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Sample records for saccharomyces cerevisiae dynein

  1. Molecular and genetic characterization of SLC1, a putative Saccharomyces cerevisiae homolog of the metazoan cytoplasmic dynein light chain 1.

    PubMed

    Dick, T; Surana, U; Chia, W

    1996-04-24

    Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of intracellular movements. As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, we have identified the first non-metazoan dynein light chain 1, SLC1, in the yeast Saccharomyces cerevisiae. The amino acid sequence of the SLC1 protein is similar to those of the human, Drosophila and Caenorhabditis cytoplasmic dynein light chains 1. The SLC1 gene lies adjacent to the YAP2 (= CAD1) transcription unit. The SLC1 coding sequence is split by two introns and its mRNA is detectable throughout the cell cycle. Tetrad analysis of heterozygotes harboring a TRP insertion in the SLC1 coding region indicate that SLC1 function is not essential for cell viability. Furthermore, we demonstrate that double mutants, defective for SLC1 and the kinesin-related CIN8 genes are non-lethal. The redundancy of SLC1 function in yeast contrasts with the cell death caused by loss-of-function mutations in the dynein light chain 1 gene in Drosophila melanogaster. PMID:8628245

  2. A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein.

    PubMed

    Gleave, Emma S; Schmidt, Helgo; Carter, Andrew P

    2014-06-01

    Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our Saccharomycescerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins. PMID:24680784

  3. Peptidase activities in Saccharomyces cerevisiae.

    PubMed Central

    Rose, B; Becker, J M; Naider, F

    1979-01-01

    At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives. Images PMID:378955

  4. Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Ostergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

    2000-01-01

    Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production. PMID:10704473

  5. Saccharomyces cerevisiae in directed evolution

    PubMed Central

    Gonzalez-Perez, David; Garcia-Ruiz, Eva; Alcalde, Miguel

    2012-01-01

    Over the past 20 years, directed evolution has been seen to be the most reliable approach to protein engineering. Emulating the natural selection algorithm, ad hoc enzymes with novel features can be tailor-made for practical purposes through iterative rounds of random mutagenesis, DNA recombination and screening. Of the heterologous hosts used in laboratory evolution experiments, the budding yeast Saccharomyces cerevisiae has become the best choice to express eukaryotic proteins with improved properties. S. cerevisiae not only allows mutant enzymes to be secreted but also, it permits a wide range of genetic manipulations to be employed, ranging from in vivo cloning to the creation of greater molecular diversity, thanks to its efficient DNA recombination apparatus. Here, we summarize some successful examples of the use of the S. cerevisiae machinery to accelerate artificial evolution, complementing the traditional in vitro methods to generate tailor-made enzymes. PMID:22572788

  6. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  7. Glucose repression in Saccharomyces cerevisiae.

    PubMed

    Kayikci, Ömur; Nielsen, Jens

    2015-09-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression. PMID:26205245

  8. Chronological Aging in Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    The two paradigms to study aging in Saccharomyces cerevisiae are the chronological life span (CLS) and the replicative life span (RLS). The chronological life span is a measure of the mean and maximum survival time of non-dividing yeast populations while the replicative life span is based on the mean and maximum number of daughter cells generated by an individual mother cell before cell division stops irreversibly. Here we review the principal discoveries associated with yeast chronological aging and how they are contributing to the understanding of the aging process and of the molecular mechanisms that may lead to healthy aging in mammals. We will focus on the mechanisms of life span regulation by the Tor/Sch9 and the Ras/adenylate cyclase/PKA pathways with particular emphasis on those implicating age-dependent oxidative stress and DNA damage/repair. PMID:22094419

  9. Glucose repression in Saccharomyces cerevisiae

    PubMed Central

    Kayikci, Ömur; Nielsen, Jens

    2015-01-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression. PMID:26205245

  10. Postreplication repair in Saccharomyces cerevisiae

    SciTech Connect

    Resnick, M.A.; Boyce, J.; Cox, B.

    1981-04-01

    Postreplication events in logarithmically growing excision-defective mutants of Saccharomyces cerevisiae were examined after low doses of ultraviolet light. Pulse-labeled deoxyribonucleic acid had interruptions, and when the cells were chased, the interruptions were no longer detected. Since the loss of interruptions was not associated with an exchange of pyrimidine dimers at a detection level of 10 to 20% of the induced dimers, it was concluded that postreplication repair in excision-defective mutants does not involve molecular recombination. Pyrimidine dimers were assayed by utilizing the ultraviolet-endonuclease activity in extracts of Micrococcus luteus and newly developed alkaline sucrose gradient techniques, which yielded chromosomal-size deoxyribonucleic acid after treatment of irradiated cells.

  11. Microarray Analysis for Saccharomyces cerevisiae

    PubMed Central

    Tighe, Scott; Hunter, Tim; Reed, Pat; Murray, Janet

    2011-01-01

    In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software. PMID:21505409

  12. Choline transport in Saccharomyces cerevisiae.

    PubMed

    Hosaka, K; Yamashita, S

    1980-07-01

    Choline transport of Saccharomyces cerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N,N-dimethylethanolamine, and beta-methylcholine were competitive inhibitors of choline transport, with Ki values of 40.1, 3.1, and 6.9 microM, respectively. Ethanolamine, phosphorylcholine, and various amino acids examined had no effect. Choline transport required metabolic energy; removal of glucose resulted in a great loss of transport activity, and the remaining activity was abolished by 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, arsenate, and cyanide. External Na+ was not required, and the transport was not effected by ionophores, valinomycin, and gramicidin D. These results indicate that S. cerevisiae possess an active choline transport system mediated by a specific carrier. This view is further supported by the isolation and characterization of a choline transport mutant. The choline transport activity in this mutant was very low, whereas the transport of L-leucine, L-methionine, D-glucose, and myo-inositol was normal. Together with the choline transport mutant, mutants defective in choline kinase were also isolated. PMID:6995427

  13. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

    Saccharomyces cerevisiae is a yeast commonly used in baking and a frequent colonizer of human mucosal surfaces. It is considered relatively nonpathogenic in immunocompetent adults. We present a case of S. cerevisiae fungemia and aortic graft infection in an immunocompetent adult. This is the first reported case of S. cerevisiue fungemia where the identity of the pathogen was confirmed by rRNA sequencing.

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... cerevisiae antibodies may aid in the diagnosis of Crohn's disease. (b) Classification. Class II (special... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  15. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... cerevisiae antibodies may aid in the diagnosis of Crohn's disease. (b) Classification. Class II (special... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  16. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... cerevisiae antibodies may aid in the diagnosis of Crohn's disease. (b) Classification. Class II (special... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  17. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... cerevisiae antibodies may aid in the diagnosis of Crohn's disease. (b) Classification. Class II (special... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  18. The genetic characteristics Saccharomyces cerevisiae aci(+) mutants.

    PubMed

    Grochowalska, Renata; Machnicka, Beata; Wysocki, Robert; Lachowicz, Tadeusz M

    2003-01-01

    A series of 30 Saccharomyces cerevisiae aci(+) mutants (characterized as acidifying Ogur's glucose medium containing bromocresol purple) were isolated after EMS mutagenesis. All the mutants excreted acid metabolites to the medium after 24 or 48 hours of incubation. The character of the aci(+) mutations was defined using classical genetic techniques. Three of the aci(+) mutants were studied by molecular genetics techniques. PMID:12813559

  19. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  20. Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. Preparation of Saccharomyces cerevisiae expression plasmids.

    PubMed

    Drew, David; Kim, Hyun

    2012-01-01

    Expression plasmids for Saccharomyces cerevisiae offer a wide choice of vector copy number, promoters of varying strength and selection markers. These expression plasmids are usually shuttle vectors that can be propagated both in yeast and bacteria, making them useful in gene cloning. For heterologous production of membrane proteins, we used the green fluorescent protein (GFP) fusion technology which was previously developed in the Escherichia coli system. We designed an expression plasmid carrying an inducible GAL1 promoter, a gene encoding a membrane protein of interest and the GFP-octa-histidine sequence. Here we describe construction of multi-copy yeast expression plasmids by homologous recombination in S. cerevisiae. PMID:22454112

  2. Progress in metabolic engineering of Saccharomyces cerevisiae.

    PubMed

    Nevoigt, Elke

    2008-09-01

    The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial ("white") biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate. PMID:18772282

  3. [Urinary infection by Saccharomyces cerevisiae: Emerging yeast?].

    PubMed

    Elkhihal, B; Elhalimi, M; Ghfir, B; Mostachi, A; Lyagoubi, M; Aoufi, S

    2015-12-01

    Saccharomyces cerevisiae is a commensal yeast of the digestive, respiratory and genito-urinary tract. It is widely used as a probiotic for the treatment of post-antibiotic diarrhea. It most often occurs in immunocompromised patients frequently causing fungemia. We report the case of an adult diabetic patient who had a urinary tract infection due to S.cerevisiae. The disease started with urination associated with urinary frequency burns without fever. The diagnosis was established by the presence of yeasts on direct examination and positivity of culture on Sabouraud-chloramphenicol three times. The auxanogramme gallery (Auxacolor BioRad()) allowed the identification of S.cerevisiae. The patient was put on fluconazole with good outcome. This observation points out that this is an opportunistic yeast in immunocompromised patients. PMID:26522963

  4. Assembly of F0 in Saccharomyces cerevisiae

    PubMed Central

    Rak, Malgorzata; Zeng, Xiaomei; Brière, Jean-Jacques; Tzagoloff, Alexander

    2009-01-01

    Respiratory deficient mutants of Saccharomyces cerevisiae have been instrumental in identifying an increasing number of nuclear gene products that promote pre- and post-translational steps of the pathway responsible for biogenesis of the mitochondrial ATP synthase. In this article we have attempted to marshal current information about the functions of such accessory factors and the roles they play in expression and assembly of the mitochondrially encoded subunits of the ATP synthase. We also discuss evidence that the ATP synthase may be build up from three separate modules corresponding to the F1 ATPase, the stator and F0. PMID:18672007

  5. Acid excreting mutants of yeast Saccharomyces cerevisiae.

    PubMed

    Machnicka, B; Grochowalska, R; Boniewska-Bernacka, E; Słomińska, L; Lachowicz, T M

    2004-12-17

    Saccharomyces cerevisiae mutants acidifying glucose medium containing bromocresol purple were shown to excrete protons when placed in unbuffered water in the absence of any external carbon source. The mutants belong to 16 different complementation groups. Most of them do not grow on glycerol and the excreted protons are associated to particular sets of organic anions such as citrate, aconitate, succinate, fumarate or malate. These novel types of respiratory mutations seem to be located in genes operating in the Krebs or glyoxylate cycle. PMID:15541392

  6. Components of microtubular structures in Saccharomyces cerevisiae.

    PubMed Central

    Pillus, L; Solomon, F

    1986-01-01

    Most studies of cytoskeletal organelles have concentrated on molecular analyses of abundant and biochemically accessible structures. In many of the classical cases, however, the nature of the system chosen has precluded a concurrent genetic analysis. The mitotic spindle of the yeast Saccharomyces cerevisiae is one example of an organelle that can be studied by both classical and molecular genetics. We show here that this microtubule structure also can be examined biochemically. The spindle can be isolated by selective extractions of yeast cells by using adaptations of methods successfully applied to animal cells. In this way, microtubule-associated proteins of the yeast spindle are identified. Images PMID:3517870

  7. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae

    PubMed Central

    Fossati, Elena; Narcross, Lauren; Ekins, Andrew; Falgueyret, Jean-Pierre; Martin, Vincent J. J.

