System (INIS)

    The response of two mutant alleles of the RAD6/sup +/ gene of Saccharomyces cerevisiae to the ochre translational suppressor SUQ5 was determined. Both the ultraviolet sensitivity phenotype and the deficiency in ultraviolet-induced mutagenesis phenotype of the rad6-1 allelle were suppressed in a [psi/sup +/] background. For the rad6-3 allelle, only the ultraviolet-sensitivity phenotype was suppressible in a [psi/sup +/] background. An SUQ5 rad6-3 [psi/sup +/] strain that was examined showed the normal rad6-3 deficiency in ultraviolet-induced mutagenesis. The authors propose that the RAD6/sup +/ gene is divided into two cistrons, RAD6A and RAD6B. RAD6A codes for an activity responsible for the error-prone repair of ultraviolet-induced lesions in deoxyribonucleic acid but is not involved in a cell's resistance to the lethal effects of ultraviolet light. RAD6B codes for an activity essential for error-free repair of potentially lethal mutagenic damage

  9. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rantasalo, Anssi; Czeizler, Elena; Virtanen, Riitta; Rousu, Juho; Lähdesmäki, Harri; Penttilä, Merja; Jäntti, Jussi; Mojzita, Dominik

    2016-01-01

    This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal. The system activation is based on the bacterial LexA-DNA-binding domain, a set of modified, modular LexA-binding sites and a selection of transcription activation domains. We show both experimentally and computationally that the tuning of the system is achieved through the selection of three separate modules, each of which enables an adjustable output signal: 1) the transcription-activation domain of the sTF, 2) the binding-site modules in the output promoter, and 3) the core promoter modules which define the transcription initiation site in the output promoter. The system has a novel bidirectional architecture that enables generation of compact, yet versatile expression modules for multiple genes with highly diversified expression levels ranging from negligible to very strong using one synthetic transcription factor. In contrast to most existing modular gene expression regulation systems, the present system is independent from externally added compounds. Furthermore, the established system was minimally affected by the several tested growth conditions. These features suggest that it can be highly useful in large scale biotechnology applications. PMID:26901642

  10. Partial Rescue of pos5 Mutants by YEF1 and UTR1 Genes in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    Yong-Fu LI; Feng SHI

    2006-01-01

    Three NAD kinase homologs, encoded by UTR1, POS5 and YEF1 genes, are found in the yeast Saccharomyces cerevisiae and proven to be important sources of NADPH for the cell. Pos5p, existing in the mitochondrial matrix, is critical for higher temperature endurance and mitochondrial functions, such as glycerol usability and arginine biosynthesis. Through constructing the high-copy expression plasmids of YEF1 and UTR1, which contained the green fluorescent protein reporter tag at their 3' terminus, and introducing them into POS5 gene deletion mutants (i.e. pos5, utr1pos5, yef1pos5 and utr1yef1pos5), the high-copy YEF1 and UTR1 plasmids carrying transformants for pos5 mutants were obtained. Their temperature sensitivity and growth phenotype on media with glycerol as the sole carbon source, or on media without arginine, were checked. Results showed the partial rescue of mitochondrial dysfunctions and temperature sensitivity of pos5 mutants by the high-copy YEF1 gene, and of glycerol growth defect and temperature sensitivity by the high-copy UTR1 gene, which confirmed the potential supplying ability of Yef1p and Utr1p for mitochondrial NADP(H) and implied the weak transport of NADP from cytosol to mitochondria. However, even through the green fluorescent protein reporter label, the subcellular localization of Yef1p and Utr1p in yeast cells could not be observed, which indicated the low expression level of these two NAD kinase homologs.

  11. Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates

    OpenAIRE

    Pereira, Francisco B.; Teixeira, Miguel C.; Mira, Nuno P.; Correia, Isabel Sá; Domingues, Lucília

    2014-01-01

    The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulo...

  12. Interaction of the Srb10 Kinase with Sip4, a Transcriptional Activator of Gluconeogenic Genes in Saccharomyces cerevisiae

    OpenAIRE

    Vincent, Olivier; Kuchin, Sergei; Hong, Seung-Pyo; Townley, Robert; Vyas, Valmik K; Carlson, Marian

    2001-01-01

    Sip4 is a Zn2Cys6 transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptio...

  13. Telomere-Mediated Plasmid Segregation in Saccharomyces Cerevisiae Involves Gene Products Required for Transcriptional Repression at Silencers and Telomeres

    OpenAIRE

    Longtine, M. S.; Enomoto, S.; Finstad, S L; Berman, J

    1993-01-01

    Plasmids that contain Saccharomyces cerevisiae TG(1-3) telomere repeat sequences (TRS plasmids) segregate efficiently during mitosis. Mutations in histone H4 reduce the efficiency of TRS-mediated plasmid segregation, suggesting that chromatin structure is involved in this process. Sir2, Sir3 and Sir4 are required for the transcriptional repression of genes located at the silent mating type loci (HML and HMR) and at telomeres (telomere position effect) and are also involved in the segregation ...

  14. Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.

    Science.gov (United States)

    Klauser, Benedikt; Atanasov, Janina; Siewert, Lena K; Hartig, Jörg S

    2015-05-15

    Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches. PMID:24871672

  15. The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Brummer, M H; Richard, P; Sundqvist, L; Väänänen, R; Keränen, S

    2001-07-01

    The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19-1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19-1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes. PMID:11447595

  16. Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity

  17. Expression of the bacterial recA gene impairs genetic recombination and sporulation in a Saccharomyces cerevisiae diploid strain

    Directory of Open Access Journals (Sweden)

    Marcos Antonio de Morais Junior

    2003-01-01

    Full Text Available The Escherichia coli RecA protein (RecAp has been demonstrated to induce mutagenesis in yeast cells, although there is still little information on the role of the RecAp in yeast recombination events. We evaluated spontaneous and induced general recombination in vegetative and meiotic cells of the XS2316 strain of the yeast Saccharomyces cerevisiae bearing the recA gene. We found that RecAp decreased reciprocal recombination, gene conversion and intrachromosomal recombination and promoted an increase in error-prone processes in both vegetative and meiotic cells, while its negative effect on meiotic recombination blocked ascospore formation.

  18. EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Niels Bjerg; Strucko, Tomas; Kildegaard, Kanchana Rueksomtawin;

    2014-01-01

    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...

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

    OpenAIRE

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

    2006-01-01

    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,...

  20. Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol.

    Science.gov (United States)

    Madhavan, Anjali; Tamalampudi, Sriappareddy; Ushida, Kazunari; Kanai, Daisuke; Katahira, Satoshi; Srivastava, Aradhana; Fukuda, Hideki; Bisaria, Virendra S; Kondo, Akihiko

    2009-04-01

    The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37 degrees C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose. PMID:19050860

  1. Assessment of the toxicity of CuO nanoparticles by using Saccharomyces cerevisiae mutants with multiple genes deleted.

    Science.gov (United States)

    Bao, Shaopan; Lu, Qicong; Fang, Tao; Dai, Heping; Zhang, Chao

    2015-12-01

    To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles. PMID:26386067

  2. A MultiSite GatewayTM vector set for the functional analysis of genes in the model Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Nagels Durand Astrid

    2012-09-01

    Full Text Available Abstract Background Recombinatorial cloning using the GatewayTM technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in GatewayTM compatible vectors. The MultiSite GatewayTM system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast Saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors. Results Here, we present a set of three-fragment MultiSite GatewayTM destination vectors that have been developed for gene expression in S. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins. Conclusion Our vectors make MultiSite GatewayTM cloning accessible in S. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous proteins in one of the most widely used model organisms for molecular biology research.

  3. Transcriptional expression of selected genes associated with excretion of carboxylic acids from aci mutants of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ewa Boniewska-Bernacka

    2013-04-01

    Full Text Available Introduction: Saccharomyces cerevisiae is an excellent model organism for studies of transcriptional regulation of metabolic processes in other eukaryotic cells including human cells. Cellular acid-base balance can be disturbed in pathologic situations such as renal acidosis or cancer. The extracellular pH of malignant solid tumors is acidic in the range of 6.5-6.9. EG07 and EG37 aci mutants of Saccharomyces cerevisiae excessively excrete carboxylic acids to glucose-containing media or distilled water. The excreted acids are Krebs and/or glyoxylate cycle intermediates. The genes restoring the wild-type phenotype have function that does not easily explain theAci phenotype.Material/Methods: In this study, using real-time PCR we measured relative mRNA expression, in the mutants compared to the wild-type strain, of selected genes associated with both carboxylic acid cycles and two cell transporters, Pma1 and Pdr12, of organic acids. Results: Unexpectedly, we found that the relative expression of the selected Krebs cycle and glyoxylate cycle genes did not change significantly. However, the expression of the two transporter genes was strongly elevated in EG37 and moderately increased in EG07.Conclusion: These results indicate that the induction of the two cell transporterg enes plays an important role in acid excretion by the aci mutants.

  4. Genome-wide analysis of the effect of histone modifications on the coexpression of neighboring genes in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Wang Jiang

    2010-10-01

    Full Text Available Abstract Background Neighboring gene pairs in the genome of Saccharomyces cerevisiae have a tendency to be expressed at the same time. The distribution of histone modifications along chromatin fibers is suggested to be an important mechanism responsible for such coexpression. However, the extent of the contribution of histone modifications to the coexpression of neighboring genes is unclear. Results We investigated the similarity of histone modification between neighboring genes using autocorrelation analysis and composite profiles. Our analysis showed that neighboring genes had similar levels or changes of histone modifications, especially those transcribed in the same direction. The similarities, however, were restricted to 1 or 2 neighboring genes. Moreover, the expression of a gene was significantly correlated with histone modification of its neighboring gene(s, but this was limited to only 1 or 2 neighbors. Using a hidden Markov model (HMM, we found more than 2000 chromatin domains with similar acetylation changes as the cultures changed and a considerable number of these domains covered 2-4 genes. Gene pairs within domains exhibited a higher level of coexpression than random pairs and shared similar functions. Conclusions The results of this study suggest that similar histone modifications occur within only a small local chromatin region in yeast. The modifications generally have an effect on coexpression with only 1 or 2 neighboring genes. Some blocking mechanism(s might strictly restrain the distribution of histone modifications in yeast.

  5. The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].

    Science.gov (United States)

    Ellul, P; Ríos, G; Atarés, A; Roig, L A; Serrano, R; Moreno, V

    2003-08-01

    An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species. PMID:12783167

  6. Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Knudsen, Jan Dines; Johanson, Ted; Eliasson Lantz, Anna; Carlquist, Magnus

    2015-01-01

    A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by p...... mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.......A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by...... growth rate of 0.3 h-1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry...

  7. Identification of dosage-sensitive genes in Saccharomyces cerevisiae using the genetic tug-of-war method.

    Science.gov (United States)

    Makanae, Koji; Kintaka, Reiko; Makino, Takashi; Kitano, Hiroaki; Moriya, Hisao

    2013-02-01

    Gene overexpression beyond a permissible limit causes defects in cellular functions. However, the permissible limits of most genes are unclear. Previously, we developed a genetic method designated genetic tug-of-war (gTOW) to measure the copy number limit of overexpression of a target gene. In the current study, we applied gTOW to the analysis of all protein-coding genes in the budding yeast Saccharomyces cerevisiae. We showed that the yeast cellular system was robust against an increase in the copy number by up to 100 copies in >80% of the genes. After frameshift and segmentation analyses, we isolated 115 dosage-sensitive genes (DSGs) with copy number limits of 10 or less. DSGs contained a significant number of genes involved in cytoskeletal organization and intracellular transport. DSGs tended to be highly expressed and to encode protein complex members. We demonstrated that the protein burden caused the dosage sensitivity of highly expressed genes using a gTOW experiment in which the open reading frame was replaced with GFP. Dosage sensitivities of some DSGs were rescued by the simultaneous increase in the copy numbers of partner genes, indicating that stoichiometric imbalances among complexes cause dosage sensitivity. The results obtained in this study will provide basic knowledge about the physiology of chromosomal abnormalities and the evolution of chromosomal composition. PMID:23275495

  8. Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates.

    Science.gov (United States)

    Pereira, Francisco B; Teixeira, Miguel C; Mira, Nuno P; Sá-Correia, Isabel; Domingues, Lucília

    2014-12-01

    The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH. PMID:25287021

  9. PAS3, a Saccharomyces cerevisiae Gene Encoding a Peroxisomal Integral Membrane Protein Essential for Peroxisome Biogenesis

    NARCIS (Netherlands)

    Höhfeld, Jörg; Veenhuis, Marten; Kunau, Wolf-H.

    1991-01-01

    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

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

    DEFF Research Database (Denmark)

    Regenberg, Birgitte; Grotkjaer, Thomas; Winther, Ole;

    2006-01-01

    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...

  11. KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae.

    Science.gov (United States)

    Staneva, Dessislava; Uccelletti, Daniela; Farina, Francesca; Venkov, Pencho; Palleschi, Claudio

    2004-01-15

    Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity. PMID:14745781

  12. KTI11 and KTI13, Saccharomyces cerevisiae genes controlling sensitivity to G1 arrest induced by Kluyveromyces lactis zymocin.

    Science.gov (United States)

    Fichtner, Lars; Schaffrath, Raffael

    2002-05-01

    The Kluyveromyces lactis zymocin and its gamma-toxin subunit inhibit cell cycle progression of Saccharomyces cerevisiae. To identify S. cerevisiae genes conferring zymocin sensitivity, we complemented the unclassified zymocin-resistant kti11 and kti13 mutations using a single-copy yeast library. Thus, we identified yeast open reading frames (ORFs) YBL071w-A and YAL020c/ATS1 as KTI11 and KTI13 respectively. Disruption of KTI11 and KTI13 results in the complex tot phenotype observed for the gamma-toxin target site mutants, tot1-7, and includes zymocin resistance, thermosensitivity, hypersensitivity to drugs and slow growth. Both loci, KTI11 and KTI13, are actively transcribed protein-encoding genes as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and in vivo HA epitope tagging. Kti11p is highly conserved from yeast to man, and Kti13p/Ats1p is related to yeast Prp20p and mammalian RCC1, components of the Ran-GTP/GDP cycle. Combining disruptions in KTI11 or KTI13 with a deletion in TOT3/ELP3 coding for the RNA polymerase II (RNAPII) Elongator histone acetyltransferase (HAT) yielded synthetic effects on slow growth phenotype expression. This suggests genetic interaction and possibly links KTI11 and KTI13 to Elongator function. PMID:11994165

  13. Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

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

    2012-01-01

    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 t...... 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....

  14. RAD24 (=R1/sup S/) gene product of Saccharomyces cerevisiae participates in two different pathways of DNA repair

    International Nuclear Information System (INIS)

    The moderately UV- and X-ray-sensitive mutant of Saccharomyces cerevisiae originally designated r1/sup s/ complements all rad and mms mutants available. Therefore, the new nomination rad24-1 according to the RAD nomenclature is suggested. RAD24 maps on chromosome V, close to RAD3 (1.3 cM). In order to associate the RAD24 gene with one of the three repair pathways, double mutants of rad24 and various representative genes of each pathway were constructed. The UV and X-ray sensitivities of the double mutants compared to the single mutants indicate that RAD24 is involved in excision repair of UV damage (RAD3 epistasis group), as well as in recombination repair of UV and X-ray damage (RAD52 epistasis group). Properties of the mutant are discussed which hint at the control of late steps in the pathways

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

    Science.gov (United States)

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

    2015-01-01

    Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products. PMID:24733517

  16. GIT1, a gene encoding a novel transporter for glycerophosphoinositol in Saccharomyces cerevisiae.

    OpenAIRE

    Patton-Vogt, J L; Henry, S A

    1998-01-01

    Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git-) defective in the uptake and metabolism of GroPIns. One ...

  17. A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae.

    Science.gov (United States)

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

    2016-07-01

    The conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae. PMID:27070284

  18. GAP1, a novel selection and counter-selection marker for multiple gene disruptions in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Regenberg, Birgitte; Hansen, J.

    2000-01-01

    We report on the use of a new homologous marker for use in multiple gene deletions in S, cerevisiae, the general amino acid permease gene (GAP1), A GAP1 strain can utilize L-citrulline as the sole nitrogen source but cannot grow in the presence of the toxic amino acid D-histidine, L-citrulline as...... well as D-histidine uptake is mediated solely by the general amino acid permease, and a gap1 strain is therefore able to grow in the presence of D-histidine but cannot utilize L-citrulline, Gene disruption is effected by transforming a gap1 strain with a gene cassette generated by PCR, containing GAP1...... the GAP1 gene. This is caused by recombination between two Salmonella typuimurium hisG direct repeats embracing GAP1, and will result in a sub-population of gap1 cells. Such cells are selected on a medium containing D-histidine, and may subsequently be used for a second gene disruption. Hence...

  19. Expression and secretion of the Candida wickerhamii extracellular beta-glucosidase gene, bglB, in Saccharomyces cerevisiae.

    Science.gov (United States)

    Skory, C D; Freer, S N; Bothast, R J

    1996-11-01

    The yeast Candida wickerhamii exports a cell-associated beta-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active beta-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-micro replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the alpha-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity. PMID:8929394

  20. The Complete Set of Predicted Genes from Saccharomyces cerevisiae in a Readily Usable Form

    OpenAIRE

    Hudson, James R.; Dawson, Elliott P.; Rushing, Kimberly L.; Jackson, Cynthia H.; Lockshon, Daniel; Conover, Diana; Lanciault, Christian; Harris, James R.; Simmons, Steven J.; Rothstein, Rodney; Fields, Stanley

    1997-01-01

    Nearly all of the open reading frames (ORFs) of the yeast Saccharomyces cerevisiae have been synthesized by PCR using a set of ∼6000 primer pairs. Each of the forward primers has a common 22-base sequence at its 5′ end, and each of the back primers has a common 20-base sequence at its 5′ end. These common termini allow reamplification of the entire set of original PCR products using a single pair of longer primers—in our case, 70 bases. The resulting 70-base elements that flank each ORF can b...

  1. Development of new USER-based cloning vectors for multiple genes expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Jensen, Niels Bjerg; Maury, Jerome;

    2013-01-01

    the production strain with the proper phenotype and product yield. However, the sequential number of metabolic engineering is time-consuming. Furthermore, the number of available selectable markers is also limiting the number of genetic modifications. To overcome these limitations, we have developed a......Saccharomyces cerevisiae is one of the most widely used cell factory in industrial biotechnology and it is used for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. Such bioprocesses frequently require multiple rounds of metabolic engineering to obtain...

  2. A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology.

    Directory of Open Access Journals (Sweden)

    Florante Ricarte

    Full Text Available Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface - ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY. Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes - MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

  3. Expression of a bacterial ice nucleation gene in a yeast Saccharomyces cerevisiae and its possible application in food freezing processes.

    Science.gov (United States)

    Hwang, W Z; Coetzer, C; Tumer, N E; Lee, T C

    2001-10-01

    A 3.6 kb ice nucleation gene (ina) isolated from Erwinia herbicola was placed under control of the galactose-inducible promoter (GAL1) and introduced into Saccharomyces cerevisiae. Yeast transformants showed increased ice nucleation activity over untransformed controls. The freezing temperature of a small volume of water droplets containing yeast cells was increased from approximately -13 degrees C in the untransformed controls to -6 degrees C in ina-expressing (Ina(+)) transformants. Lower temperature growth of Ina(+) yeast at temperatures below 25 degrees C was required for the expression of ice nucleation activity. Shift of temperature to 5-20 degrees C could induce the ice nucleation activity of Ina(+) yeast when grown at 25 degrees C, and maximum ice nucleation activity was achieved after induction at 5 degrees C for approximately 12 h. The effects of Ina(+) yeast on freezing and texturization of several food materials was also demonstrated. PMID:11600004

  4. Interactions of checkpoint-genes RAD9, RAD17, RAD24 and RAD53 determining radioresistance of Yeast Saccharomyces Cerevisiae

    International Nuclear Information System (INIS)

    The mechanisms of genetic control of progress through the division cell cycle (checkpoint-control) in yeast Saccharomyces cerevisiae have been studied intensively. To investigate the role of checkpoint-genes RAD9, RAD17, RAD24, RAD53 in cell radioresistance we have investigated cell sensitivity of double mutants to γ-ray. Double mutants involving various combinations with rad9Δ show epistatic interactions, i.e. the sensitivity of the double mutants to γ-ray was no greater than that of more sensitive of the two single mutants. This suggests that all these genes govern the same pathway. This group of genes was named RAD9-epistasis group. It is interesting to note that the genes RAD9 and RAD53 have positive effect but RAD17 and RAD24 have negative effect on radiosensitivity of yeast cells. Interactions between mutations may differ depending on the agent γ-ray or UV-light, for example mutations rad9Δ and rad24Δ show additive effect for γ-ray and epistatic effect for UV-light

  5. Comparative study of Saccharomyces cerevisiae wine strains to identify potential marker genes correlated to desiccation stress tolerance.

    Science.gov (United States)

    Capece, Angela; Votta, Sonia; Guaragnella, Nicoletta; Zambuto, Marianna; Romaniello, Rossana; Romano, Patrizia

    2016-05-01

    The most diffused formulation of starter for winemaking is active dry yeast (ADY). ADYs production process is essentially characterized by air-drying stress, a combination of several stresses, including thermal, hyperosmotic and oxidative and cell capacity to counteract such multiple stresses will determine its survival. The molecular mechanisms underlying cell stress response to desiccation have been mostly studied in laboratory and commercial yeast strains, but a growing interest is currently developing for indigenous yeast strains which represent a valuable and alternative source of genetic and molecular biodiversity to be exploited. In this work, a comparative study of different Saccharomyces cerevisiae indigenous wine strains, previously selected for their technological traits, has been carried out to identify potentially relevant genes involved in desiccation stress tolerance. Cell viability was evaluated along desiccation treatment and gene expression was analyzed by real-time PCR before and during the stress. Our data show that the observed differences in individual strain sensitivity to desiccation stress could be associated to specific gene expression over time. In particular, either the basal or the stress-induced mRNA levels of certain genes, such as HSP12, SSA3, TPS1, TPS2, CTT1 and SOD1, result tightly correlated to the strain survival advantage. This study provides a reliable and sensitive method to predict desiccation stress tolerance of indigenous wine yeast strains which could be preliminary to biotechnological applications. PMID:26882930

  6. Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the genes in the FUN38-MAK16-SPO7 region.

    OpenAIRE

    Barton, A B; Kaback, D B

    1994-01-01

    Transcribed regions on a 42-kb segment of chromosome I from Saccharomyces cerevisiae were mapped. Polyadenylated transcripts corresponding to eight previously characterized genes (MAK16, LTE1, CCR4, FUN30, FUN31, TPD3, DEP1, and CYS3) and eight new genes were identified. All transcripts were present at one to four copies per cell except for one which was significantly less abundant. This region has been sequenced, and the sizes, locations, and orientations of the transcripts were in nearly pe...

  7. Significant enhancement of methionol production by co-expression of the aminotransferase gene ARO8 and the decarboxylase gene ARO10 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yin, Sheng; Lang, Tiandan; Xiao, Xiao; Liu, Li; Sun, Baoguo; Wang, Chengtao

    2015-03-01

    Methionol is an important volatile sulfur flavor compound, which can be produced via the Ehrlich pathway in Saccharomyces cerevisiae. Aminotransferase and decarboxylase are essential enzymes catalyzing methionol biosynthesis. In this work, two aminotransferase genes ARO8 and ARO9 and one decarboxylase gene ARO10 were introduced into S. cerevisiae S288c, respectively, via an expression vector. Over-expression of ARO8 resulted in higher aminotransferase activity than that of ARO9. And the cellular decarboxylase activity was remarkably increased by over-expression of ARO10. A co-expression vector carrying both ARO8 and ARO10 was further constructed to generate the recombinant strain S810. Shaking flask experiments showed that the methionol yield from S810 reached 1.27 g L(-1), which was increased by 51.8 and 68.8% compared to that from the wild-type strain and the control strain harboring the empty vector. The fed-batch fermentation by strain S810 produced 3.24 g L(-1) of methionol after 72 h of cultivation in a bioreactor. These results demonstrated that co-expression of ARO8 and ARO10 significantly boosted the methionol production. It is the first time that more than 3.0 g L(-1) of methionol produced by genetically engineered yeast strain was reported by co-expression of the aminotransferase and decarboxylase via the Ehrlich pathway. PMID:25743068

  8. Dithizone staining of intracellular zinc: an unexpected and versatile counterscreen for auxotrophic marker genes in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Daniel S Yuan

    Full Text Available Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle.

  9. The roles of whole-genome and small-scale duplications in the functional specialization of Saccharomyces cerevisiae genes.

    Directory of Open Access Journals (Sweden)

    Mario A Fares

    Full Text Available Researchers have long been enthralled with the idea that gene duplication can generate novel functions, crediting this process with great evolutionary importance. Empirical data shows that whole-genome duplications (WGDs are more likely to be retained than small-scale duplications (SSDs, though their relative contribution to the functional fate of duplicates remains unexplored. Using the map of genetic interactions and the re-sequencing of 27 Saccharomyces cerevisiae genomes evolving for 2,200 generations we show that SSD-duplicates lead to neo-functionalization while WGD-duplicates partition ancestral functions. This conclusion is supported by: (a SSD-duplicates establish more genetic interactions than singletons and WGD-duplicates; (b SSD-duplicates copies share more interaction-partners than WGD-duplicates copies; (c WGD-duplicates interaction partners are more functionally related than SSD-duplicates partners; (d SSD-duplicates gene copies are more functionally divergent from one another, while keeping more overlapping functions, and diverge in their sub-cellular locations more than WGD-duplicates copies; and (e SSD-duplicates complement their functions to a greater extent than WGD-duplicates. We propose a novel model that uncovers the complexity of evolution after gene duplication.

  10. High-throughput phenotypic profiling of gene-environment interactions by quantitative growth curve analysis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Weiss, Andrew; Delproposto, James; Giroux, Craig N

    2004-04-01

    Cell-based assays are widely used in high-throughput screening to determine the effects of toxicants and drugs on their biological targets. To enable a functional genomics modeling of gene-environment interactions, quantitative assays are required both for gene expression and for the phenotypic responses to environmental challenge. To address this need, we describe an automated high-throughput methodology that provides phenotypic profiling of the cellular responses to environmental stress in Saccharomyces cerevisiae. Standardized assay conditions enable the use of a single metric value to quantify yeast microculture growth curves. This assay format allows precise control of both genetic and environmental determinants of the cellular responses to oxidative stress, a common mechanism of environmental insult. These yeast-cell-based assays are validated with hydrogen peroxide, a simple direct-acting oxidant. Phenotypic profiling of the oxidative stress response of a yap1 mutant strain demonstrates the mechanistic analysis of genetic susceptibility to oxidative stress. As a proof of concept for analysis of more complex gene-environment interactions, we describe a combinatorial assay design for phenotypic profiling of the cellular responses to tert-butyl hydroperoxide, a complex oxidant that is actively metabolized by its target cells. Thus, the yeast microculture assay format supports comprehensive applications in toxicogenomics. PMID:15033507

  11. A genome-wide imaging-based screening to identify genes involved in synphilin-1 inclusion formation in Saccharomyces cerevisiae

    Science.gov (United States)

    Zhao, Lei; Yang, Qian; Zheng, Ju; Zhu, Xuefeng; Hao, Xinxin; Song, Jia; Lebacq, Tom; Franssens, Vanessa; Winderickx, Joris; Nystrom, Thomas; Liu, Beidong

    2016-01-01

    Synphilin-1 is a major component of Parkinson’s disease (PD) inclusion bodies implicated in PD pathogenesis. However, the machinery controlling synphilin-1 inclusion formation remains unclear. Here, we investigated synphilin-1 inclusion formation using a systematic genome-wide, high-content imaging based screening approach (HCI) in the yeast Saccharomyces cerevisiae. By combining with a secondary screening for mutants showing significant changes on fluorescence signal intensity, we filtered out hits that significantly decreased the expression level of synphilin-1. We found 133 yeast genes that didn’t affect synphilin-1 expression but that were required for the formation of synphilin-1 inclusions. Functional enrichment and physical interaction network analysis revealed these genes to encode for functions involved in cytoskeleton organization, histone modification, sister chromatid segregation, glycolipid biosynthetic process, DNA repair and replication. All hits were confirmed by conventional microscopy. Complementation assays were performed with a selected group of mutants, results indicated that the observed phenotypic changes in synphilin-1 inclusion formation were directly caused by the loss of corresponding genes of the deletion mutants. Further growth assays of these mutants showed a significant synthetic sick effect upon synphilin-1 expression, which supports the hypothesis that matured inclusions represent an end stage of several events meant to protect cells against the synphilin-1 cytotoxicity. PMID:27440388

  12. MGA2 or SPT23 is required for transcription of the delta9 fatty acid desaturase gene, OLE1, and nuclear membrane integrity in Saccharomyces cerevisiae.

    OpenAIRE

    S. Zhang; Skalsky, Y; Garfinkel, D J

    1999-01-01

    MGA2 and SPT23 are functionally and genetically redundant homologs in Saccharomyces cerevisiae. Both genes are implicated in the transcription of a subset of genes, including Ty retrotransposons and Ty-induced mutations. Neither gene is essential for growth, but mga2 spt23 double mutants are inviable. We have isolated a gene-specific activator, SWI5, and the Delta9 fatty acid desaturase of yeast, OLE1, as multicopy suppressors of an mga2Delta spt23 temperature-sensitive mutation (spt23-ts). T...

  13. Heme regulates the expression in Saccharomyces cerevisiae of chimaeric genes containing 5'-flanking soybean leghemoglobin sequences

    DEFF Research Database (Denmark)

    Jensen, E O; Marcker, K A; Villadsen, IS

    1986-01-01

    The TM1 yeast mutant was transformed with a 2 micron-derived plasmid (YEp24) which carries a chimaeric gene containing the Escherichia coli chloramphenicol acetyl transferase (CAT) gene fused to the 5'- and 3'-flanking regions of the soybean leghemoglobin (Lb) c3 gene. Expression of the chimaeric...... CAT gene is controlled specifically by heme at a post-transcriptional level, most likely by regulating the efficiencies of translation. Expression of another chimaeric gene consisting of the neomycin phosphotransferase (NPTII) gene fused to only the 5'-flanking region of the Lbc3 gene is regulated by...

  14. Improvement of Ethanol Production in Saccharomyces cerevisiae by High-Efficient Disruption of the ADH2 Gene Using a Novel Recombinant TALEN Vector

    Science.gov (United States)

    Ye, Wei; Zhang, Weimin; Liu, Taomei; Tan, Guohui; Li, Haohua; Huang, Zilei

    2016-01-01

    Bioethanol is becoming increasingly important in energy supply and economic development. However, the low yield of bioethanol and the insufficiency of high-efficient genetic manipulation approaches limit its application. In this study, a novel transcription activator-like effector nuclease (TALEN) vector containing the left and right arms of TALEN was electroporated into Saccharomyces cerevisiae strain As2.4 to sequence the alcohol dehydrogenase gene ADH2 and the hygromycin-resistant gene hyg. Western blot analysis using anti-FLAG monoclonal antibody proved the successful expression of TALE proteins in As2.4 strains. qPCR and sequencing demonstrated the accurate knockout of the 17 bp target gene with 80% efficiency. The TALEN vector and ADH2 PCR product were electroporated into ΔADH2 to complement the ADH2 gene (ADH2+ As2.4). LC–MS and GC were employed to detect ethanol yields in the native As2.4, ΔADH2 As2.4, and ADH2+ As2.4 strains. Results showed that ethanol production was improved by 52.4 ± 5.3% through the disruption of ADH2 in As2.4. The bioethanol yield of ADH2+ As2.4 was nearly the same as that of native As2.4. This study is the first to report on the disruption of a target gene in S. cerevisiae by employing Fast TALEN technology to improve bioethanol yield. This work provides a novel approach for the disruption of a target gene in S. cerevisiae with high efficiency and specificity, thereby promoting the improvement of bioethanol production in S. cerevisiae by metabolic engineering. PMID:27462304

  15. The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene.

    OpenAIRE

    Schüller, C; Brewster, J L; M. R. Alexander; Gustin, M C; Ruis, H

    1994-01-01

    The HOG signal pathway of the yeast Saccharomyces cerevisiae is defined by the PBS2 and HOG1 genes encoding members of the MAP kinase kinase and of the MAP kinase family, respectively. Mutations in this pathway (deletions of PBS2 or HOG1, or point mutations in HOG1) almost completely abolish the induction of transcription by osmotic stress that is mediated by stress response elements (STREs). We have demonstrated previously that STREs also mediate induction of transcription by heat shock, nit...

  16. The endocytosis gene END3 is essential for the glucose-induced rapid decline of small vesicles in the extracellular fraction in Saccharomyces cerevisiae

    OpenAIRE

    Chiang, Hui-Ling; Giardina, Bennett J.; Stein, Kathryn

    2014-01-01

    Background: Protein secretion is a fundamental process in all living cells. Gluconeogenic enzymes are secreted when Saccharomyces cerevisiae are grown in media containing low glucose. However, when cells are transferred to media containing high glucose, they are internalized. We investigated whether or not gluconeogenic enzymes were associated with extracellular vesicles in glucose-starved cells. We also examined the role that the endocytosis gene END3 plays in the internalization of extracel...

  17. Differential control of Zap1-regulated genes in response to zinc deficiency in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Wu Chang-Yi

    2008-08-01

    Full Text Available Abstract Background The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. We previously used transcriptome profiling with DNA microarrays to identify 46 potential Zap1 target genes in the yeast genome. In this new study, we used complementary methods to identify additional Zap1 target genes. Results With alternative growth conditions for the microarray experiments and a more sensitive motif identification algorithm, we identified 31 new potential targets of Zap1 activation. Moreover, an analysis of the response of Zap1 target genes to a range of zinc concentrations and to zinc withdrawal over time demonstrated that these genes respond differently to zinc deficiency. Some genes are induced under mild zinc deficiency and act as a first line of defense against this stress. First-line defense genes serve to maintain zinc homeostasis by increasing zinc uptake, and by mobilizing and conserving intracellular zinc pools. Other genes respond only to severe zinc limitation and act as a second line of defense. These second-line defense genes allow cells to adapt to conditions of zinc deficiency and include genes involved in maintaining secretory pathway and cell wall function, and stress responses. Conclusion We have identified several new targets of Zap1-mediated regulation. Furthermore, our results indicate that through the differential regulation of its target genes, Zap1 prioritizes mechanisms of zinc homeostasis and adaptive responses to zinc deficiency.

