Spelling suggestions: "subject:"S. cerevisiae"" "subject:"S. serevisiae""
1 |
The SEC1 and SEC5 genes of Saccharomyces cerevisiaeEgerton, Mark January 1988 (has links)
No description available.
|
2 |
Genetic engineering of S. cerevisiae to confer xylose metabolism with a view to biofuel productionAhmed, Hassan Zubair January 2016 (has links)
Xylose is a pentose sugar that forms a substantial proportion of the monosaccharides released from lignocellulosic biomass after hydrolysis. Therefore, the economic and commercial viability of biofuel production from lignocellulosic material via microbial fermentation relies upon maximising the metabolism of monosaccharides like xylose. As such, second generation biofuels are becoming a focus of biofuel innovation because of the depleting fossil fuel reserves and increasing levels of carbon emissions. Even so the majority of current biofuel production uses glucose as a carbon source from corn, wheat or sugar cane. This conflicts with food production and has prompted the food versus fuel debate. The introduction of xylose metabolising pathways into current biofuel production microorganisms like yeast, which cannot utilize xylose, would allow xylose use from lignocellulosic biomass. The xylose-reductase (XR) pathway from fungi utilise the xylose reductase, xylose dehydrogenase and xylulokinase genes, whereas the xylose isomerase (XI) pathway from bacteria consists of xylose isomerase and xylulokinase. In this project plasmid constructs containing the two pathways were successfully introduced into yeast. The genes were further integrated into specific chromosomal sites for comparison. Depending on the type of media used, some xylose uptake and ethanol production could be demonstrated for some of these strains, but overall levels of xylose use did not reach a level likely to impact upon commercial biofuel production. As a result, several strategies were investigated with a view to increasing xylose metabolism and ethanol production from the strains. Alterations were made to the cassette design for the xylose enzyme genes, such as gene promoter replacement or removal of the epitope tag. A pentose specific transporter, GXF1, from Candida tropicalis was also introduced. However, none of these strategies improved xylose use. A further approach, which led to minor increases in xylose metabolism, was deletion of the PHO13 gene, which is thought to impact upon expression of pentose phosphate genes. One further goal of this work was to investigate whether xylose metabolism could be connected to butanol production, as butanol has superior properties as a biofuel in yeast. Unfortunately, butanol was not detected from heterologous butanol producing strains bearing the plasmid based XI pathway, presumably because the growth and health of these strains was quite poor. Overall this project has demonstrated that S. cerevisiae is able to metabolise hemicellulosic xylose to ethanol using heterologous pathways, however, the very low levels generated mean that a great deal of genetic and metabolic engineering would be required for optimisation of biofuel production for commercial viability.
|
3 |
Chemical genetic screen for inhibitors of human telomeraseWong, Lai Hong January 2013 (has links)
There remains a pressing need for the development of effective drugs that meet the clinical needs for cancer treatment, and inhibition of telomere length maintenance by disrupting human telomerase is a proven and tractable target for suppression of cancer cell growth. In response to the lack of currently available small molecules with efficacy against human telomerase, we developed a genetically and chemically tractable cell-based system in which S. cerevisiae is used to streamline the search for novel human telomerase inhibitors. Our results confirmed that yeast cell growth was rapidly inhibited upon induction of functional human telomerase at the telomere. This inducible growth arrest was used as a read-out for a high-throughput chemical screen for human telomerase inhibitors based on their ability to restore growth in the yeast system. From a library consisting of small, bioactive and cell-permeable compounds of diverse structure, we identified three novel “drug-like” compounds that inhibited the activity of native and recombinant telomerase complexes in vitro. “Validation assays” also confirmed the novel inhibitors were free of uncharacterized adverse effects against yeast and human cell models, thus confirming the specificity of these novel inhibitors against human telomerase target. This surrogate yeast model has therefore proven to be a cost-effective alternative to accelerate the search for human telomerase inhibitors, which we hope will serve to streamline the identification of further lead compounds effective against human cancer.