    2015-01-01

    Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-reticuline. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes. PMID:25905794

  8. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Nevoigt, Elke

    2008-01-01

    Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate. PMID:18772282

  9. Acidic Calcium Stores of Saccharomyces cerevisiae

    PubMed Central

    Cunningham, Kyle W.

    2011-01-01

    Fungi and animals constitute sister kingdoms in the eukaryotic domain of life. The major classes of transporters, channels, sensors, and effectors that move and respond to calcium ions were already highly networked in the common ancestor of fungi and animals. Since that time, some key components of the network have been moved, altered, relocalized, lost, or duplicated in the fungal and animal lineages and at the same time some of the regulatory circuitry has been dramatically rewired. Today the calcium transport and signaling networks in fungi provide a fresh perspective on the scene that has emerged from studies of the network in animal cells. This review provides an overview of calcium signaling networks in fungi, particularly the model yeast Saccharomyces cerevisiae, with special attention to the dominant roles of acidic calcium stores in fungal cell physiology. PMID:21377728

  10. Mechanisms of Gene Conversion in Saccharomyces Cerevisiae

    PubMed Central

    Roman, H.; Ruzinski, M. M.

    1990-01-01

    In red-white sectored colonies of Saccharomyces cerevisiae, derived from mitotic cells grown to stationary phase and irradiated with a light dose of x-rays, all of the segregational products of gene conversion and crossing over can be ascertained. Approximately 80% of convertants are induced in G(1), the remaining 20% in G(2). Crossing over, in the amount of 20%, is found among G(1) convertants but most of the crossovers are delayed until G(2). About 20% of all sectored colonies had more than one genotype in one or the other sector, thus confirming the hypothesis that conversion also occurs in G(2). The principal primary event in G(2) conversion is a single DNA heteroduplex. It is suggested that the close contact that this implies carries over to G(2) when crossing over and a second round of conversion occurs. PMID:2407607

  11. Viruses and prions of Saccharomyces cerevisiae

    PubMed Central

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

    2014-01-01

    Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses and prions. Studies of the mechanisms of virus and prion replication, virus structure and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular components: chromosomally encoded proteins necessary for or blocking the propagation of the viruses and prions, and proteins involved in expression of viral components. Here we emphasize the L-A dsRNA virus and its killer-toxin-encoding satellites, the 20S and 23S ssRNA naked viruses, and the several infectious proteins (prions) of yeast. PMID:23498901

  12. LINE Retrotransposition Assays in Saccharomyces cerevisiae.

    PubMed

    Horn, Axel V; Han, Jeffrey S

    2016-01-01

    Long interspersed nuclear element (LINE) retrotransposons make up significant parts of mammalian genomes. They alter host genomes by direct mutagenesis through integration of new transposon copies, by mobilizing non-autonomous transposons, by changes in host gene activity due to newly integrated transposons and by recombination events between different transposon copies. As a consequence, LINEs can contribute to genetic disease. Simple model systems can be useful for the study of basic molecular and cellular biology of LINE retrotransposons. Here, we describe methods for the analysis of LINE retrotransposition in the well-established model organism Saccharomyces cerevisiae. The ability to follow retrotransposition in budding yeast opens up the possibility of performing systematic screens for evolutionarily conserved interactions between LINE retrotransposons and their host cells. PMID:26895051

  13. Mitophagy and mitochondrial dynamics in Saccharomyces cerevisiae.

    PubMed

    Müller, Matthias; Lu, Kaihui; Reichert, Andreas S

    2015-10-01

    Mitochondria fulfill central cellular functions including energy metabolism, iron-sulfur biogenesis, and regulation of apoptosis and calcium homeostasis. Accumulation of dysfunctional mitochondria is observed in ageing and many human diseases such as cancer and various neurodegenerative disorders. Appropriate quality control of mitochondria is important for cell survival in most eukaryotic cells. One important pathway in this respect is mitophagy, a selective form of autophagy which removes excess and dysfunctional mitochondria. In the past decades a series of essential factors for mitophagy have been identified and characterized. However, little is known about the molecular mechanisms regulating mitophagy. The role of mitochondrial dynamics in mitophagy is controversially discussed. Here we will review recent advances in this context promoting our understanding on the molecular regulation of mitophagy in Saccharomyces cerevisiae and on the role of mitochondrial dynamics in mitochondrial quality control. PMID:25753536

  14. Acidic calcium stores of Saccharomyces cerevisiae.

    PubMed

    Cunningham, Kyle W

    2011-08-01

    Fungi and animals constitute sister kingdoms in the eukaryotic domain of life. The major classes of transporters, channels, sensors, and effectors that move and respond to calcium ions were already highly networked in the common ancestor of fungi and animals. Since that time, some key components of the network have been moved, altered, relocalized, lost, or duplicated in the fungal and animal lineages and at the same time some of the regulatory circuitry has been dramatically rewired. Today the calcium transport and signaling networks in fungi provide a fresh perspective on the scene that has emerged from studies of the network in animal cells. This review provides an overview of calcium signaling networks in fungi, particularly the model yeast Saccharomyces cerevisiae, with special attention to the dominant roles of acidic calcium stores in fungal cell physiology. PMID:21377728

  15. Transcriptional Regulatory Networks in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Lee, Tong Ihn; Rinaldi, Nicola J.; Robert, François; Odom, Duncan T.; Bar-Joseph, Ziv; Gerber, Georg K.; Hannett, Nancy M.; Harbison, Christopher T.; Thompson, Craig M.; Simon, Itamar; Zeitlinger, Julia; Jennings, Ezra G.; Murray, Heather L.; Gordon, D. Benjamin; Ren, Bing; Wyrick, John J.; Tagne, Jean-Bosco; Volkert, Thomas L.; Fraenkel, Ernest; Gifford, David K.; Young, Richard A.

    2002-10-01

    We have determined how most of the transcriptional regulators encoded in the eukaryote Saccharomyces cerevisiae associate with genes across the genome in living cells. Just as maps of metabolic networks describe the potential pathways that may be used by a cell to accomplish metabolic processes, this network of regulator-gene interactions describes potential pathways yeast cells can use to regulate global gene expression programs. We use this information to identify network motifs, the simplest units of network architecture, and demonstrate that an automated process can use motifs to assemble a transcriptional regulatory network structure. Our results reveal that eukaryotic cellular functions are highly connected through networks of transcriptional regulators that regulate other transcriptional regulators.

  16. Genetics of Expression of Asparaginase II Activity in Saccharomyces cerevisiae

    PubMed Central

    Jones, Gary E.

    1977-01-01

    Expression of asparaginase II activity in Saccharomyces cerevisiae requires the participation of the products of at least two cistrons, asp2 and asp3, which are unlinked on the yeast genetic map. PMID:320183

  17. Killer systems of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Nesterova, G.F.

    1989-01-01

    The killer systems of Saccharomyces cerevisiae are an unusual class of cytoplasmic symbionts of primitive eukaryotes. The genetic material of these symbionts is double-stranded RNA. They are characterized by the linearity of the genome, its fragmentation into a major and a minor fraction, which replicate separately, and their ability to control the synthesis of secretory mycocin proteins possessing a toxic action on closely related strains. The secretion of mycocins at the same time ensures acquiring of resistance to them. Strains containing killer symbionts are toxigenic and resistant to the action of their own toxin, but strains that are free of killer double-stranded RNAs are sensitive to the action of mycocins. The killer systems of S. cerevisiae have retained features relating them to viruses and are apparently the result of evolution of infectious viruses. The occurrences of such systems among monocellular eukaryotic organisms is an example of complication of the genome by means of its assembly from virus-like components. We discuss the unusual features of replication and the expression of killer systems and their utilization in the construction of vector molecules.

  18. Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae

    PubMed Central

    Garcia, David E.; Keasling, Jay D.

    2014-01-01

    The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni2+ column. The KM of the ATP binding site was determined to be 98.3 M at 30C, the optimal growth temperature for S. cerevisiae, and 74.3 M at 37C, the optimal growth temperature for E. coli. The KM of the mevalonate-5-phosphate binding site was determined to be 885 M at 30C and 880 M at 37C. The Vmax was determined to be 4.51 mol/min/mg enzyme at 30C and 5.33 mol/min/mg enzyme at 37C. PMK is Mg2+ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH?=?7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 M FPP. PMID:24475236

  19. Sugar and Glycerol Transport in Saccharomyces cerevisiae.

    PubMed

    Bisson, Linda F; Fan, Qingwen; Walker, Gordon A

    2016-01-01

    In Saccharomyces cerevisiae the process of transport of sugar substrates into the cell comprises a complex network of transporters and interacting regulatory mechanisms. Members of the large family of hexose (HXT) transporters display uptake efficiencies consistent with their environmental expression and play physiological roles in addition to feeding the glycolytic pathway. Multiple glucose-inducing and glucose-independent mechanisms serve to regulate expression of the sugar transporters in yeast assuring that expression levels and transporter activity are coordinated with cellular metabolism and energy needs. The expression of sugar transport activity is modulated by other nutritional and environmental factors that may override glucose-generated signals. Transporter expression and activity is regulated transcriptionally, post-transcriptionally and post-translationally. Recent studies have expanded upon this suite of regulatory mechanisms to include transcriptional expression fine tuning mediated by antisense RNA and prion-based regulation of transcription. Much remains to be learned about cell biology from the continued analysis of this dynamic process of substrate acquisition. PMID:26721273

  20. Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae.

    PubMed

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

    2003-01-01

    Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promote piecemeal microautophagy of the nucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway. Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner. PMN occurs normally in apg7-delta cells and is, therefore, not dependent on macroautophagy. Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures. These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole. PMID:12529432

  1. Stationary phase in the yeast Saccharomyces cerevisiae.

    PubMed Central

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

    1993-01-01

    Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion, (vi) membrane biosynthesis, and (vii) cell polarity. The exact nature of the relationship between these processes and survival during stationary phase remains to be elucidated. We propose that cell cycle arrest coordinated with the ability to remain viable in the absence of additional nutrients provides a good operational definition of starvation-induced stationary phase. PMID:8393130

  2. Responses of Saccharomyces cerevisiae to thermal stress.

    PubMed

    Guyot, Stéphane; Ferret, Eric; Gervais, Patrick

    2005-11-20

    We studied the mechanisms involved in heat gradient-induced thermotolerance of Saccharomyces cerevisiae. Yeasts were slowly heated in a nutrient medium from 25 to 50 degrees C at 0.5 degrees C/min or immediately heat shocked at 50 degrees C, and both sets of cultures were maintained at this temperature for 1 h. Cells that had been slowly heated showed a 50-fold higher survival rate than the rapidly heated cells. Such thermotolerance was found not to be related to protein synthesis. Indeed Hsp104 a known protein involved in yeast thermal resistance induced by a preconditioning mild heat treatment, was not synthesized and cycloheximide addition, a protein synthesis inhibitor, did not affect the thermoprotective effect. Moreover, a rapid cooling from 50 to 25 degrees C applied immediately after the heat slope treatment inhibited the mechanisms involved in thermotolerance. Such observations lead us to conclude that heat gradient-induced thermal resistance is not directly linked to mechanisms involving intracellular molecules synthesis or activity such as proteins (Hsps, enzymes) or osmolytes (trehalose). Other factors such as plasma membrane phospholipid denaturation could be involved in this phenomenon. PMID:16028292