  18. Zinc-regulated genes in Saccharomyces cerevisiae revealed by transposon tagging.

    OpenAIRE

    Yuan, D S

    2000-01-01

    The biochemistry of human nutritional zinc deficiency remains poorly defined. To characterize in genetic terms how cells respond to zinc deprivation, zinc-regulated genes (ZRG's) were identified in yeast. Gene expression was probed using random lacZ reporter gene fusions, integrated by transposon tagging into a diploid genome as previously described. About half of the genome was examined. Cells exhibiting differences in lacZ expression on low or moderate ( approximately 0. 1 vs. 10 microm) zi...

  19. Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

    Science.gov (United States)

    Wang, Jiancai; Xu, Ronghua; Wang, Ruling; Haque, Mohammad Enamul; Liu, Aizhong

    2016-06-01

    The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts. PMID:26865376

  20. Secretory Expression and Characterization of an Acidic Endo-Polygalacturonase Gene from Aspergillus niger SC323 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhou, Huoxiang; Li, Xi; Guo, Mingyue; Xu, Qingrui; Cao, Yu; Qiao, Dairong; Cao, Yi; Xu, Hui

    2015-07-01

    The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing the α-factor signal peptide of yeast. The full-length cDNA consists of 1,113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in the culture supernatant can reach up to 1,448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50°C, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca(2+), Cu(2+), and Na(+), and strongly inhibited by Pb(2+) and Mn(2+). The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in the food and feed industries. PMID:25737122

  1. Expression of the hepatitis B surface antigen gene containing the preS2 region in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yoshida,Iwao

    1991-02-01

    Full Text Available We constructed a plasmid, pBH103-ME5, in which the region encoding the 10 preS2 amino acid residues and the S domain of the hepatitis B surface antigen (HBsAg were regulated by the promoter of the yeast repressible acid phosphatase gene. Saccharomyces cerevisiae carrying pBH103-ME5 produced the HBs antigen (yHBsAg, when it was cultured in a medium containing a low concentration of phosphate. The antigen was purified to homogeneity. Its molecular weight was determined by Western blotting to be 24,000, and its amino acid composition agreed well with that deduced from the nucleotide sequence. The C-terminal amino acid sequence of yHBsAg was exactly the same as that predicted from the nucleotide sequence, while the N-terminal amino acid acetylserine, which was followed by 8 amino acid residues coded by the preS2 region. These results indicate that the recombinant yeast produced a single polypeptide consisting of the preS2 region and the subsequent S domain after being processed at the N-terminus

  2. Selection and validation of reference genes for quantitative real-time PCR studies during Saccharomyces cerevisiae alcoholic fermentation in the presence of sulfite.

    Science.gov (United States)

    Nadai, Chiara; Campanaro, Stefano; Giacomini, Alessio; Corich, Viviana

    2015-12-23

    Sulfur dioxide is extensively used during industrial fermentations and contributes to determine the harsh conditions of winemaking together with low pH, high sugar content and increasing ethanol concentration. Therefore the presence of sulfite has to be considered in yeast gene expression studies to properly understand yeast behavior in technological environments such as winemaking. A reliable expression pattern can be obtained only using an appropriate reference gene set that is constitutively expressed regardless of perturbations linked to the experimental conditions. In this work we tested 15 candidate reference genes suitable for analysis of gene expression during must fermentation in the presence of sulfite. New reference genes were selected from a genome-wide expression experiment, obtained by RNA sequencing of four Saccharomyces cerevisiae wine strains grown in enological conditions. Their performance was compared to that of the most common genes used in previous studies. The most popular software based on different statistical approaches (geNorm, NormFinder and BestKeeper) were chosen to evaluate expression stability of the candidate reference genes. Validation was obtained using other wine strains by comparing normalized gene expression data with transcriptome quantification both in the presence and absence of sulfite. Among 15 reference genes tested ALG9, FBA1, UBC6 and PFK1 appeared to be the most reliable while ENO1, PMA1, DED1 and FAS2 were the worst. The most popular reference gene ACT1, widely used for S. cerevisiae gene expression studies, showed a stability level markedly lower than those of our selected reference genes. Finally, as the expression of the new reference gene set remained constant over the entire fermentation process, irrespective of the perturbation due to sulfite addition, our results can be considered also when no sulfite is added to the must. PMID:26325600

  3. A comprehensive gene regulatory network for the diauxic shift in Saccharomyces cerevisiae

    OpenAIRE

    Geistlinger, Ludwig; Csaba, Gergely; Dirmeier, Simon; Küffner, Robert; Zimmer, Ralf

    2013-01-01

    Existing machine-readable resources for large-scale gene regulatory networks usually do not provide context information characterizing the activating conditions for a regulation and how targeted genes are affected. Although this information is essentially required for data interpretation, available networks are often restricted to not condition-dependent, non-quantitative, plain binary interactions as derived from high-throughput screens. In this article, we present a comprehensive Petri net ...

  4. Global analysis of nutrient control of gene expression in Saccharomyces cerevisiae during growth and starvation

    OpenAIRE

    Wu, Jian; Zhang, Nianshu; Hayes, Andrew; Panoutsopoulou, Kalliope; Oliver, Stephen G.

    2004-01-01

    Global gene expression in yeast was examined in five different nutrient-limited steady states and in their corresponding starvation-induced stationary phases. The use of chemostats, with their ability to generate defined and reproducible physiological conditions, permitted the exclusion of the confounding variables that frequently complicate transcriptome analyses. This approach allowed us to dissect out effects on gene expression that are specific to particular physiological states. Thus, we...

  5. Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9, the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae.

    Science.gov (United States)

    Warit, S; Walmsley, R M; Stateva, L I

    1998-09-01

    Two genomic fragments have been isolated from Candida albicans which strongly hybridize to SRB1/PSA1/VIG9, an essential gene which encodes GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae. A common 2.5 kb Xbal-Pstl fragment has been identified, which Southern analysis suggests is most likely unique in the C. albicans genome. The fragment contains an ORF, which is 82% identical and 90% homologous to the Srb1p/Psa1p/Vig9p from S. cerevisiae, contains one additional amino acid at position 254 and is able to functionally complement the major phenotypic characteristics of S. cerevisiae srb1 null and conditional mutations. The authors therefore conclude that they have cloned and sequenced from C. albicans the bona fide homologue of SRB1/PSA1/VIG9, named hereafter CaSRB1. Northern analysis data indicate that the gene is expressed in C. albicans under conditions of growth in the yeast and hyphal form and suggest that its expression might be regulated. PMID:9782489

  6. Overexpressing enzymes of the Ehrlich pathway and deleting genes of the competing pathway in Saccharomyces cerevisiae for increasing 2-phenylethanol production from glucose.

    Science.gov (United States)

    Shen, Li; Nishimura, Yuya; Matsuda, Fumio; Ishii, Jun; Kondo, Akihiko

    2016-07-01

    2-Phenylethanol (2-PE) is a higher aromatic alcohol that is used in the cosmetics and food industries. The budding yeast Saccharomyces cerevisiae is considered to be a suitable host for the industrial production of higher alcohols, including 2-PE. To produce 2-PE from glucose in S. cerevisiae, we searched for suitable 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes of the Ehrlich pathway for overexpression in strain YPH499, and found that overexpression of the ARO10 and/or ADH1 genes increased 2-PE production from glucose. Further, we screened ten BY4741 single-deletion mutants of genes involved in the competing pathways for 2-PE production, and found that strains aro8Δ and aat2Δ displayed increased 2-PE production. Based on these results, we engineered a BY4741 strain that overexpressed ARO10 and contained an aro8Δ deletion, and demonstrated that the strain produced 96 mg/L 2-PE from glucose as the sole carbon source. As this engineered S. cerevisiae strain showed a significant increase in 2-PE production from glucose without the addition of an intermediate carbon substrate, it is a promising candidate for the large-scale production of 2-PE. PMID:26975754

  7. The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.

    OpenAIRE

    Stansfield, I; Jones, K M; Kushnirov, V V; Dagkesamanskaya, A. R.; Poznyakovski, A I; Paushkin, S V; Nierras, C R; Cox, B S; Ter-Avanesyan, M D; Tuite, M F

    1995-01-01

    The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45...

  8. ATP25, a New Nuclear Gene of Saccharomyces cerevisiae Required for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

    OpenAIRE

    Zeng, Xiaomei; Barros, Mario H.; Shulman, Theodore; Tzagoloff, Alexander

    2008-01-01

    We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F0. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of...

  9. Multiple gene mediated aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae

    Science.gov (United States)

    Furfural and HMF (5-hydroxymethylfurfural) are representative inhibitors to ethanologenic yeast generated from biomass pretreatment using dilute acid hydrolysis. Few yeast strains tolerant to inhibitors are available. We have developed tolerant strains of Saccharomyces cerevisiae with enhanced bio...

  10. Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118

    OpenAIRE

    Novo, Maite; Bigey, Frederic; Beyne, Emmanuelle; Galeote, Virginie; Gavory, Frédérick; Mallet, Sandrine; Cambon, Brigitte; Legras, Jean Luc; Wincker, Patrick; Casaregola, Serge; Dequin, Sylvie

    2009-01-01

    Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1...

  11. Expression of the SOD gene from Trichoderma harzianum in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    Yang Liming; Yang Qian; Liu Pigang; Li Sen

    2008-01-01

    Superoxide dismutases are metalloproteins which play a major role in defense against oxygen radical mediated toxicity in aerobic organisms.Such proteins are important endogeneity cytoprotection factor in volving defence.A 751-bp full-length cDNA sequence of an SOD gene was isolated from the Trichoderma harzianum.The full-length cDNA of the SOD gene consists of one 465-bp open reading frame nucleotide, which encodes a 15.7-kDa polypeptide consisting of 154 amino acid residues.Sequence analysis revealed that SOD gene has more than 72%-86% amino acid sequence homology with those of other fungi.The SOD gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2-SOD.SOD expressed by pYES2-SOD was induced by galactose.We test whether SOD could offer abiotic stress resistance when it was introduced into yeast cells.A transgenic yeast harboring T.harzianum SOD was generated under the control of a constitutively expressed GAL promoter.The results indicated that SOD yeast transfonnants had significantly higher resistance to salt and drought stress.

  12. Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

    OpenAIRE

    Fahimeh Afzal-Javan; Mohsen Mobini-Dehkordi

    2013-01-01

    Background: Alpha-amylases are digestive enzymes which hydrolyze starch glycosidic bonds to glucose, maltose, maltotriose and dextrin which have diverse applications in a wide range of industries such as food, textile, paper, detergents representing approximately 30% of the world enzyme production.Objectives: In this study, the gene encoding the alpha-amylase enzyme of native isolated Bacillus subtilis was amplified with specific primers containing of NotI and AscI restriction sites by PCR and...

  13. Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1.

    OpenAIRE

    Chambers, A.; Tsang, J S; Stanway, C; Kingsman, A J; Kingsman, S M

    1989-01-01

    The promoter of the yeast glycolytic gene encoding phosphoglycerate kinase (PGK) contains an upstream activation sequence between bases -538 and -402 upstream of the initiating ATG. The upstream activation sequence contains multiple functional elements, including an essential region called the activator core (AC) sequence and three copies of the pentamer 5'-CTTCC-3'. The AC sequence shows strong homology to the consensus binding sites for the yeast proteins RAP1 (GRF1) and TUF. We have demons...

  14. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    Science.gov (United States)

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  15. Multiple chromosomal gene integration for production of pharmaceutical proteins in S. cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Malene; Mortensen, Uffe Hasbro; Gunnarsson, Nina;

    2014-01-01

    When studying protein folding and secretion the general conception is that all cells in a population express an equal amount of protein. Recent work has shown that expression levels vary greatly in cell populations which express proteins on plasmids. Hence a yeast expression platform has been...... developed at the Department of Systems Biology, DTU. The platform offers the opportunity to express genes on the chromosome in 1 to 10 copies. A comparison between the expression of CFP and RFP by the platform and by plasmids reveals the problems of plasmid expression. FACS analyses of two cell populations......, expressing CFP and RFP on the separate plasmids or expressing CFP and RFP using the yeast expression platform shows expression varies greatly in a cell population based on plasmid expression compared to the yeast expression platform. When expressed on plasmids a few cells are high performers on both proteins...

  16. Compositions and methods for modeling Saccharomyces cerevisiae metabolism

    DEFF Research Database (Denmark)

    2012-01-01

    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...

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

    DEFF Research Database (Denmark)

    Chen, Xiao; Nielsen, Kristian Fog; Borodina, Irina;

    2011-01-01

    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 reference strain, but not for the ILV2 ILV3 ILV5 overexpression strain. This result....... 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...

  18. Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Suvi Jain

    2016-04-01

    Full Text Available Correct repair of DNA double-strand breaks (DSBs is critical for maintaining genome stability. Whereas gene conversion (GC-mediated repair is mostly error-free, repair by break-induced replication (BIR is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans compared to the case when both DSB ends come from the same break (Cis. However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the "origin" of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  20. A Genomewide Screen for Tolerance to Cationic Drugs Reveals Genes Important for Potassium Homeostasis in Saccharomyces cerevisiae

    Czech Academy of Sciences Publication Activity Database

    Barreto, L.; Canadell, D.; Petrezsélyová, Silvia; Navarrete, C.; Marešová, Lydie; Peréz-Valle, J.; Herrera, R.; Olier, I.; Giraldo, J.; Sychrová, Hana; Yenush, L.; Ramos, J.; Ariňo, J.

    2011-01-01

    Roč. 10, č. 9 (2011), s. 1241-1250. ISSN 1535-9778 R&D Projects: GA MŠk(CZ) LC531; GA AV ČR(CZ) IAA500110801 Institutional research plan: CEZ:AV0Z50110509 Keywords : Potassium homeostasis * Saccharomyces cerevisiae * genomewide screen Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.604, year: 2011

  1. Improving heterologous protein secretion at aerobic conditions by activating hypoxia-induced genes in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Liu, Lifang; Zhang, Yiming; Liu, Zihe;

    2015-01-01

    cerevisiae. Intriguingly, we found a 100% increase in the recombinant fungal α-amylase yield, as well as productivity. Varied levels of improvements were also observed for the productions of the human insulin precursor and the yeast endogenous enzyme invertase. Based on the genome-wide transcriptional...

  2. Xylose utilizing recombinant Saccharomyces cerevisiae strains

    Energy Technology Data Exchange (ETDEWEB)

    Walfridsson, M.

    1996-04-01

    Through metabolic engineering, S. cerevisiae was provided with the necessary enzymes required for xylose utilisation during ethanolic fermentation of xylose-rich lignocellulose raw materials. For xylitol production, S. cerevisiae was provided with the Pichia stipitis XYL1 gene encoding xylose reductase (XR). The in-vivo reduction and the following excretion of xylitol, requires a co-substrate for maintenance and cofactor regeneration. Xylitol yields close to 100% were obtained with the XYL1 containing S. cerevisiae. Introducing P. stipitis XYL1 and XYL2 genes, encoding XR and xylitol dehydrogenase (XDH), respectively, enabled S. cerevisiae to convert xylose to xylulose, via xylitol. During the screening work of P. stipitis XDH gene, another gene encoding a polyol dehydrogenase was isolated and cloned in S. cerevisiae. The gene was identified as a D-arabinitol dehydrogenase gene. In P. stipitis it may function as a redox sink by reducing D-ribulose to D-arabinitol. The metabolism through the pentose phosphate pathway (PPP) was enhanced by over-expressing the native genes TKL1 and TAL1 encoding transketolase and transaldolase, respectively, resulting in improved xylose utilisation. The XR and XDH activities in recombinant S. cerevisiae were produced at different levels by constructing yeast vectors in which the PGK1 and ADHI promoters controlled XYL1 and XYL2. With higher XDH than XR activities, less by-products, in the form of xylitol and glycerol, were formed by the recombinant S. cerevisiae strains. The Thermus thermophilus xylA gene encoding a thermostable xylose isomerase was cloned and expressed in S. cerevisiae. The recombinant xylose isomerase was actively produced and a new functional metabolic pathway was established in S. cerevisiae resulting in ethanol production from xylose. 150 refs, 3 figs, 4 tabs

  3. Impaired Uptake and/or Utilization of Leucine by Saccharomyces cerevisiae Is Suppressed by the SPT15-300 Allele of the TATA-Binding Protein Gene

    DEFF Research Database (Denmark)

    Baerends, RJ; Qiu, Jin-Long; Rasmussen, Simon;

    2009-01-01

    mutant allele of the SPT15 gene (SPT15-300) corresponding to the three amino acid changes F177S, Y195H, and K218R has been reported (H. Alper, J. Moxley, E. Nevoigt, G. R. Fink, and G. Stephanopoulos, Science 314:1565-1568, 2006). The SPT15 gene codes for the TATA-binding protein. This finding prompted...... us to examine the effect of expression of the SPT15-300 allele in various yeast species of industrial importance. Expression of SPT15-300 in leucine-prototrophic strains of S. cerevisiae, Saccharomyces bayanus, or Saccharomyces pastorianus (lager brewing yeast), however, did not improve tolerance to...... ethanol on complex rich medium (yeast extract-peptone-dextrose). The enhanced growth of the laboratory yeast strain BY4741 expressing the SPT15-300 mutant allele was seen only on defined media with low concentrations of leucine, indicating that the apparent improved growth in the presence of ethanol was...

  4. Effects of the rad52 gene on recombination in Saccharomyces cerevisiae. [Comparison of. gamma. -, uv-induced meiotic and spontaneous mitotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, S.; Prakash, L.; Burke, W.; Montelone, B.A.

    1979-01-01

    Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, ..gamma..-ray-induced, uv-induced, and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Intra- and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the hisl-1/hisl-315 and trp5-2/trp5-48 heteroalleles. Gene-centromere recombination was also not observed in rad52/rad52 diploids. No ..gamma..-ray-induced intragenic mitotic recombination is seen in rad52/rad52 diploids and uv-induced intragenic recombination is greatly reduced. However, spontaneous mitotic recombination is not similarly affected. The RAD52 gene thus functions in recombination in meiosis and in ..gamma..-ray and uv-induced mitotic recombination but not in spontaneous mitotic recombination.

  5. Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression.

    OpenAIRE

    Ozcan, S; Dover, J; Rosenwald, A G; Wölfl, S; Johnston, M.

    1996-01-01

    Glucose is the preferred carbon source for most eukaryotic cells and has profound effects on many cellular functions. How cells sense glucose and transduce a signal into the cell is a fundamental, unanswered question. Here we describe evidence that two unusual glucose transporters in the yeast Saccharomyces cerevisiae serve as glucose sensors that generate an intracellular glucose signal. The Snf3p high-affinity glucose transporter appears to function as a low glucose sensor, since it is requ...

  6. Comparative studies of genome-wide maps of nucleosomes between deletion mutants of elp3 and hos2 genes of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available In order to elucidate the influence of histone acetylation upon nucleosomal DNA length and nucleosome position, we compared nucleosome maps of the following three yeast strains; strain BY4741 (control, the elp3 (one of histone acetyltransferase genes deletion mutant, and the hos2 (one of histone deactylase genes deletion mutant of Saccharomyces cerevisiae. We sequenced mononucleosomal DNA fragments after treatment with micrococcal nuclease. After mapping the DNA fragments to the genome, we identified the nucleosome positions. We showed that the distributions of the nucleosomal DNA lengths of the control and the hos2 disruptant were similar. On the other hand, the distribution of the nucleosomal DNA lengths of the elp3 disruptant shifted toward shorter than that of the control. It strongly suggests that inhibition of Elp3-induced histone acetylation causes the nucleosomal DNA length reduction. Next, we compared the profiles of nucleosome mapping numbers in gene promoter regions between the control and the disruptant. We detected 24 genes with low conservation level of nucleosome positions in promoters between the control and the elp3 disruptant as well as between the control and the hos2 disruptant. It indicates that both Elp3-induced acetylation and Hos2-induced deacetylation influence the nucleosome positions in the promoters of those 24 genes. Interestingly, in 19 of the 24 genes, the profiles of nucleosome mapping numbers were similar between the two disruptants.

  7. EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9.

    Science.gov (United States)

    Jessop-Fabre, Mathew M; Jakočiūnas, Tadas; Stovicek, Vratislav; Dai, Zongjie; Jensen, Michael K; Keasling, Jay D; Borodina, Irina

    2016-08-01

    Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available. PMID:27166612

  8. Conserved codon composition of ribosomal protein coding genes in Escherichia coli, Mycobacterium tuberculosis and Saccharomyces cerevisiae: lessons from supervised machine learning in functional genomics.

    Science.gov (United States)

    Lin, Kui; Kuang, Yuyu; Joseph, Jeremiah S; Kolatkar, Prasanna R

    2002-06-01

    Genomics projects have resulted in a flood of sequence data. Functional annotation currently relies almost exclusively on inter-species sequence comparison and is restricted in cases of limited data from related species and widely divergent sequences with no known homologs. Here, we demonstrate that codon composition, a fusion of codon usage bias and amino acid composition signals, can accurately discriminate, in the absence of sequence homology information, cytoplasmic ribosomal protein genes from all other genes of known function in Saccharomyces cerevisiae, Escherichia coli and Mycobacterium tuberculosis using an implementation of support vector machines, SVM(light). Analysis of these codon composition signals is instructive in determining features that confer individuality to ribosomal protein genes. Each of the sets of positively charged, negatively charged and small hydrophobic residues, as well as codon bias, contribute to their distinctive codon composition profile. The representation of all these signals is sensitively detected, combined and augmented by the SVMs to perform an accurate classification. Of special mention is an obvious outlier, yeast gene RPL22B, highly homologous to RPL22A but employing very different codon usage, perhaps indicating a non-ribosomal function. Finally, we propose that codon composition be used in combination with other attributes in gene/protein classification by supervised machine learning algorithms. PMID:12034849

  9. Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae.

    Science.gov (United States)

    Abramova, N E; Cohen, B D; Sertil, O; Kapoor, R; Davies, K J; Lowry, C V

    2001-03-01

    The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Delta allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes. PMID:11238402

  10. Effects of Gene Orientation and Use of Multiple Promoters on the Expression of XYL1 and XYL2 in Saccharomyces cerevisiae

    Science.gov (United States)

    Bae, Ju Yun; Laplaza, José; Jeffries, Thomas W.

    Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We assembled d-xylose reductase (XYL1) and d-xylitol dehydrogenase (XYL2) in four ways. Each pair of genes was placed in two different tandem (l→2→ or √1√2), convergent (1→√2), and divergent (√1 2→) orientations in autonomous plasmids. The TEF1 promoter was used to drive XYL1 and the TDH3 promoter to drive XYL2 in each of the constructs. The effects of gene orientation on growth, transcription, and enzyme activity were analyzed. The transcription level as measured by quantitative PCR (q-PCR) correlated with enzyme activities, but our data did not show a significant effect of gene orientation. To test the possible dilution of promoter strength due to multiple use of the same promoter, we examined the level of expression of XYL1 driven by either the TEF1 or TDH3 promoter when carried on a single copy plasmid. We then coexpressed XYL2 from either a single or multicopy plasmid, which was also driven by the same promoter. XYL2 transcript and enzyme expression increased with plasmid copy number, while the expression of XYLl was constant regardless of the number of other TEF1 or TDH3 promoters present in the cell. According to our data, there is no significant effect of gene orientation or multiple promoter use on gene transcription and translation when genes are expressed from plasmids; however, other factors could affect expression of adjacent genes in chromosomes.

  11. TEL2, an essential gene required for telomere length regulation and telomere position effect in Saccharomyces cerevisiae.

    OpenAIRE

    Runge, K W; Zakian, V A

    1996-01-01

    The DNA-protein complexes at the ends of linear eukaryotic chromosomes are called the telomeres. In Saccharomyces cerevisiae, telomeric DNA consists of a variable length of the short repeated sequence C1-3A. The length of yeast telomeres can be altered by mutation, by changing the levels of telomere binding proteins, or by increasing the amount of C1-3A DNA sequences. Cells bearing the tel1-1 or tel2-1 mutations, known previously to have short telomeres, did not respond to perturbations that ...

  12. The SFP1 gene product of Saccharomyces cerevisiae regulates G2/M transitions during the mitotic cell cycle and DNA-damage response

    International Nuclear Information System (INIS)

    In eukaryotic cells, checkpoint pathways arrest cell-cycle progression if a particular event has failed to complete appropriately or if an important intracellular structure is defective or damaged. Saccharomyces cerevisiae strains that lack the SFP1 gene fail to arrest at the G2 DNA-damage checkpoint in response to genomic injury, but maintain their ability to arrest at the replication and spindle-assembly checkpoints. sfp1D mutants are characterized by a premature entrance into mitosis during a normal (undamaged) cell cycle, while strains that overexpress Sfp1p exhibit delays in G2. Sfp1p therefore acts as a repressor of the G2/M transition, both in the normal cell cycle and in the G2 checkpoint pathway. Sfp1 is a nuclear protein with two Cys2His2 zinc-finger domains commonly found in transcription factors. We propose that Sfp1p regulates the expression of gene products involved in the G2/M transition during the mitotic cell cycle and the DNA-damage response. In support of this model, overexpression of Sfp1p induces the expression of the PDS1 gene, which is known to encode a protein that regulates the G2 checkpoint. (author)

  13. Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered Saccharomyces cerevisiae Strain▿ †

    OpenAIRE

    Lu, Chenfeng; Jeffries, Thomas

    2007-01-01

    We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentati...

  14. Kluyveromyces lactis maintains Saccharomyces cerevisiae intron-encoded splicing signals.

    OpenAIRE

    Deshler, J O; Larson, G P; Rossi, J J

    1989-01-01

    The actin (ACT) gene from the budding yeast Kluyveromyces lactis was cloned, and the nucleotide sequence was determined. The gene had a single intron 778 nucleotides in length which possessed the highly conserved splicing signals found in Saccharomyces cerevisiae introns. We demonstrated splicing of heterologous ACT transcripts in both K. lactis and S. cerevisiae.

  15. Genome shuffling of Saccharomyces cerevisiae for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction.

    Science.gov (United States)

    Yin, Hua; Ma, Yanlin; Deng, Yang; Xu, Zhenbo; Liu, Junyan; Zhao, Junfeng; Dong, Jianjun; Yu, Junhong; Chang, Zongming

    2016-08-01

    Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering. PMID:27302037

  16. Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: Construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

    International Nuclear Information System (INIS)

    Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinant IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with [4-3H]FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139

  17. The human homolog of S. cerevisiae CDC27, CDC27 Hs, is encoded by a highly conserved intronless gene present in multiple copies in the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Devor, E.J.; Dill-Devor, R.M. [Univ. of Iowa College of Medicine, Iowa City (United States)

    1994-09-01

    We have obtained a number of unique sequences via PCR amplification of human genomic DNA using degenerate primers under low stringency (42{degrees}C). One of these, an 853 bp product, has been identified as a partial genomic sequence of the human homolog of the S. cerevisiae CDC27 gene, CDC27Hs (GenBank No. U00001). This gene, reported by Turgendreich et al. is also designated EST00556 from Adams et al. We have undertaken a more detailed examination of our sequence, MCP34N, and have found that: 1. the genomic sequence is nearly identical to CDC27Hs over its entire 853 bp length; 2. an MCP34N-specific PCR assay of several non-human primate species reveals amplification products in chimpanzee and gorilla genomes having greater than 90% sequence identity with CDC27Hs; and 3. an MCP34N-specific PCR assay of the BIOS hybrid cell line panel gives a discordancy pattern suggesting multiple loci. Based upon these data, we present the following initial characterization: 1. the complete MCP34N sequence identity with CDC27Hs indicates that the latter is encoded by an intronless gene; 2. CDC27Hs is highly conserved among higher primates; and 3. CDC27Hs is present in multiple copies in the human genome. These characteristics, taken together with those initially reported for CDC27Hs, suggest that this is an old gene that carries out an important but, as yet, unknown function in the human brain.

  18. Genome-wide screening of the genes required for tolerance to vanillin, which is a potential inhibitor of bioethanol fermentation, in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Tokuyasu Ken

    2008-04-01

    Full Text Available Abstract Background Lignocellulosic materials are abundant and among the most important potential sources for bioethanol production. Although the pretreatment of lignocellulose is necessary for efficient saccharification and fermentation, numerous by-products, including furan derivatives, weak acids, and phenolic compounds, are generated in the pretreatment step. Many of these components inhibit the growth and fermentation of yeast. In particular, vanillin is one of the most effective inhibitors in lignocellulose hydrolysates because it inhibits fermentation at very low concentrations. To identify the genes required for tolerance to vanillin, we screened a set of diploid yeast deletion mutants, which are powerful tools for clarifying the function of particular genes. Results Seventy-six deletion mutants were identified as vanillin-sensitive mutants. The numerous deleted genes in the vanillin-sensitive mutants were classified under the functional categories for 'chromatin remodeling' and 'vesicle transport', suggesting that these functions are important for vanillin tolerance. The cross-sensitivity of the vanillin-sensitive mutants to furan derivatives, weak acids, and phenolic compounds was also examined. Genes for ergosterol biosynthesis were required for tolerance to all inhibitory compounds tested, suggesting that ergosterol is a key component of tolerance to various inhibitors. Conclusion Our analysis predicts that vanillin tolerance in Saccharomyces cerevisiae is affected by various complicated processes that take place on both the molecular and the cellular level. In addition, the ergosterol biosynthetic process is important for achieving a tolerance to various inhibitors. Our findings provide a biotechnological basis for the molecular engineering as well as for screening of more robust yeast strains that may potentially be useful in bioethanol fermentation.

  19. The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism

    International Nuclear Information System (INIS)

    Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5. (author)

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

    DEFF Research Database (Denmark)

    Förster, Jochen; Famili, I.; Fu, P.; Palsson, B.O.; Nielsen, Jens

    2003-01-01

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

  1. Cystathionine accumulation in Saccharomyces cerevisiae.

    OpenAIRE

    Ono, B; Suruga, T; Yamamoto, M.; Yamamoto, S.; Murata, K; Kimura, A; Shinoda, S; Ohmori, S.

    1984-01-01

    A cysteine-dependent strain of Saccharomyces cerevisiae and its prototrophic revertants accumulated cystathionine in cells. The cystathionine accumulation was caused by a single mutation having a high incidence of gene conversion. The mutation was designated cys3 and was shown to cause loss of gamma-cystathionase activity. Cysteine dependence of the initial strain was determined by two linked and interacting mutations, cys3 and cys1 . Since cys1 mutations cause a loss of serine acetyltransfer...

  2. Posttranscriptional regulation of the karyogamy gene by Kem1p/Xrn1p exoribonuclease and Rok1p RNA helicase of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The major biochemical activities ascribed to Kem1p/Xrn1p of Saccharomyces cerevisiae are 5'-3' exoribonuclease functioning in RNA turnover and a microtubule-binding protein. Mutational analysis has shown that Kem1p/Xrn1p participates in microtubule-related functions such as nuclear fusion (karyogamy) during mating, chromosome transmission, and spindle pole body duplication. Here, evidence is presented that Kem1p plays a specific role in nuclear fusion by affecting, at the posttranscriptional level, the pheromone induction of the karyogamy-specific transcription factor Kar4p and the expression of Rok1p, a putative RNA helicase. We found that Rok1p itself also affects the pheromone induction of Kar4p and thereby participates in nuclear fusion. Analysis of the active-site mutations, xrn1-D206A or D208A, shows that nuclear fusion as well as the Rok1p synthesis do not require the exoribonuclease activity of Kem1p. Our data provide an important insight into the gene-specific regulatory function mediated by the general RNA-modulating enzymes

  3. The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage

    International Nuclear Information System (INIS)

    In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. (author)

  4. Increased copper toxicity in Saccharomyces cerevisiae lacking VPS35, a component of the retromer and monogenic Parkinson disease gene in humans.

    Science.gov (United States)

    Sowada, Nadine; Stiller, Barbara; Kubisch, Christian

    2016-08-01

    The Saccharomyces cerevisiae gene VPS35 encodes a component of the retromer complex which is involved in vesicle transport from endosomes to the trans-Golgi network. Yeast and human VPS35 orthologs are highly conserved and mutations in human VPS35 cause an autosomal dominant form of late-onset Parkinson disease (PD). We now show that deletion of VPS35 in yeast (vps35Δ) leads to a dose-dependent growth defect towards copper. This increased sensitivity could be rescued by transformation with yeast wild-type VPS35 but not by the expression of a construct harboring the yeast equivalent (i.e. D686N) of the most commonly identified VPS35-associated PD mutation, p.D620N. In addition, we show that expression of one copy of α-synuclein, which is known to directly interact with copper, leads to a pronounced aggravation of copper toxicity in vps35Δ cells, thereby linking the regulation of copper homeostasis by Vps35p in yeast to one of the key molecules in PD pathophysiology. PMID:27262440

  5. Conditional Mutants of Rpc160, the Gene Encoding the Largest Subunit of RNA Polymerase C in Saccharomyces Cerevisiae

    OpenAIRE

    Gudenus, R; Mariotte, S; Moenne, A; Ruet, A; Memet, S; Buhler, J M; Sentenac, A; Thuriaux, P

    1988-01-01

    A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO(3) and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants ...

  6. New Aldehyde Reductase Genes of Saccharomyces cerevisiae Contribute In Situ Detoxification of Lignocellulose-to-Ethanol Conversion Inhibitiors

    Science.gov (United States)

    Furfural and 5-hydroxymethylfurfural (HMF) are inhibitory compounds commonly encountered during lignocellulose-to-ethanol conversion for cleaner transportation fuels. It is possible to in situ detoxify the aldehyde inhibitors by tolerant ethanologenic yeast strains. Multiple gene-mediated reductio...

  7. Genealogy-Based Methods for Inference of Historical Recombination and Gene Flow and Their Application in Saccharomyces cerevisiae

    OpenAIRE

    Jenkins, Paul A.; Song, Yun S.; Brem, Rachel B.

    2012-01-01

    Genetic exchange between isolated populations, or introgression between species, serves as a key source of novel genetic material on which natural selection can act. While detecting historical gene flow from DNA sequence data is of much interest, many existing methods can be limited by requirements for deep population genomic sampling. In this paper, we develop a scalable genealogy-based method to detect candidate signatures of gene flow into a given population when the source of the alleles ...

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

    Indian Academy of Sciences (India)

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

    2014-12-01

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

  9. Silencing MIG1 in Saccharomyces cerevisiae: Effects of antisense MIG1 expression and MIG1 gene disruption

    DEFF Research Database (Denmark)

    Olsson, Lisbeth; Larsen, M.E.; Rønnow, B.; Mikkelsen, J.D.; Nielsen, Jens Bredal

    1997-01-01

    repression, However, silencing of MIG1 expression was not achieved by expressing antisense MIG1, even though antisense MIG1 RNA was sufficiently stable to be detected. In the wild-type and Delta mig1 strains, the specific growth rate was 0.32 to 0.33 h(-1), whereas it was lower in the antisense strains, 0......Silencing of MIG1, a transcription factor imposing carbon catabolite repression on invertase was attempted, either by disrupting the gene or by expressing antisense copies of the gene. The performance of the recombinant strains in bioreactor batch cultivations on sucrose, in the presence of glucose...

