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Sample records for expression ratio-optimized yeast

  1. Protein expression-yeast.

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    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline. © 2014 Elsevier Inc. All rights reserved.

  2. Nanomaterials Enhanced Gene Expression in Yeast Cells

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    Su-Fang Chien

    2008-01-01

    Full Text Available Metal nanomaterials are shown to enhance gene expression for rice -galactosidase gene (-Gal in yeast cells. Au and Ag nanoparticles and their nanocomposites, silica-Au and silica-Ag, were prepared and characterized by UV-vis spectroscopy and TEM technique. The rice -galactosidase gene was cloned into the yeast chromosome, where the cloned cells were precultured and induced into a medium containing each of the testing nanomaterials. The nanomaterials were observed to incorporate inside the cells, and no cell death has been detected during the course of gene expression. The enzyme activity was determined by a synthetic substrate, p-nitrophenyl--D-galctopyranoside, and the yellow product yield was recorded in a spectrophotometer at 400 nm. When Au and Ag nanoparticles were incorporated with the culture, a 3–5 fold enhancement in -galactosidase was observed for intracellular activity as well as the secreted activity into the medium. The secreted protein was analyzed to have a pure form and displayed as a single protein band in the SDS-gel electrophoresis. The effects of size and chemical nature of nanomaterials on gene expression for the rice -galactosidase gene in yeast cells are discussed.

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

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    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.

  4. Comparative gene expression between two yeast species

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    Guan Yuanfang

    2013-01-01

    Full Text Available Abstract Background Comparative genomics brings insight into sequence evolution, but even more may be learned by coupling sequence analyses with experimental tests of gene function and regulation. However, the reliability of such comparisons is often limited by biased sampling of expression conditions and incomplete knowledge of gene functions across species. To address these challenges, we previously systematically generated expression profiles in Saccharomyces bayanus to maximize functional coverage as compared to an existing Saccharomyces cerevisiae data repository. Results In this paper, we take advantage of these two data repositories to compare patterns of ortholog expression in a wide variety of conditions. First, we developed a scalable metric for expression divergence that enabled us to detect a significant correlation between sequence and expression conservation on the global level, which previous smaller-scale expression studies failed to detect. Despite this global conservation trend, between-species gene expression neighborhoods were less well-conserved than within-species comparisons across different environmental perturbations, and approximately 4% of orthologs exhibited a significant change in co-expression partners. Furthermore, our analysis of matched perturbations collected in both species (such as diauxic shift and cell cycle synchrony demonstrated that approximately a quarter of orthologs exhibit condition-specific expression pattern differences. Conclusions Taken together, these analyses provide a global view of gene expression patterns between two species, both in terms of the conditions and timing of a gene's expression as well as co-expression partners. Our results provide testable hypotheses that will direct future experiments to determine how these changes may be specified in the genome.

  5. Expression of enzymes in yeast for lignocellulose derived oligomer CBP

    Energy Technology Data Exchange (ETDEWEB)

    McBride, John E.; Wiswall, Erin; Shikhare, Indraneel; Xu, Haowen; Thorngren, Naomi; Hau, Heidi H.; Stonehouse, Emily

    2017-08-29

    The present invention provides a multi-component enzyme system that hydrolyzes hemicellulose oligomers from hardwood which can be expressed, for example, in yeast such as Saccharomyces cerevisiae. In some embodiments, this invention provides for the engineering of a series of biocatalysts combining the expression and secretion of components of this enzymatic system with robust, rapid xylose utilization, and ethanol fermentation under industrially relevant process conditions for consolidated bioprocessing. In some embodiments, the invention utilizes co-cultures of strains that can achieve significantly improved performance due to the incorporation of additional enzymes in the fermentation system.

  6. yStreX: yeast stress expression database.

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    Wanichthanarak, Kwanjeera; Nookaew, Intawat; Petranovic, Dina

    2014-01-01

    Over the past decade genome-wide expression analyses have been often used to study how expression of genes changes in response to various environmental stresses. Many of these studies (such as effects of oxygen concentration, temperature stress, low pH stress, osmotic stress, depletion or limitation of nutrients, addition of different chemical compounds, etc.) have been conducted in the unicellular Eukaryal model, yeast Saccharomyces cerevisiae. However, the lack of a unifying or integrated, bioinformatics platform that would permit efficient and rapid use of all these existing data remain an important issue. To facilitate research by exploiting existing transcription data in the field of yeast physiology, we have developed the yStreX database. It is an online repository of analyzed gene expression data from curated data sets from different studies that capture genome-wide transcriptional changes in response to diverse environmental transitions. The first aim of this online database is to facilitate comparison of cross-platform and cross-laboratory gene expression data. Additionally, we performed different expression analyses, meta-analyses and gene set enrichment analyses; and the results are also deposited in this database. Lastly, we constructed a user-friendly Web interface with interactive visualization to provide intuitive access and to display the queried data for users with no background in bioinformatics. Database URL: http://www.ystrexdb.com. © The Author(s) 2014. Published by Oxford University Press.

  7. Molecular cloning and expression in yeast of caprine prochymosin.

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    Vega-Hernández, Maria C; Gómez-Coello, Alicia; Villar, Jesús; Claverie-Martín, Félix

    2004-10-19

    We cloned and characterized a preprochymosin cDNA from the abomasum of milk-fed kid goats. This cDNA contained an open reading frame that predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. Comparison of the caprine preprochymosin sequence with the corresponding sequences of lamb and calf revealed 99 and 94% identity at the amino acid level. The cDNA fragment encoding the mature portion of caprine prochymosin was fused in frame both to the killer toxin signal sequence and to the alpha-factor signal sequence-FLAG in two different yeast expression vectors. The recombinant plasmids were transformed into Kluyveromyces lactis and Saccharomyces cerevisiae cells, respectively. Culture supernatants of both yeast transformants showed milk-clotting activity after activation at acid pH. The FLAG-prochymosin fusion was purified from S. cerevisiae culture supernatants by affinity chromatography. Proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed kappa-casein.

  8. Expression of the house dust mite allergen Der p 2 in the baker's yeast Saccharomyces cerevisiae

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    Hakkaart, G. A.; Harmsen, M. M.; Chua, K. Y.; Thomas, W. R.; Aalberse, R. C.; van Ree, R.

    1998-01-01

    BACKGROUND AND RESULTS: The major house dust mite allergen Der p 2 was expressed as a recombinant mature protein in the baker's yeast Saccharomyces cerevisiae. The yeast produces the protein fused to the invertase signal peptide, leading to the secretion of Der p 2 as a soluble protein into the

  9. Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha

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    Moussa Manal

    2012-12-01

    Full Text Available Abstract Background Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA and streptokinase (SK. Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK, or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system. Results The development of an efficient large scale (80 L Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.. Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth of biologically active rSAK-2 of expected molecular weight was achieved. Conclusion The optimized production process

  10. Nitrogen starvation induces expression of Lg-FLO1 and flocculation in bottom-fermenting yeast.

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    Ogata, Tomoo

    2012-11-01

    When exponentially growing cells of bottom-fermenting yeast were starved for nitrogen or were grown on proline (a non-preferred nitrogen source), flocculation was induced. This flocculation was not induced by starvation for either carbon or amino acids. Expression of Lg-FLO1, which is required for flocculation of bottom-fermenting yeast, was also found to be induced by starvation for nitrogen. This suggests that the flocculation of bottom-fermenting yeast is under the control of a nitrogen catabolite repression (NCR)-like mechanism. Copyright © 2012 John Wiley & Sons, Ltd.

  11. Yeast expressing hepatitis B virus surface antigen determinants on its surface: Implications for a possible oral vaccine

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    Schreuder, M.P.; Deen, C.; Boersma, W.J.A.; Pouwels, P.H.; Klis, F.M.

    1996-01-01

    The two major hydrophilic regions of the hepatitis B virus surface antigen (HBsAg) have been expressed in the outer mannoprotein layer of the cell wall of 'Bakers Yeast', Saccharomyces cerevisiae, by fusing them between the yeast invertase signal sequence and the yeast α-agglutinin carboxyterminal

  12. Functional expression of parasite drug targets and their human orthologs in yeast.

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    Elizabeth Bilsland

    2011-10-01

    Full Text Available The exacting nutritional requirements and complicated life cycles of parasites mean that they are not always amenable to high-throughput drug screening using automated procedures. Therefore, we have engineered the yeast Saccharomyces cerevisiae to act as a surrogate for expressing anti-parasitic targets from a range of biomedically important pathogens, to facilitate the rapid identification of new therapeutic agents.Using pyrimethamine/dihydrofolate reductase (DHFR as a model parasite drug/drug target system, we explore the potential of engineered yeast strains (expressing DHFR enzymes from Plasmodium falciparum, P. vivax, Homo sapiens, Schistosoma mansoni, Leishmania major, Trypanosoma brucei and T. cruzi to exhibit appropriate differential sensitivity to pyrimethamine. Here, we demonstrate that yeast strains (lacking the major drug efflux pump, Pdr5p expressing yeast ((ScDFR1, human ((HsDHFR, Schistosoma ((SmDHFR, and Trypanosoma ((TbDHFR and (TcDHFR DHFRs are insensitive to pyrimethamine treatment, whereas yeast strains producing Plasmodium ((PfDHFR and (PvDHFR DHFRs are hypersensitive. Reassuringly, yeast strains expressing field-verified, drug-resistant mutants of P. falciparum DHFR ((Pfdhfr(51I,59R,108N are completely insensitive to pyrimethamine, further validating our approach to drug screening. We further show the versatility of the approach by replacing yeast essential genes with other potential drug targets, namely phosphoglycerate kinases (PGKs and N-myristoyl transferases (NMTs.We have generated a number of yeast strains that can be successfully harnessed for the rapid and selective identification of urgently needed anti-parasitic agents.

  13. Construction and evaluation of self-cloning bottom-fermenting yeast with high SSU1 expression.

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    Iijima, K; Ogata, T

    2010-12-01

    To construct a self-cloning brewer's yeast that can minimize the unfavourable flavours caused by oxidation and certain kinds of sulfur compounds. DNA fragments of a high-expression promoter from the TDH3 gene originating from Saccharomyces cerevisiae were integrated into the promoter regions of the S. cerevisiae-type and Saccharomyces bayanus-type SSU1 genes of bottom-fermenting brewer's yeast. PCR and sequencing confirmed the TDH3 promoter was correctly introduced into the SSU1 regions of the constructed yeasts, and no foreign DNA sequences were found. Using the constructed yeasts, the concentration of sulfite in fermenting wort was higher when compared with the parent strain. In addition, the concentrations of hydrogen sulfide, 3-methyl-2-buten-1-thiol (MBT) and 2-mercapto-3-methyl-1-butanol (2M3MB) were lower when compared with the parent strain. We successfully constructed a self-cloning brewer's yeast with high SSU1 expression that enhanced the sulfite-excreting ability and diminished the production ability of hydrogen sulfide, MBT and 2M3MB. The self-cloning brewer's yeast with high SSU1 expression would contribute to the production of superior quality beer with a high concentration of sulfite and low concentrations of hydrogen sulfide, MBT and 2M3MB. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

  14. Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

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    Miura, Takashi; Moriya, Hisao; Iwai, Sosuke

    2017-07-03

    We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. [Yeast (Saccharomyces cerevisiae) secretory expression vector maintained stably in Pro3+ transformants in rich medium].

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    Xie, H Y; Tang, Y; Jiang, W D; He, H Y; Liu, M; Kuang, D R

    2000-01-01

    A yeast (Saccharomyces cerevisiae) secretory expression vector containing PRO3 gene was constructed (pCBy310). beta HCG(Human choriogonadotropin beta subunit)-cDNA was inserted into pCBy310 to form a recombinant plasmid pCBy314. Since yeast proline auxotroph will not survive in rich medium (YPD), YPD could be used as a selection pressure, and pCBy314 could be maintained mitotically stable in transformants of yeast Pro3- auxotroph (MB299-7A) in rich medium. At an improved, yet not optimized cultural condition, the expression of beta HCG in culture medium was 650 micrograms/L. Our results showed not only that YPD could be used as a selection medium, but also that yeast grew better in YPD so as to increase the gene expression product, and that the component of YPD was simple and cheap. Our data suggested that PRO genes might be used widely in constructing vectors to clone and express foreign genes in yeast so that rich medium can be used as a selection pressure for direct selection.

  16. Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

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    I MADE ARTIKA

    2006-03-01

    Full Text Available Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody.

  17. Differential Evolution Algorithm for Gear Ratio Optimization of Vehicles

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    İlker Küçükoğlu

    2016-10-01

    Full Text Available In this study, the gear ratio optimization problem for the vehicles is considered to minimize total fuel consumption amount for a given distance and road gradient. The aim of the study is to find best gear ratio values for a gearbox that provide the best consumption value for the vehicle. In order to analyze the fuel consumption changes for a gearbox, fuel consumption formulations, which have a nonlinear structure, are presented based on the total energy requirement of the vehicle. To solve this nonlinear optimization problem, a differential evolution algorithm (DEA is proposed which is one of the popular evolutionary optimization algorithms. In computational studies, the DEA is tested on a vehicle set by considering different road conditions. The results of the experiments show that the proposed DEA is capable to find effective solutions for the problem where an improvement is obtained for each experiment according to the real fuel consumption amount of the vehicles given at their technical reports

  18. Heterologous transporter expression for improved fatty alcohol secretion in yeast

    DEFF Research Database (Denmark)

    Hu, Yating; Zhu, Zhiwei; Nielsen, Jens

    2017-01-01

    The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production...... strains. However, impaired growth caused by fatty alcohol accumulation and high cost of extraction are factors limiting large-scale production. Here, we demonstrate that the use of heterologous transporters is a promising strategy to increase fatty alcohol production. Among several plant and mammalian...

  19. Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast.

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    Kim, Yu-Young; Kim, Do-Young; Shim, Donghwan; Song, Won-Yong; Lee, Joohyun; Schroeder, Julian I; Kim, Sanguk; Moran, Nava; Lee, Youngsook

    2008-06-06

    Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.

  20. Expression of a Ripening-Related Avocado (Persea americana) Cytochrome P450 in Yeast 1

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    Bozak, Kristin R.; O'Keefe, Daniel P.; Christoffersen, Rolf E.

    1992-01-01

    One of the mRNAs that accumulates during the ripening of avocado (Persea americana Mill. cv Hass) has been previously identified as a cytochrome P450 (P450) monooxygenase and the corresponding gene designated CYP71A1. In this report we demonstrate that during ripening the accumulation of antigenically detected CYP71A1 gene product (CYP71A1) correlates with increases in total P450 and two P450-dependent enzyme activities: para-chloro-N-methylaniline demethylase, and trans-cinnamic acid hydroxylase (tCAH). To determine whether both of these activities are derived from CYP71A1, we have expressed this protein in yeast (Saccharomyces cerevisiae) using a galactose-inducible yeast promoter. Following induction, the microsomal fraction of transformed yeast cells undergoes a large increase in P450 level, attributable almost exclusively to the plant CYP71A1 protein. These membranes exhibit NADPH-dependent para-chloro-N-methylaniline demethylase activity at a rate comparable to that in avocado microsomes but have no detectable tCAH. These results demonstrate both that the CYP71A1 protein is not a tCAH and that a plant P450 is fully functional upon heterologous expression in yeast. These findings also indicate that the heterologous P450 protein can interact with the yeast NADPH:P450 reductase to produce a functional complex. Images Figure 1 Figure 3 PMID:16653226

  1. Gateway vectors for efficient artificial gene assembly in vitro and expression in yeast Saccharomyces cerevisiae.

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    Claudiu V Giuraniuc

    Full Text Available Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene's functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID. Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology.

  2. Gateway Vectors for Efficient Artificial Gene Assembly In Vitro and Expression in Yeast Saccharomyces cerevisiae

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    Giuraniuc, Claudiu V.; MacPherson, Murray; Saka, Yasushi

    2013-01-01

    Construction of synthetic genetic networks requires the assembly of DNA fragments encoding functional biological parts in a defined order. Yet this may become a time-consuming procedure. To address this technical bottleneck, we have created a series of Gateway shuttle vectors and an integration vector, which facilitate the assembly of artificial genes and their expression in the budding yeast Saccharomyces cerevisiae. Our method enables the rapid construction of an artificial gene from a promoter and an open reading frame (ORF) cassette by one-step recombination reaction in vitro. Furthermore, the plasmid thus created can readily be introduced into yeast cells to test the assembled gene’s functionality. As flexible regulatory components of a synthetic genetic network, we also created new versions of the tetracycline-regulated transactivators tTA and rtTA by fusing them to the auxin-inducible degron (AID). Using our gene assembly approach, we made yeast expression vectors of these engineered transactivators, AIDtTA and AIDrtTA and then tested their functions in yeast. We showed that these factors can be regulated by doxycycline and degraded rapidly after addition of auxin to the medium. Taken together, the method for combinatorial gene assembly described here is versatile and would be a valuable tool for yeast synthetic biology. PMID:23675537

  3. Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression.

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    Rautio, Jari J; Huuskonen, Anne; Vuokko, Heikki; Vidgren, Virve; Londesborough, John

    2007-09-01

    Brewer's yeast experiences constantly changing environmental conditions during wort fermentation. Cells can rapidly adapt to changing surroundings by transcriptional regulation. Changes in genomic expression can indicate the physiological condition of yeast in the brewing process. We monitored, using the transcript analysis with aid of affinity capture (TRAC) method, the expression of some 70 selected genes relevant to wort fermentation at high frequency through 9-10 day fermentations of very high gravity wort (25 degrees P) by an industrial lager strain. Rapid changes in expression occurred during the first hours of fermentations for several genes, e.g. genes involved in maltose metabolism, glycolysis and ergosterol synthesis were strongly upregulated 2-6 h after pitching. By the time yeast growth had stopped (72 h) and total sugars had dropped by about 50%, most selected genes had passed their highest expression levels and total mRNA was less than half the levels during growth. There was an unexpected upregulation of some genes of oxygen-requiring pathways during the final fermentation stages. For five genes, expression of both the Saccharomyces cerevisiae and S. bayanus components of the hybrid lager strain were determined. Expression profiles were either markedly different (ADH1, ERG3) or very similar (MALx1, ILV5, ATF1) between these two components. By frequent analysis of a chosen set of genes, TRAC provided a detailed and dynamic picture of the physiological state of the fermenting yeast. This approach offers a possible way to monitor and optimize the performance of yeast in a complex process environment. Copyright (c) 2007 John Wiley & Sons, Ltd.

  4. Expression divergence measured by transcriptome sequencing of four yeast species

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    Busby Michele A

    2011-12-01

    Full Text Available Abstract Background The evolution of gene expression is a challenging problem in evolutionary biology, for which accurate, well-calibrated measurements and methods are crucial. Results We quantified gene expression with whole-transcriptome sequencing in four diploid, prototrophic strains of Saccharomyces species grown under the same condition to investigate the evolution of gene expression. We found that variation in expression is gene-dependent with large variations in each gene's expression between replicates of the same species. This confounds the identification of genes differentially expressed across species. To address this, we developed a statistical approach to establish significance bounds for inter-species differential expression in RNA-Seq data based on the variance measured across biological replicates. This metric estimates the combined effects of technical and environmental variance, as well as Poisson sampling noise by isolating each component. Despite a paucity of large expression changes, we found a strong correlation between the variance of gene expression change and species divergence (R2 = 0.90. Conclusion We provide an improved methodology for measuring gene expression changes in evolutionary diverged species using RNA Seq, where experimental artifacts can mimic evolutionary effects. GEO Accession Number: GSE32679

  5. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

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    Yoichiro Ito

    Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

  6. Functional expression of the plant alternative oxidase affects growth of the yeast Schizosaccharomyces pombe.

    NARCIS (Netherlands)

    Affourtit, C.; Albury, M.S.; Krab, K.; Moore, A.L.

    1999-01-01

    We have investigated the extent to which functional expression of the plant alternative oxidase (from Sauromatum guttatum) in Schizosaccharomyces pombe affects yeast growth. When cells are cultured on glycerol, the maximum specific growth rate is decreased from 0.13 to 0.11 h

  7. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

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    Lauren E Hudson

    Full Text Available Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  8. Expression profiling of the bottom fermenting yeast Saccharomyces pastorianus orthologous genes using oligonucleotide microarrays.

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    Minato, Toshiko; Yoshida, Satoshi; Ishiguro, Tatsuji; Shimada, Emiko; Mizutani, Satoru; Kobayashi, Osamu; Yoshimoto, Hiroyuki

    2009-03-01

    The bottom fermenting yeast Saccharomyces pastorianus is reported to have arisen as a natural hybrid of two yeast strains, S. cerevisiae and S. bayanus. The S. pastorianus genome includes S. cerevisiae-type (Sc-type) genes and orthologous lager-fermenting-yeast specific-type (Lg-type) genes derived from S. cerevisiae and S. bayanus, respectively. To gain insights into the physiological properties of S. pastorianus, we developed an in situ synthesized 60-mer oligonucleotide microarray for gene expression monitoring of these orthologous genes, consisting of approximately 6600 Sc-type genes and 3200 Lg-type genes. A comparison of the transcriptional profile of orthologous genes (e.g. Sc-type and Lg-type genes) in S. cerevisiae or S. bayanus demonstrated the feasibility of performing gene expression studies with this microarray. Genome-wide analysis of S. pastorianus with this microarray could clearly distinguish more than 67% of the expressed orthologous genes. Furthermore, it was shown that the gene expression of particular Lg-type genes differed from that of the orthologous Sc-type genes, suggesting that some Lg-type and Sc-type genes may have different functional roles. We conclude that the oligonucleotide microarray that we constructed is a powerful tool for the monitoring of gene expression of the orthologous genes of S. pastorianus.

  9. Yeast expression proteomics by high-resolution mass spectrometry

    DEFF Research Database (Denmark)

    Walther, Tobias C; Olsen, Jesper Velgaard; Mann, Matthias

    2010-01-01

    . In contrast, methodologies to globally quantitate proteins are only recently becoming available. Such experiments are essential as proteins mediate the majority of biological processes and their abundance does not always correlate well with changes in gene expression. Particularly translational and post...

  10. Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.

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    Wei Wang

    Full Text Available To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.

  11. Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast

    NARCIS (Netherlands)

    Moglia, A.; Comino, C.; Lanteri, S.; Vos, de C.H.; Waard, de P.; Beek, van T.A.; Goitre, L.; Retta, S.F.; Beekwilder, M.J.

    2010-01-01

    Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker’s yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified

  12. Minimization of biosynthetic costs in adaptive gene expression responses of yeast to environmental changes.

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    Ester Vilaprinyo

    2010-02-01

    Full Text Available Yeast successfully adapts to an environmental stress by altering physiology and fine-tuning metabolism. This fine-tuning is achieved through regulation of both gene expression and protein activity, and it is shaped by various physiological requirements. Such requirements impose a sustained evolutionary pressure that ultimately selects a specific gene expression profile, generating a suitable adaptive response to each environmental change. Although some of the requirements are stress specific, it is likely that others are common to various situations. We hypothesize that an evolutionary pressure for minimizing biosynthetic costs might have left signatures in the physicochemical properties of proteins whose gene expression is fine-tuned during adaptive responses. To test this hypothesis we analyze existing yeast transcriptomic data for such responses and investigate how several properties of proteins correlate to changes in gene expression. Our results reveal signatures that are consistent with a selective pressure for economy in protein synthesis during adaptive response of yeast to various types of stress. These signatures differentiate two groups of adaptive responses with respect to how cells manage expenditure in protein biosynthesis. In one group, significant trends towards downregulation of large proteins and upregulation of small ones are observed. In the other group we find no such trends. These results are consistent with resource limitation being important in the evolution of the first group of stress responses.

  13. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    During germination of barley seeds, the mobilization of protein is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction...... of the active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. One of the key cysteine proteases in Barley...

  14. Enoate reductases from non conventional yeasts: bioconversion, cloning, and functional expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Raimondi, Stefano; Romano, Diego; Amaretti, Alberto; Molinari, Francesco; Rossi, Maddalena

    2011-12-20

    Old yellow enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the stereoselective trans-hydrogenation of the double bond, representing a promising alternative to metal-based catalysis. Bioconversion of ketoisophorone (KIP) by 28 non-conventional yeasts belonging to 16 different species was investigated. Growing cells of most of the strains reduced KIP via OYE and showed high stereoselectivity, producing R-levodione as major product. Competition by carbonyl reductase (CR) activity was observed in several strains. The best performing yeasts belong to Candida castellii, Kazachstania spencerorum and Kluyveromyces marxianus exhibited yields of levodione ≥77% up to 95% e.e., and. Candida freyschussii, the sole strain lacking the OYE gene, reduced KIP only to unsaturated alcohols via CR. Nine unedited OYE genes were cloned, sequenced, and heterologously expressed in Saccharomyces cerevisiae BY4741ΔOye2, a mutant that showed negligible OYE and CR activities. Compared with the corresponding wild-type yeasts, growing cells of the recombinant strains bioconverted KIP with improved yields of OYE products, minor competition by CR activity, and lower enantioselectivity. In particular, resting cells of recombinant S. cerevisae presented the best performance in KIP bioconversion. Based on the results herein reported, selected strains of non-conventional yeasts and novel OYE genes can be profitably used as innovative biocatalysts in asymmetric reductions. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Yeast extract from Express Five serum-free medium contains factors at about 35 kDa, essential for growth of Trichoplusia ni insect cells.

    Science.gov (United States)

    Eriksson, Ulrika; Häggström, Lena

    2005-10-01

    The yeast extract (of unknown origin) present in the commercially available serum-free medium 'Express Five' contains factors ('yeast extract factors') up to 35 kDa which are essential for growth of Trichoplusia ni insect cells. A yeast extract brand lacking these components could not support growth of T. ni cells. However, cell proliferation was restored by adding chromatographic fractions containing the yeast extract factors. The yeast extract factors were not solely responsible for the growth enhancing effect of yeast extract but some other components, which seem to be generally present in yeast extracts, are also required for T. ni proliferation.

  16. Expression of Leishmania major LmSTI1 in Yeast Pichia Pastoris

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    Mehdi Shokri

    2017-01-01

    Full Text Available Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems.Materials and Methods: To express LmSTI1 in the methylotrophic yeast         Pichia pastoris (P. pastoris, the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0.5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L.major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively.Results: The expression level was 0.2% of total soluble proteins.Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization.

  17. Significant and systematic expression differentiation in long-lived yeast strains.

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    Chao Cheng

    2007-10-01

    Full Text Available Recent studies suggest that the regulation of longevity may be partially conserved in many eukaryotes ranging from yeast to mammals. The three yeast mutants sch9Delta, ras2Delta, tor1Delta show extended chronological life span up to three folds. Our aim is to dissect the mechanisms that lead to the yeast life span extension.We obtain gene expression profiles of sch9Delta, ras2Delta, tor1Delta as well as that for a wild type at day 2.5 in SDC medium using Affymetrix Yeast2.0 arrays. To accurately estimate the expression differentiation between the wild type and the long-lived mutants, we use sub-array normalization followed by a variant of the median-polishing summarization. The results are validated by the probe sets of S. pombe on the same chips. To translate the differentiation into changes of biological activities, we make statistical inference by integrating the expression profiles with biological gene subsets defined by Gene Ontology, KEGG pathways, and cellular localization of proteins. Other than subset-versus-other comparisons, we also make local comparisons between two directly-related gene subsets such as cytosolic and mitochondrial ribosomes. Our consensus is obtained by cross-examination of these inferences. The significant and systematic differentiation in the three long-lived strains includes: lower transcriptional activities; down-regulation of TCA cycle and oxidative phosphorylation versus up-regulation of the KEGG pathway Glycolysis/Gluconeogenesis; the overall reduction of mitochondrial activities. We also report some different expression patterns such as reduction of the activities relating to mitosis in ras2Delta.The modification of energy pathways and modification of compartment activities such as down-regulation of mitochondrial ribosome proteins versus up-regulation of cytosolic ribosome proteins are directly associated with the life span extension in yeast. The results provide a new and systematic S. cerevisiae version of

  18. Cloning and expression of phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica PI12

    Science.gov (United States)

    Jaafar, Nardiah Rizwana; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad

    2015-09-01

    The conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). Better understanding of metabolic reactions performed by this enzyme has been studied extensively in prokaryotes and eukaryotes. Here, we report a phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica. cDNA encoding for PGM from G. antarctica PI12, a psychrophilic yeast isolated from sea ice at Casey Station, Antarctica was amplified. The gene was then cloned into a cloning vector and sequenced, which verified its identity as the gene putatively encoding for PGM. The recombinant protein was expressed in Escherichia coli BL21 (DE3) as inclusion bodies and this was confirmed by SDS-PAGE and Western blot.

  19. Construction of a recombinant wine yeast strain expressing beta-(1,4)-endoglucanase and its use in microvinification processes.

    Science.gov (United States)

    Pérez-González, J A; González, R; Querol, A; Sendra, J; Ramón, D

    1993-01-01

    A genetic transformation system for an industrial wine yeast strain is presented here. The system is based on the acquisition of cycloheximide resistance and is a direct adaptation of a previously published procedure for brewing yeasts (L. Del Pozo, D. Abarca, M. G. Claros, and A. Jiménez, Curr. Genet. 19:353-358, 1991). Transformants arose at an optimal frequency of 0.5 transformant per microgram of DNA, are stable in the absence of selective pressure, and produce wine in the same way as the untransformed industrial strain. By using this transformation protocol, a filamentous fungal beta-(1,4)-endoglucanase gene has been expressed in an industrial wine yeast under the control of the yeast actin gene promoter. Endoglucanolytic wine yeast secretes the fungal enzyme to the must, producing a wine with an increased fruity aroma. Images PMID:8215355

  20. Yeast genomic expression patterns in response to low-shear modeled microgravity

    Directory of Open Access Journals (Sweden)

    Altenburg Sara D

    2007-01-01

    Full Text Available Abstract The low-shear microgravity environment, modeled by rotating suspension culture bioreactors called high aspect ratio vessels (HARVs, allows investigation in ground-based studies of the effects of microgravity on eukaryotic cells and provides insights into the impact of space flight on cellular physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG causes significant phenotypic changes of a select group of Saccharomyces cerevisiae genes associated with the establishment of cell polarity, bipolar budding, and cell separation. However, the mechanisms cells utilize to sense and respond to microgravity and the fundamental gene expression changes that occur are largely unknown. In this study, we examined the global transcriptional response of yeast cells grown under LSMMG conditions using DNA microarray analysis in order to determine if exposure to LSMMG results in changes in gene expression. Results LSMMG differentially changed the expression of a significant number of genes (1372 when yeast cells were cultured for either five generations or twenty-five generations in HARVs, as compared to cells grown under identical conditions in normal gravity. We identified genes in cell wall integrity signaling pathways containing MAP kinase cascades that may provide clues to novel physiological responses of eukaryotic cells to the external stress of a low-shear modeled microgravity environment. A comparison of the microgravity response to other environmental stress response (ESR genes showed that 26% of the genes that respond significantly to LSMMG are involved in a general environmental stress response, while 74% of the genes may represent a unique transcriptional response to microgravity. In addition, we found changes in genes involved in budding, cell polarity establishment, and cell separation that validate our hypothesis that phenotypic changes observed in cells grown in microgravity are reflected in genome

  1. [Cloning and expression of VHB gene in D-arabitol producing yeast].

    Science.gov (United States)

    He, P; Lu, D; Wang, Q; Shen, A; Jiang, N

    2001-06-01

    Recombinant plasmid pVgb-EX2 containing Vitreoscilla hemoglobin gene vgb and formaldehyde resistant gene SFA1 was constructed and transformed into D-arabitol producing yeast strain Saccharomyces sp. X-62. The fact that the amount of VHb in transformant cells was considerably higher than that in control cells indicated that gene vgb was expressed in transformant cells. D-arabitol productivity and yield of fermentation by transformants were improved. The most improvement of D-arabitol productivity in repeat experiments reached 27.3%. It appeared that the fermentation productivity of D-arabitol was relative to the amount of VHb in cells under experimental conditions.

  2. Heterologous expression of cytotoxic sesquiterpenoids from the medicinal mushroom Lignosus rhinocerotis in yeast.

    Science.gov (United States)

    Yap, Hui-Yeng Yeannie; Muria-Gonzalez, Mariano Jordi; Kong, Boon-Hong; Stubbs, Keith A; Tan, Chon-Seng; Ng, Szu-Ting; Tan, Nget-Hong; Solomon, Peter S; Fung, Shin-Yee; Chooi, Yit-Heng

    2017-06-12

    Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. Metabolite profile analysis of the yeast culture extracts by GC-MS showed the production of several sesquiterpene alcohols (C 15 H 26 O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, β-elemene, β-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1 H and 13 C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC 50 value of 3.5 ± 0.58 μg/ml while α-cadinol is less active (IC 50  = 18.0 ± 3.27 μg/ml). This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.

  3. Identification of bottom-fermenting yeast genes expressed during lager beer fermentation.

    Science.gov (United States)

    Yoshida, Satoshi; Hashimoto, Kaori; Shimada, Emiko; Ishiguro, Tatsuji; Minato, Toshiko; Mizutani, Satoru; Yoshimoto, Hiroyuki; Tashiro, Kosuke; Kuhara, Satoru; Kobayashi, Osamu

    2007-07-01

    It has been proposed that bottom-fermenting yeast strains of Saccharomyces pastorianus possess at least two types of genomes. Sequences of genes of one genome [S. cerevisiae (Sc)-type] have been found to be highly homologous (more than 90% identity) to S. cerevisiae S288C sequences, while those of the other [Lager (Lg)-type] are less so. To identify and discriminate Lg-type from Sc-type genes expressed during lager beer fermentation, normalized cDNA libraries were constructed and analysed. From approximately 22 000 ESTs, 3892 Sc-type and 2695 Lg-type ORFs were identified. Expression patterns of Sc- and Lg-type genes did not correlate with particular cell functions in KEGG classification system. Moreover, 405 independent clones were isolated that have no significant homology with sequences in the S288C database, suggesting that they include the bottom-fermenting yeast-specific (BFY) genes. Most of BFY genes have significant homology with the S. bayanus genome. Copyright (c) 2007 John Wiley & Sons, Ltd.

  4. Construction and evaluation of yeast expression networks by database-guided predictions

    Directory of Open Access Journals (Sweden)

    Katharina Papsdorf

    2016-05-01

    Full Text Available DNA-Microarrays are powerful tools to obtain expression data on the genome-wide scale. We performed microarray experiments to elucidate the transcriptional networks, which are up- or down-regulated in response to the expression of toxic polyglutamine proteins in yeast. Such experiments initially generate hit lists containing differentially expressed genes. To look into transcriptional responses, we constructed networks from these genes. We therefore developed an algorithm, which is capable of dealing with very small numbers of microarrays by clustering the hits based on co-regulatory relationships obtained from the SPELL database. Here, we evaluate this algorithm according to several criteria and further develop its statistical capabilities. Initially, we define how the number of SPELL-derived co-regulated genes and the number of input hits influences the quality of the networks. We then show the ability of our networks to accurately predict further differentially expressed genes. Including these predicted genes into the networks improves the network quality and allows quantifying the predictive strength of the networks based on a newly implemented scoring method. We find that this approach is useful for our own experimental data sets and also for many other data sets which we tested from the SPELL microarray database. Furthermore, the clusters obtained by the described algorithm greatly improve the assignment to biological processes and transcription factors for the individual clusters. Thus, the described clustering approach, which will be available through the ClusterEx web interface, and the evaluation parameters derived from it represent valuable tools for the fast and informative analysis of yeast microarray data.

  5. Expression profiling reveals an unexpected growth-stimulating effect of surplus iron on the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Du, Yang; Cheng, Wang; Li, Wei-Fang

    2012-08-01

    Iron homeostasis plays a crucial role in growth and division of cells in all kingdoms of life. Although yeast iron metabolism has been extensively studied, little is known about the molecular mechanism of response to surplus iron. In this study, expression profiling of Saccharomyces cerevisiae in the presence of surplus iron revealed a dual effect at 1 and 4 h. A cluster of stress-responsive genes was upregulated via activation of the stress-resistance transcription factor Msn4, which indicated the stress effect of surplus iron on yeast metabolism. Genes involved in aerobic metabolism and several anabolic pathways are also upregulated in iron-surplus conditions, which could significantly accelerate yeast growth. This dual effect suggested that surplus iron might participate in a more complex metabolic network, in addition to serving as a stress inducer. These findings contribute to our understanding of the global response of yeast to the fluctuating availability of iron in the environment.

  6. The mammalian AMP-activated protein kinase complex mediates glucose regulation of gene expression in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Ye, Tian; Bendrioua, Loubna; Carmena, David; García-Salcedo, Raúl; Dahl, Peter; Carling, David; Hohmann, Stefan

    2014-06-05

    The AMP-activated protein kinase (AMPK) controls energy homeostasis in eukaryotic cells. Here we expressed hetero-trimeric mammalian AMPK complexes in a Saccharomyces cerevisiae mutant lacking all five genes encoding yeast AMPK/SNF1 components. Certain mammalian complexes complemented the growth defect of the yeast mutant on non-fermentable carbon sources. Phosphorylation of the AMPK α1-subunit was glucose-regulated, albeit not by the Glc7-Reg1/2 phosphatase, which performs this function on yeast AMPK/SNF1. AMPK could take over SNF1 function in glucose derepression. While indirectly acting anti-diabetic drugs had no effect on AMPK in yeast, compound 991 stimulated α1-subunit phosphorylation. Our results demonstrate a remarkable functional conservation of AMPK and that glucose regulation of AMPK may not be mediated by regulatory features of a specific phosphatase. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.

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    Daria Jacob

    Full Text Available Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV nucleoprotein (N known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs. Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen

  8. Expression of a synthetic rust fungal virus cDNA in yeast.

    Science.gov (United States)

    Cooper, Bret; Campbell, Kimberly B; Garrett, Wesley M

    2016-01-01

    Mycoviruses are viruses that infect fungi. Recently, mycovirus-like RNAs were sequenced from the fungus Phakopsora pachyrhizi, the causal agent of soybean rust. One of the RNAs appeared to represent a novel mycovirus and was designated Phakopsora pachyrhizi virus 2383 (PpV2383). The genome of PpV2383 resembles Saccharomyces cerevisiae virus L-A, a double-stranded (ds) RNA mycovirus of yeast. PpV2383 encodes two major, overlapping open reading frames with similarity to gag (capsid protein) and pol (RNA-dependent RNA polymerase), and a -1 ribosomal frameshift is necessary for the translation of a gag-pol fusion protein. Phylogenetic analysis of pol relates PpV2383 to members of the family Totiviridae, including L-A. Because the obligate biotrophic nature of P. pachyrhizi makes it genetically intractable for in vivo analysis and because PpV2383 is similar to L-A, we synthesized a DNA clone of PpV2383 and tested its infectivity in yeast cells. PpV2383 RNA was successfully expressed in yeast, and mass spectrometry confirmed the translation of gag and gag-pol fusion proteins. There was, however, no production of PpV2383 dsRNA, the evidence of viral replication. Neither the presence of endogenous L-A nor the substitution of the 5' and 3' untranslated regions with those from L-A was sufficient to rescue replication of PpV2383. Nevertheless, the proof of transcription and translation from the clone in vivo are steps toward confirming that PpV2383 is a mycovirus. Further development of a surrogate biological system for the study of rust mycoviruses is necessary, and such research may facilitate biological control of rust diseases.

  9. Temporal Expression of a Master Regulator Drives Synchronous Sporulation in Budding Yeast

    Directory of Open Access Journals (Sweden)

    Minghao Chia

    2016-11-01

    Full Text Available Yeast cells enter and undergo gametogenesis relatively asynchronously, making it technically challenging to perform stage-specific genomic and biochemical analyses. Cell-to-cell variation in the expression of the master regulator of entry into sporulation, IME1, has been implicated to be the underlying cause of asynchronous sporulation. Here, we find that timing of IME1 expression is of critical importance for inducing cells to undergo sporulation synchronously. When we force expression of IME1 from an inducible promoter in cells incubated in sporulation medium for 2 hr, the vast majority of cells exhibit synchrony during premeiotic DNA replication and meiotic divisions. Inducing IME1 expression too early or too late affects the synchrony of sporulation. Surprisingly, our approach for synchronous sporulation does not require growth in acetate-containing medium, but can be achieved in cells grown in rich medium until saturation. Our system requires solely IME1, because the expression of the N6-methyladenosine methyltransferase IME4, another key regulator of early sporulation, is controlled by IME1 itself. The approach described here can be combined easily with other stage-specific synchronization methods, and thereby applied to study specific stages of sporulation, or the complete sporulation program.

  10. Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

    Science.gov (United States)

    Zhang, Guo-Chang; Turner, Timothy L; Jin, Yong-Su

    2017-03-01

    Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD + -linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

  11. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Smialowska, Agata, E-mail: smialowskaa@gmail.com [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden); Djupedal, Ingela; Wang, Jingwen [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); Kylsten, Per [School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden); Swoboda, Peter [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); Ekwall, Karl, E-mail: Karl.Ekwall@ki.se [Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institute, Huddinge 141-83 (Sweden); School of Life Sciences, Södertörn Högskola, Huddinge 141-89 (Sweden)

    2014-02-07

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.

  12. Improved expression of Rhizopus oryzae α-amylase in the methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Li, Song; Sing, Suren; Wang, Zhengxiang

    2011-09-01

    In our previous study, the α-amylase from Rhizopus oryzae (RoAmy) was expressed in Escherichia coli and Saccharomyces cerevisiae but the obtained recombinant RoAmy (rRoAmy) yields were too low. The aim of the present research was to obtain high-level expressions of RoAmy in the methylotrophic yeast Pichia pastoris. To this end, we constructed P. pastoris strains with the capability to express recombinant α-amylase under the control of constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) and methanol-inducible alcohol oxidase 1 promoters. The levels of inducibly expressed rRoAmy were higher than those of constitutively expressed. The maximal inducible rRoAmy expression levels for the Mut(+) strains (41.1mg/l) were approximately eight times higher than those for the Mut(s) strains and 24 times higher than those expressed under the control of the GAP promoter. For both inducible and constitutive expressions, the S. cerevisiae α-prepro sequence and the native signal sequence of RoAmy were used separately to direct the secretion of rRoAmy into the culture medium of P. pastoris. Low levels of intracellular amylase activities that had been detected after shake-flask fermentation indicated that both signal sequences could effectively direct the secretion of rRoAmy under all studied conditions. In addition, the secretion levels of rRoAmy directed with its own signal peptide were 7-10% higher than those directed by the α-prepro sequence. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

    Science.gov (United States)

    Young, Travis [San Diego, CA; Schultz, Peter G [La Jolla, CA

    2014-02-11

    The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methyltrophic yeast such as Pichia pastoris are also provided.

  14. In vivo unnatural amino acid expression in the methylotrophic yeast Pichia pastoris

    Energy Technology Data Exchange (ETDEWEB)

    Young, Travis; Schultz, Peter G.

    2017-08-15

    The invention provides orthogonal translation systems for the production of polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris. Methods for producing polypeptides comprising unnatural amino acids in methylotrophic yeast such as Pichia pastoris are also provided.

  15. Molecular cloning, expression and purification of lactoferrin from Tibetan sheep mammary gland using a yeast expression system.

    Science.gov (United States)

    Li, Jianbo; Zhu, Wuzheng; Luo, Meirong; Ren, Honghui; Tang, Lu; Liao, Honghai; Wang, Yong

    2015-05-01

    This paper reports the successful expression of a lactoferrin gene-obtained from the mammary gland tissue of Tibetan sheep-in the yeast Pichia pastoris GS115 using pPICZαA as the recombinant plasmid and α-factor signal sequence for secretion. The recombinant lactoferrin was purified by ammonium sulfate precipitation, ion-exchange column chromatography and gel-filtration chromatography, and it had a molecular mass of 76kDa. We obtained an expression yield of >60mgL(-1) and specific activity of 2533.33Umg(-1). The antimicrobial activities and iron-binding behaviors of recombinant lactoferrin indicated that it was correctly folded and functional. Additionally, recombinant lactoferrin inhibited the growth of Escherichia coli JM109 and Staphylococcus aureus. These findings indicate that recombinant lactoferrin is a potential antibiotic for use on humans. This study also demonstrates the successful expression of recombinant lactoferrin using the eukaryotic host organism P. pastoris, paving the way for protein engineering using this gene. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

    Directory of Open Access Journals (Sweden)

    Hudler Petra

    2009-10-01

    Full Text Available Abstract Background Loss of DNA mismatch repair (MMR in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC. Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells. Methods Yeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested. Results The yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic. Conclusion Results of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

  17. Heterologous Expression of Membrane and Soluble Proteins Derepresses GCN4 mRNA Translation in the Yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Steffensen, L.; Pedersen, P. A.

    2006-01-01

    This paper describes the first physiological response at the translational level towards heterologous protein production in Saccharomyces cerevisiae. In yeast, the phosphorylation of eukaryotic initiation factor 2 (eIF-2 ) by Gcn2p protein kinase mediates derepression of GCN4 mRNA translation. Gcn4......p is a transcription factor initially found to be required for transcriptional induction of genes responsible for amino acid or purine biosynthesis. Using various GCN4-lacZ fusions, knockout yeast strains, and anti-eIF-2 -P/anti-eIF-2 antibodies, we observed that heterologous expression...

  18. Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

    Directory of Open Access Journals (Sweden)

    Passos Geraldo AS

    2006-08-01

    Full Text Available Abstract Background Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM, a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. Results In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i control of cell organisation – cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein, bgl (encoding for a 1,3-β-glucosidase in mycelium cells; and ags (an α-1,3-glucan synthase, cda (a chitin deacetylase and vrp (a verprolin in yeast cells; (ii ion metabolism and transport – two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells – isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. Conclusion Taken

  19. The wine yeast strain-dependent expression of genes implicated in sulfide production in response to nitrogen availability.

    Science.gov (United States)

    Mendes-Ferreira, Ana; Barbosa, Catarina; Jimenez-Marti, Elena; Del Olmo, Marcel Li; Mendes-Faia, Arlete

    2010-09-01

    Sulfur metabolism in S. cerevisiae is well established, but the mechanisms underlying the formation of sulfide remain obscure. Here we investigated by real time RT-PCR the dependence of expression levels of MET3, MET5/ECM17, MET10, MET16 and MET17 along with SSU1 on nitrogen availability in two wine yeast strains that produce divergent sulfide profiles. MET3 was the most highly expressed of the genes studied in strain PYCC4072, and SSU1 in strain UCD522. Strains behaved differently according to the sampling times, with UCD522 and PYCC4072 showing the highest expression levels at 120h and 72h, respectively. In the presence of 267mg assimilable N/l, the genes were more highly expressed in strain UCD522 than in PYCC4072. MET5/ECM17 and MET17 were only weakly expressed in both strains under any condition tested. MET10 and SSU1 in both strains, but MET16 only in PYCC4072, were consistently up-regulated when sulfide production was inhibited. This study illustrates that strain genotype could be important in determining enzyme activities and therefore the rate of sulfide liberation. This linkage, for some yeast strains, of sulfide production to expression levels of genes associated to sulfate assimilation and sulfur amino acid biosynthesis could be relevant for defining new strategies for genetic improvement of wine yeasts.

  20. Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit.

    Science.gov (United States)

    Kolotilin, Igor; Koltai, Hinanit; Bar-Or, Carmiya; Chen, Lea; Nahon, Sahadia; Shlomo, Haviva; Levin, Ilan; Reuveni, Moshe

    2011-07-01

    Tomato (Solanum lycopersicum) fruits expressing a yeast S-adenosyl methionine decarboxylase (ySAMdc) gene under control of a ripening-induced promoter show altered phytonutrient content and broad changes in gene expression. Genome-wide transcriptional alterations in pericarp tissues of the ySAMdc-expressing fruits are shown. Consistent with the ySAMdc expression pattern from the ripening-induced promoter, very minor transcriptional alterations were detected at the mature green developmental stage. At the breaker and red stages, altered levels of numerous transcripts were observed with a general tendency toward upregulation in the transgenic fruits. Ontological analysis of up- and downregulated transcript groups revealed various affected metabolic processes, mainly carbohydrate and amino acid metabolism, and protein synthesis, which appeared to be intensified in the ripening transgenic fruits. Other functional ontological categories of altered transcripts represented signal transduction, transcription regulation, RNA processing, molecular transport and stress response, as well as metabolism of lipids, glycans, xenobiotics, energy, cofactors and vitamins. In addition, transcript levels of genes encoding structural enzymes for several biosynthetic pathways showed strong correlations to levels of specific metabolites that displayed altered levels in transgenic fruits. Increased transcript levels of fatty acid biosynthesis enzymes were accompanied by a change in the fatty acid profile of transgenic fruits, most notably increasing ω-3 fatty acids at the expense of other lipids. Thus, SAMdc is a prime target in manipulating the nutritional value of tomato fruits. Combined with analyses of selected metabolites in the overripe fruits, a model of enhanced homeostasis of the pericarp tissue in the polyamine-accumulating tomatoes is proposed. Copyright © Physiologia Plantarum 2011.

  1. Enhanced citric acid production by a yeast Yarrowia lipolytica over-expressing a pyruvate carboxylase gene.

    Science.gov (United States)

    Tan, Mei-Juan; Chen, Xi; Wang, Yu-Kuan; Liu, Guang-Lei; Chi, Zhen-Ming

    2016-08-01

    In this study, after the expression of a pyruvate carboxylase gene (PYC) cloned from Meyerozyma guilliermondii in a marine-derived yeast Yarrowia lipolytica SWJ-1b, a transformant PG86 obtained had much higher PYC activity than Y. lipolytica SWJ-1b. At the same time, the PYC gene expression and citric acid (CA) production by the transformant PG86 were also greatly enhanced. When glucose concentration in the medium was 60.0 g L(-1), CA concentration formed by the transformant PG86 was 34.02 g L(-1), leading to a CA yield of 0.57 g g(-1) of glucose. During a 10-L fed-batch fermentation, the final concentration of CA was 101.0 ± 1.3 g L(-1), the yield was 0.89 g g(-1) of glucose, the productivity was 0.42 g L(-1) h(-1) and only 5.93 g L(-1) reducing sugar was left in the fermented medium within 240 h of the fed-batch fermentation. HPLC analysis showed that most of the fermentation products were CA.