  3. Classical NLS proteins from Saccharomyces cerevisiae.

    PubMed

    Hahn, Silvia; Maurer, Patrick; Caesar, Stefanie; Schlenstedt, Gabriel

    2008-06-13

    Proteins can enter the nucleus through various receptor-mediated import pathways. One class of import cargos carries a classical nuclear localization signal (cNLS) containing a short cluster of basic residues. This pathway involves importin alpha (Impalpha), which possesses the cNLS binding site, and importin beta (Impbeta), which translocates the import complex through the nuclear pore complex. The defining criteria for a cNLS protein from Saccharomyces cerevisiae are an in vivo import defect in Impalpha and Impbeta mutants, direct binding to purified Impalpha, and stimulation of this binding by Impbeta. We show for the first time that endogenous S. cerevisiae proteins Prp20, Cdc6, Swi5, Cdc45, and Clb2 fulfill all of these criteria identifying them as authentic yeast cNLS cargos. Furthermore, we found that the targeting signal of Prp20 is a bipartite cNLS and that of Cdc6 is a monopartite cNLS. Basic residues present within these motifs are of different significance for the interaction with Impalpha. We determined the binding constants for import complexes containing the five cNLS proteins by surface plasmon resonance spectrometry. The dissociation constants for cNLS/alpha/beta complexes differ considerably, ranging from 1 nM for Cdc6 to 112 nM for Swi5, suggesting that the nuclear import kinetics is determined by the strength of cNLS/Impalpha binding. Impbeta enhances the affinity of Impalpha for cNLSs approximately 100-fold. This stimulation of cNLS binding to Impalpha results from a faster association in the presence of Impbeta, whereas the dissociation rate is unaffected by Impbeta. This implies that, after entry into the nucleus, the release of Impbeta by the Ran guanosine triphosphatase (Ran GTPase) from the import complex is not sufficient to dissociate the cNLS/Impalpha subcomplex. Our observation that the nucleoporin Nup2, which had been previously shown to release the cNLS from Impalpha in vitro, is required for efficient import of all the genuine cNLS cargos supports a general role of Nup2 in import termination. PMID:18485366

  4. Regulation of Cation Balance in Saccharomyces cerevisiae

    PubMed Central

    Cyert, Martha S.; Philpott, Caroline C.

    2013-01-01

    All living organisms require nutrient minerals for growth and have developed mechanisms to acquire, utilize, and store nutrient minerals effectively. In the aqueous cellular environment, these elements exist as charged ions that, together with protons and hydroxide ions, facilitate biochemical reactions and establish the electrochemical gradients across membranes that drive cellular processes such as transport and ATP synthesis. Metal ions serve as essential enzyme cofactors and perform both structural and signaling roles within cells. However, because these ions can also be toxic, cells have developed sophisticated homeostatic mechanisms to regulate their levels and avoid toxicity. Studies in Saccharomyces cerevisiae have characterized many of the gene products and processes responsible for acquiring, utilizing, storing, and regulating levels of these ions. Findings in this model organism have often allowed the corresponding machinery in humans to be identified and have provided insights into diseases that result from defects in ion homeostasis. This review summarizes our current understanding of how cation balance is achieved and modulated in baker’s yeast. Control of intracellular pH is discussed, as well as uptake, storage, and efflux mechanisms for the alkali metal cations, Na+ and K+, the divalent cations, Ca2+ and Mg2+, and the trace metal ions, Fe2+, Zn2+, Cu2+, and Mn2+. Signal transduction pathways that are regulated by pH and Ca2+ are reviewed, as well as the mechanisms that allow cells to maintain appropriate intracellular cation concentrations when challenged by extreme conditions, i.e., either limited availability or toxic levels in the environment. PMID:23463800

  5. Analysis of the Saccharomyces cerevisiae proteome with PeptideAtlas

    PubMed Central

    King, Nichole L; Deutsch, Eric W; Ranish, Jeffrey A; Nesvizhskii, Alexey I; Eddes, James S; Mallick, Parag; Eng, Jimmy; Desiere, Frank; Flory, Mark; Martin, Daniel B; Kim, Bong; Lee, Hookeun; Raught, Brian; Aebersold, Ruedi

    2006-01-01

    We present the Saccharomyces cerevisiae PeptideAtlas composed from 47 diverse experiments and 4.9 million tandem mass spectra. The observed peptides align to 61% of Saccharomyces Genome Database (SGD) open reading frames (ORFs), 49% of the uncharacterized SGD ORFs, 54% of S. cerevisiae ORFs with a Gene Ontology annotation of 'molecular function unknown', and 76% of ORFs with Gene names. We highlight the use of this resource for data mining, construction of high quality lists for targeted proteomics, validation of proteins, and software development. PMID:17101051

  6. Invasive Saccharomyces cerevisiae infection: a friend turning foe?

    PubMed

    Pillai, Unnikrishnan; Devasahayam, Joe; Kurup, Aparna Narayana; Lacasse, Alexandre

    2014-11-01

    We report a very rare case of acute pyelonephritis in a 51-year-old female with a history of chronic kidney disease (CKD) and diabetes caused by a normally benign and a well-known human commensal organism, Saccharomyces cerevisiae that is very often prescribed as a probiotic in modern medical practice. The causal role of S. cerevisiae was confirmed by its isolation in blood, urine, stool as well as vaginal swabs thus proving its virulent nature in suitable situations. PMID:25394448

  7. Aerobic and anaerobic NAD+ metabolism in Saccharomyces cerevisiae.

    PubMed

    Panozzo, Cristina; Nawara, Magdalena; Suski, Catherine; Kucharczyka, Roza; Skoneczny, Marek; Bécam, Anne Marie; Rytka, Joanna; Herbert, Christopher J

    2002-04-24

    In Saccharomyces cerevisiae the nicotinic acid moiety of NAD+ can be synthesized from tryptophan using the kynurenine pathway or incorporated directly using nicotinate phosphoribosyl transferase (NPT1). We have identified the genes that encode the enzymes of the kynurenine pathway and for BNA5 (YLR231c) and BNA6 (YFR047c) confirmed that they encode kynureninase and quinolinate phosphoribosyl transferase respectively. We show that deletion of genes encoding kynurenine pathway enzymes are co-lethal with the Deltanpt1, demonstrating that no other pathway for the synthesis of nicotinic acid exists in S. cerevisiae. Also, we show that under anaerobic conditions S. cerevisiae is a nicotinic acid auxotroph. PMID:12062417

  8. L-methionine as an ethylene precursor in Saccharomyces cerevisiae.

    PubMed

    Thomas, K C; Spencer, M

    1977-12-01

    L-Methionine induced production of ethylene by Saccharomyces cerevisiae growing in lactate medium. The production induced by L-methionine was inhibited by pyruvate, and elevated by glucose. Labeled ethylene was produced when L-[U-14C]methionine, but not [U-14C]glucose, was fed to the yeast. The mutant S. cerevisiae G1332 (ade-, met-) did not produce significant amounts of ethylene unless L-methionine was added. Thus L-methionine acts as a precursor of ethylene in S. cerevisiae. The role of glucose appears to be other than as a precursor. PMID:340018

  9. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

    The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins. It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules. It is carried out efficiently and is exquisitely sensitive to the needs of the cell. Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed. The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies. This tandem array has led to unique ways of carrying out a number of functions. Replication is asymmetric and does not initiate from every autonomously replicating sequence. Recombination is suppressed. Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit. Genes for many ribosomal proteins have been cloned and sequenced. Few are linked; most are duplicated; most have an intron. There is extensive homology between yeast ribosomal proteins and those of other species. Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor. This factor binds also to many other places in the genome, including the telomeres. There is coordinated transcription of the ribosomal protein genes under a variety of conditions. However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein. A deficiency causes slow growth. Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min. Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins. However, in the case of ribosomal protein L32, the protein itself causes a feedback inhibition of the splicing of the transcript of its own gene. The synthesis of ribosomes involves a massive transfer of material across the nuclear envelope in both directions. Nuclear localization signals have been identified for at least three ribosomal proteins; they are similar but not identical to those identified for the simian virus 40 T antigen. There is no information about how ribosomal subunits are transported from the nucleus to the cytoplasm.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2666845

  10. Improving biomass sugar utilization by engineered Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficient utilization of all available sugars in lignocellulosic biomass, which is more abundant than available commodity crops and starch, represents one of the most difficult technological challenges for the production of bioethanol. The well-studied yeast Saccharomyces cerevisiae has played a...

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

    PubMed

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

    2015-08-01

    During wine fermentation, Saccharomyces clearly dominate over non-Saccharomyces wine yeasts, and several factors could be related to this dominance. However, the main factor causing the reduction of cultivable non-Saccharomyces populations has not yet been fully established. In the present study, various single and mixed fermentations were performed to evaluate some of the factors likely responsible for the interaction between Saccharomyces cerevisiae and Hanseniaspora uvarum. Alcoholic fermentation was performed in compartmented experimental set ups with ratios of 1:1 and 1:9 and the cultivable population of both species was followed. The cultivable H. uvarum population decreased sharply at late stages when S. cerevisiae was present in the other compartment, similarly to alcoholic fermentations in non-compartmented vessels. Thus, cell-to-cell contact did not seem to be the main cause for the lack of cultivability of H. uvarum. Other compounds related to fermentation performance (such as sugar and ethanol) and/or certain metabolites secreted by S. cerevisiae could be related to the sharp decrease in H. uvarum cultivability. When these factors were analyzed, it was confirmed that metabolites from S. cerevisiae induced lack of cultivability in H. uvarum, however ethanol and other possible compounds did not seem to induce this effect but played some role during the process. This study contributes to a new understanding of the lack of cultivability of H. uvarum populations during the late stages of wine fermentation. PMID:25956738

  12. The nucleotide sequence of Saccharomyces cerevisiae chromosome XII.

    PubMed

    Johnston, M; Hillier, L; Riles, L; Albermann, K; André, B; Ansorge, W; Benes, V; Brückner, M; Delius, H; Dubois, E; Düsterhöft, A; Entian, K D; Floeth, M; Goffeau, A; Hebling, U; Heumann, K; Heuss-Neitzel, D; Hilbert, H; Hilger, F; Kleine, K; Kötter, P; Louis, E J; Messenguy, F; Mewes, H W; Hoheisel, J D

    1997-05-29

    The yeast Saccharomyces cerevisiae is the pre-eminent organism for the study of basic functions of eukaryotic cells. All of the genes of this simple eukaryotic cell have recently been revealed by an international collaborative effort to determine the complete DNA sequence of its nuclear genome. Here we describe some of the features of chromosome XII. PMID:9169871

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

    ERIC Educational Resources Information Center

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a

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

    ERIC Educational Resources Information Center

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

  15. Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The yeast Saccharomyces cerevisiae is a superb ethanol producer, yet sensitive to ethanol at higher concentrations especially under high gravity or very high gravity fermentation conditions. Although significant efforts have been made to study ethanol-stress response in past decades, molecular mecha...

  16. Potential immobilized Saccharomyces cerevisiae as heavy metal removal

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

    Biosorption of copper ion using treated and untreated immobilized Saccharomyces cerevisiae from aqueous solution was investigate in this study. S.cerevisiae has been choosing as biosorbent due to low cost, easy and continuously available from various industries. In this study, the ability of treated and untreated immobilized S.cerevisiae in removing copper ion influence by the effect of pH solution, and initial concentration of copper ion with contact time. Besides, adsorption isotherm and kinetic model also studied. The result indicated that the copper ion uptake on treated and untreated immobilized S.cerevisiae was increased with increasing of contact time and initial concentration of copper ion. The optimum pH for copper ion uptake on untreated and treated immobilized S.cerevisiae at 4 and 6. From the data obtained of copper ion uptake, the adsorption isotherm was fitted well by Freundlich model for treated immobilized S.cerevisiae and Langmuir model for untreated immobilized S.cerevisiae according to high correlation coefficient. Meanwhile, the pseudo second order was described as suitable model present according to high correlation coefficient. Since the application of biosorption process has been received more attention from numerous researchers as a potential process to be applied in the industry, future study will be conducted to investigate the potential of immobilized S.cerevisiae in continuous process.