  10. The relationship between the "TATA" sequence and transcription initiation sites at the HIS4 gene of Saccharomyces cerevisiae.

    OpenAIRE

    Nagawa, F; Fink, G R

    1985-01-01

    Transcription of the HIS4 gene begins at a single site (I) at position -60 from the ATG that begins translation. We have made linker insertions/deletions in the 5' noncoding region to identify the elements required for the specificity of transcription initiation. Although there are four sequences that begin TATA and are near the start of transcription (-170, -132, -123, and -102) only the sequence at -123 (TATA-123) is required for transcription initiation. By inserting synthetic oligonucleot...

  11. Verification of the resistance of a LEA gene from Tamarix expression in Saccharomyces cerevisiae to abiotic stresses

    Institute of Scientific and Technical Information of China (English)

    WANG Bing-feng; WANG Yu-cheng; ZHANG Da-wei; LI Hong-yan; YANG Chuan-ping

    2008-01-01

    The role of late embryogenesis abundant (LEA) proteins in stress tolerance was examined by using a yeast expression system. LEA protein tolerance to the abotic stresses in plants involved in salt, drought and freezing stresses and additional tolerance to heat, NaHCO3 (salt-alkali) and ultraviolet radiation was also investigated. The transgenic yeast harboring the Tamarix LEA gene (DQ663481) was generated under the control of inducible GAL promoter (pYES2 vector), yeast cells transformed with pYES2 empty vector were also generated as a control. Stress tolerance tests showed that LEA yeast transformants exhibited a higher survival rates than the control transformants under high temperature, NaHCO3, ultraviolet radiation, salt (NaCl), drought and freezing, indicating that the LEA gene is tolerant to these abiotic stresses. These results suggest that the LEA gene is resistant to a wider repertoire of stresses and may play a common role in plant acclimation to the examined stress conditions.

  12. Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

    OpenAIRE

    Erickson, J. R.; Johnston, M

    1993-01-01

    We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When ye...

  13. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

    OpenAIRE

    Stålbrand, H; Saloheimo, A; Vehmaanperä, J; HENRISSAT, B.; Penttilä, M

    1995-01-01

    beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanas...

  14. Transcription regulation of AAC3 gene encoding hypoxic isoform of ADP/ATP carrier in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Two repressoric regions are present in the AAC3 promoter, termed URS1 and URS2. URS1 region is responsible for a carbon source-dependent regulation and plays a role under both, aerobic and anaerobic conditions. By deletion analysis URS1 was localized into the -322/-244 region and was found that the regulation is likely exerted by the repression by non-fermentable or non-repressing fermentable carbon sources than by the activation by repressing carbon source. By computer analysis cis sequences for two potential transcription factors, Rap1 and ERA, were identified within URS1. Rap1 binding into its consensus sequence was proved, effort to find the protein binding to the ERA cis regulatory sequences has failed. By the means of mutational analysis we revealed that the regulation pathway mediating the carbon source-dependent regulation via URS1 differs according to the presence or absence of oxygen in the growth medium. Under aerobic conditions the carbon source-dependent repression is mediated by the ERA factor and the role of Rap1 is only marginal. On the contrary, under anaerobic conditions, the repression is mediated solely by Rap1. AAC1 gene product might be involved in the regulation of the AAC3 gene, the regulation pathway has not been characterized yet. (author)

  15. Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity.

    Science.gov (United States)

    Crabeel, M; Abadjieva, A; Hilven, P; Desimpelaere, J; Soetens, O

    1997-12-01

    We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine. Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y-S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable-type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence. Yeast Arg7p shares between 32-43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay. The

  16. Overexpression of a novel gene, Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutant (mcm10-1) of budding yeast shows a growth arrest at 37℃. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37℃. Interestingly, this mcm10-1 suppressor undergoes cell cycle arrest at 14℃. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cmsl (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of transformation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14℃, indicating that overexpression of Cmsl can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.

  17. Detection of overlapping protein complexes in gene expression, phenotype and pathways of Saccharomyces cerevisiae using Prorank based Fuzzy algorithm.

    Science.gov (United States)

    Manikandan, P; Ramyachitra, D; Banupriya, D

    2016-04-15

    Proteins show their functional activity by interacting with other proteins and forms protein complexes since it is playing an important role in cellular organization and function. To understand the higher order protein organization, overlapping is an important step towards unveiling functional and evolutionary mechanisms behind biological networks. Most of the clustering algorithms do not consider the weighted as well as overlapping complexes. In this research, Prorank based Fuzzy algorithm has been proposed to find the overlapping protein complexes. The Fuzzy detection algorithm is incorporated in the Prorank algorithm after ranking step to find the overlapping community. The proposed algorithm executes in an iterative manner to compute the probability of robust clusters. The proposed and the existing algorithms were tested on different datasets such as PPI-D1, PPI-D2, Collins, DIP, Krogan Core and Krogan-Extended, gene expression such as GSE7645, GSE22269, GSE26923, pathways such as Meiosis, MAPK, Cell Cycle, phenotypes such as Yeast Heterogeneous and Yeast Homogeneous datasets. The experimental results show that the proposed algorithm predicts protein complexes with better accuracy compared to other state of art algorithms. PMID:26809099

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

    DEFF Research Database (Denmark)

    Asadollahi, Mohammadali; Maury, Jerome; Patil, Kiran Raosaheb;

    2009-01-01

    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 framewo...

  19. Mitochondrial genetics X: Effects of UV irradiation on transmission and recombination of mitochondrial genes in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    UV irradiation has been applied either to one parent prior to crossing or to newly formed zygotes. The effects of UV have been studied on the transmission of mitochondrial alleles at the loci conferring resistance to antibiotic and the frequency of recombinants between various combinations of alleles at these loci. The effects of UV depend on the nature of the cross i.e. homosexual (ω+ x ω+ or ω- x ω-) or heterosexual (ω+ x ω-). In all cases UV irradiation of one of the parents diminishes the transmission of the mitochondrial alleles originated from the irradiated parent. In homosexual crosses the decrease of transmission is the same for alleles at all the loci. In heterosexual crosses, when the ω+ parent is irradiated, there is a differential decrease of transmission depending on the distance of the resistance locus relative to the ω locus. In heterosexual crosses irradiation of the ω+ parent increases the frequency of recombinants while irradiation of the ω- parent slightly decreases it. In homosexual crosses the frequency of recombinants diminishes when a high UV dose is applied to one of the parents. No or only minor modifications of the polarity of recombination are observed. Irradiation of newly formed zygotes has no or minor effects on the transmission of alleles and recombinant frequencies. All these effects can be interpreted in terms of a general model for recombination of mitochondrial genes. UV irradiation of one of the parents leads to a modification of the input fraction in favor of the non irradiated parent. As a consequence of this modification the output of alleles and the frequency of recombinants are changed. A good quantitative agreement between the predictions calculated on the basis of the model and the experimental data is found. Relationships between the molecular events responsible for the modifications of input and the production of rho- primary clones by UV are discussed. (orig./MG)

  20. Multiple Gene Mediated NAD(P)H-Dependent Aldehyde Reduction is a Mechanism of in situ Detoxification of Furfural and HMF by Saccharomyces cerevisiae

    Science.gov (United States)

    Furfural and 5-hydroxymethylfurfural (HMF) are representative inhibitors to ethanologenic yeast generated from biomass pretreatment using dilute acid hydrolysis. Few yeast strains tolerant to inhibitors are available. In this study, we report a tolerant strain 12HF10 of Saccharomyces cerevisiae ha...

  1. Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Liu Z Lewis

    2010-11-01

    Full Text Available Abstract Background The yeast Saccharomyces cerevisiae is able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and HMF. The length of lag phase for cell growth in response to the inhibitor challenge has been used to measure tolerance of strain performance. Mechanisms of yeast tolerance at the genome level remain unknown. Using systems biology approach, this study investigated comparative transcriptome profiling, metabolic profiling, cell growth response, and gene regulatory interactions of yeast strains and selective gene deletion mutations in response to HMF challenges during the lag phase of growth. Results We identified 365 candidate genes and found at least 3 significant components involving some of these genes that enable yeast adaptation and tolerance to HMF in yeast. First, functional enzyme coding genes such as ARI1, ADH6, ADH7, and OYE3, as well as gene interactions involved in the biotransformation and inhibitor detoxification were the direct driving force to reduce HMF damages in cells. Expressions of these genes were regulated by YAP1 and its closely related regulons. Second, a large number of PDR genes, mainly regulated by PDR1 and PDR3, were induced during the lag phase and the PDR gene family-centered functions, including specific and multiple functions involving cellular transport such as TPO1, TPO4, RSB1, PDR5, PDR15, YOR1, and SNQ2, promoted cellular adaptation and survival in order to cope with the inhibitor stress. Third, expressed genes involving degradation of damaged proteins and protein modifications such as SHP1 and SSA4, regulated by RPN4, HSF1, and other co-regulators, were necessary for yeast cells to survive and adapt the HMF stress. A deletion mutation strain Δrpn4 was unable to recover the growth in the presence of HMF. Conclusions Complex gene interactions and regulatory networks as well as co-regulations exist in yeast adaptation and tolerance to the lignocellulose derived

  2. 酿酒酵母减数分裂的事件和特异性基因%Meiosis Events and Specific Genes Involved in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    李丽

    2006-01-01

    Meiosis is the most important reproduce and favoured by organisms. The key events of meiosis identified are pairing, synapsis, genetic recombination, segregation. In this article, the specific genes of meiosis and pathways are described in these events in Saccharomyces cerevisiae. Two pathways have been identified in homolog pairing and Dmc1,Hop2, Rad51 and Mnd1 have function in pairing. In synapsis Red1, Hop1 and Zip1 are the components of synaptonemal complex. Hop2 is required for synapsis. Genetic recombination is the most important event of meiosis and provides genetic diversity for organisms to adapt the environment change. The meiosis specific genes involved in gene recombination are Spo11, Rad52 epistasis group, Dmc1, Mnd1, Msh4, Msh5, Mek1, Red1, and Hop1. Rec8,Spo13 and Sgo1 are involved in segregation.%减数分裂是生物体重要的有性生殖方式,它提供来自母本和父本的基因信息,产生具有生物多样性的子代,使其能够适应环境的变化而不断进化.本文简述了现已阐明的酿酒酵母减数分裂的重要事件如同源染色体配对、联会、基因重组、染色体分裂和特异性基因.在同源染色体配对的过程中现已发现了2条途径,一条由Rad51独立完成,另一条有Dmc1、Hop2、Rad51和Mnd1参与,同时Rad51也可能参与.Red1、Hop1和Zip1是联会复合体的组成成分,而联会也要求其他减数分裂的特异性基因如Hop2的参与.基因重组是减数分裂中最重要的事件,它为子代提供了新的遗传信息,是生物多样性的基础之一.Spo11、Rad52组、Dmc1、Mnd1、Msh4、Msh5、Mek1、Red1和Hop1参与了基因重组.Spo11是发现和研究得最早的启动基因重组的基因之一;Rec8、Spo13和Sgo1参与了染色体分裂的过程.

  3. SAS1 and SAS2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae.

    OpenAIRE

    Saxe, S A; Kimmel, A R

    1990-01-01

    We have identified two novel, very closely related genes, SAS1 and SAS2, from Dictyostelium discoideum. These encode small, approximately 20-kilodaton proteins with amino acid sequences thought to be involved in interaction with guanine nucleotides. The protein sizes, spacings of GTP-binding domains, and carboxyl-terminal sequences suggest their relationship to the ubiquitous ras-type proteins. Their sequences, however, are sufficiently different to indicate that they are not true ras protein...

  4. REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability, recombination and sporulation: cell-type and life cycle stage specific expression of the rec46-1 mutation

    International Nuclear Information System (INIS)

    Studies of chromosomal recombination during mitosis and meiosis of Saccharomyces cerevisiae have demonstrated that recombination at these two distinct stages of the yeast life cycle proceeds by mechanisms that appear similar but involve discrete mitosis-specific and meiosis-specific properties. UV radiation induced REC mutants are being employed as a genetic tool to identify the partial reactions comprising recombination and the involvement of individual REC gene products in mitotic and meiotic recombination. The sequence of molecular events that results in genetic recombination in eukaryotes is presently ill-defined. Genetic characterization of REC gene mutants and biochemical analyses of them for discrete defects in DNA metabolic proteins and enzymes (in collaboration with the laboratory of Junko Hosoda) are beginning to remedy this gap in the authors knowledge. This report summarizes the genetic properties of the rec46-1 mutation

  5. Identification and characterization of a novel C20-elongase gene from the marine microalgae, Pavlova viridis, and its use for the reconstitution of two pathways of long-chain polyunsatured fatty acids biosynthesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Shi, Tonglei; Yu, Aiqun; Li, Ming; Zhang, Meng; Xing, Laijun; Li, Mingchun

    2013-08-01

    The marine microalga, Pavlova viridis, contains long-chain polyunsatured fatty acids including eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3). A full-length cDNA sequence, pvelo5, was isolated from P. viridis. From sequence alignment, the gene was homologous to fatty acyl elongases from other organisms. Heterologous expression of pvelo5 in Saccharomyces cerevisiae confirmed that it encoded a specific C20-elongase within the n-3 and n-6 pathways. Elongation activity was confined exclusively to EPA and arachidonic acid (20:4n-6). GC analysis indicated that pvelo5 could co-express with other genes for biosynthesis to reconstitute the Δ8 and Δ6 pathways. Real-time PCR results and fatty acid analysis demonstrated that long-chain polyunsatured fatty acids production by the Δ8 pathway might be more effective than that by the Δ6 pathway. PMID:23546943

  6. Changes in metabolism of yeast Saccharomyces cerevisiae caused by deletion of the two Pdr transporter genes PDR5 and SNQ2

    Czech Academy of Sciences Publication Activity Database

    Hlaváček, Otakar; Váchová, Libuše; Palková, Z.

    Dorchester : Wiley, 2007, s. 2-2. [International Conference on Yeast Genetics and Molecular Biology /23./. Melbourne (AU), 01.07.2007-06.07.2007] R&D Projects: GA ČR GP204/05/P175; GA ČR GA525/05/0297; GA MŠk(CZ) LC531 Institutional research plan: CEZ:AV0Z50200510 Keywords : saccharomyces cerevisiae Subject RIV: EE - Microbiology, Virology

  7. Effects of rpt1, rpt4 and rpt6 td mutants on GAL1/10 gene expression in Saccharomyces cerevisiae

    OpenAIRE

    Román, Lorena Casado 1991

    2012-01-01

    The aim of this project was to study the roles of Rpt1, Rpt4 and Rpt6 in transcriptional regulation of the GAL network. Therefore, three temperature sentitive degron (td) mutants were created by integrating recombinant plasmids into Saccharomyces cerevisiae chromosomes. Under galactose induction, degradation of Rpt4 caused a decrease in GAL1 and GAL10 mRNA levels, Rpt1 degradation did not cause any detectable effect and Rpt6 degradation caused an increase in GAL1 and GAL10 mRNA transcribed...

  8. Atypical yeasts identified as Saccharomyces cerevisiae by MALDI-TOF MS and gene sequencing are the main responsible of fermentation of chicha, a traditional beverage from Peru.

    Science.gov (United States)

    Vallejo, Juan Andrés; Miranda, Patricia; Flores-Félix, José David; Sánchez-Juanes, Fernando; Ageitos, José M; González-Buitrago, José Manuel; Velázquez, Encarna; Villa, Tomás G

    2013-12-01

    Chicha is a drink prepared in several Andean countries from Inca's times by maize fermentation. Currently this fermentation is carried out in familiar artesanal "chicherías" that make one of the most known types of chicha, the "chicha de jora". In this study we isolate and identify the yeasts mainly responsible of the fermentation process in this type of chicha in 10 traditional "chicherías" in Cusco region in Peru. We applied by first time MALDI-TOF MS analysis for the identification of yeast of non-clinic origin and the results showed that all of yeast strains isolated belong to the species Saccharomyces cerevisiae. These results agree with those obtained after the analysis of the D1/D2 and 5.8S-ITS regions. However the chicha strains have a phenotypic profile that differed in more than 40% as compared to that of current S. cerevisiae strains. To the best of our knowledge this is the first report concerning the yeasts involved in chicha fermentation. PMID:24120265

  9. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...... gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise...... combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no...

  10. Cloning of Xylose Reductase Gene and Site-Specific Integrating it into Saccharomyces cerevisiae Genome%木糖还原酶基因的克隆及其在酿酒酵母中的定向整合

    Institute of Scientific and Technical Information of China (English)

    高岚; 夏黎明

    2013-01-01

      酿酒酵母(Saccharomyces cerevisiae)是重要的乙醇生产菌株,但因缺少戊糖代谢途径而不能利用木糖,为了改良工业酿酒酵母利用半纤维素发酵生产乙醇的性能,利用分子生物学技术构建能够利用木糖的基因工程酵母。选取酿酒酵母染色体的rDNA重复序列作为外源基因整合位点,依此构建多拷贝染色体整合型载体pUG-LR。采用融合表达策略扩增得到含有酿酒酵母乙醇脱氢酶启动子PADH和树干毕赤酵母木糖还原酶基因xyl1的融合序列,并将其插入pUG-LR载体中,构建成含遗传霉素G418抗性标记的同源重组质粒pUG-LR-XYL1。以工业酿酒酵母ZU-01为宿主,通过优化后的电穿孔法将重组质粒导入经缓冲液处理的酵母细胞,30℃培养。通过提高YEPX复筛培养基G418浓度,得到10株生长较快的优良性状转化子。在不含G418的YEPX培养基上传代8次以上,以转化子基因组DNA为模板,进行PCR检测,均可获得目的基因片段。研究结果表明:木糖还原酶基因xyl1已定向整合于ZU-01染色体DNA上并稳定遗传,为后续构建工业酿酒酵母的木糖代谢通路、利用木糖产酒精的重组菌株奠定了基础。%  Saccharomyces cerevisiae is an important ethanol producing fungus, but it cannot be used in large-scale bioethanol production from hemicelluloses due to its lack of the metabolism pathway of pentose, and therefore the xylose can not be utilized. In order to improve its ability of utilizing hemicelluloses to produce ethand, molecular biology technology was adopted to obtain recombinant strain with ability of xylose-using in this study. The repeat region of rDNA in the S. cerevisiae chromosome was chosen as the insert site of foreign genes. Plasmid pUG6, with KanMX resistance marker, was connected with rDNA fragments to construct site-specific integration vector pUG-LR. Then a Pichia stipitis xylose reductase gene xyl1 was cloned

  11. The efflux pump MlcE from the Penicillium solitum compactin biosynthetic gene cluster increases Saccharomyces cerevisiae resistance to natural statins

    DEFF Research Database (Denmark)

    Ley, Ana; Frandsen, Rasmus John Normand

    natural producers difficult to culture in bioreactors. The production limitations associated with the use of natural producers can be overcome by heterologous expression of the biosynthetic pathway in Saccharomyces cerevisiae (1), however, it is crucial to establish a nondestructive resistance mechanism...... in yeast, which would overcome the undesirable effects of statins. One possible mechanism is an active export of statins, a mechanism that does not just provide the resistance but can also significantly ease the purification of the produced compounds. In order to establish export of statins from...... transmembrane efflux pump, capable of exporting natural and semi-natural statins from yeast, and overexpression of MlcE in a statinproducing yeast could therefore greatly improve the commercial production of natural and semi-natural statins. Reference: (1) Xu W. et al., (2013), “LovG: The Thioesterase Required...

  12. Engineering Saccharomyces cerevisiae toward n‐butanol production

    OpenAIRE

    Swidah, Reem

    2016-01-01

    Biobutanol represents a second generation biofuel, which can be producedfrom renewable resources by microorganisms. A Saccharomyces cerevisiae strainbearing the five butanol synthetic genes (hbd, adhe2, crt, ccr and ERG10) wasconstructed, where the hbd, adhe2, crt and ccr genes are derived from Clostridiumbeijerinckii, while ERG10 is a yeast gene. The genes were transformed individually onsingle cassettes, which integrated into specific chromosomal sites. The single integrantstrains were back...

  13. Expression of native and mutant extracellular lipases fromYarrowia lipolytica in Saccharomyces cerevisiae

    OpenAIRE

    Darvishi, Farshad

    2012-01-01

    Summary Saccharomyces cerevisiae cannot produce extracellular lipase and utilize low‐cost lipid substrates. This study aimed to express extracellular lipase from Yarrowia lipolytica in S. cerevisiae, construct recombinant oily substrate consumer strains, and compare the roles of native and mutant Y. lipolytica extracellular lipases in S. cerevisiae. The LIP2 gene of Y. lipolytica DSM3286 and its mutant Y. lipolytica U6 were isolated and cloned by expression vector in S. cerevisiae. New recomb...

  14. Ethanol production from acid- and alkali-pretreated corncob by endoglucanase and β-glucosidase co-expressing Saccharomyces cerevisiae subject to the expression of heterologous genes and nutrition added.

    Science.gov (United States)

    Feng, Chunying; Zou, Shaolan; Liu, Cheng; Yang, Huajun; Zhang, Kun; Ma, Yuanyuan; Hong, Jiefang; Zhang, Minhua

    2016-05-01

    Low-cost technologies to overcome the recalcitrance of cellulose are the key to widespread utilization of lignocellulosic biomass for ethanol production. Efficient enzymatic hydrolysis of cellulose requires the synergism of various cellulases, and the ratios of each cellulase are required to be regulated to achieve the maximum hydrolysis. On the other hand, engineering of cellulolytic Saccharomyces cerevisiae strains is a promising strategy for lignocellulosic ethanol production. The expression of cellulase-encoding genes in yeast would affect the synergism of cellulases and thus the fermentation ability of strains with exogenous enzyme addition. However, such researches are rarely reported. In this study, ten endoglucanase and β-glucosidase co-expressing S. cerevisiae strains were constructed and evaluated by enzyme assay and fermentation performance measurement. The results showed that: (1) maximum ethanol titers of recombinant strains exhibited high variability in YPSC medium (20 g/l peptone, 10 g/l yeast extract, 100 g/l acid- and alkali-pretreated corncob) within 10 days. However, they had relatively little difference in USC medium (100 g/l acid- and alkali-pretreated corncob, 0.33 g/l urea, pH 5.0). (2) Strains 17# and 19#, with ratio (CMCase to β-glucosidase) of 7.04 ± 0.61 and 7.40 ± 0.71 respectively, had the highest fermentation performance in YPSC. However, strains 11# and 3# with the highest titers in USC medium had a higher ratio of CMCase to β-glucosidase, and CMCase activities. These results indicated that nutrition, enzyme activities and the ratio of heterologous enzymes had notable influence on the fermentation ability of cellulase-expressing yeast. PMID:27038956

  15. Profiling of the toxicity mechanisms of coated and uncoated silver nanoparticles to yeast Saccharomyces cerevisiae BY4741 using a set of its 9 single-gene deletion mutants defective in oxidative stress response, cell wall or membrane integrity and endocytosis.

    Science.gov (United States)

    Käosaar, Sandra; Kahru, Anne; Mantecca, Paride; Kasemets, Kaja

    2016-09-01

    The widespread use of nanosilver in various antibacterial, antifungal, and antiviral products warrants the studies of the toxicity pathways of nanosilver-enabled materials toward microbes and viruses. We profiled the toxicity mechanisms of uncoated, casein-coated, and polyvinylpyrrolidone-coated silver nanoparticles (AgNPs) using Saccharomyces cerevisiae wild-type (wt) and its 9 single-gene deletion mutants defective in oxidative stress (OS) defense, cell wall/membrane integrity, and endocytosis. The 48-h growth inhibition assay in organic-rich growth medium and 24-h cell viability assay in deionized (DI) water were applied whereas AgNO3, H2O2, and SDS served as positive controls. Both coated AgNPs (primary size 8-12nm) were significantly more toxic than the uncoated (~85nm) AgNPs. All studied AgNPs were ~30 times more toxic if exposed to yeast cells in DI water than in the rich growth medium: the IC50 based on nominal concentration of AgNPs in the growth inhibition test ranged from 77 to 576mg Ag/L and in the cell viability test from 2.7 to 18.7mg Ag/L, respectively. Confocal microscopy showed that wt but not endocytosis mutant (end3Δ) internalized AgNPs. Comparison of toxicity patterns of wt and mutant strains defective in OS defense and membrane integrity revealed that the toxicity of the studied AgNPs to S. cerevisiae was not caused by the OS or cell wall/membrane permeabilization. PMID:27260961

  16. A recombination repair gene of Schizosaccharomyces pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the human XRCC3 gene.

    OpenAIRE

    Tsutsui, Y; Morishita, T; Iwasaki, H.; H. Toh; Shinagawa, H

    2000-01-01

    To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% ident...

  17. The Improvement of SAM Accumulation by Integrating the Endogenous Methionine Adenosyltransferase Gene SAM2 in Genome of the Industrial Saccharomyces cerevisiae Strain.

    Science.gov (United States)

    Zhao, Weijun; Shi, Feng; Hang, Baojian; Huang, Lei; Cai, Jin; Xu, Zhinan

    2016-03-01

    S-Adenosyl-L-methionine (SAM) plays important roles in trans-methylation, trans-sulfuration, and polyamine synthesis in all living cells, and it is also an effective cure for liver disease, depressive syndromes, and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, a multiple-copy integrative plasmid pYMIKP-SAM2 was introduced into the chromosome of wild-type Saccharomyces cerevisiae strain ZJU001 to construct the recombined strain R1-ZJU001. Further studies showed that the recombinant yeast exhibited higher enzymatic activity of methionine adenosyltransferase and improved its SAM biosynthesis. With a three-phase fed-batch strategy in 15-liter bench-top fermentor, 8.81 g/L SAM was achieved after 52 h cultivation of R1-ZJU001, about 27.1 % increase over its parent strain ZJU001, whereas the SAM content was also improved from 64.6 mg/g DCW to 91.0 mg/g DCW. Our results shall provide insights into the metabolic engineering of SAM pathway in yeast for improved productivity of SAM and subsequent industrial applications. PMID:26728652

  18. Regulation of Maltose Transport and Metabolism in Saccharomyces cerevisiae

    OpenAIRE

    Novak, Srđan; Zechner-Krpan, Vesna; Marić, Vladimir

    2004-01-01

    Maltose metabolism in the yeast Saccharomyces cerevisiae is of great importance both for academic and industrial researchers. It requires the presence of at least one of five independent MAL loci: MAL1, MAL2, MAL3, MAL4 and MAL6. Each active locus is made of three genes: two structural genes that encode intracellular enzyme maltase and transport protein for maltose, and the third gene that encodes positive regulatory protein. Maltose is transported unchanged into the cell with the help of spe...

  19. A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Nookaew, Intawat; Papini, Marta; Pornputtapong, Natapol;

    2012-01-01

    genetic variation on the estimation of gene expression level using three different aligners for read-mapping (Gsnap, Stampy and TopHat) on S288c genome, the capabilities of five different statistical methods to detect differential gene expression (baySeq, Cuffdiff, DESeq, edgeR and NOISeq) and we explored...... differential gene expression identification derived from the different statistical methods, as well as their integrated analysis results based on gene ontology annotation are in good agreement. Overall, our study provides a useful and comprehensive comparison between the two platforms (RNA-seq and microrrays...

  20. The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 genes encode a novel family of membrane-anchored lipases that are required for steryl ester hydrolysis.

    Science.gov (United States)

    Köffel, René; Tiwari, Rashi; Falquet, Laurent; Schneiter, Roger

    2005-03-01

    Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Lipase triple-mutant yeast cells are completely blocked in steryl ester hydrolysis but do not affect the mobilization of triacylglycerols, indicating that the three lipases are required for steryl ester mobilization in vivo. Lipase single mutants mobilize steryl esters to various degrees, indicating partial functional redundancy of the three gene products. Lipase double-mutant cells in which the third lipase is expressed from the inducible GAL1 promoter have greatly reduced steady-state levels of steryl esters, indicating that overexpression of any of the three lipases is sufficient for steryl ester mobilization in vivo. The three yeast enzymes constitute a novel class of membrane-anchored lipases that differ in topology and subcellular localization. PMID:15713625

  1. Genetic Basis for Saccharomyces cerevisiae Biofilm in Liquid Medium

    DEFF Research Database (Denmark)

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

    2014-01-01

    than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the S1278b background and found 71 genes that were essential for biofilm development. Quantitative...

  2. Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae

    OpenAIRE

    1993-01-01

    Abplp is a yeast cortical actin-binding protein that contains an SH3 domain similar to those found in signal transduction proteins that function at the membrane/cytoskeleton interface. Although no detectable phenotypes are associated with a disruption allele of ABP1, mutations that create a requirement for this protein have now been isolated in the previously identified gene SAC6 and in two new genes, SLA1 and SLA2. The SAC6 gene encodes yeast fimbrin, an actin filament-bundling protein. Null...

  3. Evidence That the Transcriptional Regulators Sin3 and Rpd3, and a Novel Gene (Sds3) with Similar Functions, Are Involved in Transcriptional Silencing in S. Cerevisiae

    OpenAIRE

    Vannier, D; Balderes, D; Shore, D

    1996-01-01

    In a screen for extragenic suppressors of a silencing defective rap1(s) hmrΔA strain, recessive mutations in 21 different genes were found that restored repression to HMR. We describe the characterization of three of these SDS (suppressors of defective silencing) genes. SDS16 and SDS6 are known transcriptional modifiers, SIN3(RPD1/UME4/SDI1/GAM2) and RPD3(SDI2), respectively, while the third is a novel gene, SDS3. SDS3 shares the meiotic functions of SIN3 and RPD3 in that it represses IME2 in...

  4. CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis.

    OpenAIRE

    Bulawa, C E

    1992-01-01

    In Saccharomyces cerevisiae, chitin forms the primary division septum and the bud scar in the walls of vegetative cells. Three chitin synthetic activities have been detected. Two of them, chitin synthase I and chitin synthase II, are not required for synthesis of most of the chitin present in vivo. Using a novel screen, I have identified three mutations, designated csd2, csd3, and csd4, that reduce levels of chitin in vivo by as much as 10-fold without causing any obvious perturbation of cell...

  5. Production of β-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Lopez, Javiera; Essus, Karen; Kim, Il-Kwon;

    2015-01-01

    number episomal vectors, in an engineered strain that accumulates FPP. Results: Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing...... cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of beta-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy......, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene-the highest β-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods....

  6. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    Energy Technology Data Exchange (ETDEWEB)

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy); Caligo, Maria Adelaide [Section of Genetic Oncology, University Hospital and University of Pisa, via Roma 57, 56125 Pisa (Italy); Galli, Alvaro, E-mail: alvaro.galli@ifc.cnr.it [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy)

    2015-04-15

    Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

  7. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    International Nuclear Information System (INIS)

    Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

  8. Enhancement of malate-production and increase in sensitivity to dimethyl succinate by mutation of the VID24 gene in Saccharomyces cerevisiae.

    Science.gov (United States)

    Negoro, Hiroaki; Kotaka, Atsushi; Matsumura, Kengo; Tsutsumi, Hiroko; Hata, Yoji

    2016-06-01

    Malate in sake (a Japanese alcoholic beverage) is an important component for taste that is produced by yeasts during alcoholic fermentation. To date, many researchers have developed methods for breeding high-malate-producing yeasts; however, genes responsible for the high-acidity phenotype are not known. We determined the mutated gene involved in high malate production in yeast, isolated as a sensitive mutant to dimethyl succinate. In the comparative whole genome analysis between high-malate-producing strain and its parent strain, one of the non-synonymous substitutions was identified in the VID24 gene. The mutation of VID24 resulted in enhancement of malate-productivity and sensitivity to dimethyl succinate. The mutation appeared to lead to a deficiency in Vid24p function. Furthermore, disruption of cytoplasmic malate dehydrogenase (Mdh2p) gene in the VID24 mutant inhibited the high-malate-producing phenotype. Vid24p is known as a component of the multisubunit ubiquitin ligase and participates in the degradation of gluconeogenic enzymes such as Mdh2p. We suggest that the enhancement of malate-productivity results from an accumulation of Mdh2p due to the loss of Vid24p function. These findings propose a novel mechanism for the regulation of organic acid production in yeast cells by the component of ubiquitin ligase, Vid24p. PMID:26983942

  9. Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae

    OpenAIRE

    Foland, Travis B.; Dentler, William L.; SUPRENANT, KATHY A.; Gupta, Mohan L.; Himes, Richard H.

    2005-01-01

    Wild-type Saccharomyces cerevisiae tubulin does not bind the anti-mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for β-tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule-stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated β-tubulin gene into a S. cerevisiae strain with dimini...

  10. Conserved codon composition of ribosomal protein coding genes in Escherichia coli, Mycobacterium tuberculosis and Saccharomyces cerevisiae: lessons from supervised machine learning in functional genomics

    OpenAIRE

    Lin, Kui; Kuang, Yuyu; Joseph, Jeremiah S.; Kolatkar, Prasanna R

    2002-01-01

    Genomics projects have resulted in a flood of sequence data. Functional annotation currently relies almost exclusively on inter-species sequence comparison and is restricted in cases of limited data from related species and widely divergent sequences with no known homologs. Here, we demonstrate that codon composition, a fusion of codon usage bias and amino acid composition signals, can accurately discriminate, in the absence of sequence homology information, cytoplasmic ribosomal protein gene...

  11. Identification of Saccharomyces cerevisiae genes involved in the resistance to multiple stresses during Very-High-Gravity and lignocellulosic biomass industrial fermentations

    OpenAIRE

    Pereira, Francisco B.; Guimarães, Pedro M. R.; Gomes, Daniel Gonçalves; Mira, Nuno P.; Teixeira, Miguel C; Correia, Isabel Sá; Domingues, Lucília

    2011-01-01

    Most of the current processes for bioethanol production are based on the use of Very-High-Gravity (VHG) technology and the processing of lignocellulosic biomass, limited by the high osmotic pressure and ethanol concentration in the fermentation medium, and by inhibitors resulting from biomass pre-treatments, respectively. Aiming the optimization of strains for industrial bioethanol production an integrated approach was undertaken to identify genes required for simultaneous yeast resistance...

  12. Genome-wide identification of the Fermentome; genes required for successful and timely completion of wine-like fermentation by Saccharomyces cerevisiae

    OpenAIRE

    Walker, Michelle E; Nguyen, Trung D; Liccioli, Tommaso; Schmid, Frank; Kalatzis, Nicholas; Sundstrom, Joanna F.; Gardner, Jennifer M.; Jiranek, Vladimir

    2014-01-01

    Background Wine fermentation is a harsh ecological niche to which wine yeast are well adapted. The initial high osmotic pressure and acidity of grape juice is followed by nutrient depletion and increasing concentrations of ethanol as the fermentation progresses. Yeast’s adaptation to these and many other environmental stresses, enables successful completion of high-sugar fermentations. Earlier transcriptomic and growth studies have tentatively identified genes important for high-sugar ferment...