  2. A yeast expression system for functional and pharmacological studies of the malaria parasite Ca2+/H+ antiporter

    Directory of Open Access Journals (Sweden)

    Salcedo-Sora J

    2012-08-01

    Full Text Available Abstract Background Calcium (Ca2+ signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca2+ is tightly regulated through the co-ordinated action of primary and secondary Ca2+ transporters. Identifying selective inhibitors of Ca2+ transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented. Methods The Plasmodium falciparum Ca2+/H+ antiporter (PfCHA was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese. Results Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca2+, Mg2+ and Mn2+ in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca2+ transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca2+-loading assays for further functional and pharmacological studies. Discussion The functional expression of PfCHA in S. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent

  3. A yeast's eye view of mammalian reproduction: cross-species gene co-expression in meiotic prophase

    Directory of Open Access Journals (Sweden)

    Li Yunfei

    2010-09-01

    Full Text Available Abstract Background Meiotic prophase is a critical stage in sexual reproduction. Aberrant chromosome recombination during this stage is a leading cause of human miscarriages and birth defects. However, due to the experimental intractability of mammalian gonads, only a very limited number of meiotic genes have been characterized. Here we aim to identify novel meiotic genes important in human reproduction through computational mining of cross-species and cross-sex time-series expression data from budding yeast, mouse postnatal testis, mouse embryonic ovary, and human fetal ovary. Results Orthologous gene pairs were ranked by order statistics according to their co-expression profiles across species, allowing us to infer conserved meiotic genes despite obvious differences in cellular synchronicity and composition in organisms. We demonstrated that conserved co-expression networks could successfully recover known meiotic genes, including homologous recombination genes, chromatin cohesion genes, and genes regulating meiotic entry. We also showed that conserved co-expression pairs exhibit functional connections, as evidenced by the annotation similarity in Gene Ontology and overlap with physical interactions. More importantly, we predicted six new meiotic genes through their co-expression linkages with known meiotic genes, and subsequently used the genetically more amenable yeast system for experimental validation. The deletion mutants of all six genes showed sporulation defects, equivalent to a 100% validation rate. Conclusions We identified evolutionarily conserved gene modules in meiotic prophase by integrating cross-species and cross-sex expression profiles from budding yeast, mouse, and human. Our co-expression linkage analyses confirmed known meiotic genes and identified several novel genes that might be critical players in meiosis in multiple species. These results demonstrate that our approach is highly efficient to discover evolutionarily

  4. Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds on both receptor types

    NARCIS (Netherlands)

    Bovee, T.F.H.; Helsdingen, J.R.; Rietjens, I.M.C.M.; Keijer, J.; Hoogenboom, L.A.P.

    2004-01-01

    Previously, we described the construction of a rapid yeast bioassay stably expressing human estrogen receptor (hER) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, the properties of this assay were further studied by testing a series of estrogenic

  5. Protein stickiness, rather than number of functional protein-protein interactions, predicts expression noise and plasticity in yeast

    Directory of Open Access Journals (Sweden)

    Brettner Leandra M

    2012-09-01

    Full Text Available Abstract Background A hub protein is one that interacts with many functional partners. The annotation of hub proteins, or more generally the protein-protein interaction “degree” of each gene, requires quality genome-wide data. Data obtained using yeast two-hybrid methods contain many false positive interactions between proteins that rarely encounter each other in living cells, and such data have fallen out of favor. Results We find that protein “stickiness”, measured as network degree in ostensibly low quality yeast two-hybrid data, is a more predictive genomic metric than the number of functional protein-protein interactions, as assessed by supposedly higher quality high throughput affinity capture mass spectrometry data. In the yeast Saccharomyces cerevisiae, a protein’s high stickiness, but not its high number of functional interactions, predicts low stochastic noise in gene expression, low plasticity of gene expression across different environments, and high probability of forming a homo-oligomer. Our results are robust to a multiple regression analysis correcting for other known predictors including protein abundance, presence of a TATA box and whether a gene is essential. Once the higher stickiness of homo-oligomers is controlled for, we find that homo-oligomers have noisier and more plastic gene expression than other proteins, consistent with a role for homo-oligomerization in mediating robustness. Conclusions Our work validates use of the number of yeast two-hybrid interactions as a metric for protein stickiness. Sticky proteins exhibit low stochastic noise in gene expression, and low plasticity in expression across different environments.

  6. Nuclear RNA Decay Pathways Aid Rapid Remodeling of Gene Expression in Yeast.

    Science.gov (United States)

    Bresson, Stefan; Tuck, Alex; Staneva, Desislava; Tollervey, David

    2017-03-02

    In budding yeast, the nuclear RNA surveillance system is active on all pre-mRNA transcripts and modulated by nutrient availability. To test the role of nuclear surveillance in reprogramming gene expression, we identified transcriptome-wide binding sites for RNA polymerase II and the exosome cofactors Mtr4 (TRAMP complex) and Nab3 (NNS complex) by UV crosslinking immediately following glucose withdrawal (0, 4, and 8 min). In glucose, mRNA binding by Nab3 and Mtr4 was mainly restricted to promoter-proximal sites, reflecting early transcription termination. Following glucose withdrawal, many growth-related mRNAs showed reduced transcription but increased Nab3 binding, accompanied by downstream recruitment of Mtr4, and oligo(A) tailing. We conclude that transcription termination is followed by TRAMP-mediated RNA decay. Upregulated transcripts evaded increased surveillance factor binding following glucose withdrawal. Some upregulated genes showed use of alternative transcription starts to bypass strong NNS binding sites. We conclude that nuclear surveillance pathways regulate both positive and negative responses to glucose availability. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl

    2014-02-07

    RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Synthetic Core Promoters as Universal Parts for Fine-Tuning Expression in Different Yeast Species.

    Science.gov (United States)

    Portela, Rui M C; Vogl, Thomas; Kniely, Claudia; Fischer, Jasmin E; Oliveira, Rui; Glieder, Anton

    2017-03-17

    Synthetic biology and metabolic engineering experiments frequently require the fine-tuning of gene expression to balance and optimize protein levels of regulators or metabolic enzymes. A key concept of synthetic biology is the development of modular parts that can be used in different contexts. Here, we have applied a computational multifactor design approach to generate de novo synthetic core promoters and 5' untranslated regions (UTRs) for yeast cells. In contrast to upstream cis-regulatory modules (CRMs), core promoters are typically not subject to specific regulation, making them ideal engineering targets for gene expression fine-tuning. 112 synthetic core promoter sequences were designed on the basis of the sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs. The synthetic core promoters were fused to the Pichia pastoris AOX1 CRM, and the resulting activity spanned more than a 200-fold range (0.3% to 70.6% of the wild type AOX1 level). The top-ten synthetic core promoters with highest activity were fused to six additional CRMs (three in P. pastoris and three in Saccharomyces cerevisiae). Inducible CRM constructs showed significantly higher activity than constitutive CRMs, reaching up to 176% of natural core promoters. Comparing the activity of the same synthetic core promoters fused to different CRMs revealed high correlations only for CRMs within the same organism. These data suggest that modularity is maintained to some extent but only within the same organism. Due to the conserved role of eukaryotic core promoters, this rational design concept may be transferred to other organisms as a generic engineering tool.

  9. Functional expression, production, and biochemical characterization of a laccase using yeast surface display technology.

    Science.gov (United States)

    Bertrand, Brandt; Trejo-Hernández, María R; Morales-Guzmán, Daniel; Caspeta, Luis; Suárez Rodríguez, Ramón; Martínez-Morales, Fernando

    2016-12-01

    A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19 735 ± 1719 Ug(-1) and 6.22 ± 0.53 gL(-1) respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (μmol min(-1)) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25-70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  10. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

    DEFF Research Database (Denmark)

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota

    2017-01-01

    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploi...... production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside....

  11. TORC1 signaling inhibition by rapamycin and caffeine affect lifespan, global gene expression, and cell proliferation of fission yeast

    Science.gov (United States)

    Rallis, Charalampos; Codlin, Sandra; Bähler, Jürg

    2013-01-01

    Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here, we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprograming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Rapamycin showed a much more subtle effect on global translation than did caffeine, while both drugs were effective in prolonging chronological lifespan. Rapamycin and caffeine did not affect the lifespan via the pH of the growth media. Rapamycin prolonged the lifespan of nongrowing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond. PMID:23551936

  12. Expression of Genes Related to Oxidative Stress in Yeast Treated with Ionizing Radiation and N-acetyl -L-cysteine

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-10-15

    Ionizing radiation (IR) induces water radiolysis, which generates highly reactive hydroxyl radicals. Reactive oxygen species (ROS) cause apoptosis and cell damage including DNA strand breaks (DSBs), base damage, protein damage and lipid-hydroperoxide. Detoxifying enzymes are immediately triggered for ROS scavenging. Yeast contains two forms of superoxide dismutase (SOD). SOD1 as a cytosolic copper-zinc superoxide dismutase is located in the cytoplasm and cytosol. SOD2 as a manganese containing enzyme is act in mitochondria matrix and mitochondrion. These enzymes scavenge superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Yeast contains two catalases. Catalase A (CTA1) and Cytosolic catalase T (CTT1) is located in peroxisome and cytoplasm, respectively. Yeast has two glutathione (GSH) peroxidases, which are GPX1 and GPX2. GPX1 and GPX2 are component of cellular component and cytoplasm, respectively. The biochemical function of GSH peroxidase is to reduce lipid-hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. Otherwise, chemicals and materials help ROS detoxification against oxidative damage. N-acetyl-Lcysteine (NAC) having a thiol, a precursor for glutathione (GSH), is known as one of the antioxidants. In this study, we examined the effect of NAC through gene expressions related to protective enzyme against oxidative stress in yeast

  13. A stochastic model correctly predicts changes in budding yeast cell cycle dynamics upon periodic expression of CLN2.

    Directory of Open Access Journals (Sweden)

    Cihan Oguz

    Full Text Available In this study, we focus on a recent stochastic budding yeast cell cycle model. First, we estimate the model parameters using extensive data sets: phenotypes of 110 genetic strains, single cell statistics of wild type and cln3 strains. Optimization of stochastic model parameters is achieved by an automated algorithm we recently used for a deterministic cell cycle model. Next, in order to test the predictive ability of the stochastic model, we focus on a recent experimental study in which forced periodic expression of CLN2 cyclin (driven by MET3 promoter in cln3 background has been used to synchronize budding yeast cell colonies. We demonstrate that the model correctly predicts the experimentally observed synchronization levels and cell cycle statistics of mother and daughter cells under various experimental conditions (numerical data that is not enforced in parameter optimization, in addition to correctly predicting the qualitative changes in size control due to forced CLN2 expression. Our model also generates a novel prediction: under frequent CLN2 expression pulses, G1 phase duration is bimodal among small-born cells. These cells originate from daughters with extended budded periods due to size control during the budded period. This novel prediction and the experimental trends captured by the model illustrate the interplay between cell cycle dynamics, synchronization of cell colonies, and size control in budding yeast.

  14. Intra and Interspecific Variations of Gene Expression Levels in Yeast Are Largely Neutral: (Nei Lecture, SMBE 2016, Gold Coast).

    Science.gov (United States)

    Yang, Jian-Rong; Maclean, Calum J; Park, Chungoo; Zhao, Huabin; Zhang, Jianzhi

    2017-09-01

    It is commonly, although not universally, accepted that most intra and interspecific genome sequence variations are more or less neutral, whereas a large fraction of organism-level phenotypic variations are adaptive. Gene expression levels are molecular phenotypes that bridge the gap between genotypes and corresponding organism-level phenotypes. Yet, it is unknown whether natural variations in gene expression levels are mostly neutral or adaptive. Here we address this fundamental question by genome-wide profiling and comparison of gene expression levels in nine yeast strains belonging to three closely related Saccharomyces species and originating from five different ecological environments. We find that the transcriptome-based clustering of the nine strains approximates the genome sequence-based phylogeny irrespective of their ecological environments. Remarkably, only ∼0.5% of genes exhibit similar expression levels among strains from a common ecological environment, no greater than that among strains with comparable phylogenetic relationships but different environments. These and other observations strongly suggest that most intra and interspecific variations in yeast gene expression levels result from the accumulation of random mutations rather than environmental adaptations. This finding has profound implications for understanding the driving force of gene expression evolution, genetic basis of phenotypic adaptation, and general role of stochasticity in evolution. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

    Science.gov (United States)

    Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

    2014-10-06

    ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

  16. Construction of a Genetically Modified Wine Yeast Strain Expressing the Aspergillus aculeatus rhaA Gene, Encoding an α-l-Rhamnosidase of Enological Interest

    Science.gov (United States)

    Manzanares, Paloma; Orejas, Margarita; Gil, José Vicente; de Graaff, Leo H.; Visser, Jaap; Ramón, Daniel

    2003-01-01

    The Aspergillus aculeatus rhaA gene encoding an α-l-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a β-glucosidase, show increased content mainly of the aromatic compound linalool. PMID:14660415

  17. Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation.

    Science.gov (United States)

    Teixeira, Miguel C; Godinho, Cláudia P; Cabrito, Tânia R; Mira, Nuno P; Sá-Correia, Isabel

    2012-07-27

    The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased ³H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio

  18. Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation

    Directory of Open Access Journals (Sweden)

    Teixeira Miguel C

    2012-07-01

    Full Text Available Abstract Background The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC Superfamily and Major Facilitator Superfamily (MFS in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to

  19. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

    DEFF Research Database (Denmark)

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota

    2017-01-01

    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically...... exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein...... production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene...

  20. Expression of the bovine papillomavirus type 1, 2 and 4 L1 genes in the yeast Pichia pastoris.

    Science.gov (United States)

    Jesus, A L S; Mariz, F C; Souza, H M; Cordeiro, M N; Coimbra, E C; Leitão, M C G; Nascimento, L M; Stocco, R C; Beçak, W; Freitas, A C

    2012-08-16

    Papillomaviruses are known to cause benign or malignant lesions in various animals. In cattle, bovine papillomavirus (BPV) is the etiologic agent of papillomatosis and neoplasia of the upper gastrointestinal tract and urinary bladder. Currently, there are no standard diagnostic tests or prophylactic vaccines. Protection against papillomavirus infection is conferred by neutralizing antibodies directed towards the major structural protein L1. These antibodies can be efficiently induced by immunization with virus-like particles that are formed spontaneously after L1 gene expression in recombinant systems. The yeast Pichia pastoris is known to provide an efficient system for expression of proteins due to reduced cost and high levels of protein production. We evaluated P. pastoris for expression of the L1 gene from BPV1, BPV2 and BPV4. After methanol induction, the recombinants were able to produce L1 proteins of the three different BPV types. To increase heterologous L1 protein levels, a codon optimization strategy was used for production under bioreactor conditions. The BPV1 L1 protein was identified by monoclonal antibody anti-6xHis. This is the first report of BPV L1 expression in yeast.

  1. Identification of the sulphate ion as one of the key components of yeast spoilage of a sports drink through genome-wide expression analysis.

    Science.gov (United States)

    Jayakody, Lahiru N; Tsuge, Keisuke; Suzuki, Akihiro; Shimoi, Hitoshi; Kitagaki, Hiroshi

    2013-01-01

    Because of the growing market for sports drinks, prevention of yeast contamination of these beverages is of significant concern. This research was performed to achieve insight into the physiology of yeast growing in sports drinks through a genome-wide approach to prevent microbial spoilage of sports drinks. The genome-wide gene expression profile of Saccharomyces cerevisiae growing in the representative sports drink was investigated. Genes that were relevant to sulphate ion starvation response were upregulated in the yeast cells growing in the drink. These results suggest that yeast cells are suffering from deficiency of extracellular sulphate ions during growth in the sports drink. Indeed, the concentration of sulphate ions was far lower in the sports drink than in a medium that allows the optimal growth of yeast. To prove the starvation of sulphate ions of yeast, several ions were added to the beverage and its effects were investigated. The addition of sulphate ions, but not chloride ions or sodium ions, to the beverage stimulated yeast growth in the beverage in a dose-dependent manner. Moreover, the addition of sulphate ions to the sports drink increased the biosynthesis of sulphur-containing amino acids in yeast cells and hydrogen sulphide in the beverage. These results indicate that sulphate ion concentration should be regulated to prevent microbial spoilage of sports drinks.

  2. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture

    Directory of Open Access Journals (Sweden)

    Woo Tae Park

    2016-03-01

    Full Text Available The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L and silver nitrate (30 mg/L for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to the amount of yeast extract and silver nitrate. The transcript levels of HPPR under yeast extract treatment were 1.84-, 1.97-, and 2.86-fold higher than the control treatments after 3, 6, and 12 h, respectively, whereas PAL expression under silver nitrate treatment was 52.31-fold higher than in the non-treated controls after 24 h of elicitation. The concentration of rosmarinic acid was directly proportional to the concentration of the applied elicitors. Yeast extract supplementation documented the highest amount of rosmarinic acid at 4.98 mg/g, whereas silver nitrate addition resulted in a comparatively lower amount of rosmarinic acid at 0.65 mg/g. In conclusion, addition of yeast extract to the cell cultures enhanced the accumulation of rosmarinic acid, which was evidenced by the expression levels of the phenylpropanoid biosynthetic pathway genes in A. rugosa.

  3. Yeast Extract and Silver Nitrate Induce the Expression of Phenylpropanoid Biosynthetic Genes and Induce the Accumulation of Rosmarinic Acid in Agastache rugosa Cell Culture.

    Science.gov (United States)

    Park, Woo Tae; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Yeo, Sun Kyung; Jeon, Jin; Park, Jong Seok; Lee, Sook Young; Park, Sang Un

    2016-03-29

    The present study aimed to investigate the role of yeast extract and silver nitrate on the enhancement of phenylpropanoid pathway genes and accumulation of rosmarinic acid in Agastache rugosa cell cultures. The treatment of cell cultures with yeast extract (500 mg/L) and silver nitrate (30 mg/L) for varying times enhanced the expression of genes in the phenylpropanoid pathway and the production of rosmarinic acid. The results indicated that the expression of RAS and HPPR was proportional to the amount of yeast extract and silver nitrate. The transcript levels of HPPR under yeast extract treatment were 1.84-, 1.97-, and 2.86-fold higher than the control treatments after 3, 6, and 12 h, respectively, whereas PAL expression under silver nitrate treatment was 52.31-fold higher than in the non-treated controls after 24 h of elicitation. The concentration of rosmarinic acid was directly proportional to the concentration of the applied elicitors. Yeast extract supplementation documented the highest amount of rosmarinic acid at 4.98 mg/g, whereas silver nitrate addition resulted in a comparatively lower amount of rosmarinic acid at 0.65 mg/g. In conclusion, addition of yeast extract to the cell cultures enhanced the accumulation of rosmarinic acid, which was evidenced by the expression levels of the phenylpropanoid biosynthetic pathway genes in A. rugosa.

  4. Microarray analysis of gene expression profile induced by the biocontrol yeast Cryptococcus laurentii in cherry tomato fruit.

    Science.gov (United States)

    Jiang, Feng; Zheng, Xiaodong; Chen, Jishuang

    2009-02-01

    To obtain an overall view on transcript modification during the cherry tomato fruit responding to biocontrol yeast Cryptococcus laurentii, we performed a microarrary analysis, using Affymetrix Tomato Genechip arrays, representing approximately 10,000 genes. The results showed that 194 and 312 genes were up- or down-regulated, respectively, more then ten time fold (log2 change ratio>or=2 or log2 change ratiotomato fruit as compared with control fruits. Those up-regulated included genes involved in metabolism, signal transduction, and stress response. Conversely, genes related to energy metabolism and photosynthesis were generally down-regulated. Our results suggest that biocontrol yeast treatment induces fruit resistance response, suppresses energy metabolism and photosynthesis. A number of transcripts encoding proteins/enzymes which are known to be up-regulated under some biotic and abiotic stress are also up-regulated after the applying biological control yeast to cherry tomato fruit. The expression of these proteins might increase the fruit resistance towards postharvest pathogen infection and damage.

  5. Selenium-enriched milk proteins and selenium yeast affect selenoprotein activity and expression differently in mouse colon.

    Science.gov (United States)

    Hu, Ying; McIntosh, Graeme H; Le Leu, Richard K; Young, Graeme P

    2010-07-01

    Certain forms of dietary Se may have an advantage in improving Se status and reducing cancer risk. The present study compared the effects of an Se-enriched milk protein product (dairy-Se) with an Se yeast (yeast-Se) on selenoprotein activity and expression in the mouse colon. Mice were fed four diets for 4 weeks: a control milk protein diet (Se at 0.068 parts per million (ppm)), dairy-Se diets with Se at 0.5 and 1 ppm, and a yeast-Se diet with Se at 1 ppm. Cytosolic glutathione peroxidase-1 (GPx-1) activity, mRNA of selenoprotein P (SeP), GPx-1, gastrointestinal glutathione peroxidase-2 (GPx-2) and thioredoxin reductase-1 (TrxR-1) were examined in the mouse colon. Dairy-Se diets did not significantly affect GPx-1 mRNA and GPx-1 activity but produced a dose-dependent increase in SeP and GPx-2 mRNA, with a significantly higher level achieved at 1 ppm Se (P supplement had any effect on TrxR-1. The present study indicates that selenoprotein levels in the mouse colon are regulated differently depending on the Se supplement. As we have previously shown that dairy-Se at 1 ppm was protective against colorectal cancer (CRC) in an azoxymethane-induced CRC mouse model, this up-regulation of colonic GPx-2 and SeP with Se supplementation may be crucial to its chemopreventive action.

  6. Thermotolerant yeasts selected by adaptive evolution express heat stress response at 30ºC

    DEFF Research Database (Denmark)

    Caspeta, Luis; Chen, Yun; Nielsen, Jens

    2016-01-01

    Exposure to long-term environmental changes across >100s of generations results in adapted phenotypes, but little is known about how metabolic and transcriptional responses are optimized in these processes. Here, we show that thermotolerant yeast strains selected by adaptive laboratory evolution ...

  7. Expression of the Salmonella Spp. Virulence Factor SifA in Yeast Alters Rho1 Activity on Peroxisomes

    Science.gov (United States)

    Vinh, Dani B. N.; Ko, Dennis C.; Rachubinski, Richard A.; Aitchison, John D.

    2010-01-01

    The Salmonella typhimurium effector protein SifA regulates the assembly and tubulation of the Salmonella phagosome. SifA localizes to the phagosome and interacts with the membrane via its prenylated tail. SifA is a structural homologue of another bacterial effector that acts as a GTP-exchange factor for Rho family GTPases and can bind GDP-RhoA. When coexpressed with a bacterial lipase that is activated by RhoA, SifA can induce tubulation of mammalian endosomes. In an effort to develop a genetic system to study SifA function, we expressed SifA and characterized its activity in yeast. GFP-SifA predominantly localized to yeast peroxisomal membranes. Under peroxisome-inducing conditions, GFP-SifA reduced the number of free peroxisomes and promoted the formation of large peroxisomes with membrane invaginations. GFP-SifA activity depended on the recruitment to peroxisomes of wild-type Rho1p and Pex25p, a receptor for Rho1p. GFP-SifA could also rescue the actin organization defects in pex25Δ and rho1 mutants, suggesting that SifA may recruit and potentiate Rho1p activity. We reexamined the distribution of GFP-SifA in mammalian cells and found the majority colocalizing with LAMP1-positive compartment and not with the peroxisomal marker PMP70. Together, these data suggest that SifA may use a similar mode of action via Rho proteins to alter yeast peroxisomal and mammalian endosomal membranes. Further definition of SifA activity on yeast peroxisomes could provide more insight into its role in regulating host membrane dynamics and small GTPases. PMID:20739463

  8. Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Hsien-Wei Meng

    Full Text Available SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. Particularly, selected DNA aptamers against VEGF were specific and of high affinity (K(D  = ∼ 1 nM and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.

  9. UV-dependent production of 25-hydroxyvitamin D2 in the recombinant yeast cells expressing human CYP2R1.

    Science.gov (United States)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Sakaki, Toshiyuki

    2013-05-03

    CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D3 or vitamin D2 was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D2 was produced without additional vitamin D2. Endogenous ergosterol was likely converted into vitamin D2 by UV irradiation and thermal isomerization, and then the resulting vitamin D2 was converted to 25-hydroxyvitamin D2 by CYP2R1. This novel method for producing 25-hydroxyvitamin D2 without a substrate could be useful for practical purposes. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Quantitative measurement of allele-specific protein expression in a diploid yeast hybrid by LC-MS.

    Science.gov (United States)

    Khan, Zia; Bloom, Joshua S; Amini, Sasan; Singh, Mona; Perlman, David H; Caudy, Amy A; Kruglyak, Leonid

    2012-01-01

    Understanding the genetic basis of gene regulatory variation is a key goal of evolutionary and medical genetics. Regulatory variation can act in an allele-specific manner (cis-acting) or it can affect both alleles of a gene (trans-acting). Differential allele-specific expression (ASE), in which the expression of one allele differs from another in a diploid, implies the presence of cis-acting regulatory variation. While microarrays and high-throughput sequencing have enabled genome-wide measurements of transcriptional ASE, methods for measurement of protein ASE (pASE) have lagged far behind. We describe a flexible, accurate, and scalable strategy for measurement of pASE by liquid chromatography-coupled mass spectrometry (LC-MS). We apply this approach to a hybrid between the yeast species Saccharomyces cerevisiae and Saccharomyces bayanus. Our results provide the first analysis of the relative contribution of cis-acting and trans-acting regulatory differences to protein expression divergence between yeast species.

  11. Effect of heterologous expression of acyl-CoA-binding protein on acyl-CoA level and composition in yeast

    DEFF Research Database (Denmark)

    Mandrup, S; Jepsen, R; Skøtt, H

    1993-01-01

    We have expressed a bovine synthetic acyl-CoA-binding protein (ACBP) gene in yeast (Saccharomyces cerevisiae) under the control of the GAL1 promoter. The heterologously expressed bovine ACBP constituted up to 6.4% of total cellular protein and the processing was identical with that of native bovine...... ACBP, i.e. the initiating methionine was removed and the following serine residue was N-acetylated. The expression of this protein did not affect the growth rate of the cells. Determination of the yeast acyl-CoA pool size showed a close positive correlation between the ACBP content of the cells...

  12. Production of L-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases.

    Science.gov (United States)

    Ilmén, Marja; Koivuranta, Kari; Ruohonen, Laura; Rajgarhia, Vineet; Suominen, Pirkko; Penttilä, Merja

    2013-05-25

    Polylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding L-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expressed in a new host organism, the non-conventional yeast Candida sonorensis, with or without the competing ethanol fermentation pathway. Each LDH strain produced substantial amounts of lactate, but the properties of the heterologous LDH affected the distribution of carbon between lactate and by-products significantly, which was reflected in extra-and intracellular metabolite concentrations. Under neutralizing conditions C. sonorensis expressing L. helveticus LDH accumulated lactate up to 92 g/l at a yield of 0.94 g/g glucose, free of ethanol, in minimal medium containing 5 g/l dry cell weight. In rich medium with a final pH of 3.8, 49 g/l lactate was produced. The fermentation pathway was modified in some of the strains studied by deleting either one or both of the pyruvate decarboxylase encoding genes, PDC1 and PDC2. The deletion of both PDC genes together abolished ethanol production and did not result in significantly reduced growth characteristic to Saccharomyces cerevisiae deleted of PDC1 and PDC5. We developed an organism without previous record of genetic engineering to produce L-lactic acid to a high concentration, introducing a novel host for the production of an industrially important metabolite, and opening the way for exploiting C. sonorensis in additional biotechnological applications. Comparison of metabolite production, growth, and enzyme activities in a representative set of transformed strains expressing different LDH genes in the presence and absence of a functional

  13. Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containing materials.

    Science.gov (United States)

    Zhao, Chun-Hai; Cui, Wei; Liu, Xiao-Yan; Chi, Zhen-Ming; Madzak, Catherine

    2010-11-01

    Yarrowia lipolytica ACA-DC 50109 has been reported to be an oleaginous yeast and significant quantities of lipids were accumulated inside the yeast cells. In this study, the INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the expression plasmid pINA1317 and expressed in the cells of the oleaginous yeast. The activity of the inulinase with 6 × His tag secreted by the transformant Z31 obtained was found to be 41.7U mL(-1) after cell growth for 78 h. After optimization of the medium and cultivation conditions for single cell oil production, the transformant could accumulate 46.3% (w/w) oil from inulin in its cells and cell dry weight was 11.6 g L(-1) within 78 h at the flask level. During the 2-L fermentation, the transformant could accumulate 48.3% (w/w) oil from inulin in its cells and cell dry weight was 13.3 g L(-1) within 78 h while the transformant could accumulate 50.6% (w/w) oil from extract of Jerusalem artichoke tubers in its cells and cell dry weight was 14.6 g L(-1) within 78 h. At the end of fermentation, most of the added sugar was utilized by the transformant cells. Over 91.5% of the fatty acids from the transformant cultivated in the extract of Jerusalem artichoke tubercles was C(16:0), C(18:1) and C(18:2), especially C(18:1) (58.5%). Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

    Science.gov (United States)

    Lamping, Erwin; Niimi, Masakazu; Cannon, Richard D

    2013-07-29

    A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited

  15. Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

    Science.gov (United States)

    2013-01-01

    Background A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Results Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (

  16. Construction of a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA gene, encoding an -L-Rhamnosidase of enological interest

    NARCIS (Netherlands)

    Manzanares, P.; Orejas, M.; Vicente Gil, J.; Graaff, de L.H.; Visser, J.; Ramon, D.

    2003-01-01

    The Aspergillus aculeatus rhaA gene encoding an alpha-L-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain

  17. A PCR-based strategy to generate yeast strains expressing endogenous levels of amino-terminal epitope-tagged proteins.

    Science.gov (United States)

    Booher, Keith R; Kaiser, Peter

    2008-04-01

    An epitope tag introduced to a gene of interest (GOI) greatly increases the ease of studying cellular proteins. Rapid PCR-based strategies for epitope tagging a protein's C-terminus at its native gene locus are widely used in yeast. C-terminal epitope tagging is not suitable for all proteins, however. Epitope tags fused to the C-terminus can interfere with function of some proteins or can even be removed by C-terminal protein processing. To overcome such problems, proteins can be tagged with epitopes at their amino-termini, but generating yeast strains expressing N-terminal epitope tagged genes under control of the endogenous promoter at the native locus is comparatively more difficult. Strategies to introduce N-terminal epitope tags have been reported previously but often introduce additional sequences other than the epitope tag into the genome. Furthermore, N-terminal tagging of essential genes by current methods requires formation of diploid strains followed by tetrad dissection or expression of an additional copy of the GOI from a plasmid. The strategies described here provide a quick, facile means of epitope tagging the N-terminus of both essential and nonessential genes in a two-step PCR-based procedure. The procedure has the significant advantage of leaving tagged genes under the control of their endogenous promoters, and no additional sequences other than the epitope tag encoding nucleotides are inserted into the genome.

  18. Expression of heterologous transporters in Saccharomyces kudriavzevii: A strategy for improving yeast salt tolerance and fermentation performance.

    Science.gov (United States)

    Dibalova-Culakova, Hana; Alonso-Del-Real, Javier; Querol, Amparo; Sychrova, Hana

    2018-03-02

    S. kudriavzevii has potential for fermentations and other biotechnological applications, but is sensitive to many types of stress. We tried to increase its tolerance and performance via the expression of various transporters from different yeast species. Whereas the overexpression of Z. rouxii fructose uptake systems (ZrFfz1 and ZrFsy1) or a glycerol importer (ZrStl1) did not improve the ability of S. kudriavzevii to consume fructose and survive osmotic stress, the expression of alkali-metal-cation exporters (ScEna1, ScNha1, YlNha2) improved S. kudriavzevii salt tolerance, and that of ScNha1 also the fermentation performance. The level of improvement depended on the type and activity of the transporter suggesting that the natural sensitivity of S. kudriavzevii cells to salts is based on a non-optimal functioning of its own transporters. Copyright © 2018. Published by Elsevier B.V.

  19. UV-dependent production of 25-hydroxyvitamin D{sub 2} in the recombinant yeast cells expressing human CYP2R1

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi; Kamakura, Masaki [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan); Ohta, Miho [Department of Food and Nutrition Management Studies, Faculty of Human Development, Soai University, 4-4-1 Nanko-naka, Suminoe-ku, Osaka 559-0033 (Japan); Sakaki, Toshiyuki, E-mail: tsakaki@pu-toyama.ac.jp [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan)

    2013-05-03

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D{sub 3} or vitamin D{sub 2} was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D{sub 2} was produced without additional vitamin D{sub 2}. Endogenous ergosterol was likely converted into vitamin D{sub 2} by UV irradiation and thermal isomerization, and then the resulting vitamin D{sub 2} was converted to 25-hydroxyvitamin D{sub 2} by CYP2R1. This novel method for producing 25-hydroxyvitamin D{sub 2} without a substrate could be useful for practical purposes.

  20. Inferring condition-specific modulation of transcription factor activity in yeast through regulon-based analysis of genomewide expression.

    Directory of Open Access Journals (Sweden)

    André Boorsma

    Full Text Available BACKGROUND: A key goal of systems biology is to understand how genomewide mRNA expression levels are controlled by transcription factors (TFs in a condition-specific fashion. TF activity is frequently modulated at the post-translational level through ligand binding, covalent modification, or changes in sub-cellular localization. In this paper, we demonstrate how prior information about regulatory network connectivity can be exploited to infer condition-specific TF activity as a hidden variable from the genomewide mRNA expression pattern in the yeast Saccharomyces cerevisiae. METHODOLOGY/PRINCIPAL FINDINGS: We first validate experimentally that by scoring differential expression at the level of gene sets or "regulons" comprised of the putative targets of a TF, we can accurately predict modulation of TF activity at the post-translational level. Next, we create an interactive database of inferred activities for a large number of TFs across a large number of experimental conditions in S. cerevisiae. This allows us to perform TF-centric analysis of the yeast regulatory network. CONCLUSIONS/SIGNIFICANCE: We analyze the degree to which the mRNA expression level of each TF is predictive of its regulatory activity. We also organize TFs into "co-modulation networks" based on their inferred activity profile across conditions, and find that this reveals functional and mechanistic relationships. Finally, we present evidence that the PAC and rRPE motifs antagonize TBP-dependent regulation, and function as core promoter elements governed by the transcription regulator NC2. Regulon-based monitoring of TF activity modulation is a powerful tool for analyzing regulatory network function that should be applicable in other organisms. Tools and results are available online at http://bussemakerlab.org/RegulonProfiler/.

  1. Global Effects on Gene Expression in Fission Yeast by Silencing and RNA Interference Machineries

    DEFF Research Database (Denmark)

    Hansen, Klavs R.; Burns, G.; Mata, J.

    2005-01-01

    sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr...... by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing....

  2. Gln3-Gcn4 hybrid transcriptional activator determines catabolic and biosynthetic gene expression in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Hernández, Hugo; Aranda, Cristina; Riego, Lina; González, Alicia

    2011-01-21

    The yeast Saccharomyces cerevisiae is able to sense the availability and quality of nitrogen sources and the intrinsic variation of amino acid disponibility for protein synthesis. When this yeast is provided with secondary nitrogen sources, transcription of genes encoding enzymes involved in their catabolism is elicited through the action of Gln3, which constitutes the main activator of the Nitrogen Catabolite Repression network (NCR). Activation of genes encoding enzymes involved in the amino acid biosynthetic pathways is achieved through the action of the GCN4-encoded transcriptional modulator whose transcriptional activation is induced at the translational level by limitation for any amino acid. Thus the role of each one of these activators had been secluded to either catabolic or biosynthetic pathways. However, some observations have suggested that under peculiar physiological conditions, Gln3 and Gcn4 could act simultaneously in order to contemporaneously increase expression of both sets of genes. This paper addresses the question of whether Gln3 and Gcn4 cooperatively determine expression of their target genes. Results presented herein show that induced expression of catabolic and biosynthetic genes when cells are grown under nitrogen derepressive conditions and amino acid deprivation is dependent on the concurrent action of Gln3 and Gcn4, which form part of a unique transcriptional complex. We propose that the combination of Gln3 and Gcn4 results in the constitution of a hybrid modulator which elicits a novel transcriptional response, not evoked when these modulators act in a non-combinatorial fashion. Copyright © 2010 Elsevier Inc. All rights reserved.

  3. Integrative Expression of Glucoamylase Gene in a Brewer’s Yeast Saccharomyces pastorianus Strain

    Directory of Open Access Journals (Sweden)

    Guangyi Zhang

    2008-01-01

    Full Text Available The recombinant brewer’s yeast Saccharomyces pastorianus strain was constructed byintroducing the ilv2:GLA fragment released from pMGI6, carrying glucoamylase gene (GLA and using the yeast α-acetolactate synthase gene (ILV2 as the recombination sequence. The strain was able to utilise starch as the sole carbon source, its glucoamylase activity was 6.3 U/mL and its α-acetolactate synthase activity was lowered by 33.3 %. The introduced GLA gene was integrated at the recipient genomic ILV2 gene, one copy of ILV2 gene was disrupted and the other copy remained intact. Primary wort fermentation test confirmed that the diacetyl and residual sugar concentration in the wort fermented by the recombinant strain were reduced by 65.6 and 34.2 % respectively, compared to that of the recipient strain. Under industrial operating conditions, the maturation time of beer fermented by the recombinant strain was reduced from 7 to 4 days, there were no significant differences in the appearance and mouthfeel, and the beer satisfied the high quality demands. That is why the strain could be used in beer production safely.

  4. Novel wine-mediated FLO11 flocculation phenotype of commercial Saccharomyces cerevisiae wine yeast strains with modified FLO gene expression.

    Science.gov (United States)

    Govender, Patrick; Kroppenstedt, Sven; Bauer, Florian F

    2011-04-01

    Depending on the genetic background of Saccharomyces strains, a wide range of phenotypic adhesion identities can be directly attributed to the FLO11-encoded glycoprotein, which includes asexual flocculation, invasive growth and pseudohyphal formation, flor formation and adhesion to biotic and abiotic surfaces. In a previous study, we reported that HSP30-mediated stationary-phase expression of the native chromosomal FLO11 ORF in two nonflocculent commercial Saccharomyces cerevisiae wine yeast strains, BM45 or VIN13 did not generate a flocculent phenotype under either standard laboratory media or synthetic MS300 must fermentation conditions. In the present study, the BM45- and VIN13-derived HSP30p-FLO11 wine yeast transformants were observed to be exclusively and strongly flocculent under authentic red wine-making conditions, thus suggesting that this specific fermentation environment specifically contributes to the development of a flocculent phenotype, which is insensitive to either glucose or mannose. Furthermore, irrespective of the strain involved this phenotype displayed both Ca(2+)-dependent and Ca(2+)-independent flocculation characteristics. A distinct advantage of this unique FLO11-based phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by BM45-F11H and VIN13-F11H transformants were significantly less turbid than those produced by their wild-type parental strains. © 2011 University of Stellenbosch. FEMS Microbiology Letters © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

  5. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    Science.gov (United States)

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L

    2003-02-01

    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

  6. The suppressor of AAC2 Lethality SAL1 modulates sensitivity of heterologously expressed artemia ADP/ATP carrier to bongkrekate in yeast.

    Science.gov (United States)

    Wysocka-Kapcinska, Monika; Torocsik, Beata; Turiak, Lilla; Tsaprailis, George; David, Cynthia L; Hunt, Andrea M; Vekey, Karoly; Adam-Vizi, Vera; Kucharczyk, Roza; Chinopoulos, Christos

    2013-01-01

    The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner.

  7. Differential gene expression and Hog1 interaction with osmoresponsive genes in the extremely halotolerant black yeast Hortaea werneckii

    Directory of Open Access Journals (Sweden)

    Plemenitaš Ana

    2007-08-01

    Full Text Available Abstract Background Fluctuations in external salinity force eukaryotic cells to respond by changes in the gene expression of proteins acting in protective biochemical processes, thus counteracting the changing osmotic pressure. The high-osmolarity glycerol (HOG signaling pathway is essential for the efficient up-regulation of the osmoresponsive genes. In this study, the differential gene expression of the extremely halotolerant black yeast Hortaea werneckii was explored. Furthermore, the interaction of mitogen-activated protein kinase HwHog1 and RNA polymerase II with the chromatin in cells adapted to an extremely hypersaline environment was analyzed. Results A cDNA subtraction library was constructed for H. werneckii, adapted to moderate salinity or an extremely hypersaline environment of 4.5 M NaCl. An uncommon osmoresponsive set of 95 differentially expressed genes was identified. The majority of these had not previously been connected with the adaptation of salt-sensitive S. cerevisiae to hypersaline conditions. The transcriptional response in hypersaline-adapted and hypersaline-stressed cells showed that only a subset of the identified genes responded to acute salt-stress, whereas all were differentially expressed in adapted cells. Interaction with HwHog1 was shown for 36 of the 95 differentially expressed genes. The majority of the identified osmoresponsive and HwHog1-dependent genes in H. werneckii have not been previously reported as Hog1-dependent genes in the salt-sensitive S. cerevisiae. The study further demonstrated the co-occupancy of HwHog1 and RNA polymerase II on the chromatin of 17 up-regulated and 2 down-regulated genes in 4.5 M NaCl-adapted H. werneckii cells. Conclusion Extremely halotolerant H. werneckii represents a suitable and highly relevant organism to study cellular responses to environmental salinity. In comparison with the salt-sensitive S. cerevisiae, this yeast shows a different set of genes being expressed at

  8. Combining Spinach-tagged RNA and gene localization to image gene expression in live yeast.

    Science.gov (United States)

    Guet, David; Burns, Laura T; Maji, Suman; Boulanger, Jérôme; Hersen, Pascal; Wente, Susan R; Salamero, Jean; Dargemont, Catherine

    2015-11-19

    Although many factors required for the formation of export-competent mRNPs have been described, an integrative view of the spatiotemporal coordinated cascade leading mRNPs from their site of transcription to their site of nuclear exit, at a single cell level, is still partially missing due to technological limitations. Here we report that the RNA Spinach aptamer is a powerful tool for mRNA imaging in live S. cerevisiae with high spatial-temporal resolution and no perturbation of the mRNA biogenesis properties. Dedicated image processing workflows are developed to allow detection of very low abundance of transcripts, accurate quantitative dynamic studies, as well as to provide a localization precision close to 100 nm at consistent time scales. Combining these approaches has provided a state-of-the-art analysis of the osmotic shock response in live yeast by localizing induced transcription factors, target gene loci and corresponding transcripts.

  9. Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Weninger, Astrid; Hatzl, Anna-Maria; Schmid, Christian; Vogl, Thomas; Glieder, Anton

    2016-10-10

    The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is one of the most commonly used expression systems for heterologous protein production. However the recombination machinery in P. pastoris is less effective in contrast to Saccharomyces cerevisiae, where efficient homologous recombination naturally facilitates genetic modifications. The lack of simple and efficient methods for gene disruption and specifically integrating cassettes has remained a bottleneck for strain engineering in P. pastoris. Therefore tools and methods for targeted genome modifications are of great interest. Here we report the establishment of CRISPR/Cas9 technologies for P. pastoris and demonstrate targeting efficiencies approaching 100%. However there appeared to be a narrow window of optimal conditions required for efficient CRISPR/Cas9 function for this host. We systematically tested combinations of various codon optimized DNA sequences of CAS9, different gRNA sequences, RNA Polymerase III and RNA Polymerase II promoters in combination with ribozymes for the expression of the gRNAs and RNA Polymerase II promoters for the expression of CAS9. Only 6 out of 95 constructs were functional for efficient genome editing. We used this optimized CRISPR/Cas9 system for gene disruption studies, to introduce multiplexed gene deletions and to test the targeted integration of homologous DNA cassettes. This system allows rapid, marker-less genome engineering in P. pastoris enabling unprecedented strain and metabolic engineering applications. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

    Science.gov (United States)

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

  11. 5´-UTR introns enhance protein expression in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Hoshida, Hisashi; Kondo, Masaki; Kobayashi, Takafumi; Yarimizu, Tohru; Akada, Rinji

    2017-01-01

    Saccharomyces cerevisiae is one of the most suitable microorganisms for recombinant protein production. To enhance protein production, various expression systems have been intensively studied. However, the effect of introns on protein expression has not been examined deeply in S. cerevisiae. In this study, we analyzed the effect of some introns on protein expression. RPS25A, RPS26A, and RPS26B contain single introns within the 5´-untranslated regions (5´-UTRs), and RPS24A has an intron just downstream of the initiation codon. Expression activity of the promoter regions containing introns (intron promoters) were analyzed by luciferase reporter assays. These intron promoters showed higher expression than the TDH3 promoter (TDH3p), which is one of the strongest promoters in S. cerevisiae. Deletion of the introns from these promoters decreased luciferase expression, indicating that introns have a role in enhancing protein expression. To develop artificial strong intron promoters, several chimeric promoters were constructed using the TDH3p and the RPS25A intron promoter. A construct containing the entire TDH3p followed by the RPS25A intron showed about 50-fold higher expression than the TDH3p alone. Inducible expressions driven by the GAL10 promoter and the CUP1 promoter were also enhanced by the RPS25A intron. However, enhancement of mRNA accumulation by the TDH3p and the GAL10 promoter with the RPS25A intron was lower than the effect on luciferase activity, suggesting that the intron affects post-transcriptionally. The chimeric promoter, TDH3p-RPS25A-intron, enhanced expressions of some, but not all proteins examined, indicating that 5'-UTR introns increase production of a certain type of recombinant proteins in S. cerevisiae.

  12. Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

    Directory of Open Access Journals (Sweden)

    Farnaz Nikzad Jamnani

    2013-11-01

    Full Text Available Introduction: Fossil fuel is an expensive and finite energy source. Therefore, the use of renewable energy and biofuels production has been taken into consideration. One of the most suitable raw materials for biofuels is cellulosic compounds. Only microorganisms that contain cellulose enzymes can decompose cellulose and fungus of Trichodermareesei is the most important producer of this enzyme. Methods: In this study the nucleotide sequence of endoglucanase II, which is the starter of attack to cellulose chains, synthesized from amino acid sequence of this enzyme in fungus T.reesei and based on codon usage in the host; yeast Pichiapastoris. To produce optimized enzyme and to decrease the production time and enzyme price, protein engineering will be used. There are some methods to improve the enzymatic properties like site-directed mutagenesis in which amino-acid replacement occur. In this study two mutations were induced in endoglucanase enzyme gene by PCR in which free syctein positions 169 and 393 were switched to valine and histidine respectively. Then this gene was inserted into the pPinka expression vector and cloned in Escherichia coli. The recombinant plasmids were transferred into P.pastoris competent cells with electroporation, recombinant yeasts were cultured in BMMY medium and induced with methanol. Results: The sequencing of gene proved the induction of the two mutations and the presence of recombinant enzyme was confirmed by dinitrosalicilic acid method and SDS-PAGE. Conclusion: Examination of biochemical properties revealed that the two mutations simultaneously decreased catalytic power, thermal stability and increased the affinity of enzyme and substrate.

  13. Effects of N-acetyl-L-cysteine on gene expression of antioxidant enzymes in yeast cells after irradiation

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-04-15

    Ionizing radiation induces water radiolysis, which generates highly reactive hydroxyl radicals. Reactive oxygen species (ROS) cause apoptosis and cell damage. When exposed to ionizing radiation, cells activates ROS scavenging detoxifying enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase. SOD scavenges superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen. The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen. Yeast has two catalase and three GPx proteins. The biochemical function of GPx is to reduce lipid-hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. N-acetylL-cysteine (NAC) having a thiol, a precursor for glutathione (GSH), is known as one of the antioxidants. NAC prevents the depletion of GSH by radiation, increases the production of GSH, and improves enzymes activity and alkaline phosphatase. In this study, the role of NAC as an antioxidant and a radioprotector was examined on cell survival, transcriptional level, and protein level. through observing viability of cells, analyzing the gene expression of antioxidant enzyme, measuring the SOD activity and intracellular GSH levels in yeast W303-1A strain The cell viability of haploid S. cerevisiae W303-1A strain was reduced significantly at the low dose (10∼30 Gy). The half-lethal dose of the strain was about 20 Gy. The CFU assay result confirmed that NAC could not rescue the cells from radiation-induced death. When irradiated with 100 Gy, an increase in the transcriptional expression was observed in the antioxicant genes. The expression of these genes decreased by treatment of NAC in irradiated cells. NAC decline SOD activity and intracellular GSH levels. The present study shows that NAC can directly scavenge

  14. Natural genetic variation impacts expression levels of coding, non-coding, and antisense transcripts in fission yeast

    DEFF Research Database (Denmark)

    Clément-Ziza, Mathieu; Marsellach, Francesc X.; Codlin, Sandra

    2014-01-01

    Our current understanding of how natural genetic variation affects gene expression beyond well-annotated coding genes is still limited. The use of deep sequencing technologies for the study of expression quantitative trait loci (eQTLs) has the potential to close this gap. Here, we generated...... to be affected by eQTLs as protein-coding RNAs. We identified a genetic variation of swc5 that modifies the levels of 871 RNAs, with effects on both sense and antisense transcription, and show that this effect most likely goes through a compromised deposition of the histone variant H2A.Z. The strains, methods...... the first recombinant strain library for fission yeast and conducted an RNA-seq-based QTL study of the coding, non-coding, and antisense transcriptomes. We show that the frequency of distal effects (trans-eQTLs) greatly exceeds the number of local effects (cis-eQTLs) and that non-coding RNAs are as likely...

  15. Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

    Directory of Open Access Journals (Sweden)

    Athmaram TN

    2011-11-01

    Full Text Available Abstract Currently available vaccines for the pandemic Influenza A (H1N1 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

  16. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric; Suominen, Pirkko

    2010-12-07

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. Methods of producing ethanol include utilizing these modified yeast strains. ##STR00001##

  17. Global gene expression in recombinant and non-recombinant yeast Saccharomyces cerevisiae in three different metabolic states.