  17. Protective effect of vitamins against trichothecene toxicity towards Saccharomyces cerevisiae.

    PubMed

    Yagen, B; Halevy, S

    1987-08-15

    Several trichothecene mycotoxins were shown to inhibit the growth of Saccharomyces cerevisiae. This effect was most pronounced with the macrocyclic trichothecenes, especially verrucarin A. Much less growth inhibition was observed with T-2 toxin. Verrucarol, diacetoxyscirpenol, acetyl T-2 toxin, HT-2 toxin, T-2 tetraol and neosolaniol were inactive at a concentration of 75 micrograms of toxin per disc. Incubation of S. cerevisiae with verrucarin A together with vitamins resulted in a decrease in toxicity. Pyridoxine-HCl, Ca-pantothenate, thiamine-HCl and alpha-tocopheryl acetate were amongst the most potent of the vitamins tested which reversed growth inhibition, overcoming the inhibitory potential of the toxins. PMID:3305064

  18. Saccharomyces cerevisiae: a sexy yeast with a prion problem.

    PubMed

    Kelly, Amy C; Wickner, Reed B

    2013-01-01

    Yeast prions are infectious proteins that spread exclusively by mating. The frequency of prions in the wild therefore largely reflects the rate of spread by mating counterbalanced by prion growth slowing effects in the host. We recently showed that the frequency of outcross mating is about 1% of mitotic doublings with 23-46% of total matings being outcrosses. These findings imply that even the mildest forms of the [PSI+], [URE3] and [PIN+] prions impart > 1% growth/survival detriment on their hosts. Our estimate of outcrossing suggests that Saccharomyces cerevisiae is far more sexual than previously thought and would therefore be more responsive to the adaptive effects of natural selection compared with a strictly asexual yeast. Further, given its large effective population size, a growth/survival detriment of > 1% for yeast prions should strongly select against prion-infected strains in wild populations of Saccharomyces cerevisiae. PMID:23764836

  19. Purification of Arp2/3 complex from Saccharomyces cerevisiae

    PubMed Central

    Doolittle, Lynda K.; Rosen, Michael K.; Padrick, Shae B.

    2014-01-01

    Summary Much of cellular control over actin dynamics comes through regulation of actin filament initiation. At the molecular level, this is accomplished through a collection of cellular protein machines, called actin nucleation factors, which position actin monomers to initiate a new actin filament. The Arp2/3 complex is a principal actin nucleation factor used throughout the eukaryotic family tree. The budding yeast Saccharomyces cerevisiae has proven to be not only an excellent genetic platform for the study of the Arp2/3 complex, but also an excellent source for the purification of endogenous Arp2/3 complex. Here we describe a protocol for the preparation of endogenous Arp2/3 complex from wild type Saccharomyces cerevisiae. This protocol produces material suitable for biochemical study, and yields milligram quantities of purified Arp2/3 complex. PMID:23868593

  20. The reference genome sequence of Saccharomyces cerevisiae: then and now.

    PubMed

    Engel, Stacia R; Dietrich, Fred S; Fisk, Dianna G; Binkley, Gail; Balakrishnan, Rama; Costanzo, Maria C; Dwight, Selina S; Hitz, Benjamin C; Karra, Kalpana; Nash, Robert S; Weng, Shuai; Wong, Edith D; Lloyd, Paul; Skrzypek, Marek S; Miyasato, Stuart R; Simison, Matt; Cherry, J Michael

    2014-03-01

    The genome of the budding yeast Saccharomyces cerevisiae was the first completely sequenced from a eukaryote. It was released in 1996 as the work of a worldwide effort of hundreds of researchers. In the time since, the yeast genome has been intensively studied by geneticists, molecular biologists, and computational scientists all over the world. Maintenance and annotation of the genome sequence have long been provided by the Saccharomyces Genome Database, one of the original model organism databases. To deepen our understanding of the eukaryotic genome, the S. cerevisiae strain S288C reference genome sequence was updated recently in its first major update since 1996. The new version, called "S288C 2010," was determined from a single yeast colony using modern sequencing technologies and serves as the anchor for further innovations in yeast genomic science. PMID:24374639

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

    PubMed Central

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

    2016-01-01

    The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae. PMID:27148191

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

    PubMed Central

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

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

    PubMed

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines. PMID:26887243

  4. Alternative Splicing in Next Generation Sequencing Data of Saccharomyces cerevisiae

    PubMed Central

    Schreiber, Konrad; Csaba, Gergely; Haslbeck, Martin; Zimmer, Ralf

    2015-01-01

    mRNA splicing is required in about 4% of protein coding genes in Saccharomyces cerevisiae. The gene structure of those genes is simple, generally comprising two exons and one intron. In order to characterize the impact of alternative splicing on the S. cerevisiae transcriptome, we perform a systematic analysis of mRNA sequencing data. We find evidence of a pervasive use of alternative splice sites and detect several novel introns both within and outside protein coding regions. We also find a predominance of alternative splicing on the 3’ side of introns, a finding which is consistent with existing knowledge on conservation of exon-intron boundaries in S. cerevisiae. Some of the alternatively spliced transcripts allow for a translation into different protein products. PMID:26469855

  5. Genetic engineering of industrial strains of Saccharomyces cerevisiae.

    PubMed

    Le Borgne, Sylvie

    2012-01-01

    Genetic engineering has been successfully applied to Saccharomyces cerevisiae laboratory strains for different purposes: extension of substrate range, improvement of productivity and yield, elimination of by-products, improvement of process performance and cellular properties, and extension of product range. The potential of genetically engineered yeasts for the massive production of biofuels as bioethanol and other nonfuel products from renewable resources as lignocellulosic biomass hydrolysates has been recognized. For such applications, robust industrial strains of S. cerevisiae have to be used. Here, some relevant genetic and genomic characteristics of industrial strains are discussed in relation to the problematic of the genetic engineering of such strains. General molecular tools applicable to the manipulation of S. cerevisiae industrial strains are presented and examples of genetically engineered industrial strains developed for the production of bioethanol from lignocellulosic biomass are given. PMID:22160914

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

    PubMed

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

    2010-01-01

    Lactose is an interesting carbon source for the production of several bio-products by fermentation, primarily because it is the major component of cheese whey, the main by-product of dairy activities. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, does not have a lactose metabolization system. Therefore, several metabolic engineering approaches have been used to construct lactose-consuming S. cerevisiae strains, particularly involving the expression of the lactose genes of the phylogenetically related yeast Kluyveromyces lactis, but also the lactose genes from Escherichia coli and Aspergillus niger, as reviewed here. Due to the existing large amounts of whey, the production of bio-ethanol from lactose by engineered S. cerevisiae has been considered as a possible route for whey surplus. Emphasis is given in the present review on strain improvement for lactose-to-ethanol bioprocesses, namely flocculent yeast strains for continuous high-cell-density systems with enhanced ethanol productivity. PMID:21326922

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

    PubMed

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

    2012-03-01

    Saccharomyces cerevisiae has become a favorite production organism in industrial biotechnology presenting new challenges to yeast engineers in terms of introducing advantageous traits such as stress tolerances. Exploring subspecies diversity of S. cerevisiae has identified strains that bear industrially relevant phenotypic traits. Provided that the genetic basis of such phenotypic traits can be identified inverse engineering allows the targeted modification of production strains. Most phenotypic traits of interest in S. cerevisiae strains are quantitative, meaning that they are controlled by multiple genetic loci referred to as quantitative trait loci (QTL). A straightforward approach to identify the genetic basis of quantitative traits is QTL mapping which aims at the allocation of the genetic determinants to regions in the genome. The application of high-density oligonucleotide arrays and whole-genome re-sequencing to detect genetic variations between strains has facilitated the detection of large numbers of molecular markers thus allowing high-resolution QTL mapping over the entire genome. This review focuses on the basic principle and state of the art of QTL mapping in S. cerevisiae. Furthermore we discuss several approaches developed during the last decade that allow down-scaling of the regions identified by QTL mapping to the gene level. We also emphasize the particular challenges of QTL mapping in nonlaboratory strains of S. cerevisiae. PMID:22150948

  8. Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae.

    PubMed

    Mormeneo, María; Pastor, Fi Javier; Zueco, Jesús

    2012-01-01

    The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western immunoblot using Pir-specific antibodies, while enzymatic activity on carboxymethyl cellulose was demonstrated on plate assays, zymographic analysis and colorimetric assays. Hyperglycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not affect activity, and values of 1.2 U/ml were obtained in growth medium supernatants in ordinary batch cultures after 24 h. These values compare quite favorably with those described for other recombinant endoglucanases expressed in S. cerevisiae. This is one of the few reports describing the expression of Bacillus cellulases in S. cerevisiae, since yeast expressed recombinant cellulases have been mostly of fungal origin. It is also the first report of the yeast expression of this particular endoglucanase. PMID:21701899

  9. Saccharomyces cerevisiae: a nomadic yeast with no niche?

    PubMed Central

    Goddard, Matthew R.; Greig, Duncan

    2015-01-01

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

  10. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

  11. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  12. A Saccharomyces cerevisiae Internet protein resource now available.

    PubMed

    Latter, G I; Boutell, T; Monardo, P J; Kobayashi, R; Futcher, B; Mclaughlin, C S; Garrels, J I

    1995-07-01

    The QUEST Protein Database Center is now making available two Saccharomyces cerevisiae protein databases via the Internet. The yeast electrophoretic protein database (YEPD) is a database of approximately one hundred protein identifications on two-dimensional gels. The yeast protein database (YPD) is a database of gene names and properties of over 3500 yeast proteins of known sequence. These databases can be accessed via a World-Wide Web (WWW) server (URL http:@siva.cshl.org). YPD is available via public ftp (isis.cshl.org) as well, in a spreadsheet format, and in ASCII format. When accessed via WWW, both of these databases have hypertext links to other biological data, such as the SWISS-PROT protein sequence database and the Saccharomyces Genome Database (SacchDB), and to each other. PMID:7498160

  13. Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae

    SciTech Connect

    Batt, C.A.; Carvallo, S.; Easson, D.D.; Akedo, M.; Sinskey, A.J.

    1986-04-01

    Xylose transport, xylose reductase, and xylitol dehydrogenase activities are demonstrated in Saccharomyces cerevisiae. The enzymes in the xylose catabolic pathway necessary for the conversion of xylose xylulose are present, although S. cerevisiae cannot grow on xylose as a sole carbon source. Xylose transport is less efficient than glucose transport, and its rate is dependent upon aeration. Xylose reductase appears to be a xylose inducible enzyme and xylitol dehydrogenase activity is constitutive, although both are repressed by glucose. Both xylose reductase and xylitol dehydrogenase activities are five- to tenfold lower in S. cerevisie as compared to Candida utilis. In vivo conversion of /sup 14/C-xylose in S. cerevisiage is demonstrated and xylitol is detected, although no significant levels of any other /sup 14/C-labeled metabolites (e.g., ethanol) are observed. 22 references.