  13. Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of PMS1-1 and PMS1-2

    International Nuclear Information System (INIS)

    The PMS1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked HIS4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in PMS1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered

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

    Science.gov (United States)

    2010-04-01

    ...) antibody (ASCA) test systems. 866.5785 Section 866.5785 Food and Drugs FOOD AND DRUG ADMINISTRATION... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  15. Candida albicans AGE3, the ortholog of the S. cerevisiae ARF-GAP-encoding gene GCS1, is required for hyphal growth and drug resistance.

    Directory of Open Access Journals (Sweden)

    Thomas Lettner

    Full Text Available BACKGROUND: Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Delta mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Delta cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Delta cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Delta mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p. CONCLUSIONS/SIGNIFICANCE: The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Delta cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Delta cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Delta cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it

  16. Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene

    International Nuclear Information System (INIS)

    Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1

  17. The Bioconversion of Red Ginseng Ethanol Extract into Compound K by Saccharomyces cerevisiae HJ-014

    OpenAIRE

    Choi, Hak Joo; Kim, Eun A.; Kim, Dong Hee; Shin, Kwang-Soo

    2014-01-01

    A β-glucosidase producing yeast strain was isolated from Korean traditional rice wine. Based on the sequence of the YCL008c gene and analysis of the fatty acid composition, the isolate was identified as Saccharomyces cerevisiae strain HJ-014. S. cerevisiae HJ-014 produced ginsenoside Rd, F2, and compound K from the ethanol extract of red ginseng. The production was increased by shaking culture, where the bioconversion efficiency was increased 2-fold compared to standing culture. The productio...

  18. Horizontal and vertical growth of S. cerevisiae metabolic network.

    KAUST Repository

    Grassi, Luigi

    2011-10-14

    BACKGROUND: The growth and development of a biological organism is reflected by its metabolic network, the evolution of which relies on the essential gene duplication mechanism. There are two current views about the evolution of metabolic networks. The retrograde model hypothesizes that a pathway evolves by recruiting novel enzymes in a direction opposite to the metabolic flow. The patchwork model is instead based on the assumption that the evolution is based on the exploitation of broad-specificity enzymes capable of catalysing a variety of metabolic reactions. RESULTS: We analysed a well-studied unicellular eukaryotic organism, S. cerevisiae, and studied the effect of the removal of paralogous gene products on its metabolic network. Our results, obtained using different paralog and network definitions, show that, after an initial period when gene duplication was indeed instrumental in expanding the metabolic space, the latter reached an equilibrium and subsequent gene duplications were used as a source of more specialized enzymes rather than as a source of novel reactions. We also show that the switch between the two evolutionary strategies in S. cerevisiae can be dated to about 350 million years ago. CONCLUSIONS: Our data, obtained through a novel analysis methodology, strongly supports the hypothesis that the patchwork model better explains the more recent evolution of the S. cerevisiae metabolic network. Interestingly, the effects of a patchwork strategy acting before the Euascomycete-Hemiascomycete divergence are still detectable today.

  19. Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae

    Science.gov (United States)

    The yeast Saccharomyces cerevisiae is able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and hydroxymethylfurfural (HMF). The length of lag phase for cell growth in response to the inhibitor challenge has been used to measure tolerance of strain performance. Mechan...

  20. 葡萄酒酵母car1基因表达量与EC含量相关性的研究%Study on correlation between the expression of car1 gene of different Saccharomyces cerevisiae and the content of carbamate ethgl in wine

    Institute of Scientific and Technical Information of China (English)

    魏玉洁; 付方圆; 武顺; 邹弯; 薛洁; 闫寅卓; 武运

    2015-01-01

    酿酒酵母(Saccharomyces cerevisiae)代谢精氨酸产生尿素对葡萄酒中氨基甲酸乙酯(carbamate ethyl,EC)含量的影响非常重要.利用实时荧光PCR技术,比较分析了18种酿酒酵母的发酵性能以及发酵期间酵母car1基因的表达活性与精氨酸、EC含量间的关系,结果表明:发酵期间,酵母car1基因的表达量均呈现先增加后降低的趋势,与发酵液中的精氨酸呈负相关关系,相关系数在0.9903~0.9977;而与EC含量呈现正相关关系,相关系数在0.6249~0.995 8之间;不同酵母car1基因的相对表达量存在显著差异,筛选精氨酸酶活力低的酵母菌株可以有效降低葡萄酒中的EC含量.%Urea produced by arginine metabolites from Saccharomyces cerevisiae has very important effect on the content of ethyl carbamate in wine.In this paper,the fermentation performance of 18 S.cerevisiae strains and the correlation between the expression level of car1 gene and the content of EC and arginine were analyzed.Real-time fluorescence PCR showed that the expression of car1 gene increased at the beginning of fermentation and decreased two days after fermentation.The expression level of car1 gene was significantly negatively related with the content of arginine with the correlation coefficient between 0.990 3 ~ 0.997 7,while it was positively related with the content of carbamate ethyl with the correlation coefficient between 0.624 9 ~ 0.995 8,which was obviously different between different yeasts.Selection of S.cerevisiae strains containing arginase with low activity could effectively reduce the content of Ethyl carbamate in wine.

  1. Draft Genome Sequence of Saccharomyces cerevisiae Strain NCIM3186 Used in the Production of Bioethanol from Sweet Sorghum

    OpenAIRE

    Sravanthi Goud, Burragoni; Ulaganathan, Kandasamy

    2015-01-01

    Here, we report the draft genome sequence of Saccharomyces cerevisiae strain NCIM3186 used in bioethanol production from sweet sorghum. The size of the genome is approximately 11.9 Mb and contains 5,347 protein-coding genes.

  2. Automated Yeast Mating Protocol Using Open Reading Frames from Saccharomyces cerevisiae Genome to Improve Yeast Strains for Cellulosic Ethanol Production

    Science.gov (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...

  3. The DNA-damage signature in Saccharomyces cerevisiae is associated with single-strand breaks in DNA

    OpenAIRE

    Begley Thomas J; Cosgrove Joseph P; DeMott Michael S; Fry Rebecca C; Samson Leona D; Dedon Peter C

    2006-01-01

    Abstract Background Upon exposure to agents that damage DNA, Saccharomyces cerevisiae undergo widespread reprogramming of gene expression. Such a vast response may be due not only to damage to DNA but also damage to proteins, RNA, and lipids. Here the transcriptional response of S. cerevisiae specifically induced by DNA damage was discerned by exposing S. cerevisiae to a panel of three "radiomimetic" enediyne antibiotics (calicheamicin γ1I, esperamicin A1 and neocarzinostatin) that bind speci...

  4. Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae.

    OpenAIRE

    Seifert, H S; Chen, E Y; So, M; Heffron, F

    1986-01-01

    We have extended the method of transposon mutagenesis to the eukaryote, Saccharomyces cerevisiae. A bacterial transposon containing a selectable yeast gene can be transposed into a cloned fragment of yeast DNA in Escherichia coli, and the transposon insertion can be returned to the yeast genome by homologous recombination. Initially, the cloned yeast DNA fragment to be mutagenized was transformed into an E. coli strain containing an F factor derivative carrying the transposable element. The c...

  5. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

    OpenAIRE

    DiCarlo, James; Norville, Julie; Mali, Prashant; Rios Villanueva, Xavier; Aach, John Dennis; Church, George McDonald

    2013-01-01

    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...

  6. Constitutive Optimized Production of Streptokinase in Saccharomyces cerevisiae Utilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter of Pichia pastoris

    OpenAIRE

    Vellanki, Ravi N.; Ravichandra Potumarthi; Doddapaneni, Kiran K.; Naveen Anubrolu; Lakshmi N. Mangamoori

    2013-01-01

    A novel expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was co...

  7. [Production of β-carotene by metabolically engineered Saccharomyces cerevisiae].

    Science.gov (United States)

    Wang, Beibei; Shi, Mingyu; Wang, Dong; Xu, Jiaoyang; Liu, Yi; Yang, Hongjiang; Dai, Zhubo; Zhang, Xueli

    2014-08-01

    β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production. PMID:25507473

  8. 人醛糖还原酶基因在酿酒酵母中的诱导表达及活性分析%Induced expression and activity analysis of human aldose reductase gene in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    翟冰; 叶玲; 刘静; 刘建伟

    2011-01-01

    Objective To construct the Saccharvmyces cerevisiae cell strains expressing aldose reductase(AR) and AR-green fluorescent protein(AR-GFP) fusion protein, and to detect the target gene expression and the AR enzyme activity. Methods The yeast expression plasmids pYEX-BX inserted with AR and AR::GFP fusion gene were transformed into the yeast strain ENVScl, which were named as XAR and XAG strains respectively. The blank pYEX-BX strain was used as the normal control. The growth curves and the fluorescence were determined in all strains. Northern blot, Western blot and the fluorescent method were used to detect the AR mRNA transcription, AR protein expression, and the AR activity respectively. Results There was no significant difference in the growth rates among three strains. There was a linear relationship between relative fluorescence of the XAG and the growth time. The mRNA transcription and protein expression of AR and AR::GFP were sustainable and stable in XAR and XAG strains. The AR activities in the two strains were both proved by the fluorescent method. Conclusion The yeast expression strain of AR was constructed successfully, which lays basis for its application in the researches on pathogenic mechanism of AR and preliminary screening of new AR inhibitors.%目的 建立人醛糖还原酶(AR)及其与绿色荧光蛋白(GFP)融合表达的酿酒酵母细胞表达株,检测细胞中目的基因的表达及蛋白活性.方法 将含目的基因AR及融合基因AR::GFP的重组质粒pYEX-BX转化酵母细胞INVScl,得到宿主菌株XAR,XAG及对照菌株YEX.观察酵母细胞生长曲线及GFP荧光信号;Northern blot检测细胞中AR mRNA转录;Western blot检测AR蛋白表达;荧光法测定AR活性.结果 三菌株生长速率无明显差异;XAG菌株相对荧光强度与生长时间成线性关系;XAR,XAG菌株分别有持续、稳定的目的基因mRNA转录及目的蛋白表达,并具有AR活性.结论 成功构建了AR基因的酵母表达株,为

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

    DEFF Research Database (Denmark)

    Fazio, Alessandro

    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...... 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...

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

    Directory of Open Access Journals (Sweden)

    Maksim I. Sorokin

    2014-01-01

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

  11. Applied systems biology - vanillin production in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Strucko, Tomas; Eriksen, Carsten; Nielsen, J.;

    2012-01-01

    Vanillin is the most important aroma compound based on market value, and natural vanillin is extracted from the cured seed pods of the Vanilla orchid. Most of the world’s vanillin, however, is obtained by chemical synthesis from petrochemicals or wood pulp lignins. As an alternative, de novo...... biosynthesis of vanillin in baker’s yeast Saccharomyces cerevisiae was recently demonstrated by successfully introducing the metabolic pathway for vanillin production in yeast. Nevertheless, the amount of vanillin produced in this S. cerevisiae strain is insufficient for commercial production and improvements...... need to be done. We have introduced the genes necessary for vanillin production in an identical manner in two different yeast strains S288c and CEN.PK,where comprehensive – omics datasets are available, hence, allowing vanillin production in the two strain backgrounds to be evaluated and compared in a...

  12. Redox balancing in recombinant strains of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Anderlund, M.

    1998-09-01

    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

  13. Utilizing an endogenous pathway for 1-butanol production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Si, Tong; Luo, Yunzi; Xiao, Han; Zhao, Huimin

    2014-03-01

    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 adh1Δ, 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

  14. Improvement of Xylose Uptake and Ethanol Production in Recombinant Saccharomyces cerevisiae through an Inverse Metabolic Engineering Approach

    OpenAIRE

    Jin, Yong-Su; Alper, Hal; Yang, Yea-Tyng; Stephanopoulos, Gregory

    2005-01-01

    We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from t...

  15. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    OpenAIRE

    Nevoigt, Elke

    2008-01-01

    Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic...

  16. Sequence Analysis and Subcellular Localization of a Novel Nosema bombycis Gene in Saccharomyces cerevisiae Cells%一个家蚕微孢子虫新基因的序列分析及蛋白质在酿酒酵母细胞中的定位

    Institute of Scientific and Technical Information of China (English)

    罗洁; 邓远洪; 黄为; 李田; 杨琼; 殷梅; 潘国庆; 李春峰; 周泽扬

    2013-01-01

    Microsporidian is typified by its high rate of gene evolution but low rate of genome evolution. There are many synteny blocks among different microsporidian species. Through comparative genomic analysis, we found a novel gene (numbered as NBO32g0035) with unknown function from Nosema bombycis CQ1 isolate. But no orthologous gene was found in genome of Encephalitozoon cuniculiand Nosema ceranae. This gene encodes 521 amino acids, among which 131 have acidic and 76 have basic side chains. The content of lysine is the highest. This protein has 6 cysteine residues and was predicted to have no peptide signal by SignalP3.0. RT-PCR results indicated that this gene was transcribed in silkworm midgut tissue on the 1 st day to 10th day after infection by N. bombycis CQ1 isolate, and it could only be cloned from genome of N. bombycis. Nucleotide polymorphism on this gene was characterized by cloning and sequencing. After being transfected into Sacchromyces cerevisiae (a unicellular model organism), it was revealed that the protein encoded by this gene was not located in a specific region of the cell. Instead, it was mainly distributed in cytoplasm of S. cerevisiae cells. The obtained result showed that this novel N. bombycis gene had the characteristics of expression at early stage of infection and of nucleotide polymorphism, and its encoded protein was located in cytoplasm of S. cerevisiae cells.%微孢子虫具有基因组进化速度慢,但基因进化速度快的特征,不同微孢子虫间存在较多的共线性区域.通过比较基因组分析在家蚕微孢子虫(Nosema bombycis,Nb) CQ1株中发现一个编号为NBO32g0035的功能未知新基因,而在兔脑炎微孢子虫(Encephalitozoon cuniculi和蜜蜂微孢子虫(Nosema ceranae)均无该基因的存在.该基因编码521个氨基酸,其中有131个酸性氨基酸和76个碱性氨基酸,赖氨酸含量最高,半胱氨酸有6个,SignalP3.0预测该基因编码的蛋白质无信号

  17. Detoxification of Aflatoxin B1 by Saccharomyces cerevisiae Mutants of Anti-Oxidative Relating Genes%酿酒酵母抗氧化相关基因突变体对黄曲霉毒素B1的清除作用

    Institute of Scientific and Technical Information of China (English)

    史锋; 黄宇啸; 李永富

    2012-01-01

    黄曲霉毒素(AF)是粮食作物和饲料原料中容易污染的一种强毒性和强致癌性物质,酿酒酵母具有毒素清除功能.利用HPLC分析了酿酒酵母野生菌BY4742及三株关键的抗氧化相关基因缺失茵zwf1△、sod2△、glr1△对黄曲霉毒素B1的清除能力.结果表明,在PBS缓冲液中存活和死亡的细胞对AFB1的清除率分别为74%~76%和71%~73%,说明酵母细胞对AFB1的清除以生物吸附作用为主.在培养基中,3种突变菌活细胞对AFB1的清除率发生不同程度的降低,其中glr1△的AFB1清除能力下降最明显,其次是sod2△,而zwf1△下降最少,说明这些关键的抗氧化基因的缺失会影响细胞在生长状态下对AFB1的清除作用.%Aflatoxins are a group of mycotoxins with strong mutagenic and carcinogenic properties. Various commodities including crop and feed materials are easy to be contaminated with aflatoxin. Saccharomyces cerevisiae have been reported to bind or degrade aflatoxin. Here, detoxification of aflatoxin B1 (AFB1 ) by wild-type strain of S. cerevisiae (BY4742) and three mutants of anti-oxidative relating genes (zw/l△, sod2△and girl △) were determined by HPLC. In PBS buffer, AFBi binding abilities of viable and dead cells were 74% -76% and 71% — 73%, respectively, indicating AFB1 was removed by yeast cells mainly through cell adsorption. In YPD medium, clearance of AFB1 by three mutant viable cells reduced, while that by wild-type BY4742 remaining high. AFB1 binding ability of g/rlA decreased most seriously, then was that of sod2△ and zwfl△. Thus, the deletion of critical anti-oxidative relating genes would decrease the AFB1 binding ability of S. cerevisiae growing cells.

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

    Science.gov (United States)

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

    2015-09-01

    Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae. PMID:26164646

  19. Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

    Directory of Open Access Journals (Sweden)

    Serge Feyder

    2015-01-01

    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.

  20. Fungal genomics beyond Saccharomyces cerevisiae?

    DEFF Research Database (Denmark)

    Hofmann, Gerald; Mcintyre, Mhairi; Nielsen, Jens

    2003-01-01

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

  1. Molecular Basis for Saccharomyces cerevisiae Biofilm Development

    DEFF Research Database (Denmark)

    Andersen, Kaj Scherz

    In this study, I sought to identify genes regulating the global molecular program for development of sessile multicellular communities, also known as biofilm, of the eukaryotic microorganism, Saccharomyces cerevisiae (yeast). Yeast biofilm has a clinical interest, as biofilms can cause chronic...

  2. Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids.

    Science.gov (United States)

    Yu, Ai-Qun; Juwono, Nina Kurniasih Pratomo; Foo, Jee Loon; Leong, Susanna Su Jan; Chang, Matthew Wook

    2016-03-01

    Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement. PMID:26721212

  3. Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces cerevisiae Strains

    NARCIS (Netherlands)

    Zelle, R.M.; Harrison, J.C.; Pronk, J.T.; Van Maris, A.J.A.

    2010-01-01

    Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2. The Saccharomyces cerevisiae MAE1 gene encodes a mitochondrial malic enzyme whose proposed physiological roles are related to the oxidative, malate-decarboxylating reaction. Hitherto, the inability of pyru

  4. Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass

    Science.gov (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...

  5. Hyper- and hyporesponsive mutant forms of the Saccharomyces cerevisiae Ssy1 amino acid sensor

    DEFF Research Database (Denmark)

    Poulsen, Peter; Gaber, Richard F.; Kielland-Brandt, Morten

    2008-01-01

    The Saccharomyces cerevisiae integral membrane protein Ssy1p functions with Ssy5p and Ptr3p to sense extracellular amino acids. Signal transduction leads to processing and nuclear localization of Stp1p and Stp2p, transcriptional activators of many amino acid transporter genes. Ssy1p is structurally...

  6. Genomic approaches for identifying DNA damage response pathways in S. cerevisiae

    NARCIS (Netherlands)

    Chang, Michael; Parsons, Ainslie B; Sheikh, Bilal H; Boone, Charles; Brown, Grant W

    2006-01-01

    DNA damage response pathways have been studied extensively in the budding yeast Saccharomyces cerevisiae, yet new genes with roles in the DNA damage response are still being identified. In this chapter we describe the use of functional genomic approaches in the identification of DNA damage response

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

    NARCIS (Netherlands)

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

    2004-01-01

    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

  8. A repressor activator protein1 homologue from an oleaginous strain of Candida tropicalis increases storage lipid production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chattopadhyay, Atrayee; Dey, Prabuddha; Barik, Amita; Bahadur, Ranjit P; Maiti, Mrinal K

    2015-06-01

    The repressor activator protein1 (Rap1) has been studied over the years as a multifunctional regulator in Saccharomyces cerevisiae. However, its role in storage lipid accumulation has not been investigated. This report documents the identification and isolation of a putative transcription factor CtRap1 gene from an oleaginous strain of Candida tropicalis, and establishes the direct effect of its expression on the storage lipid accumulation in S. cerevisiae, usually a non-oleaginous yeast. In silico analysis revealed that the CtRap1 polypeptide binds relatively more strongly to the promoter of fatty acid synthase1 (FAS1) gene of S. cerevisiae than ScRap1. The expression level of CtRap1 transcript in vivo was found to correlate directly with the amount of lipid produced in oleaginous native host C. tropicalis. Heterologous expression of the CtRap1 gene resulted in ∼ 4-fold enhancement of storage lipid content (57.3%) in S. cerevisiae. We also showed that the functionally active CtRap1 upregulates the endogenous ScFAS1 and ScDGAT genes of S. cerevisiae, and this, in turn, might be responsible for the increased lipid production in the transformed yeast. Our findings pave the way for the possible utility of the CtRap1 gene in suitable microorganisms to increase their storage lipid content through transcription factor engineering. PMID:25805842

  9. Global Transcriptional and Physiological Responses of Saccharomyces cerevisiae to Ammonium, L-Alanine, or L-Glutamine Limitation

    DEFF Research Database (Denmark)

    Usaite, Renata; Patil, Kiran Raosaheb; Grotkjær, Thomas;

    2006-01-01

    The yeast Saccharomyces cerevisiae encounters a range of nitrogen sources at various concentrations in its environment. The impact of these two parameters on transcription and metabolism was studied by growing S. cerevisiae in chemostat cultures with L-glutamine, L-alanine, or L-ammonium in...... repression (NCR) may be responsible for this regulation. Ninety-one genes had transcript levels on both L-glutamine and ammonium that were decreased compared to those on L-alanine, independent of the concentration. The GATAAG element in these genes suggests two groups of NCR-responsive genes, those that...

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

    DEFF Research Database (Denmark)

    Hou, Jin; Österlund, Tobias; Liu, Zihe;

    2013-01-01

    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...... 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...

  11. Non-Coding RNA Prediction and Verification in Saccharomyces cerevisiae

    OpenAIRE

    Kavanaugh, Laura A.; Dietrich, Fred S.

    2009-01-01

    Non-coding RNA (ncRNA) play an important and varied role in cellular function. A significant amount of research has been devoted to computational prediction of these genes from genomic sequence, but the ability to do so has remained elusive due to a lack of apparent genomic features. In this work, thermodynamic stability of ncRNA structural elements, as summarized in a Z-score, is used to predict ncRNA in the yeast Saccharomyces cerevisiae. This analysis was coupled with comparative genomics ...

  12. Expression of a lipid-inducible, self-regulating form of Yarrowia lipolytica lipase LIP2 in Saccharomyces cerevisiae

    Science.gov (United States)

    The Yarrowia lipolytica lipase 2 gene (YlLIP2) was cloned into galactose- and fatty acid-inducible Saccharomyces cerevisiae expression vectors and used to generate yeast strains that secrete active LIP2 enzyme activity, as evidenced by results from gene expression analysis and tributyrin turbidity c...

  13. Switching the mode of sucrose utilization by Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Miletti Luiz C

    2008-02-01

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

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

    Science.gov (United States)

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

    2015-11-01

    During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation. PMID:26279142

  15. Glycolipids of Saccharomyces cerevisiae Cell

    Directory of Open Access Journals (Sweden)

    Renuka Malhotra

    2005-01-01

    Full Text Available Total lipids of Saccharomyces cerevisiae were isolated by chloroform and methanol (2:1. Glycolipids were separated from total lipids by silicic acid chromatography. Glycolipid’s constituent sugars and fatty acids were analyzed by using Gas Liquid Chromatography. Galactose was the prominent sugar followed by mannose. Relative concentrations of fucose, mannose, galactose and glucose in the glycolipid were 5.3, 35.2, 55.1 and 4.2%. 16:0, 18:0, 18:1, 18:2 and 18:3 were the major fatty acids of the total glycolipids. Oleic acid was the dominating fatty acid followed by linoliec acid. They were separated into different fractions by using DEAE-Sephadex ion exchange chromatography. Glycolipids were fractionated and identified as cerebrosides, ceramide polyhexosides, sulfatides, monoglucosyldiglycerides and diglucosyldiglycerides. Ceramide polyhexosides were present in higher concentration as compared to other fractions.

  16. Glucose repression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kayikci, Omur; Nielsen, Jens

    2015-01-01

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

  17. Methionine catabolism in Saccharomyces cerevisiae.

    Science.gov (United States)

    Perpète, Philippe; Duthoit, Olivier; De Maeyer, Simon; Imray, Louise; Lawton, Andrew I; Stavropoulos, Konstantinos E; Gitonga, Virginia W; Hewlins, Michael J E; Dickinson, J Richard

    2006-01-01

    The catabolism of methionine to methionol and methanethiol in Saccharomyces cerevisiae was studied using (13)C NMR spectroscopy, GC-MS, enzyme assays and a number of mutants. Methionine is first transaminated to alpha-keto-gamma-(methylthio)butyrate. Methionol is formed by a decarboxylation reaction, which yields methional, followed by reduction. The decarboxylation is effected specifically by Ydr380wp. Methanethiol is formed from both methionine and alpha-keto-gamma-(methylthio)butyrate by a demethiolase activity. In all except one strain examined, demethiolase was induced by the presence of methionine in the growth medium. This pathway results in the production of alpha-ketobutyrate, a carbon skeleton, which can be re-utilized. Hence, methionine catabolism is more complex and economical than the other amino acid catabolic pathways in yeast, which use the Ehrlich pathway and result solely in the formation of a fusel alcohol. PMID:16423070

  18. High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ahlgren Simon

    2011-09-01

    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.

  19. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek; Schölz, Christian; Wagner, Sebastian A; Magnes, Christoph; Zechner, Rudolf; Choudhary, Chuna Ram

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  20. TOTAL ANTIOXIDANT ACTIVITY OF YEAST SACCHAROMYCES CEREVISIAE

    OpenAIRE

    Blažena Lavová; Dana Urminská

    2013-01-01

    Antioxidants are health beneficial compounds that can protect cells and macromolecules (e.g. fats, lipids, proteins and DNA) from the damage of reactive oxygen species (ROS). Sacchamomyces cerevisiae are know as organisms with very important antioxidative enzyme systems such as superoxide dismutase or catalase. The total antioxidant activity (mmol Trolox equivalent – TE.g-1 d.w.) of Saccharomyces cerevisiae was measured by 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) during the yeas...

  1. Transposon Mutagenesis To Improve the Growth of Recombinant Saccharomyces cerevisiae on d-Xylose▿

    OpenAIRE

    Ni, Haiying; Laplaza, José M.; Jeffries, Thomas W.

    2007-01-01

    Saccharomyces cerevisiae L2612 transformed with genes for xylose reductase and xylitol dehydrogenase (XYL1 and XYL2) grows well on glucose but very poorly on d-xylose. When a gene for d-xylulokinase (XYL3 or XKS1) is overexpressed, growth on glucose is unaffected, but growth on xylose is blocked. Spontaneous or chemically induced mutants of this engineered yeast that would grow on xylose could, however, be obtained. We therefore used insertional transposon mutagenesis to identify two loci tha...

  2. Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance▿

    OpenAIRE

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

    2009-01-01

    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)...

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

    Science.gov (United States)

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

    2016-04-01

    The budding yeast Saccharomyces cerevisiae is widely used for brewing and ethanol production. The ethanol sensitivity of yeast cells is still a serious problem during ethanol fermentation, and a variety of genetic approaches (e.g., random mutant screening under selective pressure of ethanol) have been developed to improve ethanol tolerance. In this study, we developed a strategy for improving ethanol tolerance of yeast cells based on metabolomics as a high-resolution quantitative phenotypic analysis. We performed gas chromatography-mass spectrometry analysis to identify and quantify 36 compounds on 14 mutant strains including knockout strains for transcription factor and metabolic enzyme genes. A strong relation between metabolome of these mutants and their ethanol tolerance was observed. Data mining of the metabolomic analysis showed that several compounds (such as trehalose, valine, inositol and proline) contributed highly to ethanol tolerance. Our approach successfully detected well-known ethanol stress related metabolites such as trehalose and proline thus, to further prove our strategy, we focused on valine and inositol as the most promising target metabolites in our study. Our results show that simultaneous deletion of LEU4 and LEU9 (leading to accumulation of valine) or INM1 and INM2 (leading to reduction of inositol) significantly enhanced ethanol tolerance. This study shows the potential of the metabolomic approach to identify target genes for strain improvement of S. cerevisiae with higher ethanol tolerance. PMID:26344121

  4. Identification of the Xenopus laevis Homolog of Saccharomyces cerevisiae DNA2 and Its Role in DNA Replication

    OpenAIRE

    Liu, Qingquan; Choe, Won-chae; Campbell, Judith L.

    2000-01-01

    The DNA2 gene of Saccharomyces cerevisiae is essential for growth and appears to be required for a late stage of chromosomal DNA replication. S. cerevisiae Dna2p (ScDna2p) is a DNA helicase and also a nuclease. We have cloned and sequenced the homologous gene from Xenopus (Xenopus Dna2). Xenopus Dna2p (XDna2p) is 32% identical to ScDna2p, and the similarity extends over the entire length, including but not limited to the five conserved helicase motifs. XDna2p is even more closely related (60%...

  5. Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2016-03-01

    Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces. PMID:26671616

  6. SACCHAROMYCES CEREVISIAE AND ITS VALIDATION

    Directory of Open Access Journals (Sweden)

    Miroslav Ondrejovič

    2015-02-01

    Full Text Available The aim of this study was to optimize of independent variables as temperature, time and reaction ratio to output parameter of simultaneous enzyme saccharification and fermentation by Saccharomyces cerevisiae of pretreated wheat straw as model substrate via RSM (response surface methodology approach. As dependent variable, it was chosen ethanol yields characterizing effectivity of process. The optimal conditions were approximately temperature 100 °C, time 1 hour and reaction ratio 26 mL to 1 g of treated wheat straw with ethanol yields 141.9 mg.g-1. After calculating the optimal values, the validation analyze was carried out and it was found out that the predicted and experimentally verified dependent variable was in agreement with the optimal parameters (~ 95 %. Proposed model was tested for three lignocellulosic materials (winter wheat straw, alfalfa hay and maize straw as wheat straw used as model substrate and it was confirmed the possibility of its use for other agricultural residues with similar content of lignocellulose.

  7. Xylitol production by Saccharomyces cerevisiae overexpressing different xylose reductases using non-detoxified hemicellulosic hydrolysate of corncob

    OpenAIRE

    Kogje, Anushree; Ghosalkar, Anand

    2016-01-01

    Xylitol production was compared in fed batch fermentation by Saccharomyces cerevisiae strains overexpressing xylose reductase (XR) genes from Candida tropicalis, Pichia stipitis, Neurospora crassa, and an endogenous gene GRE3. The gene encoding a xylose specific transporter (SUT1) from P. stipitis was cloned to improve xylose transport and fed batch fermentation was used with glucose as a cosubstrate to regenerate NADPH. Xylitol yield was near theoretical for all the strains in fed batch ferm...

  8. A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin

    Directory of Open Access Journals (Sweden)

    Zhang Tingting

    2012-12-01

    Full Text Available Abstract Background Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products. Results We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice. Conclusions Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.

  9. Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

    Science.gov (United States)

    Hong, Soo-Jeong; Kim, Hyo Jin; Kim, Jin-Woo; Lee, Dae-Hee; Seo, Jin-Ho

    2015-02-01

    Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity. PMID:25142154

  10. Characterization of the Viable but Nonculturable (VBNC State in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Mohammad Salma

    Full Text Available The Viable But Non Culturable (VBNC state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to "resuscitate". The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO2-induced VBNC state in S. cerevisiae and that the stress removal allows the "resuscitation" of VBNC cells during the VBNC state.

  11. 高效表达木糖醇脱氢酶基因酿酒酵母的构建及木酮糖发酵的初步研究%Construction of Saccharomyces cerevisiae Strain Expressing Xylitol Dehydrogenase Gene Efficiently and Primary Study of Its Xylulose Fermentation

    Institute of Scientific and Technical Information of China (English)

    陈高云; 刘敏; 叶凯; 张元忠; 涂振东; 于孟斌

    2011-01-01

    通过RT—PCR方法克隆得到Candidatropicalis木糖醇脱氢酶基因xyl2,将该基因连入酵母表达载体pYES2的诱导型启动子GAL1下,构建表达质粒pYES2-xyl2;同时用从Pichiapastoris中克隆获取的甘油醛磷酸脱氢酶基因GAP换下GAL1基因,构建含组成型启动子GAP基因的表达质粒pYES2-GAP—xyl2;通过电转化法将其依次转入酿酒酵母S.cerevisiaeINVSc1,山梨醇培养基上筛选的转化子经木糖醇梯度驯化培养,筛选出1株耐木糖醇浓度为20%的酿酒酵母重组菌株ZCX4和1株在半乳糖诱导下耐木糖醇浓度为15%的重组菌株YDX2。酶活测定表明。重组菌株ZCX4比酶活0.621U/mg(蛋白),是YDX2比酶活的2.29倍。摇瓶发酵结果显示,重组菌株ZCX4木糖醇消耗76.46g/L,木糖醇消耗率为76.46%,是重组茵株YDX2木糖醇消耗率的1.63倍,说明木糖醇脱氢酶实现了高效表达。%Yeast expression vector pYES2-xyl2 was constructed by cloning xylitol dehydrogenase gene xyl2, which originated from Candida tropicalis and placed under the inducible promoter GALl of the vector. Meanwhile, the other yeast expression vector pYES2-GAP-xyI2 containing the constitutive strong promoter GAP gene instead ofGAL gene was constructed. The plasmids containing xyl2 gene were transformed into industrial strain of S.cerevisiae INVScl by electroporation. The recombinant transformants ZCX4 and YDX2 grew well on plates in condition of high-concentration xylitol. The xylitol dehydrogenase specific activity of recombinant strain ZCX4 was 0.621 U/mg protein, 2.39 times as much as the recombinant strain YDX2, In addition, flask-shaking fermentation results revealed that the consumption of xylitol for ZCX4 was 76.46 g/L, 1.63 times as much as the recombinant strain YDX2. The results demonstrated that the recombinant stain could utilize xylitol efficiently by xylulose fermentation.

  12. Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Thomsen, Rune; Libri, Domenico; Boulay, Jocelyne;

    2003-01-01

    In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retain......In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated...... in retaining RNAs in these foci; on deletion of the exosome component Rrp6p, the RNA is released. To determine the exact nuclear location of retained as well as released mRNAs, we have used mRNA export mutant strains to analyze the spatial relationship between newly synthesized heat shock mRNA, the chromosomal...

  13. Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

    2015-12-01

    Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production. PMID:26470683

  14. Replication and transcription of human papillomavirus type 58 genome in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Wang Xin

    2010-12-01

    Full Text Available Abstract Background To establish a convenient system for the study of human papillomavirus (HPV, we inserted a Saccharomyces cerevisiae selectable marker, Ura, into HPV58 genome and transformed it into yeast. Results HPV58 genome could replicate extrachromosomally in yeast, with transcription of its early and late genes. However, with mutation of the viral E2 gene, HPV58 genome lost its mitotic stability, and the transcription levels of E6 and E7 genes were upregulated. Conclusions E2 protein could participate in viral genome maintenance, replication and transcription regulation. This yeast model could be used for the study of certain aspects of HPV life cycle.