    Science.gov (United States)

    Díaz, H; Andrews, B A; Hayes, A; Castrillo, J; Oliver, S G; Asenjo, J A

    2009-01-01

    Global gene expression of two strains of Saccharomyces cerevisiae, one recombinant (P+), accumulating large amounts of an intracellular protein Superoxide Dismutase (SOD) and one non-recombinant (P-) which does not contain the recombinant plasmid, were compared in batch culture during diauxic growth when cells were growing exponentially on glucose, when they were growing exponentially on ethanol, and in the early stationary phase when glycerol was being utilized. When comparing the gene expression for P- (and P+) during growth on ethanol to that on glucose (Eth/Gluc), overexpression is related to an increase in consumption of glycerol, activation of the TCA cycle, degradation of glycogen and metabolism of ethanol. Furthermore, 97.6% of genes (80 genes) involved in the central metabolic pathway are overexpressed. This is similar to that observed by DeRisi et al. [DeRisi, J.L., Iyer, V.R. & Brown, P.O. 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680-686.] but very different from was observed for Metabolic Flux Analysis (MFA), where the specific growth rate is lowered to ca. 40%, the fluxes in the TCA cycle are reduced to ca. 40% (to 30% in P+), glycolysis is reduced to virtually 0 and protein synthesis to ca. 50% (to 40% in P+). Clearly it is not possible to correlate in a simple or direct way, quantitative mRNA expression levels with cell function which is shown by the Metabolic Flux Analysis (MFA). When comparing the two strains in the 3 growth stages, 4 genes were found to be under or overexpressed in all cases. The products of all of these genes are expressed at the plasma membrane or cell wall of the yeast. While comparing the strains (P+/P-) when growing on glucose, ethanol and in the early stationary phase, many of the genes of the central metabolic pathways are underexpressed in P+, which is similar to the behaviour of the metabolic fluxes of both strains (MFA). Comparing the gene expression for P- (and

  18. Salt-dependent expression of ammonium assimilation genes in the halotolerant yeast, Debaryomyces hansenii.

    Science.gov (United States)

    Guerrero, Carlos A; Aranda, Cristina; Deluna, Alexander; Filetici, Patrizia; Riego, Lina; Anaya, Víctor Hugo; González, Alicia

    2005-03-01

    Debaryomyces hansenii is adapted to grow in saline environments, accumulating high intracellular Na(+) concentrations. Determination of the DhGDH1-encoded NADP-glutamate dehydrogenase enzymatic activity showed that it increased in a saline environment. Thus, it was proposed that, in order to overcome Na(+) inhibition of enzyme activity, this organism possessed salt-dependent mechanisms which resulted in increased activity of enzymes pertaining to the central metabolic pathways. However, the nature of the mechanisms involved in augmented enzyme activity were not analyzed. To address this matter, we studied the expression of DhGDH1 and DhGLN1 encoding glutamine synthetase, which constitute the central metabolic circuit involved in ammonium assimilation. It was found that: (1) expression of DhGDH1 is increased when D. hansenii is grown in the presence of high NaCl concentrations, while that of DhGLN1 is reduced, (2) DhGDH1 expression in Saccharomyces cerevisiae takes place in a GLN3- and HAP2,3-dependent manner and (3) salt-dependent DhGDH1 and DhGLN1 expression involves mechanisms which are limited to D. hansenii and are not present in S. cerevisiae. Thus, salt-dependent regulation of the genes involved in central metabolic pathways could form part of a strategy leading to the ability to grow under hypersaline conditions.

  19. (H5N1) strain neuraminidase expressed in yeast Pichia pastoris

    Indian Academy of Sciences (India)

    2014-03-20

    Mar 20, 2014 ... The colonies were subcultured on YPD broth and gene integration was confirmed as described at ww.invitrogen.com/content/sfs/manuals/pich_man.pdf. 2.4 Expression of rNA in Pichia pastoris. The positive transformants were inoculated into 2 mL of. Buffered Glycerol-complex Medium (BMGY) media and.

  20. Novel functional residues in the core domain of histone H2B regulate yeast gene expression and silencing and affect the response to DNA damage.

    Science.gov (United States)

    Kyriss, McKenna N M; Jin, Yi; Gallegos, Isaura J; Sanford, James A; Wyrick, John J

    2010-07-01

    Previous studies have identified novel modifications in the core fold domain of histone H2B, but relatively little is known about the function of these putative histone modification sites. We have mutated core modifiable residues that are conserved in Saccharomyces cerevisiae histone H2B and characterized the effects of the mutants on yeast silencing, gene expression, and the DNA damage response. We identified three histone H2B core modifiable residues as functionally important. We find that mutating H2B K49 in yeast confers a UV sensitivity phenotype, and we confirm that the homologous residue in human histone H2B is acetylated and methylated in human cells. Our results also indicate that mutating H2B K111 impairs the response to methyl methanesulfonate (MMS)-induced DNA lesions and disrupts telomeric silencing and Sir4 binding. In contrast, mutating H2B R102 enhances silencing at yeast telomeres and the HML silent mating loci and increases Sir4 binding to these regions. The H2B R102A mutant also represses the expression of endogenous genes adjacent to yeast telomeres, which is likely due to the ectopic spreading of the Sir complex in this mutant strain. We propose a structural model by which H2B R102 and K111 regulate the binding of the Sir complex to the nucleosome.

  1. Novel Functional Residues in the Core Domain of Histone H2B Regulate Yeast Gene Expression and Silencing and Affect the Response to DNA Damage ▿

    Science.gov (United States)

    Kyriss, McKenna N. M.; Jin, Yi; Gallegos, Isaura J.; Sanford, James A.; Wyrick, John J.

    2010-01-01

    Previous studies have identified novel modifications in the core fold domain of histone H2B, but relatively little is known about the function of these putative histone modification sites. We have mutated core modifiable residues that are conserved in Saccharomyces cerevisiae histone H2B and characterized the effects of the mutants on yeast silencing, gene expression, and the DNA damage response. We identified three histone H2B core modifiable residues as functionally important. We find that mutating H2B K49 in yeast confers a UV sensitivity phenotype, and we confirm that the homologous residue in human histone H2B is acetylated and methylated in human cells. Our results also indicate that mutating H2B K111 impairs the response to methyl methanesulfonate (MMS)-induced DNA lesions and disrupts telomeric silencing and Sir4 binding. In contrast, mutating H2B R102 enhances silencing at yeast telomeres and the HML silent mating loci and increases Sir4 binding to these regions. The H2B R102A mutant also represses the expression of endogenous genes adjacent to yeast telomeres, which is likely due to the ectopic spreading of the Sir complex in this mutant strain. We propose a structural model by which H2B R102 and K111 regulate the binding of the Sir complex to the nucleosome. PMID:20479120

  2. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Sofie eVan Rossom

    2012-07-01

    Full Text Available DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine-nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2 to 6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human HEK293T cells. Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  3. Expression and characterization of the antimicrobial peptide ABP-dHC-cecropin A in the methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Sang, Ming; Wei, Hui; Zhang, Jiaxin; Wei, Zhiheng; Wu, Xiaolong; Chen, Yan; Zhuge, Qiang

    2017-12-01

    ABP-dHC-cecropin A is a linear cationic peptide that exhibits antimicrobial properties. To explore a new approach for expression of ABP-dHC-cecropin A using the methylotrophic yeast Pichia pastoris, we cloned the ABP-dHC-cecropin A gene into the vector pPICZαA. The SacI-linearized plasmid pPICZαA-ABP-dHC-cecropin A was then transformed into P. pastoris GS115 by electroporation. Expression was induced after a 96-h incubation with 0.5% methanol at 20 °C in a culture supplied with 2% casamino acids to avoid proteolysis. Under these conditions, approximately 48 mg of ABP-dHC-cecropin A was secreted into 1L (4 × 250-mL)of medium. Recombinant ABP-dHC-cecropin A was purified using size-exclusion chromatography, and 21 mg of pure active ABP-dHC-cecropin A was obtained from 1L (4 × 250-mL)of culture. Electrophoresis on 4-20% gradient gels indicated that recombinant ABP-dHC-cecropin A was secreted as a protein approximately 4 kDa in size. Recombinant ABP-dHC-cecropin A was successfully expressed, as the product displayed antibacterial and antifungal activities (based on an antibacterial assay, scanning electron microscopy, and antifungal assay) indistinguishable from those of the synthesized protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Effect of environmental stress on regulation of gene expression in the yeast

    Science.gov (United States)

    Gross, Eitan

    2015-07-01

    Several mathematical models have been proposed to predict the activation state of a transcription factor (TF) from the expression levels of its target genes. This inference problem is complicated however due to the fact that different genes may be regulated by different activation schemes (linear, exponential, sigmoidal, etc.). In addition to transcription regulation, the rate of gene expression at any instantaneous point in time is also determined by the independent rates of baseline production and degradation. Consequently, the set of solutions to any model equations describe an infinite number of trajectories in probability space, thus rendering the problem NP-hard. In the current study we used a Gaussian process (GP) approach to address this inverse problem. Experimental gene expression data were modeled by a putative linear activation scheme and discrepancy between theory and experiment was modeled by a GP. Model hyperparameters were calculated using maximum likelihood estimates to generate continuous TF state-space profiles. Identifiability of model parameters was optimized by obtaining TF state-space functions for multiple genes simultaneously. We found that model parameters were sensitive to environmental stress conditions, producing different state-space profiles for different stresses.

  5. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

    Science.gov (United States)

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-12-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

  6. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes*

    Science.gov (United States)

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-01-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  7. Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols.

    Directory of Open Access Journals (Sweden)

    Nawel Haïli

    Full Text Available The membrane proteins acyl-CoA:diacylglycerol acyltransferases (DGAT are essential actors for triglycerides (TG biosynthesis in eukaryotic organisms. Microbial production of TG is of interest for producing biofuel and value-added novel oils. In the oleaginous yeast Yarrowia lipolytica, Dga1p enzyme from the DGAT2 family plays a major role in TG biosynthesis. Producing recombinant DGAT enzymes pure and catalytically active is difficult, hampering their detailed functional characterization. In this report, we expressed in Escherichia coli and purified two soluble and active forms of Y. lipolytica Dga1p as fusion proteins: the first one lacking the N-terminal hydrophilic segment (Dga1pΔ19, the second one also devoid of the N-terminal putative transmembrane domain (Dga1pΔ85. Most DGAT assays are performed on membrane fractions or microsomes, using radiolabeled substrates. We implemented a fluorescent assay in order to decipher the substrate specificity of purified Dga1p enzymes. Both enzyme versions prefer acyl-CoA saturated substrates to unsaturated ones. Dga1pΔ85 preferentially uses long-chain saturated substrates. Dga1p activities are inhibited by niacin, a specific DGAT2 inhibitor. The N-terminal transmembrane domain appears important, but not essential, for TG biosynthesis. The soluble and active proteins described here could be useful tools for future functional and structural studies in order to better understand and optimize DGAT enzymes for biotechnological applications.

  8. Definition of culture conditions for Arxula adeninivorans, a rational basis for studying heterologous gene expression in this dimorphic yeast.

    Science.gov (United States)

    Stöckmann, Christoph; Palmen, Thomas G; Schroer, Kirsten; Kunze, Gotthard; Gellissen, Gerd; Büchs, Jochen

    2014-06-01

    The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l(-1) was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l(-1). Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h(-1) and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.

  9. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

    Science.gov (United States)

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  10. Expression, purification, and characterization of Sss1p, an essential component of the yeast Sec61p protein translocation complex.

    Science.gov (United States)

    Beswick, V; Brodsky, J L; Képès, F; Neumann, J M; Sanson, A; Garrigos, M

    1998-08-01

    Sss1p, a 8.9-kDa membrane protein, is an essential component of the protein translocation complex involved in the transport of secretory proteins across the Saccharomyces cerevisiae endoplasmic reticulum membrane. In order to determine the high resolution structure of Sss1p by NMR, we have undertaken its overexpression and purification. We first inserted the yeast SSS1 gene into the pGEX-2T plasmid expression vector. Sss1p was expressed as fusions with Schistosoma japonica glutathione S-transferase (GST-Sss1p) in MC1061 Escherichia coli cells. Maximum yield of GST-Sss1p was obtained from cells harvested 2 h after induction at 37 degreesC in Luria broth medium. GST-Sss1p was found associated predominantly with the membrane pool and was readily extracted with Triton X-100. Detergent-solubilized GST-Sss1p was isolated by adsorption on glutathione-agarose beads. Sss1p was released from its GST carrier by cleavage with thrombin and its recovery was maximized by addition of dodecyl maltoside. Desorbed Sss1p was loaded on a high-performance liquid chromatography hydroxyapatite column equilibrated in phosphate buffer supplemented with dodecyl maltoside and the fractions containing Sss1p were subsequently purified to homogeneity by reverse-phase chromatography on a C4 column. The entire purification protocol can be completed in 5-6 h and yields about 0.4 mg of Sss1p per gram of transformed cells. CD and preliminary 1H NMR experiments show that purified Sss1p solubilized in SDS micelles is very stable and adopts a helical secondary structure. Copyright 1998 Academic Press.

  11. Multiple abiotic stress tolerance of the transformants yeast cells and the transgenic Arabidopsis plants expressing a novel durum wheat catalase.

    Science.gov (United States)

    Feki, Kaouthar; Kamoun, Yosra; Ben Mahmoud, Rihem; Farhat-Khemakhem, Ameny; Gargouri, Ali; Brini, Faiçal

    2015-12-01

    Catalases are reactive oxygen species scavenging enzymes involved in response to abiotic and biotic stresses. In this study, we described the isolation and functional characterization of a novel catalase from durum wheat, designed TdCAT1. Molecular Phylogeny analyses showed that wheat TdCAT1 exhibited high amino acids sequence identity to other plant catalases. Sequence homology analysis showed that TdCAT1 protein contained the putative calmodulin binding domain and a putative conserved internal peroxisomal targeting signal PTS1 motif around its C-terminus. Predicted three-dimensional structural model revealed the presence of four putative distinct structural regions which are the N-terminal arm, the β-barrel, the wrapping and the α-helical domains. TdCAT1 protein had the heme pocket that was composed by five essential residues. TdCAT1 gene expression analysis showed that this gene was induced by various abiotic stresses in durum wheat. The expression of TdCAT1 in yeast cells and Arabidopsis plants conferred tolerance to several abiotic stresses. Compared with the non-transformed plants, the transgenic lines maintained their growth and accumulated more proline under stress treatments. Furthermore, the amount of H2O2 was lower in transgenic lines, which was due to the high CAT and POD activities. Taken together, these data provide the evidence for the involvement of durum wheat catalase TdCAT1 in tolerance to multiple abiotic stresses in crop plants. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  12. Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases

    Directory of Open Access Journals (Sweden)

    Brooks Wynse S

    2011-09-01

    Full Text Available Abstract Background The trichothecene mycotoxin deoxynivalenol (DON may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON, and may reduce its toxicity in plants and animals. Results Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201 were cloned and expressed in yeast (Saccharomyces cerevisiae during a series of small-scale ethanol fermentations using barley (Hordeum vulgare. DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash. Conclusions This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.

  13. The expression of glycerol facilitators from various yeast species improves growth on glycerol of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Klein, Mathias; Islam, Zia ul; Knudsen, Peter Boldsen

    2016-01-01

    cerevisiae grow poorly in synthetic glycerol medium. It has been hypothesized that the uptake of glycerol could be a major bottleneck for the utilization of glycerol in S. cerevisiae. This species exclusively relies on an active transport system for glycerol uptake. This work demonstrates that the expression......Glycerol is an abundant by-product during biodiesel production and additionally has several assets compared to sugars when used as a carbon source for growing microorganisms in the context of biotechnological applications. However, most strains of the platform production organism Saccharomyces....... cerevisiae wild-type strain (CBS 6412-13A). The maximum specific growth rate increased from 0.13 up to 0.18 h−1 and a biomass yield coefficient of 0.56 gDW/gglycerol was observed. These results pave the way for exploiting the assets of glycerol in the production of fuels, chemicals and pharmaceuticals based...

  14. Heterologous expression of human carnitine palmitoyltransferase (CPT) II in yeast: A model for the molecular analysis of mitochondrial fatty acid oxidation defects

    Energy Technology Data Exchange (ETDEWEB)

    Cavadini, P.; Invernizzi, F.; Baratta, S. [Istituto Nazionale Neurologico Carlo Besta, Milano (Italy)] [and others

    1994-09-01

    The CPT enzyme system, which is composed of two distinct mitochondrial membrane-bound proteins (CPT I and CPT II), provides the mechanism whereby long-chain fatty acids are transferred from the cytosol to the mitochondrial matrix to undergo {beta}-oxidation. Here, we report the development of an expression system for investigating genotype/phenotype correlations in CPT II deficiency and, potentially, other mitochondrial fatty acid oxidation defects. To explore yeast as an expression system, we introduced a cDNA encoding the entire human CPT II precursor into Saccharomyces cerevisiae. Expression was programmed by using an inducible galactose operon promoter (GAL1). Following induction, human CPT II was expressed at high levels, with activity 4- to 16-fold greater than in human fibroblasts. Levels of expression paralleled those of respiration, being higher in cells grown on a nonfermentable carbon source than in those grown on glucose. Immunoprecipitation of pulse-labeled transformed cells demonstrated that human CPT II expressed in yeast was targeted to mitochondria with correct proteolytic processing of its 25-residue mitochondrial leader sequence. Preliminary results on the expression of a number of mutant CPT II alleles associated with different clinical phenotypes demonstrated the value of this system for examining the functional consequences of disease-causing mutations and investigating genotype/phenotype correlations in patients with CPT II deficiency.

  15. L-arabinose fermenting yeast

    Science.gov (United States)

    Zhang, Min; Singh, Arjun; Suominen, Pirkko; Knoshaug, Eric; Franden, Mary Ann; Jarvis, Eric

    2013-02-12

    An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes. L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

  16. Molecular Cloning and Yeast Expression of Cinnamate 4-Hydroxylase from Ornithogalum saundersiae Baker

    Directory of Open Access Journals (Sweden)

    Jian-Qiang Kong

    2014-01-01

    Full Text Available OSW-1, isolated from the bulbs of Ornithogalum saundersiae Baker, is a steroidal saponin endowed with considerable antitumor properties. Biosynthesis of the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically via the phenylpropanoid biosynthetic pathway, but molecular biological characterization of the related genes has been insufficient. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11, catalyzing the hydroxylation of trans-cinnamic acid to p-coumaric acid, plays a key role in the ability of phenylpropanoid metabolism to channel carbon to produce the 4-methoxybenzoyl group on the disaccharide moiety of OSW-1. Molecular isolation and functional characterization of the C4H genes, therefore, is an important step for pathway characterization of 4-methoxybenzoyl group biosynthesis. In this study, a gene coding for C4H, designated as OsaC4H, was isolated according to the transcriptome sequencing results of Ornithogalum saundersiae. The full-length OsaC4H cDNA is 1,608-bp long, with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 55-bp 5′ non-coding region and a 35-bp 3'-untranslated region. OsaC4H was functionally characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of trans-cinnamic acid to p-coumaric acid, which was identified by high performance liquid chromatography with diode array detection (HPLC-DAD, HPLC-MS and nuclear magnetic resonance (NMR analysis. The identification of the OsaC4H gene was expected to open the way to clarification of the biosynthetic pathway of OSW-1.

  17. From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

    Science.gov (United States)

    Kouprina, Natalay; Lee, Nicholas C O; Kononenko, Artem V; Samoshkin, Alexander; Larionov, Vladimir

    2015-01-01

    Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.

  18. Aberrant ORM (yeast)–like protein isoform 3 (ORMDL3) expression dysregulates ceramide homeostasis in cells and ceramide exacerbates allergic asthma in mice

    Science.gov (United States)

    Oyeniran, Clement; Sturgill, Jamie L.; Hait, Nitai C.; Huang, Wei-Ching; Avni, Dorit; Maceyka, Michael; Newton, Jason; Allegood, Jeremy C.; Montpetit, Alison; Conrad, Daniel H.; Milstien, Sheldon; Spiegel, Sarah

    2015-01-01

    Background Asthma, a chronic inflammatory condition defined by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern affecting more than 250 million persons. Genome-wide association studies have identified ORM (yeast)–like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma. Although its yeast ortholog is a negative regulator of de novo ceramide biosynthesis, how ORMDL3 contributes to asthma pathogenesis is not known. Objectives We sought to decipher the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis. Methods We determined the relationship between expression of ORMDL3 and ceramide in epithelial and inflammatory cells and in asthma pathogenesis in mice. Results Although small increases in ORMDL3 expression decrease ceramide levels, remarkably, higher expression in lung epithelial cells and macrophages in vitro and in vivo increased ceramide production, which promoted chronic inflammation, airway hyperresponsiveness, and mucus production during house dust mite–induced allergic asthma. Moreover, nasal administration of the immunosuppressant drug FTY720/fingolimod reduced ORMDL3 expression and ceramide levels and mitigated airway inflammation and hyperreactivity and mucus hypersecretion in house dust mite–challenged mice. Conclusions Our findings demonstrate that overexpression of ORMDL3 regulates ceramide homeostasis in cells in a complex manner and suggest that local FTY720 administration might be a useful therapeutic intervention for the control of allergic asthma. PMID:25842287

  19. Aberrant ORM (yeast)-like protein isoform 3 (ORMDL3) expression dysregulates ceramide homeostasis in cells and ceramide exacerbates allergic asthma in mice.

    Science.gov (United States)

    Oyeniran, Clement; Sturgill, Jamie L; Hait, Nitai C; Huang, Wei-Ching; Avni, Dorit; Maceyka, Michael; Newton, Jason; Allegood, Jeremy C; Montpetit, Alison; Conrad, Daniel H; Milstien, Sheldon; Spiegel, Sarah

    2015-10-01

    Asthma, a chronic inflammatory condition defined by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern affecting more than 250 million persons. Genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma. Although its yeast ortholog is a negative regulator of de novo ceramide biosynthesis, how ORMDL3 contributes to asthma pathogenesis is not known. We sought to decipher the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis. We determined the relationship between expression of ORMDL3 and ceramide in epithelial and inflammatory cells and in asthma pathogenesis in mice. Although small increases in ORMDL3 expression decrease ceramide levels, remarkably, higher expression in lung epithelial cells and macrophages in vitro and in vivo increased ceramide production, which promoted chronic inflammation, airway hyperresponsiveness, and mucus production during house dust mite-induced allergic asthma. Moreover, nasal administration of the immunosuppressant drug FTY720/fingolimod reduced ORMDL3 expression and ceramide levels and mitigated airway inflammation and hyperreactivity and mucus hypersecretion in house dust mite-challenged mice. Our findings demonstrate that overexpression of ORMDL3 regulates ceramide homeostasis in cells in a complex manner and suggest that local FTY720 administration might be a useful therapeutic intervention for the control of allergic asthma. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  20. Biotin starvation with adequate glucose provision causes paradoxical changes in fuel metabolism gene expression similar in rat (Rattus norvegicus), nematode (Caenorhabditis elegans) and yeast (Saccharomyces cerevisiae).

    Science.gov (United States)

    Ortega-Cuellar, D; Hernandez-Mendoza, A; Moreno-Arriola, E; Carvajal-Aguilera, K; Perez-Vazquez, V; Gonzalez-Alvarez, R; Velazquez-Arellano, A

    2010-01-01

    Biotin affects the genetic expression of several glucose metabolism enzymes, besides being a cofactor of carboxylases. To explore how extensively biotin affects the expression of carbon metabolism genes, we studied the effects of biotin starvation and replenishment in 3 distantly related eukaryotes: yeast Saccharomyces cerevisiae, nematode Caenorhabditis elegans and rat Rattus norvegicus. Biotin starvation was produced in Wistar rats, in C. elegans N2 and S. cerevisiae W303A fed with abundant glucose. High-density oligonucleotide microarrays were used to find gene expression changes. Glucose consumption, lactate and ethanol were measured by conventional tests. In spite of abundant glucose provision, the expression of fatty oxidation and gluconeogenic genes was augmented, and the transcripts for glucose utilization and lipogenesis were diminished in biotin starvation. These results were associated with diminished glucose consumption and glycolysis products (lactate and ethanol in yeast), which was consistent across 3 very different eukaryotes. The results point toward a strongly selected role of biotin in the control of carbon metabolism, and in adaptations to variable availability of carbon, conceivably mediated by signal transduction including soluble guanylate cyclase, cGMP and a cGMP-dependent protein kinase (PKG) and/or biotin-dependent processes. Copyright © 2010 S. Karger AG, Basel.

  1. Differentially expressed genes under simulated deep-sea conditions in the psychrotolerant yeast Cryptococcus sp. NIOCCPY13

    Digital Repository Service at National Institute of Oceanography (India)

    Singh, P.; Raghukumar, C.; Verma, A.K.; Meena, R.M.

    these sequences have been submitted to EST database of NCBI GenBank. Their GenBank Accession numbers and dbEST_Ids are listed in Table 3. Results Culturable yeasts were isolated by incubation of the deep-sea sediment sample in nutrient media at 5°C for a... 85-130 14    Legends to the figures: Fig. 1. Pie chart showing percentage of downregulated genes in a deep-sea psychrotolerant yeast NIOCC#PY13, at a) 50 MPa and b) 50 MPa/5°C. 15    16    Clone ID NCBI database...

  2. Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli.

    Science.gov (United States)

    Horinouchi, Nobuyuki; Ogawa, Jun; Kawano, Takako; Sakai, Takafumi; Saito, Kyota; Matsumoto, Seiichiro; Sasaki, Mie; Mikami, Yoichi; Shimizu, Sakayu

    2006-06-01

    2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.

  3. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris

    NARCIS (Netherlands)

    Shen, SG; Sulter, G; Jeffries, TW; Cregg, JM

    1998-01-01

    In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the

  4. Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic

    DEFF Research Database (Denmark)

    Dimitrova, Irina; Toby, Garabet G; Tili, Esmerina

    2004-01-01

    Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S-transfer...

  5. Long-chain acyl-CoA-dependent regulation of gene expression in bacteria, yeast and mammals

    DEFF Research Database (Denmark)

    Black, P N; Færgeman, Nils J.; DiRusso, C C

    2000-01-01

    Saccharomyces cerevisiae, genes encoding peroxisomal proteins are activated in response to exogenously supplied fatty acids. In contrast, growth of yeast cells in media containing exogenous fatty acids results in repression of a number of genes, including that encoding the delta9-fatty acid desaturase (OLE1...

  6. Expression of bacterial Rho factor in yeast identifies new factors involved in the functional interplay between transcription and mRNP biogenesis.

    Science.gov (United States)

    Mosrin-Huaman, Christine; Honorine, Romy; Rahmouni, A Rachid

    2009-08-01

    In eukaryotic cells, the nascent pre-mRNA molecule is coated sequentially with a large set of processing and binding proteins that mediate its transformation into an export-competent ribonucleoprotein particle (mRNP) that is ready for translation in the cytoplasm. We have implemented an original assay that monitors the dynamic interplay between transcription and mRNP biogenesis and that allows the screening for new factors linking mRNA synthesis to translation in Saccharomyces cerevisiae. The assay is based on the perturbation of gene expression induced by the bacterial Rho factor, an RNA-dependent helicase/translocase that acts as a competitor at one or several steps of mRNP biogenesis in yeast. We show that the expression of Rho in yeast leads to a dose-dependent growth defect that stems from its action on RNA polymerase II-mediated transcription. Rho expression induces the production of aberrant transcripts that are degraded by the nuclear exosome. A screen for dosage suppressors of the Rho-induced growth defect identified several genes that are involved in the different steps of mRNP biogenesis and export, as well as other genes with both known functions in transcription regulation and unknown functions. Our results provide evidence for an extensive cross talk between transcription, mRNP biogenesis, and export. They also uncover new factors that potentially are involved in these interconnected events.

  7. Expression of Bacterial Rho Factor in Yeast Identifies New Factors Involved in the Functional Interplay between Transcription and mRNP Biogenesis▿ †

    Science.gov (United States)

    Mosrin-Huaman, Christine; Honorine, Romy; Rahmouni, A. Rachid

    2009-01-01

    In eukaryotic cells, the nascent pre-mRNA molecule is coated sequentially with a large set of processing and binding proteins that mediate its transformation into an export-competent ribonucleoprotein particle (mRNP) that is ready for translation in the cytoplasm. We have implemented an original assay that monitors the dynamic interplay between transcription and mRNP biogenesis and that allows the screening for new factors linking mRNA synthesis to translation in Saccharomyces cerevisiae. The assay is based on the perturbation of gene expression induced by the bacterial Rho factor, an RNA-dependent helicase/translocase that acts as a competitor at one or several steps of mRNP biogenesis in yeast. We show that the expression of Rho in yeast leads to a dose-dependent growth defect that stems from its action on RNA polymerase II-mediated transcription. Rho expression induces the production of aberrant transcripts that are degraded by the nuclear exosome. A screen for dosage suppressors of the Rho-induced growth defect identified several genes that are involved in the different steps of mRNP biogenesis and export, as well as other genes with both known functions in transcription regulation and unknown functions. Our results provide evidence for an extensive cross talk between transcription, mRNP biogenesis, and export. They also uncover new factors that potentially are involved in these interconnected events. PMID:19451224

  8. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

    Directory of Open Access Journals (Sweden)

    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  9. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and gamma-rays.

    Science.gov (United States)

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (gamma)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and gamma-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and gamma-rays). Similarly, for X- and gamma-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and gamma-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-à-vis their energy levels.

  10. ESCRT components regulate the expression of the ER/Golgi calcium pump gene PMR1 through the Rim101/Nrg1 pathway in budding yeast.

    Science.gov (United States)

    Zhao, Yunying; Du, Jingcai; Xiong, Bing; Xu, Huihui; Jiang, Linghuo

    2013-10-01

    The endosomal sorting complex required for transport (ESCRT) complexes function to form multivesicular bodies for sorting of proteins destined for the yeast vacuole or the mammalian lysosome. ESCRT components are well conserved in eukaryotes, and their mutations cause neurodegenerative diseases and other cellular pathologies in humans. PMR1 is the orthologous gene of two human genes for calcium pumps secretory pathway Ca(2+)-ATPase (SPCA1, ATP2C1) and sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA, ATP2A2), which are mutated in Hailey-Hailey and Darier genetic diseases, respectively. Here we show that deletion mutation of ESCRT components Snf7, Snf8, Stp22, Vps20, Vps25, Vps28, or Vps36 activates the calcium/calcineurin signaling in yeast cells, but surprisingly leads to a nearly 50% reduction in expression of the ER/Golgi calcium pump gene PMR1 independent of calcium stress. These ESCRT mutants are known to have a defect in Rim101 activation. Ectopic expression of a constitutively active form of Rim101 or further deletion of NRG1 in these mutants partially suppresses their calcium hypersensitivity. Deletion of NRG1 also completely rescues the expression of PMR1 in these mutants to the level of the wild type. Promoter mutagenesis, gel electrophoretic mobility shift assay, and chromatin immunoprecipitation analysis demonstrate that Nrg1 binds to two motifs in the PMR1 promoter. In addition, expression of PMR1 under the control of its promoters with mutated Nrg1-binding motifs suppresses the calcium hypersensitivity of these ESCRT mutants. Collectively, these data have uncovered a function of ESCRT components in regulating PMR1 expression through the Nrg1/Rim101 pathway. Our findings provide important clues for understanding human diseases related to calcium homeostasis.

  11. Forces in yeast flocculation

    Science.gov (United States)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Vincent, Stéphane P.; Abellán Flos, Marta; Hols, Pascal; Lipke, Peter N.; Dufrêne, Yves F.

    2015-01-01

    In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (``flocculation'') is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding.

  12. The Physiological and Biochemical Mechanisms Providing the Increased Constitutive Cold Resistance in the Potato Plants, Expressing the Yeast SUC2 Gene Encoding Apoplastic Invertase

    Directory of Open Access Journals (Sweden)

    A.N. Deryabin

    2016-05-01

    Full Text Available The expression of heterologous genes in plants is an effective method to improve our understanding of plant resistance mechanisms. The purpose of this work was to investigate the involvement of cell-wall invertase and apoplastic sugars into constitutive cold resistance of potato (Solanum tuberosum L., cv. Dйsirйe plants, which expressed the yeast SUC2 gene encoding apoplastic invertase. WT-plants of a potato served as the control. The increase in the essential cell-wall invertase activity in the leaves of transformed plants indicates significant changes in the cellular carbohydrate metabolism and regulatory function of this enzyme. The activity of yeast invertase changed the composition of intracellular sugars in the leaves of the transformed potato plant. The total content of sugars (sucrose, glucose, fructose in the leaves and apoplast was higher in the transformants, in comparison by WT-plants. Our data indicate higher constitutive resistance of transformants to severe hypothermia conditions compared to WT-plants. This fact allows us to consider cell-wall invertase as a enzyme of carbohydrate metabolism playing an important regulatory role in the metabolic signaling upon forming increased plant resistance to low temperature. Thus, the potato line with the integrated SUC2 gene is a convenient tool to study the role of the apoplastic invertase and the products of its activity during growth, development and formation constitutive resistance to hypothermia.

  13. Nitrile Metabolizing Yeasts

    Science.gov (United States)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  14. Thioester-containing proteins of the tick Ixodes ricinus: gene expression, response to microbial challenge and their role in phagocytosis of the yeast Candida albicans.

    Science.gov (United States)

    Urbanová, Veronika; Šíma, Radek; Šauman, Ivo; Hajdušek, Ondřej; Kopáček, Petr

    2015-01-01

    The ability of ticks to act as vectors for a wide range of serious human and animal infectious diseases is apparently linked to the insufficiency of the tick immune system to effectively eliminate pathogens they transmit. At the tick-pathogen interface, an important role is presumably played by components of an ancient complement system that includes a repertoire of thioester-containing proteins (TEPs), which in Ixodes sp. comprises three α2-macroglobulins (A2M), three C3 complement component-related molecules (C3), two macroglobulin complement-related (Mcr) and one insect-type TEPs (Tep). In order to assess the function of TEPs in tick immunity, a quantitative real-time PCR expression analysis of tick TEPs was performed at various developmental stages of Ixodes ricinus, and in tissues dissected from adult females. Expression of TEP genes was mostly tissue specific; IrA2M1, IrC3-1, IrC3-3 were found to be expressed in cells of tick fat body adjacent to the tracheal trunks, IrA2M2 in hemocytes, IrTep in ovaries, IrMcr1 in salivary glands and only IrA2M3, IrC3-2 and IrMcr2 mRNAs were present in multiple organs. Expression of tick TEPs was further examined in response to injection of model microbes representing Gram-negative, Gram-positive bacteria and yeast. The greatest expression induction was observed for IrA2M1 and IrC3-1 after challenge with the yeast Candida albicans. Phagocytosis of the yeast was strongly dependent on an active thioester bond and the subsequent silencing of individual tick TEPs by RNA interference demonstrated the involvement of IrC3-1 and IrMcr2. This result suggests the existence of a distinct complement-like pathway, different from that leading to phagocytosis of Gram-negative bacteria. Understanding of the tick immune response against model microbes should provide new concepts for investigating interactions between ticks and relevant tick-borne pathogens. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Expression of GPD1 and SIP18 genes during rehydration in active dry industrial Saccharomyces cerevisiae cider-making yeast strains (ADY).

    Science.gov (United States)

    Goncerzewicz, Anna; Kamińska-Wojteczek, Karolina; Młynarczyk, Izabella; Misiewicz, Anna

    2017-01-01

    In this study we determined the influence of different sugar concentration in media, time of rehydration and type of strain on relative expression level of GPD1 and SIP18 genes of active dry cider-making yeast strains, followed by the assessment of the impact of rehydration on the fermentation process. High expression of SIP18 at the beginning of rehydration was shown to be due to high transcription of the gene during the drying process. High sugar concentrations of media initiated transcription of the GPD1 gene and triggered the cellular glycerol biosynthesis pathway in examined strains. Rehydration time and type of strain showed to have no statistically significant impact on the course of the fermentation; RT qPCR results depended mainly on the time of rehydration and sugar concentration of the medium. This is the first attempt to confront rehydration time and molecular mechanisms acting upon rehydration with the course of the fermentation process.

  16. Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters.

    Science.gov (United States)

    Verstrepen, Kevin J; Van Laere, Stijn D M; Vanderhaegen, Bart M P; Derdelinckx, Guy; Dufour, Jean-Pierre; Pretorius, Isak S; Winderickx, Joris; Thevelein, Johan M; Delvaux, Freddy R

    2003-09-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Delta atf2Delta double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.

  17. The glucose metabolite methylglyoxal inhibits expression of the glucose transporter genes by inactivating the cell surface glucose sensors Rgt2 and Snf3 in yeast.

    Science.gov (United States)

    Roy, Adhiraj; Hashmi, Salman; Li, Zerui; Dement, Angela D; Cho, Kyu Hong; Kim, Jeong-Ho

    2016-03-01

    Methylglyoxal (MG) is a cytotoxic by-product of glycolysis. MG has inhibitory effect on the growth of cells ranging from microorganisms to higher eukaryotes, but its molecular targets are largely unknown. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of expression of genes encoding glucose transporters (HXTs). Here we provide evidence that these glucose sensors are primary targets of MG in yeast. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. These results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. In addition, the inhibitory effect of MG on the glucose sensors is greatly enhanced in cells lacking Glo1, a key component of the MG detoxification system. Thus the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell-surface glucose sensors and thus identify MG as a potential glycolytic inhibitor. © 2016 Roy, Hashmi, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. Biotechnical Microbiology, yeast and bacteria

    DEFF Research Database (Denmark)

    Villadsen, Ingrid Stampe

    1999-01-01

    This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular Compart...

  19. Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin.

    Science.gov (United States)

    Liu, Xiao-Yan; Chi, Zhe; Liu, Guang-Lei; Madzak, Catherine; Chi, Zhen-Ming

    2013-02-01

    In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.

  20. Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

    Directory of Open Access Journals (Sweden)

    Huijun Xue

    2017-10-01

    Full Text Available Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA and origin recognition complex (ORC as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.

  1. Constitutive expression of the DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation.

    Science.gov (United States)

    Wu, Dianhui; Li, Xiaomin; Lu, Jian; Chen, Jian; Zhang, Liang; Xie, Guangfa

    2016-01-01

    Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Plasmids with E2 epitope tags: tagging modules for N- and C-terminal PCR-based gene targeting in both budding and fission yeast, and inducible expression vectors for fission yeast.

    Science.gov (United States)

    Tamm, Tiina

    2009-01-01

    A single-step PCR-based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10-amino acids (epitope E2a: SSTSSDFRDR)- and 12 amino acids (epitope E2b: GVSSTSSDFRDR)-long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C-terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM-series plasmid. The N-terminal E2a and E2b tagging modules were based on pOM-series plasmid. The pOM-series plasmids were selected for this study because of their use of the Cre-loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N-terminal or C-terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti-E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2-tagged strains.

  3. Yeasts associated with Manteca.

    Science.gov (United States)

    Suzzi, Giovanna; Schirone, Maria; Martuscelli, Maria; Gatti, Monica; Fornasari, Maria Emanuela; Neviani, Erasmo

    2003-04-01

    Manteca is a traditional milk product of southern Italy produced from whey deriving from Caciocavallo Podolico cheese-making. This study was undertaken to obtain more information about the microbiological properties of this product and particularly about the presence, metabolic activities, and technological significance of the different yeast species naturally occurring in Manteca. High numbers of yeasts were counted after 7 days ripening (10(4)-10(5) cfu g(-1)) and then decreased to 10(2) at the end. A total of 179 isolates were identified and studied for their phenotypic and genotypic characteristics. The most frequently encountered species were Trichosporon asahii (45), Candida parapsilosis (33), Rhodotorula mucilaginosa (32), Candida inconspicua (29). Some of these yeasts showed lipolytic activity (32 strains) and proteolytic activity (29 strains), NaCl resistance up to 10% and growth up to 45 degrees C (42 strains). Biogenic amines were formed by proteolytic strains, in particular phenylethylamine, putrescine and spermidine. Spermidine was produced by all the yeasts tested in this work, but only Trichosporon produced a great quantity of this compound. Histamine was not detectable. Caseinolytic activity was common to almost all strains, corresponding to the ability to efficiently split off amino-terminal amino acids. The highest and most constant activity expressed by all species was X-prolyl-dipeptidyl aminopeptidase. The findings suggest that the presence of yeasts may play a significant role in justifying interactions with lactic acid bacteria, and consequently with their metabolic activity in the definition of the peculiar characteristics of Manteca cheese.

  4. A novel treatment for weight reduction by the recombinant "Pichia pastoris" yeast expressing the hybrid protein of "irisin-furin-transferrin".

    Science.gov (United States)

    Jalili, Mahsa; Bazi, Zahra; Hekmatdoost, Azita

    2016-01-01

    Obesity is a major health problem across the world, but there are few ways to effectively treat or manage it in the long term. Researchers are searching for more convenient, cost-effective and noninvasive therapies for overweight and obese people. Recent studies have illustrated that the microbiome of the body's different organs can be used as a vehicle for in-situ gene therapy. We suggest that the recombinant form of "Pichia pastoris" yeast expressing the hybrid protein of "irisin-furin-transferrin" under the control of the enolase 1 promoter is a new nutraceutical strategy to absorb fewer calories from intestinal nutrients, and induce a higher metabolic rate to expend more calories, similar to that from engaging in physical activity. By comparison, this method can be a long-term, noninvasive treatment and can be used for obese patients who have movement limitations.

  5. Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50▿

    Science.gov (United States)

    Basanta, Antonio; Gómez-Sala, Beatriz; Sánchez, Jorge; Diep, Dzung B.; Herranz, Carmen; Hernández, Pablo E.; Cintas, Luis M.

    2010-01-01

    In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast. PMID:20348300

  6. Extracellular expression of YlLip11 with a native signal peptide from Yarrowia lipolytica MSR80 in three different yeast hosts.

    Science.gov (United States)

    Kumari, Arti; Baronian, Keith; Kunze, Gotthard; Gupta, Rani

    2015-06-01

    Lipase YlLip11 from Yarrowia lipolytica was expressed with a signal peptide encoding sequence in Arxula adeninivorans, Saccharomyces cerevisiae and Hansenula polymorpha using the Xplor®2 transformation/expression platform and an expression module with the constitutive Arxula-derived TEF1 promoter. The YlLip11 signal peptide was functional in all of the yeast hosts with 97% of the recombinant enzyme being secreted into the culture medium. However, recombinant YlLip11 with His Tag fused at C-terminal was not active. The best recombinant YlLip11 producing A. adeninivorans G1212/YRC102-YlLip11 transformant cultivated in shake flasks produced 2654 U/L lipase, followed by S. cerevisiae SEY6210/YRC103-YlLip11 (1632U/L) and H. polymorpha RB11/YRC103-YlLip11 (1144U/L). Although the biochemical parameters of YlLip11 synthesized in different hosts were similar, their glycosylation level and thermo stability differed. The protein synthesized by the H. polymorpha transformant had the highest degree of glycosylation and with a t1/2 of 60min at 70°C, exhibited the highest thermostability. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Expression of hydroxynitrile lyase from Manihot esculenta in yeast and its application in (S)-mandelonitrile production using an immobilized enzyme reactor.

    Science.gov (United States)

    Semba, Hisashi; Dobashi, Yukio; Matsui, Toru

    2008-06-01

    Hydroxynitrile lyase from cassava, Manihot esculenta (MeHNL), catalyzes the formation of (S)-cyanohydrins from HCN and aldehydes or ketones. (S)-Mandelonitrile was produced on a bench scale with immobilized MeHNL, after optimizing the enzyme expression system using recombinant technology. MeHNL was cloned from a cDNA library prepared from a leaf of Manihot esculenta, and then expressed in a multi-auxotrophic mutant of Saccharomyces cerevisiae cells. The maximum yield of active MeHNL was obtained by integrating transformation 4 times with a tandemly repeated expression cassette. Silica gel was the most suitable support for immobilization of the prepared enzyme from the recombinant yeast. Using this immobilized enzyme, 22 batches of (S)-mandelonitrile synthesis were performed in a 20 liters bioreactor (1 M benzaldehyde and 1.5 M HCN). During this operation, about 29 kg of (S)-mandelonitrile was produced from 23.3 kg of benzaldehyde, giving 98 mol % yield and a mean enantio excess of 98.9% ee.

  8. The Drosophila gene twister, an orthologue of the yeast helicase SKI2, is differentially expressed during development.

    Science.gov (United States)

    Seago, J E; Chernukhin, I V; Newbury, S F

    2001-08-01

    We have identified and characterized a Drosophila orthologue of SKI2, which, in Saccharomyces cerevisiae, is one of the key components in the cytoplasmic 3'-5' decay of mRNA. The Drosophila orthologue (twister, tst), is expressed as two transcripts which differ in the lengths of their 3'-UTRs, with the smaller transcript being particularly abundant in 0-2 h embryos and the larger transcript reaching its highest levels in 6-8 h embryos. TST protein is expressed in two forms which are differentially expressed in adult tissues and throughout development. Differential expression of TST may modulate activity of the mRNA turnover pathway and could have a major impact on the expression of target RNAs.

  9. Yeast Interacting Proteins Database: YFR015C, YFR015C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entr...ression induced by glucose limitation, nitrogen starvation, environmental stress, and entry into stationary ...tion, nitrogen starvation, environmental stress, and entry into stationary phase Rows with this bait as bait..., the more highly expressed yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental

  10. Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases

    Directory of Open Access Journals (Sweden)

    Artem G. Lada

    2017-12-01

    Full Text Available DNA editing deaminases (APOBECs are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis. We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive

  11. In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production.

    Directory of Open Access Journals (Sweden)

    Evamaria Gruchattka

    Full Text Available Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo.Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1 was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production.Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes

  12. In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production.

    Science.gov (United States)

    Gruchattka, Evamaria; Kayser, Oliver

    2015-01-01

    Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo. Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1) was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production. Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes are essential to

  13. Yeast-expressed recombinant As16 protects mice against Ascaris suum infection through induction of a Th2-skewed immune response.

    Science.gov (United States)

    Wei, Junfei; Versteeg, Leroy; Liu, Zhuyun; Keegan, Brian; Gazzinelli-Guimarães, Ana Clara; Fujiwara, Ricardo T; Briggs, Neima; Jones, Kathryn M; Strych, Ulrich; Beaumier, Coreen M; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin

    2017-07-01

    Ascariasis remains the most common helminth infection in humans. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development targeting Ascaris, hookworm, and Trichuris infections. As16 and As14 have previously been described as two genetically related proteins from Ascaris suum that induced protective immunity in mice when formulated with cholera toxin B subunit (CTB) as an adjuvant, but the exact protective mechanism was not well understood. As16 and As14 were highly expressed as soluble recombinant proteins (rAs16 and rAs14) in Pichia pastoris. The yeast-expressed rAs16 was highly recognized by immune sera from mice infected with A. suum eggs and elicited 99.6% protection against A. suum re-infection. Mice immunized with rAs16 formulated with ISA720 displayed significant larva reduction (36.7%) and stunted larval development against A. suum eggs challenge. The protective immunity was associated with a predominant Th2-type response characterized by high titers of serological IgG1 (IgG1/IgG2a > 2000) and high levels of IL-4 and IL-5 produced by restimulated splenocytes. A similar level of protection was observed in mice immunized with rAs16 formulated with alum (Alhydrogel), known to induce mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both known to induce a Th1-type biased response, were not significantly protected against A. suum infection. The rAs14 protein was not recognized by A. suum infected mouse sera and mice immunized with rAs14 formulated with ISA720 did not show significant protection against challenge infection, possibly due to the protein's inaccessibility to the host immune system or a Th1-type response was induced which would counter a protective Th2-type response. Yeast-expressed rAs16 formulated with ISA720 or alum induced significant protection in mice against A. suum egg challenge that associates with a Th2-skewed immune response

  14. Analysis of cellular responses to aflatoxin B{sub 1} in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Guo Yingying [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Breeden, Linda L. [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Fan, Wenhong [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zhao Lueping [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Eaton, David L. [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zarbl, Helmut [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States) and Fred Hutchinson Cancer Research Center, Seattle, WA (United States)]. E-mail: hzarbl@fhcrc.org

    2006-01-29

    Aflatoxin B1 (AFB{sub 1}) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB{sub 1} is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N{sup 7}-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB{sub 1}, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB{sub 1} that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB{sub 1} treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB{sub 1}-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific

  15. Yeast-expressed recombinant As16 protects mice against Ascaris suum infection through induction of a Th2-skewed immune response

    Science.gov (United States)

    Liu, Zhuyun; Keegan, Brian; Gazzinelli-Guimarães, Ana Clara; Fujiwara, Ricardo T.; Briggs, Neima; Jones, Kathryn M.; Strych, Ulrich; Beaumier, Coreen M.; Bottazzi, Maria Elena; Zhan, Bin

    2017-01-01

    Background Ascariasis remains the most common helminth infection in humans. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development targeting Ascaris, hookworm, and Trichuris infections. As16 and As14 have previously been described as two genetically related proteins from Ascaris suum that induced protective immunity in mice when formulated with cholera toxin B subunit (CTB) as an adjuvant, but the exact protective mechanism was not well understood. Methodology/Principal findings As16 and As14 were highly expressed as soluble recombinant proteins (rAs16 and rAs14) in Pichia pastoris. The yeast-expressed rAs16 was highly recognized by immune sera from mice infected with A. suum eggs and elicited 99.6% protection against A. suum re-infection. Mice immunized with rAs16 formulated with ISA720 displayed significant larva reduction (36.7%) and stunted larval development against A. suum eggs challenge. The protective immunity was associated with a predominant Th2-type response characterized by high titers of serological IgG1 (IgG1/IgG2a > 2000) and high levels of IL-4 and IL-5 produced by restimulated splenocytes. A similar level of protection was observed in mice immunized with rAs16 formulated with alum (Alhydrogel), known to induce mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both known to induce a Th1-type biased response, were not significantly protected against A. suum infection. The rAs14 protein was not recognized by A. suum infected mouse sera and mice immunized with rAs14 formulated with ISA720 did not show significant protection against challenge infection, possibly due to the protein’s inaccessibility to the host immune system or a Th1-type response was induced which would counter a protective Th2-type response. Conclusions/Significance Yeast-expressed rAs16 formulated with ISA720 or alum induced significant protection in mice against A. suum

  16. Yeast-expressed recombinant As16 protects mice against Ascaris suum infection through induction of a Th2-skewed immune response.