  14. ROG1 encodes a monoacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Vishnu Varthini, Lakshmanaperumal; Selvaraju, Kandasamy; Srinivasan, Malathi; Nachiappan, Vasanthi

    2015-01-01

    Lipid metabolism is extensively studied in Saccharomyces cerevisiae. Here, we report that revertant of glycogen synthase kinase mutation-1 (Rog1p) possesses monoacylglycerol (MAG) lipase activity in S. cerevisiae. The lipase activity of Rog1p was confirmed in two ways: through analysis of a strain with a double deletion of ROG1 and monoglyceride lipase YJU3 (yju3Δrog1Δ) and by site-directed mutagenesis of the ROG1 lipase motif (GXSXG). Rog1p is localized in both the cytosol and the nucleus. Overexpression of ROG1 in a ROG1-deficient strain resulted in an accumulation of reactive oxygen species. These results suggest that Rog1p is a MAG lipase that regulates lipid homeostasis. PMID:25433290

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. Uptake and intracellular compartmentation of thorium in Saccharomyces cerevisiae.

    PubMed

    Gadd, G M; White, C

    1989-01-01

    When Saccharomyces cerevisiae was cultured in the presence of thorium, the element was accumulated by the cells and was visible in electron micrographs as electron dense granules. When thorium was present during exponential growth, these granules were located mainly in the vacuole, with some present in the cytosol. Where thorium was present only during the stationary phase, there appeared to be greater thorium deposition in the cell wall than during exponential growth and some vacuolar deposits were also evident. Thorium uptake by exponential-phase cells was not stimulated by glucose and was thus independent of metabolic energy. PMID:15092359

  18. Properties of partially purified endopolyphosphatase of the yeast Saccharomyces cerevisiae.

    PubMed

    Lichko, L P; Kulakovskaya, T V; Kulaev, I S

    2010-11-01

    Partially purified endopolyphosphatase from cytosol of the yeast Saccharomyces cerevisiae with inactivated genes PPX1 and PPN1 encoding exopolyphosphatases was obtained with ion-exchange and affinity chromatography. The enzyme activity was estimated by decrease of polyphosphate chain length determined by PAGE. The enzyme cleaved inorganic polyphosphate without the release of orthophosphate (P(i)) and was inhibited by heparin and insensitive to fluoride. Mg2+, Mn2+, and Co2+ (1.5 mM) stimulated the activity, and Ca2+ was ineffective. The molecular mass of the endopolyphosphatase determined by gel filtration was of ~20 kDa. PMID:21314609

  19. Fermentation of whey and starch by transformed Saccharomyces cerevisiae cells.

    PubMed

    Compagno, C; Porro, D; Smeraldi, C; Ranzi, B M

    1995-10-01

    Among the main agro-industrial wastes, whey and starch are of prime importance. In previous work we showed that strains of Saccharomyces cerevisiae transformed with the episomal plasmid pM1 allow production of yeast biomass and ethanol from whey/lactose. Ethanol production from whey and derivatives has been improved in computer-controlled bioreactors, while fermentation studies showed that the composition of the medium greatly modulates the productivity (g ethanol produced/l in 1 h of fermentation). A yeast strain for the simultaneous utilization of lactose and starch has also been developed. Biotechnological perspectives are discussed. PMID:7576548

  20. DNA sequence analysis of spontaneous mutagenesis in Saccharomyces cerevisiae.

    PubMed Central

    Kunz, B A; Ramachandran, K; Vonarx, E J

    1998-01-01

    To help elucidate the mechanisms involved in spontaneous mutagenesis, DNA sequencing has been applied to characterize the types of mutation whose rates are increased or decreased in mutator or antimutator strains, respectively. Increased spontaneous mutation rates point to malfunctions in genes that normally act to reduce spontaneous mutation, whereas decreased rates are associated with defects in genes whose products are necessary for spontaneous mutagenesis. In this article, we survey and discuss the mutational specificities conferred by mutator and antimutator genes in the budding yeast Saccharomyces cerevisiae. The implications of selected aspects of the data are considered with respect to the mechanisms of spontaneous mutagenesis. PMID:9560369

  1. Expression levels of transdominant peptides and proteins in Saccharomyces cerevisiae.

    PubMed

    Sandrock, Tanya; Poritz, Mark; Kim, Marianne; Feldhaus, Michael J; Roth, Bruce; Caponigro, Giordano; Kamb, Alexander

    2002-01-15

    From libraries of peptides and protein fragments, several inhibitors that block pheromone response in Saccharomyces cerevisiae have been isolated previously. In many cases, the inhibitors are displayed as part of a scaffold, such as green fluorescent protein. Each of the inhibitors has a characteristic physiological strength or genetic penetrance. In this report, the roles of expression level and display scaffold on the activities of a subset of pheromone-response pathway inhibitors were examined. Special consideration was given to the relationship between expression levels of specific inhibitors, which may exceed 50 microM in some instances, and penetrance. PMID:11754477

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

    PubMed

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

    2016-02-01

    Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering. PMID:26658003

  3. Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae.

    PubMed

    Van Hoek, P; Modesti, A; Ramponi, G; Kötter, P; van Dijken, J P; Pron, J T

    2001-10-01

    Human acylphosphatase (h-AP, EC 3.6.1.7) has been reported to catalyse the hydrolysis of the 1-phosphate group of 1,3-diphosphoglycerate. In vivo operation of this reaction in the yeast Saccharomyces cerevisiae would bypass phosphoglycerate kinase and thus reduce the ATP yield from glycolysis. To investigate whether h-AP can indeed replace the S. cerevisiae phosphoglycerate kinase, a multi-copy plasmid carrying the h-AP gene under control of the yeast TDH3 promoter was introduced into a pgk1 delta mutant of S. cerevisiae. A strain carrying the expression vector without the h-AP cassette was used as a reference. For both strains, steady-state carbon- and energy-limited chemostat cultures were obtained at a dilution rate of 0.10 h(-1) on a medium containing a mixture of glucose and ethanol (15% and 85% on a carbon basis, respectively). Although the h-AP strain exhibited a high acylphosphatase activity in cell extracts, switching to glucose as sole carbon and energy source resulted in a complete arrest of glucose consumption and growth. The lack of a functional glycolytic pathway was further evident from the absence of ethanol formation in the presence of excess glucose in the culture. As h-AP cannot replace yeast phosphoglycerate kinase in vivo, the enzyme is not a useful tool to modify the ATP yield of glycolysis in S. cerevisiae. PMID:11761363

  4. Genetic Variation of the Repeated Mal Loci in Natural Populations of Saccharomyces Cerevisiae and Saccharomyces Paradoxus

    PubMed Central

    Naumov, G. I.; Naumova, E. S.; Michels, C. A.

    1994-01-01

    In Saccharomyces cerevisiae, the gene functions required to ferment the disaccharide maltose are encoded by the MAL loci. Any one of five highly sequence homologous MAL loci identified in various S. cerevisiae strains (called MAL1, 2, 3, 4 and 6) is sufficient to ferment maltose. Each is a complex of three genes encoding maltose permease, maltase and a transcription activator. This family of loci maps to telomere-linked positions on different chromosomes and most natural strains contain more than one MAL locus. A number of naturally occurring, mutant alleles of MAL1 and MAL3 have been characterized which lack one or more of the gene functions encoded by the fully functional MAL loci. Loss of these gene functions appears to have resulted from mutation and/or rearrangement within the locus. Studies to date concentrated on the standard maltose fermenting strains of S. cerevisiae available from the Berkeley Yeast Stock Center collection. In this report we extend our genetic analysis of the MAL loci to a number of maltose fermenting and nonfermenting natural strains of S. cerevisiae and Saccharomyces paradoxus. No new MAL loci were discovered but several new mutant alleles of MAL1 were identified. The evolution of this gene family is discussed. PMID:8005435

  5. Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

    PubMed Central

    Erratt, J A; Nasim, A

    1986-01-01

    A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae. Images PMID:3009402

  6. Cytoplasmic dynein is required for normal nuclear segregation in yeast.

    PubMed Central

    Eshel, D; Urrestarazu, L A; Vissers, S; Jauniaux, J C; van Vliet-Reedijk, J C; Planta, R J; Gibbons, I R

    1993-01-01

    We have identified the gene DYN1, which encodes the heavy chain of cytoplasmic dynein in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence (M(r) 471,305) reveals the presence of four P-loop motifs, as in all dyneins known so far, and has 28% overall identity to the dynein heavy chain of Dictyostelium [Koonce, M. P., Grissom, P. M. & McIntosh, J. R. (1992) J. Cell Biol. 119, 1597-1604] with 40% identity in the putative motor domain. Disruption of DYN1 causes misalignment of the spindle relative to the bud neck during cell division and results in abnormal distribution of the dividing nuclei between the mother cell and the bud. Cytoplasmic dynein, by generating force along cytoplasmic microtubules, may play an important role in the proper alignment of the mitotic spindle in yeast. Images Fig. 3 Fig. 4 PMID:8248224

  7. Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae

    SciTech Connect

    Yannai, S.; Berdicevsky, I.; Duek, L. )

    1991-01-01

    Saccharomyces cerevisiae and Candida albicans were incubated with 0.25, 0.5, or 0.75 {mu}g of Hg (as HgCl{sub 2}) per ml of Nelson's medium in the presence of trace amounts of oxygen at 28{degree}C for 12 days. Two control media were used, one without added Hg and one without yeast inoculum. Yeast cell growth was estimated after 1, 2, 3, and 8 days of incubation. The contents of organomercury in the system and of elemental mercury released from the media and collected in traps were determined at the end of the experiments. The results were as follows: (1) C. albicans was the more mercury-resistant species, but both yeast species failed to grown in the media containing 0.75 {mu}g of Hg per ml.; (2) The amounts of organomercury produced by the two species were proportional to the amount of HgCl{sub 2} added to the medium. In all cases C. albicans produced considerably larger amounts of methylmercury than S. cerevisiae; (3) The amounts of elemental Hg produced were inversely proportional to the HgCl{sub 2} level added in the case of S. cerevisiae but were all similar in the case of C. albicans;and (4) Neither organomercury nor elemental Hg was produced in any of the control media.