  15. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

    Science.gov (United States)

    Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

  16. YEASTRACT-DISCOVERER: new tools to improve the analysis of transcriptional regulatory associations in Saccharomyces cerevisiae

    OpenAIRE

    Monteiro, Pedro T.; Mendes, Nuno D; Teixeira, Miguel C.; d’Orey, Sofia; Tenreiro, Sandra; Mira, Nuno P; Pais, Hélio; Francisco, Alexandre P.; Alexandra M. Carvalho; Lourenço, Artur B.; Sá-Correia, Isabel; Oliveira, Arlindo L.; Freitas, Ana T.

    2007-01-01

    The Yeast search for transcriptional regulators and consensus tracking (YEASTRACT) information system (www.yeastract.com) was developed to support the analysis of transcription regulatory associations in Saccharomyces cerevisiae. Last updated in September 2007, this database contains over 30 990 regulatory associations between Transcription Factors (TFs) and target genes and includes 284 specific DNA binding sites for 108 characterized TFs. Computational tools are also provided to facilitate ...

  17. Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair.

    OpenAIRE

    Teo, S H; Jackson, S P

    1997-01-01

    DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I-IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae. Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian ...

  18. Transcriptional Response of Saccharomyces cerevisiae to Different Nitrogen Concentrations during Alcoholic Fermentation▿ †

    OpenAIRE

    Mendes-Ferreira A; Olmo Muñoz, Marcel·lí del; García Martínez, José; Jiménez Martí, Elena; Mendes-Faia A; Pérez Ortín, José Enrique; Leão C.

    2007-01-01

    Gene expression profiles of a wine strain of Saccharomyces cerevisiae PYCC4072 were monitored during alcoholic fermentations with three different nitrogen supplies: (i) control fermentation (with enough nitrogen to complete sugar fermentation), (ii) nitrogen-limiting fermentation, and (iii) the addition of nitrogen to the nitrogen-limiting fermentation (refed fermentation). Approximately 70% of the yeast transcriptome was altered in at least one of the fermentation stages studied, revealing t...

  19. The YEASTRACT database: a tool for the analysis of transcription regulatory associations in Saccharomyces cerevisiae

    OpenAIRE

    Teixeira, Miguel C; Monteiro, Pedro; Jain, Pooja; Tenreiro, Sandra; Fernandes, Alexandra R.; Mira, Nuno P.; Alenquer, Marta; Freitas, Ana T.; Oliveira, Arlindo L.; Sá-Correia, Isabel

    2005-01-01

    We present the YEAst Search for Transcriptional Regulators And Consensus Tracking (YEASTRACT; ) database, a tool for the analysis of transcription regulatory associations in Saccharomyces cerevisiae. This database is a repository of 12 346 regulatory associations between transcription factors and target genes, based on experimental evidence which was spread throughout 861 bibliographic references. It also includes 257 specific DNA-binding sites for more than a hundred characterized transcript...

  20. Old Yellow Enzymes Protect against Acrolein Toxicity in the Yeast Saccharomyces cerevisiae

    OpenAIRE

    Trotter, Eleanor W; Collinson, Emma J.; Dawes, Ian W.; Grant, Chris M.

    2006-01-01

    Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but parti...

  1. Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets

    OpenAIRE

    Reimand, Jüri; Vaquerizas, Juan M.; Todd, Annabel E.; Vilo, Jaak; Luscombe, Nicholas M.

    2010-01-01

    Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here...

  2. Members of the Hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae.

    OpenAIRE

    López-Ribot, J L; Chaffin, W L

    1996-01-01

    Western blot (immunoblot) analysis of cell wall and cytosolic extracts obtained from parental and ssa1 and ssa2 single- and double-mutant strains of Saccharomyces cerevisiae showed that the heat shock protein 70 (Hsp70) products of these genes, previously thought to be restricted to the cell interior, are also present in the cell wall. A cell wall location was further confirmed by indirect immunofluorescence with intact cells and biotinylation of extracellular Hsp70. Hsp70s have been implicat...

  3. Nitrogen Catabolite Repression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hofman-Bang, H Jacob Peider

    1999-01-01

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

  4. Oscillations in glycolysis in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kloster, Antonina; Olsen, Lars Folke

    Wehave investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations. The...

  5. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    Science.gov (United States)

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

    2008-01-01

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

  6. [Construction of Saccharomyces cerevisiae cell factories for lycopene production].

    Science.gov (United States)

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

    2014-10-01

    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

  7. Genetic Basis for Saccharomyces cerevisiae Biofilm in Liquid Medium

    DEFF Research Database (Denmark)

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

    2014-01-01

    Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics...... than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the S1278b background and found 71 genes that were essential for biofilm development. Quantitative...... 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...

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

    Science.gov (United States)

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

    2015-08-01

    Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains). PMID:26220934

  9. NCW2, a Gene Involved in the Tolerance to Polyhexamethylene Biguanide (PHMB), May Help in the Organisation of β-1,3-Glucan Structure of Saccharomyces cerevisiae Cell Wall.

    Science.gov (United States)

    Elsztein, Carolina; de Lima, Rita de Cássia Pereira; de Barros Pita, Will; de Morais, Marcos Antonio

    2016-09-01

    In the present work, we provide biological evidences supporting the participation of NCW2 gene in the mechanism responsible for cell tolerance to polyhexamethylene biguanide (PHMB), an antifungal agent. The growth rate of yeast cells exposed to this agent was significantly reduced in ∆ncw2 strain and the mRNA levels of NCW2 gene in the presence of PHMB showed a 7-fold up-regulation. Moreover, lack of NCW2 gene turns yeast cell more resistant to zymolyase treatment, indicating that alterations in the β-glucan network do occur when Ncw2p is absent. Computational analysis of the translated protein indicated neither catalytic nor transmembrane sites and reinforced the hypothesis of secretion and anchoring to cell surface. Altogether, these results indicated that NCW2 gene codes for a protein which participates in the cell wall biogenesis in yeasts and that Ncw2p might play a role in the organisation of the β-glucan assembly. PMID:27246500

  10. Catabolite degradation of fructose-1,6-bisphosphatase in the yeast Saccharomyces cerevisiae: a genome-wide screen identifies eight novel GID genes and indicates the existence of two degradation pathways

    Czech Academy of Sciences Publication Activity Database

    Regelmann, J.; Schüle, T.; Josupeit, F. S.; Horák, Jaroslav; Rose, M.; Entian, K. D.; Thumm, M.; Wolf, D. H.

    2003-01-01

    Roč. 14, č. 4 (2003), s. 1652-1663. ISSN 1059-1524 R&D Projects: GA ČR GA204/01/0272; GA ČR GA204/02/1240 Institutional research plan: CEZ:AV0Z5011922 Keywords : degradation * fructose -1 * 6-bisphosphatase * GID genes Subject RIV: CE - Biochemistry Impact factor: 7.454, year: 2003

  11. AGAPE (Automated Genome Analysis PipelinE for pan-genome analysis of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Giltae Song

    Full Text Available The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which integrates the steps of assembly, annotation, and variation calling. To assign strain-specific functional annotations, we identified genes that were not present in the reference genome. We classified these according to their presence or absence across strains and characterized each group of genes with known functional and phenotypic features. The functional roles of novel genes not found in the reference genome and associated with strains or groups of strains appear to be consistent with anticipated adaptations in specific lineages. As more S. cerevisiae strain genomes are released, our analysis can be used to collate genome data and relate it to lineage-specific patterns of genome evolution. Our new tool set will enhance our understanding of genomic and functional evolution in S. cerevisiae, and will be available to the yeast genetics and molecular biology community.

  12. Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

    2016-05-01

    Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes. PMID:26883346

  13. Synthesis of Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae by Using Intermediates of Fatty Acid β-Oxidation

    OpenAIRE

    Poirier, Yves; Erard, Nadine; Petétot, Jean MacDonald-Comber

    2001-01-01

    Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expre...

  14. Aumento de dosis génica de los genes DPL1, SSD1 y SRP101 en Saccharomyces cerevisiae y fenotipo de tolerancia a acidificación intracelular

    OpenAIRE

    BERNABEU LORENZO, MANUEL

    2015-01-01

    [ES] El pH alto intracelular es una señal promotora del crecimiento y proliferación de las células pero sus mecanismos no son bien conocidos. En un trabajo previo se había identificado una región genómica de levadura que al ser transformada en plásmido de copia simple aumenta el crecimiento de la levadura en condiciones de acidificación intracelular. Esta región contiene tres genes, DPL1, SSD1 y SRP101 y en este trabajo hemos identificado el gen SSD1 como el responsable del fen...

  15. Expression of an alpha-galactosidase from Saccharomyces cerevisiae in Aspergillus awamori and Aspergillus oryzae.

    Science.gov (United States)

    Murphy, R A; Power, R F G

    2002-02-01

    A gene encoding alpha-galactosidase activity was isolated by polymerase chain reaction (PCR) from Saccharomyces cerevisiae NCYC686 and separately placed under the control of transcriptional elements regulating alpha-amylase expression in Aspergillus oryzae and glucoamylase expression in A. awamori. Following transformation of both A. oryzae and A. awamori with their respective expression vectors, induction of heterologous alpha-galactosidase from positively selected clones was effected through the addition of soluble starch (10% wt/vol) to the growth medium. Upon induction in A. oryzae, a transcriptional instability resulted in degradation of mRNA encoding heterologous alpha-galactosidase, thus preventing expression of the active enzyme. The use of a gene fusion strategy in A. awamori overcame this instability and resulted in stable expression of S. cerevisiae alpha-galactosidase. Subsequent to initial (shake flask) experiments, a series of scale-up and optimisation studies led to heterologous expression of the recombinant enzyme in batch fermentation at 51 U mg(-1) total extracellular protein. This was higher than previously published works, which reported extracellular levels of heterologous alpha-galactosidase up to 38 U mg(-1) total protein. Analysis of crude extracts of the fermentation medium revealed significant differences between the activity parameters reported previously in the literature for this enzyme and those observed here. The recombinant enzyme exhibited thermostability properties not previously reported for S. cerevisiae alpha-galactosidase, a trait which would make it suitable for use in processes requiring high temperatures. PMID:12074058

  16. Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans

    Science.gov (United States)

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2016-01-01

    Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds. PMID:21815095

  17. Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening.

    Science.gov (United States)

    Viña-Gonzalez, Javier; Gonzalez-Perez, David; Alcalde, Miguel

    2016-01-01

    Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps. PMID:27077451

  18. Improving 2-phenylethanol production via Ehrlich pathway using genetic engineered Saccharomyces cerevisiae strains.

    Science.gov (United States)

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

    2015-05-01

    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

  19. TOTAL ANTIOXIDANT ACTIVITY OF YEAST SACCHAROMYCES CEREVISIAE

    Directory of Open Access Journals (Sweden)

    Blažena Lavová

    2013-02-01

    Full Text Available Antioxidants are health beneficial compounds that can protect cells and macromolecules (e.g. fats, lipids, proteins and DNA from the damage of reactive oxygen species (ROS. Sacchamomyces cerevisiae are know as organisms with very important antioxidative enzyme systems such as superoxide dismutase or catalase. The total antioxidant activity (mmol Trolox equivalent – TE.g-1 d.w. of Saccharomyces cerevisiae was measured by 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid during the yeast cultivation. It was found that the total antioxidant activity was the highest (1.08 mmol TE.g-1 d.w. in the strain Kolín after 32 hours of cultivation and the lowest (0.26 mmol TE.g-1 d.w. in the strain Gyöng after 12 hours of cultivation.

  20. SCREENING AND CHARACTERIZATION OF STRESS TOLERANT SACCHAROMYCES CEREVISIAE ISOLATED FROM BREWERY EFFLUENTS FOR ANIMAL PROBIOTIC APPLICATIONS

    Directory of Open Access Journals (Sweden)

    Bhukya Bhima, Sudhakara Reddy Marrivada, Tangutur Anjana Devi , Yerradoddi Ramana Reddy, and Linga Venkateswar Rao*

    2010-12-01

    Full Text Available Based on the colony morphology and microscopic characteristics, 26 yeasts were isolated from different sources including brewery effluents. Initially they were screened for their thermotolerance at 40 0C and only 5 strains were selected. They were later grown in yeast extract peptone dextrose medium to screen their stress tolerance at five different temperatures; at different concentrations of a mixture of acetic, propionic and butyric acids; at different pH; at different concentrations of glucose and bile salts. Based on the growth at different stress conditions, yeast OBV9 was selected and characterized as Saccharomyces cerevisiae by sequencing its 5.8S rRNA gene and internal transcribed spacer (ITS 1 and 2. The sequence obtained was most similar (99% to S. cerevisiae, when it was blast searched in NCBI database and showed a separate branch in phylogenetic analysis.

  1. Specialization of B-Type Cyclins for Mitosis or Meiosis in S. Cerevisiae

    OpenAIRE

    Dahmann, C.; Futcher, B.

    1995-01-01

    The CLB1, CLB2, and CLB3 genes encode B-type cyclins important for mitosis in Saccharomyces cerevisiae, while a fourth B-type cyclin gene, CLB4, has no clear role. The effects of homozygous clb mutations on meiosis were examined. Mutants homozygous for clb1 clb3, or for clb1 clb4, gave high levels of sporulation, but produced mainly two-spored asci instead of four-spored asci. The cells had completed meiosis I but not meiosis II, producing viable diploid ascospores. CLB1 and CLB4 seem to be m...

  2. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

    OpenAIRE

    Jones, S; Vignais, M L; Broach, J R

    1991-01-01

    The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also o...

  3. Phosphate transport and sensing in Saccharomyces cerevisiae.

    OpenAIRE

    Wykoff, D D; O'Shea, E K

    2001-01-01

    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...

  4. Viruses and prions of Saccharomyces cerevisiae

    OpenAIRE

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

    2013-01-01

    Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses, and prions. Studies of the mechanisms of virus and prion replication, virus structure, and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular compone...

  5. Stationary phase in the yeast Saccharomyces cerevisiae.

    OpenAIRE

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

    1993-01-01

    Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant c...

  6. Identification of coated vesicles in Saccharomyces cerevisiae

    OpenAIRE

    1984-01-01

    Clathrin-coated vesicles were found in yeast, Saccharomyces cerevisiae, and enriched from spheroplasts by a rapid procedure utilizing gel filtration on Sephacryl S-1000. The coated vesicles (62-nm diam) were visualized by negative stain electron microscopy and clathrin triskelions were observed by rotary shadowing. The contour length of a triskelion leg was 490 nm. Coated vesicle fractions contain a prominent band with molecular weight of approximately 185,000 when analyzed by SDS PAGE. The p...

  7. Cell Wall Assembly in Saccharomyces cerevisiae

    OpenAIRE

    Lesage, Guillaume; Bussey, Howard

    2006-01-01

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

  8. EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Maury, Jerome; Germann, Susanne Manuela; Jacobsen, Simo Abdessamad;

    2016-01-01

    Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred...... integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a...

  9. Investigation of centers sensitive to S1-nuclease in the genoma of the yeast S. cerevisiae after in-vivo exposure to gamma radiation

    International Nuclear Information System (INIS)

    The structure, distribution and repair of basal damage in DNS after exposure to 60Co gamma radiation were investigated in S. cerevisiae cells. Small DNS regions with mispaired or unpaired bases of rather high stability were found whose rate of incidence and linear dose dependence appear to be similar to those of double strand breaks. In contrast to double strand breaks, they showed no statistical' distribution pattern across the genoma. Liquid holding experiments showed that centers sensitive to S1-nuclease will be repaired in S. cerevisiae by a combined process of recombination and postreplication repair; the gene products of the genes RAD50 and RAD18 are involved. (orig./AJ)

  10. Genetic redundancy between SPT23 and MGA2: regulators of Ty-induced mutations and Ty1 transcription in Saccharomyces cerevisiae.

    OpenAIRE

    S. Zhang; Burkett, T J; Yamashita, I; Garfinkel, D J

    1997-01-01

    SPT23 was isolated as a dosage-dependent suppressor of Ty-induced mutations in Saccharomyces cerevisiae. SPT23 shows considerable sequence homology with MGA2, a gene identified as a dosage-dependent suppressor of a snf2-imposed block on STA1 transcription in S. cerevisiae var. diastaticus. Although single mutations in either of these genes have only modest effects on cell growth, spt23 mga2 double mutants are inviable. Unlike SPT23, multicopy expression of a truncated form of MGA2 suppresses ...

  11. Increase of betulinic acid production in Saccharomyces cerevisiae by balancing fatty acids and betulinic acid forming pathways.

    Science.gov (United States)

    Li, Jing; Zhang, Yansheng

    2014-04-01

    Betulinic acid is a plant-based triterpenoid that has been recognized for its antitumor and anti-HIV activities. The level of betulinic acid in its natural hosts is extremely low. In the present study, we constructed betulinic acid biosynthetic pathway in Saccharomyces cerevisiae by metabolic engineering. Given the betulinic acid forming pathways sharing the common substrate acetyl-CoA with fatty acid synthesis, the metabolic fluxes between the two pathways were varied by changing gene expressions, and their effects on betulinic acid production were investigated. We constructed nine S. cerevisiae strains representing nine combinations of the flux distributions between betulinic acid and fatty acid pathways. Our results demonstrated that it was possible to improve the betulinic acid production in S. cerevisiae while keeping a desirable growth phenotype by optimally balancing the carbon fluxes of the two pathways. Through modulating the expressions of the key genes on betulinic acid and fatty acid pathways, the difference in betulinic acid yield varied largely in the range of 0.01-1.92 mg L(-1) OD(-1). The metabolic engineering approach used in this study could be extended for synthesizing other triterpenoids in S. cerevisiae. PMID:24389702

  12. A Saccharomyces cerevisiae Wine Strain Inhibits Growth and Decreases Ochratoxin A Biosynthesis by Aspergillus carbonarius and Aspergillus ochraceus

    Directory of Open Access Journals (Sweden)

    Marilena Budroni

    2012-12-01

    Full Text Available The aim of this study was to select wine yeast strains as biocontrol agents against fungal contaminants responsible for the accumulation of ochratoxin A (OTA in grape and wine and to dissect the mechanism of OTA detoxification by a Saccharomyces cerevisiae strain (DISAABA1182, which had previously been reported to reduce OTA in a synthetic must. All of the yeast strains tested displayed an ability to inhibit the growth of Aspergillus carbonarius both in vivo and in vitro and addition of culture filtrates from the tested isolates led to complete inhibition of OTA production. S. cerevisiae DISAABA1182 was selected and further tested for its capacity to inhibit OTA production and pks (polyketide synthase transcription in A. carbonarius and Aspergillus ochraceus in vitro. In order to dissect the mechanism of OTA detoxification, each of these two fungi was co-cultured with living yeast cells exposed to yeast crude or to autoclaved supernatant: S. cerevisiae DISAABA1182 was found to inhibit mycelial growth and OTA production in both Aspergilli when co-cultured in the OTA-inducing YES medium. Moreover, a decrease in pks transcription was observed in the presence of living cells of S. cerevisiae DISAABA1182 or its supernatant, while no effects were observed on transcription of either of the constitutively expressed calmodulin and β-tubulin genes. This suggests that transcriptional regulation of OTA biosynthetic genes takes place during the interaction between DISAABA1182 and OTA-producing Aspergilli.

  13. Horizontally acquired oligopeptide transporters favour adaptation of Saccharomyces cerevisiae wine yeast to oenological environment.

    Science.gov (United States)

    Marsit, Souhir; Sanchez, Isabelle; Galeote, Virginie; Dequin, Sylvie

    2016-04-01

    In the past decade, horizontal gene transfer (HGT) has emerged as a major evolutionary process that has shaped the genome of Saccharomyces cerevisiae wine yeasts. We recently showed that a large Torulaspora microellipsoides genomic island carrying two oligopeptide transporters encoded by FOT genes increases the fitness of wine yeast during fermentation of grape must. However, the impact of these genes on the metabolic network of S. cerevisiae remained uncharacterized. Here we show that Fot-mediated peptide uptake substantially affects the glutamate node and the NADPH/NADP(+) balance, resulting in the delayed uptake of free amino acids and altered profiles of metabolites and volatile compounds. Transcriptome analysis revealed that cells using a higher amount of oligopeptides from grape must are less stressed and display substantial variation in the expression of genes in the central pathways of carbon and nitrogen metabolism, amino acid and protein biosynthesis, and the oxidative stress response. These regulations shed light on the molecular and metabolic mechanisms involved in the higher performance and fitness conferred by the HGT-acquired FOT genes, pinpointing metabolic effects that can positively affect the organoleptic balance of wines. PMID:26549518

  14. Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Soracom Chardwiriyapreecha

    Full Text Available In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.

  15. Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Suortti Tapani

    2006-02-01

    Full Text Available Abstract Background Secretion stress is caused by compromised folding, modification or transport of proteins in the secretory pathway. In fungi, induction of genes in response to secretion stress is mediated mainly by the unfolded protein response (UPR pathway. This study aims at uncovering transcriptional responses occurring in the filamentous fungi Trichoderma reesei exposed to secretion stress and comparing these to those found in the yeast Saccharomyces cerevisiae. Results Chemostat cultures of T. reesei expressing human tissue plasminogen activator (tPA and batch bioreactor cultures treated with dithiothreitol (DTT to prevent correct protein folding were analysed with cDNA subtraction and cDNA-amplified fragment length polymorphism (AFLP experiments. ESTs corresponding to 457 unique genes putatively induced under secretion stress were isolated and the expression pattern of 60 genes was confirmed by Northern analysis. Expression of these genes was also studied in a strain over-expressing inositol-requiring enzyme 1 (IREI protein, a sensor for the UPR pathway. To compare the data with that of S. cerevisiae, published transcriptome profiling data on various stress responses in S. cerevisiae was reanalysed. The genes up-regulated in response to secretion stress included a large number of secretion related genes in both organisms. In addition, analysis of T. reesei revealed up regulation of the cpc1 transcription factor gene and nucleosomal genes. The induction of the cpcA and histone gene H4 were shown to be induced also in cultures of Aspergillus nidulans treated with DTT. Conclusion Analysis of the genes induced under secretion stress has revealed novel features in the stress response in T. reesei and in filamentous fungi. We have demonstrated that in addition to the previously rather well characterised induction of genes for many ER proteins or secretion related proteins also other types of responses exist.

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

    Science.gov (United States)

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

    2016-09-01

    Dissection of the hereditary architecture underlying Saccharomyces cerevisiae tolerance to acetic acid is essential for ethanol fermentation. In this work, a genomics approach was used to dissect hereditary variations in acetic acid tolerance between two phenotypically different strains. A total of 160 segregants derived from these two strains were obtained. Phenotypic analysis indicated that the acetic acid tolerance displayed a normal distribution in these segregants, and suggested that the acetic acid tolerant traits were controlled by multiple quantitative trait loci (QTLs). Thus, 220 SSR markers covering the whole genome were used to detect QTLs of acetic acid tolerant traits. As a result, three QTLs were located on chromosomes 9, 12, and 16, respectively, which explained 38.8-65.9 % of the range of phenotypic variation. Furthermore, twelve genes of the candidates fell into the three QTL regions by integrating the QTL analysis with candidates of acetic acid tolerant genes. These results provided a novel avenue to obtain more robust strains. PMID:27430512

  17. Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

    Directory of Open Access Journals (Sweden)

    Danuza Nogueira Moysés

    2016-02-01

    Full Text Available Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review.

  18. Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects.

    Science.gov (United States)

    Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

    2016-01-01

    Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review. PMID:26927067

  19. Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

    Science.gov (United States)

    Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves

    2016-01-01

    Many years have passed since the first genetically modified Saccharomyces cerevisiae strains capable of fermenting xylose were obtained with the promise of an environmentally sustainable solution for the conversion of the abundant lignocellulosic biomass to ethanol. Several challenges emerged from these first experiences, most of them related to solving redox imbalances, discovering new pathways for xylose utilization, modulation of the expression of genes of the non-oxidative pentose phosphate pathway, and reduction of xylitol formation. Strategies on evolutionary engineering were used to improve fermentation kinetics, but the resulting strains were still far from industrial application. Lignocellulosic hydrolysates proved to have different inhibitors derived from lignin and sugar degradation, along with significant amounts of acetic acid, intrinsically related with biomass deconstruction. This, associated with pH, temperature, high ethanol, and other stress fluctuations presented on large scale fermentations led the search for yeasts with more robust backgrounds, like industrial strains, as engineering targets. Some promising yeasts were obtained both from studies of stress tolerance genes and adaptation on hydrolysates. Since fermentation times on mixed-substrate hydrolysates were still not cost-effective, the more selective search for new or engineered sugar transporters for xylose are still the focus of many recent studies. These challenges, as well as under-appreciated process strategies, will be discussed in this review. PMID:26927067

  20. Evolutionary advantage conferred by an eukaryote-to-eukaryote gene transfer event in wine yeasts

    OpenAIRE

    Marsit, Souhir; Mena, Adriana; Bigey, Frederic; Sauvage, Francois Xavier; Couloux, Arnaud; Guy, Julie; Legras, Jean Luc; Barrio, Eladio; Dequin, Sylvie

    2015-01-01

    Although an increasing number of horizontal gene transfers have been reported in eukaryotes, experimental evidence for their adaptive value is lacking. Here, we report the recent transfer of a 158-kb genomic region between Torulaspora microellipsoides and Saccharomyces cerevisiae wine yeasts or closely related strains. This genomic region has undergone several rearrangements in S. cerevisiae strains, including gene loss and gene conversion between two tandemly duplicated FOT genes encoding ol...

  1. De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

    Science.gov (United States)

    2012-01-01

    Background Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules. Result Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations (Synthesis of aromatic amino acids was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40-fold increase of extracellular naringenin titers (to approximately 200 μM) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 μM were reached. Conclusion The results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions. PMID:23216753

  2. Investigation of autonomous cell cycle oscillation in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hansen, Morten Skov

    2007-01-01

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

  3. The DNA-damage signature in Saccharomyces cerevisiae is associated with single-strand breaks in DNA

    Directory of Open Access Journals (Sweden)

    Begley Thomas J

    2006-12-01

    Full Text Available Abstract Background Upon exposure to agents that damage DNA, Saccharomyces cerevisiae undergo widespread reprogramming of gene expression. Such a vast response may be due not only to damage to DNA but also damage to proteins, RNA, and lipids. Here the transcriptional response of S. cerevisiae specifically induced by DNA damage was discerned by exposing S. cerevisiae to a panel of three "radiomimetic" enediyne antibiotics (calicheamicin γ1I, esperamicin A1 and neocarzinostatin that bind specifically to DNA and generate varying proportions of single- and double-strand DNA breaks. The genome-wide responses were compared to those induced by the non-selective oxidant γ-radiation. Results Given well-controlled exposures that resulted in similar and minimal cell death (~20–25% across all conditions, the extent of gene expression modulation was markedly different depending on treatment with the enediynes or γ-radiation. Exposure to γ-radiation resulted in more extensive transcriptional changes classified both by the number of genes modulated and the magnitude of change. Common biological responses were identified between the enediynes and γ-radiation, with the induction of DNA repair and stress response genes, and the repression of ribosomal biogenesis genes. Despite these common responses, a fraction of the response induced by gamma radiation was repressed by the enediynes and vise versa, suggesting that the enediyne response is not entirely "radiomimetic." Regression analysis identified 55 transcripts with gene expression induction associated both with double- or single-strand break formation. The S. cerevisiae "DNA damage signature" genes as defined by Gasch et al. 1 were enriched among regulated transcripts associated with single-strand breaks, while genes involved in cell cycle regulation were associated with double-strand breaks. Conclusion Dissection of the transcriptional response in yeast that is specifically signaled by DNA strand

  4. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

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

    Directory of Open Access Journals (Sweden)

    Xiaoling Tang

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

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

    Directory of Open Access Journals (Sweden)

    Myers Samuel

    2008-12-01

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

  7. Recombination-stable multimeric green fluorescent protein for characterization of weak promoter outputs in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rugbjerg, Peter; Knuf, Christoph; Förster, Jochen; Sommer, Morten O A

    2015-12-01

    Green fluorescent proteins (GFPs) are widely used for visualization of proteins to track localization and expression dynamics. However, phenotypically important processes can operate at too low expression levels for routine detection, i.e. be overshadowed by autofluorescence noise. While GFP functions well in translational fusions, the use of tandem GFPs to amplify fluorescence signals is currently avoided in Saccharomyces cerevisiae and many other microorganisms due to the risk of loop-out by direct-repeat recombination. We increased GFP fluorescence by translationally fusing three different GFP variants, yeast-enhanced GFP, GFP+ and superfolder GFP to yield a sequence-diverged triple GFP molecule 3vGFP with 74-84% internal repeat identity. Unlike a single GFP, the brightness of 3vGFP allowed characterization of a weak promoter in S. cerevisiae. Utilizing 3vGFP, we further engineered a less leaky Cu(2+)-inducible promoter based on CUP1. The basal expression level of the new promoter was approximately 61% below the wild-type CUP1 promoter, thus expanding the absolute range of Cu(2+)-based gene control. The stability of 3vGFP towards direct-repeat recombination was assayed in S. cerevisiae cultured for 25 generations under strong and slightly toxic expression after which only limited reduction in fluorescence was detectable. Such non-recombinogenic GFPs can help quantify intracellular responses operating a low copy number in recombination-prone organisms. PMID:26392044

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-11-25

    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.

  9. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.

    Science.gov (United States)

    Liu, Hong-Zhi; Wang, Qiang; Liu, Xiao-Yong; Tan, Sze-Sze

    2008-12-01

    Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production. PMID:18797865

  10. A single heterochromatin boundary element imposes position-independent antisilencing activity in Saccharomyces cerevisiae minichromosomes.

    Directory of Open Access Journals (Sweden)

    Sangita A Chakraborty

    Full Text Available Chromatin boundary elements serve as cis-acting regulatory DNA signals required to protect genes from the effects of the neighboring heterochromatin. In the yeast genome, boundary elements act by establishing barriers for heterochromatin spreading and are sufficient to protect a reporter gene from transcriptional silencing when inserted between the silencer and the reporter gene. Here we dissected functional topography of silencers and boundary elements within circular minichromosomes in Saccharomyces cerevisiae. We found that both HML-E and HML-I silencers can efficiently repress the URA3 reporter on a multi-copy yeast minichromosome and we further showed that two distinct heterochromatin boundary elements STAR and TEF2-UASrpg are able to limit the heterochromatin spreading in circular minichromosomes. In surprising contrast to what had been observed in the yeast genome, we found that in minichromosomes the heterochromatin boundary elements inhibit silencing of the reporter gene even when just one boundary element is positioned at the distal end of the URA3 reporter or upstream of the silencer elements. Thus the STAR and TEF2-UASrpg boundary elements inhibit chromatin silencing through an antisilencing activity independently of their position or orientation in S. cerevisiae minichromosomes rather than by creating a position-specific barrier as seen in the genome. We propose that the circular DNA topology facilitates interactions between the boundary and silencing elements in the minichromosomes.

  11. The genetic basis of natural variation in oenological traits in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Francisco Salinas

    Full Text Available Saccharomyces cerevisiae is the main microorganism responsible for wine alcoholic fermentation. The oenological phenotypes resulting from fermentation, such as the production of acetic acid, glycerol, and residual sugar concentration are regulated by multiple genes and vary quantitatively between different strain backgrounds. With the aim of identifying the quantitative trait loci (QTLs that regulate oenological phenotypes, we performed linkage analysis using three crosses between highly diverged S. cerevisiae strains. Segregants from each cross were used as starter cultures for 20-day fermentations, in synthetic wine must, to simulate actual winemaking conditions. Linkage analysis on phenotypes of primary industrial importance resulted in the mapping of 18 QTLs. We tested 18 candidate genes, by reciprocal hemizygosity, for their contribution to the observed phenotypic variation, and validated five genes and the chromosome II right subtelomeric region. We observed that genes involved in mitochondrial metabolism, sugar transport, nitrogen metabolism, and the uncharacterized ORF YJR030W explained most of the phenotypic variation in oenological traits. Furthermore, we experimentally validated an exceptionally strong epistatic interaction resulting in high level of succinic acid between the Sake FLX1 allele and the Wine/European MDH2 allele. Overall, our work demonstrates the complex genetic basis underlying wine traits, including natural allelic variation, antagonistic linked QTLs and complex epistatic interactions between alleles from strains with different evolutionary histories.

  12. Inhibition effect of expression of Cu/Zn superoxide dismutase from rice on synthesis of Glutathione in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    AI Yu-zhuo; DU Ye-jie; ZU Yuan-gang; AN Zhi-gang

    2008-01-01

    The expression of a rice Cu/Zn superoxide dismutase (Cu/Zn-SOD) in Saccharomyces cerevisiae regulated by GAPDH promoter, involved in the inhibition of endogenous Glutathione (GSH) synthesis, and the competitive expression was detected by constructing the expression vector transferred Cu/Zn-SOD gene into wild-type S. Cerevisiae. Transcription and expression of the Cu/Zn-SOD gene in S. Cerevisiawere were confirmed by northern blot and SDS-PAGE, respectively, and activity of the Cu/Zn-SOD from crude extracts was enzymatically detected based on the effect of nitroblue tetrazolium (NBT) after running a native polyacrylamide gel. The GSH synthesis was also tested by DTNB (5, 5′-Dithiobis (2-nitrobenzoic acid)) method. Results showed that GSH synthesis was evidently suppressed by the expression of Cu/Zn-SOD gene in both control and heat shock strains. It implied that the expression of the Cu/Zn-SOD gene in S. Cerevisiae has more potential facility in response to oxidative exposure than that of endogenous GSH, although Cu/Zn-SOD and GSH were both contributed to the function of oxygen radical oxidoreduction.

  13. Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic.

    Science.gov (United States)

    Todorova, Tatina T; Kujumdzieva, Anna V; Vuilleumier, Stéphane

    2010-11-01

    The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2-2.5-fold upon addition of either arsenate (As(V)) or arsenite (As(III)). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3'-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation. PMID:20740275

  14. A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Dai Wei

    2009-11-01

    Full Text Available Abstract Background The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for Saccharomyces cerevisiae to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid S. cerevisiae gene deletion strains (the entire set of nonessential genes for this organism to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO4. Results We have identified 149 genes whose gene deletion causes sensitivity to NiSO4 and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of S. cerevisiae to NiSO4. Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO4 include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells. Conclusion We have undertaken a whole genome approach in order to further understand the mechanism(s regulating the cell

  15. 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)

  16. Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration.

    Science.gov (United States)

    de Jong, Bouke Wim; Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2015-03-01

    Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE (L641P) ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 (S1157A,S659A) ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE (L641P) , ACC1 (S1157A,S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and β-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae. PMID:25422103

  17. DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jakob Madsen Pedersen

    Full Text Available To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

  18. Genetic engineering to enhance the Ehrlich pathway and alter carbon flux for increased isobutanol production from glucose by Saccharomyces cerevisiae.