    Directory of Open Access Journals (Sweden)

    Junfei Wei

    2017-07-01

    Full Text Available Ascariasis remains the most common helminth infection in humans. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development targeting Ascaris, hookworm, and Trichuris infections. As16 and As14 have previously been described as two genetically related proteins from Ascaris suum that induced protective immunity in mice when formulated with cholera toxin B subunit (CTB as an adjuvant, but the exact protective mechanism was not well understood.As16 and As14 were highly expressed as soluble recombinant proteins (rAs16 and rAs14 in Pichia pastoris. The yeast-expressed rAs16 was highly recognized by immune sera from mice infected with A. suum eggs and elicited 99.6% protection against A. suum re-infection. Mice immunized with rAs16 formulated with ISA720 displayed significant larva reduction (36.7% and stunted larval development against A. suum eggs challenge. The protective immunity was associated with a predominant Th2-type response characterized by high titers of serological IgG1 (IgG1/IgG2a > 2000 and high levels of IL-4 and IL-5 produced by restimulated splenocytes. A similar level of protection was observed in mice immunized with rAs16 formulated with alum (Alhydrogel, known to induce mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both known to induce a Th1-type biased response, were not significantly protected against A. suum infection. The rAs14 protein was not recognized by A. suum infected mouse sera and mice immunized with rAs14 formulated with ISA720 did not show significant protection against challenge infection, possibly due to the protein's inaccessibility to the host immune system or a Th1-type response was induced which would counter a protective Th2-type response.Yeast-expressed rAs16 formulated with ISA720 or alum induced significant protection in mice against A. suum egg challenge that associates with a Th2-skewed immune

  17. Xuezhikang, extract of red yeast rice, improved abnormal hemorheology, suppressed caveolin-1 and increased eNOS expression in atherosclerotic rats.

    Directory of Open Access Journals (Sweden)

    Xin-Yuan Zhu

    Full Text Available BACKGROUND: Xuezhikang is the extract of red yeast rice, which has been widely used for the management of atherosclerotic disease, but the molecular basis of its antiatherosclerotic effects has not yet been fully identified. Here we investigated the changes of eNOS in vascular endothelia and RBCs, eNOS regulatory factor Caveolin-1 in endothelia, and hemorheological parameters in atherosclerotic rats to explore the protective effects of Xuezhikang. METHODOLOGY/PRINCIPAL FINDINGS: Wistar rats were divided into 4 groups (n = 12/group group C, controls; group M, high-cholesterol diet (HCD induced atherosclerotic models; group X, HCD+Xuezhikang; and group L, HCD +Lovastatin. In group X, Xuezhikang inhibited oxidative stress, down-regulated caveolin-1 in aorta wall (P<0.05, up-regulated eNOS expression in vascular endothelia and erythrocytes (P<0.05, increased NOx (nitrite and nitrate in plasma and cGMP in erythrocyte plasma and aorta wall (P<0.05, increased erythrocyte deformation index (EDI, and decreased whole blood viscosity and plasma viscosity (P<0.05, with the improvement of arterial pathology. CONCLUSIONS/SIGNIFICANCE: Xuezhikang up-regulated eNOS expression in vascular endothelia and RBCs, increased plasma NOx and improved abnormal hemorheology in high cholesterol diet induced atherosclerotic rats. The elevated eNOS/NO and improved hemorheology may be beneficial to atherosclerotic disease.

  18. A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active in cellulo.

    Science.gov (United States)

    Legaz, Sophie; Exposito, Jean-Yves; Borel, Agnès; Candusso, Marie-Pierre; Megy, Simon; Montserret, Roland; Lahaye, Vincent; Terzian, Christophe; Verrier, Bernard

    2015-09-01

    Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Gene expression and yeast two-hybrid studies of transcription factors mediating drought stress response in root tissues of chickpea (Cicer arietinum L.

    Directory of Open Access Journals (Sweden)

    Abirami eRamalingam

    2015-12-01

    Full Text Available Drought stress has been one of the serious constraints affecting chickpea productivity to a great extent. Genomic assisted breeding in chickpea has been effective in providing a yield advantage of up to 24 %, thus having a potential to accelerate breeding precisely and efficiently. In order to do so, understanding the molecular mechanisms for drought tolerance and identification of candidate genes are crucial. Transcription factors (TFs have important roles in the regulation of plant stress related genes. In this context, quantitative real time-PCR (qRT-PCR was used to study the differential gene expression of selected TFs, identified from large-scale gene expression analysis, in contrasting drought responsive genotypes. Root tissues of ICC 4958 (tolerant, ICC 1882 (sensitive, JG 11 (elite and JG 11+ (introgression line were used for the study. Subsequently, a candidate single repeat MYB gene (1R-MYB that was remarkably induced in the drought tolerant genotypes under drought stress was cloned and subjected to Y2H analysis by screening a root cDNA library. The protein-protein interaction study identified three interacting peptides, a galactinol-sucrose galactosyltransferase 2, a CBL (Calcineurin B-like-interacting serine/threonine-protein kinase 25 and an ABA responsive 17-like, which were confirmed by the co-transformation of candidate plasmids in yeast. These findings provide preliminary insights into the ability of 1R-MYB TF to co-regulate drought tolerance mechanism in chickpea roots.

  20. Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12

    Directory of Open Access Journals (Sweden)

    Norsyuhada Alias

    2014-01-01

    Full Text Available Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE strategy with an open reading frame (ORF of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml was obtained from P. pastoris GS115 host (GpPro2 at 20°C after 72 hours of postinduction time with 0.5% (v/v of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa.

  1. Molecular Cloning and Optimization for High Level Expression of Cold-Adapted Serine Protease from Antarctic Yeast Glaciozyma antarctica PI12

    Science.gov (United States)

    Ahmad Mazian, Mu'adz; Salleh, Abu Bakar; Basri, Mahiran; Rahman, Raja Noor Zaliha Raja Abd.

    2014-01-01

    Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa. PMID:25093119

  2. Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

    Science.gov (United States)

    Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

    2015-12-01

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains. © 2015 Society for Laboratory Automation and Screening.

  3. Time-dependent regulation analysis dissects shifts between metabolic and gene-expression regulation during nitrogen starvation in baker's yeast

    NARCIS (Netherlands)

    van Eunen, Karen; Bouwman, Jildau; Lindenbergh, Alexander; Westerhoff, Hans V.; Bakker, Barbara M.

    2009-01-01

    Time-dependent regulation analysis is a new methodology that allows us to unravel, both quantitatively and dynamically, how and when functional changes in the cell are brought about by the interplay of gene expression and metabolism. In this first experimental implementation, we dissect the initial

  4. Interaction Between Yeasts and Zinc

    Science.gov (United States)

    Nicola, Raffaele De; Walker, Graeme

    Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

  5. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.

    Science.gov (United States)

    Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

    2014-01-01

    Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.

  6. A PROP1-binding factor, AES cloned by yeast two-hybrid assay represses PROP1-induced Pit-1 gene expression.

    Science.gov (United States)

    Sugiyama, Yuka; Ikeshita, Nobuko; Shibahara, Hiromi; Yamamoto, Daisuke; Kawagishi, Mayuko; Iguchi, Genzo; Iida, Keiji; Takahashi, Yutaka; Kaji, Hidesuke; Chihara, Kazuo; Okimura, Yasuhiko

    2013-08-25

    PROP1 mutation causes combined pituitary hormone deficiency (CPHD). Several mutations are located in a transactivation domain (TAD) of Prop1, and the loss of TAD binding to cofactors is likely the cause of CPHD. PROP1 cofactors have not yet been identified. In the present study, we aimed to identify the PROP1-interacting proteins from the human brain cDNA library. Using a yeast two-hybrid assay, we cloned nine candidate proteins that may bind to PROP1. Of those nine candidates, amino-terminal enhancer of split (AES) was the most abundant, and we analyzed the AES function. AES dose-dependently decreased the PROP1-induced Pit-1 reporter gene expression. An immunoprecipitation assay revealed the relationship between AES and PROP1. In a mammalian two-hybrid assay, a leucine zipper-like motif of the AES Q domain was identified as a region that interacted with TAD. These results indicated that AES was a corepressor of PROP1. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis.

    Science.gov (United States)

    Han, Gil-Soo; Carman, George M

    2017-08-11

    The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Heterologous expression of three Camellia sinensis small heat shock protein genes confers temperature stress tolerance in yeast and Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Mingle; Zou, Zhongwei; Li, Qinghui; Xin, Huahong; Zhu, Xujun; Chen, Xuan; Li, Xinghui

    2017-07-01

    CsHSP17.7, CsHSP18.1, and CsHSP21.8 expressions are induced by heat and cold stresses, and CsHSP overexpression confers tolerance to heat and cold stresses in transgenic Pichia pastoris and Arabidopsis thaliana. Small heat shock proteins (sHSPs) are crucial for protecting plants against biotic and abiotic stresses, especially heat stress. However, knowledge concerning the functions of Camellia sinensis sHSP in heat and cold stresses remains poorly understood. In this study, three C. sinensis sHSP genes (i.e., CsHSP17.7, CsHSP18.1, and CsHSP21.8) were isolated and characterized using suppression subtractive hybridization (SSH) technology. The CsHSPs expression levels in C. sinensis leaves were significantly up-regulated by heat and cold stresses. Phylogenetic analyses revealed that CsHSP17.7, CsHSP18.1, and CsHSP21.8 belong to sHSP Classes I, II, and IV, respectively. Heterologous expression of the three CsHSP genes in Pichia pastoris cells enhanced heat and cold stress tolerance. When exposed to heat and cold treatments, transgenic Arabidopsis thaliana plants overexpressing CsHSP17.7, CsHSP18.1, and CsHSP21.8 had lower malondialdehyde contents, ion leakage, higher proline contents, and transcript levels of stress-related genes (e.g., AtPOD, AtAPX1, AtP5CS2, and AtProT1) compared with the control line. In addition, improved seed germination vigor was also observed in the CsHSP-overexpressing seeds under heat stress. Taken together, our results suggest that the three identified CsHSP genes play key roles in heat and cold tolerance.

  9. Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform

    DEFF Research Database (Denmark)

    Mikkelsen, Michael Dalgaard; Buron, Line Due; Salomonsen, Bo

    2012-01-01

    Epidemiological studies have shown that consumption of cruciferous vegetables, such as, broccoli and cabbages, is associated with a reduced risk of developing cancer. This phenomenon has been attributed to specific glucosinolates among the ∼30 glucosinolates that are typically present as natural...... products characteristic of cruciferous plants. Accordingly, there has been a strong interest to produce these compounds in microbial cell factories as it will allow production of selected beneficial glucosinolates. We have developed a versatile platform for stable expression of multi-gene pathways...

  10. Yeast phospholipase C is required for stability of casein kinase I Yck2p and expression of hexose transporters.

    Science.gov (United States)

    Zhang, Tiantian; Galdieri, Luciano; Hasek, Jiri; Vancura, Ales

    2017-12-01

    Phospholipase C (Plc1p) in Saccharomyces cerevisiae is required for normal degradation of repressor Mth1p and expression of the HXT genes encoding cell membrane transporters of glucose. Plc1p is also required for normal localization of glucose transporters to the cell membrane. Consequently, plc1Δ cells display histone hypoacetylation and transcriptional defects due to reduced uptake and metabolism of glucose to acetyl-CoA, a substrate for histone acetyltransferases. In the presence of glucose, Mth1p is phosphorylated by casein kinase I Yck1/2p, ubiquitinated by the SCFGrr1 complex and degraded by the proteasome. Here, we show that while Plc1p does not affect the function of the SCFGrr1 complex or the proteasome, it is required for normal protein level of Yck2p. Since stability of Yck1/2p is regulated by a glucose-dependent mechanism, PLC1 inactivation results in destabilization of Yck1/2p and defect in Mth1p degradation. Based on our results and published data, we propose a model in which plc1Δ mutation causes increased internalization of glucose transporters, decreased transport of glucose into the cells, and consequently decreased stability of Yck1/2p, increased stability of Mth1p and decreased expression of the HXT genes. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. A microfluidic device for temporally controlled gene expression and long-term fluorescent imaging in unperturbed dividing yeast cells.

    Directory of Open Access Journals (Sweden)

    Gilles Charvin

    Full Text Available BACKGROUND: Imaging single cells with fluorescent markers over multiple cell cycles is a powerful tool for unraveling the mechanism and dynamics of the cell cycle. Over the past ten years, microfluidic techniques in cell biology have emerged that allow for good control of growth environment. Yet the control and quantification of transient gene expression in unperturbed dividing cells has received less attention. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a microfluidic flow cell to grow Saccharomyces Cerevisiae for more than 8 generations (approximately 12 hrs starting with single cells, with controlled flow of the growth medium. This setup provides two important features: first, cells are tightly confined and grow in a remarkably planar array. The pedigree can thus be determined and single-cell fluorescence measured with 3 minutes resolution for all cells, as a founder cell grows to a micro-colony of more than 200 cells. Second, we can trigger and calibrate rapid and transient gene expression using reversible administration of inducers that control the GAL1 or MET3 promoters. We then show that periodic 10-20 minutes gene induction pulses can drive many cell division cycles with complete coherence across the cell cluster, with either a G1/S trigger (cln1 cln2 cln3 MET3-CLN2 or a mitotic trigger (cdc20 GALL-CDC20. CONCLUSIONS/SIGNIFICANCE: In addition to evident cell cycle applications, this device can be used to directly measure the amount and duration of any fluorescently scorable signal-transduction or gene-induction response over a long time period. The system allows direct correlation of cell history (e.g., hysteresis or epigenetics or cell cycle position with the measured response.

  12. Molecular characterization, expression analysis and RNAi knock-down of elongation factor 1α and 1γ from Nilaparvata lugens and its yeast-like symbiont.

    Science.gov (United States)

    Wang, W X; Zhu, T H; Li, K L; Chen, L F; Lai, F X; Fu, Q

    2017-06-01

    In the present paper, four cDNAs encoding the alpha and gamma subunits of elongation factor 1 (EF-1) were cloned and sequenced from Nilaparvata lugens, named NlEF-1α, NlEF-1γ, and its yeast-like symbiont (YLS), named YsEF-1α and YsEF-1γ, respectively. Comparisons with sequences from other species indicated a greater conservation for EF-1α than for EF-1γ. NlEF-1α has two identical copies. The deduced amino acid sequence homology of NlEF-1α and NlEF-1γ is 96 and 64%, respectively, compared with Homalodisca vitripennis and Locusta migratoria. The deduced amino acid sequence homology of YsEF-1α and YsEF-1γ is 96 and 74%, respectively, compared with Metarhizium anisopliae and Ophiocordyceps sinensis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the expression level of NlEF-1α and NlEF-1γ mRNA in hemolymph, ovary, fat body and salivary glands were higher than the midgut and leg tissue. YsEF-1α and YsEF-1γ was highly expressed in fat body. The expression level of NlEF-1α was higher than that of NlEF-1γ. Through RNA interference (RNAi) of the two genes, the mortality of nymph reached 92.2% at the 11th day after treatment and the ovarian development was severely hindered. The RT-qPCR analysis verified the correlation between mortality, sterility and the down-regulation of the target genes. The expression and synthesis of vitellogenin (Vg) protein in insects injected with NlEF-1α and NlEF-1γ double-stranded RNA (dsRNA) was significantly lower than control groups. Attempts to knockdown the YsEF-1 genes in the YLS was unsuccessful. However, the phenotype of N. lugens injected with YsEF-1α dsRNA was the same as that injected with NlEF-1α dsRNA, possibly due to the high similarity (up to 71.9%) in the nucleotide sequences between NlEF-1α and YsEF-1α. We demonstrated that partial silencing of NlEF-1α and NlEF-1γ genes caused lethal and sterility effect on N. lugens. NlEF-1γ shares low identity with that of

  13. Yeast Genetics and Biotechnological Applications

    Science.gov (United States)

    Mishra, Saroj; Baranwal, Richa

    Yeast can be recognized as one of the very important groups of microorganisms on account of its extensive use in the fermentation industry and as a basic eukaryotic model cellular system. The yeast Saccharomyces cerevisiae has been extensively used to elucidate the genetics and regulation of several key functions in the cell such as cell mating, electron transport chain, protein trafficking, cell cycle events and others. Even before the genome sequence of the yeast was out, the structural organization and function of several of its genes was known. With the availability of the origin of replication from the 2 μm plasmid and the development of transformation system, it became the host of choice for expression of a number of important proteins. A large number of episomal and integrative shuttle vectors are available for expression of mammalian proteins. The latest developments in genomics and micro-array technology have allowed investigations of individual gene function by site-specific deletion method. The application of metabolic profiling has also assisted in understanding the cellular network operating in this yeast. This chapter is aimed at reviewing the use of this system as an experimental tool for conducting classical genetics. Various vector systems available, foreign genes expressed and the limitations as a host will be discussed. Finally, the use of various yeast enzymes in biotechnology sector will be reviewed.

  14. Vaginal Yeast Infections

    Science.gov (United States)

    ... Yeast Infections Print A A A en español Infecciones vaginales por hongos What Are Vaginal Yeast Infections? ... keep the amount in a person's body under control. But yeast in the vagina can sometimes "overgrow" ...

  15. Yeast Infection (Vaginal)

    Science.gov (United States)

    ... vaginal discharge with a cottage cheese appearance Complicated yeast infection You might have a complicated yeast infection ... have an uncomplicated or a complicated infection. Uncomplicated yeast infection For mild to moderate symptoms and infrequent ...

  16. Expression in yeast of the T-urf13 protein from Texas male-sterile maize mitochondria confers sensitivity to methomyl and to Texas-cytoplasm-specific fungal toxins.

    Science.gov (United States)

    Huang, J; Lee, S H; Lin, C; Medici, R; Hack, E; Myers, A M

    1990-01-01

    The mitochondrial gene T-urf13 from maize (Zea mays L.) with Texas male-sterile (T) cytoplasm codes for a unique 13 kd polypeptide, T-URF13, which is implicated in cytoplasmic male sterility and sensitivity to the insecticide methomyl and to host-specific fungal toxins produced by Helminthosporium maydis race T (HmT toxin) and Phyllosticta maydis (Pm toxin). A chimeric gene coding for T-URF13 fused to the mitochondrial targeting peptide from the Neurospora crassa ATP synthase subunit 9 precursor was constructed. Expression of this gene in the yeast Saccharomyces cerevisiae yielded a polypeptide that was translocated into the membrane fraction of mitochondria and processed to give a protein the same size as maize T-URF13. Methomyl, HmT toxin and Pm toxin inhibited growth of yeast cells expressing the gene fusion on medium containing glycerol as sole carbon source and stimulated respiration with NADH as substrate by isolated mitochondria from these cells. These effects were not observed in yeast cells expressing T-URF13 without a targeting peptide. The results show that T-URF13 is sufficient to confer sensitivity to methomyl and the fungal toxins in a heterologous eukaryotic system, and suggest that mitochondrial localization of T-URF13 is critical for these functions. Images Fig. 2. Fig. 3. Fig. 5. PMID:2303028

  17. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  18. Yeast Interacting Proteins Database: YFR015C, YLR258W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entr...; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entry into statio

  19. Signature gene expressions of cell wall integrity pathway concur with tolerance response of industrial yeast Saccharomyces cerevisiae against biomass pretreatment inhibitors

    Science.gov (United States)

    Traditional industrial ethanologenic yeast Saccharomyces cerevisiae has a robust performance under various environmental conditions and can be served as a candidate for the next-generation biocatalyst development for advanced biofuels production using lignocellulose mateials. Overcoming toxic compou...

  20. Biotechnological Applications of Dimorphic Yeasts

    Science.gov (United States)

    Doiphode, N.; Joshi, C.; Ghormade, V.; Deshpande, M. V.

    The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic Saccharomyces, Mucor, Rhizopus and Benjaminiella, oil degradation and biotransformation with yeast-form of Yarrowia species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of Aureobasidium pullulans, to name a few. Myrothecium verrucaria can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while Beauveria bassiana and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make Benjaminiella poitrasii, Basidiobolus sp., and Mucor rouxii strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

  1. Insight into the Recent Genome Duplication of the Halophilic Yeast Hortaea werneckii: Combining an Improved Genome with Gene Expression and Chromatin Structure

    Directory of Open Access Journals (Sweden)

    Sunita Sinha

    2017-07-01

    Full Text Available Extremophilic organisms demonstrate the flexibility and adaptability of basic biological processes by highlighting how cell physiology adapts to environmental extremes. Few eukaryotic extremophiles have been well studied and only a small number are amenable to laboratory cultivation and manipulation. A detailed characterization of the genome architecture of such organisms is important to illuminate how they adapt to environmental stresses. One excellent example of a fungal extremophile is the halophile Hortaea werneckii (Pezizomycotina, Dothideomycetes, Capnodiales, a yeast-like fungus able to thrive at near-saturating concentrations of sodium chloride and which is also tolerant to both UV irradiation and desiccation. Given its unique lifestyle and its remarkably recent whole genome duplication, H. werneckii provides opportunities for testing the role of genome duplications and adaptability to extreme environments. We previously assembled the genome of H. werneckii using short-read sequencing technology and found a remarkable degree of gene duplication. Technology limitations, however, precluded high-confidence annotation of the entire genome. We therefore revisited the H. wernickii genome using long-read, single-molecule sequencing and provide an improved genome assembly which, combined with transcriptome and nucleosome analysis, provides a useful resource for fungal halophile genomics. Remarkably, the ∼50 Mb H. wernickii genome contains 15,974 genes of which 95% (7608 are duplicates formed by a recent whole genome duplication (WGD, with an average of 5% protein sequence divergence between them. We found that the WGD is extraordinarily recent, and compared to Saccharomyces cerevisiae, the majority of the genome’s ohnologs have not diverged at the level of gene expression of chromatin structure.

  2. Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11 and MDR3 (ABCB4 expressed in the yeast Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    Philipp Ellinger

    Full Text Available The human liver ATP-binding cassette (ABC transporters bile salt export pump (BSEP/ABCB11 and the multidrug resistance protein 3 (MDR3/ABCB4 fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.

  3. Oral application of freeze-dried yeast particles expressing the PCV2b Cap protein on their surface induce protection to subsequent PCV2b challenge in vivo.

    Science.gov (United States)

    Patterson, Robert; Eley, Thomas; Browne, Christopher; Martineau, Henny M; Werling, Dirk

    2015-11-17

    Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCV-associated disease (PCVAD), linked with large scale economic losses. Current vaccination strategies are based on the capsid protein of the virus and are reasonably successful in preventing PCVAD but fail to induce sterile immunity. Additionally, vaccinating whole herds is expensive and time consuming. In the present study a "proof of concept" vaccine trial was employed to test the effectiveness of powdered freeze-dried recombinant Saccharomyces cerevisiae yeast stably expressing the capsid protein of PCV2b on its surface as an orally applied vaccine. PCV2-free pigs were given 3 doses of vaccine or left un-vaccinated before challenge with a defined PCV2b strain. Rectal temperatures were measured and serum and faeces samples were collected weekly. At the end of the study, pigs were euthanized, tissue samples taken and tested for PCV2b load by qPCR and immunohistochemistry. The peak of viraemia in sera and faeces of unvaccinated pigs was higher than that of vaccinated pigs. Additionally more sIgA was found in faeces of vaccinated pigs than unvaccinated. Vaccination was associated with lower serum concentrations of TNFα and IL-1β but higher concentrations of IFNα and IFNγ in comparison to the unvaccinated animals. At the end of the trial, a higher viral load was found in several lymphatic tissues and the ileum of unvaccinated pigs in comparison to vaccinated pigs. The difference between groups was especially apparent in the ileum. The results presented here demonstrate a possible use for recombinant S. cerevisiae expressing viral proteins as an oral vaccine against PCV2. A powdered freeze-dried recombinant S. cerevisiae used as an oral vaccine could be mixed with feed and may offer a cheap and less labour intensive alternative to inoculation with the additional advantage that no cooling chain would be required for vaccine transport and storage. Copyright © 2015 The

  4. The Use of HIS6 Gene as a Selectable Marker for Yeast Vector

    OpenAIRE

    IMADEARTIKA,

    2009-01-01

    The yeast Saccharomyces cerevisiae HIS6 gene has been shown to be functional as a selectable marker for selecting and maintaining a yeast vector in yeast S. cerevisiae host cells. The yeast HIS6 gene encodes an enzyme involved in the yeast histidine biosynthesis. The yeast HIS6 gene was cloned into a yeast expression vector. The resultant recombinant plasmid was introduced into yeast host cells defective in endogenous HIS6 gene. The functionality of the HIS6 gene as a selectable marker was te...

  5. The Use of HIS6 Gene as a Selectable Marker for Yeast Vector

    Directory of Open Access Journals (Sweden)

    IMADEARTIKA

    2009-03-01

    Full Text Available The yeast Saccharomyces cerevisiae HIS6 gene has been shown to be functional as a selectable marker for selecting and maintaining a yeast vector in yeast S. cerevisiae host cells. The yeast HIS6 gene encodes an enzyme involved in the yeast histidine biosynthesis. The yeast HIS6 gene was cloned into a yeast expression vector. The resultant recombinant plasmid was introduced into yeast host cells defective in endogenous HIS6 gene. The functionality of the HIS6 gene as a selectable marker was tested by growing transformed cells on selective minimum medium lacking histidine supplementation.

  6. Prions in yeast

    OpenAIRE

    Bezdíčka, Martin

    2013-01-01

    The thesis describes yeast prions and their biological effects on yeast in general. It defines the basic characteristics of yeast prions, that distinguish prions from other proteins. The thesis introduces various possibilities of prion formation, and propagation as well as specific types of yeast prions, including various functions of most studied types of prions. The thesis also focuses on chaperones that affect the state of yeast prions in cells. Lastly, the thesis indicates similarities be...

  7. Cloning, characterization and heterologous expression of epoxide hydrolase-encoding cDNA sequences from yeasts belonging to the genera Rhodotorula and Rhodosporidium

    NARCIS (Netherlands)

    Visser, H.; Weijers, C.A.G.M.; Ooyen, van A.J.J.; Verdoes, J.C.

    2002-01-01

    Epoxide hydrolase-encoding cDNA sequences were isolated from the basidiomycetous yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031 in order to evaluate the molecular data and potential application of this type of enzymes. The

  8. Phytase-producing capacity of yeasts isolated from traditional African fermented food products and PHYPk gene expression of Pichia kudriavzevii strains

    DEFF Research Database (Denmark)

    Greppi, Anna; Krych, Lukasz; Costantini, Antonella

    2015-01-01

    Phytate is known as a strong chelate of minerals causing their reduced uptake by the human intestine. Ninety-three yeast isolates from traditional African fermented food products, belonging to nine species (Pichia kudriavzevii, Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces...... marxianus, Millerozyma farinosa, Candida glabrata, Wickerhamomyces anomalus, Hanseniaspora guilliermondii and Debaryomyces nepalensis) were screened for phytase production on solid and liquid media. 95% were able to grow in the presence of phytate as sole phosphate source, P. kudriavzevii being the best...... growing species. A phytase coding gene of P. kudriavzevii (PHYPk) was identified and its expression was studied during growth by RT-qPCR. The expression level of PHYPk was significantly higher in phytate-medium, compared to phosphate-medium. In phytate-medium expression was seen in the lag phase...

  9. Yeast Interacting Proteins Database: YFR015C, YJL137C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entr...pression induced by glucose limitation, nitrogen starvation, environmental stress, and entry into stationary

  10. Yeast Infection during Pregnancy

    Science.gov (United States)

    ... OK? What's the best way to treat a yeast infection during pregnancy? Answers from Yvonne Butler Tobah, M.D. You can safely treat a yeast infection during pregnancy with various over-the-counter ...

  11. [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies].

    Science.gov (United States)

    Aydın, Cevahir; Ataoğlu, Haluk

    2015-01-01

    Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method

  12. Accelerating Yeast Prion Biology using Droplet Microfluidics

    Science.gov (United States)

    Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

    2012-02-01

    Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

  13. Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

    OpenAIRE

    Verstrepen, Kevin J; Van Laere, Stijn D. M.; Vanderhaegen, Bart M. P.; Derdelinckx, Guy; Dufour, Jean-Pierre; Pretorius, Isak S.; Winderickx, Joris; Thevelein, Johan?M.; Delvaux, Freddy R

    2003-01-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing str...

  14. Yeast cell factories on the horizon

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2015-01-01

    been engineered to make chemicals at industrial scale (e.g., succinic acid, lactic acid, resveratrol) and advanced biofuels (e.g., isobutanol) (1). On page 1095 of this issue, Galanie et al. (2) demonstrate that yeast can now be engineered to produce opioids (2), a major class of compounds used...... for treating severe pain. Their study represents a tour de force in the metabolic engineering of yeast, as it involved the expression of genes for more than 20 enzymatic activities from plants, mammals, bacteria, and yeast itself. It clearly represents a breakthrough advance for making complex natural products...

  15. Combinatorial pathway assembly in yeast

    Directory of Open Access Journals (Sweden)

    Khalil Essani

    2015-10-01

    Full Text Available With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reactions, the challenge nowadays is to implement whole natural or synthetic pathways into microorganisms. For this purpose balanced enzyme activities throughout the pathway need to be achieved in addition to simple functional gene expression to avoid bottlenecks and to obtain high titers of the desired product. As the optimization of pathways in a specific biological context is often hard to achieve by rational design, combinatorial approaches have been developed to address this issue. Here, current strategies and proof of concepts for combinatorial pathway assembly in yeasts are reviewed. By exploiting its ability to join multiple DNA fragments in a very efficient and easy manner, the yeast Saccharomyces cerevisiae does not only constitute an attractive host for heterologous pathway expression, but also for assembling pathways by recombination in vivo.

  16. Yeast for virus research

    Science.gov (United States)

    Zhao, Richard Yuqi

    2017-01-01

    Budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) are two popular model organisms for virus research. They are natural hosts for viruses as they carry their own indigenous viruses. Both yeasts have been used for studies of plant, animal and human viruses. Many positive sense (+) RNA viruses and some DNA viruses replicate with various levels in yeasts, thus allowing study of those viral activities during viral life cycle. Yeasts are single cell eukaryotic organisms. Hence, many of the fundamental cellular functions such as cell cycle regulation or programed cell death are highly conserved from yeasts to higher eukaryotes. Therefore, they are particularly suited to study the impact of those viral activities on related cellular activities during virus-host interactions. Yeasts present many unique advantages in virus research over high eukaryotes. Yeast cells are easy to maintain in the laboratory with relative short doubling time. They are non-biohazardous, genetically amendable with small genomes that permit genome-wide analysis of virologic and cellular functions. In this review, similarities and differences of these two yeasts are described. Studies of virologic activities such as viral translation, viral replication and genome-wide study of virus-cell interactions in yeasts are highlighted. Impacts of viral proteins on basic cellular functions such as cell cycle regulation and programed cell death are discussed. Potential applications of using yeasts as hosts to carry out functional analysis of small viral genome and to develop high throughput drug screening platform for the discovery of antiviral drugs are presented. PMID:29082230

  17. Establishment of Salvia castanea Diels f. tomentosa Stib. hairy root cultures and the promotion of tanshinone accumulation and gene expression with Ag⁺, methyl jasmonate, and yeast extract elicitation.

    Science.gov (United States)

    Li, Bo; Wang, Bangqing; Li, Hongyan; Peng, Liang; Ru, Mei; Liang, Zongsuo; Yan, Xijun; Zhu, Yonghong

    2016-01-01

    Salvia castanea Diels f. tomentosa Stib. is an endemic medicinal plant distributed in China, and the notably high content of tanshinone IIA in the root is proven effective for the therapy of heart diseases. Hairy root induction of this Salvia species was inoculated with Agrobacterium rhizogenes strain ATCC 15834. Transformed hairy root was cultured in 6,7-V liquid medium for growth kinetics assessment and elicitation. An S curve was present in the hairy root cultures based on the fresh and dry weights with an interval of 3 days. An optimum concentration of the applied elicitors (15 μM Ag(+), 200 μM methyl jasmonate, and 200 mg l(-1) yeast extract elicitor) benefitted both the growth status and tanshinone accumulation in the hairy root cultures. Tanshinone IIA contents were mostly stimulated 1.8-fold and 1.99-fold compared with the control by Ag(+) and methyl jasmonate elicitation, respectively. Yeast extract dramatically enhanced dry mass accumulation, while it promoted cryptotanshinone content of 2.84 ± 0.33 mg g(-1) dry weight at most in the hairy root cultures. Selected elicitors diversely influenced tanshinone accumulation in the time courses of hairy root cultures within 7 days. Furthermore, transcripts of selected genes in the tanshinone biosynthetic pathway were remarkably upregulated with elicitation. Yeast extract elicitor heightened 13.9-fold of isopentenyl diphosphate isomerase expression level at 12 h, while it increased 16.7-fold of geranylgeranyl diphosphate synthase transcript at 24 h compared with that of the control, which was more effective than Ag(+) and methyl jasmonate. This study provided a convenient hairy root culture system of S. castanea Diels f. tomentosa Stib. for tanshinone production for the first time.

  18. Yeast metabolic engineering for hemicellulosic ethanol production

    Science.gov (United States)

    Jennifer Van Vleet; Thomas W. Jeffries

    2009-01-01

    Efficient fermentation of hemicellulosic sugars is critical for the bioconversion of lignocellulosics to ethanol. Efficient sugar uptake through the heterologous expression of yeast and fungal xylose/glucose transporters can improve fermentation if other metabolic steps are not rate limiting. Rectification of cofactor imbalances through heterologous expression of...

  19. Yeast-based biosensors: design and applications.

    Science.gov (United States)

    Adeniran, Adebola; Sherer, Michael; Tyo, Keith E J

    2015-02-01

    Yeast-based biosensing (YBB) is an exciting research area, as many studies have demonstrated the use of yeasts to accurately detect specific molecules. Biosensors incorporating various yeasts have been reported to detect an incredibly large range of molecules including but not limited to odorants, metals, intracellular metabolites, carcinogens, lactate, alcohols, and sugars. We review the detection strategies available for different types of analytes, as well as the wide range of output methods that have been incorporated with yeast biosensors. We group biosensors into two categories: those that are dependent upon transcription of a gene to report the detection of a desired molecule and those that are independent of this reporting mechanism. Transcription-dependent biosensors frequently depend on heterologous expression of sensing elements from non-yeast organisms, a strategy that has greatly expanded the range of molecules available for detection by YBBs. Transcription-independent biosensors circumvent the problem of sensing difficult-to-detect analytes by instead relying on yeast metabolism to generate easily detected molecules when the analyte is present. The use of yeast as the sensing element in biosensors has proven to be successful and continues to hold great promise for a variety of applications. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  20. Expression of a Human Cytochrome P450 in Yeast Permits Analysis of Pathways for Response to and Repair of Aflatoxin-Induced DNA Damage†

    Science.gov (United States)

    Guo, Yingying; Breeden, Linda L.; Zarbl, Helmut; Preston, Bradley D.; Eaton, David L.

    2005-01-01

    Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity. PMID:15988000

  1. Expression of a human cytochrome p450 in yeast permits analysis of pathways for response to and repair of aflatoxin-induced DNA damage.

    Science.gov (United States)

    Guo, Yingying; Breeden, Linda L; Zarbl, Helmut; Preston, Bradley D; Eaton, David L

    2005-07-01

    Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity.

  2. Biomedical applications of yeast- a patent view, part one: yeasts as workhorses for the production of therapeutics and vaccines.

    Science.gov (United States)

    Roohvand, Farzin; Shokri, Mehdi; Abdollahpour-Alitappeh, Meghdad; Ehsani, Parastoo

    2017-08-01

    Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall β-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.

  3. Effects of albusin B (a bacteriocin) of Ruminococcus albus 7 expressed by yeast on growth performance and intestinal absorption of broiler chickens--its potential role as an alternative to feed antibiotics.

    Science.gov (United States)

    Wang, Han-Tsung; Yu, Chi; Hsieh, Ya-Hui; Chen, Shiau-Wei; Chen, Bao-Ji; Chen, Ching-Yi

    2011-10-01

    Bacteriocins with antimicrobial activity are considered as potential alternatives to antibiotics. The aim of this study was to investigate the effect of albusin B (bacteriocin) of Ruminococcus albus 7 expressed by yeast on the growth performance of broiler chickens. Ninety 1-day-old healthy broiler chickens were randomly divided into three groups: control, albusin B (2.5 g kg(-1)) and nosiheptide (2.5 mg kg(-1), antibiotic control). Growth performance and intestinal functions were measured at 5 weeks of age. Albusin B-supplemented broilers showed increased body weight gain compared with control broilers (54.7 ± 5.3 vs 48.5 ± 6.1 g day(-1) per bird, P alternatives to antibiotics in broiler chicken feed. Copyright © 2011 Society of Chemical Industry.

  4. Chemotropism during yeast mating.

    Science.gov (United States)

    Follette, Peter J; Arkowitz, Robert A

    2009-01-01

    Virtually all eukaryotic cells can grow in a polarized fashion in response to external signals. Cells can respond to gradients of chemoattractants or chemorepellents by directional growth, a process referred to as chemotropism. The budding yeast Saccharomyces cerevisiae undergoes chemotropic growth during mating, in which two haploid cells of opposite mating type grow toward one another. We have shown that mating pheromone gradients are essential for efficient mating in yeast and have examined the chemotropism defects of different yeast mutants. Two methods of assessing the ability of yeast strains to respond to pheromone gradients are presented here.

  5. Prions in Yeast

    Science.gov (United States)

    Liebman, Susan W.; Chernoff, Yury O.

    2012-01-01

    The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

  6. The yeast Golgi apparatus.

    Science.gov (United States)

    Suda, Yasuyuki; Nakano, Akihiko

    2012-04-01

    The Golgi apparatus is an organelle that has been extensively studied in the model eukaryote, yeast. Its morphology varies among yeast species; the Golgi exists as a system of dispersed cisternae in the case of the budding yeast Saccharomyces cerevisiae, whereas the Golgi cisternae in Pichia pastoris and Schizosaccharomyces pombe are organized into stacks. In spite of the different organization, the mechanism of trafficking through the Golgi apparatus is believed to be similar, involving cisternal maturation, in which the resident Golgi proteins are transported backwards while secretory cargo proteins can stay in the cisternae. Questions remain regarding the organization of the yeast Golgi, the regulatory mechanisms that underlie cisternal maturation of the Golgi and transport machinery of cargo proteins through this organelle. Studies using different yeast species have provided hints to these mechanisms. © 2011 John Wiley & Sons A/S.

  7. Biofuels. Altered sterol composition renders yeast thermotolerant

    DEFF Research Database (Denmark)

    Caspeta, Luis; Chen, Yun; Ghiaci, Payam

    2014-01-01

    adaptive laboratory evolution to select yeast strains with improved growth and ethanol production at ≥40°C. Sequencing of the whole genome, genome-wide gene expression, and metabolic-flux analyses revealed a change in sterol composition, from ergosterol to fecosterol, caused by mutations in the C-5 sterol...

  8. yeast transformation of Mucor circinelloides Tieghe

    African Journals Online (AJOL)

    GRACE

    2006-05-02

    May 2, 2006 ... In a study monitored at 24 h intervals, profiling gave 2-phase expression in some treatments and this was ..... observed in the broths, although minimal in contrast to the observation in the 2- phase growth pattern described in ... Microscopic examination showed that the morphology of note was the yeast form ...

  9. Ureohydrolases as dominant selectable markers in yeast

    NARCIS (Netherlands)

    Daran, J.G.; Pronk, J.T.; Romagnoli, G.

    2015-01-01

    The invention relates to a nucleic acid molecule encoding a novel selection marker. Said marker is a guanidinobutyrase from Kluyveromyces lactis, which, when expressed in Saccharomyces, allows the growth of the yeast in the presence of guanidinobutyrate as the sole nitrogen source. Said marker can

  10. Tools for genetic engineering of the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Saraya, Ruchi; Gidijala, Loknath; Veenhuis, Marten; van der Klei, Ida J; Mapelli, Valeria

    2014-01-01

    Hansenula polymorpha is a methylotrophic yeast species that has favorable properties for heterologous protein production and metabolic engineering. It provides an attractive expression platform with the capability to secrete high levels of commercially important proteins. Over the past few years

  11. [Penicillium-inhibiting yeasts].

    Science.gov (United States)

    Benítez Ahrendts, M R; Carrillo, L

    2004-01-01

    The objective of this work was to establish the in vitro and in vivo inhibition of post-harvest pathogenic moulds by yeasts in order to make a biocontrol product. Post-harvest pathogenic moulds Penicillium digitatum, P. italicum, P. ulaiense, Phyllosticta sp., Galactomyces geotrichum and yeasts belonging to genera Brettanomyces, Candida, Cryptococcus, Kloeckera, Pichia, Rhodotorula were isolated from citrus fruits. Some yeasts strains were also isolated from other sources. The yeasts were identified by their macro and micro-morphology and physiological tests. The in vitro and in vivo activities against P. digitatum or P. ulaiense were different. Candida cantarellii and one strain of Pichia subpelliculosa produced a significant reduction of the lesion area caused by the pathogenic moulds P. digitatum and P. ulaiense, and could be used in a biocontrol product formulation.

  12. Kinetic analysis of gene expression during mycelium to yeast transition and yeast to mycelium germination in Paracoccidioides brasiliensis Análisis de la cinética de expresión de genes durante la transición de micelio a levadura y la germinación levadura a micelio en Paracoccidioides brasiliensis

    Directory of Open Access Journals (Sweden)

    Ana María García

    2011-06-01

    Full Text Available Introduction.Paracoccidioidomycosis is an endemic systemic mycosis caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus that in tissues and cultures at 37ºC grows as a yeast while at lower temperatures (less than 24 ºC it becomes a mold; however the genes that rule these processes and their expression are poorly understood.
    Objective.This research focused on the kinetic expression of certain genes in P. brasiliensis throughout the dimorphic process, one that involves the transition from the mycelium-to-yeast(M-Y forms and the germination from the yeast-to-mycelium(Y- M form.
    Materials and methods. A real-time quantitative polymerase chain reaction(RT-qPCR was optimized to measure the expression of ten genes connected with diverse cellular functions including cell synthesis and wall structure, oxidative stress response, heat shock response, metabolism, proteins' processing, solute transport across the cell membrane and signal transduction pathways at different time points during the M-Y transition, as well as in the Y-M germination processes.
    Results.Genes involved in cell synthesis and wall structure, metabolism and signal transduction were differentially expressed and highly up-regulated during the Y-M germination process; on the other hand, genes involved in heat shock response, cell synthesis and wall structure were highly up-regulated during the M-Y transition process. The remaining genes were differentially regulated during both processes.
    Conclusion.In this work the up-regulation of certain genes involved in the morphological changes occurring in P. brasiliensis yeast and mycelia forms were confirmed, indicating that these biological processes play an important role during the host-pathogen interactions, as well as in the fungus adaptation to environmental conditions
    Introducción. La paracoccidioidomicosis es una micosis sistémica causada por el hogo termo-dimórfico Paracoccidioides

  13. The glucose signaling network in yeast

    Science.gov (United States)

    Kim, Jeong-Ho; Roy, Adhiraj; Jouandot, David; Cho, Kyu Hong

    2013-01-01

    Background Most cells possess a sophisticated mechanism for sensing glucose and responsing to it appropriately. Glucose sensing and signaling in the budding yeast Saccharomyces cerevisiae represents an important paradigm for understanding how extracellular signals lead to changes in the gene expression program in eukaryotes. Scope of review This review focuses on the yeast glucose sensing and signaling pathways that operate in a highly regulated and cooperative manner to bring about glucose-induction of HXT gene expression. Major conclusions The yeast cells possess a family of glucose transporters (HXTs), with different kinetic properties. They employ three major glucose signaling pathways— Rgt2/Snf3, AMPK, and cAMP-PKA—to express only those transporters best suited for the amounts of glucose available. We discuss the current understanding of how these pathways are integrated into a regulatory network to ensure efficient uptake and utilization of glucose. General significance Elucidating the role of multiple glucose signals and pathways involved in glucose uptake and metabolism in yeast may reveal the molecular basis of glucose homeostasis in humans, especially under pathological conditions, such as hyperglycemia in diabetics and the elevated rate of glycolysis observed in many solid tumors. PMID:23911748

  14. Yeast flocculation: what brewers should know.

    Science.gov (United States)

    Verstrepen, K J; Derdelinckx, G; Verachtert, H; Delvaux, F R

    2003-05-01

    For many industrial applications in which the yeast Saccharomyces cerevisiae is used, e.g. beer, wine and alcohol production, appropriate flocculation behaviour is certainly one of the most important characteristics of a good production strain. Yeast flocculation is a very complex process that depends on the expression of specific flocculation genes such as FLO1, FLO5, FLO8 and FLO11. The transcriptional activity of the flocculation genes is influenced by the nutritional status of the yeast cells as well as other stress factors. Flocculation is also controlled by factors that affect cell wall composition or morphology. This implies that, during industrial fermentation processes, flocculation is affected by numerous parameters such as nutrient conditions, dissolved oxygen, pH, fermentation temperature, and yeast handling and storage conditions. Theoretically, rational use of these parameters offers the possibility of gaining control over the flocculation process. However, flocculation is a very strain-specific phenomenon, making it difficult to predict specific responses. In addition, certain genes involved in flocculation are extremely variable, causing frequent changes in the flocculation profile of some strains. Therefore, both a profound knowledge of flocculation theory as well as close monitoring and characterisation of the production strain are essential in order to gain maximal control over flocculation. In this review, the various parameters that influence flocculation in real-scale brewing are critically discussed. However, many of the conclusions will also be useful in various other industrial processes where control over yeast flocculation is desirable.

  15. Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

    Directory of Open Access Journals (Sweden)

    Reza Saberianfar

    Full Text Available In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD. Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis.

  16. Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

    Science.gov (United States)

    Shi, Nianci; Mao, Weian; He, Xiaoxia; Chi, Zhe; Chi, Zhenming; Liu, Guanglei

    2017-11-18

    Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

  17. Biotechnology of non-Saccharomyces yeasts--the ascomycetes.

    Science.gov (United States)

    Johnson, Eric A

    2013-01-01

    Saccharomyces cerevisiae and several other yeast species are among the most important groups of biotechnological organisms. S. cerevisiae and closely related ascomycetous yeasts are the major producer of biotechnology products worldwide, exceeding other groups of industrial microorganisms in productivity and economic revenues. Traditional industrial attributes of the S. cerevisiae group include their primary roles in food fermentations such as beers, cider, wines, sake, distilled spirits, bakery products, cheese, sausages, and other fermented foods. Other long-standing industrial processes involving S. cerevisae yeasts are production of fuel ethanol, single-cell protein (SCP), feeds and fodder, industrial enzymes, and small molecular weight metabolites. More recently, non-Saccharomyces yeasts (non-conventional yeasts) have been utilized as industrial organisms for a variety of biotechnological roles. Non-Saccharomyces yeasts are increasingly being used as hosts for expression of proteins, biocatalysts and multi-enzyme pathways for the synthesis of fine chemicals and small molecular weight compounds of medicinal and nutritional importance. Non-Saccharomyces yeasts also have important roles in agriculture as agents of biocontrol, bioremediation, and as indicators of environmental quality. Several of these products and processes have reached commercial utility, while others are in advanced development. The objective of this mini-review is to describe processes currently used by industry and those in developmental stages and close to commercialization primarily from non-Saccharomyces yeasts with an emphasis on new opportunities. The utility of S. cerevisiae in heterologous production of selected products is also described.

  18. Effect of salt hyperosmotic stress on yeast cell viability

    Directory of Open Access Journals (Sweden)

    Logothetis Stelios

    2007-01-01

    Full Text Available During fermentation for ethanol production, yeasts are subjected to different kinds of physico-chemical stresses such as: initially high sugar concentration and low temperature; and later, increased ethanol concentrations. Such conditions trigger a series of biological responses in an effort to maintain cell cycle progress and yeast cell viability. Regarding osmostress, many studies have been focused on transcriptional activation and gene expression in laboratory strains of Saccharomyces cerevisiae. The overall aim of this present work was to further our understanding of wine yeast performance during fermentations under osmotic stress conditions. Specifically, the research work focused on the evaluation of NaCl-induced stress responses of an industrial wine yeast strain S. cerevisiae (VIN 13, particularly with regard to yeast cell growth and viability. The hypothesis was that osmostress conditions energized specific genes to enable yeast cells to survive under stressful conditions. Experiments were designed by pretreating cells with different sodium chloride concentrations (NaCl: 4%, 6% and 10% w/v growing in defined media containing D-glucose and evaluating the impact of this on yeast growth and viability. Subsequent fermentation cycles took place with increasing concentrations of D-glucose (20%, 30%, 40% w/v using salt-adapted cells as inocula. We present evidence that osmostress induced by mild salt pre-treatments resulted in beneficial influences on both cell viability and fermentation performance of an industrial wine yeast strain.

  19. Yeast genome sequencing:

    DEFF Research Database (Denmark)

    Piskur, Jure; Langkjær, Rikke Breinhold

    2004-01-01

    they are short and degenerate and occupy different positions. Comparative genomics helps to understand the origin of yeasts and points out crucial molecular events in yeast evolutionary history, such as whole-genome duplication and horizontal gene transfer(s). In addition, the accumulating sequence data provide...... that the minimum number of genes from each species that need to be compared to produce a reliable phylogeny is about 20. Yeast has also become an attractive model to study speciation in eukaryotes, especially to understand molecular mechanisms behind the establishment of reproductive isolation. Comparison...... of closely related species helps in gene annotation and to answer how many genes there really are within the genomes. Analysis of non-coding regions among closely related species has provided an example of how to determine novel gene regulatory sequences, which were previously difficult to analyse because...