|
4 |
C. elegans MAP Kinase Mutants Show Enhanced Susceptibility to Infection by the Yeast S. cerevisiaeYun, Meijiang 14 May 2010 (has links)
C. elegans is as an extremely powerful model for the study of innate immunity. MAP kinase signaling pathways in C. elegans are involved in the response of C. elegans to infection by pathogenic bacteria. The yeast S. cerevisiae can infect C. elegans, producing pathogenic effects. In this project, we tested whether several MAP kinase pathways are important for C. elegans¡¯ resistance to yeast infection. We tested members of several MAP kinase pathways including tir-1, nsy-1, sek-1 and pmk-1 in the p38 pathway, mek-1, jnk-1 and kgb-1 in JNK pathway and mek-2 and mpk-1 in the ERK pathway. We used survival assays to compare the responses of mutants of components of these pathways to the control responses of wild-type C. elegans. In the survival assay, we found that mutants in all three MAP kinase pathways showed a decreased survival relative to wild type; therefore all three pathways are important for innate immunity against the yeast pathogen. With respect to the p38 pathway, mutations affected survival but not the deformed anal region (Dar) phenotype, a putative defensive response induced by yeast in wild-type C. elegans. This indicates that for the p38 pathway, survival depends on some other immune response besides Dar. Finally, we hypothesize that cross talk occurs between p38 and JNK MAPK pathways in the C. elegans immune responses.
|
5 |
Evaluation of five saccharomyces cerevisiae promoter during growth on xyloseMande, Livhuwani January 2015 (has links)
Thesis (MSc. (Microbiology)) -- University of Limpopo, 2015 / S. cerevisiae has many properties which have made it the preferred host for the expression and production of a number of recombinant proteins. Xylose is the second most abundant sugar in nature and S. cerevisiae has been engineered to grow on this abundant sugar. Therefore, identifying S. cerevisiae promoters that are strongly induced during growth on xylose will be important in the production of recombinant proteins for the biofuel and other industries. Since xylose is not a native substrate for S. cerevisiae, it is not known how S. cerevisiae promoters will react during growth on xylose. The objective of the study was to evaluate the expression of a reporter gene, the Trichoderma reesei xylanase 2 (XYN2), under the control of five commonly used expression promoters (GPD3, ENO2, PGK1, ADH2 and YG100). Five episomal expression vectors were constructed for this purpose. These vectors were transformed to a recombinant xylose utilizing S. cerevisiae. Xylanase activity assays were used to determine the expression level from each of these promoters. The PGK1 promoter was observed to be the strongest promoter with average activity/OD of 130 nkat/ml/OD on both xylose and glucose. The GPD3 promoter showed the highest average activity/OD of 150 nkat/ml, but xylanase was only produced during growth on glucose. The data presented show that xylose is not a better carbon source than glucose for recombinant protein production in terms of the S. cerevisie promoters evaluated. Further research is required to obtain a yeast strain that grows well on xylose and promoters that show higher level on protein production.
Keywords: xylose, promoter, expression, recombinant, S. cerevisiae
|
6 |
Produção biotecnológica de L-asparaginase (ASP1) de Saccharomyces cerevisiae em sistema de expressão heterólogo Pichia pastoris / Biotechnological production of L-asparaginase (ASP1) of Saccharomyces cerevisiae in a heterologous expression system Pichia pastoris.Divino, Bruna de Souza 24 November 2015 (has links)
Utilizada amplamente no tratamento de Leucemias Linfoblásticas Agudas, a L-Asparaginase é uma enzima que atua diminuindo a concentração de L-asparagina livre no plasma e, dessa forma, impede a proliferação de células cancerígenas. Isso ocorre porque tais células, ao contrário das células saudáveis, não conseguem sintetizar o aminoácido L-asparagina que necessitam para a síntese proteica por não possuírem a enzima asparagina sintetase devido a um silenciamento genético. A L-Asparaginase utilizada no Brasil era importada e produzida em bactéria, o que gerava problemas com custo e abastecimento, além de causar algumas reações imunológicas aos usuários. Isso impulsiona a encontrar alternativas para produção nacional de tal enzima e, se possível, com menor potencial alergênico. Para isso, foi estudada a produção da enzima L-Asparaginase do gene ASP1 de Saccharomyces cerevisiae em sistema de expressão heteróloga Pichia pastoris através da clonagem do gene sintético (ASP1), com códons