    Science.gov (United States)

    Kondo, Takashi; Tezuka, Hironori; Ishii, Jun; Matsuda, Fumio; Ogino, Chiaki; Kondo, Akihiko

    2012-05-31

    The production of higher alcohols by engineered bacteria has received significant attention. The budding yeast, Saccharomyces cerevisiae, has considerable potential as a producer of higher alcohols because of its capacity to naturally fabricate fusel alcohols, in addition to its robustness and tolerance to low pH. However, because its natural productivity is not significant, we considered a strategy of genetic engineering to increase production of the branched-chain higher alcohol isobutanol, which is involved in valine biosynthesis. Initially, we overexpressed 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) in S. cerevisiae to enhance the endogenous activity of the Ehrlich pathway. We then overexpressed Ilv2, which catalyzes the first step in the valine synthetic pathway, and deleted the PDC1 gene encoding a major pyruvate decarboxylase with the intent of altering the abundant ethanol flux via pyruvate. Through these engineering steps, along with modification of culture conditions, the isobutanol titer of S. cerevisiae was elevated 13-fold, from 11 mg/l to 143 mg/l, and the yield was 6.6 mg/g glucose, which is higher than any previously reported value for S. cerevisiae. PMID:22342368

  19. Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae.

    Science.gov (United States)

    Fujita, Ken-Ichi; Tatsumi, Miki; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

    2014-02-01

    trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae. PMID:24393541

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

    Directory of Open Access Journals (Sweden)

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

    2014-06-01

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

  1. N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tang, Hongting; Wang, Shenghuan; Wang, Jiajing; Song, Meihui; Xu, Mengyang; Zhang, Mengying; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2016-01-01

    Saccharomyces cerevisiae is a robust host for heterologous protein expression. The efficient expression of cellulases in S. cerevisiae is important for the consolidated bioprocess that directly converts lignocellulose into valuable products. However, heterologous proteins are often N-hyperglycosylated in S. cerevisiae, which may affect protein activity. In this study, the expression of three heterologous proteins, β-glucosidase, endoglucanase and cellobiohydrolase, was found to be N-hyperglycosylated in S. cerevisiae. To block the formation of hypermannose glycan, these proteins were expressed in strains with deletions in key Golgi mannosyltransferases (Och1p, Mnn9p and Mnn1p), respectively. Their extracellular activities improved markedly in the OCH1 and MNN9 deletion strains. Interestingly, truncation of the N-hypermannose glycan did not increase the specific activity of these proteins, but improved the secretion yield. Further analysis showed OCH1 and MNN9 deletion up-regulated genes in the secretory pathway, such as protein folding and vesicular trafficking, but did not induce the unfolded protein response. The cell wall integrity was also affected by OCH1 and MNN9 deletion, which contributed to the release of secretory protein extracellularly. This study demonstrated that mannosyltransferases disruption improved protein secretion through up-regulating secretory pathway and affecting cell wall integrity and provided new insights into glycosylation engineering for protein secretion. PMID:27156860

  2. Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering

    Directory of Open Access Journals (Sweden)

    Ng Chiam Yu

    2012-05-01

    Full Text Available Abstract Background 2,3-Butanediol is a chemical compound of increasing interest due to its wide applications. It can be synthesized via mixed acid fermentation of pathogenic bacteria such as Enterobacter aerogenes and Klebsiella oxytoca. The non-pathogenic Saccharomyces cerevisiae possesses three different 2,3-butanediol biosynthetic pathways, but produces minute amount of 2,3-butanediol. Hence, we attempted to engineer S. cerevisiae strain to enhance 2,3-butanediol production. Results We first identified gene deletion strategy by performing in silico genome-scale metabolic analysis. Based on the best in silico strategy, in which disruption of alcohol dehydrogenase (ADH pathway is required, we then constructed gene deletion mutant strains and performed batch cultivation of the strains. Deletion of three ADH genes, ADH1, ADH3 and ADH5, increased 2,3-butanediol production by 55-fold under microaerobic condition. However, overproduction of glycerol was observed in this triple deletion strain. Additional rational design to reduce glycerol production by GPD2 deletion altered the carbon fluxes back to ethanol and significantly reduced 2,3-butanediol production. Deletion of ALD6 reduced acetate production in strains lacking major ADH isozymes, but it did not favor 2,3-butanediol production. Finally, we introduced 2,3-butanediol biosynthetic pathway from Bacillus subtilis and E. aerogenes to the engineered strain and successfully increased titer and yield. Highest 2,3-butanediol titer (2.29 g·l-1 and yield (0.113 g·g-1 were achieved by Δadh1 Δadh3 Δadh5 strain under anaerobic condition. Conclusions With the aid of in silico metabolic engineering, we have successfully designed and constructed S. cerevisiae strains with improved 2,3-butanediol production.

  3. Biosorption of cesium by saccharomyces cerevisia

    International Nuclear Information System (INIS)

    The characteristics of Cs+ biosorption by Saccharornyces cerevisia was investigated, including the biosorption kinetics, biosorption equilibrium, isotherm as well as the IR spectrum of biomass pre- and post-biosorption. The experimental results show that the process of Cs+ biosorption onto the biomass of Saccharornyces cerevisia can be devided into two stages, the first stage is physical sorption and the sorption equilibrium is very quickly reached (within 20 min). The biosorption kinetics can be described by the pseudo second-order equation quite well (R2=0.989), the kinetic parameters k2 and qe are 3.56 x 10-3 g/(mg·min) and 7.18 mg/g, respectively. The equilibrium isotherm data can be fitted with Langmuir and Freundlich models, with the maximum biosorptive capacity of 10.13 mg/g. Both the IR spectra of the biomass pre- and post-biosorption almost are same, and it indicates that the biosorption of Cs+ does not change the structure of the biomass, however, some adsorptive peaks shift. (authors)

  4. Response of Saccharomyces cerevisiae to cadmium stress

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-07-01

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

  5. Induction of S.cerevisiae MAG 3-methyladenine DNA glycosylase transcript levels in response to DNA damage.

    OpenAIRE

    J. Chen; Samson, L

    1991-01-01

    We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E. coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents. Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide. Unlike some other yeast DNA-damage-inducible genes, MAG ...

  6. Mechanisms of DNA repair, recombination and mutagenesis in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Full text. 1. It was confirmed that from the six DNA polymerases discovered in yeast cells, only DNA polymerases δ, ε and ζ are engaged in dark repair of lesions caused by UV-light and MMS. DNA polymerase δ is involved in the repair of both types of lesions, while DNA polymerase ε and ζ only in lesions caused by UV and MMS, respectively. Other polymerases are not involved or play only a minor role in repair. The results obtained are being prepared for publication. 2. Studies on the involvement of the three replicative DNA polymerases in mitotic gene conversion induced by mono- and bifunctional psoralens (and also by UV- light or MMS) revealed that DNA polymerases α and δ are the main polymerases responsible for induced intragenic conversion. DNA polymerase ε seems to play minor role in this process. It is possible that DNA polymerase α may also be involved in DNA repair synthesis but only in cases when the opening of new replication forks is necessary for repair. 3. Studies on the influence of mutations in the replicative and nonreplicative DNA polymerases on adaptive mutations in the cells of Saccharomyces cerevisiae were continued. We found that thermosensitive mutation in the POL2 gene encoding DNA polymerase ε increased the frequency of adaptive mutation in a similar manner as found earlier for DNA polymerase δ. A similar effect was observed also in strains with deletions in the MSH3 gene responsible for mismatch repair. Mutations in other DNA polymerases, including the essential DNA polymerase α and the inessential DNA polymerases β and ζ revealed no effect on this process. Analysis of DNA sequences in the revertants showed that in all cases the obtained reversions resulted from a single nucleotide deletion most often in sequences having short homopolymer tracts. The results obtained suggest that errors arising during DNA elongation and their persistence in mutants deficient in mismatch repair activity seem to be the source of the adaptive

  7. Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

    Science.gov (United States)

    Rich, Matthew S; Payen, Celia; Rubin, Alan F; Ong, Giang T; Sanchez, Monica R; Yachie, Nozomu; Dunham, Maitreya J; Fields, Stanley

    2016-05-01

    In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene's promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness >5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1 Mutations that create new Met32- or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology-saturation mutagenesis, chemostat selection, and DNA sequencing to track variants-should be a broadly applicable approach. PMID:26936925

  8. Changes of telomere length cause reciprocal changes in the lifespan of mother cells in Saccharomyces cerevisiae

    OpenAIRE

    Austriaco, Nicanor R.; Guarente, Leonard P.

    1997-01-01

    Budding yeast cells divide asymmetrically, giving rise to a mother and its daughter. Mother cells have a limited division potential, called their lifespan, which ends in proliferation-arrest and lysis. In this report we mutate telomerase in Saccharomyces cerevisiae to shorten telomeres and show that, rather than shortening lifespan, this leads to a significant extension in lifespan. This extension requires the product of the SIR3 gene, an essential component of the silencing machinery which b...

  9. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

    OpenAIRE

    Guo, Yakun; Dong, Junkai; Zhou, Tong; Auxillos, Jamie; Li, Tianyi; Zhang, Weimin; Wang, LiHui; Shen, Yue; Luo, Yisha; Zheng, Yijing; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Cai, Yizhi; Dai, Junbiao

    2015-01-01

    It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genet...

  10. Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies

    OpenAIRE

    Paulius Lukas Tamošiūnas; Rasa Petraitytė-Burneikienė; Rita Lasickienė; Artiomas Akatov; Gabrielis Kundrotas; Vilimas Sereika; Raimundas Lelešius; Aurelija Žvirblienė; Kęstutis Sasnauskas

    2014-01-01

    Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revea...

  11. Distinct Subsets of Sit4 Holophosphatases Are Required for Inhibition of Saccharomyces cerevisiae Growth by Rapamycin and Zymocin ▿ †

    OpenAIRE

    Jablonowski, Daniel; Täubert, Jens-Eike; Bär, Christian; Stark, Michael J. R.; Schaffrath, Raffael

    2009-01-01

    Protein phosphatase Sit4 is required for growth inhibition of Saccharomyces cerevisiae by the antifungals rapamycin and zymocin. Here, we show that the rapamycin effector Tap42, which interacts with Sit4, is dispensable for zymocin action. Although Tap42 binding-deficient sit4 mutants are resistant to zymocin, these mutations also block interaction between Sit4 and the Sit4-associating proteins Sap185 and Sap190, previously shown to mediate zymocin toxicity. Among the four different SAP genes...

  12. Growth and Glucose Repression Are Controlled by Glucose Transport in Saccharomyces cerevisiae Cells Containing Only One Glucose Transporter

    OpenAIRE

    Ye, Ling; Kruckeberg, Arthur L.; Berden, Jan A.; van Dam, Karel

    1999-01-01

    A set of Saccharomyces cerevisiae strains with variable expression of only the high-affinity Hxt7 glucose transporter was constructed by partial deletion of the HXT7 promoter in vitro and integration of the gene at various copy numbers into the genome of an hxt1-7 gal2 deletion strain. The glucose transport capacity increased in strains with higher levels of HXT7 expression. The consequences for various physiological properties of varying the glucose transport capacity were examined. The cont...

  13. Sodium Orthovanadate-Resistant Mutants of Saccharomyces Cerevisiae Show Defects in Golgi-Mediated Protein Glycosylation, Sporulation and Detergent Resistance

    OpenAIRE

    Kanik-Ennulat, C.; Montalvo, E.; Neff, N

    1995-01-01

    Orthovanadate is a small toxic molecule that competes with the biologically important oxyanion orthophosphate. Orthovanadate resistance arises spontaneously in Saccharomyces cerevisiae haploid cells by mutation in a number of genes. Mutations selected at 3 mM sodium orthovanadate have different degrees of vanadate resistance, hygromycin sensitivity, detergent sensitivity and sporulation defects. Recessive vanadate-resistant mutants belong to at least six genetic loci. Most mutants are defecti...

  14. A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

    OpenAIRE

    Firmenich, A A; Elias-Arnanz, M; Berg, P

    1995-01-01

    To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endon...

  15. Molecular Basis of Fructose Utilization by the Wine Yeast Saccharomyces cerevisiae: a Mutated HXT3 Allele Enhances Fructose Fermentation▿

    OpenAIRE

    Guillaume, Carole; Delobel, Pierre; Sablayrolles, Jean-Marie; Blondin, Bruno

    2007-01-01

    Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allel...

  16. Common features and interesting differences in transcriptional responses to secretion stress in the fungi Trichoderma reesei and Saccharomyces cerevisiae

    OpenAIRE

    Suortti Tapani; Valkonen Mari; Saloheimo Markku; Lanthaler Karin; Pakula Tiina; Arvas Mikko; Robson Geoff; Penttilä Merja

    2006-01-01

    Abstract Background Secretion stress is caused by compromised folding, modification or transport of proteins in the secretory pathway. In fungi, induction of genes in response to secretion stress is mediated mainly by the unfolded protein response (UPR) pathway. This study aims at uncovering transcriptional responses occurring in the filamentous fungi Trichoderma reesei exposed to secretion stress and comparing these to those found in the yeast Saccharomyces cerevisiae. Results Chemostat cult...

  17. Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene▿

    OpenAIRE

    Perlińska-Lenart, Urszula; Orłowski, Jacek; Laudy, Agnieszka E.; Zdebska, Ewa; Palamarczyk, Grażyna; Kruszewska, Joanna S.

    2006-01-01

    Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increas...

  18. Characterization of transcription site-associated mRNP retention in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    In a variety of S. cerevisiae mutants with defective mRNP maturation and/or export, heat shock (hs) mRNPs are retained at or near their sites of transcription. For example, mutants of the THO complex display an intense hs-mRNA FISH signal, which co-localizes with the hs-gene after transcriptional...... underrepresented in recovered fractions from mutant cells. This bias is abolished when a THO mutation is combined with a second site mutation alleviating the mRNA export block. Thus, the bias parallels transcription-site retention of the mRNP and suggests the existence of a complex specifically formed at the 3...

  19. Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Cells of Saccharomyces cerevisiae with mutations in the RAD52 gene have previously been shown to be defective in meiotic and mitotic recombination, in sporulation, and in repair of radiation-induced damage to DNA. In this study we show that diploid cells homozygous for rad52 lose chromosomes at high frequencies and that these frequencies of loss can be increased dramatically by exposure of these cells to x-rays. Genetic analyses of survivors of x-ray treatment demonstrate that chromosome loss events result in the conversion of diploid cells to cells with near haploid chromosome numbers

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

    OpenAIRE

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

    1999-01-01

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

  1. Reproducibility of oligonucleotide microarray transcriptome analyses - An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Piper, M.D.W.; Daran-Lapujade, P.; Bro, Christoffer;

    2002-01-01

    -microarray analysis in functional genomics and metabolic engineering, we designed a set of experiments to specifically address this issue. Saccharomyces cerevisiae CEN.PK113-7D was grown under defined,conditions in, glucose-limited chemostats, followed by transcriptome analysis with Affymetrix Gene-Chip arrays. In...... each of the laboratories, three independent replicate cultures were grown aerobically as well as anaerobically. Although variations introduced by in vitro handling steps were small and unbiased, greater variation from replicate cultures underscored that, to obtain reliable information, experimental...

  2. Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Pedersen, Mette Louise; Krogh, Berit Olsen;

    2012-01-01

    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...... in mutants carrying a deletion of the SGS1 helicase-encoding gene. Using our experimental conditions, a gap-repair efficiency of > 10(6) plasmid-harbouring colonies/µg gapped vector DNA is obtained in a single transformation, with a recombination fidelity > 90%....

  3. Evidence for Domesticated and Wild Populations of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    2005-07-01

    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.

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

    Science.gov (United States)

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

    2016-03-01

    There is an increasing demand for microbial production of lactic acid (LA) as a monomer of biodegradable poly lactic acid (PLA). Both optical isomers, D-LA and L-LA, are required to produce stereocomplex PLA with improved properties. In this study, we developed Saccharomyces cerevisiae strains for efficient production of D-LA. D-LA production was achieved by expressing highly stereospecific D-lactate dehydrogenase gene (ldhA, LEUM_1756) from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 in S. cerevisiae lacking natural LA production activity. D-LA consumption after glucose depletion was inhibited by deleting DLD1 encoding D-lactate dehydrogenase and JEN1 encoding monocarboxylate transporter. In addition, ethanol production was reduced by deleting PDC1 and ADH1 genes encoding major pyruvate decarboxylase and alcohol dehydrogenase, respectively, and glycerol production was eliminated by deleting GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase. LA tolerance of the engineered D-LA-producing strain was enhanced by adaptive evolution and overexpression of HAA1 encoding a transcriptional activator involved in weak acid stress response, resulting in effective D-LA production up to 48.9 g/L without neutralization. In a flask fed-batch fermentation under neutralizing condition, our evolved strain produced 112.0 g/L D-LA with a yield of 0.80 g/g glucose and a productivity of 2.2 g/(L · h). PMID:26596574

  5. Activation of futile cycles as an approach to increase ethanol yield during glucose fermentation in Saccharomyces cerevisiae.

    Science.gov (United States)

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

    2016-04-01

    An increase in ethanol yield by yeast from the fermentation of conventional sugars such as glucose and sucrose is possible by reducing the production of a key byproduct such as cellular biomass. Previously we have reported that overexpression of PHO8 gene encoding non-specific ATP-hydrolyzing alkaline phosphatase can lead to a decrease in cellular ATP content and to an increase in ethanol yield during glucose fermentation by Saccharomyces cerevisiae. In this work we further report on 2 new successful approaches to reduce cellular levels of ATP that increase ethanol yield and productivity. The first approach is based on the overexpression of the heterologous Escherichia coli apy gene encoding apyrase or SSB1 part of the chaperon that exhibit ATPase activity in yeast. In the second approach we constructed a futile cycle by the overexpression of S. cerevisiae genes encoding pyruvate carboxylase and phosphoenolpyruvate carboxykinase in S. cerevisiae. These genetically engineered strains accumulated more ethanol compared to the wild-type strain during alcoholic fermentation. PMID:26890808

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

    Directory of Open Access Journals (Sweden)

    Shuhei Noda

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

  7. The genetic basis of variation in clean lineages of Saccharomyces cerevisiae in response to stresses encountered during bioethanol fermentations.

    Directory of Open Access Journals (Sweden)

    Darren Greetham

    Full Text Available Saccharomyces cerevisiae is the micro-organism of choice for the conversion of monomeric sugars into bioethanol. Industrial bioethanol fermentations are intrinsically stressful environments for yeast and the adaptive protective response varies between strain backgrounds. With the aim of identifying quantitative trait loci (QTL's that regulate phenotypic variation, linkage analysis on six F1 crosses from four highly divergent clean lineages of S. cerevisiae was performed. Segregants from each cross were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Tolerance levels within populations of F1 segregants to stress conditions differed and displayed transgressive variation. Linkage analysis resulted in the identification of QTL's for tolerance to weak acid and osmotic stress. We tested candidate genes within loci identified by QTL using reciprocal hemizygosity analysis to ascertain their contribution to the observed phenotypic variation; this approach validated a gene (COX20 for weak acid stress and a gene (RCK2 for osmotic stress. Hemizygous transformants with a sensitive phenotype carried a COX20 allele from a weak acid sensitive parent with an alteration in its protein coding compared with other S. cerevisiae strains. RCK2 alleles reveal peptide differences between parental strains and the importance of these changes is currently being ascertained.

  8. Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt.

    Science.gov (United States)

    Zhou, Pingping; Ye, Lidan; Xie, Wenping; Lv, Xiaomei; Yu, Hongwei

    2015-10-01

    Astaxanthin is a highly valued carotenoid with strong antioxidant activity and has wide applications in aquaculture, food, cosmetic, and pharmaceutical industries. The market demand for natural astaxanthin promotes research in metabolic engineering of heterologous hosts for astaxanthin production. In this study, an astaxanthin-producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis β-carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed β-carotene hyperproducer. Further integration of strategies including codon optimization, gene copy number adjustment, and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in S. cerevisiae reported to date. Besides, the substrate specificity of H. pluvialis CrtZ and BKT and the probable formation route of astaxanthin from β-carotene in S. cerevisiae were figured out by expressing the genes separately and in combination. The yeast strains engineered in this work provide a basis for further improving biotechnological production of astaxanthin and might offer a useful general approach to the construction of heterologous biosynthetic pathways for other natural products. PMID:26156241

  9. CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Rajkumar, Arun Stephen; Zhang, Jie;

    2015-01-01

    Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here...... method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking...... out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae....

  10. Systematic identification of balanced transposition polymorphisms in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Dina A Faddah

    2009-06-01

    Full Text Available High-throughput techniques for detecting DNA polymorphisms generally do not identify changes in which the genomic position of a sequence, but not its copy number, varies among individuals. To explore such balanced structural polymorphisms, we used array-based Comparative Genomic Hybridization (aCGH to conduct a genome-wide screen for single-copy genomic segments that occupy different genomic positions in the standard laboratory strain of Saccharomyces cerevisiae (S90 and a polymorphic wild isolate (Y101 through analysis of six tetrads from a cross of these two strains. Paired-end high-throughput sequencing of Y101 validated four of the predicted rearrangements. The transposed segments contained one to four annotated genes each, yet crosses between S90 and Y101 yielded mostly viable tetrads. The longest segment comprised 13.5 kb near the telomere of chromosome XV in the S288C reference strain and Southern blotting confirmed its predicted location on chromosome IX in Y101. Interestingly, inter-locus crossover events between copies of this segment occurred at a detectable rate. The presence of low-copy repetitive sequences at the junctions of this segment suggests that it may have arisen through ectopic recombination. Our methodology and findings provide a starting point for exploring the origins, phenotypic consequences, and evolutionary fate of this largely unexplored form of genomic polymorphism.

  11. Protective Effects of Arginine on Saccharomyces cerevisiae Against Ethanol Stress

    Science.gov (United States)

    Cheng, Yanfei; Du, Zhaoli; Zhu, Hui; Guo, Xuena; He, Xiuping

    2016-01-01

    Yeast cells are challenged by various environmental stresses in the process of industrial fermentation. As the currently main organism for bio-ethanol production, Saccharomyces cerevisiae suffers from ethanol stress. Some amino acids have been reported to be related to yeast tolerance to stresses. Here the relationship between arginine and yeast response to ethanol stress was investigated. Marked inhibitions of ethanol on cell growth, expression of genes involved in arginine biosynthesis and intracellular accumulation of arginine were observed. Furthermore, extracellular addition of arginine can abate the ethanol damage largely. To further confirm the protective effects of arginine on yeast cells, yeast strains with different levels of arginine content were constructed by overexpression of ARG4 involved in arginine biosynthesis or CAR1 encoding arginase. Intracellular arginine was increased by 18.9% or 13.1% respectively by overexpression of ARG4 or disruption of CAR1, which enhanced yeast tolerance to ethanol stress. Moreover, a 41.1% decrease of intracellular arginine was observed in CAR1 overexpressing strain, which made yeast cells keenly sensitive to ethanol. Further investigations indicated that arginine protected yeast cells from ethanol damage by maintaining the integrity of cell wall and cytoplasma membrane, stabilizing the morphology and function of organellae due to low ROS generation. PMID:27507154

  12. Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

    Science.gov (United States)

    Qi, Xin; Zha, Jian; Liu, Gao-Gang; Zhang, Weiwen; Li, Bing-Zhi; Yuan, Ying-Jin

    2015-01-01

    Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold. By repetitive adaptation, the xylose utilization rate was further increased by ∼10-fold in the resultant strain SyBE003. Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003. PMID:26539187

  13. Saccharomyces cerevisiae Bat1 and Bat2 aminotransferases have functionally diverged from the ancestral-like Kluyveromyces lactis orthologous enzyme.

    Directory of Open Access Journals (Sweden)

    Maritrini Colón

    Full Text Available BACKGROUND: Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism. PRINCIPAL FINDINGS: Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs. This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1, while catabolic substrates are accumulated in the cytosol (Bat2. Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition. CONCLUSIONS: Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the

  14. Substrate specificity of thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Romagnoli, Gabriele; Luttik, Marijke A H; Kötter, Peter; Pronk, Jack T; Daran, Jean-Marc

    2012-11-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols. PMID:22904058

  15. Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Poulsen, Allan K.; Andersen, Ann Zahle; Brasen, Jens Christian; Scharff-Poulsen, Anne Marie; Olsen, Lars Folke

    2008-01-01

    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...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose...

  17. Biosorption of 241Am by immobilized Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Americium-241 is one of the most serious radioactive contaminating nuclides due to its high toxicity and long half-life. The encouraging biosorption of 241Am from aqueous solutions by free Saccharomyces cerevisiae (S. cerevisiae) has been observed in our previous experiments. 241Am biosorption by immobilized S. cerevisiae and the effect of the various experimental conditions on the adsorption were investigated. The results indicated that the 241Am biosorption by immobilized S. cerevisiae is still very efficient, and immobilized S. cerevisiae can be used repeatedly or continuously. The biosorption equilibrium was achieved within 2 hours, and more than 92% of 241Am was removed by immobilized S. cerevisiae in the pH 1-4 range. No significant differences in 241Am biosorption were observed at 15-45 deg C. The immobilized S. cerevisiae, even after used repeatedly for 6 times, still could adsorb more than 90% of 241Am in solutions of 1.08 MBq/l (8.5 μg/l). At this moment, the total adsorption capacity for 241Am was more than 63.3 KBq/g globe (0.5 μg/g), but has not reached saturation yet. The 241Am left in solutions with initial concentration of 1.08 MBq/l (8.5 μg/l) was noted as low as ∼10 Bq/l (∼8.0 x 10-5 μg/l) after adsorption by the immobilized S. cerevisiae for 3 times. (author)

  18. Research on biosorption of uranium by saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The effects of pH and the granularity of S. cerevisiae on the biosorption capacity were examined in order to study the properties of the biosorption of uranium from effluent by Saccharomyces cerevisiae. The isotherm was drawn. From the isotherm, the equations of Langmuir and Freundlich were achieved. The results showed the highest biosorption capacity was obtained when the pH value was about 6 and the granularity was 0.15-0.13 mm

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

    DEFF Research Database (Denmark)

    Rødkaer, Steven V; Færgeman, Nils J.

    2014-01-01

    steps and by numerous different regulators. As numerous of these regulating proteins, biochemical mechanisms, and cellular pathways are evolutionary conserved, complex biochemical information relevant to humans can be obtained by studying simple organisms. Thus, the yeast Saccharomyces cerevisiae has...... been recognized as a powerful model system to study fundamental biochemical processes. In the present review, we highlight central signaling pathways and molecular circuits conferring nitrogen- and glucose sensing in S. cerevisiae....

  20. Recovery of Saccharomyces cerevisiae from ethanol-induced growth inhibition.

    OpenAIRE

    Walker-Caprioglio, H M; Rodriguez, R J; Parks, L. W.

    1985-01-01

    Ethanol caused altered mobility of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene in plasma membrane preparations of Saccharomyces cerevisiae. Because lipids had been shown to protect yeast cells against ethanol toxicity, sterols, fatty acids, proteins, and combinations of these were tested; however, protection from growth inhibition was not seen. Ethanol-induced, prolonged lag periods and diminished growth rates in S. cerevisiae were reduced by an autoconditioning of the medium by the in...

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

    OpenAIRE

    Hana Šuranská; Dana Vránová; Jiřina Omelková

    2016-01-01

    Abstract In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typ...

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

    OpenAIRE

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were ...

  3. Contribution of PRS3, RPB4 and ZWF1 to the resistance of industrial Saccharomyces cerevisiae CCUG53310 and PE-2 strains to lignocellulosic hydrolysate-derived inhibitors

    OpenAIRE

    Cunha, J. T.; Aguiar, Tatiana Quinta; Romaní, Aloia; Oliveira, Carla Cristina Marques de; Domingues, Lucília

    2015-01-01

    PRS3, RPB4 and ZWF1 were previously identified as key genes for yeast tolerance to lignocellulose-derived inhibitors. To better understand their contribution to yeast resistance to the multiple stresses occurring during lignocellulosic hydrolysate fermentations, we overexpressed these genes in two industrial Saccharomyces cerevisiae strains, CCUG53310 and PE-2, and evaluated their impact on the fermentation of Eucalyptus globulus wood and corn cob hydrolysates. PRS3 overexpression improved...

  4. Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.

    OpenAIRE

    Bushman, J L; Asuru, A I; Matts, R L; Hinnebusch, A G

    1993-01-01

    Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent o...

  5. Size and position of intervening sequences are critical for the splicing efficiency of pre-mRNA in the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Klinz, F. J.; Gallwitz, D

    1985-01-01

    The size of the 309 bp actin gene intron of the yeast Saccharomyces cerevisiae was enlarged by inserting DNA fragments of different lengths and sequence. Enlarging the intron above 551 bp, the largest known yeast intron, led to a decrease in splicing efficiency. The effect on transcript splicing was dependent on the length of the inserted fragments rather than their sequence. It was also observed that insertion of the actin gene intron into different regions of the normally unsplit yeast YP2 ...

  6. KONSTRUKSI MUTAN PROTEIN FOSFATASE ptc2D Saccharomyces cerevisiae DENGAN METODE PENGGANTIAN GEN TARGET DENGAN POLYMERASE CHAIN REACTION (PCR

    Directory of Open Access Journals (Sweden)

    Hermansyah

    2011-05-01

    Full Text Available Yeast Saccharomyces cerevisiae is an excellent model to studi genes function of eukarotic cells such as study of gene encoding protein phosphatase PTC2. Novel phenotypic caused by mutated gene is an important step to study function of gene. In this study constructed mutant of PTC2 gene encoding protein phosphatase. Method that used in this construction was replacement of target gene (PTC2 with auxotroph marker Candida albicans HIS3 by Polymer Chain Reaction (PCR or called by PCR-mediated disruption. Mutant colonies which grew in selective medium SC without histidine were confirmed by PCR amplification. By using 1% Agarose gel electrophoresis the result showed that size of ptc2D::CgHIS3 transformant was 3.52 kb while wild type strain was 2.9 kb, indicated that ptc2D::CgHIS3 has integrated on chromosome V replacing PTC2 wild type.

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

    Science.gov (United States)

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

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

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

    Science.gov (United States)

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hana Šuranská

    2016-03-01

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

  10. Reconstruction and evaluation of the synthetic bacterial MEP pathway in Saccharomyces cerevisiae.

    Science.gov (United States)

    Partow, Siavash; Siewers, Verena; Daviet, Laurent; Schalk, Michel; Nielsen, Jens

    2012-01-01

    Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH. PMID:23285068

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  12. Evolutionary engineering of Saccharomyces cerevisiae for efficient conversion of red algal biosugars to bioethanol.

    Science.gov (United States)

    Lee, Hye-Jin; Kim, Soo-Jung; Yoon, Jeong-Jun; Kim, Kyoung Heon; Seo, Jin-Ho; Park, Yong-Cheol

    2015-09-01

    The aim of this work was to apply the evolutionary engineering to construct a mutant Saccharomyces cerevisiae HJ7-14 resistant on 2-deoxy-D-glucose and with an enhanced ability of bioethanol production from galactose, a mono-sugar in red algae. In batch and repeated-batch fermentations, HJ7-14 metabolized 5-fold more galactose and produced ethanol 2.1-fold faster than the parental D452-2 strain. Transcriptional analysis of genes involved in the galactose metabolism revealed that moderate relief from the glucose-mediated repression of the transcription of the GAL genes might enable HJ7-14 to metabolize galactose rapidly. HJ7-14 produced 7.4 g/L ethanol from hydrolysates of the red alga Gelidium amansii within 12 h, which was 1.5-times faster than that observed with D452-2. We demonstrate conclusively that evolutionary engineering is a promising tool to manipulate the complex galactose metabolism in S. cerevisiae to produce bioethanol from red alga. PMID:25804535

  13. Physiological and transcriptional characterization of Saccharomyces cerevisiae engineered for production of fatty acid ethyl esters.

    Science.gov (United States)

    de Jong, Bouke Wim; Siewers, Verena; Nielsen, Jens

    2016-02-01

    Saccharomyces cerevisiae has previously been engineered to become a cell factory for the production of fatty acid ethyl esters (FAEEs), molecules suitable for crude diesel replacement. To find new metabolic engineering targets for the improvement of FAEE cell factories, three different FAEE-producing strains of S. cerevisiae, constructed previously, were compared and characterized by quantification of key fluxes and genome-wide transcription analysis. From both the physiological and the transcriptional data, it was indicated that strain CB2I20, with high expression of a heterologous wax ester synthase gene (ws2) and strain BdJ15, containing disruptions of genes DGA1, LRO1, ARE1, ARE2 and POX1, which prevent the conversion of acyl-CoA to sterol esters, triacylglycerides and the degradation to acetyl-CoA, triggered oxidative stress that consequently influenced cellular growth. In the latter strain, stress was possibly triggered by disabling the buffering capacity of lipid droplets in encapsulating toxic fatty acids such as oleic acid. Additionally, it was indicated that there was an increased demand for NADPH required for the reduction steps in fatty acid biosynthesis. In conclusion, our analysis clearly shows that engineering of fatty acid biosynthesis results in transcriptional reprogramming and has a significant effect on overall cellular metabolism. PMID:26590613

  14. Comparative genomic analysis reveals a critical role of de novo nucleotide biosynthesis for Saccharomyces cerevisiae virulence.

    Directory of Open Access Journals (Sweden)

    Roberto Pérez-Torrado

    Full Text Available In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

  15. RNA complementary to the 5' UTR of mRNA triggers effective silencing in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Conditional silencing of target genes in Saccharomyces cerevisiae by antisense RNAs expressed in vivo has been challenged. The MFα1::lacZ fusion present in S. cerevisiae SF51-3 was chosen as a model target, and fragments of this gene were cloned in reverse orientation into the expression vector pYES2, bearing the GAL1 promoter. Among the different antisense constructs tested, only the one complementary to the 5' UTR of target mRNA featured effective silencing. Nevertheless, the expression in vivo of this antisense RNA could not be properly tuned by the absence or presence of galactose in the culture medium. Accordingly, conditional silencing could not be attained by this antisense hosted into pYES2. On the contrary, cloning the same antisense construct into the expression vector pSAL4 yielded a fully conditional silencing linked to the control of antisense expression by the absence or presence of Cu2+ into the culture medium

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    nontumorigenic porcine jejunal epithelial cell line (IPEC-J2) was investigated by incorporation of H-3-methionine into the yeast cells and use of liquid scintillation counting. Only few of the food-borne S. cerevisiae strains exhibited noteworthy adhesiveness with the strongest levels of adhesion (13...... effects hence indicating that food-borne strains of S. cerevisiae may possess probiotic properties in spite of low adhesiveness. © 2004 Elsevier B.V. All rights reserved....