  20. Immunogenicity and antigenicity of the recombinant EMA-1 protein of Theileria equi expressed in the yeast Pichia pastoris Imunogenicidade e antigenicidade da proteína recombinante EMA-1 de Theileria equi expressa em Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Leandro Q. Nizoli

    2009-06-01

    Full Text Available The equine piroplasmosis caused by Theileria equi is one of the most important parasitic diseases of the equine, causing damage to animal health and economic losses. In T. equi, 2 merozoite surface proteins, equi merozoite antigen EMA-1 and EMA-2, have been identified as the most immunodominant antigens. This suggests that these antigens might be used as immunobiological tools. The EMA-1 of Theileria equi was cloned and expressed in the yeast Pichia pastoris. The transformed yeast was grown at high cell density, expressing up to 389 mg.L-1 of recombinant protein. The protein was concentrated and detected in Dot blot. The recombinant product was antigenically similar to the native protein as determined using monoclonal antibodies, and polyclonal antibodies obtained from equines naturally infected with T. equi. The immunogenicity of rEMA-1 protein was demonstrated by IFAT using sera from recombinant-protein-immunized mice using aluminum hydroxide as adjuvant. All animals vaccinated with rEMA-1 developed a high specific antibody response. This results suggest that rEMA-1expressed in P. pastoris might be a strong candidate to be used as an antigen for immune diagnostics as well as a vaccine antigen.A piroplasmose equina causada por Theileria equi é uma das mais importantes doenças parasitárias de equídeos, causando danos a saúde animal e perdas econômicas. Em T. equi, 2 proteínas de superfície de merozoítos, equi merozoite antigen EMA-1 e EMA-2, têm sido identificadas como antígenos imunodominantes. Sugerindo que estes antígenos possam ser usados como produtos imunobiológicos. O gene EMA-1 de T. equi foi clonado e expressado na levedura Pichia pastoris. As leveduras transformadas foram cultivadas a altas densidades celulares expressando 389 mg.L-1 de proteína recombinante. A proteína foi concentrada e detectada em Dot blot. O produto recombinante foi antigenicamente similar à proteína nativa quando determinado usando anticorpo

  1. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    Science.gov (United States)

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  2. Genetics of Yeasts

    Science.gov (United States)

    Querol, Amparo; Fernández-Espinar, M. Teresa; Belloch, Carmela

    The use of yeasts in biotechnology processes dates back to ancient days. Before 7000 BC, beer was produced in Sumeria. Wine was made in Assyria in 3500 BC, and ancient Rome had over 250 bakeries, which were making leavened bread by 100 BC. And milk has been made into Kefyr and Koumiss in Asia for many centuries (Demain, Phaff, & Kurtzman, 1999). However, the importance of yeast in the food and beverage industries was only realized about 1860, when their role in food manufacturing became evident.

  3. Validation and application of a robust yeast estrogen bioassay for the screening of estrogenic activity in animal feed

    NARCIS (Netherlands)

    Bovee, T.F.H.; Bor, G.; Heskamp, H.H.; Hoogenboom, L.A.P.; Nielen, M.W.F.

    2006-01-01

    Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor ¿ (hER¿) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to estrogens. In the present study this yeast estrogen assay was validated as a

  4. Opportunistic Pathogenic Yeasts

    Science.gov (United States)

    Banerjee, Uma

    Advances in medical research, made during the last few decades, have improved the prophylactic, diagnostic and therapeutic capabilities for variety of infections/diseases. However, many of the prophylactic and therapeutic procedures have been seen in many instances to exact a price of host-vulnerability to an expanding group of opportunistic pathogens and yeasts are one of the important members in it. Fortunately amongst the vast majority of yeasts present in nature only few are considered to have the capability to cause infections when certain opportunities predisposes and these are termed as ‘opportunistic pathogenic yeasts.’ However, the term ‘pathogenic’ is quite tricky, as it depends of various factors of the host, the ‘bug’ and the environment to manifest the clinical infection. The borderline is expanding. In the present century with unprecedented increase in number of immune-compromised host in various disciplines of health care settings, where any yeast, which has the capability to grow at 37 ° C (normal body temperature of human), can be pathogenic and cause infection in particular situation

  5. Recombinant wine yeasts

    OpenAIRE

    González García, Ramón; González Ramos, Daniel

    2008-01-01

    The invention relates to a method for obtaining strains that secrete a higher concentration of mannoproteins to the medium, a Saccharomyces cerevisiae yeast strain deposited at the Spanish Type Culture Collection (CECT) as CECT 13012, and to the uses of said strains.

  6. Enhanced tolerance to NaCl and LiCl stresses by over-expressing Caragana korshinskii sodium/proton exchanger 1 (CkNHX1) and the hydrophilic C terminus is required for the activity of CkNHX1 in Atsos3-1 mutant and yeast

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Da-Hai, E-mail: gresea_young@hotmail.com [Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101 (China); Department of Plant Physiology, Institute of General Botany and Plant Physiology, Friedrich-Schiller-University, Dornburger Strasse 159, 07743 Jena (Germany); Song, Li-Ying, E-mail: lysong@genetics.ac.cn [Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101 (China); Hu, Jun, E-mail: jhu@genetics.ac.cn [Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101 (China); Yin, Wei-Bo, E-mail: wbyin@genetics.ac.cn [Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101 (China); Li, Zhi-Guo, E-mail: gzhi@genetics.ac.cn [Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101 (China); Chen, Yu-Hong, E-mail: yhchen@genetics.ac.cn [Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101 (China); Su, Xiao-Hua, E-mail: suxh@caf.ac.cn [Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091 (China); Wang, Richard R.-C., E-mail: Richard.Wang@ARS.USDA.GOV [USDA-ARS, FRRL, Utah State University, Logan, UT 84322-6300 (United States); Hu, Zan-Min, E-mail: zmhu@genetics.ac.cn [Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101 (China)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer CkNHX1 was isolated from Caragana korshinskii. Black-Right-Pointing-Pointer CkNHX1 was expressed mainly in roots, and significantly induced by NaCl in stems. Black-Right-Pointing-Pointer Expression of CkNHX1 enhanced the resistance to NaCl and LiCl in yeast and Atsos3-1. Black-Right-Pointing-Pointer Expression of CkNHX1-{Delta}C had little effect on NaCl/LiCl tolerance in Atsos3-1. Black-Right-Pointing-Pointer C-terminal region of CkNHX1 is required for its Na{sup +} and Li{sup +} transporting activity. -- Abstract: Sodium/proton exchangers (NHX antiporters) play important roles in plant responses to salt stress. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na{sup +}/H{sup +} transporting activity. In this study, CkNHX1 were isolated from Caragana korshinskii, a pea shrub with high tolerance to salt, drought, and cold stresses. Transcripts of CkNHX1 were detected predominantly in roots, and were significantly induced by NaCl stress in stems. Transgenic yeast and Arabidopsisthalianasos3-1 (Atsos3-1) mutant over-expressing CkNHX1 and its hydrophilic C terminus-truncated derivative, CkNHX1-{Delta}C, were generated and subjected to NaCl and LiCl stresses. Expression of CkNHX1 significantly enhanced the resistance to NaCl and LiCl stresses in yeast and Atsos3-1 mutant. Whereas, compared with expression of CkNHX1, the expression of CkNHX1-{Delta}C had much less effect on NaCl tolerance in Atsos3-1 and LiCl tolerance in yeast and Atsos3-1. All together, these results suggest that the predominant expression of CkNHX1 in roots might contribute to keep C. korshinskii adapting to the high salt condition in this plant's living environment; CkNHX1 could recover the phenotype of Atsos3-1 mutant; and the hydrophilic C-terminal region of CkNHX1 should be required for Na{sup +}/H{sup +} and Li{sup +}/H{sup +} exchanging activity of CkNHX1.

  7. Proteolytic activities in yeast.

    Science.gov (United States)

    Saheki, T; Holzer, H

    1975-03-28

    Studies on the mechanism and time course of the activation of proteinases A (EC 3.4.23.8), B (EC 3.4.22.9) and C (EC 3.4.12.--) in crude yeast extracts at pH 5.1 and 25 degrees C showed that the increase in proteinase B activity is paralleled with the disappearance of proteinase B inhibitor. Addition of purified proteinase A to fresh crude extracts accelerates the inactivation of the proteinase B inhibitor and the appearance of maximal activities of proteinases B and C. The decrease of proteinase B inhibitor activity and the increase of proteinase B activity are markedly retarded by the addition of pepstatin. Because 10-minus 7 M pepstatin completely inhibits proteinase A without affecting proteinase B activity, this is another indication for the role of proteinase A during the activation of proteinase B. Whereas extracts of yeast grown on minimal medium reached maximal activation of proteinases B and C after 20 h of incubation at pH 5.1 and 25 degrees C, extracts of yeast grown on complete medium had to be incubated for about 100 h. In the latter case, the addition of proteinas A results in maximal activation of proteinases B and C and disappearance of proteinase B inhibitor activity only after 10--20 h of incubation. With the optimal conditions, the maximal activities of proteinases A, B and C, as well as of the proteinase B inhibitor, were determined in crude extracts of yeast that had been grown batchwise for different lengths of time either on minimal or on complete medium. Upon incubation, all three proteinases were activated by several times their initial activity. This reflects the existence of proteolytically degradable inhibitors of the three proteinases and together with the above mentioned observations it demonstrates that the "activation" of yeast proteinases A, B and C upon incubation results from the proteolytic digestion of inhibitors rather than from activation of inactive zymogens by limited proteolysis.

  8. Yeast ecology of Kombucha fermentation.

    Science.gov (United States)

    Teoh, Ai Leng; Heard, Gillian; Cox, Julian

    2004-09-01

    Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.

  9. A standardized toolkit for genetic engineering of CTG clade yeasts.

    Science.gov (United States)

    Defosse, Tatiana A; Courdavault, Vincent; Coste, Alix T; Clastre, Marc; de Bernonville, Thomas Dugé; Godon, Charlotte; Vandeputte, Patrick; Lanoue, Arnaud; Touzé, Antoine; Linder, Tomas; Droby, Samir; Rosa, Carlos A; Sanglard, Dominique; d'Enfert, Christophe; Bouchara, Jean-Philippe; Giglioli-Guivarc'h, Nathalie; Papon, Nicolas

    2018-01-01

    We have developed a series of synthetic constructs suitable to genetically manipulate a broad range of yeast species belonging to the fungal CTG clade. This molecular toolbox notably allows heterologous gene expression, single or dual fluorescence labeling and construction of luciferase-expressing strains for bioluminescence imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  11. An engineered yeast efficiently secreting penicillin.

    Directory of Open Access Journals (Sweden)

    Loknath Gidijala

    Full Text Available This study aimed at developing an alternative host for the production of penicillin (PEN. As yet, the industrial production of this beta-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS delta-(L-alpha-aminoadipyl-L-cysteinyl-D-valine synthetase (ACVS in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT and phenylacetyl CoA ligase (PCL resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L. PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel beta-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents, whose production involves NRPS's.

  12. Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

    Science.gov (United States)

    van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

    Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

  13. Yeast Methylotrophy and Autophagy in a Methanol-Oscillating Environment on Growing Arabidopsis thaliana Leaves

    OpenAIRE

    Kosuke Kawaguchi; Hiroya Yurimoto; Masahide Oku; Yasuyoshi Sakai

    2011-01-01

    The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes ...

  14. Induction and construct UV protective yeast plasmid.

    Science.gov (United States)

    Cuero, Raul; McKay, David S

    2013-07-10

    In this study, we apply concepts of synthetic biology in combination with conventional methods to assemble different genetic components to construct yeast resistant to UV radiation, and to induce production of anti-UV proteins. This work combines sequences of different promoters, STRESS-proteins, heat shock protein (HSP), kinase proteins, alcohol dehydrogenase protein (ADH), ribosomal binding sites, fluorescent reporter proteins, terminators, and a synthetic ribosomal switch. The aim of this investigation was to induce an anti-UV proteins, and to construct an anti-UV yeast plasmid to be used for protection of skin cells against UV radiation. This investigation demonstrates induction and construction of anti-UV genes and production of their corresponding proteins. Cultures of Saccharomyces cerevisiae (ATCC # 66348) were exposed to short-wave UV radiation and were then subjected to time-PCR to assess specific gene expression. Proteins were identified using two dimensional difference gel electrophoresis (2D DIGE) and LC-MS/MS. Different up-regulated and down-regulated proteins were identified. Highly expressed identified proteins were cloned into S. cerevisiae using a synthetic biology approach. Extracts from UV-induced genetically transformed yeasts were used to protect skin cell cultures (ATCC #2522-CRL) in vitro. Both microscopic analysis and an apoptosis assay showed protection of the skin cell cultures against UV radiation. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Yeasts in Hevea brasiliensis Latex.

    Science.gov (United States)

    Glushakova, A M; Kachalkin, A V; Maksimova, I A; Chernov, I Yu

    2016-07-01

    Yeast abundance and species diversity in the latex of caoutchouc tree Hevea brasiliensis (Willd. ex Juss.) M611. Arg., on its green leaves, and in soil below the plant Was studied. The yeasts present in the fresh latex in concentrations of up to 5.5 log(CFU/g) were almost exclusively represented by the species Candida heveicola, which was previously isolated from Hevea latex in China. In the course of natural modification of the latex yeast diversity increased, while yeast abundance decreased. The yeasts of thickened and solidified latex were represented by typical epiphytic and ubiquitous species: Kodamea ohmeri, Debaryomyces hansenii, Rhodotorula mucilaginosa, and synanthropic species Candida parapsilosis and Cutaneotrichosporon arbori- formis. The role of yeasts in latex modification at the initial stages of succession and their probable role in de- velopment of antifungal activity in the latex are discussed.

  16. Yeast interactions and wine flavour.

    Science.gov (United States)

    Fleet, Graham H

    2003-09-01

    Wine is the product of complex interactions between fungi, yeasts and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Although grape cultivar and cultivation provide the foundations of wine flavour, microorganisms, especially yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it is important to identify and understand the ecological interactions that occur between the different microbial groups, species and strains. These interactions encompass yeast-yeast, yeast-filamentous fungi and yeast-bacteria responses. The surface of healthy grapes has a predominance of Aureobasidium pullulans, Metschnikowia, Hanseniaspora (Kloeckera), Cryptococcus and Rhodotorula species depending on stage of maturity. This microflora moderates the growth of spoilage and mycotoxigenic fungi on grapes, the species and strains of yeasts that contribute to alcoholic fermentation, and the bacteria that contribute to malolactic fermentation. Damaged grapes have increased populations of lactic and acetic acid bacteria that impact on yeasts during alcoholic fermentation. Alcoholic fermentation is characterised by the successional growth of various yeast species and strains, where yeast-yeast interactions determine the ecology. Through yeast-bacterial interactions, this ecology can determine progression of the malolactic fermentation, and potential growth of spoilage bacteria in the final product. The mechanisms by which one species/strain impacts on another in grape-wine ecosystems include: production of lytic enzymes, ethanol, sulphur dioxide and killer toxin/bacteriocin like peptides; nutrient depletion including removal of oxygen, and production of carbon dioxide; and release of cell autolytic components. Cell-cell communication through quorum sensing molecules needs investigation.

  17. Flavour-active wine yeasts

    OpenAIRE

    Cordente, Antonio G.; Curtin, Christopher D.; Varela, Cristian; Pretorius, Isak S.

    2012-01-01

    The flavour of fermented beverages such as beer, cider, saké and wine owe much to the primary fermentation yeast used in their production, Saccharomyces cerevisiae. Where once the role of yeast in fermented beverage flavour was thought to be limited to a small number of volatile esters and higher alcohols, the discovery that wine yeast release highly potent sulfur compounds from non-volatile precursors found in grapes has driven researchers to look more closely at how choice of yeast can infl...

  18. Divergence of iron metabolism in wild Malaysian yeast.

    Science.gov (United States)

    Lee, Hana N; Mostovoy, Yulia; Hsu, Tiffany Y; Chang, Amanda H; Brem, Rachel B

    2013-12-09

    Comparative genomic studies have reported widespread variation in levels of gene expression within and between species. Using these data to infer organism-level trait divergence has proven to be a key challenge in the field. We have used a wild Malaysian population of S. cerevisiae as a test bed in the search to predict and validate trait differences based on observations of regulatory variation. Malaysian yeast, when cultured in standard medium, activated regulatory programs that protect cells from the toxic effects of high iron. Malaysian yeast also showed a hyperactive regulatory response during culture in the presence of excess iron and had a unique growth defect in conditions of high iron. Molecular validation experiments pinpointed the iron metabolism factors AFT1, CCC1, and YAP5 as contributors to these molecular and cellular phenotypes; in genome-scale sequence analyses, a suite of iron toxicity response genes showed evidence for rapid protein evolution in Malaysian yeast. Our findings support a model in which iron metabolism has diverged in Malaysian yeast as a consequence of a change in selective pressure, with Malaysian alleles shifting the dynamic range of iron response to low-iron concentrations and weakening resistance to extreme iron toxicity. By dissecting the iron scarcity specialist behavior of Malaysian yeast, our work highlights the power of expression divergence as a signpost for biologically and evolutionarily relevant variation at the organismal level. Interpreting the phenotypic relevance of gene expression variation is one of the primary challenges of modern genomics.

  19. Yeast Infections: MedlinePlus Health Topic

    Science.gov (United States)

    ... Vaginal yeast infection (Medical Encyclopedia) Also in Spanish Topic Image MedlinePlus Email Updates Get Yeast Infections updates ... gram stain Thrush Vaginal yeast infection Related Health Topics Fungal Infections Vaginitis National Institutes of Health The ...

  20. Paraphyly and (yeast) classification.

    Science.gov (United States)

    Lachance, Marc-André

    2016-12-01

    Yeast systematics has wholeheartedly embraced the phylogenetic approach. Central to this has been the unspoken convention that taxa at all ranks be strictly monophyletic. This can result in a proliferation of small genera and instances of nomenclatural instability, counter to the expected benefit of phylogenetic systematics. But the literature abounds with examples, at all taxonomic levels, where paraphyly is a reality that can no longer be ignored. The very concepts of Bacteria or Archaea, under the constraint of monophyly, are in peril. It is therefore desirable to effect a shift in practices that will recognize the existence of paraphyletic taxa.

  1. Modelling Biolog profiles' evolution for yeast growth monitoring in alcoholic fermentation.

    Science.gov (United States)

    DeNittis, M; Zanoni, B; Minati, J L; Gorra, R; Ambrosoli, R

    2011-02-01

    A research was undertaken to explore the possibility to express with suitable mathematical models Biolog metabolic curves obtained for oenological yeasts and to use such models for monitoring yeast growth in alcoholic fermentation. Experimental curves of metabolic activity in Biolog YT microplates, obtained in a previous work for various oenological yeast strains in pure cultures and mixed populations, at various cell concentrations, have been modelled with Gompertz's, Gompertz's modified and Lindstrom's mathematical equations. Lindstrom's model proved to be the most suitable to fit the curves of the oenological yeasts under study, providing the highest correlation coefficients between experimental and calculated data. The model made it possible to recognize, in mixed yeast populations, the presence of active dry yeasts used for guided fermentations. Model's constant parameters were used for a numerical characterization of yeast curves.   The application of the model to the experimental data resulted to be suitable for an early prediction of the successive evolution of yeast growth. The results obtained indicate the possibility to develop protocols for monitoring yeast presence during alcoholic fermentation, with an early assessment of the correct evolution of their growth, especially when active dry yeasts are employed. © 2010 The Authors. Letters in Applied Microbiology © 2010 The Society for Applied Microbiology.

  2. Biodiversity of cold-adapted yeasts from glacial meltwater rivers in Patagonia, Argentina.

    Science.gov (United States)

    de García, Virginia; Brizzio, Silvia; Libkind, Diego; Buzzini, Pietro; van Broock, María

    2007-02-01

    The occurrence of culturable yeasts in glacial meltwater from the Frías, Castaño Overo and Río Manso glaciers, located on Mount Tronador in the Nahuel Huapi National Park (Northwestern Patagonia, Argentina) is presented. Subsurface water samples were filtered for colony counting and yeast isolation. The total yeast count ranged between 6 and 360 CFU L(-1). Physiologic and molecular methods were employed to identify 86 yeast isolates. In agreement with yeast diversity data from studies for Antarctic and Alpine glaciers, the genera Cryptococcus, Leucosporidiella, Dioszegia, Rhodotorula, Rhodosporidium, Mrakia, Sporobolomyces, Udeniomyces and Candida were found. Cryptococcus and Leucosporidiella accounted for 50% and 20% of the total number of strains, respectively. Among 21 identified yeast species, Cryptococcus sp. 1 and Leucosporidiella fragaria were the most frequent. The typically psychrophilic Mrakia yeast strain and three new yeast species, yet to be described, were also isolated. All yeast strains were able to grow at 5, 10, and 15 degrees C. Among yeast strains expressing extracellular enzymatic activity, higher proteolytic and lipolytic activities were obtained at 4 degrees C than at 20 degrees C.

  3. Inheritance of the yeast mitochondrial genome

    DEFF Research Database (Denmark)

    Piskur, Jure

    1994-01-01

    Mitochondrion, extrachromosomal genetics, intergenic sequences, genome size, mitochondrial DNA, petite mutation, yeast......Mitochondrion, extrachromosomal genetics, intergenic sequences, genome size, mitochondrial DNA, petite mutation, yeast...

  4. Yeasts preservation: alternatives for lyophilisation

    NARCIS (Netherlands)

    Nyanga, L.K.; Nout, M.J.R.; Smid, E.J.; Boekhout, T.; Zwietering, M.H.

    2012-01-01

    The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts

  5. Sociobiology of the budding yeast

    Indian Academy of Sciences (India)

    ... the unicellular yeast Saccharomyces cerevisiae, for sociobiological research. I discuss the problems connected with clear classification of yeast behaviour based on the fitness-based Hamilton paradigm. Relevant traits include different types of communities, production of flocculins, invertase and toxins, and the presence ...

  6. Yeast signaling pathways in the oxidative stress response

    Energy Technology Data Exchange (ETDEWEB)

    Ikner, Aminah [Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA 95616 (United States); Shiozaki, Kazuhiro [Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA 95616 (United States)]. E-mail: kshiozaki@ucdavis.edu

    2005-01-06

    Oxidative stress that generates the reactive oxygen species (ROS) is one of the major causes of DNA damage and mutations. The 'DNA damage checkpoint' that arrests cell cycle and repairs damaged DNA has been a focus of recent studies, and the genetically amenable model systems provided by yeasts have been playing a leading role in the eukaryotic checkpoint research. However, means to eliminate ROS are likely to be as important as the DNA repair mechanisms in order to suppress mutations in the chromosomal DNA, and yeasts also serve as excellent models to understand how eukaryotes combat oxidative stress. In this article, we present an overview of the signaling pathways that sense oxidative stress and induce expression of various anti-oxidant genes in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the pathogenic yeast Candida albicans. Three conserved signaling modules have been identified in the oxidative stress response of these diverse yeast species: the stress-responsive MAP kinase cascade, the multistep phosphorelay and the AP-1-like transcription factor. The structure and function of these signaling modules are discussed.

  7. Mitochondria autophagy in yeast.

    Science.gov (United States)

    Kanki, Tomotake; Klionsky, Daniel J; Okamoto, Koji

    2011-05-15

    The mitochondrion is an organelle that carries out a number of important metabolic processes such as fatty acid oxidation, the citric acid cycle, and oxidative phosphorylation. However, this multitasking organelle also generates reactive oxygen species (ROS), which can cause oxidative stress resulting in self-damage. This type of mitochondrial damage can lead to the further production of ROS and a resulting downward spiral with regard to mitochondrial capability. This is extremely problematic because the accumulation of dysfunctional mitochondria is related to aging, cancer, and neurodegenerative diseases. Accordingly, appropriate quality control of this organelle is important to maintain proper cellular homeostasis. It has been thought that selective mitochondria autophagy (mitophagy) contributes to the maintenance of mitochondrial quality by eliminating damaged or excess mitochondria, although little is known about the mechanism. Recent studies in yeast identified several mitophagy-related proteins, which have been characterized with regard to their function and regulation. In this article, we review recent advances in the physiology and molecular mechanism of mitophagy and discuss the similarities and differences of this degradation process between yeast and mammalian cells.

  8. Cell wall trapping of autocrine peptides for human G-protein-coupled receptors on the yeast cell surface.

    Directory of Open Access Journals (Sweden)

    Jun Ishii

    Full Text Available G-protein-coupled receptors (GPCRs regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP strategy. In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

  9. Root : shoot ratios, optimization and nitrogen productivity.

    Science.gov (United States)

    Agren, Göran I; Franklin, Oskar

    2003-12-01

    Plants respond to nitrogen availability by changing their root : shoot ratios. One hypothesis used to explain this allocation is that plants optimize their behaviour by maximizing their relative growth rate. The consequences of this hypothesis were investigated by formulating two models for root : shoot allocation, with and without explicit inclusion of maintenance respiration. The models also took into account that relative growth rate is a linear function of plant nitrogen concentration. The model without respiration gave qualitatively reasonable results when predictions were compared with observed results from growth experiments with birch and tomato. The explicit inclusion of maintenance respiration improved considerably the agreement between prediction and observation, and for birch was within the experimental accuracy. Further improvements will require additional details in the description of respiratory processes and the nitrogen uptake function. Plants growing under extreme nutrient stress may also optimize their behaviour with respect to other variables in addition to relative growth rate.

  10. Production of Food Grade Yeasts

    Directory of Open Access Journals (Sweden)

    Argyro Bekatorou

    2006-01-01

    Full Text Available Yeasts have been known to humans for thousands of years as they have been used in traditional fermentation processes like wine, beer and bread making. Today, yeasts are also used as alternative sources of high nutritional value proteins, enzymes and vitamins, and have numerous applications in the health food industry as food additives, conditioners and flavouring agents, for the production of microbiology media and extracts, as well as livestock feeds. Modern scientific advances allow the isolation, construction and industrial production of new yeast strains to satisfy the specific demands of the food industry. Types of commercial food grade yeasts, industrial production processes and raw materials are highlighted. Aspects of yeast metabolism, with respect to carbohydrate utilization, nutritional aspects and recent research advances are also discussed.

  11. Red yeast rice for dysipidemia.

    Science.gov (United States)

    Shamim, Shariq; Al Badarin, Firas J; DiNicolantonio, James J; Lavie, Carl J; O'Keefe, James H

    2013-01-01

    Red yeast rice is an ancient Chinese food product that contains monacolins, chemical substances that are similar to statins in their mechanisms of action and lipid lowering properties. Several studies have found red yeast rice to be moderately effective at improving the lipid profile, particularly for lowering the low-density lipoprotein cholesterol levels. One large randomized controlled study from China found that red yeast rice significantly improved risk of major adverse cardiovascular events and overall survival in patients following myocardial infarction. Thus, red yeast rice is a potentially useful over-the-counter cholesterol-lowering agent. However, many red yeast rice formulations are non-standardized and unregulated food supplements, and there is a need for further research and regulation of production.

  12. Evolutionary History of Ascomyceteous Yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Haridas, Sajeet; Riley, Robert; Salamov, Asaf; Goker, Markus; Klenk, Hans-Peter; Kurtzman, Cletus P.; Blackwell, Meredith; Grigoriev, Igor; Jeffries, Thomas W.

    2014-06-06

    Yeasts are important for many industrial and biotechnological processes and show remarkable diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. A comparison of these with several other previously published yeast genomes have added increased confidence to the phylogenetic positions of previously poorly placed species including Saitoella complicata, Babjeviella inositovora and Metschnikowia bicuspidata. Phylogenetic analysis also showed that yeasts with alternative nuclear codon usage where CUG encodes serine instead of leucine are monophyletic within the Saccharomycotina. Most of the yeasts have compact genomes with a large fraction of single exon genes with Lipomyces starkeyi and the previously published Pneumocystis jirovecii being notable exceptions. Intron analysis suggests that early diverging species have more introns. We also observed a large number of unclassified lineage specific non-simple repeats in these genomes.

  13. The yeast Ski complex is a hetero-tetramer

    NARCIS (Netherlands)

    Synowsky, S.A.; Heck, A.J.R.

    2008-01-01

    The yeast Ski complex assists the exosome in the degradation of mRNA. The Ski complex consists of three components; Ski2, Ski3, and Ski8, believed to be present in a 1:1:1 stoichiometry. Measuring the mass of intact isolated endogenously expressed Ski complexes by native mass spectrometry we

  14. Sporangiospore-to-yeast conversion: Model for morphogenesis ...

    African Journals Online (AJOL)

    This report reviewed the specific characteristics of Mucor circinelloides in solid cultures and its capacity for multiple anamorphic expressions in synthetic broth, including holoblastic, holothallic, enterothallic conidia, and vesicular conidia head group as well as yeast cells. Attempt was made to show that the sequence of ...

  15. Bioprotective Role of Yeasts

    Science.gov (United States)

    Muccilli, Serena; Restuccia, Cristina

    2015-01-01

    The yeasts constitute a large group of microorganisms characterized by the ability to grow and survive in different and stressful conditions and then to colonize a wide range of environmental and human ecosystems. The competitive traits against other microorganisms have attracted increasing attention from scientists, who proposed their successful application as bioprotective agents in the agricultural, food and medical sectors. These antagonistic activities rely on the competition for nutrients, production and tolerance of high concentrations of ethanol, as well as the synthesis of a large class of antimicrobial compounds, known as killer toxins, which showed clearly a large spectrum of activity against food spoilage microorganisms, but also against plant, animal and human pathogens. This review describes the antimicrobial mechanisms involved in the antagonistic activity, their applications in the processed and unprocessed food sectors, as well as the future perspectives in the development of new bio-drugs, which may overcome the limitations connected to conventional antimicrobial and drug resistance. PMID:27682107

  16. Bioprotective Role of Yeasts

    Directory of Open Access Journals (Sweden)

    Serena Muccilli

    2015-10-01

    Full Text Available The yeasts constitute a large group of microorganisms characterized by the ability to grow and survive in different and stressful conditions and then to colonize a wide range of environmental and human ecosystems. The competitive traits against other microorganisms have attracted increasing attention from scientists, who proposed their successful application as bioprotective agents in the agricultural, food and medical sectors. These antagonistic activities rely on the competition for nutrients, production and tolerance of high concentrations of ethanol, as well as the synthesis of a large class of antimicrobial compounds, known as killer toxins, which showed clearly a large spectrum of activity against food spoilage microorganisms, but also against plant, animal and human pathogens. This review describes the antimicrobial mechanisms involved in the antagonistic activity, their applications in the processed and unprocessed food sectors, as well as the future perspectives in the development of new bio-drugs, which may overcome the limitations connected to conventional antimicrobial and drug resistance.

  17. Lager Yeast Comes of Age

    Science.gov (United States)

    2014-01-01

    Alcoholic fermentations have accompanied human civilizations throughout our history. Lager yeasts have a several-century-long tradition of providing fresh beer with clean taste. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. We summarize the initial findings on this hybrid nature, the genomics/transcriptomics of lager yeasts, and established targets of strain improvements. Next-generation sequencing has provided fast access to yeast genomes. Its use in population genomics has uncovered many more hybridization events within Saccharomyces species, so that lager yeast hybrids are no longer the exception from the rule. These findings have led us to propose network evolution within Saccharomyces species. This “web of life” recognizes the ability of closely related species to exchange DNA and thus drain from a combined gene pool rather than be limited to a gene pool restricted by speciation. Within the domesticated lager yeasts, two groups, the Saaz and Frohberg groups, can be distinguished based on fermentation characteristics. Recent evidence suggests that these groups share an evolutionary history. We thus propose to refer to the Saaz group as Saccharomyces carlsbergensis and to the Frohberg group as Saccharomyces pastorianus based on their distinct genomes. New insight into the hybrid nature of lager yeast will provide novel directions for future strain improvement. PMID:25084862

  18. An IPTG‐inducible derivative of the fission yeast nmt promoter

    National Research Council Canada - National Science Library

    Kjærulff, Søren; Nielsen, Olaf

    2015-01-01

    We here describe an IPTG‐inducible system that reveals that the lac repressor alone can function as a potent transmodulator to regulate gene expression in the fission yeast, Schizosaccharomyces pombe...

  19. Extension of Yeast Chronological Lifespan by Methylamine

    NARCIS (Netherlands)

    Kumar, Sanjeev; Lefevre, Sophie D.; Veenhuis, Marten; van der Klei, Ida J.

    2012-01-01

    Background: Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast

  20. Oxidative stress response pathways: Fission yeast as archetype.

    Science.gov (United States)

    Papadakis, Manos A; Workman, Christopher T

    2015-01-01

    Schizosaccharomyces pombe is a popular model eukaryotic organism to study diverse aspects of mammalian biology, including responses to cellular stress triggered by redox imbalances within its compartments. The review considers the current knowledge on the signaling pathways that govern the transcriptional response of fission yeast cells to elevated levels of hydrogen peroxide. Particular attention is paid to the mechanisms that yeast cells employ to promote cell survival in conditions of intermediate and acute oxidative stress. The role of the Sty1/Spc1/Phh1 mitogen-activated protein kinase in regulating gene expression at multiple levels is discussed in detail.

  1. How does yeast respond to pressure?

    Directory of Open Access Journals (Sweden)

    P.M.B. Fernandes

    2005-08-01

    Full Text Available The brewing and baking yeast Saccharomyces cerevisiae has been used as a model for stress response studies of eukaryotic cells. In this review we focus on the effect of high hydrostatic pressure (HHP on S. cerevisiae. HHP exerts a broad effect on yeast cells characteristic of common stresses, mainly associated with protein alteration and lipid bilayer phase transition. Like most stresses, pressure induces cell cycle arrest. Below 50 MPa (500 atm yeast cell morphology is unaffected whereas above 220 MPa wild-type cells are killed. S. cerevisiae cells can acquire barotolerance if they are pretreated with a sublethal stress due to temperature, ethanol, hydrogen peroxide, or pressure. Nevertheless, pressure only leads to protection against severe stress if, after pressure pretreatment, the cells are also re-incubated at room pressure. We attribute this effect to the inhibition of the protein synthesis apparatus under HHP. The global genome expression analysis of S. cerevisiae cells submitted to HHP revealed a stress response profile. The majority of the up-regulated genes are involved in stress defense and carbohydrate metabolism while most repressed genes belong to the cell cycle progression and protein synthesis categories. However, the signaling pathway involved in the pressure response is still to be elucidated. Nitric oxide, a signaling molecule involved in the regulation of a large number of cellular functions, confers baroprotection. Furthermore, S. cerevisiae cells in the early exponential phase submitted to 50-MPa pressure show induction of the expression level of the nitric oxide synthase inducible isoform. As pressure becomes an important biotechnological tool, studies concerning this kind of stress in microorganisms are imperative.

  2. Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Nakanishi, Akihito; Bae, Jun Gu; Fukai, Kotaro; Tokumoto, Naoki; Kuroda, Kouichi; Ogawa, Jun; Shimizu, Sakayu; Ueda, Mitsuyoshi [Kyoto Univ. (Japan). Div. of Applied Life Sciences; Nakatani, Masato [Daiwa Kasei, Shiga (Japan)

    2012-05-15

    A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2{sup '}-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and {beta}-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and {beta}-glucosidase. (orig.)

  3. Red Yeast Rice: An Introduction

    Science.gov (United States)

    ... mg twice daily) in patients with previous statin intolerance . American Journal of Cardiology . 2010;105:198–204. ... to Avoid Red Yeast Rice Products Promoted on Internet as Treatments for High Cholesterol: Products Found to ...

  4. Sexual differentiation in fission yeast

    DEFF Research Database (Denmark)

    Egel, R; Nielsen, O; Weilguny, D

    1990-01-01

    The regulation of sexual reproduction in yeast constitutes the highest level of differentiation observed in these unicellular organisms. The various ramifications of this system involve DNA rearrangement, transcriptional control, post-translational modification (such as protein phosphorylation) a...

  5. Genetically engineered yeast

    DEFF Research Database (Denmark)

    2014-01-01

    A genetically modified Saccharomyces cerevisiae comprising an active fermentation pathway producing 3-HP expresses an exogenous gene expressing the aminotransferase YhxA from Bacillus cereus AH1272 catalysing a transamination reaction between beta-alanine and pyruvate to produce malonate semialde......A genetically modified Saccharomyces cerevisiae comprising an active fermentation pathway producing 3-HP expresses an exogenous gene expressing the aminotransferase YhxA from Bacillus cereus AH1272 catalysing a transamination reaction between beta-alanine and pyruvate to produce malonate...

  6. Mucositis Grades and Yeast Species

    OpenAIRE

    Ognjenović, Marina; Milatić, Katja; Parat, Katica; KOVAČIĆ, IVAN; Ježina Bušelić, Marina A.; Božić, Joško

    2013-01-01

    Surgically treated patients with oral, head and neck cancer commonly develop mucositis during additional irradiation therapy. Oral mucosa inflammation other than irradiation is mostly caused by Candida albicans, yeast of Candida genus. This study evaluated possible connection between grades of oral mucositis and oral yeast profile in irradiated patients before, during and after irradiation. In 25 examined patients mucosits grades »0« to »2« before irradiation with 20% positive smears and o...

  7. Reconstitution of the mitochondrial calcium uniporter in yeast.

    Science.gov (United States)

    Kovács-Bogdán, Erika; Sancak, Yasemin; Kamer, Kimberli J; Plovanich, Molly; Jambhekar, Ashwini; Huber, Robert J; Myre, Michael A; Blower, Michael D; Mootha, Vamsi K

    2014-06-17

    The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits--mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)--and two soluble regulatory components--MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.

  8. Transporter engineering in biomass utilization by yeast.

    Science.gov (United States)

    Hara, Kiyotaka Y; Kobayashi, Jyumpei; Yamada, Ryosuke; Sasaki, Daisuke; Kuriya, Yuki; Hirono-Hara, Yoko; Ishii, Jun; Araki, Michihiro; Kondo, Akihiko

    2017-11-01

    Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Whole Genome Analysis of a Wine Yeast Strain

    Science.gov (United States)

    Hauser, Nicole C.; Fellenberg, Kurt; Gil, Rosario; Bastuck, Sonja; Hoheisel, Jörg D.

    2001-01-01

    Saccharomyces cerevisiae strains frequently exhibit rather specific phenotypic features needed for adaptation to a special environment. Wine yeast strains are able to ferment musts, for example, while other industrial or laboratory strains fail to do so. The genetic differences that characterize wine yeast strains are poorly understood, however. As a first search of genetic differences between wine and laboratory strains, we performed DNA-array analyses on the typical wine yeast strain T73 and the standard laboratory background in S288c. Our analysis shows that even under normal conditions, logarithmic growth in YPD medium, the two strains have expression patterns that differ significantly in more than 40 genes. Subsequent studies indicated that these differences correlate with small changes in promoter regions or variations in gene copy number. Blotting copy numbers vs. transcript levels produced patterns, which were specific for the individual strains and could be used for a characterization of unknown samples. PMID:18628902

  10. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    Directory of Open Access Journals (Sweden)

    Fernando eAleman

    2014-09-01

    Full Text Available Potassium (K+ is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter.

  11. Red Yeast Rice

    Directory of Open Access Journals (Sweden)

    Thu Nguyen

    2017-03-01

    Full Text Available Red yeast rice (RYR, produced by the fermentation of the Monascus purpureus mold, has been used for a long time in Asian cuisine and traditional medicine. It consists of multiple bioactive substances, including monacolins, which potentially can be used as a nutraceutical. Monacolin K, which is chemically identical to lovastatin, has been recognized as responsible for the cholesterolreducing effect of this compound. While the European Food Safety Authority maintains that the use of monacolin K from RYR preparations of at least 10 mg can produce a normal blood cholesterol level, the United States Food and Drug Administration considers monacolin K, due to its similarity with lovastatin, an unapproved drug, and therefore marketing of products that label the monacolin content is prohibited. This mini-review summarizes the benefit of RYR in hyperlipidemia, maintains RYR use as a food, and addresses the importance of regulation regarding RYR and the need for clinical data and clear label information for consumers with reference to a toxin-free, nonaugmented, standardized amount of monacolins.

  12. Red Yeast Rice.

    Science.gov (United States)

    Nguyen, Thu; Karl, Mitchell; Santini, Antonello

    2017-03-01

    Red yeast rice (RYR), produced by the fermentation of the Monascus purpureus mold, has been used for a long time in Asian cuisine and traditional medicine. It consists of multiple bioactive substances, including monacolins, which potentially can be used as a nutraceutical. Monacolin K, which is chemically identical to lovastatin, has been recognized as responsible for the cholesterolreducing effect of this compound. While the European Food Safety Authority maintains that the use of monacolin K from RYR preparations of at least 10 mg can produce a normal blood cholesterol level, the United States Food and Drug Administration considers monacolin K, due to its similarity with lovastatin, an unapproved drug, and therefore marketing of products that label the monacolin content is prohibited. This mini-review summarizes the benefit of RYR in hyperlipidemia, maintains RYR use as a food, and addresses the importance of regulation regarding RYR and the need for clinical data and clear label information for consumers with reference to a toxin-free, nonaugmented, standardized amount of monacolins.

  13. Synthetic Yeast Cooperation

    Science.gov (United States)

    Shou, Wenying; Burton, Justin

    2010-03-01

    Cooperation is wide-spread and has been postulated to drive major transitions in evolution. However, Darwinian selection favors ``cheaters'' that consume benefits without paying a fair cost. How did cooperation evolve against the threat of cheaters? To investigate the evolutionary trajectories of cooperation, we created a genetically tractable system that can be observed as it evolves from inception. The system consists of two engineered yeast strains -- a red-fluorescent strain that requires adenine and releases lysine and a yellow-fluorescent strain that requires lysine and releases adenine. Cells that consume but not supply metabolites would be cheaters. From the properties of two cooperating strains, we calculated and experimentally verified the minimal initial cell densities required for the viability of the cooperative system in the absence of exogenously added adenine and lysine. Strikingly, evolved cooperative systems were viable at 100-fold lower initial cell densities than their ancestors. We are investigating the nature and diversity of pro-cooperation changes, the dynamics of cooperator-cheater cocultures, and the effects of spatial environment on cooperation and cheating.

  14. Development and evaluation of consolidated bioprocessing yeast for ethanol production from ionic liquid-pretreated bagasse.

    Science.gov (United States)

    Amoah, Jerome; Ishizue, Naoya; Ishizaki, Miki; Yasuda, Misa; Takahashi, Kenji; Ninomiya, Kazuaki; Yamada, Ryosuke; Kondo, Akihiko; Ogino, Chiaki

    2017-12-01

    This work aimed to study the use of consolidated bioprocess (CBP) yeast expressing five cellulase genes (BGL, XYNII, EGII, CBHI and CBHII) for ethanol production from ionic liquid-pretreated bagasse and Laubholz unbleached Kraft pulp (LUKP). A proposed screening method shows that the optimal cellulase ratio varies for each biomass substrate, and thus it is essential to breed CBP yeast having optimal cellulase-displaying ratio for the target biomass. CBP yeast specialized towards bagasse produced 0.93g/l ethanol whiles that for LUKP produced 0.71g/l ethanol, which is approximately 4 and 2-fold, respectively, higher than that of the wild type. The cell-surface displayed enzymes synergistically contributed to the degradation of the biomass. The developed CBP yeast is a potential cheap source for consolidated bioprocessing of ethanol and the proposed screening method can be used for matching CBP yeast to a target biomass. Copyright © 2017. Published by Elsevier Ltd.

  15. Yeast methylotrophy and autophagy in a methanol-oscillating environment on growing Arabidopsis thaliana leaves.

    Directory of Open Access Journals (Sweden)

    Kosuke Kawaguchi

    Full Text Available The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels.

  16. Yeast Methylotrophy and Autophagy in a Methanol-Oscillating Environment on Growing Arabidopsis thaliana Leaves

    Science.gov (United States)

    Kawaguchi, Kosuke; Yurimoto, Hiroya; Oku, Masahide; Sakai, Yasuyoshi

    2011-01-01

    The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels. PMID:21966472

  17. Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters

    Directory of Open Access Journals (Sweden)

    Laila Berg

    2013-01-01

    Full Text Available Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the promoter DNA sequence. We demonstrate that this approach can be used to select for promoter variants with abolished glucose repression in large mutant libraries. We also selected promoter variants with elevated expression level under induced conditions. The properties of the selected promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.

  18. Characterization of specialized flocculent yeasts to improve sparkling wine fermentation.

    Science.gov (United States)

    Tofalo, R; Perpetuini, G; Di Gianvito, P; Arfelli, G; Schirone, M; Corsetti, A; Suzzi, G

    2016-06-01

    Flocculent wine yeasts were characterized for the expression of FLO1, FLO5, FLO8, AMN1 and RGA1 genes, growth kinetics and physicochemical properties of the cell surface during a 6-month sparkling wine fermentation period. The expression of FLO1, FLO5, FLO8, AMN1 and RGA1 genes was determined by RT-qPCR. The physicochemical characterization of yeast surface properties was evaluated by the microbial adhesion to solvents method. FLO5 gene was the most expressed one and a linear correlation with the flocculent degree was found. Flocculent strains were more hydrophobic than the commercial wine strain EC1118. Gene expressions and the ability to face secondary wine fermentation conditions were strain dependent. The importance of FLO5 gene in developing the high flocculent characteristic of wine yeasts was highlighted. Cell surface properties depended on the time of fermentation. Better knowledge about the expression of some genes encoding the flocculent phenotype which could be useful to select suitable starter cultures to improve sparkling wine technology was achieved. A step forward in understanding the complexity and strain-specific nature of flocculation phenotype was done. © 2016 The Society for Applied Microbiology.

  19. Population FBA predicts metabolic phenotypes in yeast.

    Directory of Open Access Journals (Sweden)

    Piyush Labhsetwar

    2017-09-01

    Full Text Available Using protein counts sampled from single cell proteomics distributions to constrain fluxes through a genome-scale model of metabolism, Population flux balance analysis (Population FBA successfully described metabolic heterogeneity in a population of independent Escherichia coli cells growing in a defined medium. We extend the methodology to account for correlations in protein expression arising from the co-regulation of genes and apply it to study the growth of independent Saccharomyces cerevisiae cells in two different growth media. We find the partitioning of flux between fermentation and respiration predicted by our model agrees with recent 13C fluxomics experiments, and that our model largely recovers the Crabtree effect (the experimentally known bias among certain yeast species toward fermentation with the production of ethanol even in the presence of oxygen, while FBA without proteomics constraints predicts respirative metabolism almost exclusively. The comparisons to the 13C study showed improvement upon inclusion of the correlations and motivated a technique to systematically identify inconsistent kinetic parameters in the literature. The minor secretion fluxes for glycerol and acetate are underestimated by our method, which indicate a need for further refinements to the metabolic model. For yeast cells grown in synthetic defined (SD medium, the calculated broad distribution of growth rates matches experimental observations from single cell studies, and we characterize several metabolic phenotypes within our modeled populations that make use of diverse pathways. Fast growing yeast cells are predicted to perform significant amount of respiration, use serine-glycine cycle and produce ethanol in mitochondria as opposed to slow growing cells. We use a genetic algorithm to determine the proteomics constraints necessary to reproduce the growth rate distributions seen experimentally. We find that a core set of 51 constraints are essential but

  20. Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Smith, S; Blobel, G

    1994-01-01

    We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin ...

  1. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    Science.gov (United States)

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

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

  2. PGASO: A synthetic biology tool for engineering a cellulolytic yeast

    Directory of Open Access Journals (Sweden)

    Chang Jui-Jen

    2012-07-01

    Full Text Available Abstract Background To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. Results A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO, that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei, a beta-glucosidase (from a cow rumen fungus, a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. Conclusions This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

  3. Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast

    DEFF Research Database (Denmark)

    Kleinow, T.; Bhalerao, R.; Breuer, F.

    2000-01-01

    . By expression of an Arabidopsis cDNA library in yeast, heterologous multicopy snf4 suppressors were isolated. In addition to AKIN10 and AKIN11, the deficiency of yeast snf4 mutant to grown on non-fermentable carbon source was suppressed by Arabidopsis Myb30, CAAT-binding factor Hap3b, casein kinase I, zinc...

  4. Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis.

    Science.gov (United States)

    Pericolini, Eva; Gabrielli, Elena; Ballet, Nathalie; Sabbatini, Samuele; Roselletti, Elena; Cayzeele Decherf, Amélie; Pélerin, Fanny; Luciano, Eugenio; Perito, Stefano; Jüsten, Peter; Vecchiarelli, Anna

    2017-01-02

    Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection.

  5. Indole-3-Acetic Acid-Producing Yeasts in the Phyllosphere of the Carnivorous Plant Drosera indica L

    Science.gov (United States)

    Shin, Li-Ying; Wei, Jyuan-Yu; Fu, Shih-Feng; Chou, Jui-Yu

    2014-01-01

    Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA) when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture. PMID:25464336

  6. Indole-3-acetic acid-producing yeasts in the phyllosphere of the carnivorous plant Drosera indica L.

    Directory of Open Access Journals (Sweden)

    Pei-Feng Sun

    Full Text Available Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture.

  7. Indole-3-acetic acid-producing yeasts in the phyllosphere of the carnivorous plant Drosera indica L.

    Science.gov (United States)

    Sun, Pei-Feng; Fang, Wei-Ta; Shin, Li-Ying; Wei, Jyuan-Yu; Fu, Shih-Feng; Chou, Jui-Yu

    2014-01-01

    Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA) when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture.

  8. Emulsifying activity of hydrocarbonoclastic marine yeasts

    Digital Repository Service at National Institute of Oceanography (India)

    Gupta, R.

    Marine yeast growth on four petroleum hydrocarbons induced the production of extracellular emulsifying agents (biosurfactants). Out of the 17 marine yeast isolates tested, 7 isolates, i.e., Candida parapsilosis, C. cantarelli, C. membranae...