otimizados para expressão em P. pastoris, estudo este nunca antes realizado ao que se sabe. Além disso, a produção da enzima foi realizada com auxílio de um planejamento experimental fatorial em agitador orbital levando em conta as variáveis: pH, temperatura e concentração do indutor. A melhor condição encontrada dentre as estudadas neste trabalho foi, a temperatura de 20°C, pH 6,0 e concentração de indutor de 1,5% que alcançou uma atividade de 6,9 U/g de célula e apesar do esforços, o peptídeo sinal utilizado (alfa da S. cerevisiae) não foi capaz de promover a secreção da enzima para o meio extracelular.Deve-se salientar que a realização de um maior número de ensaios através de um planejamento experimental fatorial fracionário para encontrar uma região de rendimentos máximos talvez melhore o ajuste dos modelos mostrados. Este trabalho foi a primeira tentativa de expressão do gene ASP1 de S. cerevisiae em Pichia pastoris até o momento, e pretende-se aprofundar mais este estudo. / Used widely in the treatment of Acute lymphoblastic leukemia, L-Asparaginase is an enzyme that acts by decreasing the concentration of L-asparagine free in the plasma and thus prevents the proliferation of cancer cells. This is because such cells, in contrast to healthy cells can not synthesize L-asparagine amino acid for protein synthesis need not possess the enzyme asparagine synthetase due to a gene silencing. The L-asparaginase used in Brazil was imported and produced in bacteria, which caused problems with cost and supply, as well as cause some immune responses to users. This boosts find alternatives to domestic production of this enzyme and, if possible, less allergenic potential. For this, the production of L-asparaginase enzyme ASP1 Saccharomyces cerevisiae gene heterologous to Pichia pastoris expression system was investigated by cloning the synthetic gene (ASP1), with codons optimized for expression in P. pastoris, this study never before achieved to what is known. Furthermore, the enzyme production was performed with the aid of a factorial design in an orbital shaker taking into account the following variables: pH, temperature and concentration of inducer. The best condition found among those studied in this work was a temperature of 20 ° C, pH 6.0 and concentration of 1.5% inductor attained an activity of 6.9 U / g cell and despite the efforts, used signal peptide (S. cerevisiae alpha) was not able to promote the secretion of the enzyme into the medium extracelular.Deve be noted that the achievement of a greater number of tests using a fractional factorial design yields to find a region maximum might improve the fit of the models shown. This work was the first attempt to ASP1 gene expression of S. cerevisiae in Pichia pastoris to date, and is intended to further deepen this study.
|
7 |
Produção biotecnológica de L-asparaginase (ASP1) de Saccharomyces cerevisiae em sistema de expressão heterólogo Pichia pastoris / Biotechnological production of L-asparaginase (ASP1) of Saccharomyces cerevisiae in a heterologous expression system Pichia pastoris.Bruna de Souza Divino 24 November 2015 (has links)
Utilizada amplamente no tratamento de Leucemias Linfoblásticas Agudas, a L-Asparaginase é uma enzima que atua diminuindo a concentração de L-asparagina livre no plasma e, dessa forma, impede a proliferação de células cancerígenas. Isso ocorre porque tais células, ao contrário das células saudáveis, não conseguem sintetizar o aminoácido L-asparagina que necessitam para a síntese proteica por não possuírem a enzima asparagina sintetase devido a um silenciamento genético. A L-Asparaginase utilizada no Brasil era importada e produzida em bactéria, o que gerava problemas com custo e abastecimento, além de causar algumas reações imunológicas aos usuários. Isso impulsiona a encontrar alternativas para produção nacional de tal enzima e, se possível, com menor potencial alergênico. Para isso, foi estudada a produção da enzima L-Asparaginase do gene ASP1 de Saccharomyces cerevisiae em sistema de expressão heteróloga Pichia pastoris através da clonagem do gene sintético (ASP1), com códons otimizados para expressão em P. pastoris, estudo este nunca antes realizado ao que se sabe. Além disso, a produção da enzima foi realizada com auxílio de um planejamento experimental fatorial em agitador orbital levando em conta as variáveis: pH, temperatura e concentração do indutor. A melhor condição encontrada dentre as estudadas neste trabalho foi, a temperatura de 20°C, pH 6,0 e concentração de indutor de 1,5% que alcançou uma atividade de 6,9 U/g de célula e apesar do esforços, o peptídeo sinal utilizado (alfa da S. cerevisiae) não foi capaz