  17. Similarity between the association factor of ribosomal subunits and the protein Stm1p from Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Heriberto Correia

    2004-11-01

    Full Text Available A ribosome association factor (AF was isolated from the yeast Sacchharomyces cerevisiae. Partial amino acid sequence of AF was determined from its fragment of 25 kDa isolated by treating AF with 2-(2-nitrophenylsulfenyl-3-methyl-3'-Bromoindolenine (BNPS-skatole. This sequence has a 86% identity to the product of the single-copy S. cerevisiae STM1 gene that is apparently involved in several events like binding to quadruplex and triplex nucleic acids and participating in apoptosis, stability of telomere structures, cell cycle, and ribosomal function. Here we show that AF and Stm1p share some characteristics: both bind to quadruplex and Pu triplex DNA, associates ribosomal subunits, and are thermostable. These observations suggest that these polypeptides belong to a family of proteins that may have roles in the translation process.

  18. Industrial systems biology of Saccharomyces cerevisiae enables novel succinic acid cell factory.

    Directory of Open Access Journals (Sweden)

    José Manuel Otero

    Full Text Available Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol, and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the α-keto-glutarate dehydrogenase catalyzed conversion of α-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2(nd-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we

  19. Synergistic effects of TAL1 over-expression and PHO13 deletion on the weak acid inhibition of xylose fermentation by industrial Saccharomyces cerevisiae strain.

    Science.gov (United States)

    Li, Yun-Cheng; Gou, Zi-Xi; Liu, Ze-Shen; Tang, Yue-Qin; Akamatsu, Takashi; Kida, Kenji

    2014-10-01

    In the industrial production of bioethanol from lignocellulosic biomass, a strain of Saccharomyces cerevisiae that can ferment xylose in the presence of inhibitors is of utmost importance. The recombinant, industrial-flocculating S. cerevisiae strain NAPX37, which can ferment xylose, was used as the parent to delete the gene encoding p-nitrophenylphosphatase (PHO13) and overexpress the gene encoding transaldolase (TAL1) to evaluate the synergistic effects of these two genes on xylose fermentation in the presence of weak acid inhibitors, including formic, acetic, or levulinic acids. TAL1 over-expression or PHO13 deletion improved xylose fermentation as well as the tolerance of NAPX37 to all three weak acids. The simultaneous deletion of PHO13 and the over-expression of TAL1 had synergistic effects and improved ethanol production and reduction of xylitol accumulation in the absence and presence of weak acid inhibitors. PMID:24966040

  20. Vanillin causes the activation of Yap1 and mitochondrial fragmentation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nguyen, Trinh Thi My; Iwaki, Aya; Ohya, Yoshikazu; Izawa, Shingo

    2014-01-01

    Vanillin and furfural are derived from lignocellulosic biomass and inhibit yeast growth and fermentation as biomass conversion inhibitors. Furfural has been shown to induce oxidative stress in Saccharomyces cerevisiae. Since there has been no report on the relationship between vanillin and oxidative stress, we investigated whether vanillin caused oxidative stress in yeast cells. We showed that vanillin caused the nuclear accumulation of Yap1, an oxidative stress responsive transcription factor, and subsequent transcriptional activation of Yap1-target genes. The growth of the null mutant of the YAP1 gene (yap1Δ) was delayed in the presence of vanillin, which indicated that Yap1 plays a role in the acquisition of tolerance to vanillin. We also demonstrated that vanillin facilitated the fragmentation of mitochondria. These findings suggest that the toxicity of vanillin involves damage induced by oxidative stress. PMID:23850265

  1. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN; Xiangling

    2005-01-01

    [1]Brunelli, J. P., Pall, M. L., A series of yeast vectors for expression of cDNAs and other DNA sequences, Yeast, 1993, 9: 1299―1308.[2]Sikorski, R. S., Hieter, P., A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics, 1989, 122: 19―27.[3]Bonneaud, N., Ozier-Kalogerogoulos, O., Li, G. et al., A family of low and high copy replicative, integrative and single-stranded S. cerevisiae /E. coli shuttle vector, Yeast, 1991, 7: 609―615.[4]Huo, K. K., Yu, L. L., Chen, X. J., Li, Y. Y., A stable vector for high-level expression and secretion of human interferon alpha A in yeast, Science in China, Ser. B, 1993, 36(5): 557―567.[5]Zhou, Z. X., Yuan, H. Y., He, W. et al., Expression of the modified HBsAg gene SA-28 directed by a constitutive promoter, Journal of Fudan university (Natural Science), 2000, 39(3): 264―268.[6]Paques, F., Haber, J. E., Multiple pathways of recombination induces by double-strand breaks in Saccharomyces cerevisiae, Microbiology and Molecular Biology Reviews, 1999, 63(2): 349―404.[7]Martin, K., Damage-induced recombination in the yeast Saccharomyces cerevisiae, Mutation Research, 2000, 451: 91―105.[8]Alira, S., Tomoko, O., Homologous recombination and the roles of double-strand breaks, TIBS, 1995, 20: 387―391.[9]Patrick, S., Kelly, M. T., Stephen, V. K., Recombination factor of Saccharomyces cerevisiae, Mutation Research, 2000, 451: 257―275.[10]Manivasakam, P., Weber, S. C., McElver, J., Schiestl, R. H., Micro-homology mediated PCR targeting in Saccharomyces cerevisiae, Nucleic Acids Res., 1995, 23(14): 2799―2800.[11]Baudin, A., Lacroute, F., Cullin, C., A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, Nucleic Acids Res., 1993, 21(14): 3329―3330.[12]Hua, S. B., Qiu, M., Chan, E., Zhu, L., Luo, Y., Minimum length of sequence homology required for in vivo cloning by homolo-gous recombination in yeast, Plasmid, 1997, 38

  2. Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response

    Science.gov (United States)

    Hoffman, Kyle S.; Duennwald, Martin L.; Karagiannis, Jim; Genereaux, Julie; McCarton, Alexander S.; Brandl, Christopher J.

    2016-01-01

    The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2. The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae. We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly. PMID:27172216

  3. Ploidy influences cellular responses to gross chromosomal rearrangements in saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Lemoine Sophie

    2011-06-01

    Full Text Available Abstract Background Gross chromosomal rearrangements (GCRs such as aneuploidy are key factors in genome evolution as well as being common features of human cancer. Their role in tumour initiation and progression has not yet been completely elucidated and the effects of additional chromosomes in cancer cells are still unknown. Most previous studies in which Saccharomyces cerevisiae has been used as a model for cancer cells have been carried out in the haploid context. To obtain new insights on the role of ploidy, the cellular effects of GCRs were compared between the haploid and diploid contexts. Results A total number of 21 haploid and diploid S. cerevisiae strains carrying various types of GCRs (aneuploidies, nonreciprocal translocations, segmental duplications and deletions were studied with a view to determining the effects of ploidy on the cellular responses. Differences in colony and cell morphology as well as in the growth rates were observed between mutant and parental strains. These results suggest that cells are impaired physiologically in both contexts. We also investigated the variation in genomic expression in all the mutants. We observed that gene expression was significantly altered. The data obtained here clearly show that genes involved in energy metabolism, especially in the tricarboxylic acid cycle, are up-regulated in all these mutants. However, the genes involved in the composition of the ribosome or in RNA processing are down-regulated in diploids but up-regulated in haploids. Over-expression of genes involved in the regulation of the proteasome was found to occur only in haploid mutants. Conclusion The present comparisons between the cellular responses of strains carrying GCRs in different ploidy contexts bring to light two main findings. First, GCRs induce a general stress response in all studied mutants, regardless of their ploidy. Secondly, the ploidy context plays a crucial role in maintaining the stoichiometric balance

  4. Incipient balancing selection through adaptive loss of aquaporins in natural Saccharomyces cerevisiae populations.

    Directory of Open Access Journals (Sweden)

    Jessica L Will

    2010-04-01

    Full Text Available A major goal in evolutionary biology is to understand how adaptive evolution has influenced natural variation, but identifying loci subject to positive selection has been a challenge. Here we present the adaptive loss of a pair of paralogous genes in specific Saccharomyces cerevisiae subpopulations. We mapped natural variation in freeze-thaw tolerance to two water transporters, AQY1 and AQY2, previously implicated in freeze-thaw survival. However, whereas freeze-thaw-tolerant strains harbor functional aquaporin genes, the set of sensitive strains lost aquaporin function at least 6 independent times. Several genomic signatures at AQY1 and/or AQY2 reveal low variation surrounding these loci within strains of the same haplotype, but high variation between strain groups. This is consistent with recent adaptive loss of aquaporins in subgroups of strains, leading to incipient balancing selection. We show that, although aquaporins are critical for surviving freeze-thaw stress, loss of both genes provides a major fitness advantage on high-sugar substrates common to many strains' natural niche. Strikingly, strains with non-functional alleles have also lost the ancestral requirement for aquaporins during spore formation. Thus, the antagonistic effect of aquaporin function-providing an advantage in freeze-thaw tolerance but a fitness defect for growth in high-sugar environments-contributes to the maintenance of both functional and nonfunctional alleles in S. cerevisiae. This work also shows that gene loss through multiple missense and nonsense mutations, hallmarks of pseudogenization presumed to emerge after loss of constraint, can arise through positive selection.

  5. A unique ecological niche fosters hybridization of oak-tree and vineyard isolates of Saccharomyces cerevisiae.

    Science.gov (United States)

    Clowers, Katie J; Will, Jessica L; Gasch, Audrey P

    2015-12-01

    Differential adaptation to distinct niches can restrict gene flow and promote population differentiation within a species. However, in some cases the distinction between niches can collapse, forming a hybrid niche with features of both environments. We previously reported that distinctions between vineyards and oak soil present an ecological barrier that restricts gene flow between lineages of Saccharomyces cerevisiae. Vineyard isolates are tolerant to stresses associated with grapes while North American oak strains are particularly tolerant to freeze-thaw cycles. Here, we report the isolation of S. cerevisiae strains from Wisconsin cherry trees, which display features common to vineyards (e.g. high sugar concentrations) and frequent freeze-thaw cycles. Genome sequencing revealed that the isolated strains are highly heterozygous and represent recent hybrids of the oak × vineyard lineages. We found that the hybrid strains are phenotypically similar to vineyard strains for some traits, but are more similar to oak strains for other traits. The cherry strains were exceptionally good at growing in cherry juice, raising the possibility that they have adapted to this niche. We performed transcriptome profiling in cherry, oak and vineyard strains and show that the cherry-tree hybrids display vineyard-like or oak-like expression, depending on the gene sets, and in some cases, the expression patterns linked back to shared stress tolerances. Allele-specific expression in these natural hybrids suggested concerted cis-regulatory evolution at sets of functionally regulated genes. Our results raise the possibility that hybridization of the two lineages provides a genetic solution to the thriving in this unique niche. PMID:26518477

  6. Discrete dynamical system modelling for gene regulatory networks of 5-hydroxymethylfural tolerance for ethanologenic yeast

    Science.gov (United States)

    Composed of linear difference equations, a discrete dynamic system model was designed to reconstruct transcriptional regulations in gene regulatory networks in response to 5-hydroxymethylfurfural, a bioethanol conversion inhibitor for ethanologenic yeast Saccharomyces cerevisiae. The modeling aims ...

  7. Removing cadmium from electroplating wastewater by waste saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  8. N-acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Du, Xiaoyi [Fukui Prefectural Univ., Fukui (Japan). Dept. of Bioscience; Takagi, Hiroshi [Nara Inst. of Science and Technology, Ikoma, Nara (Japan). Graduate School of Biological Sciences

    2007-07-15

    N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H{sub 2}O{sub 2}, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H{sub 2}O{sub 2} or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H{sub 2}O{sub 2}. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains. (orig.)

  9. Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

    OpenAIRE

    Katie E Hyma; Saerens, Sofie M; Verstrepen, Kevin J.; Justin C Fay

    2011-01-01

    The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea t...

  10. Genome Sequence and Analysis of a Stress-Tolerant, Wild-Derived Strain of Saccharomyces cerevisiae Used in Biofuels Research

    Directory of Open Access Journals (Sweden)

    Sean J. McIlwain

    2016-06-01

    Full Text Available The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3, a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.

  11. Genome Sequence and Analysis of a Stress-Tolerant, Wild-Derived Strain of Saccharomyces cerevisiae Used in Biofuels Research.

    Science.gov (United States)

    McIlwain, Sean J; Peris, David; Sardi, Maria; Moskvin, Oleg V; Zhan, Fujie; Myers, Kevin S; Riley, Nicholas M; Buzzell, Alyssa; Parreiras, Lucas S; Ong, Irene M; Landick, Robert; Coon, Joshua J; Gasch, Audrey P; Sato, Trey K; Hittinger, Chris Todd

    2016-01-01

    The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics. PMID:27172212

  12. Crystallization and preliminary crystallographic studies of the NAD+-dependent deacetylase HST1 from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The Saccharomyces cerevisiae NAD+-dependent deacetylase HST1156–503 was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.90 Å resolution. The Saccharomyces cerevisiae NAD+-dependent deacetylase HST1 belongs to the class III HDAC family; it acts as a transcriptional corepressor for the specific middle sporulation and de novo NAD+-biosynthesis genes and also takes part in the SET3C and SUM1–RFM1–HST1 complexes. Structural information on HST1 and its related complexes would be helpful in order to understand the structural basis of its deacetylation mechanism and the assembly of these complexes. Here, HST1156–503 was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.90 Å resolution and belonged to space group P21, with unit-cell parameters a = 40.2, b = 101.7, c = 43.9 Å, β = 103.9°. Both Matthews coefficient analysis and the self-rotation function suggested the presence of four molecules per asymmetric unit in the crystal, with a solvent content of 49.76% (VM = 2.45 Å3 Da−1)

  13. Construction of novel Saccharomyces cerevisiae strains for bioethanol active dry yeast (ADY production.

    Directory of Open Access Journals (Sweden)

    Daoqiong Zheng

    Full Text Available The application of active dry yeast (ADY in bioethanol production simplifies operation processes and reduces the risk of bacterial contamination. In the present study, we constructed a novel ADY strain with improved stress tolerance and ethanol fermentation performances under stressful conditions. The industrial Saccharomyces cerevisiae strain ZTW1 showed excellent properties and thus subjected to a modified whole-genome shuffling (WGS process to improve its ethanol titer, proliferation capability, and multiple stress tolerance for ADY production. The best-performing mutant, Z3-86, was obtained after three rounds of WGS, producing 4.4% more ethanol and retaining 2.15-fold higher viability than ZTW1 after drying. Proteomics and physiological analyses indicated that the altered expression patterns of genes involved in protein metabolism, plasma membrane composition, trehalose metabolism, and oxidative responses contribute to the trait improvement of Z3-86. This work not only successfully developed a novel S. cerevisiae mutant for application in commercial bioethanol production, but also enriched the current understanding of how WGS improves the complex traits of microbes.

  14. Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase.

    Science.gov (United States)

    Greer, Michael S; Truksa, Martin; Deng, Wei; Lung, Shiu-Cheung; Chen, Guanqun; Weselake, Randall J

    2015-03-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to produce triacylglycerol (TAG). This enzyme, which is critical to numerous facets of oilseed development, has been highlighted as a genetic engineering target to increase storage lipid production in microorganisms designed for biofuel applications. Here, four transcriptionally active DGAT1 genes were identified and characterized from the oil crop Brassica napus. Overexpression of each BnaDGAT1 in Saccharomyces cerevisiae increased TAG biosynthesis. Further studies showed that adding an N-terminal tag could mask the deleterious influence of the DGATs' native N-terminal sequences, resulting in increased in vivo accumulation of the polypeptides and an increase of up to about 150-fold in in vitro enzyme activity. The levels of TAG and total lipid fatty acids in S. cerevisiae producing the N-terminally tagged BnaDGAT1.b at 72 h were 53 and 28 % higher than those in cultures producing untagged BnaA.DGAT1.b, respectively. These modified DGATs catalyzed the synthesis of up to 453 mg fatty acid/L by this time point. The results will be of benefit in the biochemical analysis of recombinant DGAT1 produced through heterologous expression in yeast and offer a new approach to increase storage lipid content in yeast for industrial applications. PMID:25520169

  15. Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Turner, Timothy L; Zhang, Guo-Chang; Oh, Eun Joong; Subramaniam, Vijay; Adiputra, Andrew; Subramaniam, Vimal; Skory, Christopher D; Jang, Ji Yeon; Yu, Byung Jo; Park, In; Jin, Yong-Su

    2016-05-01

    Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, β-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast. PMID:26524688

  16. Real-time Monitoring of Non-specific Toxicity Using a Saccharomyces cerevisiae Reporter System

    Directory of Open Access Journals (Sweden)

    Matti Karp

    2008-10-01

    Full Text Available Baker’s yeast, Saccharomyces cerevisiae, is the simplest and most well-known representative of eukaryotic cells and thus a convenient model organism for evaluating toxic effects in human cells and tissues. Yeast cell sensors are easy to maintain with short generation times, which makes the analytical method of assessing antifungal toxicity cheap and less-time consuming. In this work, the toxicity of test compounds was assessed in bioassays based on bioluminescence inhibition and on traditional growth inhibition on agar plates. The model organism in both tests was a modified S. cerevisiae sensor strain that produces light when provided with D-luciferin in an insect luciferase reporter gene activity assay. The bioluminescence assay showed toxic effects for yeast cell sensor of 5,6-benzo-flavone, rapamycin, nystatin and cycloheximide at concentrations of nM to µM. In addition, arsenic compounds, cadmium chloride, copper sulfate and lead acetate were shown to be potent non-specific inhibitors of the reporter organism described here. The results from a yeast agar diffusion assay correlated with the bioluminescence assay results.

  17. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

  18. Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases.

    Science.gov (United States)

    Favaro, Lorenzo; Viktor, Marko J; Rose, Shaunita H; Viljoen-Bloom, Marinda; van Zyl, Willem H; Basaglia, Marina; Cagnin, Lorenzo; Casella, Sergio

    2015-09-01

    The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch-based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon-optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α-amylase (SFA1) genes were δ-integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1-SFA1] and MEL2[TLG1-SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch-to-ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre-treatment or commercial enzyme addition) using industrial yeast strains co-secreting both a glucoamylase and α-amylase. PMID:25786804

  19. Cell Surface Display of Four Types of Solanum nigrum Metallothionein on Saccharomyces cerevisiae for Biosorption of Cadmium.

    Science.gov (United States)

    Wei, Qinguo; Zhang, Honghai; Guo, Dongge; Ma, Shisheng

    2016-05-28

    We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd(2+) in this study. The transformed yeast strains showed much higher resistance ability to Cd(2+) compared with the control. Strikingly, their Cd(2+) accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd(2+) under a wide range of pH levels, from 3 to 7, without disturbing the Cu(2+) and Hg(2+). Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd(2+)-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants. PMID:26838339

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

    KAUST Repository

    Aouida, Mustapha

    2015-04-01

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

  1. Implication of Ccr4-Not complex function in mRNA quality control in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Assenholt, Jannie; Mouaikel, John; Saguez, Cyril;

    2011-01-01

    Production of messenger ribonucleoprotein particles (mRNPs) is subjected to quality control (QC). In Saccharomyces cerevisiae, the RNA exosome and its cofactors are part of the nuclear QC machinery that removes, or stalls, aberrant molecules, thereby ensuring that only correctly formed mRNPs are ......Production of messenger ribonucleoprotein particles (mRNPs) is subjected to quality control (QC). In Saccharomyces cerevisiae, the RNA exosome and its cofactors are part of the nuclear QC machinery that removes, or stalls, aberrant molecules, thereby ensuring that only correctly formed m......RNPs are exported to the cytoplasm. The Ccr4-Not complex, which constitutes the major S. cerevisiae cytoplasmic deadenylase, has recently been implied in nuclear exosome–related processes. Consistent with a possible nuclear function of the complex, the deletion or mutation of Ccr4-Not factors also elicits...... transcription phenotypes. Here we use genetic depletion of the Mft1p protein of the THO transcription/mRNP packaging complex as a model system to link the Ccr4-Not complex to nuclear mRNP QC. We reveal strong genetic interactions between alleles of the Ccr4-Not complex with both the exosomal RRP6 and MFT1 genes...

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

    Science.gov (United States)

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

    2016-03-01

    The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae. PMID:26724864

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

    Science.gov (United States)

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

    2015-10-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S. cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S. cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. PMID:25907574

  4. Directed Evolution towards Increased Isoprenoid Production in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Carlsen, Simon; Nielsen, Michael Lynge; Kielland-Brandt, Morten;

    Due to declining drug discovery rates from organic synthetic libraries, pharmaceutical companies are turning their attention towards secondary metabolites. Isoprenoids, also known as terpenoids, constitute the largest known group of secondary metabolites isolated to date, encompassing more than 55...... pyrophosphate and geranylgeranyl pyrophosphate for large-scale microbial production of terpenoids. Saccharomyces cerevisiae was chosen as production platform due to its widespread use in industrial production and the waste number of molecular biology tools which is available for its manipulation. The effort...... for discovering new genetic perturbations, which would results in and increased production of isoprenoids by S. cerevisiae has been very limited. This project is focus on creating diversity within a lycopene producing S. cerevisiae strain by construction of gDNA-, cDNA-, and transposon-libraries. The diversified...

  5. A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae.

    Science.gov (United States)

    Piirainen, Mari A; Boer, Harry; de Ruijter, Jorg C; Frey, Alexander D

    2016-04-01

    N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae. PMID:26983412

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

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Juncker, Agnieszka; Hallstrom, Bjorn; Jensen, Niels Bjerg; Maury, Jerome; Nielsen, Jen; Förster, Jochen; Borodina, Irina

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

  7. Accumulation of gold using Baker's yeast, Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Authors have reported preconcentration of 152Eu, a long-lived fission product, by yeast cells, Saccharomyces cerevisiae. Gold being a precious metal is used in electroplating, hydrogenation catalyst, etc. Heterogeneous composition of samples and low concentration offers renewed interest in its selective extraction of gold using various extractants. Gold can be recovered from different solutions using various chemical reagents like amines, organophosphorus compounds, and extractants containing sulphur as donor atom, etc. In the present work, two different strains of baker's yeast, Saccharomyces cerevisiae have been used to study the preconcentration of gold at various experimental conditions

  8. Structure of Ynk1 from the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The crystal structure of Ynk1, an NDPK from the yeast Saccharomyces cerevisiae, has been solved at 3.1 Å resolution. Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates. In addition to biochemical studies, a number of crystal structures of NDPK from various organisms, including both native proteins and complexes with nucleotides or nucleotide analogues, have been determined. Here, the crystal structure of Ynk1, an NDPK from the yeast Saccharomyces cerevisiae, has been solved at 3.1 Å resolution. Structural analysis strongly supports the oligomerization state of this protein being hexameric rather than tetrameric

  9. Origin of co-expression patterns in E. coli and S. cerevisiae emerging from reverse engineering algorithms.

    Directory of Open Access Journals (Sweden)

    Mattia Zampieri

    Full Text Available BACKGROUND: The concept of reverse engineering a gene network, i.e., of inferring a genome-wide graph of putative gene-gene interactions from compendia of high throughput microarray data has been extensively used in the last few years to deduce/integrate/validate various types of "physical" networks of interactions among genes or gene products. RESULTS: This paper gives a comprehensive overview of which of these networks emerge significantly when reverse engineering large collections of gene expression data for two model organisms, E. coli and S. cerevisiae, without any prior information. For the first organism the pattern of co-expression is shown to reflect in fine detail both the operonal structure of the DNA and the regulatory effects exerted by the gene products when co-participating in a protein complex. For the second organism we find that direct transcriptional control (e.g., transcription factor-binding site interactions has little statistical significance in comparison to the other regulatory mechanisms (such as co-sharing a protein complex, co-localization on a metabolic pathway or compartment, which are however resolved at a lower level of detail than in E. coli. CONCLUSION: The gene co-expression patterns deduced from compendia of profiling experiments tend to unveil functional categories that are mainly associated to stable bindings rather than transient interactions. The inference power of this systematic analysis is substantially reduced when passing from E. coli to S. cerevisiae. This extensive analysis provides a way to describe the different complexity between the two organisms and discusses the critical limitations affecting this type of methodologies.

  10. Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

    DEFF Research Database (Denmark)

    Nissen, T.L.; Anderlund, M.; Nielsen, Jens;

    2001-01-01

    systems is made possible by expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii. We therefore cloned sth, encoding this enzyme and expressed it under the control of a S, cerevisiae promoter in a strain derived from the industrial model strain S, cerevisiae CBS8066, Anaerobic batch...... cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein was...... measured in the extracts from the strain expressing the sth gene from A. vinelandii, while no transhydrogenase activity could be detected in control strains without the gene. Production of the transhydrogenase caused a significant increase in formation of glycerol and 2-oxoglutarate. Since NADPH is used to...

  11. Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae.

    OpenAIRE

    Southgate, V J; Steyn, A J; Pretorius, I. S.; van Vuuren, H J

    1993-01-01

    Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae. However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.

  12. The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae

    OpenAIRE

    Wiederkehr, Andreas; Avaro, Sandrine; Prescianotto-Baschong, Cristina; Haguenauer-Tsapis, Rosine; Riezman, Howard

    2000-01-01

    In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the ac...

  13. Análisis estructural y modificación funcional de la glucoamilasa de Saccharomyces cerevisiae var diastaticus.

    OpenAIRE

    Latorre García, Lorena

    2008-01-01

    RESUMEN La glucoamilasa (GA) es uno de los enzimas producidos en mayor cantidad por la industria biotecnológica. Se emplea en el procesado del almidón degradándolo y liberando residuos de glucosa al medio. Saccharomyces cerevisiae (var. diastaticus) posee genes denominados STA que codifican GAs (Yamashita et al 1987). Este enzima presenta una estructura atípica ya que posee un dominio N-terminal, rico en residuos de serina y treonina (STRD o Ser and Thr rich-domain) ausente en las otras...

  14. Enhanced citrate production through gene insertion in Aspergillus niger

    DEFF Research Database (Denmark)

    Jongh, Wian de; Nielsen, Jens

    2007-01-01

    The effect of inserting genes involved in the reductive branch of the tricarboxylic acid (TCA) cycle on citrate production by Aspergillus niger was evaluated. Several different genes were inserted individually and in combination, i.e. malate dehydrogenase (mdh2) from Saccharomyces cerevisiae, two...

  15. Genetiese manipulering van die gis Saccharomyces cerevisiae betreffende polisakkariedbenutting

    Directory of Open Access Journals (Sweden)

    I. S. Pretoruis

    1992-07-01

    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.

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

    Science.gov (United States)

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

    2015-08-01

    During wine fermentation, Saccharomyces clearly dominate over non-Saccharomyces wine yeasts, and several factors could be related to this dominance. However, the main factor causing the reduction of cultivable non-Saccharomyces populations has not yet been fully established. In the present study, various single and mixed fermentations were performed to evaluate some of the factors likely responsible for the interaction between Saccharomyces cerevisiae and Hanseniaspora uvarum. Alcoholic fermentation was performed in compartmented experimental set ups with ratios of 1:1 and 1:9 and the cultivable population of both species was followed. The cultivable H. uvarum population decreased sharply at late stages when S. cerevisiae was present in the other compartment, similarly to alcoholic fermentations in non-compartmented vessels. Thus, cell-to-cell contact did not seem to be the main cause for the lack of cultivability of H. uvarum. Other compounds related to fermentation performance (such as sugar and ethanol) and/or certain metabolites secreted by S. cerevisiae could be related to the sharp decrease in H. uvarum cultivability. When these factors were analyzed, it was confirmed that metabolites from S. cerevisiae induced lack of cultivability in H. uvarum, however ethanol and other possible compounds did not seem to induce this effect but played some role during the process. This study contributes to a new understanding of the lack of cultivability of H. uvarum populations during the late stages of wine fermentation. PMID:25956738

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

    Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering. PMID:26658003

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

    Science.gov (United States)

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

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

  20. Improving biomass sugar utilization by engineered Saccharomyces cerevisiae

    Science.gov (United States)

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

  1. Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Solis Escalante, D.

    2015-01-01

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

  2. Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.

    Science.gov (United States)

    Roloff, Gabrielle A; Henry, Michael F

    2015-08-15

    Three mitochondrial DNA-encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1. PMID:26108620

  3. Statistically Significant Strings are Related to Regulatory Elements in the Promoter Regions of Saccharomyces cerevisiae

    CERN Document Server

    Hu, R; Hu, Rui; Wang, Bin

    2000-01-01

    Finding out statistically significant words in DNA and protein sequences forms the basis for many genetic studies. By applying the maximal entropy principle, we give one systematic way to study the nonrandom occurrence of words in DNA or protein sequences. Through comparison with experimental results, it was shown that patterns of regulatory binding sites in Saccharomyces cerevisiae(yeast) genomes tend to occur significantly in the promoter regions. We studied two correlated gene family of yeast. The method successfully extracts the binding sites varified by experiments in each family. Many putative regulatory sites in the upstream regions are proposed. The study also suggested that some regulatory sites are a ctive in both directions, while others show directional preference.

  4. Electrochemical Probing of in Vivo 5-Hydroxymethyl Furfural Reduction in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kostesha, Natalie; Almeida, J.R.M.; Heiskanen, Arto;

    2009-01-01

    In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NADPH dependent 5-hydroxymethyl-2-furaldehyde...... amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: K-M,cell(app), V-MAX, k(cat,cell), and k(cat,cell)/K-M,cell(app). The results indicated that the overexpression of the ADH6 gene gave a...... 68% decrease in K-M,cell(app), and 42% increase in V-MAX, resulting in a 4-fold increase in k(cat,cell)/K-M,cell(app). These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzyme kinetic...

  5. Reducing alcohol levels in wines through rational and evolutionary engineering of Saccharomyces cerevisiae.

    Science.gov (United States)

    Tilloy, Valentin; Cadière, Axelle; Ehsani, Maryam; Dequin, Sylvie

    2015-11-20

    Over the past two decades, the level of ethanol in wine has increased in most wine-producing regions, raising a number of issues related to consumer health, prevention policies, the effectiveness of the fermentation and wine sensorial quality. This review focuses on metabolic challenges and recent achievements in the development of Saccharomyces cerevisiae wine strains with reduced ethanol yield. Metabolic engineering approaches that have been successfully used to optimize endogenous pathways have been gradually replaced in recent years by evolutionary engineering strategies, which can generate strains with improved phenotypes using new circuits and can be put to immediate commercial use. The power of adaptive evolutionary strategies is expected to increase with the rapid development of whole-genome sequencing, which, combined with gene expression and metabolic flux analysis, enables the identification of the genetic basis of improved phenotypes and the transfer of such phenotypes between strains. PMID:26219842

  6. Functional replacement of the Saccharomyces cerevisiae fatty acid synthase with a bacterial type II system allows flexible product profiles.

    Science.gov (United States)

    Fernandez-Moya, Ruben; Leber, Christopher; Cardenas, Javier; Da Silva, Nancy A

    2015-12-01

    The native yeast type I fatty acid synthase (FAS) is a complex, rigid enzyme, and challenging to engineer for the production of medium- or short-chain fatty acids. Introduction of a type II FAS is a promising alternative as it allows expression control for each discrete enzyme and the addition of heterologous thioesterases. In this study, the native Saccharomyces cerevisiae FAS was functionally replaced by the Escherichia coli type II FAS (eFAS) system. The E. coli acpS + acpP (together), fabB, fabD, fabG, fabH, fabI, fabZ, and tesA were expressed in individual S. cerevisiae strains, and enzyme activity was confirmed by in vitro activity assays. Eight genes were then integrated into the yeast genome, while tesA or an alternate thioesterase gene, fatB from Ricinus communis or TEII from Rattus novergicus, was expressed from a multi-copy plasmid. Native FAS activity was eliminated by knocking out the yeast FAS2 gene. The strains expressing only the eFAS as de novo fatty acid source grew without fatty acid supplementation demonstrating that this type II FAS is able to functionally replace the native yeast FAS. The engineered strain expressing the R. communis fatB thioesterase increased total fatty acid titer 1.7-fold and shifted the fatty acid profile towards C14 production, increasing it from fatty acids, and reducing C18 production from 39% to 8%. PMID:26084339

  7. Expression of protein engineered NADP{sup +}-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki [National Institute of Advanced Industrial Science and Technology, Hiroshima (Japan). Biomass Technology Research Center; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke [Kyoto Univ. (Japan). Inst. of Advanced Energy

    2008-11-15

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD{sup +}-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP{sup +}. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP{sup +}-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP{sup +}-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain. (orig.)

  8. EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jérôme Maury

    Full Text Available Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP. The best 3HP producing clone, with 5.45 g.L(-1 of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

  9. AMIODARONE INDUCES THE SYNTHESIS OF HSPS IN SACCHAROMYCES CEREVISIAE AND ARABIDOPSIS THALIANA CELLS

    Directory of Open Access Journals (Sweden)

    Pyatrikas D.V.

    2012-08-01

    Full Text Available Many biotic and abiotic stresses cause an increase of cytosolic Ca2+ level in cells. Calcium is one of the most important second messengers, regulating many various activities in the cell and was known to affect expression of stress activated genes. Mild heat shock induces the expression of heat shock proteins (Hsps which protect cell from drastic heat shock exposure. There are some literature data permitting to suggest that transient elevation of cytosolic Ca2+ level in plant cells is important for activation of Hsps expression. On the other hand mitochondria are known to regulate the intracellular calcium and reactive oxygen species signaling. It has been shown recently that mild heat shock induces hyperpolarization of inner mitochondrial membrane in plant and yeast cells and this event is critically important for activation of Hsps expression. To reveal the relationship between mitochondrial activity, intracellular calcium homeostasis and Hsps expression an antiarrhythmic drug amiodarone (AMD have been used. AMD is known to cause transient increase of cytosolic Ca2+ level in Saccharomyces cerevisiae. Obtained results have showed that AMD treatment induced the synthesis of Hsp104p in S. cerevisiae cells and Hsp101p in A. thaliana cell culture. Induction of Hsp104p synthesis leads to enhanced yeast capability to survive lethal heat shock exposure. Development of S. cerevisiae thermotolerance depended significantly on the presence of Hsp104p. Elevation of Hsp104p level in the result of AMD treatment was shown to be governed by activity of Msn2p and Msn4p transcription factors. Deletion of the MSN2 and MSN4 genes abrogated the AMD ability to induce Hsp104p synthesis. Mild heat shock and AMD treatment induced the hyperpolarization of the inner mitochondrial membrane in yeast and Arabidopsis cells which accompanied by HSP synthesis and development of thermotolerance. It was suggested that increase of cytosolic Ca2+ level after AMD treatment

  10. Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae.