  9. Histone modification pattern evolution after yeast gene duplication

    Directory of Open Access Journals (Sweden)

    Zou Yangyun

    2012-07-01

    Full Text Available Abstract Background Gene duplication and subsequent functional divergence especially expression divergence have been widely considered as main sources for evolutionary innovations. Many studies evidenced that genetic regulatory network evolved rapidly shortly after gene duplication, thus leading to accelerated expression divergence and diversification. However, little is known whether epigenetic factors have mediated the evolution of expression regulation since gene duplication. In this study, we conducted detailed analyses on yeast histone modification (HM, the major epigenetics type in this organism, as well as other available functional genomics data to address this issue. Results Duplicate genes, on average, share more common HM-code patterns than random singleton pairs in their promoters and open reading frames (ORF. Though HM-code divergence between duplicates in both promoter and ORF regions increase with their sequence divergence, the HM-code in ORF region evolves slower than that in promoter region, probably owing to the functional constraints imposed on protein sequences. After excluding the confounding effect of sequence divergence (or evolutionary time, we found the evidence supporting the notion that in yeast, the HM-code may co-evolve with cis- and trans-regulatory factors. Moreover, we observed that deletion of some yeast HM-related enzymes increases the expression divergence between duplicate genes, yet the effect is lower than the case of transcription factor (TF deletion or environmental stresses. Conclusions Our analyses demonstrate that after gene duplication, yeast histone modification profile between duplicates diverged with evolutionary time, similar to genetic regulatory elements. Moreover, we found the evidence of the co-evolution between genetic and epigenetic elements since gene duplication, together contributing to the expression divergence between duplicate genes.

  10. Chromatin and Transcription in Yeast

    Science.gov (United States)

    Rando, Oliver J.; Winston, Fred

    2012-01-01

    Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

  11. Mutations in yeast calmodulin cause defects in spindle pole body functions and nuclear integrity

    OpenAIRE

    1992-01-01

    Yeast calmodulin (CaM) is required for the progression of nuclear division (Ohya, Y. and Y. Anraku. 1989. Curr. Genet. 15:113-120), although the precise mechanism and physiological role of CaM in this process are unclear. In this paper we have characterized the phenotype caused by a temperature-sensitive lethal mutation (cmdl-101) in the yeast CaM. The cmdl-101 mutation expresses a carboxyl-terminal half of the yeast CaM (Met72-Cys147) under the control of an inducible GAL1 promoter. Incubati...

  12. Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

    Science.gov (United States)

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-02-01

    In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  13. Characteristics of fermentation yeast isolated from traditional ...

    African Journals Online (AJOL)

    A relatively higher amount of propan-1-ol (43 mg/l) was found in the honey wine than in those made with wine yeast W4 and sake yeast K7. The aroma characteristics of honey wine made with yeast ET99 were acceptable, as determined by organoleptic tests, and were found to be applicable to ethanol fermentation.

  14. 21 CFR 73.355 - Phaffia yeast.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Phaffia yeast. 73.355 Section 73.355 Food and... ADDITIVES EXEMPT FROM CERTIFICATION Foods § 73.355 Phaffia yeast. (a) Identity. (1) The color additive phaffia yeast consists of the killed, dried cells of a nonpathogenic and nontoxicogenic strain of the...

  15. Building terpene production platforms in yeast.

    Science.gov (United States)

    Zhuang, Xun; Chappell, Joe

    2015-09-01

    Plants and microbes commonly make terpenes and terpenoids in small amounts and as complex mixtures, and their chemical synthesis is often costly and inefficient. Hence, there are many efforts to create robust and efficient biological production platforms for this interesting class of molecules. In this study, our effort was directed towards building a yeast production platform using an unbiased genetic selection approach. Yeast strain BY4741 was subjected to EMS mutagenesis, followed by selection for growth in the presence of nystatin, squalestatin, and exogenous cholesterol. This unbiased screen selected for mutant yeast lines having a dispensable mevalonate pathway and containing uncharacterized SUE (sterol uptake enhancement) mutations supporting aerobic uptake of exogenous sterol. These mutants were next screened for high level accumulation of farnesol (FOH), an indicator for high level accumulation of the key intermediate FPP, farnesyl diphosphate. To further improve the FPP pool in these mutants, insertional mutations into the ERG9 gene (coding for squalene synthase) were introduced into those lines capable of accumulating ≥50 mg farnesol/L. This generated another series of lines that accumulated farnesol levels over 70 mg/L in small-scale shake cultures. To evaluate the utility of these lines as a general production platform for specific terpenes, select SUE/erg9 lines were transformed with a vector harboring the Hyoscyamus muticus premnaspirodiene synthase (HPS) gene encoding for a sesquiterpene synthase. The new yeast line ZX178-08 accumulated the highest level of premnaspirodiene, up to 116 mg/L, with FOH levels of 23.6 mg/L. In comparison, the parental line BY4741 accumulated 10 times less premnaspirodiene, 10.94 mg/L, with no farnesol detectable. Co-expression of the HPS gene with an amino-terminal truncated, catalytic form of the hamster HMGR gene, tHMGR, increased premnaspirodiene accumulation to 170.23 ± 30.44 mg/L, almost a 50

  16. Yeast genomics on food flavours

    NARCIS (Netherlands)

    Schoondermark-Stolk, Sung Ah

    2005-01-01

    The appearance and concentration of the fusel alcohol 3-methyl-1-butanol is important for the flavour of fermented foods. 3-Methyl-1-butanol is formed by yeast during the conversion of L-leucine. Identification of the enzymes and genes involved in the formation of 3-methyl-1-butanol is a major

  17. Nucleotide excision repair in yeast

    NARCIS (Netherlands)

    Eijk, Patrick van

    2012-01-01

    Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

  18. Surplus yeast tank failing catastrophically

    DEFF Research Database (Denmark)

    Hedlund, Frank Huess

    2016-01-01

    GOOD REASON FOR CAUTION I A large surplus yeast tank shot into the air leaving the floor plate and the contents behind. Although not designed for overpressure, the tank was kept at “very slight overpressure” to suppress nuisance foaming. The brewery was unaware of the hazards of compressed air...

  19. Yeast as a Heterologous Model System to Uncover Type III Effector Function.

    Directory of Open Access Journals (Sweden)

    Crina Popa

    2016-02-01

    Full Text Available Type III effectors (T3E are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. "Favourite" targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure-function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations

  20. Differential response to UV stress and DNA damage during the yeast replicative life span.

    Science.gov (United States)

    Kale, S P; Jazwinski, S M

    1996-01-01

    The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death.

  1. An engineered yeast as a potential biosensor of environmental stress

    Energy Technology Data Exchange (ETDEWEB)

    Godon, C.; Quemeneur, E.; Chagvardieff, P.

    2004-07-01

    We developed a simple quantitative assay to detect stress activated nuclear translocation of nucleo-cytoplasmic protein in the yeast S. cerevisiae. The assay relies on the fusion of a transcription factor comprising the bacterial DNA binding domain (bDBD) and the activation domain of the yeast (yAD) to YAP1 and the {beta}-galactosidase gene placed under the promoter inducible by bDBD-yAD transcription factor. YAPI (yeast) is known to shuttle between the nucleus and the cytoplasm during oxidative stress. After stress, bDBD-yAD-YAP1 chimeric proteins localization can be simply quantified by measuring {beta}-galactosidase expression. We analysed H2O and mercury response. Very low mercury (down to 5 10-7 M) and H2O2 (5 10-5 M) concentrations activated the YAP1 translocation system. This quick and easy to use assay, based on yeast engineered for detection and quantization of translocation factor, may represent a suitable bio indicator to quantified environmental heavy metal exposure. (Author) 4 refs.

  2. Yeast synthetic biology toolbox and applications for biofuel production.

    Science.gov (United States)

    Tsai, Ching-Sung; Kwak, Suryang; Turner, Timothy L; Jin, Yong-Su

    2015-02-01

    Yeasts are efficient biofuel producers with numerous advantages outcompeting bacterial counterparts. While most synthetic biology tools have been developed and customized for bacteria especially for Escherichia coli, yeast synthetic biological tools have been exploited for improving yeast to produce fuels and chemicals from renewable biomass. Here we review the current status of synthetic biological tools and their applications for biofuel production, focusing on the model strain Saccharomyces cerevisiae We describe assembly techniques that have been developed for constructing genes, pathways, and genomes in yeast. Moreover, we discuss synthetic parts for allowing precise control of gene expression at both transcriptional and translational levels. Applications of these synthetic biological approaches have led to identification of effective gene targets that are responsible for desirable traits, such as cellulosic sugar utilization, advanced biofuel production, and enhanced tolerance against toxic products for biofuel production from renewable biomass. Although an array of synthetic biology tools and devices are available, we observed some gaps existing in tool development to achieve industrial utilization. Looking forward, future tool development should focus on industrial cultivation conditions utilizing industrial strains. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  3. Effects of maternal and dietary selenium (Se-enriched yeast) on the expression of Sel P and apoER2 of germ cells of their offspring in goats.

    Science.gov (United States)

    Wang, Qian; Shi, Lei; Ren, Youshe; Yue, Wenbin; Zhang, Chunxiang; Lei, Fulin

    2012-11-01

    In this experiment the effect of maternal dietary selenium on the expression of Sel P and apoER2 of goat offspring was studied. The experiment was conducted on 119 Taihang Black Goats randomly divided into 4 groups which were fed with a basal diet, supplemented with 0 (control), 0.5, 2 and 4 mg kg(-1) DM Se. Testis samples were collected from young male of each treatment group at the end of the study (30 d after weaning) for mRNA expression using real-time PCR and for protein expression by immunohistochemistry assay. A significant decrease was observed in mRNA expression of Sel P and apoER2 in the testis of the Se-deficient (Group 1) and the Se-excess (Group 4) compared with that in Groups 2 and 3. A similar trend of the protein expression of Sel P and apoER2 was also found. These data indicate that maternal and dietary selenium has an effect on the expression of Sel P and apoER2 in testis of their offspring. In addition, both groups were similar suggesting that the relationship between Sel P and apoER2, and apoER2 is a receptor of Sel P in the seminiferous epithelium to uptake the selenium. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Yeasts in an industrial malting ecosystem.

    Science.gov (United States)

    Laitila, A; Wilhelmson, A; Kotaviita, E; Olkku, J; Home, S; Juvonen, R

    2006-11-01

    The malting ecosystem consists of two components: the germinating cereal grains and the complex microbial community. Yeasts and yeast-like fungi are an important part of this ecosystem, but the composition and the effects of this microbial group have been largely unknown. In this study we surveyed the development of yeasts and yeast-like fungi in four industrial scale malting processes. A total of 136 malting process samples were collected and examined for the presence of yeasts growing at 15, 25 and 37 degrees C. More than 700 colonies were isolated and characterized. The isolates were discriminated by PCR-fingerprinting with microsatellite primer (M13). Yeasts representing different fingerprint types were identified by sequence analysis of the D1/D2 domain of the 26S rRNA gene. Furthermore, identified yeasts were screened for the production of alpha-amylase, beta-glucanase, cellulase and xylanase. A numerous and diverse yeast community consisting of both ascomycetous (25) and basidiomycetous (18) species was detected in the various stages of the malting process. The most frequently isolated ascomycetous yeasts belonged to the genera Candida, Clavispora, Galactomyces, Hanseniaspora, Issatchenkia, Pichia, Saccharomyces and Williopsis and the basidiomycetous yeasts to Bulleromyces, Filobasidium, Cryptococcus, Rhodotorula, Sporobolomyces and Trichosporon. In addition, two ascomycetous yeast-like fungi (black yeasts) belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Knowledge of the microbial diversity provides a basis for microflora management and understanding of the role of microbes in the cereal germination process.

  5. Metabolic regulation rather than de novo enzyme synthesis dominates the osmo-adaptation of yeast

    NARCIS (Netherlands)

    Bouwman, Jildau; Kiewiet, Jose; Lindenbergh, Alexander; van Eunen, Karen; Siderius, Marco; Bakker, Barbara M.

    Intracellular accumulation of glycerol is essential for yeast cells to survive hyperosmotic stress. Upon hyperosmotic stress the gene expression of enzymes in the glycerol pathway is strongly induced. Recently, however, it was shown that this gene-expression response is not essential for survival of

  6. Metabolic regulation rather than de novo enzyme synthesis dominates the osmo-adaptation of yeast.

    NARCIS (Netherlands)

    Bouwman, J.; Kiewiet, J.A.L.; Lindenbergh, A; Van Eunen, K.; Siderius, M.H.; Bakker, B.M.

    2011-01-01

    Intracellular accumulation of glycerol is essential for yeast cells to survive hyperosmotic stress. Upon hyperosmotic stress the gene expression of enzymes in the glycerol pathway is strongly induced. Recently, however, it was shown that this gene-expression response is not essential for survival of

  7. Metabolic regulation rather than de novo enzyme synthesis dominates the osmo-adaptation of yeast

    NARCIS (Netherlands)

    Bouwman, J; Kiewiet, J; Lindenbergh, A; Eunen, K, van; Siderius, M; Bakker, BM

    2011-01-01

    Intracellular accumulation of glycerol is essential for yeast cells to survive hyperosmotic stress. Upon hyperosmotic stress the gene expression of enzymes in the glycerol pathway is strongly induced. Recently, however, it was shown that this gene-expression response is not essential for survival of

  8. An IPTG-inducible derivative of the fission yeast nmt promoter

    DEFF Research Database (Denmark)

    Kjærulff, Søren; Nielsen, Olaf

    2015-01-01

    We here describe an IPTG-inducible system that reveals that the lac repressor alone can function as a potent transmodulator to regulate gene expression in the fission yeast, Schizosaccharomyces pombe. This expression system is a derivative of the Sz. pombe nmt promoter, which normally is strongly...

  9. Antioxidant defense parameters as predictive biomarkers for fermentative capacity of active dried wine yeast.

    Science.gov (United States)

    Gamero-Sandemetrio, Esther; Gómez-Pastor, Rocío; Matallana, Emilia

    2014-08-01

    The production of active dried yeast (ADY) is a common practice in industry for the maintenance of yeast starters and as a means of long term storage. The process, however, causes multiple cell injuries, with oxidative damage being one of the most important stresses. Consequentially, dehydration tolerance is a highly appreciated property in yeast for ADY production. In this study we analyzed the cellular redox environment in three Saccharomyces cerevisiae wine strains, which show markedly different fermentative capacities after dehydration. To measure/quantify the effect of dehydration on the S. cerevisiae strains, we used: (i) fluorescent probes; (ii) antioxidant enzyme activities; (ii) intracellular damage; (iii) antioxidant metabolites; and (iv) gene expression, to select a minimal set of biochemical parameters capable of predicting desiccation tolerance in wine yeasts. Our results show that naturally enhanced antioxidant defenses prevent oxidative damage after wine yeast biomass dehydration and improve fermentative capacity. Based on these results we chose four easily assayable parameters/biomarkers for the selection of industrial yeast strains of interest for ADY production: trehalose and glutathione levels, and glutathione reductase and catalase enzymatic activities. Yeast strains selected in accordance with this process display high levels of trehalose, low levels of oxidized glutathione, a high induction of glutathione reductase activity, as well as a high basal level and sufficient induction of catalase activity, which are properties inherent in superior ADY strains. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Yeast Isolation for Bioethanol Production

    Directory of Open Access Journals (Sweden)

    EKA RURIANI

    2012-09-01

    Full Text Available We have isolated 12 yeast isolates from five different rotten fruits by using a yeast glucose chloramphenicol agar (YGCA medium supplemented with tetracycline. From pre-screening assay, four isolates exhibited higher substrate (glucose-xylose consumption efficiency in the reaction tube fermentation compared to Saccharomyces cerevisiae dan Saccharomyces ellipsoids as the reference strains. Based on the fermentation process in gooseneck flasks, we observed that two isolates (K and SB showed high fermentation efficiency both in sole glucose and mixed glucose-xylose substrate. Moreover, isolates K and SB produced relatively identical level of ethanol concentration compared to the reference strains. Isolates H and MP could only produce high levels of ethanol in glucose fermentation, while only half of that amount of ethanol was detected in glucose-xylose fermentation. Isolate K and SB were identified as Pichia kudriavzeevii (100% based on large sub unit (LSU ribosomal DNA D1/D2 region.

  11. Mechanisms of yeast resistance to environmental stress 

    Directory of Open Access Journals (Sweden)

    Agata Piecuch

    2013-04-01

    Full Text Available Changes in environmental conditions might be a stress factor for yeast cells. There are several mechanisms of stress tolerance, developed by the cell, which activate when the stress appears. Different transcription factors coordinate the expression of stress response genes. Msn2/4p regulate the expression of the general stress response. Heat shock defense involves heat shock proteins (Hsp, controlled by Hsf1p. Osmotic shock induces the MAP kinase cascade (HOG, whereas the oxidative stress response requires the YAP network. Fungicide resistance is mediated mainly by the activity of membrane transporters and changes in the structure of the plasma membrane. 

  12. Tools for genetic engineering of the yeast Hansenula polymorpha.

    Science.gov (United States)

    Saraya, Ruchi; Gidijala, Loknath; Veenhuis, Marten; van der Klei, Ida J

    2014-01-01

    Hansenula polymorpha is a methylotrophic yeast species that has favorable properties for heterologous protein production and metabolic engineering. It provides an attractive expression platform with the capability to secrete high levels of commercially important proteins. Over the past few years many efforts have led to advances in the development of this microbial host including the generation of expression vectors containing strong constitutive or inducible promoters and a large array of dominant and auxotrophic markers. Moreover, highly efficient transformation procedures used to generate genetically stable strains are now available. Here, we describe these tools as well as the methods for genetic engineering of H. polymorpha.

  13. Yeasts colonizing the leaf surfaces.

    Science.gov (United States)

    Sláviková, Elena; Vadkertiová, Renata; Vránová, Dana

    2007-08-01

    The yeasts were isolated from the leaf surfaces of ten species of trees. The study site was a forest park (Zelezná Studnicka) of the Small Carpathians mountain range. One hundred and thirty seven yeast strains belonging to 13 genera were isolated from 320 samples of leaves and needles. Seventeen yeast species were isolated, but only seven occurred regularly: Aureobasidium pullulans, Cryptococcus laurentii, Pichia anomala, Metschnikowia pulcherrima, Saccharomyces sp., Lachancea thermotolerans, and Rhodotorula glutinis. The remaining species were isolated from the leaves and needles of three or less tree species. A. pullulans, Cr. laurentii, and P. anomala were the most frequently found species and they occurred on leaves and needles of all ten tree species. Saccharomyces sp. occurred in leaf samples collected from eight kinds of trees. M. pulcherrima and L. thermotolerans were found in samples collected from six species of trees. Both these species occurred almost always on the leaves of deciduous trees. Rh. glutinis was the most frequently isolated carotenoids producing species. We have found out that the ascomycetous and basidiomycetous species were present in the leaf samples in approximately equal frequency, contrary to the soil samples taken from this forest park, where the ascomycetous species were found rarely.

  14. Yeast: A new oil producer?

    Directory of Open Access Journals (Sweden)

    Beopoulos Athanasios

    2012-01-01

    Full Text Available The increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives for the oleochemical field (such as lubricants, adhesives or plastics have created price imbalance in both the alimentary and energy field. Moreover, the lack of non-edible oil feedstock has given rise to concerns on land-use practices and on oil production strategies. Recently, much attention has been paid to the exploitation of microbial oils. Most of them present lipid profiles similar in type and composition to plants and could therefore have many advantages as are no competitive with food, have short process cycles and their cultivation is independent of climate factors. Among microorganisms, yeasts seem to be very promising as they can be easily genetically enhanced, are suitable for large-scale fermentation and are devoid of endotoxins. This review will focus on the recent understanding of yeasts lipid metabolism, the succeeding genetic engineering of the lipid pathways and the recent developments on fermentation techniques that pointed out yeasts as promising alternative producers for oil or plastic.

  15. Deciphering the molecular basis of wine yeast fermentation traits using a combined genetic and genomic approach.

    Science.gov (United States)

    Ambroset, Chloé; Petit, Maud; Brion, Christian; Sanchez, Isabelle; Delobel, Pierre; Guérin, Cyprien; Chiapello, Hélène; Nicolas, Pierre; Bigey, Frédéric; Dequin, Sylvie; Blondin, Bruno

    2011-09-01

    The genetic basis of the phenotypic diversity of yeast is still poorly understood. Wine yeast strains have specific abilities to grow and ferment under stressful conditions compared with other strains, but the genetic basis underlying these traits is unknown. Understanding how sequence variation influences such phenotypes is a major challenge to address adaptation mechanisms of wine yeast. We aimed to identify the genetic basis of fermentation traits and gain insight into their relationships with variations in gene expression among yeast strains. We combined fermentation trait QTL mapping and expression profiling of fermenting cells in a segregating population from a cross between a wine yeast derivative and a laboratory strain. We report the identification of QTL for various fermentation traits (fermentation rates, nitrogen utilization, metabolites production) as well as expression QTL (eQTL). We found that many transcripts mapped to several eQTL hotspots and that two of them overlapped with QTL for fermentation traits. A QTL controlling the maximal fermentation rate and nitrogen utilization overlapping with an eQTL hotspot was dissected. We functionally demonstrated that an allele of the ABZ1 gene, localized in the hotspot and involved in p-aminobenzoate biosynthesis, controls the fermentation rate through modulation of nitrogen utilization. Our data suggest that the laboratory strain harbors a defective ABZ1 allele, which triggers strong metabolic and physiological alterations responsible for the generation of the eQTL hotspot. They also suggest that a number of gene expression differences result from some alleles that trigger major physiological disturbances.

  16. Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  17. Construction of dextrin and isomaltose-assimilating brewer's yeasts for production of low-carbohydrate beer.

    Science.gov (United States)

    Park, Jin-Yeong; Lee, Ja-Yeon; Choi, Seung-Hyun; Ko, Hyun-Mi; Kim, Il-Chul; Lee, Hwanghee Blaise; Bai, Suk

    2014-08-01

    Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer's bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer's yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both α-1,4- and α-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.

  18. Mechanisms of patulin toxicity under conditions that inhibit yeast growth.

    Science.gov (United States)

    Iwahashi, Yumiko; Hosoda, Hiroshi; Park, Ji-Hyun; Lee, Joo-Hee; Suzuki, Yoshiteru; Kitagawa, Emiko; Murata, Satomi Mizukami; Jwa, Nam-Soo; Gu, Man-Bock; Iwahashi, Hitoshi

    2006-03-08

    Patulin, 4-hydroxy-4H-furo[3,2c]pyran-2(6H)-one, is one of the best characterized and most widely disseminated mycotoxins found in agricultural products. Nonetheless, the mechanisms by which patulin causes toxicity are not well understood. Thus, the cytotoxicity of patulin was characterized by analysis of the yeast transcriptome upon challenge with patulin. Interestingly, patulin-induced yeast gene expression profiles were found to be similar to gene expression patterns obtained after treatment with the antifungal agricultural chemicals thiuram, maneb, and zineb. Moreover, patulin treatment was found to activate protein degradation, especially proteasome activities, sulfur amino acid metabolism, and the defense system for oxidative stress. Damage to DNA by alkylation was also suggested, and this seemed to be repaired by recombinational and excision repair mechanisms. Furthermore, the results provide potential biomarker genes for the detection of patulin in agricultural products. The results suggest the possibility of applying the yeast transcriptome system for the evaluation of chemicals, especially for natural chemicals that are difficult to get by organic synthesis.

  19. Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

    Directory of Open Access Journals (Sweden)

    Sean A Gray

    Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

  20. Yeasts Diversity in Fermented Foods and Beverages

    Science.gov (United States)

    Tamang, Jyoti Prakash; Fleet, Graham H.

    People across the world have learnt to culture and use the essential microorganisms for production of fermented foods and alcoholic beverages. A fermented food is produced either spontaneously or by adding mixed/pure starter culture(s). Yeasts are among the essential functional microorganisms encountered in many fermented foods, and are commercially used in production of baker's yeast, breads, wine, beer, cheese, etc. In Asia, moulds are predominant followed by amylolytic and alcohol-producing yeasts in the fermentation processes, whereas in Africa, Europe, Australia and America, fermented products are prepared exclusively using bacteria or bacteria-yeasts mixed cultures. This chapter would focus on the varieties of fermented foods and alcoholic beverages produced by yeasts, their microbiology and role in food fermentation, widely used commercial starters (pilot production, molecular aspects), production technology of some common commercial fermented foods and alcoholic beverages, toxicity and food safety using yeasts cultures and socio-economy

  1. Yeast Interacting Proteins Database: YEL005C, YGL079W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available endosome; identified as a transcriptional activator in a high-throughput yeast one-hybrid assay Rows with th...protein localizes to the endosome; identified as a transcriptional activator in a high-throughput yeast one-

  2. Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation.

    Directory of Open Access Journals (Sweden)

    Roger W Kramer

    2007-02-01

    Full Text Available Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.

  3. Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation.

    Science.gov (United States)

    Kramer, Roger W; Slagowski, Naomi L; Eze, Ngozi A; Giddings, Kara S; Morrison, Monica F; Siggers, Keri A; Starnbach, Michael N; Lesser, Cammie F

    2007-02-01

    Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.

  4. Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker's yeast.

    Science.gov (United States)

    Awan, Ali R; Blount, Benjamin A; Bell, David J; Shaw, William M; Ho, Jack C H; McKiernan, Robert M; Ellis, Tom

    2017-05-04

    Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker's yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

  5. Improved vanillin production in baker's yeast through in silico design

    DEFF Research Database (Denmark)

    Brochado, Ana Rita; Matos, Cláudia; Møller, Birger L.

    2010-01-01

    Background: Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has...... recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis. Results: Expression of a glycosyltransferase from Arabidopsis...... thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets...

  6. Automated multiplex genome-scale engineering in yeast

    Science.gov (United States)

    Si, Tong; Chao, Ran; Min, Yuhao; Wu, Yuying; Ren, Wen; Zhao, Huimin

    2017-01-01

    Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems. Here we report an automated platform for multiplex genome-scale engineering in Saccharomyces cerevisiae, an important eukaryotic model and widely used microbial cell factory. Standardized genetic parts encoding overexpression and knockdown mutations of >90% yeast genes are created in a single step from a full-length cDNA library. With the aid of CRISPR-Cas, these genetic parts are iteratively integrated into the repetitive genomic sequences in a modular manner using robotic automation. This system allows functional mapping and multiplex optimization on a genome scale for diverse phenotypes including cellulase expression, isobutanol production, glycerol utilization and acetic acid tolerance, and may greatly accelerate future genome-scale engineering endeavours in yeast. PMID:28469255

  7. Incompatibility with Formin Cdc12p Prevents Human Profilin from Substituting for Fission Yeast Profilin: Insights from Crystal Structures of Fission Yeast Proflin

    Energy Technology Data Exchange (ETDEWEB)

    Ezezika, O.; Younger, N; Lu, J; Kaiser, D; Corbin, Z; Nolen, B; Kovar, D; Pollard, T

    2009-01-01

    Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-l-proline, but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider 'wings' that interact with actin.

  8. Effect of long term selenium yeast intervention on activity and gene expression of antioxidant and xenbiotic metabolising enzymes in healthy elderly volunteers from the Danish Prevention of Cancer by Intervention by Selenium (PRECISE) Pilot Study

    DEFF Research Database (Denmark)

    Ravn-Haren, Gitte; Krath, Britta; Overvad, Kim

    2008-01-01

    Numerous mechanisms have been proposed to explain the anti-carcinogenic effects of Se, among them altered carcinogen metabolism. We investigated the effect of Se supplementation on activities of glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) in differ...... genes could increase the risk of cancer. However, further studies are needed to establish whether the observed effect in leucocytes reflects a similar expression pattern in target tissues....

  9. Revaluation of Waste Yeast from Beer Production

    OpenAIRE

    Nicoleta Suruceanu; Sonia Socaci; Teodora Coldea; Elena Mudura

    2013-01-01

    Brewing yeast is an important waste product from beer production. The valorification of slurry yeast mainly consists of separation of vitamins and important nitrogen compounds. The hops compounds, one of the most important raw materials in beer technology are removed beforehand valorification. The prenylflavonoids compounds from hops are important bioactive compounds that can be revaluation with proper technology. Revaluation of prenylflavonoids from waste yeast into dietary supplement, ident...

  10. Expression of the rgMT gene, encoding for a rice metallothionein ...

    Indian Academy of Sciences (India)

    protein, as well as the impact of gene expression in yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana under heavy metal ion, salt ... Yeast cells transgenic for rgMT showed vigorous growth compared to the nontransgenic controls when exposed to 7 mM .... precultured in liquid YPD medium (1% yeast extract + 2%.

  11. Did Gause Have a Yeast Infection?

    Science.gov (United States)

    Pritchard, Jonathon O; Porter, Alice H M; Montagnes, David J S

    2016-09-01

    We planned to develop predator-prey models using Paramecium and yeast, but they have not been empirically examined since work by Gause in the 1930s. Therefore, we evaluated if Paramecium aurelia ingests and grows on eight yeasts. Recognising that it ingested yeasts but could not grow, we assessed if it might grow on other yeasts, by empirically parameterising a predator-prey model that relies on ingestion, not growth. Simulations were compared to P. aurelia-yeast time-series data, from Gause. We hypothesised that if the model simulated predator-prey dynamics that mimicked the original data, then possibly P. aurelia could grow on yeast; simulations did not mimic the original data. Reviewing works by Gause exposed two issues: experiments were undoubtedly contaminated with bacteria, allowing growth on bacteria, not yeast; and the population cycle data cannot be considered a self-sustaining time series, as they were manipulated by adding yeast and ciliates. We conclude that past and future work should not rely on this system, for either empirical or theoretical evaluations. Finally, although we show that P. aurelia, P. caudatum, Euplotes patella, and Blepharisma sp. cannot grow on yeast, Tetrahymena pyriformis and Colpidium striatum can; these may provide models to explore predator-prey dynamics. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

  12. Evaluation of Automated Yeast Identification System

    Science.gov (United States)

    McGinnis, M. R.

    1996-01-01

    One hundred and nine teleomorphic and anamorphic yeast isolates representing approximately 30 taxa were used to evaluate the accuracy of the Biolog yeast identification system. Isolates derived from nomenclatural types, environmental, and clinica isolates of known identity were tested in the Biolog system. Of the isolates tested, 81 were in the Biolog database. The system correctly identified 40, incorrectly identified 29, and was unable to identify 12. Of the 28 isolates not in the database, 18 were given names, whereas 10 were not. The Biolog yeast identification system is inadequate for the identification of yeasts originating from the environment during space program activities.

  13. Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis.

    Science.gov (United States)

    Jiménez, Juan J; Borrero, Juan; Diep, Dzung B; Gútiez, Loreto; Nes, Ingolf F; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2013-09-01

    Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.

  14. Yeast Infection Test: MedlinePlus Lab Test Information

    Science.gov (United States)

    ... this page: https://medlineplus.gov/labtests/yeastinfectiontest.html Yeast Infection Test To use the sharing features on this page, please enable JavaScript. What is a Yeast Test? Yeast is a type of fungus that ...

  15. Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation.

    Science.gov (United States)

    Nambu-Nishida, Yumiko; Sakihama, Yuri; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-08-28

    To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, PTDH3, PFBA1, and PTDH1 were favorable for high expression, and PSED1, PHXT7, PPDC1, PTEF1, PTPI1, and PPGK1 were acceptable for medium-high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. PTEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium-high expression in glucose media. PZWF1 and PSOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. PALD3 and PTKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Cell size control in yeast.

    Science.gov (United States)

    Turner, Jonathan J; Ewald, Jennifer C; Skotheim, Jan M

    2012-05-08

    Cell size is an important adaptive trait that influences nearly all aspects of cellular physiology. Despite extensive characterization of the cell-cycle regulatory network, the molecular mechanisms coupling cell growth to division, and thereby controlling cell size, have remained elusive. Recent work in yeast has reinvigorated the size control field and suggested provocative mechanisms for the distinct functions of setting and sensing cell size. Further examination of size-sensing models based on spatial gradients and molecular titration, coupled with elucidation of the pathways responsible for nutrient-modulated target size, may reveal the fundamental principles of eukaryotic cell size control. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. FLO gene-dependent phenotypes in industrial wine yeast strains.

    Science.gov (United States)

    Govender, Patrick; Bester, Michael; Bauer, Florian F

    2010-04-01

    Most commercial yeast strains are nonflocculent. However, controlled flocculation phenotypes could provide significant benefits to many fermentation-based industries. In nonflocculent laboratory strains, it has been demonstrated that it is possible to adjust flocculation and adhesion phenotypes to desired specifications by altering expression of the otherwise silent but dominant flocculation (FLO) genes. However, FLO genes are characterized by high allele heterogeneity and are subjected to epigenetic regulation. Extrapolation of data obtained in laboratory strains to industrial strains may therefore not always be applicable. Here, we assess the adhesion phenotypes that are associated with the expression of a chromosomal copy of the FLO1, FLO5, or FLO11 open reading frame in two nonflocculent commercial wine yeast strains, BM45 and VIN13. The chromosomal promoters of these genes were replaced with stationary phase-inducible promoters of the HSP30 and ADH2 genes. Under standard laboratory and wine making conditions, the strategy resulted in expected and stable expression patterns of these genes in both strains. However, the specific impact of the expression of individual FLO genes showed significant differences between the two wine strains and with corresponding phenotypes in laboratory strains. The data suggest that optimization of the flocculation pattern of individual commercial strains will have to be based on a strain-by-strain approach.

  18. The wine and beer yeast Dekkera bruxellensis.

    Science.gov (United States)

    Schifferdecker, Anna Judith; Dashko, Sofia; Ishchuk, Olena P; Piškur, Jure

    2014-09-01

    Recently, the non-conventional yeast Dekkera bruxellensis has been gaining more and more attention in the food industry and academic research. This yeast species is a distant relative of Saccharomyces cerevisiae and is especially known for two important characteristics: on the one hand, it is considered to be one of the main spoilage organisms in the wine and bioethanol industry; on the other hand, it is 'indispensable' as a contributor to the flavour profile of Belgium lambic and gueuze beers. Additionally, it adds to the characteristic aromatic properties of some red wines. Recently this yeast has also become a model for the study of yeast evolution. In this review we focus on the recently developed molecular and genetic tools, such as complete genome sequencing and transformation, to study and manipulate this yeast. We also focus on the areas that are particularly well explored in this yeast, such as the synthesis of off-flavours, yeast detection methods, carbon metabolism and evolutionary history. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd.

  19. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

    . Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  20. Yeast (Saccharomyces cereveresiae) Supplementation In High ...

    African Journals Online (AJOL)

    A four-week trial to assess the impact of yeast supplementation on the performance characteristics of broiler starters fed high levels of rice bran with or without yeast addition, was conducted using two hundred and forty day old broilers of the Bova nera strain. The chicks were divided into 15 groups of 16 chicks each.

  1. Virgin olive oil yeasts: A review.

    Science.gov (United States)

    Ciafardini, Gino; Zullo, Biagi Angelo

    2018-04-01

    This review summarizes current knowledge on virgin olive oil yeasts. Newly produced olive oil contains solid particles and micro drops of vegetation water in which yeasts reproduce to become the typical microbiota of olive oil. To date, about seventeen yeast species have been isolated from different types of olive oils and their by-products, of which six species have been identified as new species. Certain yeast species contribute greatly to improving the sensorial characteristics of the newly produced olive oil, whereas other species are considered harmful as they can damage the oil quality through the production of unpleasant flavors and triacylglycerol hydrolysis. Studies carried out in certain yeast strains have demonstrated the presence of defects in olive oil treated with Candida adriatica, Nakazawaea wickerhamii and Candida diddensiae specific strains, while other olive oil samples treated with other Candida diddensiae strains were defect-free after four months of storage and categorized as extra virgin. A new acetic acid producing yeast species, namely, Brettanomyces acidodurans sp. nov., which was recently isolated from olive oil, could be implicated in the wine-vinegary defect of the product. Other aspects related to the activity of the lipase-producing yeasts and the survival of the yeast species in the flavored olive oils are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Yeast evolution and ecology meet genomics

    OpenAIRE

    Dunham, Maitreya J.; Louis, Edward J.

    2010-01-01

    The EMBO Conference on Experimental Approaches to Evolution and Ecology in Yeast covered a broad range of interests. The applications of genomic methods to ecological and evolutionary questions emphasize that the yeasts are poised to make significant contributions to these fields.

  3. Exobiopolymer from polyhydroxyalkanoate-producing transgenic yeast

    African Journals Online (AJOL)

    Recently, the wild type yeast Kloeckera sp. strain KY1 was equipped in their cytoplasm with the phaABC operon containing genes phbA, phbB and phbC of the PHA biosynthetic pathway of Ralstonia eutropha. Unpredicted, resulted transgenic yeast strain KY1/PHA was able to synthesize another exopolymer beside the ...

  4. Biosynthesis of polyhydroxyalkanotes in wildtype yeasts | Desuoky ...

    African Journals Online (AJOL)

    Biosynthesis of the biodegradable polymers polyhydroxyalkanotes (PHAs) are studied extensively in wild type and genetically modified prokaryotic cells, however the content and structure of PHA in wild type yeasts are not well documented. The purpose of this study was to screen forty yeast isolates collected from different ...

  5. Yeasts in sustainable bioethanol production: A review

    Directory of Open Access Journals (Sweden)

    Siti Hajar Mohd Azhar

    2017-07-01

    Full Text Available Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.

  6. Sporangiospore - Yeast Transformation of Mucor circinelloides

    African Journals Online (AJOL)

    Charles

    2012-02-16

    Feb 16, 2012 ... Measurement of intracellular ion concentration during sporangiospores-yeast transformation of Mucor circinelloides .... Intercellular ion variation (K+, 0.90 g/l; Na+, 0.05 to 0.20 g/l) during sporangiospore-to-yeast transformation of M. circinelloides ...... progressive replication of DNA and, hence nucleation.

  7. Measurement of yeast invertase during alcoholic fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Naudin, O.; Boudarel, M.J.; Ramirez, A.

    1986-01-01

    In continuous alcoholic fermentation of molasses by Saccharomyces cerevisiae, it is important but difficult to know the variation of yeast physiological state with time, so as to maintain maximum yeast productivity. We decided to quantify invertase activity, for which there are few if any appropriate methods (Vitolo and Borzani, Analytical Biochemistry 130, 469-470, 1983). 1 reference.

  8. Biodiesel generation from oleaginous yeast Rhodotorula glutinis ...

    African Journals Online (AJOL)

    This study explored a strategy to convert agricultural and forestry residues into microbial lipid, which could be further transformed into biodiesel. Among the 250 yeast strains screened for xylose assimilating capacity, eight oleaginous yeasts were selected by Sudan Black B test. The lipid content of these 8 strains was ...

  9. Growth requirements of san francisco sour dough yeasts and bakers' yeast.

    Science.gov (United States)

    Henry, N

    1976-03-01

    The growth requirements of several yeasts isolated from San Francisco sour dough mother sponges were compared with those of bakers' yeast. The sour dough yeasts studied were one strain of Saccharomyces uvarum, one strain of S. inusitatus, and four strains of S. exiguus. S. inusitatus was the only yeast found to have an amino acid requirement, namely, methionine. All of the yeasts had an absolute requirement for pantothenic acid and a partial requirement for biotin. Inositol was stimulatory to all except bakers' yeast. All strains of S. exiguus required niacin and thiamine. Interestingly, S. inusitatus, the only yeast that required methionine, also needed folic acid. For optimal growth of S. exiguus in a molasses medium, supplementation with thiamine was required.

  10. YEAST β-MANNANASE ACTIVITY

    Directory of Open Access Journals (Sweden)

    N. V. Borzova

    2017-02-01

    Full Text Available The aim of the research was to determine the mannan-degrading activity of yeasts cultures isolated from various sources and select strains with high β-mannanase activity. As a result of screening of 245 yeast strains, which are the representatives of 7 genera and 14 species, the active producers of extracellular β-mannanase were identified. To increase β-mannanase activity, the cultures were grown under submerged conditions using guar gum galactomannan as a carbon source and an inducer. β-Mannanase activity was determined by dinitrosalicylic method. The most active biosynthetic species were Cryptococcus albidus, C. gastricus, C. magnus, C. terreus, C. laurentii, Saccharomyces cerevisiae, Williopsis californica, Metschnikowia pulcherrima, Pichia anomala and P. guilliermondii. The activity in culture supernatant was ranged from 0.2 to 75 U/ml. α-Galactosidase activity was found in two strains (Debaryomyces polymorphus UCM Y-152 and Debaryomyces hansenii var. fabryi UCM Y-2400. None of the tested cultures demonstrated both β-mannanase and α-galactosidase activity, that is, they are unable to attack both the main and side chains of galactomannan.

  11. Engineering yeasts as platform organisms for cannabinoid biosynthesis.

    Science.gov (United States)

    Zirpel, Bastian; Degenhardt, Friederike; Martin, Chantale; Kayser, Oliver; Stehle, Felix

    2017-10-10

    Δ9-tetrahydrocannabinolic acid (THCA) is a plant derived secondary natural product from the plant Cannabis satival. The discovery of the human endocannabinoid system in the late 1980s resulted in a growing number of known physiological functions of both synthetic and plant derived cannabinoids. Thus, manifold therapeutic indications of cannabinoids currently comprise a significant area of research. Here we reconstituted the final biosynthetic cannabinoid pathway in yeasts. The use of the soluble prenyltransferase NphB from Streptomyces sp. strain CL190 enables the replacement of the native transmembrane prenyltransferase cannabigerolic acid synthase from C. sativa. In addition to the desired product cannabigerolic acid, NphB catalyzes an O-prenylation leading to 2-O-geranyl olivetolic acid. We show for the first time that the bacterial prenyltransferase and the final enzyme of the cannabinoid pathway tetrahydrocannabinolic acid synthase can both be actively expressed in the yeasts Saccharomyces cerevisiae and Komagataella phaffii simultaneously. While enzyme activities in S. cerevisiae were insufficient to produce THCA from olivetolic acid and geranyl diphosphate, genomic multi-copy integrations of the enzyme's coding sequences in K. phaffii resulted in successful synthesis of THCA from olivetolic acid and geranyl diphosphate. This study is an important step toward total biosynthesis of valuable cannabinoids and derivatives and demonstrates the potential for developing a sustainable and secure yeast bio-manufacturing platform. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Ethyl acetate production by the elusive alcohol acetyltransferase from yeast.

    Science.gov (United States)

    Kruis, Aleksander J; Levisson, Mark; Mars, Astrid E; van der Ploeg, Max; Garcés Daza, Fernando; Ellena, Valeria; Kengen, Servé W M; van der Oost, John; Weusthuis, Ruud A

    2017-05-01

    Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the α/β hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  13. Lipase production in lipolytic yeast from Wonorejo mangrove area

    Science.gov (United States)

    Alami, Nur Hidayatul; Nasihah, Liziyatin; Umar, Rurin Luswidya Artaty; Kuswytasari, Nengah Dwianita; Zulaika, Enny; Shovitri, Maya

    2017-06-01

    Lipase is an enzyme that is often used in industry and become a commercial enzyme. One group of microorganisms capable of producing lipase is a yeast. This study aims to screen yeast from Wonorejo mangrove that potential to produce lipase and to optimize the production of these enzymes. Screening test include the measurement of lipolytic index and value of fatty acid. Yeast with the best value of fatty acid will be continued to the measurement of lipase activity. It is affected by several environmental factors, such as pH, temperature, and incubation time. This research was conducted to observe the optimization variation on environmental factors combination to produce lipase. Lipase activity was tested by using p-Nitrophenyl Palmitate (pNPP). Absorbency was measured by spectrofotometer on wavelength of 410 nm. Measurement of the enzyme activity was done by interpolating the absorbance values on the p-nitrophenol standard curve then calculated by the formula. All data were analyzed by using descriptive quantitative method. The results show that the highest lypolityc index was 2.08. The highest value of fatty acid was 0.49 that was reached on 168 hours of incubation. Candida W3.8 expressed the highest lypolylitic potential. The optimum environment to produce lipase by Candida W 3.8 was on 120 hours of incubation time, in temperature range of 27°C - 45°C and pH range of 4,5 - 7.

  14. The yeast Dekkera bruxellensis genome contains two orthologs of the ARO10 gene encoding for phenylpyruvate decarboxylase.

    Science.gov (United States)

    de Souza Liberal, Anna Theresa; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante; Simões, Diogo Ardaillon; de Morais, Marcos Antonio

    2012-07-01

    The yeast Dekkera bruxellensis possesses important physiological traits that enable it to grow in industrial environments as either spoiling yeast of wine production or a fermenting strain used for lambic beer, or fermenting yeast in the bioethanol production process. In this work, in silico analysis of the Dekkera genome database allowed the identification of two paralogous genes encoding for phenylpyruvate decarboxylase (DbARO10) that represents a unique trait among the hemiascomycetes. The molecular analysis of the theoretical protein confirmed its protein identity. Upon cultivation of the cell in medium containing phenylpyruvate, both increases in gene expression and in phenylpyruvate decarboxylase activity were observed. Both genes were differentially expressed depending on the culture condition and the type of metabolism, which indicated the difference in the biological function of their corresponding proteins. The importance of the duplicated DbARO10 genes in the D. bruxellensis genome was discussed and represents the first effort to understand the production of flavor by this yeast.

  15. SREBP controls oxygen-dependent mobilization of retrotransposons in fission yeast.

    Directory of Open Access Journals (Sweden)

    Alfica Sehgal

    2007-08-01

    Full Text Available Retrotransposons are mobile genetic elements that proliferate through an RNA intermediate. Transposons do not encode transcription factors and thus rely on host factors for mRNA expression and survival. Despite information regarding conditions under which elements are upregulated, much remains to be learned about the regulatory mechanisms or factors controlling retrotransposon expression. Here, we report that low oxygen activates the fission yeast Tf2 family of retrotransposons. Sre1, the yeast ortholog of the mammalian membrane-bound transcription factor sterol regulatory element binding protein (SREBP, directly induces the expression and mobilization of Tf2 retrotransposons under low oxygen. Sre1 binds to DNA sequences in the Tf2 long terminal repeat that functions as an oxygen-dependent promoter. We find that Tf2 solo long terminal repeats throughout the genome direct oxygen-dependent expression of adjacent coding and noncoding sequences, providing a potential mechanism for the generation of oxygen-dependent gene expression.

  16. Mycotoxins - prevention and decontamination by yeasts.

    Science.gov (United States)

    Pfliegler, Walter P; Pusztahelyi, Tünde; Pócsi, István

    2015-07-01

    The application of yeasts has great potential in reducing the economic damage caused by toxigenic fungi in the agriculture. Some yeasts may act as biocontrol agents inhibiting the growth of filamentous fungi. These species may also gain importance in the preservation of agricultural products and in the reduction of their mycotoxin contamination, yet the extent of mycotoxin production in the presence of biocontrol agents is relatively less understood. The application of yeasts in various technological processes may have a direct inhibitory effect on the toxin production of certain molds, which is independent of their growth suppressing effect. Furthermore, several yeast species are capable of accumulating mycotoxins from agricultural products, thereby effectively decontaminating them. Probiotic yeasts or products containing yeast cell wall are also applied to counteract mycotoxicosis in livestock. Several yeast strains are also able to degrade toxins to less-toxic or even non-toxic substances. This intensively researched field would greatly benefit from a deeper knowledge on the genetic and molecular basis of toxin degradation. Moreover, yeasts and their biotechnologically important enzymes may exhibit sensitivity to certain mycotoxins, thereby mounting a considerable problem for the biotechnological industry. It is noted that yeasts are generally regarded as safe; however, there are reports of toxin degrading species that may cause human fungal infections. The aspects of yeast-mycotoxin relations with a brief consideration of strain improvement strategies and genetic modification for improved detoxifying properties and/or mycotoxin resistance are reviewed here. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. SUMO expression shortens the lag phase of Saccharomyces cerevisiae yeast growth caused by complex interactive effects of major mixed fermentation inhibitors found in hot-compressed water-treated lignocellulosic hydrolysate.

    Science.gov (United States)

    Jayakody, Lahiru N; Kadowaki, Masafumi; Tsuge, Keisuke; Horie, Kenta; Suzuki, Akihiro; Hayashi, Nobuyuki; Kitagaki, Hiroshi

    2015-01-01

    The complex inhibitory effects of inhibitors present in lignocellulose hydrolysate suppress the ethanol fermentation of Saccharomyces cerevisiae. Although the interactive inhibitory effects play important roles in the actual hydrolysate, few studies have investigated glycolaldehyde, the key inhibitor of hot-compressed water-treated lignocellulose hydrolysate. Given this challenge, we investigated the interactive effects of mixed fermentation inhibitors, including glycolaldehyde. First, we confirmed that glycolaldehyde was the most potent inhibitor in the hydrolysate and exerted interactive inhibitory effects in combination with major inhibitors. Next, through genome-wide analysis and megavariate data modeling, we identified SUMOylation as a novel potential mechanism to overcome the combinational inhibitory effects of fermentation inhibitors. Indeed, overall SUMOylation was increased and Pgk1, which produces an ATP molecule in glycolysis by substrate-level phosphorylation, was SUMOylated and degraded in response to glycolaldehyde. Augmenting the SUMO-dependent ubiquitin system in the ADH1-expressing strain significantly shortened the lag phase of growth, released cells from G2/M arrest, and improved energy status and glucose uptake in the inhibitor-containing medium. In summary, our study was the first to establish SUMOylation as a novel platform for regulating the lag phase caused by complex fermentation inhibitors.

  18. Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

    Directory of Open Access Journals (Sweden)

    Vasundhara Sharma

    Full Text Available The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD, a chimeric construct containing the TAD derived from p65 was also generated (p50TAD to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

  19. FTIR spectroscopic characterization of differently cultivated food related yeasts.

    Science.gov (United States)

    Shapaval, V; Walczak, B; Gognies, S; Møretrø, T; Suso, H P; Wold Åsli, A; Belarbi, A; Kohler, A

    2013-07-21

    The application of Fourier Transform Infrared Spectroscopy for characterization of yeasts is growing rapidly. Since it is known that the phenotypic expression of yeast cells depends sensitively on the nutrients that are available in the growth medium, one standardized growth medium is usually used for identification and characterization purposes in order to obtain reproducible FTIR signals. Since our recently developed high-throughput micro-cultivation protocol has the capacity to use more than one standardized growth medium, we wanted to investigate if the parallel use of multiple growth media can improve identification results. For this purpose, five different cultivation media (YP, YPD, YMB, SAB and SD) were used. In total 91 food spoilage yeast strains of 12 different genera were cultivated in different cultivation media and subsequently characterized by FTIR spectroscopy. For spectral identifications, Radial Basis Function-Partial Least Squares (RBF-PLS) was used in combination with cross-model validation where an inner cross-validation loop was used to optimize the model, while in an outer loop an independent test set was kept aside to test the optimized model. Sensitivity and specificity were evaluated for each studied genus class. The results show that the YMB selective medium gave the best discrimination results for 9 of the 12 genera with sensitivity above 90%. Only three genera showed better identification results on other media (Clavispora and Metschnikowia on medium SD, Debaryomyces on medium YPD). We therefore suggest to use the media SD, YPD in combination with the YMB medium for the identification of food spoilage yeasts.