  8. Biogeographical characterization of Saccharomyces cerevisiae wine yeast by molecular methods

    PubMed Central

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

    2013-01-01

    Biogeography is the descriptive and explanatory study of spatial patterns and processes involved in the distribution of biodiversity. Without biogeography, it would be difficult to study the diversity of microorganisms because there would be no way to visualize patterns in variation. Saccharomyces cerevisiae, “the wine yeast,” is the most important species involved in alcoholic fermentation, and in vineyard ecosystems, it follows the principle of “everything is everywhere.” Agricultural practices such as farming (organic versus conventional) and floor management systems have selected different populations within this species that are phylogenetically distinct. In fact, recent ecological and geographic studies highlighted that unique strains are associated with particular grape varieties in specific geographical locations. These studies also highlighted that significant diversity and regional character, or ‘terroir,’ have been introduced into the winemaking process via this association. This diversity of wild strains preserves typicity, the high quality, and the unique flavor of wines. Recently, different molecular methods were developed to study population dynamics of S. cerevisiae strains in both vineyards and wineries. In this review, we will provide an update on the current molecular methods used to reveal the geographical distribution of S. cerevisiae wine yeast. PMID:23805132

  9. Multiparameter analysis of apoptosis in puromycin-treated Saccharomyces cerevisiae.

    PubMed

    Citterio, Barbara; Albertini, Maria Cristina; Ghibelli, Lina; Falcieri, Elisabetta; Battistelli, Michela; Canonico, Barbara; Rocchi, Marco B L; Teodori, Laura; Ciani, Maurizio; Piatti, Elena

    2015-08-01

    In Saccharomyces cerevisiae, a typical apoptotic phenotype is induced by some stress factors such as sugars, acetic acid, hydrogen peroxide, aspirin and age. Nevertheless, no data have been reported for apoptosis induced by puromycin, a damaging agent known to induce apoptosis in mammalian cells. We treated S. cerevisiae with puromycin to induce apoptosis and evaluated the percentage of dead cells by using Hoechst 33342 staining, transmission electron microscopy (TEM) and Annexin V flow cytometry (FC) analysis. Hoechst 33342 fluorescence images were processed to acquire parameters to use for multiparameter analysis [and perform a principal component analysis, (PCA)]. Cell viability was evaluated by Rhodamine 123 (Rh 123) and Acridine Orange microscope fluorescence staining. The results show puromycin-induced apoptosis in S. cerevisiae, and the PCA analysis indicated that the increasing percentage of apoptotic cells delineated a well-defined graph profile. The results were supported by TEM and FC. This study gives new insights into yeast apoptosis using puromycin as inducer agent, and PCA analysis may complement molecular analysis facilitating further studies to its detection. PMID:25868793

  10. Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

    PubMed Central

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

    2015-01-01

    The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes. PMID:25584613

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

    PubMed

    Stefanini, Irene; Dapporto, Leonardo; Legras, Jean-Luc; Calabretta, Antonio; Di Paola, Monica; De Filippo, Carlotta; Viola, Roberto; Capretti, Paolo; Polsinelli, Mario; Turillazzi, Stefano; Cavalieri, Duccio

    2012-08-14

    Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vector and natural reservoir of S. cerevisiae during all seasons. We provide experimental evidence that queens of social wasps overwintering as adults (Vespa crabro and Polistes spp.) can harbor yeast cells from autumn to spring and transmit them to their progeny. This result is mirrored by field surveys of the genetic variability of natural strains of yeast. Microsatellites and sequences of a selected set of loci able to recapitulate the yeast strain's evolutionary history were used to compare 17 environmental wasp isolates with a collection of strains from grapes from the same region and more than 230 strains representing worldwide yeast variation. The wasp isolates fall into subclusters representing the overall ecological and industrial yeast diversity of their geographic origin. Our findings indicate that wasps are a key environmental niche for the evolution of natural S. cerevisiae populations, the dispersion of yeast cells in the environment, and the maintenance of their diversity. The close relatedness of several wasp isolates with grape and wine isolates reflects the crucial role of human activities on yeast population structure, through clonal expansion and selection of specific strains during the biotransformation of fermented foods, followed by dispersal mediated by insects and other animals. PMID:22847440

  12. Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts.

    PubMed Central

    Makarow, M

    1985-01-01

    When vesicular stomatitis virus was incubated with Saccharomyces cerevisiae spheroplasts at 37 degrees C, part of the virus was internalized by the spheroplasts as shown by the following criteria. (i) The spheroplast-associated virus was protected from proteinase K digestion, which releases surface-bound virus by degrading the envelope glycoproteins. (ii) The spheroplast-associated virus was resistant to mild Triton X-100 treatment, which readily solubilizes the virus. The same results were obtained with Semliki Forest virus. Internalization of the two viruses followed linear kinetics up to 90 min at 37 degrees C. Internalization was concentration- and temperature-dependent. At 11 degrees C no uptake could be detected for at least 2 h. Homogenization and organelle fractionation protocols were designed for the S. cerevisiae spheroplasts to study the compartments into which the virions were internalized. Three compartments containing both marker viruses could be separated in density gradients. One coincided with vacuole markers, one banded at a slightly higher and one at a similar density to the plasma membrane markers. Thus, S. cerevisiae spheroplasts appear to have the capability of endocytosing particulate markers like viruses. The companion paper describes internalization of two soluble macromolecules, alpha-amylase and fluorescent dextran, into intact cells. Images Fig. 2. Fig. 4. PMID:2992948

  13. Sucrose fermentation by Saccharomyces cerevisiae lacking hexose transport.

    PubMed

    Batista, Anderson S; Miletti, Luiz C; Stambuk, Boris U

    2004-01-01

    Sucrose is the major carbon source used by Saccharomyces cerevisiae during production of baker's yeast, fuel ethanol and several distilled beverages. It is generally accepted that sucrose fermentation proceeds through extracellular hydrolysis of the sugar, mediated by the periplasmic invertase, producing glucose and fructose that are transported into the cells and metabolized. In the present work we analyzed the contribution to sucrose fermentation of a poorly characterized pathway of sucrose utilization by S. cerevisiae cells, the active transport of the sugar through the plasma membrane and its intracellular hydrolysis. A yeast strain that lacks the major hexose transporters (hxt1-hxt7 and gal2) is incapable of growing on or fermenting glucose or fructose. Our results show that this hxt-null strain is still able to ferment sucrose due to direct uptake of the sugar into the cells. Deletion of the AGT1 gene, which encodes a high-affinity sucrose-H(+) symporter, rendered cells incapable of sucrose fermentation. Since sucrose is not an inducer of the permease, expression of the AGT1 must be constitutive in order to allow growth of the hxt-null strain on sucrose. The molecular characterization of active sucrose transport and fermentation by S. cerevisiae cells opens new opportunities to optimize yeasts for sugarcane-based industrial processes. PMID:15741738

  14. Comparative proteomic analysis of Saccharomyces cerevisiae under different nitrogen sources.

    PubMed

    Zhao, Shaohui; Zhao, Xinrui; Zou, Huijun; Fu, Jianwei; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2014-04-14

    In cultures containing multiple sources of nitrogen, Saccharomyces cerevisiae exhibits a sequential use of nitrogen sources through a mechanism known as nitrogen catabolite repression (NCR). To identify proteins differentially expressed due to NCR, proteomic analysis of S. cerevisiae S288C under different nitrogen source conditions was performed using two-dimensional gel electrophoresis (2-DE), revealing 169 candidate protein spots. Among these 169 protein spots, 121 were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF). The identified proteins were closely associated with four main biological processes through Gene Ontology (GO) categorical analysis. The identification of the potential proteins and cellular processes related to NCR offer a global overview of changes elicited by different nitrogen sources, providing clues into how yeast adapt to different nutritional conditions. Moreover, by comparing our proteomic data with corresponding mRNA data, proteins regulated at the transcriptional and post-transcriptional level could be distinguished. Biological significance In S. cerevisiae, different nitrogen sources provide different growth characteristics and generate different metabolites. The nitrogen catabolite repression (NCR) process plays an important role for S. cerevisiae in the ordinal utilization of different nitrogen sources. NCR process can result in significant shift of global metabolic networks. Previous works on NCR primarily focused on transcriptomic level. The results obtained in this study provided a global atlas of the proteome changes triggered by different nitrogen sources and would facilitate the understanding of mechanisms for how yeast could adapt to different nutritional conditions. PMID:24530623

  15. A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis.

    PubMed Central

    Efimov, V P; Morris, N R

    1998-01-01

    Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution. PMID:9584089

  16. Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

    PubMed Central

    2012-01-01

    Background Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. Methods We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. Results We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. Conclusions In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagy associated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism whereby propolis causes cell death in this organism at the concentration herein tested. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi. PMID:23092287

  17. Effects of Fusariotoxin T-2 on Saccharomyces cerevisiae and Saccharomyces carlsbergensis

    PubMed Central

    Schappert, Keith T.; Khachatourians, George G.

    1983-01-01

    A Fusarium metabolite, T-2 toxin, inhibits the growth of Saccharomyces carlsbergensis and Saccharomyces cerevisiae. The growth inhibitory concentrations of T-2 toxin were 40 and 100 μg/ml, respectively, for exponentially growing cultures of the two yeasts. S. carlsbergensis was more sensitive to the toxin and exhibited a biphasic dose-response curve. Addition of the toxin at 10 μg/ml of S. carlsbergensis culture resulted in a retardation of growth as measured turbidimetrically, after only 30 to 40 min. This action was reversible upon washing the cells free of the toxin. The sensitivity of the yeasts to the toxin was dependent upon the types and concentrations of carbohydrates used in the growth media. The sensitivity of the cells to the toxin decreased in glucose-repressed cultures. These results suggest that T-2 toxin interferes with mitochondrial functions of these yeasts. Images PMID:16346249

  18. Exposure to benzene metabolites causes oxidative damage in Saccharomyces cerevisiae.

    PubMed

    Raj, Abhishek; Nachiappan, Vasanthi

    2016-06-01

    Hydroquinone (HQ) and benzoquinone (BQ) are known benzene metabolites that form reactive intermediates such as reactive oxygen species (ROS). This study attempts to understand the effect of benzene metabolites (HQ and BQ) on the antioxidant status, cell morphology, ROS levels and lipid alterations in the yeast Saccharomyces cerevisiae. There was a reduction in the growth pattern of wild-type cells exposed to HQ/BQ. Exposure of yeast cells to benzene metabolites increased the activity of the anti-oxidant enzymes catalase, superoxide dismutase and glutathione peroxidase but lead to a decrease in ascorbic acid and reduced glutathione. Increased triglyceride level and decreased phospholipid levels were observed with exposure to HQ and BQ. These results suggest that the enzymatic antioxidants were increased and are involved in the protection against macromolecular damage during oxidative stress; presumptively, these enzymes are essential for scavenging the pro-oxidant effects of benzene metabolites. PMID:27016252

  19. Protein production in Saccharomyces cerevisiae for systems biology studies.

    PubMed

    Malys, Naglis; Wishart, Jill A; Oliver, Stephen G; McCarthy, John E G

    2011-01-01

    Proteins together with metabolites, nucleic acids, lipids, and other intracellular molecules form biological systems that involve networks of functional and physical interactions. To understand these interactions and the many other characteristics of proteins in the context of biochemical networks and systems biology, research aimed at studying medium and large sets of proteins is required. This either involves an investigation focused on individual protein activities in the mixture (e.g., cell extracts) or a protein characterization in the isolated form. This chapter provides an overview on the currently available resources and strategies for isolation of proteins from Saccharomyces cerevisiae. The use of standardized gene expression systems is discussed, and protein production protocols applied to the data generation pipeline for systems biology are described in detail. PMID:21943899

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

    PubMed

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

    2014-03-01

    Bioaccumulation via growing cells is a potential technique for heavy metal removal from food materials. The cadmium bioaccumulation characteristics by growing Zygosaccharomyces rouxii and Saccharomyces cerevisiae were investigated. Z. rouxii displayed powerful cadmium removal ability at low cadmium concentrations, which mainly depended on the intracellular cadmium bioaccumulation. The percentage of intracellular cadmium bioaccumulation of both yeasts obviously decreased with the increase of initial biomass and cadmium concentrations. Low pH and elevated concentrations of zinc and copper significantly decreased the intracellular cadmium bioaccumulation of both yeasts but improved the cadmium tolerance and the cell-surface cadmium bioaccumulation of Z. rouxii. Cadmium removal of Z. rouxii was improved by zinc and copper conditionally. Z. rouxii that possessed more powerful cadmium tolerance and removal ability at low pH and high concentration of competing ions can be developed into a potential cadmium removal agent using in complex food environment in future. PMID:24440489

  1. Nucleotide sequence of the GDS1 gene of Saccharomyces cerevisiae.

    PubMed

    Konopinska, A; Szczesniak, B; Boguta, M

    1995-12-01

    We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae. The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database. GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms. Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product. PMID:8750239

  2. Isolation and Partial Purification of the Saccharomyces cerevisiae Cytokinetic Apparatus

    PubMed Central

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

    2009-01-01

    Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested—actin, myosin heavy and light chain, and IQGAP—as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis. PMID:19790107

  3. Hormetic Effect of H2O2 in Saccharomyces cerevisiae

    PubMed Central

    Valishkevych, Bohdana V.