de promover a secreção da enzima para o meio extracelular.Deve-se salientar que a realização de um maior número de ensaios através de um planejamento experimental fatorial fracionário para encontrar uma região de rendimentos máximos talvez melhore o ajuste dos modelos mostrados. Este trabalho foi a primeira tentativa de expressão do gene ASP1 de S. cerevisiae em Pichia pastoris até o momento, e pretende-se aprofundar mais este estudo. / Used widely in the treatment of Acute lymphoblastic leukemia, L-Asparaginase is an enzyme that acts by decreasing the concentration of L-asparagine free in the plasma and thus prevents the proliferation of cancer cells. This is because such cells, in contrast to healthy cells can not synthesize L-asparagine amino acid for protein synthesis need not possess the enzyme asparagine synthetase due to a gene silencing. The L-asparaginase used in Brazil was imported and produced in bacteria, which caused problems with cost and supply, as well as cause some immune responses to users. This boosts find alternatives to domestic production of this enzyme and, if possible, less allergenic potential. For this, the production of L-asparaginase enzyme ASP1 Saccharomyces cerevisiae gene heterologous to Pichia pastoris expression system was investigated by cloning the synthetic gene (ASP1), with codons optimized for expression in P. pastoris, this study never before achieved to what is known. Furthermore, the enzyme production was performed with the aid of a factorial design in an orbital shaker taking into account the following variables: pH, temperature and concentration of inducer. The best condition found among those studied in this work was a temperature of 20 ° C, pH 6.0 and concentration of 1.5% inductor attained an activity of 6.9 U / g cell and despite the efforts, used signal peptide (S. cerevisiae alpha) was not able to promote the secretion of the enzyme into the medium extracelular.Deve be noted that the achievement of a greater number of tests using a fractional factorial design yields to find a region maximum might improve the fit of the models shown. This work was the first attempt to ASP1 gene expression of S. cerevisiae in Pichia pastoris to date, and is intended to further deepen this study.
|
8 |
Mise en évidence d’une nouvelle voie impliquée dans l’homéostasie de la taille cellulaire chez S. cerevisiae / A new pathway involved in cell size homeostasis in yeast S. cerevisiaeMoretto, Fabien 17 December 2012 (has links)
L’homéostasie de la taille des cellules implique l’existence de mécanismes capables de coordonner la croissance (l’augmentation du volume) avec la prolifération (l’augmentation du nombre de cellules). Il est clairement établi que la taille des cellules est affectée par la disponibilité en nutriment du milieu de culture et par la ploïdie, mais les mécanismes sous-jacents demeurent inconnus. Une étude à l’échelle du génome réalisée chez la levure par l’équipe de M. Tyers a révélée que l’inactivation d’environ 400 gènes conduisait à un volume cellulaire moyen significativement différent de celui de la souche sauvage isogénique. Le contrôle de la taille des cellules est ainsi une situation intéressante dans laquelle de nombreux loci contribuent à un caractère quantitatif complexe. La plupart de ces loci demeurant orphelin de voie de signalisation distincte, leur influence respective reste à élucider. Nous avons commencé cette étude en partant de l’observation qu’un mutant sir2 présentait un volume cellulaire augmenté. De manière cohérente, un traitement au nicotinamide (Nam), un inhibiteur de Sir2, reproduit le défaut de taille de sir2 par un mécanisme dépendant de Sir2p. Nous avons alors pu, par une approche d’épistasie chimique, identifier 22 mutants non affectés par le traitement de la Nam, parmi ~200 mutants de petite taille. De manière surprenante, 16 de ces 22 mutants de taille insensibles à la NAM, sont affectés dans la biogenèse de la grande sous unité du ribosome (60S). Une drogue capable de bloquer spécifiquement la biogenèse tardive de la 60S, la diazaborine, mime le phénotype de taille des mutants de la 60S, produisant des cellules sauvages plus petites. Un ensemble de ~200 mutants de grande taille a été traité à la diazaborine et leur volume mesuré. Cette approche chimiogénétique nous a permis d’identifier 31 mutants insensibles à la diazaborine, incluant swi4 et swi6, deux régulateurs majeurs du cycle cellulaire, critiques pour le contrôle de la taille des cellules. Ces résultats furent confirmés par la construction de double mutants. Ce travail montre qu’il est possible d’organiser des mutants de taille au sein de voies spécifiques et de définir