  20. The yeast Ski complex is a hetero-tetramer

    OpenAIRE

    Synowsky, Silvia A.; Heck, Albert J.R.

    2008-01-01

    The yeast Ski complex assists the exosome in the degradation of mRNA. The Ski complex consists of three components; Ski2, Ski3, and Ski8, believed to be present in a 1:1:1 stoichiometry. Measuring the mass of intact isolated endogenously expressed Ski complexes by native mass spectrometry we unambiguously demonstrate that the Ski complex has a hetero-tetrameric stoichiometry consisting of one copy of Ski2 and Ski3 and two copies of Ski8. To validate the stoichiometry of the Ski complex, we pe...

  1. Recent advances in bioengineering of the oleaginous yeast Yarrowia lipolytica

    Directory of Open Access Journals (Sweden)

    Murtaza Shabbir Hussain

    2016-12-01

    Full Text Available The oleaginous yeast, Yarrowia lipolytica, is becoming increasing popular for metabolic engineering applications. Advances in synthetic biology and metabolic engineering have allowed microorganisms such as Y. lipolytica to be tailored for specific chemical production. Significant progress has been made to understand the genetics of Y. lipolytica and towards developing novel genetic engineering tools, leading to accelerated metabolic engineering efforts for a variety of different products. In this review, we discuss recent advances in genetic engineering tools and metabolic engineering achievements specific to Y. lipolytica. Topics covered in this review include genetic manipulation and expression systems, lipid-based products, peroxisome-based products and alternative sugar utilization.

  2. Nucleosome structure of the yeast CHA1 promoter

    DEFF Research Database (Denmark)

    Moreira, José Manuel Alfonso; Holmberg, S

    1998-01-01

    conditions. Five yeast TBP mutants defective in different steps in activated transcription abolished CHA1 expression, but failed to affect induction-dependent chromatin rearrangement of the promoter region. Progressive truncations of the RNA polymerase II C-terminal domain caused a progressive reduction...... in CHA1 transcription, but no difference in chromatin remodeling. Analysis of swi1, swi3, snf5 and snf6, as well as gcn5, ada2 and ada3 mutants, suggested that neither the SWI/SNF complex nor the ADA/GCN5 complex is involved in efficient activation and/or remodeling of the CHA1 promoter. Interestingly...

  3. A novel platform for the production of nonhydroxylated gelatins based on the methylotrophic yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Geerlings, Torsten H.; de Boer, Arjo L.; Lunenborg, Marcel G. J.; Veenhuis, Marten; van der Klei, Ida J.

    2007-01-01

    The use of yeast as a host for heterologous expression of proteins that are normally derived from animal tissue is a promising way to ensure defined products that are devoid of potential harmful animal side products. Here we report on the production and secretion of a custom-designed gelatin,

  4. Repression of a mating type cassette in the fission yeast by four DNA elements

    DEFF Research Database (Denmark)

    Ekwall, K; Nielsen, O; Ruusala, T

    1991-01-01

    The fission yeast, Schizosaccharomyces pombe, expresses one of two alternative mating types. They are specified by one of two determinants (M or P) present at the mat1 locus. In addition, silent copies of M and P are present on the same chromosome. In the present work we demonstrate that the diff...... partitioning in mitosis to Schizosaccharomyces pombe ars plasmids....

  5. Transcriptional, proteomic, and metabolic responses to lithium in galactose-grown yeast cells

    DEFF Research Database (Denmark)

    Bro, Christoffer; Regenberg, Birgitte; Lagniel, G.

    2003-01-01

    Lithium is highly toxic to yeast when grown in galactose medium mainly because phosphoglucomutase, a key enzyme of galactose metabolism, is inhibited. We studied the global protein and gene expression profiles of Saccharomyces cerevisiae grown in galactose in different time intervals after addition...

  6. Rheologically interesting polysaccharides from yeasts

    Science.gov (United States)

    Petersen, G. R.; Nelson, G. A.; Cathey, C. A.; Fuller, G. G.

    1989-01-01

    We have examined the relationships between primary, secondary, and tertiary structures of polysaccharides exhibiting the rheological property of friction (drag) reduction in turbulent flows. We found an example of an exopolysaccharide from the yeast Cryptococcus laurentii that possessed high molecular weight but exhibited lower than expected drag reducing activity. Earlier correlations by Hoyt showing that beta 1 --> 3, beta 2 --> 4, and alpha 1 --> 3 linkages in polysaccharides favored drag reduction were expanded to include correlations to secondary structure. The effect of sidechains in a series of gellan gums was shown to be related to sidechain length and position. Disruption of secondary structure in drag reducing polysaccharides reduced drag reducing activity for some but not all exopolysaccharides. The polymer from C. laurentii was shown to be more stable than xanthan gum and other exopolysaccharides under the most vigorous of denaturing conditions. We also showed a direct relationship between extensional viscosity measurements and the drag reducing coefficient for four exopolysaccharides.

  7. Functional conservation between Schizosaccharomyces pombe ste8 and Saccharomyces cerevisiae STE11 protein kinases in yeast signal transduction

    DEFF Research Database (Denmark)

    Styrkársdóttir, U; Egel, R; Nielsen, O

    1992-01-01

    In fission yeast (Schizosaccharomyces pombe), the mat1-Pm gene, which is required for entry into meiosis, is expressed in response to a pheromone signal. Cells carrying a mutation in the ste8 gene are unable to induce transcription of mat1-Pm in response to pheromone, suggesting that the ste8 gene...... in signal transduction in budding yeast. Expression of the S. cerevisiae STE11 gene in S. pombe ste8 mutants restores the ability to transcribe mat1-Pm in response to pheromone. Also, such cells become capable of conjugation and sporulation. When mat1-Pm is artifically expressed from a heterologous promoter...

  8. 21 CFR 172.325 - Bakers yeast protein.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bakers yeast protein. 172.325 Section 172.325 Food... Special Dietary and Nutritional Additives § 172.325 Bakers yeast protein. Bakers yeast protein may be safely used in food in accordance with the following conditions: (a) Bakers yeast protein is the...

  9. Chemical gradients and chemotropism in yeast.

    Science.gov (United States)

    Arkowitz, Robert A

    2009-08-01

    Chemical gradients of peptide mating pheromones are necessary for directional growth, which is critical for yeast mating. These gradients are generated by cell-type specific secretion or export and specific degradation in receiving cells. Spatial information is sensed by dedicated seven-transmembrane G-protein coupled receptors and yeast cells are able to detect extremely small differences in ligand concentration across their approximately 5-microm cell surface. Here, I will discuss our current knowledge of how cells detect and respond to such shallow chemical gradients and in particular what is known about the proteins that are involved in directional growth and the establishment of the polarity axis during yeast mating.

  10. Identification of the Transcription Factor Znc1p, which Regulates the Yeast-to-Hypha Transition in the Dimorphic Yeast Yarrowia lipolytica

    Science.gov (United States)

    Martinez-Vazquez, Azul; Gonzalez-Hernandez, Angelica; Domínguez, Ángel; Rachubinski, Richard; Riquelme, Meritxell; Cuellar-Mata, Patricia; Guzman, Juan Carlos Torres

    2013-01-01

    The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica. PMID:23826133

  11. A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

    DEFF Research Database (Denmark)

    Kampranis, S C; Damianova, R; Atallah, M

    2000-01-01

    for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed...... in Escherichia coli and found to possess glutathione S-transferase (GST) and weak glutathione peroxidase (GPX) activity. Expression of Bax in yeast decreases the intracellular levels of total glutathione, causes a substantial reduction of total cellular phospholipids, diminishes the mitochondrial membrane...... potential, and alters the intracellular redox potential. Co-expression of the BI-GST/GPX protein brought the total glutathione levels back to normal and re-established the mitochondrial membrane potential but had no effect on the phospholipid alterations. Moreover, expression of BI-GST/GPX in yeast...

  12. YeastWeb: a workset-centric web resource for gene family analysis in yeast

    Directory of Open Access Journals (Sweden)

    Bao Haihua

    2010-07-01

    Full Text Available Abstract Background Currently, a number of yeast genomes with different physiological features have been sequenced and annotated, which provides invaluable information to investigate yeast genetics, evolutionary mechanism, structure and function of gene families. Description YeastWeb is a novel database created to provide access to gene families derived from the available yeast genomes by assigning the genes into putative families. It has many useful features that complement existing databases, such as SGD, CYGD and Génolevures: 1 Detailed computational annotation was conducted with each entry with InterProScan, EMBOSS and functional/pathway databases, such as GO, COG and KEGG; 2 A well established user-friendly environment was created to allow users to retrieve the annotated genes and gene families using functional classification browser, keyword search or similarity-based search; 3 Workset offers users many powerful functions to manage the retrieved data efficiently, associate the individual items easily and save the intermediate results conveniently; 4 A series of comparative genomics and molecular evolution analysis tools are neatly implemented to allow users to view multiple sequence alignments and phylogenetic tree of gene families. At present, YeastWeb holds the gene families clustered from various MCL inflation values from a total of 13 available yeast genomes. Conclusions Given the great interest in yeast research, YeastWeb has the potential to become a useful resource for the scientific community of yeast biologists and related researchers investigating the evolutionary relationship of yeast gene families. YeastWeb is available at http://centre.bioinformatics.zj.cn/Yeast/.

  13. Tuning the Sensitivity of the PDR5 Promoter-Based Detection of Diclofenac in Yeast Biosensors

    Directory of Open Access Journals (Sweden)

    Astrid Schuller

    2017-06-01

    Full Text Available The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

  14. The fission yeast inhibitor of growth (ING) protein Png1p functions in response to DNA damage.

    Science.gov (United States)

    Chen, Jian-Qiang; Li, Yang; Pan, Xian; Lei, Bing-Kun; Chang, Cheng; Liu, Zheng-Xun; Lu, Hong

    2010-05-21

    In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.

  15. Effects of FSGS-associated mutations on the stability and function of myosin-1 in fission yeast

    Directory of Open Access Journals (Sweden)

    Jing Bi

    2015-08-01

    Full Text Available Point mutations in the human MYO1E gene, encoding class I myosin Myo1e, are associated with focal segmental glomerulosclerosis (FSGS, a primary kidney disorder that leads to end-stage kidney disease. In this study, we used a simple model organism, fission yeast Schizosaccharomyces pombe, to test the effects of FSGS-associated mutations on myosin activity. Fission yeast has only one class I myosin, Myo1, which is involved in actin patch assembly at the sites of endocytosis. The amino acid residues mutated in individuals with FSGS are conserved between human Myo1e and yeast Myo1, which allowed us to introduce equivalent mutations into yeast myosin and use the resulting mutant strains for functional analysis. Yeast strains expressing mutant Myo1 exhibited defects in growth and endocytosis similar to those observed in the myo1 deletion strain. These mutations also disrupted Myo1 localization to endocytic actin patches and resulted in mis-localization of Myo1 to eisosomes, linear membrane microdomains found in yeast cells. Although both mutants examined in this study exhibited loss of function, one of these mutants was also characterized by the decreased protein stability. Thus, using the yeast model system, we were able to determine that the kidney-disease-associated mutations impair myosin functional activity and have differential effects on protein stability.

  16. NetPhosYeast: prediction of protein phosphorylation sites in yeast

    DEFF Research Database (Denmark)

    Ingrell, C.R.; Miller, Martin Lee; Jensen, O.N.

    2007-01-01

    We here present a neural network-based method for the prediction of protein phosphorylation sites in yeast-an important model organism for basic research. Existing protein phosphorylation site predictors are primarily based on mammalian data and show reduced sensitivity on yeast phosphorylation...... sites compared to those in humans, suggesting the need for an yeast-specific phosphorylation site predictor. NetPhosYeast achieves a correlation coefficient close to 0.75 with a sensitivity of 0.84 and specificity of 0.90 and outperforms existing predictors in the identification of phosphorylation sites...

  17. Methanol regulated yeast promoters: production vehicles and toolbox for synthetic biology.

    Science.gov (United States)

    Gasser, Brigitte; Steiger, Matthias G; Mattanovich, Diethard

    2015-12-02

    Promoters are indispensable elements of a standardized parts collection for synthetic biology. Regulated promoters of a wide variety of well-defined induction ratios and expression strengths are highly interesting for many applications. Exemplarily, we discuss the application of published genome scale transcriptomics data for the primary selection of methanol inducible promoters of the yeast Pichia pastoris (Komagataella sp.). Such a promoter collection can serve as an excellent toolbox for cell and metabolic engineering, and for gene expression to produce heterologous proteins.

  18. Systems biology of energy homeostasis in yeast.

    Science.gov (United States)

    Zhang, Jie; Vemuri, Goutham; Nielsen, Jens

    2010-06-01

    The yeast Saccharomyces cerevisiae attains energy homeostasis through complex regulatory events that are predominantly controlled by the Snf1 kinase. This master regulator senses the stress and energy starvation and activates the metabolic processes to produce ATP and inhibits biosynthesis. In doing so, Snf1 controls the switch between catabolism and anabolism accordingly, and regulates the cellular growth and development in coordination with other signaling pathways. Since its mammalian ortholog AMPK, a drug target for obesity and type II diabetes, also exerts analogous control of metabolism, there has been extensive interest recently to understand the chemical and biological aspects of Snf1 activation and regulation in yeast to expedite human disease studies as well as fundamental understanding of yeast. This review will focus on how Snf1 regulates lipid metabolism based on the cellular energy status in yeast and drawing parallels with the mammalian system. Copyright 2010 Elsevier Ltd. All rights reserved.

  19. [Red yeast rice: An unsafe food supplement?

    Science.gov (United States)

    Steffen, Christian

    2017-03-01

    Red yeast rice is the fermentation product of the mould Monascus ruber and is traditionally used in East Asia to dye and conserve food. Its main pharmacologically active compound, monakolin K, was isolated from red yeast rice and is used as an inhibitor of cholesterol synthesis under the INN lovastatin. Lovastatin and several other statins are marketed as drugs whereas red yeast rice is offered as a food supplement. As statins can cause severe side effects, such as muscle damage and kidney failure, the dosing and information about interactions with drugs and food is essential for the use of these products. Furthermore, red yeast rice can contain the mycotoxin citrinin and several other substances that are not yet toxicologically evaluated.

  20. Sporulation in the budding yeast Saccharomyces cerevisiae

    National Research Council Canada - National Science Library

    Neiman, Aaron M

    2011-01-01

    In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores...

  1. Genomic Evolution of the Ascomycete Yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Riley, Robert; Haridas, Sajeet; Salamov, Asaf; Boundy-Mills, Kyria; Goker, Markus; Hittinger, Chris; Klenk, Hans-Peter; Lopes, Mariana; Meir-Kolthoff, Jan P.; Rokas, Antonis; Rosa, Carlos; Scheuner, Carmen; Soares, Marco; Stielow, Benjamin; Wisecaver, Jennifer H.; Wolfe, Ken; Blackwell, Meredith; Kurtzman, Cletus; Grigoriev, Igor; Jeffries, Thomas

    2015-03-16

    Yeasts are important for industrial and biotechnological processes and show remarkable metabolic and phylogenetic diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. Phylogenetic analysis of these and previously published yeast genomes helped resolve the placement of species including Saitoella complicata, Babjeviella inositovora, Hyphopichia burtonii, and Metschnikowia bicuspidata. Moreover, we find that alternative nuclear codon usage, where CUG encodes serine instead of leucine, are monophyletic within the Saccharomycotina. Most of the yeasts have compact genomes with a large fraction of single exon genes, and a tendency towards more introns in early-diverging species. Analysis of enzyme phylogeny gives insights into the evolution of metabolic capabilities such as methanol utilization and assimilation of alternative carbon sources.

  2. Structure and function of yeast alcohol dehydrogenase

    Directory of Open Access Journals (Sweden)

    VLADIMIR LESKOVAC

    2000-04-01

    Full Text Available 1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer

  3. Propagation of Mammalian Prions in Yeast

    National Research Council Canada - National Science Library

    Harris, David A

    2006-01-01

    ...: the budding yeast Saccharomyces cerevisiae. This unicellular organism offers a number of potential advantages for the study of prion biology, including rapid generation time, ease of culturing, and facile genetics...

  4. Live Cell Imaging in Fission Yeast.

    Science.gov (United States)

    Mulvihill, Daniel P

    2017-10-03

    Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast. © 2017 Cold Spring Harbor Laboratory Press.

  5. Multidrug resistant yeasts in synanthropic wild birds

    Directory of Open Access Journals (Sweden)

    Somanath Sushela

    2010-03-01

    Full Text Available Abstract Background The aim of this study was to investigate the presence of multidrug resistant yeasts in the faeces of synanthropic wild birds from the Bangsar suburb of Kuala Lumpur. Methods Species characterisations of yeast isolates and determinations of antimycotic susceptibility profiles were undertaken using the commercial characterization kit, Integral System Yeasts Plus (Liofilchem, Italy. Results Fourteen species of yeasts were detected in the bird faecal samples.Candida albicans was present in 28.89% of bird faecal samples, Candida krusei (13.33%, Candida tropicalis (4.44%, Candida glabrata (4.44%, Candida parapsilosis (2.22%, Candida lambica (2.22%, Candida stellatoidea (2.22%, Candida rugosa (2.22% and Candida lusitaniae (2.22%. Amongst the non-candidal yeast isolates, Cryptococcus laurentii was present in 6.67% of bird faecal samples, Cryptococcus uniguttulatus (4.44%, Saccharomyces cerevisiae (4.44%, Trichosporon pullulans (2.22%, Trichosporon pullulans/Cryptococcus albidus (8.89% and Rhodotorula rubra/Rhodotorula glutinis (4.44%. Of the isolated yeasts, 18.1% (or 26/144 were found to be resistant to all 11 antimycotic agents they were tested against i.e. Nystatin, Amphotericin B, Flucytosine, Econazole, Ketoconazole, Clotrimazole, Miconazole, Itraconazole, Voriconazole, Fluconazole 16 and Fluconazole 64. 45.8% (or 66/144 of the bird faecal yeast isolates were resistant to four or more of the 11 antimycotic agents they were tested against. Conclusions This finding is of public health significance as these synanthropic wild birds may be reservoirs for transmission of drug resistant yeast infections to humans.

  6. Yeasts are essential for cocoa bean fermentation.

    Science.gov (United States)

    Ho, Van Thi Thuy; Zhao, Jian; Fleet, Graham

    2014-03-17

    Cocoa beans (Theobroma cacao) are the major raw material for chocolate production and fermentation of the beans is essential for the development of chocolate flavor precursors. In this study, a novel approach was used to determine the role of yeasts in cocoa fermentation and their contribution to chocolate quality. Cocoa bean fermentations were conducted with the addition of 200ppm Natamycin to inhibit the growth of yeasts, and the resultant microbial ecology and metabolism, bean chemistry and chocolate quality were compared with those of normal (control) fermentations. The yeasts Hanseniaspora guilliermondii, Pichia kudriavzevii and Kluyveromyces marxianus, the lactic acid bacteria Lactobacillus plantarum and Lactobacillus fermentum and the acetic acid bacteria Acetobacter pasteurianus and Gluconobacter frateurii were the major species found in the control fermentation. In fermentations with the presence of Natamycin, the same bacterial species grew but yeast growth was inhibited. Physical and chemical analyses showed that beans fermented without yeasts had increased shell content, lower production of ethanol, higher alcohols and esters throughout fermentation and lesser presence of pyrazines in the roasted product. Quality tests revealed that beans fermented without yeasts were purplish-violet in color and not fully brown, and chocolate prepared from these beans tasted more acid and lacked characteristic chocolate flavor. Beans fermented with yeast growth were fully brown in color and gave chocolate with typical characters which were clearly preferred by sensory panels. Our findings demonstrate that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

  7. Yeast evolution and ecology meet genomics.

    Science.gov (United States)

    Dunham, Maitreya J; Louis, Edward J

    2011-01-01

    The first EMBO Conference on Experimental Approaches to Evolution and Ecology in Yeast was held in Heidelberg, Germany, at the end of September 2010. What might sound like a rather narrow topic actually covered a broad range of interests, approaches, and systems and generated a great deal of excitement among participants. The applications of genomic methods to ecological and evolutionary questions emphasize that the yeasts are poised to make significant contributions to these fields.

  8. Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen.

    Directory of Open Access Journals (Sweden)

    Anthony Arnoldo

    2008-02-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS, a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.

  9. Flavor impacts of glycerol in the processing of yeast fermented beverages: a review.

    Science.gov (United States)

    Zhao, Xiangdong; Procopio, Susanne; Becker, Thomas

    2015-12-01

    Glycerol contributes to the beverage body and fullness. Moreover, it also influences the flavor intensity. As a major byproduct, glycerol not only serves critical roles in yeast osmoregulation and redox balancing, but also acts as the carbon competitor against ethanol in alcoholic fermentation. Therefore, increasing glycerol yield benefits both the flavor and ethanol reduction for the fermented beverages. Glycerol yield has been elevated either by fermentation optimization or by yeast genetic modification. The fermentation optimizations reached maximum 14 g/L glycerol through screening yeast strains and optimizing fermentation parameters. Meanwhile the yeast overexpressing GPD1 (encoding glycerol-3-phosphate dehydrogenase) produced up to 6 folds more glycerol for beer and wine. Except for glycerol improvement, the genetically modified yeasts accumulated dramatically undesirable compounds such as acetaldehyde, acetate and acetoin which are detrimental for beverage flavor. In comparison, the natural high glycerol producers showed strain-specific manner on the yeast-derived aroma compounds like volatile acids, fusel alcohols, esters, and aldehydes. Temperature, sugar concentration, nitrogen composition, oxygen and pH-value, which influence glycerol biosynthesis, also obtained various effects on the production of aromatic compounds. In the current review, we firstly deliberate the organoleptic contributions of glycerol for fermented beverages. Furthermore, glycerol optimization strategies are discussed regarding to the yield improvement, the genes expressions, the overall flavor impacts and the feasibilities in beverage applications. Lastly, for improving beverage flavor by glycerol optimization, a high-throughput platform is proposed to increase the screening capacity of yeast strains and parameters in the processing of fermented beverages.

  10. Fermentative Stability of Wine Yeast Saccharomyces Sensu Stricto Complex and Their Hybrids

    Directory of Open Access Journals (Sweden)

    Katarzyna Rajkowska

    2012-01-01

    Full Text Available The objective of this paper is to investigate the technological usefulness of selected industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus and their intra- and interspecific hybrids responsible for excessively acidic musts. The stability of yeast fermentation profiles in apple musts was assessed after 90–170 generations, following previous subculturing under aerobic or anaerobic conditions in media with or without L-malic acid. During this study, 35 apple wines produced by wild strains and their segregates were statistically evaluated according to 12 chemical parameters. Although the wines met the official standards for basic chemical parameters, their total acidity was too low. Both the yeasts and their segregates metabolized from 66.3 to 77.0 % of malic acid present in the must. The industrial wine yeasts and their hybrids exhibited marked polymorphism of fermentation profiles in apple must with elevated L-malic acid content. At the same time, the level of demalication activity made it possible to clearly differentiate segregates from the wild strains, which may suggest that malic acid is probably one of the principal factors in the adaptive evolution of yeasts. Our study proves that among industrial wine yeasts, there are both, strains expressing very high stability (Saccharomyces cerevisiae W-13 and labile ones (S. cerevisiae Syrena. The interspecific hybrids S. cerevisiae × S. bayanus showed low stability of technological features, while the intraspecific hybrid of S. cerevisiae preserved its fermentative capacity. The presented results indicate that fermentative stability assessment under environmental stress can help to select the yeast strains best suited for the fermentation of specific musts.

  11. Yeast cell surface display for lipase whole cell catalyst and its applications

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

    2014-08-01

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

  12. Integrative analysis of the mitochondrial proteome in yeast.

    Directory of Open Access Journals (Sweden)

    Holger Prokisch

    2004-06-01

    Full Text Available In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.

  13. Metabolic Engineering of Oleaginous Yeasts for Fatty Alcohol Production

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei; Wei, Hui; Knoshaug, Eric; Van Wychen, Stefanie; Xu, Qi; Himmel, Michael E.; Zhang, Min

    2016-04-25

    To develop pathways for advanced biological upgrading of sugars to hydrocarbons, we are seeking biological approaches to produce high carbon efficiency intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels. In this study, we successfully demonstrated fatty alcohol production by oleaginous yeasts Yarrowia lipolytica and Lipomyces starkeyi by expressing a bacteria-derived fatty acyl-CoA reductase (FAR). Moreover, we find higher extracellular distribution of fatty alcohols produced by FAR-expressing L. starkeyi strain as compared to Y. lipolytica strain, which would benefit the downstream product recovery process. In both oleaginous yeasts, long chain length saturated fatty alcohols were predominant, accounting for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Taken together, our work demonstrates that in addition to Y. lipolytica, L. starkeyi can also serve as a platform organism for production of fatty acid-derived biofuels and bioproducts via metabolic engineering. We believe strain and process development both will significantly contribute to our goal of producing scalable and cost-effective fatty alcohols from renewable biomass.

  14. Yeast communities in a natural tequila fermentation.

    Science.gov (United States)

    Lachance, M A

    1995-08-01

    Fresh and cooked agave, Drosophila spp., processing equipment, agave molasses, agave extract, and fermenting must at a traditional tequila distillery (Herradura, Amatitan, Jalisco, México) were studied to gain insight on the origin of yeasts involved in a natural tequila fermentations. Five yeast communities were identified. (1) Fresh agave contained a diverse mycobiota dominated by Clavispora lusitaniae and an endemic species, Metschnikowia agaveae. (2) Drosophila spp. from around or inside the distillery yielded typical fruit yeasts, in particular Hanseniaspora spp., Pichia kluyveri, and Candida krusei. (3) Schizosaccharomyces pombe prevailed in molasses. (4) Cooked agave and extract had a considerable diversity of species, but included Saccharomyces cerevisiae. (5) Fermenting juice underwent a gradual reduction in yeast heterogeneity. Torulaspora delbrueckii, Kluyveromyces marxianus, and Hanseniaspora spp. progressively ceded the way to S. cerevisiae, Zygosaccharomyces bailii, Candida milleri, and Brettanomyces spp. With the exception of Pichia membranaefaciens, which was shared by all communities, little overlap existed. That separation was even more manifest when species were divided into distinguishable biotypes based on morphology or physiology. It is concluded that crushing equipment and must holding tanks are the main source of significant inoculum for the fermentation process. Drosophila species appear to serve as internal vectors. Proximity to fruit trees probably contributes to maintaining a substantial Drosophila community, but the yeasts found in the distillery exhibit very little similarity to those found in adjacent vegetation. Interactions involving killer toxins had no apparent direct effects on the yeast community structure.

  15. Revaluation of Waste Yeast from Beer Production

    Directory of Open Access Journals (Sweden)

    Nicoleta Suruceanu

    2013-11-01

    Full Text Available Brewing yeast is an important waste product from beer production. The valorification of slurry yeast mainly consists of separation of vitamins and important nitrogen compounds. The hops compounds, one of the most important raw materials in beer technology are removed beforehand valorification. The prenylflavonoids compounds from hops are important bioactive compounds that can be revaluation with proper technology. Revaluation of prenylflavonoids from waste yeast into dietary supplement, identification and quantification of xanthohumol by HPLC method. Waste yeast from brewery pilot plant of USAMV Cluj Napoca it was dried by atomization and the powder was analyzed on xanthohumol content by HPLC method. For quantification a calibration curve it was used. The process of drying by atomisation lead to a powder product. It was used malt dextrin powder for stabilisation. The final product it was encapsulated. The xanthohumol content of powdered yeast it was 1.94 µg/ml. In conclusion the slurry yeast from beer production it is an important source of prenylflavonoids compounds.

  16. Flor yeast: new perspectives beyond wine ageing

    Directory of Open Access Journals (Sweden)

    Jean-luc eLegras

    2016-04-01

    Full Text Available The most important dogma in white-wine production is the preservation of the wine aroma and the limitation of the oxidative action of oxygen. In contrast, the ageing of Sherry and Sherry-like wines is an aerobic process that depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Under depletion of nitrogen and fermentable carbon sources, these yeast produce aggregates of floating cells and form an air-liquid biofilm on the wine surface, which is also known as the velum or flor. This behaviour is due to genetic and metabolic peculiarities that differentiate flor yeast from other wine yeast. This review will focus first on the most updated data obtained through the analysis of flor yeast with -omic tools. Comparative genomics, proteomics and metabolomics of flor and wine yeast strains are shedding new light on several features of these special yeast, and in particular, they have revealed the extent of proteome remodelling imposed by the biofilm life-style. Finally, new insights in terms of promotion and inhibition of biofilm formation through small molecules, amino acids and di/tri-peptides, and novel possibilities for the exploitation of biofilm immobilisation within a fungal hyphae framework, will be discussed.

  17. Evolution. Systematic humanization of yeast genes reveals conserved functions and genetic modularity.

    Science.gov (United States)

    Kachroo, Aashiq H; Laurent, Jon M; Yellman, Christopher M; Meyer, Austin G; Wilke, Claus O; Marcotte, Edward M

    2015-05-22

    To determine whether genes retain ancestral functions over a billion years of evolution and to identify principles of deep evolutionary divergence, we replaced 414 essential yeast genes with their human orthologs, assaying for complementation of lethal growth defects upon loss of the yeast genes. Nearly half (47%) of the yeast genes could be successfully humanized. Sequence similarity and expression only partly predicted replaceability. Instead, replaceability depended strongly on gene modules: Genes in the same process tended to be similarly replaceable (e.g., sterol biosynthesis) or not (e.g., DNA replication initiation). Simulations confirmed that selection for specific function can maintain replaceability despite extensive sequence divergence. Critical ancestral functions of many essential genes are thus retained in a pathway-specific manner, resilient to drift in sequences, splicing, and protein interfaces. Copyright © 2015, American Association for the Advancement of Science.

  18. Hypotonic shocks activate rat TRPV4 in Yeast in the Absence of Polyunsaturated Fatty Acids

    Science.gov (United States)

    Loukin, Stephen H.; Su, Zhenwei; Kung, Ching

    2010-01-01

    Transient-receptor-potential channels (TRPs) underlie the sensing of chemicals, heat, and mechanical force. We expressed the rat TRPV1 and TRPV4 subtypes in yeast and monitored their activities in vivo as Ca2+ rise using transgenic aequorin. Heat and capsaicin activate TRPV1 but not TRPV4 in yeast. Hypotonic shocks activate TRPV4 but not TRPV1. Osmotic swelling is modeled to activate enzyme(s), producing polyunsaturated fatty acids (PUFAs) to open TRPV4 in mammalian cells. This model relegates mechanosensitivity to the enzyme and not the channel. Yeast has only a single Δ9 fatty-acid monodesaturase and cannot make PUFAs suggesting an alternative mechanism for TRPV4 activation. We discuss possible explanations of this difference. PMID:19174160

  19. Effects of wort gravity and nitrogen level on fermentation performance of brewer's yeast and the formation of flavor volatiles.

    Science.gov (United States)

    Lei, Hongjie; Zhao, Haifeng; Yu, Zhimin; Zhao, Mouming

    2012-03-01

    Normal gravity wort and high gravity wort with different nitrogen levels were used to examine their effects on the fermentation performance of brewer's yeast and the formation of flavor volatiles. Results showed that both the wort gravity and nitrogen level had significant impacts on the growth rate, viability, flocculation, and gene expression of brewer's yeast and the levels of flavor volatiles. The sugar (glucose, maltose, and maltotriose) consumption rates and net cell growth decreased when high gravity worts were used, while these increased with increasing nitrogen level. Moreover, high gravity resulted in lower expression levels of ATF1, BAP2, BAT1, HSP12, and TDH, whereas the higher nitrogen level caused higher expression levels for these genes. Furthermore, the lower nitrogen level resulted in increases in the levels of higher alcohols and esters at high wort gravity. All these results demonstrated that yeast physiology and flavor balance during beer brewing were significantly affected by the wort gravity and nitrogen level.

  20. Identification of mouse sphingomyelin synthase 1 as a suppressor of Bax-mediated cell death in yeast.

    Science.gov (United States)

    Yang, Zhao; Khoury, Chamel; Jean-Baptiste, Gaël; Greenwood, Michael T

    2006-08-01

    We have identified mouse sphingomyelin synthase 1 as a novel suppressor of the growth inhibitory effect of heterologously expressed Bax. Yeast cells expressing sphingomyelin synthase 1 were also found to show an increased resistance to a variety of cytotoxic stimuli including hydrogen peroxide, osmotic stress and elevated temperature. Sphingomyelin synthase 1 functions by catalyzing the conversion of ceramide and phosphatidylcholine to sphingomyelin and diacylglycerol. Ceramide is an antiproliferative and proapoptotic sphingolipid whose level increases in response to a variety of stresses. Consistent with its biochemical function, yeast cells expressing sphingomyelin synthase 1 have an enhanced ability to grow in media containing the cell-permeable C2-ceramide analog as well as the ceramide precursor phytosphingosine. We also show that overexpression of AUR1, a potential yeast functional homolog of sphingomyelin synthase, also protects cells from osmotic stress. Taken together, these results suggest that sphingomyelin synthase 1 likely prevents cell death by counteracting stress-mediated accumulation of endogenous sphingolipids.

  1. Impact of pitching rate on yeast fermentation performance and beer flavour.

    Science.gov (United States)

    Verbelen, P J; Dekoninck, T M L; Saerens, S M G; Van Mulders, S E; Thevelein, J M; Delvaux, F R

    2009-02-01

    The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.

  2. Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance.

    Science.gov (United States)

    Chen, Xianzhong

    2017-03-04

    The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.

  3. Regulation of the yeast metabolic cycle by transcription factors with periodic activities

    Directory of Open Access Journals (Sweden)

    Pellegrini Matteo

    2011-10-01

    Full Text Available Abstract Background When growing budding yeast under continuous, nutrient-limited conditions, over half of yeast genes exhibit periodic expression patterns. Periodicity can also be observed in respiration, in the timing of cell division, as well as in various metabolite levels. Knowing the transcription factors involved in the yeast metabolic cycle is helpful for determining the cascade of regulatory events that cause these patterns. Results Transcription factor activities were estimated by linear regression using time series and genome-wide transcription factor binding data. Time-translation matrices were estimated using least squares and were used to model the interactions between the most significant transcription factors. The top transcription factors have functions involving respiration, cell cycle events, amino acid metabolism and glycolysis. Key regulators of transitions between phases of the yeast metabolic cycle appear to be Hap1, Hap4, Gcn4, Msn4, Swi6 and Adr1. Conclusions Analysis of the phases at which transcription factor activities peak supports previous findings suggesting that the various cellular functions occur during specific phases of the yeast metabolic cycle.

  4. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned...

  5. Purification and crystallization of the yeast translation elongation factor eEF3

    DEFF Research Database (Denmark)

    Andersen, Christian Folsted; Anand, Monika; Boesen, Thomas

    2004-01-01

    A Saccharomyces cerevisiae strain expressing full-length histidine-tagged translation elongation factor 3 (eEF3) as the only form of the protein facilitated purification of the factor for both structural and functional studies. Additionally, an identical full-length form has been successfully...... expressed in Escherichia coli and a C-terminally truncated form of histidine-tagged eEF3 has been successfully expressed in E. coli and S. cerevisiae. Both forms have been crystallized and crystals of the truncated protein expressed in yeast diffract synchrotron radiation to a maximum resolution of 2.3 A...

  6. Building Better Biosensors for Exploration into Deep-Space, Using Humanized Yeast

    Science.gov (United States)

    Liddell, Lauren; Santa Maria, Sergio; Tieze, Sofia; Bhattacharya, Sharmila

    2017-01-01

    1.BioSentinel is 1 of 13 secondary payloads hitching a ride beyond Low Earth Orbit on Exploration Mission 1 (EM-1), set to launch from NASAs Space Launch System in 2019. EM-1 is our first opportunity to investigate the effects of the deep space environment on a eukaryotic biological system, the budding yeast S. cerevisiae. Though separated by a billion years of evolution we share hundreds of genes important for basic cell function, including responses to DNA damage. Thus, yeast is an ideal biosensor for detecting typesextent of damage induced by deep-space radiation.We will fly desiccated cells, then rehydrate to wake them up when the automated payload is ready to initiate the experiment. Rehydration solution contains SC (Synthetic Complete) media and alamarBlue, an indicator for changes in growth and metabolism. Telemetry of LED readings will then allow us to detect how cells respond throughout the mission. The desiccation-rehydration process can be extremely damaging to cells, and can severely diminish our ability to accurately measure and model cellular responses to deep-space radiation. The aim of this study is to develop a better biosensor: yeast strains that are more resistant to desiccation stress. We will over-express known cellular protectants, including hydrophilin Sip18, the protein disaggregase Hsp104, and thioredoxin Trx2, a responder to oxidative stress, then measure cell viability after desiccation to determine which factors improve stress tolerance. Over-expression of SIP18 in wine yeast starter cultures was previously reported to increase viability following desiccation stress by up to 70. Thus, we expect similar improvements in our space-yeast strains. By designing better yeast biosensors we can better prepare for and mitigate the potential dangers of deep-space radiation for future missions.This work is funded by NASAs AES program.

  7. Quantification and characterization of cell wall polysaccharides released by non-Saccharomyces yeast strains during alcoholic fermentation.

    Science.gov (United States)

    Giovani, Giovanna; Rosi, Iolanda; Bertuccioli, Mario

    2012-11-15

    In order to improve knowledge about the oenological characteristics of non-Saccharomyces yeast strains, and to reconsider their contribution to wine quality, we studied the release of polysaccharides by 13 non-Saccharomyces strains of different species (three wine yeasts, six grape yeasts, and three spoilage yeasts) during alcoholic fermentation in synthetic must. Three Saccharomyces cerevisiae strains were included for comparison. All of the non-Saccharomyces strains released polysaccharides into fermentation medium; the amount released depended on the yeast species, the number of cells formed and their physiological conditions. Normalizing the quantity of macromolecules released to the cell biomass revealed that most non-Saccharomyces strains produced a greater quantity of polysaccharides compared to S. cerevisiae strains after 7 and 14days of fermentation. This capacity was particularly expressed in the studied wine spoilage yeasts (Saccharomycodes ludwigii, Zygosaccharomyces bailii, and Brettanomyces bruxellensis). Chemical characterization of exocellular polysaccharides produced by non-Saccharomyces yeasts revealed them to essentially be mannoproteins with high mannose contents, ranging from 93% for S'codes. ludwigii to 73-74% for Pichia anomala and Starmerella bombicola. Protein contents varied from 9% for P. anomala to 29% for Z. bailii. These compositions were very similar to those of the S. cerevisiae strains, and to the chemical composition of the cell wall mannoproteins of different yeast species. The presence of galactose, in addition to mannose and glucose, in the exocellular polysaccharides released by Schizosaccharomyces pombe, confirmed the parietal nature of the polysaccharides released by non-Saccharomyces yeasts; only this species has a galactomannan located in the outer layer of the cell wall. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Expression of the rgMT gene, encoding for a rice metallothionein ...

    Indian Academy of Sciences (India)

    ... 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 ...

  9. Yeast Interacting Proteins Database: YGR113W, YGL079W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available sion protein localizes to the endosome; identified as a transcriptional activator in a high-throughput yeast... a transcriptional activator in a high-throughput yeast one-hybrid assay Rows with this prey as prey Rows wi

  10. Discussion of teleomorphic and anamorphic Ascomycetous yeasts and yeast-like taxa

    Science.gov (United States)

    The relationship of ascomycetous yeasts with other members of the ascomycete fungi (Ascomycota) has been controversial for over 100 years. Because yeasts are morphologically simple, it was proposed that they represent primitive forms of ascomycetes (e.g., Guilliermond 1912). Alternatively, the ide...

  11. Boolean model of yeast apoptosis as a tool to study yeast and human apoptotic regulations.

    Science.gov (United States)

    Kazemzadeh, Laleh; Cvijovic, Marija; Petranovic, Dina

    2012-01-01

    Programmed cell death (PCD) is an essential cellular mechanism that is evolutionary conserved, mediated through various pathways and acts by integrating different stimuli. Many diseases such as neurodegenerative diseases and cancers are found to be caused by, or associated with, regulations in the cell death pathways. Yeast Saccharomyces cerevisiae, is a unicellular eukaryotic organism that shares with human cells components and pathways of the PCD and is therefore used as a model organism. Boolean modeling is becoming promising approach to capture qualitative behavior and describe essential properties of such complex networks. Here we present large literature-based and to our knowledge first Boolean model that combines pathways leading to apoptosis (a type of PCD) in yeast. Analysis of the yeast model confirmed experimental findings of anti-apoptotic role of Bir1p and pro-apoptotic role of Stm1p and revealed activation of the stress protein kinase Hog proposing the maximal level of activation upon heat stress. In addition we extended the yeast model and created an in silico humanized yeast in which human pro- and anti-apoptotic regulators Bcl-2 family and Valosin-contain protein (VCP) are included in the model. We showed that accumulation of Bax in silico humanized yeast shows apoptotic markers and that VCP is essential target of Akt Signaling. The presented Boolean model provides comprehensive description of yeast apoptosis network behavior. Extended model of humanized yeast gives new insights of how complex human disease like neurodegeneration can initially be tested.

  12. Differences between flocculating yeast and regular industrial yeast in transcription and metabolite profiling during ethanol fermentation

    Directory of Open Access Journals (Sweden)

    Lili Li

    2017-03-01

    Full Text Available Objectives: To improve ethanolic fermentation performance of self-flocculating yeast, difference between a flocculating yeast strain and a regular industrial yeast strain was analyzed by transcriptional and metabolic approaches. Results: The number of down-regulated (industrial yeast YIC10 vs. flocculating yeast GIM2.71 and up-regulated genes were 4503 and 228, respectively. It is the economic regulation for YIC10 that non-essential genes were down-regulated, and cells put more “energy” into growth and ethanol production. Hexose transport and phosphorylation were not the limiting-steps in ethanol fermentation for GIM2.71 compared to YIC10, whereas the reaction of 1,3-disphosphoglycerate to 3-phosphoglycerate, the decarboxylation of pyruvate to acetaldehyde and its subsequent reduction to ethanol were the most limiting steps. GIM2.71 had stronger stress response than non-flocculating yeast and much more carbohydrate was distributed to other bypass, such as glycerol, acetate and trehalose synthesis. Conclusions: Differences between flocculating yeast and regular industrial yeast in transcription and metabolite profiling will provide clues for improving the fermentation performance of GIM2.71.

  13. Accelerated alcoholic fermentation caused by defective gene expression related to glucose derepression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Watanabe, Daisuke; Hashimoto, Naoya; Mizuno, Megumi; Zhou, Yan; Akao, Takeshi; Shimoi, Hitoshi

    2013-01-01

    Sake yeast strains maintain high fermentation rates, even after the stationary growth phase begins. To determine the molecular mechanisms underlying this advantageous brewing property, we compared the gene expression profiles of sake and laboratory yeast strains of Saccharomyces cerevisiae during the stationary growth phase. DNA microarray analysis revealed that the sake yeast strain examined had defects in expression of the genes related to glucose derepression mediated by transcription factors Adr1p and Cat8p. Furthermore, deletion of the ADR1 and CAT8 genes slightly but statistically significantly improved the fermentation rate of a laboratory yeast strain. We also identified two loss-of-function mutations in the ADR1 gene of existing sake yeast strains. Taken together, these results indicate that the gene expression program associated with glucose derepression for yeast acts as an impediment to effective alcoholic fermentation under glucose-rich fermentative conditions.

  14. Human COQ9 Rescues a coq9 Yeast Mutant by Enhancing Coenzyme Q Biosynthesis from 4-Hydroxybenzoic Acid and Stabilizing the CoQ-Synthome

    Directory of Open Access Journals (Sweden)

    Cuiwen H. He

    2017-07-01

    Full Text Available Coq9 is required for the stability of a mitochondrial multi-subunit complex, termed the CoQ-synthome, and the deamination step of Q intermediates that derive from para-aminobenzoic acid (pABA in yeast. In human, mutations in the COQ9 gene cause neonatal-onset primary Q10 deficiency. In this study, we determined whether expression of human COQ9 could complement yeast coq9 point or null mutants. We found that expression of human COQ9 rescues the growth of the temperature-sensitive yeast mutant, coq9-ts19, on a non-fermentable carbon source and increases the content of Q6, by enhancing Q biosynthesis from 4-hydroxybenzoic acid (4HB. To study the mechanism for the rescue by human COQ9, we determined the steady-state levels of yeast Coq polypeptides in the mitochondria of the temperature-sensitive yeast coq9 mutant expressing human COQ9. We show that the expression of human COQ9 significantly increased steady-state levels of yeast Coq4, Coq6, Coq7, and Coq9 at permissive temperature. Human COQ9 polypeptide levels persisted at non-permissive temperature. A small amount of the human COQ9 co-purified with tagged Coq6, Coq6-CNAP, indicating that human COQ9 interacts with the yeast Q-biosynthetic complex. These findings suggest that human COQ9 rescues the yeast coq9 temperature-sensitive mutant by stabilizing the CoQ-synthome and increasing Q biosynthesis from 4HB. This finding provides a powerful approach to studying the function of human COQ9 using yeast as a model.

  15. New yeast/E. coli/Drosophila triple shuttle vectors for efficient generation of Drosophila P element transformation constructs.

    Science.gov (United States)

    Paululat, Achim; Heinisch, Jürgen J

    2012-12-15

    We have generated a set of novel triple shuttle vectors that facilitate the construction of Drosophila-P-element transformations vectors. These YED-vectors allow the insertion of any kind of sequence at any chosen position due to the presence of a yeast casette which ensures replication and allows for homologous recombination in Saccharomyces cerevisiae. As a proof of principle we generated several reporter constructs and tested them in transgenic flies for expression and correct subcellular localization. YED-vectors can be used for many purposes including promoter analysis or the expression of tagged or truncated proteins. Thus, time-consuming conventional restriction site based multi-step cloning procedures can be circumvented by using the new YED-vectors. The new set of triple shuttle vectors will be highly beneficial for the rapid construction of complex Drosophila transformation plasmids. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...... fermentation processes in which the escape of genetically modified cells would be considered highly risky....

  17. Transcriptional regulation and the diversification of metabolism in wine yeast strains.

    Science.gov (United States)

    Rossouw, Debra; Jacobson, Dan; Bauer, Florian F

    2012-01-01

    Transcription factors and their binding sites have been proposed as primary targets of evolutionary adaptation because changes to single transcription factors can lead to far-reaching changes in gene expression patterns. Nevertheless, there is very little concrete evidence for such evolutionary changes. Industrial wine yeast strains, of the species Saccharomyces cerevisiae, are a geno- and phenotypically diverse group of organisms that have adapted to the ecological niches of industrial winemaking environments and have been selected to produce specific styles of wine. Variation in transcriptional regulation among wine yeast strains may be responsible for many of the observed differences and specific adaptations to different fermentative conditions in the context of commercial winemaking. We analyzed gene expression profiles of wine yeast strains to assess the impact of transcription factor expression on metabolic networks. The data provide new insights into the molecular basis of variations in gene expression in industrial strains and their consequent effects on metabolic networks important to wine fermentation. We show that the metabolic phenotype of a strain can be shifted in a relatively predictable manner by changing expression levels of individual transcription factors, opening opportunities to modify transcription networks to achieve desirable outcomes.

  18. Characterization of two genes encoding metal tolerance proteins from Beta vulgaris subspecies maritima that confers manganese tolerance in yeast

    DEFF Research Database (Denmark)

    Erbasol, Isil; Bozdag, Gonensin Ozan; Koc, Ahmet

    2013-01-01

    of excess metal ions. In this study, we identified and characterized two MTP genes from Beta vulgaris spp. maritima (B. v. ssp. maritima). Overexpression of these two genes provided Mn tolerance in yeast cells. Sequence analyses displayed BmMTP10 and BmMTP11as members of the Mn-CDF family. Functional...... analyses of these proteins indicated that they are specific to Mn with a role in reducing excess cellular Mn levels when expressed in yeast. GFP-fusion constructs of both proteins localized to the Golgi apparatus as a punctuated pattern. Finally, Q-RT-PCR results showed that BmMTP10 expression was induced...

  19. Improved vanillin production in baker's yeast through in silico design

    Directory of Open Access Journals (Sweden)

    Hansen Jørgen

    2010-11-01

    Full Text Available Abstract Background Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis. Results Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1 were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast. Conclusion Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories.

  20. Influence of pesticides on yeasts colonizing leaves.

    Science.gov (United States)

    Vadkertiová, Renata; Sláviková, Elena

    2011-01-01

    The effect of nine different pesticides on the growth of yeasts isolated from the leaves of fruit and forest trees was investigated. Four insecticides (with the active ingredients: thiacloprid, deltamethrin, lambdacyhalothrin, and thiamethoxam) and five fungicides (with the effective substances: bitertanol, kresoxim-methyl, mancozeb, trifloxystrobin, and cupric oxychloride) were tested. The concentrations of chemicals were those recommended by the manufacturers for the spraying of trees. The yeast strains isolated from the leaves of fruit trees were not sensitive to any of the insecticides. The majority of yeast strains isolated from the leaves of forest trees were either not sensitive or only to a small extent. While Rhodotorula mucilaginosa and Pichia anomala were not affected by any insecticide, the strains of Cryptococcus laurentii and Rhodotorula glutinis showed the highest sensitivity. The effects of fungicides on the growth of isolated yeasts were more substantial. The fungicide Dithane DG (mancozeb) completely inhibited the growth of all yeasts. All strains isolated from fruit tree leaves were more resistant to the tested fungicides than those isolated from the leaves of forest trees. The most resistant strains from the leaves of fruit trees belonged to the species Metschnikowia pulcherrima, Pichia anomala, and Saccharomyces cerevisiae, whereas Cryptococcus albidus and C. laurentii, originating from the leaves of forest trees, showed the highest sensitivity to fungicides.