    2016-01-01

    In this study, we investigated the relationship between target of rapamycin (TOR) and H2O2-induced hormetic response in the budding yeast Saccharomyces cerevisiae grown on glucose or fructose. In general, our data suggest that: (1) hydrogen peroxide (H2O2) induces hormesis in a TOR-dependent manner; (2) the H2O2-induced hormetic dose–response in yeast depends on the type of carbohydrate in growth medium; (3) the concentration-dependent effect of H2O2 on yeast colony growth positively correlates with the activity of glutathione reductase that suggests the enzyme involvement in the H2O2-induced hormetic response; and (4) both TOR1 and TOR2 are involved in the reciprocal regulation of the activity of glucose-6-phosphate dehydrogenase and glyoxalase 1. PMID:27099601

  4. Nucleotide sequence of the RAD10 gene of Saccharomyces cerevisiae.

    PubMed Central

    Reynolds, P; Prakash, L; Dumais, D; Perozzi, G; Prakash, S

    1985-01-01

    The RAD10 gene is one of several genes in Saccharomyces cerevisiae required for incision of u.v.-irradiated or cross-linked DNA. We have determined the nucleotide sequence of the RAD10 gene and its flanking regions. The RAD10 nucleotide sequence presented here differs significantly from that recently reported. The RAD10 protein predicted from the nucleotide sequence contains 210 amino acids with a calculated mol. wt. of 24 310. The middle portion of the RAD10 protein, which is highly basic and also contains eight of the total of 10 tyrosine residues present in the protein, may be involved in DNA binding by ionic interactions and tyrosine intercalation between the bases of DNA. A genomic deletion of the entire RAD10 gene does not affect viability; however, the rad10 deletion mutant is highly u.v. sensitive. Images Fig. 4. PMID:3912171

  5. Characterization of Encapsulated Berberine in Yeast Cells of Saccharomyces cerevisiae.

    PubMed

    Salari, Roshanak; Rajabi, Omid; Khashyarmanesh, Zahra; Fathi Najafi, Mohsen; Fazly Bazzaz, BiBi Sedigheh

    2015-01-01

    Berberine was loaded in yeast cells of Saccharomyces cerevisiaeas a novel pharmaceutical carrier to improve the treatment ofmany diseases. The yeast-encapsulated active materialsshowedhigh stability and bioavailability due to the enhanced solubility and sustained releasing. In this study, different characteristics of prepared berberine loaded yeast cells (loading capacity, release kinetic order, MIC and stability) were evaluatedby different analytical methods (fluorescence spectroscopy, HPLC and SEM).The loading capacity was about 78% 0.6%.Berberine release patterns of microcapsules happened in two different stages and followed by zero and first-order kinetic,respectively. About 99% of all active material released during 34 h. MIC was improved by berberine loaded microcapsules in comparison withberberine powder. The microcapsules were completely stable. Berberine loaded Sac. Cerevisiae could be considered as a favorite sustained release drug delivery system. The yeast would be applied as an efficient carrier to improve various properties of different active materials. PMID:26664393

  6. Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae

    SciTech Connect

    Hayashi, Naoyuki Kobayashi, Masahiko; Shimizu, Hiroko; Yamamoto, Ken-ichi; Murakami, Seishi; Nishimoto, Takeharu

    2007-11-23

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

  7. Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

    PubMed

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

    1988-01-01

    An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures. PMID:3278685

  8. Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

    PubMed Central

    Symington, Lorraine S.; Rothstein, Rodney; Lisby, Michael

    2014-01-01

    Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell. PMID:25381364

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    Our studies on the tolerance of plants and animals against very high pressure of several GPa have been extended to a smaller sized fungus, the budding yeast Saccharomyces cerevisiae. Several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate, and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar. It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for longer than 12 h were found dead. The high pressure tolerance of budding yeast is a little weaker than that of tardigrades.

  10. Characterization of Encapsulated Berberine in Yeast Cells of Saccharomyces cerevisiae

    PubMed Central

    Salari, Roshanak; Rajabi, Omid; Khashyarmanesh, Zahra; Fathi Najafi, Mohsen; Fazly Bazzaz, BiBi Sedigheh

    2015-01-01

    Berberine was loaded in yeast cells of Saccharomyces cerevisiaeas a novel pharmaceutical carrier to improve the treatment ofmany diseases. The yeast-encapsulated active materialsshowedhigh stability and bioavailability due to the enhanced solubility and sustained releasing. In this study, different characteristics of prepared berberine loaded yeast cells (loading capacity, release kinetic order, MIC and stability) were evaluatedby different analytical methods (fluorescence spectroscopy, HPLC and SEM).The loading capacity was about 78% ± 0.6%.Berberine release patterns of microcapsules happened in two different stages and followed by zero and first-order kinetic,respectively. About 99% of all active material released during 34 h. MIC was improved by berberine loaded microcapsules in comparison withberberine powder. The microcapsules were completely stable. Berberine loaded Sac. Cerevisiae could be considered as a favorite sustained release drug delivery system. The yeast would be applied as an efficient carrier to improve various properties of different active materials. PMID:26664393

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

    PubMed Central

    Liu, Ling-ling; Jia, Bo; Zhao, Fang; Huang, Wei-dong; Zhan, Ji-cheng

    2015-01-01

    At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu2+ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu2+ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu2+ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress. PMID:26030864

  12. Catabolism of bis(5'-nucleosidyl) tetraphosphates in Saccharomyces cerevisiae.

    PubMed Central

    Plateau, P; Fromant, M; Schmitter, J M; Blanquet, S

    1990-01-01

    Bis(5'-adenosyl) tetraphosphate (Ap4A) phosphorylase II (P. Plateau, M. Fromant, J. M. Schmitter, J. M. Buhler, and S. Blanquet, J. Bacteriol. 171:6437-6445, 1989) was obtained in a homogeneous form through a 40,000-fold purification, starting from a Saccharomyces cerevisiae strain devoid of Ap4A phosphorylase I activity. The former enzyme behaves as a 36.8K monomer. As with Ap4A phosphorylase I, the addition of divalent cations is required for the expression of activity. Mn2+, Mg2+, and Ca2+ sustain phosphorolysis by the two enzymes, whereas Co2+ and Cd2+ stimulate only phosphorylase II activity. All bis(5'-nucleosidyl) tetraphosphates assayed (Ap4A, Ap4C, Ap4G, Ap4U, Gp4G, and Gp4U) are substrates of the two enzymes. However, Ap4A phosphorylase II shows a marked preference for A-containing substrates. The two enzymes catalyze adenosine 5'-phosphosulfate phosphorolysis or an exchange reaction between Pi and the beta-phosphate of any nucleoside diphosphate. They can also produce Ap4A at the expense of ATP and ADP. The gene (APA2) encoding Ap4A phosphorylase II was isolated and sequenced. The deduced amino acid sequence shares 60% identity with that of Ap4A phosphorylase I. Disruption of APA2 and/or APA1 shows that none of these genes is essential for the viability of Saccharomyces cerevisiae. The concentrations of all bis(5'-nucleosidyl) tetraphosphates are increased in an apa1 apa2 double mutant, as compared with the parental wild-type strain. The factor of increase is 5 to 50 times, depending on the nucleotide. This observation supports the conclusion that, in vivo, Ap4A phosphorylase II, like Ap4A phosphorylase I, participates in the catabolism rather than the synthesis of the bis(5'-nucleosidyl) tetraphosphates. PMID:2174863

  13. Water treatment process and system for metals removal using Saccharomyces cerevisiae

    DOEpatents

    Krauter, Paula A. W.; Krauter, Gordon W.

    2002-01-01

    A process and a system for removal of metals from ground water or from soil by bioreducing or bioaccumulating the metals using metal tolerant microorganisms Saccharomyces cerevisiae. Saccharomyces cerevisiae is tolerant to the metals, able to bioreduce the metals to the less toxic state and to accumulate them. The process and the system is useful for removal or substantial reduction of levels of chromium, molybdenum, cobalt, zinc, nickel, calcium, strontium, mercury and copper in water.

  14. Genomic Evolution of Saccharomyces cerevisiae under Chinese Rice Wine Fermentation

    PubMed Central

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

    2014-01-01

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

  15. Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.

    PubMed Central

    Galibert, F; Alexandraki, D; Baur, A; Boles, E; Chalwatzis, N; Chuat, J C; Coster, F; Cziepluch, C; De Haan, M; Domdey, H; Durand, P; Entian, K D; Gatius, M; Goffeau, A; Grivell, L A; Hennemann, A; Herbert, C J; Heumann, K; Hilger, F; Hollenberg, C P; Huang, M E; Jacq, C; Jauniaux, J C; Katsoulou, C; Karpfinger-Hartl, L

    1996-01-01

    The complete nucleotide sequence of Saccharomyces cerevisiae chromosome X (745 442 bp) reveals a total of 379 open reading frames (ORFs), the coding region covering approximately 75% of the entire sequence. One hundred and eighteen ORFs (31%) correspond to genes previously identified in S. cerevisiae. All other ORFs represent novel putative yeast genes, whose function will have to be determined experimentally. However, 57 of the latter subset (another 15% of the total) encode proteins that show significant analogy to proteins of known function from yeast or other organisms. The remaining ORFs, exhibiting no significant similarity to any known sequence, amount to 54% of the total. General features of chromosome X are also reported, with emphasis on the nucleotide frequency distribution in the environment of the ATG and stop codons, the possible coding capacity of at least some of the small ORFs (<100 codons) and the significance of 46 non-canonical or unpaired nucleotides in the stems of some of the 24 tRNA genes recognized on this chromosome. Images PMID:8641269

  16. The nucleotide sequence of Saccharomyces cerevisiae chromosome IV.

    PubMed

    Jacq, C; Alt-Mörbe, J; Andre, B; Arnold, W; Bahr, A; Ballesta, J P; Bargues, M; Baron, L; Becker, A; Biteau, N; Blöcker, H; Blugeon, C; Boskovic, J; Brandt, P; Brückner, M; Buitrago, M J; Coster, F; Delaveau, T; del Rey, F; Dujon, B; Eide, L G; Garcia-Cantalejo, J M; Goffeau, A; Gomez-Peris, A; Zaccaria, P

    1997-05-29

    The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome IV has been determined. Apart from chromosome XII, which contains the 1-2 Mb rDNA cluster, chromosome IV is the longest S. cerevisiae chromosome. It was split into three parts, which were sequenced by a consortium from the European Community, the Sanger Centre, and groups from St Louis and Stanford in the United States. The sequence of 1,531,974 base pairs contains 796 predicted or known genes, 318 (39.9%) of which have been previously identified. Of the 478 new genes, 225 (28.3%) are homologous to previously identified genes and 253 (32%) have unknown functions or correspond to spurious open reading frames (ORFs). On average there is one gene approximately every two kilobases. Superimposed on alternating regional variations in G+C composition, there is a large central domain with a lower G+C content that contains all the yeast transposon (Ty) elements and most of the tRNA genes. Chromosome IV shares with chromosomes II, V, XII, XIII and XV some long clustered duplications which partly explain its origin. PMID:9169867