    Science.gov (United States)

    Leber, Christopher; Polson, Brian; Fernandez-Moya, Ruben; Da Silva, Nancy A

    2015-03-01

    The production of fuels and chemicals from biorenewable resources is important to alleviate the environmental concerns, costs, and foreign dependency associated with the use of petroleum feedstock. Fatty acids are attractive biomolecules due to the flexibility of their iterative biosynthetic pathway, high energy content, and suitability for conversion into other secondary chemicals. Free fatty acids (FFAs) that can be secreted from the cell are particularly appealing due to their lower harvest costs and straightforward conversion into a broad range of biofuel and biochemical products. Saccharomyces cerevisiae was engineered to overproduce extracellular FFAs by targeting three native intracellular processes. β-oxidation was disrupted by gene knockouts in FAA2, PXA1 and POX1, increasing intracellular fatty acids levels up to 55%. Disruptions in the acyl-CoA synthetase genes FAA1, FAA4 and FAT1 allowed the extracellular detection of free fatty acids up to 490mg/L. Combining these two disrupted pathways, a sextuple mutant (Δfaa1 Δfaa4 Δfat1 Δfaa2 Δpxa1 Δpox1) was able to produce 1.3g/L extracellular free fatty acids. Further diversion of carbon flux into neutral lipid droplet formation was investigated by the overexpression of DGA1 or ARE1 and by the co-overexpression of a compatible lipase, TGL1, TGL3 or TGL5. The sextuple mutant overexpressing the diacylglycerol acyltransferase, DGA1, and the triacylglycerol lipase, TGL3, yielded 2.2g/L extracellular free fatty acids. This novel combination of pathway interventions led to 4.2-fold higher extracellular free fatty acid levels than previously reported for S. cerevisiae. PMID:25461829

  11. The transcriptional response of Saccharomyces cerevisiae to proapoptotic concentrations of Pichia membranifaciens killer toxin.

    Science.gov (United States)

    Santos, A; Marquina, D

    2011-10-01

    PMKT (Pichia membranifaciens killer toxin) reportedly has antimicrobial activity against yeasts and filamentous fungi. In previous research we posited that high PMKT concentrations pose a serious challenge for cell survival by disrupting plasma membrane electrochemical gradients, inducing a transcriptional response similar to that of certain stimuli such as hyperosmotic shock. This response was related to the HOG-pathway with Hog1p phosphorylation and a transitional increase in intracellular glycerol accumulation. Such a response was consistent with the notion that the effect induced by high PMKT concentrations lies in an alteration to the ionic homeostasis of the sensitive cell. By contrast, the evidence presented here shows that low PMKT doses lead to a cell death process in Saccharomyces cerevisiae accompanied by cytological and biochemical indicators of apoptotic programmed cell death, namely, the production of reactive oxygen species, DNA strand breaks, metacaspase activation and cytochrome c release. Furthermore, dying cells progressed from an apoptotic state to a secondary necrotic state, and the rate at which this change occurred was proportional to the intensity of the stimulus. We have explored the global gene expression response of S. cerevisiae during that stimulus. The results obtained from DNA microarrays indicate that genes related with an oxidative stress response were induced in response to proapoptotic concentrations of PMKT, showing that the coordinated transcriptional response is not coincident with that obtained when ionophoric concentrations of PMKT are used. By contrast, cwp2Δ mutants showed no signs of apoptosis, indicating that the initial steps of the killer mechanism coincide when proapoptotic (low) or ionophoric (high) PMKT concentrations are used. Additionally, low dosages of PMKT promoted Hog1p phosphorylation and glycerol accumulation. PMID:21801845

  12. Genome Sequencing and Comparative Analysis of Saccharomyces cerevisiae Strains of the Peterhof Genetic Collection

    Science.gov (United States)

    Drozdova, Polina B.; Tarasov, Oleg V.; Matveenko, Andrew G.; Radchenko, Elina A.; Sopova, Julia V.; Polev, Dmitrii E.; Inge-Vechtomov, Sergey G.; Dobrynin, Pavel V.

    2016-01-01

    The Peterhof genetic collection of Saccharomyces cerevisiae strains (PGC) is a large laboratory stock that has accumulated several thousands of strains for over than half a century. It originated independently of other common laboratory stocks from a distillery lineage (race XII). Several PGC strains have been extensively used in certain fields of yeast research but their genomes have not been thoroughly explored yet. Here we employed whole genome sequencing to characterize five selected PGC strains including one of the closest to the progenitor, 15V-P4, and several strains that have been used to study translation termination and prions in yeast (25-25-2V-P3982, 1B-D1606, 74-D694, and 6P-33G-D373). The genetic distance between the PGC progenitor and S288C is comparable to that between two geographically isolated populations. The PGC seems to be closer to two bakery strains than to S288C-related laboratory stocks or European wine strains. In genomes of the PGC strains, we found several loci which are absent from the S288C genome; 15V-P4 harbors a rare combination of the gene cluster characteristic for wine strains and the RTM1 cluster. We closely examined known and previously uncharacterized gene variants of particular strains and were able to establish the molecular basis for known phenotypes including phenylalanine auxotrophy, clumping behavior and galactose utilization. Finally, we made sequencing data and results of the analysis available for the yeast community. Our data widen the knowledge about genetic variation between Saccharomyces cerevisiae strains and can form the basis for planning future work in PGC-related strains and with PGC-derived alleles. PMID:27152522

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

    Directory of Open Access Journals (Sweden)

    Hiren Karathia

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

  14. CRISPR–Cas system enables fast and simple genome editing of industrial Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Vratislav Stovicek

    2015-12-01

    Full Text Available There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of Saccharomyces cerevisiae will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid. Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR–Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously. Our study provides a foundation for efficient engineering of industrial yeast cell factories.

  15. Splicing-Mediated Autoregulation Modulates Rpl22p Expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Gabunilas, Jason; Chanfreau, Guillaume

    2016-04-01

    In Saccharomyces cerevisiae, splicing is critical for expression of ribosomal protein genes (RPGs), which are among the most highly expressed genes and are tightly regulated according to growth and environmental conditions. However, knowledge of the precise mechanisms by which RPG pre-mRNA splicing is regulated on a gene-by-gene basis is lacking. Here we show that Rpl22p has an extraribosomal role in the inhibition of splicing of the RPL22B pre-mRNA transcript. A stem loop secondary structure within the intron is necessary for pre-mRNA binding by Rpl22p in vivo and splicing inhibition in vivo and in vitro and can rescue splicing inhibition in vitro when added in trans to splicing reactions. Splicing inhibition by Rpl22p may be partly attributed to the reduction of co-transcriptional U1 snRNP recruitment to the pre-mRNA at the RPL22B locus. We further demonstrate that the inhibition of RPL22B pre-mRNA splicing contributes to the down-regulation of mature transcript during specific stress conditions, and provide evidence hinting at a regulatory role for this mechanism in conditions of suppressed ribosome biogenesis. These results demonstrate an autoregulatory mechanism that fine-tunes the expression of the Rpl22 protein and by extension Rpl22p paralog composition according to the cellular demands for ribosome biogenesis. PMID:27097027

  16. Metabolic engineering of Saccharomyces cerevisiae for the production of isobutanol and 3-methyl-1-butanol.

    Science.gov (United States)

    Park, Seong-Hee; Kim, Sujin; Hahn, Ji-Sook

    2014-11-01

    Saccharomyces cerevisiae naturally produces small amounts of isobutanol and 3-methyl-1-butanol via Ehrlich pathway from the catabolism of valine and leucine, respectively. In this study, we engineered CEN.PK2-1C, a leucine auxotrophic strain having a LEU2 gene mutation, for the production of isobutanol and 3-methyl-1-butanol. First, ALD6 encoding aldehyde dehydrogenase and BAT1 involved in valine synthesis were deleted to eliminate competing pathways. We also increased transcription of endogenous genes in the valine and leucine biosynthetic pathways by expressing Leu3Δ601, a constitutively active form of Leu3 transcriptional activator. For the production of isobutanol, genes involved in isobutanol production (ILV2, ILV3, ILV5, ARO10, and ADH2) were additionally overexpressed in ald6Δbat1Δ strain expressing LEU3Δ601, resulting in 376.9 mg/L isobutanol production from 100 g/L glucose. To increase 3-methyl-1-butanol production, leucine biosynthetic genes were additionally overexpressed in the final isobutanol-production strain. The resulting strain overexpressing LEU2 and LEU4 (D578Y) , a feedback inhibition-insensitive mutant of LEU4, showed a 34-fold increase in 3-methyl-1-butanol synthesis compared with CEN.PK2-1C control strain, producing 765.7 mg/L 3-methyl-1-butanol. PMID:25280745

  17. Biodiversity of autolytic ability in flocculent Saccharomyces cerevisiae strains suitable for traditional sparkling wine fermentation.

    Science.gov (United States)

    Perpetuini, Giorgia; Di Gianvito, Paola; Arfelli, Giuseppe; Schirone, Maria; Corsetti, Aldo; Tofalo, Rosanna; Suzzi, Giovanna

    2016-07-01

    Yeasts involved in secondary fermentation of traditional sparkling wines should show specific characteristics, such as flocculation capacity and autolysis. Recently it has been postulated that autophagy may contribute to the outcome of autolysis. In this study, 28 flocculent wine Saccahromyces cerevisiae strains characterized by different flocculation degrees were studied for their autolytic and autophagic activities. Autolysis was monitored in synthetic medium through the determination of amino acid nitrogen and total proteins released. At the same time, novel primer sets were developed to determine the expression of the genes ATG1, ATG17 and ATG29. Twelve strains were selected on the basis of their autolytic rate and ATG gene expressions in synthetic medium and were inoculated in a base wine. After 30, 60 and 180 days the autolytic process and ATG gene expressions were evaluated. The obtained data showed that autolysis and ATG gene expressions differed among strains and were independent of the degree of flocculation. This biodiversity could be exploited to select new starter stains to improve sparkling wine production. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26804203

  18. A Genetics Laboratory Module Involving Selection and Identification of Lysine Synthesis Mutants in the Yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Jill B. Keeney

    2009-12-01

    Full Text Available We have developed a laboratory exercise, currently being used with college sophomores, which uses the yeast Saccharomyces cerevisiae to convey the concepts of amino acid biosynthesis, mutation, and gene complementation. In brief, selective medium is used to isolate yeast cells carrying a mutation in the lysine biosynthesis pathway. A spontaneous mutation in any one of three separate genetic loci will allow for growth on selective media; however, the frequency of mutations isolated from each locus differs. Following isolation of a mutated strain, students use complementation analysis to identify which gene contains the mutation. Since the yeast genome has been mapped and sequenced, students with access to the Internet can then research and develop hypotheses to explain the differences in frequencies of mutant genes obtained.

  19. Structure of the Schizosaccharomyces pombe cytochrome c gene.

    OpenAIRE

    Russell, P R; Hall, B. D.

    1982-01-01

    The cytochrome c gene of the fission yeast Schizosaccharomyces pombe has been cloned by using the Saccharomyces cerevisiae iso-1-cytochrome c gene as a molecular hybridization probe. The DNA sequence and the 5' termini of the mRNA transcripts of the gene have been determined. The DNA sequence has confirmed, with two exceptions, the previously determined protein sequence. The nonrandom distribution of silent third base differences which was observed between the two cytochrome c genes of S. cer...

  20. The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining.

    Directory of Open Access Journals (Sweden)

    Mary W Corrigan

    Full Text Available In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC in two closely related species of yeast - Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ, the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%, while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.

  1. Improving L-arabinose utilization of pentose fermenting Saccharomyces cerevisiae cells by heterologous expression of L-arabinose transporting sugar transporters

    Directory of Open Access Journals (Sweden)

    Boles Eckhard

    2011-10-01

    Full Text Available Abstract Background Hydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations. Results Here we described the cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations. Conclusions Using a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations.

  2. Saccharomyces cerevisiae Genetics Predicts Candidate Therapeutic Genetic Interactions at the Mammalian Replication Fork

    Science.gov (United States)

    van Pel, Derek M.; Stirling, Peter C.; Minaker, Sean W.; Sipahimalani, Payal; Hieter, Philip

    2013-01-01

    The concept of synthetic lethality has gained popularity as a rational guide for predicting chemotherapeutic targets based on negative genetic interactions between tumor-specific somatic mutations and a second-site target gene. One hallmark of most cancers that can be exploited by chemotherapies is chromosome instability (CIN). Because chromosome replication, maintenance, and segregation represent conserved and cell-essential processes, they can be modeled effectively in simpler eukaryotes such as Saccharomyces cerevisiae. Here we analyze and extend genetic networks of CIN cancer gene orthologs in yeast, focusing on essential genes. This identifies hub genes and processes that are candidate targets for synthetic lethal killing of cancer cells with defined somatic mutations. One hub process in these networks is DNA replication. A nonessential, fork-associated scaffold, CTF4, is among the most highly connected genes. As Ctf4 lacks enzymatic activity, potentially limiting its development as a therapeutic target, we exploited its function as a physical interaction hub to rationally predict synthetic lethal interactions between essential Ctf4-binding proteins and CIN cancer gene orthologs. We then validated a subset of predicted genetic interactions in a human colorectal cancer cell line, showing that siRNA-mediated knockdown of MRE11A sensitizes cells to depletion of various replication fork-associated proteins. Overall, this work describes methods to identify, predict, and validate in cancer cells candidate therapeutic targets for tumors with known somatic mutations in CIN genes using data from yeast. We affirm not only replication stress but also the targeting of DNA replication fork proteins themselves as potential targets for anticancer therapeutic development. PMID:23390603

  3. A Random Screen Using a Novel Reporter Assay System Reveals a Set of Sequences That Are Preferred as the TATA or TATA-Like Elements in the CYC1 Promoter of Saccharomyces cerevisiae

    OpenAIRE

    Watanabe, Kiyoshi; Yabe, Makoto; Kasahara, Koji; Kokubo, Tetsuro

    2015-01-01

    In Saccharomyces cerevisiae, the core promoters of class II genes contain either TATA or TATA-like elements to direct accurate transcriptional initiation. Genome-wide analyses show that the consensus sequence of the TATA element is TATAWAWR (8 bp), whereas TATA-like elements carry one or two mismatches to this consensus. The fact that several functionally distinct TATA sequences have been identified indicates that these elements may function, at least to some extent, in a gene-specific manner...

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

    Directory of Open Access Journals (Sweden)

    Míriam Cristina Sakuragui Matuo

    2010-09-01

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

  5. Improved gene disruption method for Torulaspora delbrueckii.

    Science.gov (United States)

    Pacheco, Andreia; Almeida, Maria Judite; Sousa, Maria João

    2009-02-01

    PCR-based disruption cassettes are one of the most commonly used strategies for gene targeting in Saccharomyces cerevisiae. The efficiencies of gene disruption using this conventional method are highly variable among species, and often quite low with nonconventional yeasts. Here we describe an improved strategy to obtain deletion mutants in baker's yeast Torulaspora delbrueckii, one of the most abundant non-Saccharomyces species, present in home-made corn and rye bread dough. PMID:19016885

  6. RPG: the Ribosomal Protein Gene database

    OpenAIRE

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and informa...

  7. Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Freeman, Kathryn M. [Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395 (United States); Hoffmann, George R. [Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395 (United States)]. E-mail: ghoffmann@holycross.edu

    2007-03-01

    Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, {beta}-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv{sup +} revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state.

  8. Nucleosome alterations caused by mutations at modifiable histone residues in Saccharomyces cerevisiae

    Science.gov (United States)

    Liu, Hongde; Wang, Pingyan; Liu, Lingjie; Min, Zhu; Luo, Kun; Wan, Yakun

    2015-01-01

    Nucleosome organization exhibits dynamic properties depending on the cell state and environment. Histone proteins, fundamental components of nucleosomes, are subject to chemical modifications on particular residues. We examined the effect of substituting modifiable residues of four core histones with the non-modifiable residue alanine on nucleosome dynamics. We mapped the genome-wide nucleosomes in 22 histone mutants of Saccharomyces cerevisiae and compared the nucleosome alterations relative to the wild-type strain. Our results indicated that different types of histone mutation resulted in different phenotypes and a distinct reorganization of nucleosomes. Nucleosome occupancy was altered at telomeres, but not at centromeres. The first nucleosomes upstream (−1) and downstream (+1) of the transcription start site (TSS) were more dynamic than other nucleosomes. Mutations in histones affected the nucleosome array downstream of the TSS. Highly expressed genes, such as ribosome genes and genes involved in glycolysis, showed increased nucleosome occupancy in many types of histone mutant. In particular, the H3K56A mutant exhibited a high percentage of dynamic genomic regions, decreased nucleosome occupancy at telomeres, increased occupancy at the +1 and −1 nucleosomes, and a slow growth phenotype under stress conditions. Our findings provide insight into the influence of histone mutations on nucleosome dynamics. PMID:26498326

  9. Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, β-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv+ revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state

  10. Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets.

    Science.gov (United States)

    Reimand, Jüri; Vaquerizas, Juan M; Todd, Annabel E; Vilo, Jaak; Luscombe, Nicholas M

    2010-08-01

    Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here, we present a reanalysis of the original data using improved statistical techniques freely available from the BioConductor project. We identify over 100,000 differentially expressed genes-nine times the total reported by Hu et al. We validate the biological significance of these genes by assessing their functions, the occurrence of upstream TF-binding sites, and the prevalence of protein-protein interactions. The reanalysed dataset outperforms the original across all measures, indicating that we have uncovered a vastly expanded list of relevant targets. In summary, this work presents a high-quality reanalysis that maximizes the information contained in the Hu et al. compendium. The dataset is available from ArrayExpress (accession: E-MTAB-109) and it will be invaluable to any scientist interested in the yeast transcriptional regulatory system. PMID:20385592

  11. Crystallization and preliminary crystallographic analysis of Gre2p, an NADP+-dependent alcohol dehydrogenase from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The alcohol dehydrogenase Gre2p from S. cerevisiae catalyses the stereospecific reduction of a variety of different keto compounds and can therefore be applied as a valuable biocatalyst. The crystallization of the complex of Gre2p with NADP+ and its preliminary X-ray analysis are described. Gre2p [Genes de respuesta a estres (stress-response gene)] from Saccharomyces cerevisiae is a monomeric enzyme of 342 amino acids with a molecular weight of 38.1 kDa. The enzyme catalyses both the stereospecific reduction of keto compounds and the oxidation of various hydroxy compounds and alcohols by the simultaneous consumption of the cofactor NADPH and formation of NADP+. Crystals of a Gre2p complex with NADP+ were grown using PEG 8000 as a precipitant. They belong to the monoclinic space group P21. The current diffraction resolution is 3.2 Å. In spite of the monomeric nature of Gre2p in solution, packing and self-rotation calculations revealed the existence of two Gre2p protomers per asymmetric unit related by a twofold noncrystallographic axis

  12. Transcriptomes of a xylose-utilizing industrial flocculating Saccharomyces cerevisiae strain cultured in media containing different sugar sources.

    Science.gov (United States)

    Zeng, Wei-Yi; Tang, Yue-Qin; Gou, Min; Xia, Zi-Yuan; Kida, Kenji

    2016-12-01

    Lignocellulosic hydrolysates used for bioethanol production contain a mixture of sugars, with xylose being the second most abundant after glucose. Since xylose is not a natural substrate for Saccharomyces cerevisiae, recombinant S. cerevisiae strongly prefers glucose over xylose, and the fermentation rate and ethanol yield with xylose are both lower than those with glucose. To determine the molecular basis for glucose and xylose fermentation, we used microarrays to investigate the transcriptional difference of a xylose-utilizing industrial strain cultured in both single sugar media and a mixed sugar medium of glucose and xylose. The transcriptomes were nearly identical between glucose metabolizing cells in the glucose alone medium and those in the glucose fermentation phase in the mixed-sugar medium. Whereas the transcriptomes highly differed between the xylose metabolizing cells in the xylose alone medium and those in the xylose fermentation phase in the mixed sugar medium, and the differences mainly involved sulfur metabolism. When the transcriptional profiles were compared between glucose fermentation state and xylose fermentation state, we found the expression patterns of hexose transporters and glucose signaling pathway differed in response to different sugar sources, and the expression levels of the genes involved in gluconeogenesis, the glyoxylate and tricarboxylic acid cycles and respiration increased with xylose, indicating that the xylose-metabolizing cells had high requirements for maintenance energy and lacked the carbon catabolite repression capability. The effect of carbon catabolite repression by glucose lasted after glucose depletion for specific genes to different extents. PMID:27485516

  13. Transcriptomics in human blood incubation reveals the importance of oxidative stress response in Saccharomyces cerevisiae clinical strains

    Directory of Open Access Journals (Sweden)

    Llopis Silvia

    2012-08-01

    Full Text Available Abstract Background In recent years an increasing number of yeast infections in humans have been related to certain clinical isolates of Saccharomyces cerevisiae. Some clinical strains showed in vivo and in vitro virulence traits and were able to cause death in mice whereas other clinical strains were avirulent. Results In this work, we studied the transcriptional profiles of two S. cerevisiae clinical strains showing virulent traits and two control non-virulent strains during a blood incubation model and detected a specific transcriptional response of clinical strains. This response involves an mRNA levels increase of amino acid biosynthesis genes and especially oxidative stress related genes. We observed that the clinical strains were more resistant to reactive oxygen species in vitro. In addition, blood survival of clinical isolates was high, reaching similar levels to pathogenic Candida albicans strain. Furthermore, a virulent strain mutant in the transcription factor Yap1p, unable to grow in oxidative stress conditions, presented decreased survival levels in human blood compared with the wild type or YAP1 reconstituted strain. Conclusions Our data suggest that this enhanced oxidative stress response in virulent clinical isolates, presumably induced in response to oxidative burst from host defense cells, is important to increase survival in human blood and can help to infect and even produce death in mice models.

  14. An organic acid-tolerant HAA1-overexpression mutant of an industrial bioethanol strain of Saccharomyces cerevisiae and its application to the production of bioethanol from sugarcane molasses.

    Science.gov (United States)

    Inaba, Takuya; Watanabe, Daisuke; Yoshiyama, Yoko; Tanaka, Koichi; Ogawa, Jun; Takagi, Hiroshi; Shimoi, Hitoshi; Shima, Jun

    2013-01-01

    Bacterial contamination is known as a major cause of the reduction in ethanol yield during bioethanol production by Saccharomyces cerevisiae. Acetate is an effective agent for the prevention of bacterial contamination, but it negatively affects the fermentation ability of S. cerevisiae. We have proposed that the combined use of organic acids including acetate and lactate and yeast strains tolerant to organic acids may be effective for the elimination of principally lactic acid bacterial (LAB) contamination. In a previous study employing laboratory S. cerevisiae strains, we showed that overexpression of the HAA1 gene, which encodes a transcriptional activator, could be a useful molecular breeding method for acetate-tolerant yeast strains. In the present study, we constructed a HAA1-overexpressing diploid strain (MATa/α, named ER HAA1-OP) derived from the industrial bioethanol strain Ethanol Red (ER). ER HAA1-OP showed tolerance not only to acetate but also to lactate, and this tolerance was dependent on the increased expression of HAA1 gene. The ethanol production ability of ER HAA1-OP was almost equivalent to that of the parent strain during the bioethanol production process from sugarcane molasses in the absence of acetate. The addition of acetate at 0.5% (w/v, pH 4.5) inhibited the fermentation ability of the parent strain, but such an inhibition was not observed in the ethanol production process using ER HAA1-OP. PMID:24373204

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

    Directory of Open Access Journals (Sweden)

    Yao Xiao-Jian

    2004-08-01

    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 α-helix I (17–33 and α-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 α-helix I (17–33 and α-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.

  16. Investigation of nutrient sensing in the yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine

    2006-01-01

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

  17. Membrane Protein Production in the Yeast, S. cerevisiae.

    Science.gov (United States)

    Cartwright, Stephanie P; Mikaliunaite, Lina; Bill, Roslyn M

    2016-01-01

    The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca(2+)-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes. PMID:27485327

  18. Advanced biofuel production by the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

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

    2013-06-01

    Replacement of conventional transportation fuels with biofuels will require production of compounds that can cover the complete fuel spectrum, ranging from gasoline to kerosene. Advanced biofuels are expected to play an important role in replacing fossil fuels because they have improved properties compared with ethanol and some of these may have the energy density required for use in heavy duty vehicles, ships, and aviation. Moreover, advanced biofuels can be used as drop-in fuels in existing internal combustion engines. The yeast cell factory Saccharomyces cerevisiae can be turned into a producer of higher alcohols (1-butanol and isobutanol), sesquiterpenes (farnesene and bisabolene), and fatty acid ethyl esters (biodiesel), and here we discusses progress in metabolic engineering of S. cerevisiae for production of these advanced biofuels. PMID:23628723

  19. Introduction of a bacterial acetyl-CoA synthesis pathway improves lactic acid production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Song, Ji-Yoon; Park, Joon-Song; Kang, Chang Duk; Cho, Hwa-Young; Yang, Dongsik; Lee, Seunghyun; Cho, Kwang Myung

    2016-05-01

    Acid-tolerant Saccharomyces cerevisiae was engineered to produce lactic acid by expressing heterologous lactate dehydrogenase (LDH) genes, while attenuating several key pathway genes, including glycerol-3-phosphate dehydrogenase1 (GPD1) and cytochrome-c oxidoreductase2 (CYB2). In order to increase the yield of lactic acid further, the ethanol production pathway was attenuated by disrupting the pyruvate decarboxylase1 (PDC1) and alcohol dehydrogenase1 (ADH1) genes. Despite an increase in lactic acid yield, severe reduction of the growth rate and glucose consumption rate owing to the absence of ADH1 caused a considerable decrease in the overall productivity. In Δadh1 cells, the levels of acetyl-CoA, a key precursor for biologically applicable components, could be insufficient for normal cell growth. To increase the cellular supply of acetyl-CoA, we introduced bacterial acetylating acetaldehyde dehydrogenase (A-ALD) enzyme (EC 1.2.1.10) genes into the lactic acid-producing S. cerevisiae. Escherichia coli-derived A-ALD genes, mhpF and eutE, were expressed and effectively complemented the attenuated acetaldehyde dehydrogenase (ALD)/acetyl-CoA synthetase (ACS) pathway in the yeast. The engineered strain, possessing a heterologous acetyl-CoA synthetic pathway, showed an increased glucose consumption rate and higher productivity of lactic acid fermentation. The production of lactic acid was reached at 142g/L with production yield of 0.89g/g and productivity of 3.55gL(-1)h(-1) under fed-batch fermentation in bioreactor. This study demonstrates a novel approach that improves productivity of lactic acid by metabolic engineering of the acetyl-CoA biosynthetic pathway in yeast. PMID:26384570

  20. L-Histidine Inhibits Biofilm Formation and FLO11-Associated Phenotypes in Saccharomyces cerevisiae Flor Yeasts

    OpenAIRE

    Marc Bou Zeidan; Giacomo Zara; Carlo Viti; Francesca Decorosi; Ilaria Mannazzu; Marilena Budroni; Luciana Giovannetti; Severino Zara

    2014-01-01

    Flor yeasts of Saccharomyces cerevisiae have an innate diversity of FLO11 which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling FLO11 alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce FLO11p. The flor strains generally metabolized amino acids and ...

  1. L-Histidine Inhibits Biofilm Formation and FLO11-Associated Phenotypes in Saccharomyces cerevisiae Flor Yeasts

    OpenAIRE

    Marc Bou Zeidan; Giacomo Zara; Carlo Viti; Francesca Decorosi; Ilaria Mannazzu; Marilena Budroni; Luciana Giovannetti; Severino Zara

    2014-01-01

    Flor yeasts of Saccharomyces cerevisiae have an innate diversity of Flo11p which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling Flo11p alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce Flo11p. The flor strains generally metabolized amino acids and dipeptides...

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

    OpenAIRE

    Bajaj, Bijender K.; S Sharma

    2010-01-01

    Saccharomyces cerevisiae HAU-1, a time tested industrial yeast possesses most of the desirable fermentation characteristics like fast growth and fermentation rate, osmotolerance, high ethanol tolerance, ability to ferment molasses, and to ferment at elevated temperatures etc. However, this yeast was found to be sensitive against the killer strains of Saccharomyces cerevisiae. In the present study, killer trait was introduced into Saccharomyces cerevisiae HAU-1 by protoplast fusion with Saccha...

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

    OpenAIRE

    Bajaj, Bijender K.; S Sharma

    2010-01-01

    Saccharomyces cerevisiae HAU-1, a time tested industrial yeast possesses most of the desirable fermentation characteristics like fast growth and fermentation rate, osmotolerance, high ethanol tolerance, ability to ferment molasses, and to ferment at elevated temperatures etc. However, this yeast was found to be sensitive against the killer strains of Saccharomyces cerevisiae. In the present study, killer trait was introduced into Saccharomyces cerevisiae HAU-1 by protoplast fusion with Saccha...

  4. Translation and stability of an Escherichia coli beta-galactosidase mRNA expressed under the control of pyruvate kinase sequences in Saccharomyces cerevisiae.

    OpenAIRE

    Purvis, I J; Loughlin, L; Bettany, A J; Brown, A. J.

    1987-01-01

    Plasmids were assembled in which the coding region of the pyruvate kinase (PYK) gene of Saccharomyces cerevisiae was replaced by that of the B-galactosidase (LacZ) gene from Escherichia coli. Analysis of the resultant, chimaeric transcripts from low copy number, centromeric plasmids indicated that this substitution caused a dramatic reduction in the steady-state level of the messenger RNA (mRNA). This fluctuation cannot be wholly accounted for by the 2-fold decrease in mRNA stability observed...

  5. The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

    OpenAIRE

    Dang, V D; Bohn, C.; Bolotin-Fukuhara, M.; Daignan-Fornier, B

    1996-01-01

    In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on a...

  6. A linear discrete dynamic system model for temporal gene interaction and regulatory network influence in response to bioethanol conversion inhibitor HMF for ethanologenic yeast

    Science.gov (United States)

    A linear discrete dynamic system model is constructed to represent the temporal interactions among significantly expressed genes in response to bioethanol conversion inhibitor 5-hydroxymethylfurfural for ethanologenic yeast Saccharomyces cerevisiae. This study identifies the most significant linear...

  7. Studies of the Saccharomyces cerevisiae Cultivation under Oscillatory Mixing Conditions

    OpenAIRE

    M?ris Rikmanis; Stoyan Tzonkov; Uldis Viesturs; Andr?js B?rzi??

    2005-01-01

    Saccharomyces cerevisiae was cultivated under non-aerated conditions in a 5 l laboratory bioreactor. Using the experimental data and the regression analysis method, some mathematical correlations for stirrer rotational speed oscillation frequency and the reaction of the yeast were established. It has been found that different growth parameters are influenced variously by stirrer rotational speed and stirrer rotational speed oscillation frequency. Stirring oscillations can be among the methods...

  8. Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

    OpenAIRE

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

    1988-01-01

    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...

  9. Applied systems biology - vanillin production in Saccharomyces cerevisiae

    OpenAIRE

    Strucko, Tomas; Eriksen, Carsten; Nielsen, J.; Mortensen, Uffe Hasbro

    2012-01-01

    Vanillin is the most important aroma compound based on market value, and natural vanillin is extracted from the cured seed pods of the Vanilla orchid. Most of the world’s vanillin, however, is obtained by chemical synthesis from petrochemicals or wood pulp lignins. As an alternative, de novo biosynthesis of vanillin in baker’s yeast Saccharomyces cerevisiae was recently demonstrated by successfully introducing the metabolic pathway for vanillin production in yeast. Nevertheless, the amount of...

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

    OpenAIRE

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

    2014-01-01

    Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. ...

  11. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    OpenAIRE

    van der Rest, M E; Kamminga, A H; Nakano, A.; Anraku, Y; Poolman, B; Konings, W N

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow o...

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

    OpenAIRE

    VANDERREST, ME; KAMMINGA, AH; Nakano, A.; Anraku, Y; Poolman, B; KONINGS, WN

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive pur of lipids fi om these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow o...

  13. Magnetically altered ethanol fermentation capacity of Saccharomyces cerevisiae

    OpenAIRE

    Galonja-Corghill Tamara; Kostadinović Ljiljana M.; Bojat Nenad C.

    2009-01-01

    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...

  14. Mead production: selection and characterization assays of Saccharomyces cerevisiae

    OpenAIRE

    de Pereira, Ana Paula; Dias, Teresa; Andrade, João Verdial; Ramalhosa, Elsa; Mendes-Ferreira, Ana; Mendes-Faia, Arlete; Leticia M. Estevinho

    2009-01-01

    Mead is a traditional alcoholic drink which results from the fermentation of diluted honey. Yeasts used in mead production are, usually, wine Saccharomyces cerevisiae strains. Most of these yeasts are not adapted to the conditions of mead production namely, high sugar levels, low pH values and reduced nitrogen concentrations. The inability of yeast strains to respond and adapt to unfavorable stressful growth conditions, leads to several problems, such as lack of uniformity of the final ...

  15. Expression of Pneumocystis jirovecii Major Surface Glycoprotein in Saccharomyces cerevisiae

    OpenAIRE

    Kutty, Geetha; England, Katherine J.; Kovacs, Joseph A.

    2013-01-01

    The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis orga...

  16. Comprehensive polymorphism survey elucidates population structure of S. cerevisiae

    OpenAIRE

    Schacherer, Joseph; Shapiro, Joshua A.; Ruderfer, Douglas M.; Kruglyak, Leonid

    2009-01-01

    Comprehensive identification of polymorphisms among individuals within a species is essential both for studying the genetic basis of phenotypic differences and for elucidating the evolutionary history of the species. Large-scale polymorphism surveys have recently been reported for human1, mouse2, and Arabidopsis thaliana3. Here we report a nucleotide-level survey of genome variation in a diverse collection of 63 S. cerevisiae strains sampled from different ecological niches (beer, bread, vine...

  17. Adaption of Saccharomyces cerevisiae expressing a heterologous protein

    DEFF Research Database (Denmark)

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

    2008-01-01

    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 ...... 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....

  18. Hydrogen peroxide removal with magnetically responsive Saccharomyces cerevisiae cells

    Czech Academy of Sciences Publication Activity Database

    Šafařík, Ivo; Maděrová, Zdeňka; Šafaříková, Miroslava

    2008-01-01

    Roč. 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

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

    Directory of Open Access Journals (Sweden)

    HASSAN TAJIK

    2006-09-01

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

  20. A novel selection system for chromosome translocations in Saccharomyces cerevisiae.

    OpenAIRE

    Tennyson, Rachel B; Ebran, Nathalie; Herrera, Anissa E; Lindsley, Janet E.

    2002-01-01

    Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors. While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements. This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae. A translocation YAC was created that contains the breakpoint cluster regi...