  1. Extension of yeast chronological lifespan by methylamine.

    Directory of Open Access Journals (Sweden)

    Sanjeev Kumar

    Full Text Available BACKGROUND: Chronological aging of yeast cells is commonly used as a model for aging of human post-mitotic cells. The yeast Saccharomyces cerevisiae grown on glucose in the presence of ammonium sulphate is mainly used in yeast aging research. We have analyzed chronological aging of the yeast Hansenula polymorpha grown at conditions that require primary peroxisome metabolism for growth. METHODOLOGY/PRINCIPAL FINDINGS: The chronological lifespan of H. polymorpha is strongly enhanced when cells are grown on methanol or ethanol, metabolized by peroxisome enzymes, relative to growth on glucose that does not require peroxisomes. The short lifespan of H. polymorpha on glucose is mainly due to medium acidification, whereas most likely ROS do not play an important role. Growth of cells on methanol/methylamine instead of methanol/ammonium sulphate resulted in further lifespan enhancement. This was unrelated to medium acidification. We show that oxidation of methylamine by peroxisomal amine oxidase at carbon starvation conditions is responsible for lifespan extension. The methylamine oxidation product formaldehyde is further oxidized resulting in NADH generation, which contributes to increased ATP generation and reduction of ROS levels in the stationary phase. CONCLUSION/SIGNIFICANCE: We conclude that primary peroxisome metabolism enhanced chronological lifespan of H. polymorpha. Moreover, the possibility to generate NADH at carbon starvation conditions by an organic nitrogen source supports further extension of the lifespan of the cell. Consequently, the interpretation of CLS analyses in yeast should include possible effects on the energy status of the cell.

  2. Molecular basis of fructose utilization by the wine yeast Saccharomyces cerevisiae: a mutated HXT3 allele enhances fructose fermentation.

    Science.gov (United States)

    Guillaume, Carole; Delobel, Pierre; Sablayrolles, Jean-Marie; Blondin, Bruno

    2007-04-01

    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 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Delta strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

  3. MFS transportome of the human pathogenic yeast Candida albicans.

    Science.gov (United States)

    Gaur, Manisha; Puri, Nidhi; Manoharlal, Raman; Rai, Versha; Mukhopadhayay, Gauranga; Choudhury, Devapriya; Prasad, Rajendra

    2008-12-03

    The major facilitator superfamily (MFS) is one of the two largest superfamilies of membrane transporters present ubiquitously in bacteria, archaea, and eukarya and includes members that function as uniporters, symporters or antiporters. We report here the complete transportome of MFS proteins of a human pathogenic yeast Candida albicans. Computational analysis of C. albicans genome enabled us to identify 95 potential MFS proteins which clustered into 17 families using Saier's Transport Commission (TC) system. Among these SP, DHA1, DHA2 and ACS represented major families consisting of 22, 22, 9 and 16 members, respectively. Family designations in C. albicans were validated by subjecting Saccharomyces cerevisiae genome to TC system. Based on the published available genomics/proteomics data, 87 of the putative MFS genes of C. albicans were found to express either at mRNA or protein levels. We checked the expression of the remaining 8 genes by using RT-PCR and observed that they are not expressed under basal growth conditions implying that either these 8 genes are expressed under specific growth conditions or they may be candidates for pseudogenes. The in silico characterisation of MFS transporters in Candida albicans genome revealed a large complement of MFS transporters with most of them showing expression. Considering the clinical relevance of C. albicans and role of MFS members in antifungal resistance and nutrient transport, this analysis would pave way for identifying their physiological relevance.

  4. A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis.

    Science.gov (United States)

    Rani, Sapa Hima; Saha, Saikat; Rajasekharan, Ram

    2013-01-01

    The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.

  5. Signaling pathways and posttranslational modifications of tau in Alzheimer's disease: the humanization of yeast cells.

    Science.gov (United States)

    Heinisch, Jürgen J; Brandt, Roland

    2016-03-25

    In the past decade, yeast have been frequently employed to study the molecular mechanisms of human neurodegenerative diseases, generally by means of heterologous expression of genes encoding the relevant hallmark proteins. However, it has become evident that substantial posttranslational modifications of many of these proteins are required for the development and progression of potentially disease relevant changes. This is exemplified by the neuronal tau proteins, which are critically involved in a class of neuro-degenerative diseases collectively called tauopathies and which includes Alz-heimer's disease (AD) as its most common representative. In the course of the disease, tau changes its phosphorylation state and becomes hyperphosphory-lated, gets truncated by proteolytic cleavage, is subject to O-glycosylation, sumoylation, ubiquitinylation, acetylation and some other modifications. This poses the important question, which of these posttranslational modifications are naturally occurring in the yeast model or can be reconstituted by heterol-ogous gene expression. Here, we present an overview on common modifica-tions as they occur in tau during AD, summarize their potential relevance with respect to disease mechanisms and refer to the native yeast enzyme orthologs capable to perform these modifications. We will also discuss potential approaches to humanize yeast in order to create modification patterns resembling the situation in mammalian cells, which could enhance the value of Saccharomyces cerevisiae and Kluyveromyces lactis as disease models.

  6. NY-ESO-1 protein glycosylated by yeast induces enhanced immune responses.

    Science.gov (United States)

    Wadle, Andreas; Mischo, Axel; Strahl, Sabine; Nishikawa, Hiroyoshi; Held, Gerhard; Neumann, Frank; Wullner, Beate; Fischer, Eliane; Kleber, Sascha; Karbach, Julia; Jager, Elke; Shiku, Hiroshi; Odunsi, Kunle; Shrikant, Protul A; Knuth, Alexander; Cerundolo, Vincenzo; Renner, Christoph

    2010-11-01

    Vaccine strategies that target dendritic cells to elicit potent cellular immunity are the subject of intense research. Here we report that the genetically engineered yeast Saccharomyces cerevisiae, expressing the full-length tumour-associated antigen NY-ESO-1, is a versatile host for protein production. Exposing dendritic cells (DCs) to soluble NY-ESO-1 protein linked to the yeast a-agglutinin 2 protein (Aga2p) protein resulted in protein uptake, processing and MHC class I cross-presentation of NY-ESO-1-derived peptides. The process of antigen uptake and cross-presentation was dependent on the glycosylation pattern of NY-ESO-1-Aga2p protein and the presence of accessible mannose receptors. In addition, NY-ESO-1-Aga2p protein uptake by dendritic cells resulted in recognition by HLA-DP4 NY-ESO-1-specific CD4(+) T cells, indicating MHC class II presentation. Finally, vaccination of mice with yeast-derived NY-ESO-1-Aga2p protein led to an enhanced humoral and cellular immune response, when compared to the bacterially expressed NY-ESO-1 protein. Together, these data demonstrate that yeast-derived full-length NY-ESO-1-Aga2p protein is processed and presented efficiently by MHC class I and II complexes and warrants clinical trials to determine the potential value of S. cerevisiae as a host for cancer vaccine development. Copyright © 2010 John Wiley & Sons, Ltd.

  7. Acetyltransferase SAS2 and sirtuin SIR2, respectively, control flocculation and biofilm formation in wine yeast.

    Science.gov (United States)

    Rodriguez, María E; Orozco, Helena; Cantoral, Jesús M; Matallana, Emilia; Aranda, Agustín

    2014-09-01

    Cell-to-cell and cell-to-environment interactions of microorganisms are of substantial relevance for their biotechnological use. In the yeast Saccharomyces cerevisiae, flocculation can be an advantage to clarify final liquid products after fermentation, and biofilm formation may be relevant for the encapsulation of strains of interest. The adhesion properties of wine yeast strains can be modified by the genetic manipulation of transcriptional regulatory proteins, such as histone deacetylases, and acetylases. Sirtuin SIR2 is essential for the formation of mat structures, a kind of biofilm that requires the expression of cell-wall protein FLO11 as its deletion reduces FLO11 expression, and adhesion of cells to themselves and to agar in a commercial wine strain. Deletion of acetyltransferase GCN5 leads to a similar phenotype. A naturally flocculant wine yeast strain called P2 was characterized. Its flocculation happens only during grape juice fermentation and is due to the presence of a highly transcribed version of flocculin FLO5, linked to the presence of a δ sequence in the promoter. Deletion of acetyltransferase SAS2 enhances this phenotype and maltose fermentation even more. Therefore, the manipulation of acetylation/deacetylation machinery members is a valid way to alter the interaction of industrial yeast to their environment. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  8. Expression and Purification of Soluble, Biologically Active ...

    African Journals Online (AJOL)

    Purpose: To investigate Pichia pastoris expression system for producing clinically usable, high-quality dipeptidyl peptidase 4 recombinant protein. Methods: The yeast, Pichia pastoris, expression system was used for the production of the human recombinant dipeptidyl peptidase 4 as a secreted form. The full-length human ...

  9. Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids

    Directory of Open Access Journals (Sweden)

    Johnson Christopher B

    2011-01-01

    Full Text Available Abstract Background Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. Results S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330 in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1 also improved production levels, pointing to an additional means to improve production. Conclusions Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.

  10. Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids

    Science.gov (United States)

    2011-01-01

    Background Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. Results S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production. Conclusions Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields. PMID:21276210

  11. Dissecting the fission yeast regulatory network reveals phase-specific control elements of its cell cycle

    Directory of Open Access Journals (Sweden)

    Liu Liwen

    2009-09-01

    Full Text Available Abstract Background Fission yeast Schizosaccharomyces pombe and budding yeast Saccharomyces cerevisiae are among the original model organisms in the study of the cell-division cycle. Unlike budding yeast, no large-scale regulatory network has been constructed for fission yeast. It has only been partially characterized. As a result, important regulatory cascades in budding yeast have no known or complete counterpart in fission yeast. Results By integrating genome-wide data from multiple time course cell cycle microarray experiments we reconstructed a gene regulatory network. Based on the network, we discovered in addition to previously known regulatory hubs in M phase, a new putative regulatory hub in the form of the HMG box transcription factor SPBC19G7.04. Further, we inferred periodic activities of several less known transcription factors over the course of the cell cycle, identified over 500 putative regulatory targets and detected many new phase-specific and conserved cis-regulatory motifs. In particular, we show that SPBC19G7.04 has highly significant periodic activity that peaks in early M phase, which is coordinated with the late G2 activity of the forkhead transcription factor fkh2. Finally, using an enhanced Bayesian algorithm to co-cluster the expression data, we obtained 31 clusters of co-regulated genes 1 which constitute regulatory modules from different phases of the cell cycle, 2 whose phase order is coherent across the 10 time course experiments, and 3 which lead to identification of phase-specific control elements at both the transcriptional and post-transcriptional levels in S. pombe. In particular, the ribosome biogenesis clusters expressed in G2 phase reveal new, highly conserved RNA motifs. Conclusion Using a systems-level analysis of the phase-specific nature of the S. pombe cell cycle gene regulation, we have provided new testable evidence for post-transcriptional regulation in the G2 phase of the fission yeast cell cycle

  12. Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal); Monti, Paola; Fronza, Gilberto [Mutagenesis Unit, Istituto di Ricerca e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa (Italy); Pereira, Clara [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal); Saraiva, Lucília, E-mail: lucilia.saraiva@ff.up.pt [REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira n. 164, 4050-313 Porto (Portugal)

    2015-01-01

    In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.

  13. Yeast culture supplement during nursing and transport affects immunity and intestinal microbial ecology of weanling pigs.

    Science.gov (United States)

    Weedman, S M; Rostagno, M H; Patterson, J A; Yoon, I; Fitzner, G; Eicher, S D

    2011-06-01

    The objectives of this study were to determine the influence of a Saccharomyces cerevisiae fermentation product on innate immunity and intestinal microbial ecology after weaning and transport stress. In a randomized complete block design, before weaning and in a split-plot analysis of a 2 × 2 factorial arrangement of yeast culture (YY) and transport (TT) after weaning, 3-d-old pigs (n = 108) were randomly assigned within litter (block) to either a control (NY, milk only) or yeast culture diet (YY; delivered in milk to provide 0.1 g of yeast culture product/kg of BW) from d 4 to 21. At weaning (d 21), randomly, one-half of the NY and YY pigs were assigned to a 6-h transport (NY-TT and YY-TT) before being moved to nursery housing, and the other one-half were moved directly to nursery housing (NY-NT and YY-NT, where NT is no transport). The yeast treatment was a 0.2% S. cerevisiae fermentation product and the control treatment was a 0.2% grain blank in feed for 2 wk. On d 1 before transport and on d 1, 4, 7, and 14 after transport, blood was collected for leukocyte assays, and mesenteric lymph node, jejunal, and ileal tissue, and jejunal, ileal, and cecal contents were collected for Toll-like receptor expression (TLR); enumeration of Escherichia coli, total coliforms, and lactobacilli; detection of Salmonella; and microbial analysis. After weaning, a yeast × transport interaction for ADG was seen (P = 0.05). Transport affected (P = 0.09) ADFI after weaning. Yeast treatment decreased hematocrit (P = 0.04). A yeast × transport interaction was found for counts of white blood cells (P = 0.01) and neutrophils (P = 0.02) and for the neutrophil-to-lymphocyte ratio (P = 0.02). Monocyte counts revealed a transport (P = 0.01) effect. Interactions of yeast × transport (P = 0.001) and yeast × transport × day (P = 0.09) for TLR2 and yeast × transport (P = 0.08) for TLR4 expression in the mesenteric lymph node were detected. Day affected lactobacilli, total coliform, and E

  14. Cell polarization in budding and fission yeasts.

    Science.gov (United States)

    Martin, Sophie G; Arkowitz, Robert A

    2014-03-01

    Polarization is a fundamental cellular property, which is essential for the function of numerous cell types. Over the past three to four decades, research using the best-established yeast systems in cell biological research, Saccharomyces cerevisiae (or budding yeast) and Schizosaccharomyces pombe (or fission yeast), has brought to light fundamental principles governing the establishment and maintenance of a polarized, asymmetric state. These two organisms, though both ascomycetes, are evolutionarily very distant and exhibit distinct shapes and modes of growth. In this review, we compare and contrast the two systems. We first highlight common cell polarization pathways, detailing the contribution of Rho GTPases, the cytoskeleton, membrane trafficking, lipids, and protein scaffolds. We then contrast the major differences between the two organisms, describing their distinct strategies in growth site selection and growth zone dimensions and compartmentalization, which may be the basis for their distinct shapes. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  15. Yeast interactions in inoculated wine fermentation

    Directory of Open Access Journals (Sweden)

    Maurizio eCiani

    2016-04-01

    Full Text Available The use of selected starter culture is widely diffused in winemaking. In pure fermentation, the ability of inoculated Saccharomyces cerevisiae to suppress the wild microflora is one of the most important feature determining the starter ability to dominate the process. Since the wine is the result of the interaction of several yeast species and strains, many studies are available on the effect of mixed cultures on the final wine quality. In mixed fermentation the interactions between the different yeasts composing the starter culture can led the stability of the final product and the analytical and aromatic profile. In the present review, we will discuss the recent developments regarding yeast interactions in pure and in mixed fermentation, focusing on the influence of interactions on growth and dominance in the process.

  16. [Molecular taxonomy techniques used for yeast identification].

    Science.gov (United States)

    Ghindea, Raluca; Csutak, Ortansa; Stoica, Ileana; Ionescu, Robertina; Soare, Simona; Pelinescu, Diana; Nohit, Ana-Maria; Creangă, Oana; Vassu, Tatiana

    2004-01-01

    Due to the major impact of yeasts in human life based on the existence of pathogen yeast species and of species with biotechnological abilities, in the last few years new molecular techniques are performed for an accurate identification of natural isolates. Our study is aimed to review some of these techniques such as electrokariotyping by PFGE (Pulsed Field Gel Electrophoresis), estimation of the molar percentage of guanine and cytosine, the applications of PCR reaction in yeast identification using RAPD (Random amplified polymorphic DNA), UP-PCR (Universally Primed Polymerase Chain Reaction), MLST (Multilocus sequence typing) techniques, mtDNA and rDNA homology studies. Such molecular techniques complete the phenotypical characterization based on classical taxonomical tests allowing thus the polyphasic identification of the microorganisms.

  17. Bioadsorption strategies with yeast molecular display technology.

    Science.gov (United States)

    Shibasaki, Seiji; Ueda, Mitsuyoshi

    2014-01-01

    Molecular display techniques using microbial cell surfaces have been widely developed in the past twenty years, and are useful tools as whole cell catalysts for various applications such as bioconversion, bioremediation, biosensing, and the screening system of protein libraries. Furthermore, different types of microbial cells among eukaryotic and prokaryotic strains have been investigated for their use in surface display technologies. Recently, several kinds of protein-displaying yeasts have been utilized as bioadsorbents in this platform technology. In particular, these trials have successfully expanded the possibility of applications to metal binding, affinity purification, and receptor-ligand interaction by using the yeast cell surface. In this mini review, we describe the general principles of molecular display technology using yeast cells and its applications, with a particular focus on bioadsorption.

  18. Probiotic Properties of Non-Saccharomyces Yeasts

    DEFF Research Database (Denmark)

    Smith, Ida Mosbech

    to harmless luminal substances is a key feature of the intestinal immune system. In this context, dendritic cells (DCs) present in the tissues lining the human gut are central players involved in microbial sensing and shaping of appropriate adaptive immune responses. Probiotics are live microorganisms which...... when administered in adequate amounts confer a health benefit on the host. While the majority of probiotic microorganisms studied to date are lactic acid bacteria, research in yeasts with potentially beneficial influences on human health has mainly revolved around Saccharomyces boulardii. This yeast...... has shown a positive impact on disease outcome in clinical studies of inflammatory bowel disease, indicating an ability of S. boulardii to influence human immune responses underlying intestinal inflammation. Consequent to this focus on S. boulardii as the fundamental probiotic yeast, very little...

  19. Yeasts and yeast-like organisms associated with fruits and blossoms of different fruit trees.

    Science.gov (United States)

    Vadkertiová, Renáta; Molnárová, Jana; Vránová, Dana; Sláviková, Elena

    2012-12-01

    Yeasts are common inhabitants of the phyllosphere, but our knowledge of their diversity in various plant organs is still limited. This study focused on the diversity of yeasts and yeast-like organisms associated with matured fruits and fully open blossoms of apple, plum, and pear trees, during 2 consecutive years at 3 localities in southwest Slovakia. The occurrence of yeasts and yeast-like organisms in fruit samples was 2½ times higher and the yeast community more diverse than that in blossom samples. Only 2 species (Aureobasidium pullulans and Metschnikowia pulcherrima) occurred regularly in the blossom samples, whereas Galactomyces candidus, Hanseniaspora guilliermondii, Hanseniaspora uvarum, M. pulcherrima, Pichia kluyveri, Pichia kudriavzevii, and Saccharomyces cerevisiae were the most frequently isolated species from the fruit samples. The ratio of the number of samples where only individual species were present to the number of samples where 2 or more species were found (consortium) was counted. The occurrence of individual species in comparison with consortia was much higher in blossom samples than in fruit samples. In the latter, consortia predominated. Aureobasidium pullulans, M. pulcherrima, and S. cerevisiae, isolated from both the fruits and blossoms, can be considered as resident yeast species of various fruit tree species cultivated in southwest Slovakia localities.

  20. Cold tolerance of potato plants transformed with yeast invertase gene

    Directory of Open Access Journals (Sweden)

    Alexander N. Deryaabin

    2013-12-01

    Full Text Available Our study was carried out with potato plants (Solanum tuberosun L.,cv. Désirée transformed with the yeast invertase gene under the control of the B33 class I patatin promoter and with the proteinase inhibitor II leader peptide sequence providing for the apoplastic enzyme localization (B33-inv plants and with the plants transformed with the reporter gene encoding bb-glucuronidase under the control of the 35S CaMV promoter (control plants. Exposure to 5°C during 6 days caused an increase in invertase activity and sugar content in B33-inv leaves in comparison with the control plants. Cell membranes of B33-inv plant cells showed greater cold tolerance under low temperature conditions than control plants that was recorded by electrolyte release. We supposed that higher cold tolerance of B33-inv plants was caused by stabilizing effect of sugar on the membranes, because B33-inv plants differ from the control plants in higher invertase activity, induced by expression of yeast invertase gene, and high content of sugars.

  1. Nonselective enrichment for yeast adenine mutants by flow cytometry

    Science.gov (United States)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  2. Stochastic Polynomial Dynamic Models of the Yeast Cell Cycle

    Science.gov (United States)

    Mitra, Indranil; Dimitrova, Elena; Jarrah, Abdul S.

    2010-03-01

    In the last decade a new holistic approach for tackling biological problems, systems biology, which takes into account the study of the interactions between the components of a biological system to predict function and behavior has emerged. The reverse-engineering of biochemical networks from experimental data have increasingly become important in systems biology. Based on Boolean networks, we propose a time-discrete stochastic framework for the reverse engineering of the yeast cell cycle regulatory network from experimental data. With a suitable choice of state set, we have used powerful tools from computational algebra, that underlie the reverse-engineering algorithm, avoiding costly enumeration strategies. Stochasticity is introduced by choosing at each update step a random coordinate function for each variable, chosen from a probability space of update functions. The algorithm is based on a combinatorial structure known as the Gr"obner fans of a polynomial ideal which identifies the underlying network structure and dynamics. The model depicts a correct dynamics of the yeast cell cycle network and reproduces the time sequence of expression patterns along the biological cell cycle. Our findings indicate that the methodolgy has high chance of success when applied to large and complex systems to determine the dynamical properties of corresponding networks.

  3. Novel Wine Yeast for Improved Utilisation of Proline during Fermentation

    Directory of Open Access Journals (Sweden)

    Danfeng Long

    2018-02-01

    Full Text Available Proline is the predominant amino acid in grape juice, but it is poorly assimilated by wine yeast under the anaerobic conditions typical of most fermentations. Exploiting the abundance of this naturally occurring nitrogen source to overcome the need for nitrogen supplementation and/or the risk of stuck or sluggish fermentations would be most beneficial. This study describes the isolation and evaluation of a novel wine yeast isolate, Q7, obtained through ethyl methanesulfonate (EMS mutagenesis. The utilisation of proline by the EMS isolate was markedly higher than by the QA23 wild type strain, with approximately 700 and 300 mg/L more consumed under aerobic and self-anaerobic fermentation conditions, respectively, in the presence of preferred nitrogen sources. Higher intracellular proline contents in the wild type strain implied a lesser rate of proline catabolism or incorporation by this strain, but with higher cell viability after freezing treatment. The expression of key genes (PUT1, PUT2, PUT3, PUT4, GAP1 and URE2 involved in proline degradation, transport and repression were compared between the parent strain and the isolate, revealing key differences. The application of these strains for efficient conduct for nitrogen-limited fermentations is a possibility.

  4. Osmotic stress response in the wine yeast Dekkera bruxellensis.

    Science.gov (United States)

    Galafassi, Silvia; Toscano, Marco; Vigentini, Ileana; Piškur, Jure; Compagno, Concetta

    2013-12-01

    Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response. Gene Accession numbers in GenBank: DbHOG1: JX65361, DbSTL1: JX965362. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. A CTG Clade Candida Yeast Genetically Engineered for the Genotype-Phenotype Characterization of Azole Antifungal Resistance in Human-Pathogenic Yeasts.

    Science.gov (United States)

    Accoceberry, Isabelle; Rougeron, Amandine; Biteau, Nicolas; Chevrel, Pauline; Fitton-Ouhabi, Valérie; Noël, Thierry

    2018-01-01

    A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase ERG11 mutations on azole resistance. Candida lusitaniae was chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of the Candida species pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at the ERG11 locus by the reconstitution of a functional auxotrophic marker in the 3' intergenic region of ERG11 Homologous and heterologous ERG11 alleles are expressed from the resident ERG11 promoter of C. lusitaniae , allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in C. lusitaniae different ERG11 alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, C. albicans and Kluyveromyces marxianus Candida lusitaniae constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the ERG11 donor strain. This work led us to characterize the phenotypic effect of two kinds of mutation: mutation conferring decreased fluconazole susceptibility in a species-specific manner and mutation conferring fluconazole resistance in several yeast species. In particular, a missense mutation affecting amino acid K143 of Erg11p in Candida species, and the equivalent position K151 in K. marxianus , plays a critical role in fluconazole resistance. Copyright © 2017 American Society for Microbiology.

  6. ISOLATION OF PROTEOLYTIC PSYCHROTROPHIC YEASTS FROM FRESH RAW SEAFOODS

    NARCIS (Netherlands)

    KOBATAKE, M; KREGERVANRIJ, NJW; PLACIDO, MTLC; VANUDEN, N

    A total of 103 cultures of yeasts were isolated from seven kinds of fresh raw seafoods. The isolates comprised six genera, Candida, Cryptococcus, Debaryomyces. Rhodotorula, Sterigmatomyces and Trichosporon, and included 21 different species. All the isolates were psychrotrophic yeasts. Proteolytic

  7. A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation.

    Science.gov (United States)

    Duc, Camille; Pradal, Martine; Sanchez, Isabelle; Noble, Jessica; Tesnière, Catherine; Blondin, Bruno

    2017-01-01

    Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid) in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation.

  8. A set of nutrient limitations trigger yeast cell death in a nitrogen-dependent manner during wine alcoholic fermentation.

    Directory of Open Access Journals (Sweden)

    Camille Duc

    Full Text Available Yeast cell death can occur during wine alcoholic fermentation. It is generally considered to result from ethanol stress that impacts membrane integrity. This cell death mainly occurs when grape musts processing reduces lipid availability, resulting in weaker membrane resistance to ethanol. However the mechanisms underlying cell death in these conditions remain unclear. We examined cell death occurrence considering yeast cells ability to elicit an appropriate response to a given nutrient limitation and thus survive starvation. We show here that a set of micronutrients (oleic acid, ergosterol, pantothenic acid and nicotinic acid in low, growth-restricting concentrations trigger cell death in alcoholic fermentation when nitrogen level is high. We provide evidence that nitrogen signaling is involved in cell death and that either SCH9 deletion or Tor inhibition prevent cell death in several types of micronutrient limitation. Under such limitations, yeast cells fail to acquire any stress resistance and are unable to store glycogen. Unexpectedly, transcriptome analyses did not reveal any major changes in stress genes expression, suggesting that post-transcriptional events critical for stress response were not triggered by micronutrient starvation. Our data point to the fact that yeast cell death results from yeast inability to trigger an appropriate stress response under some conditions of nutrient limitations most likely not encountered by yeast in the wild. Our conclusions provide a novel frame for considering both cell death and the management of nutrients during alcoholic fermentation.

  9. Decolorization of a recalcitrant organic compound (Melanoidin by a novel thermotolerant yeast, Candida tropicalis RG-9

    Directory of Open Access Journals (Sweden)

    Tiwari Soni

    2012-06-01

    Full Text Available Abstract Background Sugarcane distilleries use molasses for ethanol production and generate large volume of effluent containing high biological oxygen demand (BOD and chemical oxygen demand (COD along with melanoidin pigment. Melanoidin is a recalcitrant compound that causes several toxic effects on living system, therefore, may be treated before disposal. The aim of this study was to isolate a potential thermotolerant melanoidin decolorizing yeast from natural resources, and optimized different physico-chemical and nutritional parameters. Results Total 24 yeasts were isolated from the soil samples of near by distillery site, in which isolate Y-9 showed maximum decolorization and identified as Candida tropicalis by Microbial Type Culture Collection (MTCC Chandigarh, India. The decolorization yield was expressed as the decrease in the absorbance at 475 nm against initial absorbance at the same wavelength. Uninoculated medium served as control. Yeast showed maximum decolorization (75% at 45°C using 0.2%, glucose; 0.2%, peptone; 0.05%, MgSO4; 0.01%, KH2PO4; pH-5.5 within 24 h of incubation under static condition. Decolorizing ability of yeast was also confirmed by high performance liquid chromatography (HPLC analysis. Conclusion The yeast strain efficiently decolorized melanoidin pigment of distillery effluent at higher temperature than the other earlier reported strains of yeast, therefore, this strain could also be used at industrial level for melanoidin decolorization as it tolerated a wide range of temperature and pH with very small amount of carbon and nitrogen sources.

  10. Fermentation performance of lager yeast in high gravity beer fermentations with different sugar supplementations.

    Science.gov (United States)

    Lei, Hongjie; Xu, Huaide; Feng, Li; Yu, Zhimin; Zhao, Haifeng; Zhao, Mouming

    2016-11-01

    The effects of glucose, sucrose and maltose supplementations on the fermentation performance and stress tolerance of lager yeast (Saccharomyces pastorianus) during high gravity (18°P) and very high gravity (24°P) fermentations were studied. Results showed that throughout 18°P wort fermentation, fermentation performance of lager yeast was significantly improved by glucose or sucrose supplementation, compared with maltose supplementation, especially for sucrose supplementation increasing wort fermentability and ethanol production by 6% and 8%, respectively. However, in the later stage of 24°P wort fermentation, fermentation performance of lager yeast was dramatically improved by maltose supplementation, which increased wort fermentability and ethanol production by 14% and 10%, respectively, compared with sucrose supplementation. Furthermore, higher HSP12 expression level and more intracellular trehalose accumulation in yeast cells were observed by maltose supplementation with increase of the wort gravity from 18°P to 24°P, indicating higher stress response of yeast cells. The excretion of Gly and Ala, and the absorption of Pro in the later stage of fermentation were promoted by maltose supplementation. In addition, with increase of the wort gravity from 18°P to 24°P, higher alcohols level was decreased with maltose supplementation, while esters formation was increased significantly with glucose supplementation. This study suggested that the choice of optimal fermentable sugars maintaining better fermentation performance of lager yeast should be based on not only strain specificity, but also wort gravity. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Metabolic regulation rather than de novo enzyme synthesis dominates the osmo-adaptation of yeast.

    Science.gov (United States)

    Bouwman, Jildau; Kiewiet, José; Lindenbergh, Alexander; van Eunen, Karen; Siderius, Marco; Bakker, Barbara M

    2011-01-01

    Intracellular accumulation of glycerol is essential for yeast cells to survive hyperosmotic stress. Upon hyperosmotic stress the gene expression of enzymes in the glycerol pathway is strongly induced. Recently, however, it was shown that this gene-expression response is not essential for survival of an osmotic shock [Mettetal JT et al. (2008) Science 319: 482–484 and Westfall PJ et al. (2008) Proc Natl Acad Sci 105: 12212–12217]. Instead, pure metabolic adaptation can rescue the yeast. The existence of two alternative mechanisms urged the question which of these mechanisms dominates time-dependent adaptation of wild-type yeast to osmotic stress under physiological conditions. The regulation of the glycerol pathway was analysed in aerobic, glucose-limited cultures upon addition of 1 M of sorbitol, leading to a hyperosmotic shock. In agreement with earlier studies, the mRNA levels of the glycerol-producing enzymes as well as their catalytic capacities increased. Qualitatively this induction followed a similar time course to the increase of the glycerol flux. However, a quantitative regulation analysis of the data revealed an initial regulation by metabolism alone. After only a few minutes gene expression came into play, but even after an hour, 80% of the increase in the glycerol flux was explained by metabolic changes in the cell, and 20% by induction of gene expression. This demonstrates that the novel metabolic mechanism is not just a secondary rescue mechanism, but the most important mechanism to regulate the glycerol flux under physiological conditions.

  12. Autophagy: one more Nobel Prize for yeast

    Directory of Open Access Journals (Sweden)

    Andreas Zimmermann

    2016-12-01

    Full Text Available The recent announcement of the 2016 Nobel Prize in Physiology or Medicine, awarded to Yoshinori Ohsumi for the discoveries of mechanisms governing autophagy, underscores the importance of intracellular degradation and recycling. At the same time, it further cements yeast, in which this field decisively developed, as a prolific model organism. Here we provide a quick historical overview that mirrors both the importance of autophagy as a conserved and essential process for cellular life and death as well as the crucial role of yeast in its mechanistic characterization.

  13. Autophagy: one more Nobel Prize for yeast.

    Science.gov (United States)

    Zimmermann, Andreas; Kainz, Katharina; Andryushkova, Aleksandra; Hofer, Sebastian; Madeo, Frank; Carmona-Gutierrez, Didac

    2016-12-05

    The recent announcement of the 2016 Nobel Prize in Physiology or Medicine, awarded to Yoshinori Ohsumi for the discoveries of mechanisms governing autophagy, underscores the importance of intracellular degradation and recycling. At the same time, it further cements yeast, in which this field decisively developed, as a prolific model organism. Here we provide a quick historical overview that mirrors both the importance of autophagy as a conserved and essential process for cellular life and death as well as the crucial role of yeast in its mechanistic characterization.

  14. Flux control through protein phosphorylation in yeast

    DEFF Research Database (Denmark)

    Chen, Yu; Nielsen, Jens

    2016-01-01

    Protein phosphorylation is one of the most important mechanisms regulating metabolism as it can directly modify metabolic enzymes by the addition of phosphate groups. Attributed to such a rapid and reversible mechanism, cells can adjust metabolism rapidly in response to temporal changes. The yeast...... describe the development of phosphoproteomics in yeast as well as approaches to analysing the phosphoproteomics data. Finally, we focus on integrated analyses with other omics data sets and genome-scale metabolic models. Despite the advances, future studies improving both experimental technologies...

  15. Analysis of RNA metabolism in fission yeast

    DEFF Research Database (Denmark)

    Wise, Jo Ann; Nielsen, Olaf

    2017-01-01

    Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles.......Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles....

  16. DNA replication in yeast is stochastic

    Science.gov (United States)

    Cheng-Hsin Yang, Scott; Rhind, Nicholas; Bechhoefer, John

    2010-03-01

    Largely on the basis of a simple --- perhaps too simple --- analysis of microarray-chip experiments, people have concluded that DNA replication in budding yeast (S. cerevisiae) is a nearly deterministic process, in which the position and activation time of each origin of replication is pre-determined. In this talk, we introduce a more quantitative approach to the analysis of microarray data. Applying our new methods to budding yeast, we show that the microarray data imply a picture of replication where the timing of origin activation is highly stochastic. We then propose a physical model (the ``multiple-initiator model") to account for the observed probability distributions of origin- activation timing.

  17. Yeast-based genome mining, production and mechanistic studies of the biosynthesis of fungal polyketide and peptide natural products.

    Science.gov (United States)

    Tsunematsu, Yuta; Ishiuchi, Kan'ichiro; Hotta, Kinya; Watanabe, Kenji

    2013-08-01

    In this article, we review recent successful efforts to engineer biosynthesis of several important fungal natural products through heterologous expression of relevant biosynthetic genes in Saccharomyces cerevisiae. We also describe an innovative method of rapidly cloning fungal polyketide synthase or nonribosomal peptide synthetase genes, which can be 5-20 kb or longer, from a pool of total RNA obtained from the fungus of interest using the technique we termed the "overlap extension PCR-yeast homologous recombination (ExRec)" method. The process concomitantly incorporates the cloned genes into yeast expression vectors for biosynthesis of corresponding polyketide and nonribosomal peptide compounds in our engineered S. cerevisiae strain, allowing detailed chemical characterizations to identify the activities of those previously uncharacterized biosynthetic megaenzymes. Studies reviewed here highlight yeast as a useful and versatile host not only for production of various natural products and mechanistic investigation of biosynthetic enzymes, but also for mining of uncharacterized fungal genomes for novel secondary metabolite biosynthetic pathways.

  18. Overwintering of vineyard yeasts: survival of interacting yeast communities in grapes mummified on vines

    Directory of Open Access Journals (Sweden)

    Matthias eSipiczki

    2016-02-01

    Full Text Available The conversion of grape must into wine involves the development and succession of yeast populations differing in species composition. The initial population is formed by vineyard strains which are washed into the must from the crushed grapes and then completed with yeasts coming from the cellar environment. As the origin and natural habitat of the vineyard yeasts are not fully understood, this study addresses the possibility that grape yeasts can be preserved in berries left behind on vines at harvest until the spring of the next year. These berries become mummified during the winter on the vines. To investigate whether yeasts can survive in these overwintering grapes, mummified berries were collected in 16 localities in the Tokaj wine region (Hungary-Slovakia in early March. The collected berries were rehydrated to recover viable yeasts by plating samples onto agar plates. For the detection of minority species which would not be detected by direct plating, an enrichment step repressing the propagation of alcohol-sensitive yeasts was also included in the process. The morphological, physiological and molecular analysis identified 13 basidiomycetous and 23 ascomycetous species including fermentative yeasts of wine-making relevance among the 3879 isolates. The presence of viable strains of these species demonstrates that the grapes mummified on the vine can serve as a safe reservoir of yeasts, and may contribute to the maintenance of grape-colonizing yeast populations in the vineyard over years, parallel with other vectors and habitats. All basidiomycetous species were known phylloplane yeasts. Three Hanseniaspora species and pigmented Metschnikowia strains were the most frequent ascomycetes. Other fermentative yeasts of wine-making relevance were detected only in the enrichment cultures. Saccharomyces (S. paradoxus, S. cerevisiae and S. uvarum were recovered from 13 % of the samples. No Candida zemplinina was found. The isolates with Aureobasidium

  19. Occurrence of Killer Yeast Strains in Fruit and Berry Wine Yeast Populations

    Directory of Open Access Journals (Sweden)

    Gintare Gulbiniene

    2004-01-01

    Full Text Available Apple, cranberry, chokeberry and Lithuanian red grape wine yeast populations were used for the determination of killer yeast occurrence. According to the tests of the killer characteristics and immunity the isolated strains were divided into seven groups. In this work the activity of killer toxins purified from some typical strains was evaluated. The analysed strains produced different amounts of active killer toxin and some of them possessed new industrially significant killer properties. Total dsRNA extractions in 11 killer strains of yeast isolated from spontaneous fermentations revealed that the molecular basis of the killer phenomenon was not only dsRNAs, but also unidentified genetic determinants.

  20. Sex-ratio optimization with helpers at the nest

    NARCIS (Netherlands)

    Pen, I.R.; Weissing, F.J.

    2000-01-01

    In many cooperatively breeding animals, offspring produced earlier in life assist their parents in raising subsequent broods. Such helping behaviour is often confined to offspring of one sex. Sex-allocation theory predicts that parents overproduce offspring of the helping sex, but the expected

  1. Root : Shoot Ratios, Optimization and Nitrogen Productivity

    OpenAIRE

    ÅGREN, GÖRAN I.; FRANKLIN, OSKAR

    2003-01-01

    Plants respond to nitrogen availability by changing their root : shoot ratios. One hypothesis used to explain this allocation is that plants optimize their behaviour by maximizing their relative growth rate. The consequences of this hypothesis were investigated by formulating two models for root : shoot allocation, with and without explicit inclusion of maintenance respiration. The models also took into account that relative growth rate is a linear function of plant nitrogen concentration. Th...

  2. Newly identified prions in budding yeast, and their possible functions

    OpenAIRE

    Crow, Emily T.; Li, Liming

    2011-01-01

    Yeast prions are atypical genetic elements that are transmitted as heritable protein conformations. [PSI+], [URE3], and [PIN+] are three well-studied prions in the budding yeast, Saccharomyces cerevisiae. In the last three years, several additional prions have been reported in yeast, including [SWI+], [OCT+], [MCA], [GAR+], [MOT3+], [ISP+], and [NSI+]. The growing number of yeast prions suggests that protein-based inheritance might be a widespread biological phenomenon. In this review, we sum...

  3. Effect of increasing growth temperature on yeast fermentation ...

    African Journals Online (AJOL)

    The effect of increasing growth temperature on yeast fermentation was studied at approximately 5 oC intervals over a range of 18 – 37 oC, using one strain each of ale, lager and wine yeast. The ale and wine yeasts grew at all the temperatures tested, but lager yeast failed to grow at 37 oC. All these strains gave lower ...

  4. Construction of yeast strains useful for screening drugs that inhibit glucose uptake and glycolysis.

    Science.gov (United States)

    Roy, Adhiraj; Shin, Yong Jae; Kim, Jeong-Ho

    2013-05-01

    The budding yeast Saccharomyces cerevisiae expresses different isoforms of glucose transporters (HXTs) in response to different levels of glucose. Here, we constructed reporter strains in which the nourseothricin (NAT) resistance gene is expressed under the control of the HXT1, 2, or 3 promoter. The resulting HXT-NAT reporter strains exhibited a strict growth dependence on glucose, and their growth could be easily controlled and optimized by adjusting glucose concentration, demonstrating the value of the reporter strains for studying the molecular basis of differential expression of HXT genes, as well as for screening drugs that inhibit glucose uptake and glycolysis. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Differential Adsorption of Ochratoxin A and Anthocyanins by Inactivated Yeasts and Yeast Cell Walls during Simulation of Wine Aging

    National Research Council Canada - National Science Library

    Petruzzi, Leonardo; Baiano, Antonietta; De Gianni, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria; Bevilacqua, Antonio

    2015-01-01

    The adsorption of ochratoxin A (OTA) by yeasts is a promising approach for the decontamination of musts and wines, but some potential competitive or interactive phenomena between mycotoxin, yeast cells, and anthocyanins might modify...

  6. Drosophila-associated yeast species in vineyard ecosystems.

    Science.gov (United States)

    Lam, Samuel S T H; Howell, Kate S

    2015-10-01

    Yeast activity during wine fermentation directly contributes to wine quality, but the source and movement of yeasts in vineyards and winery environments have not been resolved. Here, we investigate the yeast species associated with the Drosophila insect vector to help understand yeast dispersal and persistence. Drosophila are commonly found in vineyards and are known to have a mutualistic relationship with yeasts in other ecosystems. Drosophilids were collected from vineyards, grape waste (marc) piles and wineries during grape harvest. Captured flies were identified morphologically, and their associated yeasts were identified. Drosophila melanogaster/D. simulans, D. hydei and Scaptodrosophila lativittata were identified in 296 captured Drosophila flies. These flies were associated with Metschnikowia pulcherrima, Hanseniaspora uvarum, Torulaspora delbrueckii and H. valbyensis yeasts. Yeast and Drosophila species diversity differed between collection locations (vineyard and marc: R = 0.588 for Drosophila and R = 0.644 for yeasts). Surprisingly, the primary wine fermentation yeast, Saccharomyces cerevisiae, was not isolated. Drosophila flies are preferentially associated with different yeast species in the vineyard and winery environments, and this association may help the movement and dispersal of yeast species in the vineyard and winery ecosystem. © FEMS 2015. All rights reserved.

  7. Performance of baker's yeast produced using date syrup substrate ...

    African Journals Online (AJOL)

    Baker's yeast was produced from three selected baker's yeast strains using date syrup as a substrate at low and high flow rate compared to those produced using molasses substrates. Performance of the produced baker's yeasts on Arabic bread quality was investigated. Baking tests showed a positive relationship between ...

  8. Effect of yeast extract and chitosan on shoot proliferation ...

    African Journals Online (AJOL)

    This paper reported the effect of yeast extract and chitosan with combination of yeast extract on the growth and morphological changes and production of phenolics in the in vitro plantlets of Curcuma mangga. Yeast extract did not show any effect on the biomass and shoot proliferation of in vitro plantlets. However, the ...

  9. Effects of chlorine and temperature on yeasts isolatedfrom a soft ...

    African Journals Online (AJOL)

    Yeasts isolated from sugar and filling valves in a bottling process were exposed to different chlorine concentrations and various high temperatures. It was found that growth of yeasts decreased with increase in chlorine concentration. The maximum chlorine concentration that inhibited both types of yeasts was 60mg/l while ...

  10. Bright stable luminescent yeast using bacterial luciferase as a sensor.

    NARCIS (Netherlands)

    Szittner, R; Jansen, G.; Thomas, DY; Meighen, E

    2003-01-01

    24h while luminescence of yeast with decanal decayed to less than 0.01% of that with Z-9-tetradecenal after 2min. Moreover, yeast survived in 0.5% (v/v) Z-9-tetradecenal while 0.005% (v/v) decanal was lethal. Luminescence of yeast (+luxAB) was also stimulated 100-fold by transformation with the

  11. Screening of yeasts capable of producing cellulase-free xylanase

    African Journals Online (AJOL)

    Professor

    2015-06-10

    Jun 10, 2015 ... medium and the enzymatic activities of endo-xylanase, β-xylosidase, carboxymetilcellulase, and filter paper cellulose ... yeasts, parts of the fruits and vegetables (stems, leaves, roots) were evaluated separately. Yeast isolation. For enrichment, about 2.5 g of each sample was inoculated into. 25 mL yeast ...

  12. (FOS)-fermenting yeast or bacterial strains as potential

    African Journals Online (AJOL)

    ltrujillo

    or solid growth medium containing these “prebiotic” ... Saccharomyces cerevisiae L/25-7-82, S. cerevisiae L/25-7-76, ... Culture media. The commonly used minimal Yeast Nitrogen base (YNB) and rich media YP (Yeast extract and Peptone) and LB (Luria Bertani) for yeast and bacterial grow, respectively, were prepared ...

  13. Quality evaluation of some commercial baker's yeasts in Nigeria ...

    African Journals Online (AJOL)

    Seven different brands of commercial baker's yeast commonly found in Nigerian markets were evaluated for their acidity, viability and leavening activity. The baking and staling qualities of bread produced using the yeasts were also determined. Acidity and viability of yeast cells in the samples ranged from 2.09 to 2.78 and ...

  14. Phenotypic characters of yeasts isolated from kpete-kpete, a ...

    African Journals Online (AJOL)

    USER

    2015-07-08

    Jul 8, 2015 ... Key words: Sorghum beer, tchoukoutou, kpete-kpete, yeast, Saccharomyces cerevisiae. INTRODUCTION. Fermented .... Physicochemical and microbiological characteristics of the traditional starter kpete-kpete. Samples origin. Yeasts ... Phenotypic characteristics of yeasts isolates. Results (Table 2) show ...

  15. Yeast Contamination Potential in a Carbonated Soft Drink Industry ...

    African Journals Online (AJOL)

    MICHAEL

    species of Saccharomyces cerevisiae (Thrall, 2004). Yeasts are useful in bakery and breweries but undesirable in carbonated soft drink industries due to ... characteristics compared to yeast colonies described in Cheesebrough (1985). RESULTS AND DISCUSSION. The yeasts isolated had some budding cells. The.

  16. 21 CFR 172.590 - Yeast-malt sprout extract.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Yeast-malt sprout extract. 172.590 Section 172.590... CONSUMPTION Flavoring Agents and Related Substances § 172.590 Yeast-malt sprout extract. Yeast-malt sprout extract, as described in this section, may be safely used in food in accordance with the following...

  17. 21 CFR 184.1983 - Bakers yeast extract.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bakers yeast extract. 184.1983 Section 184.1983 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD... Listing of Specific Substances Affirmed as GRAS § 184.1983 Bakers yeast extract. (a) Bakers yeast extract...

  18. 21 CFR 573.750 - Pichia pastoris dried yeast.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Pichia pastoris dried yeast. 573.750 Section 573... Food Additive Listing § 573.750 Pichia pastoris dried yeast. (a) Identity. The food additive Pichia pastoris dried yeast may be used in feed formulations of broiler chickens as a source of protein not to...

  19. Translation efficiency in yeasts correlates with nucleosome formation in promoters.

    Science.gov (United States)

    Matushkin, Yu G; Levitsky, V G; Orlov, Yu L; Likhoshvai, V A; Kolchanov, N A

    2013-01-01

    Elongation efficiency index (EEI) was suggested earlier to estimate gene expression efficiency by nucleotide context of coding sequence in unicellular organisms. We have analyzed association between EEI and nucleosome formation potential (NFP) in 5' regulatory regions upstream translation initiation site (TIS) from two yeast species. Theoretical estimations of NFP based on DNA sequence were obtained by Recon method. Experimental estimation of nucleosome occupancy was obtained by high-throughput sequencing data of nucleosomal DNA in Saccharomyces cerevisiae . For the sample of all genes correlation coefficient was calculated between two vectors: vector of NFP values for fixed position relative to TIS and vector of EEI values. Profiles of correlation coefficients of NFP and EEI were counted in (-600; +600) regions relative to TIS for gene sequences extracted from GenBank. We found regions of strong negative dependence between NFP and EEI for all genes as well as for 10% highly expressed genes in Schizosaccharomyces pombe (10% of EEI-highest genes). At the same time, we found positive dependence between NFP and EEI for all genes and for low expressed genes in S. cerevisiae (10% of EEI-lowest genes). The association between NFP and EEI could be explained by evolutionary selection of context characteristics of nucleotide sequences for gene expression optimization.

  20. Identification of yeast transcriptional regulation networks using multivariate random forests.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Xiao

    2009-06-01

    Full Text Available The recent availability of whole-genome scale data sets that investigate complementary and diverse aspects of transcriptional regulation has spawned an increased need for new and effective computational approaches to analyze and integrate these large scale assays. Here, we propose a novel algorithm, based on random forest methodology, to relate gene expression (as derived from expression microarrays to sequence features residing in gene promoters (as derived from DNA motif data and transcription factor binding to gene promoters (as derived from tiling microarrays. We extend the random forest approach to model a multivariate response as represented, for example, by time-course gene expression measures. An analysis of the multivariate random forest output reveals complex regulatory networks, which consist of cohesive, condition-dependent regulatory cliques. Each regulatory clique features homogeneous gene expression profiles and common motifs or synergistic motif groups. We apply our method to several yeast physiological processes: cell cycle, sporulation, and various stress conditions. Our technique displays excellent performance with regard to identifying known regulatory motifs, including high order interactions. In addition, we present evidence of the existence of an alternative MCB-binding pathway, which we confirm using data from two independent cell cycle studies and two other physioloigical processes. Finally, we have uncovered elaborate transcription regulation refinement mechanisms involving PAC and mRRPE motifs that govern essential rRNA processing. These include intriguing instances of differing motif dosages and differing combinatorial motif control that promote regulatory specificity in rRNA metabolism under differing physiological processes.