des relations d’épistasie claires entre eux. Nos données indiquent que le contrôle de la taille par cette voie “Sir2-60S” est indépendant des effets de la ploïdie et du contrôle nutritionnel sur la taille des cellules. / Cell size homeostasis implies that specific mechanisms are devoted to coordinating growth and proliferation. It is well established that cell size is affected by nutrient availability and ploidy but the underlying mechanisms are not elucidated. A genome wide search for yeast mutants affected for cell volume homeostasis, conducted in the Tyers’lab, revealed that the inactivation of about 400 genes leads to a median cell volume diverging from the isogenic wild-type. The cell size control process is thus a very interesting situation where multiple loci contributing to a complex quantitative trait have been identified but their organisation into distinct pathways and their respective influence remain largely to be elucidated. To address this issue, we started from the observation that a sir2 mutant shows an increased cell size. Consistently, nicotinamide (NAM), a Sir2 inhibitor, mimics the sir2 size defect in a Sir2p-dependent manner. This allowed us to identify among ~200 small size mutants, 22 mutants that were clearly not affected by NAM treatment. Strikingly, 16 out of the 22 NAM unresponsive mutants affected biogenesis of the large ribosomal subunit (named 60S below). Consistently, diazaborine, a drug that blocks the large ribosomal subunit assembly and therefore mimics 60S mutants, rendered wild-type yeast cells smaller. A set of ~200 large mutants were treated with diazaborine and their cell volume was measured. This chemogenetics approach allowed us to identify 31 diazaborine-unresponsive mutants, including swi4 and swi6, two major cell cycle regulators that are critical for cell size control. These results were confirmed by constructing double mutants. This work shows that it is possible to organize cell size mutants in specific pathways and to define clear epistasis relationships between them. Our data indicate that the control of cell size by the “Sir2-60S” pathway is independent of both the ploidy and the nutritional control of cell size.
|
9 |
Développement de nouvelles souches de levures œnologiques à faible rendement en éthanol par évolution adaptative / Development of new oenological yeast strains with reduced ethanol yield, by using a combination of various approaches based on adaptative evolutionTilloy, Valentin 23 April 2013 (has links)
Il existe une forte demande de l'industrie pour des technologies permettant de réduire la teneur en alcool des vins. Bien que des levures à faible rendement alcool aient été développées par ingénierie génétique, les approches non-OGM sont aujourd'hui largement privilégiées. Nous avons mis en œuvre et comparé différentes stratégies d'évolution adaptative, à partir d'une souche œnologique commerciale, afin de réorienter le flux carboné vers la formation de glycérol aux dépens de l'éthanol. Après 200 générations en conditions de stress salin, nous avons obtenu des souches évoluées présentant une augmentation de la production de glycérol de 50 à 70 %, capables de diminuer de 0,45 à 0,80 % (v/v) la teneur en alcool de vins naturels ou synthétiques. Cette réorientation s'accompagne d'une accumulation de succinate et de 2,3-butanediol et d'une réduction de la vitesse fermentaire. Les mutants ont une survie accrue en conditions de stress salin et carence en glucose. Afin d'identifier les mécanismes sous-jacents, nous avons réalisé des analyses du transcriptome, du métabolome (exo- et endo-) et du génome des souches évoluées. Nous avons montré que le phénotype des mutants n'est pas dû à une dérégulation ou à des mutations des gènes de la voie de synthèse du glycérol mais à de larges modifications du métabolisme carboné, énergétique et redox. Le génome des souches évoluées présente des pertes d'hétérozygotie qui pourraient contribuer au phénotype observé. L'étude génétique d'une souche évoluée montre une origine multigénique des traits métaboliques. Une analyse de cartographie de QTL en utilisant une approche « bulk sequencing » a été initiée pour identifier les mutations impliquées dans le caractère fort glycérol/faible éthanol. Ces travaux ont ainsi permis de développer et caractériser des souches œnologiques faibles productrices d'alcool et de fournir un cadre pour l'identification des bases moléculaires impliquées.Mots-clefs : S. cerevisiae, fermentation œnologique, évolution adaptative, éthanol, glycérol, transcriptome, métabolome, génome. / There is a strong demand from the industry for technologies to reduce the alcohol content of wine. Although low-alcohol yield yeasts have been developed by genetic engineering, GMO-free approaches are now widely preferred. We have implemented and compared different strategies for adaptive evolution to redirect the carbon flux towards glycerol formation at the expense of ethanol. After 200 generations salt stress conditions, we obtained evolved strains with glycerol production increased by 50 to 70%, able to decrease from 0.45 to 0.80% (v/v) the alcohol content of natural or synthetic wines. This shift is accompanied by an accumulation of succinate and 2,3-butanediol and a reduced fermentation rate. Mutants also exhibit a better survival under salt stress and glucose restriction conditions. To identify the underlying mechanisms, we analysed the transcriptome, metabolome (endo- and exo-) and genome of the evolved strains. We showed that the evolved phenotype is not due to deregulation or mutations of genes of involved in the glycerol synthesis pathway but to major changes in carbon, energy and redox metabolism. The genome of the evolved strains revealed loss of heterozygosity which could contribute to the observed phenotype. The genetic study of an evolved strain shows that the metabolic traits are under multigenic control. A QTL mapping analysis using a "bulk sequencing" approach was initiated to identify mutations involved in the high glycerol/low ethanol trait. This work has enabled the development and characterization of low alcohol wine yeast strains and has provided a framework for the identification of the underlying molecular bases.Key-words: S. cerevisiae, wine fermentation, adaptative evolution, ethanol, glycerol, transcriptome, metabolome, genome.
|
10 |
Identification des bases moléculaires de propriétés technologiques de levures oenologiques / Identification of molecular basis of technological properties of wine yeastsNoble, Jessica 30 June 2011 (has links)
Les bases moléculaires responsables des propriétés technologiques des levures œnologiques sont pas ou peu connues. Or, ces connaissances sont nécessaires pour mettre en œuvre des stratégies d'amélioration génétique des capacités fermentaires ou de l'impact organoleptique des levures par croisements et une meilleure exploitation de la biodiversité. Ces travaux de thèse ont visé à l'identification des bases génétiques de plusieurs propriétés d'intérêt, telles que la production de sulfites, la capacité fermentaire en milieu carencé en azote ou la formation d'esters. Une approche de recherche de QTL a été mise en œuvre en s'appuyant sur la création d'une population de ségrégants méiotiques issus du croisement de deux souches œnologiques aux caractéristiques contrastées. Une caractérisation des phénotypes des ségrégants associé à leur génotypage a permis d'identifier des QTL pour les différents traits étudiés. L'implication d'une région du génome dans le contrôle de la voie d'assimilation des sulfates a été démontrée. Ce locus est responsable de la variation phénotypique de plusieurs métabolites de cette voie et de métabolites indirectement connectés. D'autres QTL ont également pu être mis en évidence pour les capacités fermentaires sur un milieu carencé en azote et la production de différents composés volatils. Leur analyse a permis d'identifier plusieurs gènes candidats dont les fonctions sont liées directement ou indirectement aux phénotypes étudiés. / The molecular basis of technological properties of wine yeasts are not or poorly known. However, this knowledge is required for the improvement of the fermentation capacity and of the organoleptic impact of wine yeasts by breeding strategies and a better exploitation of the yeast biodiversity. This thesis aimed at identifying the genetic bases of several traits of interest such as: sulfite production, fermentation ability in low nitrogen medium or esters formation. A QTL mapping approach has been implemented from a population of meiotic segregants derived from the cross of two strains with contrasting oenological characteristics. Phenotyping of the a population of meiotic segregants was combined with a genotyping to identify QTL for these traits. The involvement of a region of the genome in the control of sulphate assimilation pathway has been demonstrated. This locus is responsible for the phenotypic variation of several metabolites of this pathway and indirectly connected metabolites. Other QTLs have also been demonstrated for the fermentation capacities on a nitrogen limiting medium and production of various volatile compounds. Their analysis revealed several candidate genes whose functions are related directly or indirectly to the studied phenotypes.
|
Page generated in 0.0327 seconds