  17. Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae

    PubMed Central

    Aimanianda, Vishukumar; Clavaud, Cécile; Simenel, Catherine; Fontaine, Thierry; Delepierre, Muriel; Latgé, Jean-Paul

    2009-01-01

    Despite its essential role in the yeast cell wall, the exact composition of the β-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant β-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ∼38 kDa and could be hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR (1H and 13C) analyses confirmed it to be a β-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two β-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[14C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for β-(1,6)-glucan synthetic activity were defined. PMID:19279004

  18. Identification of a Saccharomyces cerevisiae glucosidase that hydrolyzes flavonoid glucosides.

    PubMed

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

    2011-03-01

    Baker's yeast (Saccharomyces cerevisiae) whole-cell bioconversions of naringenin 7-O-β-glucoside revealed considerable β-glucosidase activity, which impairs any strategy to generate or modify flavonoid glucosides in yeast transformants. Up to 10 putative glycoside hydrolases annotated in the S. cerevisiae genome database were overexpressed with His tags in yeast cells. Examination of these recombinant, partially purified polypeptides for hydrolytic activity with synthetic chromogenic α- or β-glucosides identified three efficient β-glucosidases (EXG1, SPR1, and YIR007W), which were further assayed with natural flavonoid β-glucoside substrates and product verification by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). Preferential hydrolysis of 7- or 4'-O-glucosides of isoflavones, flavonols, flavones, and flavanones was observed in vitro with all three glucosidases, while anthocyanins were also accepted as substrates. The glucosidase activities of EXG1 and SPR1 were completely abolished by Val168Tyr mutation, which confirmed the relevance of this residue, as reported for other glucosidases. Most importantly, biotransformation experiments with knockout yeast strains revealed that only EXG1 knockout strains lost the capability to hydrolyze flavonoid glucosides. PMID:21216897

  19. Quantifying separation and similarity in a Saccharomyces cerevisiae metapopulation

    PubMed Central

    Knight, Sarah; Goddard, Matthew R

    2015-01-01

    Eukaryotic microbes are key ecosystem drivers; however, we have little theory and few data elucidating the processes influencing their observed population patterns. Here we provide an in-depth quantitative analysis of population separation and similarity in the yeast Saccharomyces cerevisiae with the aim of providing a more detailed account of the population processes occurring in microbes. Over 10 000 individual isolates were collected from native plants, vineyards and spontaneous ferments of fruit from six major regions spanning 1000 km across New Zealand. From these, hundreds of S. cerevisiae genotypes were obtained, and using a suite of analytical methods we provide comprehensive quantitative estimates for both population structure and rates of gene flow or migration. No genetic differentiation was detected within geographic regions, even between populations inhabiting native forests and vineyards. We do, however, reveal a picture of national population structure at scales above ∼100 km with distinctive populations in the more remote Nelson and Central Otago regions primarily contributing to this. In addition, differential degrees of connectivity between regional populations are observed and correlate with the movement of fruit by the New Zealand wine industry. This suggests some anthropogenic influence on these observed population patterns. PMID:25062126

  20. Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Yumoto, N; Kawata, Y; Noda, S; Tokushige, M

    1991-03-01

    Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000. The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis. PMID:1897932

  1. Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining.

    PubMed

    Yu, Xin; Gabriel, Abram

    2004-02-01

    Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells. Under these conditions, reciprocal translocations via nonhomologous end joining (NHEJ) occur at frequencies of approximately 2-7 x 10(-5)/cell exposed to the DSBs. Yku80p is a component of the cell's NHEJ machinery. In its absence, reciprocal translocations still occur, but the junctions are associated with deletions and extended overlapping sequences. After induction of a single DSB, translocations and inversions are recovered in wild-type and rad52 strains. In these rearrangements, a nonrandom assortment of sites have fused to the DSB, and their junctions show typical signs of NHEJ. The sites tend to be between open reading frames or within Ty1 LTRs. In some cases the translocation partner is formed by a break at a cryptic HO recognition site. Our results demonstrate that NHEJ-mediated reciprocal translocations can form in S. cerevisiae as a consequence of DSB repair. PMID:15020464

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

    PubMed

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

    2016-04-01

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

  3. Exploring improved endoglucanase expression in Saccharomyces cerevisiae strains.

    PubMed

    du Plessis, Lisa; Rose, Shaunita H; van Zyl, Willem H

    2010-05-01

    The endoglucanase I and II genes (egI or Cel7B and egII or Cel5A) of Trichoderma reesei QM6a were successfully cloned and expressed in Saccharomyces cerevisiae under the transcriptional control of the yeast ENO1 promoter and terminator sequences. Random mutagenesis of the egI-bearing plasmid resulted in a twofold increase in extracellular EGI activity. Both endoglucanase genes were co-expressed with the synthetic, codon-optimised cellobiohydrolase gene (s-cbhI) from T. reesei as well as the beta-glucosidase gene (bgl1) from Saccharomycopsis fibuligera in S. cerevisiae. Extracellular endoglucanase activity was lower when co-expressed with s-cbhI or bgl1. Recombinant strains were able to hydrolyse phosphoric acid swollen cellulose, generating mainly cellotriose, cellobiose and glucose. Cellobiose accumulated, suggesting the beta-glucosidase activity to be the rate-limiting factor. As a consequence, the recombinant strains were unable to produce enough glucose for growth on amorphous cellulose. The results of this study provide insight into further optimisation of recombinantly expressed cellulase combinations for saccharification and fermentation of cellulose to ethanol. PMID:20041241

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

    PubMed

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

    2015-06-01

    In the past decade industrial-scale production of renewable transportation biofuels has been developed as an alternative to fossil fuels, with ethanol as the most prominent biofuel and yeast as the production organism of choice. However, ethanol is a less efficient substitute fuel for heavy-duty and maritime transportation as well as aviation due to its low energy density. Therefore, new types of biofuels, such as alkanes, are being developed that can be used as drop-in fuels and can substitute gasoline, diesel, and kerosene. Here, we describe for the first time the heterologous biosynthesis of long-chain alkanes by the yeast Saccharomyces cerevisiae. We show that elimination of the hexadecenal dehydrogenase Hfd1 and expression of a redox system are essential for alkane biosynthesis in yeast. Deletion of HFD1 together with expression of an alkane biosynthesis pathway resulted in the production of the alkanes tridecane, pentadecane, and heptadecane. Our study provides a proof of principle for producing long-chain alkanes in the industrial workhorse S. cerevisiae, which was so far limited to bacteria. We anticipate that these findings will be a key factor for further yeast engineering to enable industrial production of alkane based drop-in biofuels, which can allow the biofuel industry to diversify beyond bioethanol. PMID:25545362

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

    PubMed

    Hou, Jin; Osterlund, Tobias; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2013-04-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously. PMID:23208612

  6. High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. Results We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. Conclusions Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome. PMID:21910902

  7. Physiological adaptations of Saccharomyces cerevisiae evolved for improved butanol tolerance

    PubMed Central

    2013-01-01

    Background Butanol is a chemical with potential uses as biofuel and solvent, which can be produced by microbial fermentation. However, the end product toxicity is one of the main obstacles for developing the production process irrespective of the choice of production organism. The long-term goal of the present project is to produce 2-butanol in Saccharomyces cerevisiae. Therefore, unraveling the toxicity mechanisms of solvents such as butanol and understanding the mechanisms by which tolerant strains of S. cerevisiae adapt to them would be an important contribution to the development of a bio-based butanol production process. Results A butanol tolerant S. cerevisiae was achieved through a series of sequential batch cultures with gradual increase of 2-butanol concentration. The final mutant (JBA-mut) tolerates all different alcohols tested at higher concentrations compared to the wild type (JBA-wt). Proteomics analysis of the two strains grown under mild butanol-stress revealed 46 proteins changing their expression by more than 1.5-fold in JBA-mut, 34 of which were upregulated. Strikingly, 21 out of the 34 upregulated proteins were predicted constituents of mitochondria. Among the non-mitochondrial up-regulated proteins, the minor isoform of Glycerol-3-phosphatase (Gpp2) was the most notable, since it was the only tested protein whose overexpression was found to confer butanol tolerance. Conclusion The study demonstrates several differences between the butanol tolerant mutant and the wild type. Upregulation of proteins involved in the mitochondrial ATP synthesizing machinery constituents and glycerol biosynthesis seem to be beneficial for a successful adaptation of yeast cells to butanol stress. PMID:23855998

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

    PubMed Central

    Kavanaugh, Laura A.; Dietrich, Fred S.

    2009-01-01

    Non-coding RNA (ncRNA) play an important and varied role in cellular function. A significant amount of research has been devoted to computational prediction of these genes from genomic sequence, but the ability to do so has remained elusive due to a lack of apparent genomic features. In this work, thermodynamic stability of ncRNA structural elements, as summarized in a Z-score, is used to predict ncRNA in the yeast Saccharomyces cerevisiae. This analysis was coupled with comparative genomics to search for ncRNA genes on chromosome six of S. cerevisiae and S. bayanus. Sets of positive and negative control genes were evaluated to determine the efficacy of thermodynamic stability for discriminating ncRNA from background sequence. The effect of window sizes and step sizes on the sensitivity of ncRNA identification was also explored. Non-coding RNA gene candidates, common to both S. cerevisiae and S. bayanus, were verified using northern blot analysis, rapid amplification of cDNA ends (RACE), and publicly available cDNA library data. Four ncRNA transcripts are well supported by experimental data (RUF10, RUF11, RUF12, RUF13), while one additional putative ncRNA transcript is well supported but the data are not entirely conclusive. Six candidates appear to be structural elements in 5? or 3? untranslated regions of annotated protein-coding genes. This work shows that thermodynamic stability, coupled with comparative genomics, can be used to predict ncRNA with significant structural elements. PMID:19119416

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

    PubMed

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

    2012-10-01

    The aim of this study was to develop a method to optimize expression levels of xylose-metabolizing enzymes to improve xylose utilization capacity of Saccharomyces cerevisiae. A xylose-utilizing recombinant S. cerevisiae strain YY2KL, able to express nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-dependent xylose reductase (XR), nicotinamide adenine dinucleotide (NAD(+))-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK), showed a low ethanol yield and sugar consumption rate. To optimize xylose utilization by YY2KL, a recombinant expression plasmid containing the XR gene was transformed and integrated into the aur1 site of YY2KL. Two recombinant expression plasmids containing an nicotinamide adenine dinucleotide phosphate (NADP(+))-dependent XDH mutant and XK genes were dually transformed and integrated into the 5S ribosomal DNA (rDNA) sites of YY2KL. This procedure allowed systematic construction of an S. cerevisiae library with different ratios of genes for xylose-metabolizing enzymes, and well-grown colonies with different xylose fermentation capacities could be further selected in yeast protein extract (YPX) medium (1 % yeast extract, 2 % peptone, and 2 % xylose). We successfully isolated a recombinant strain with a superior xylose fermentation capacity and designated it as strain YY5A. The xylose consumption rate for strain YY5A was estimated to be 2.32 g/gDCW/h (g xylose/g dry cell weight/h), which was 2.34 times higher than that for the parent strain YY2KL (0.99 g/gDCW/h). The ethanol yield was also enhanced 1.83 times by this novel method. Optimal ratio and expression levels of xylose-metabolizing enzymes are important for efficient conversion of xylose to ethanol. This study provides a novel method that allows rapid and effective selection of ratio-optimized xylose-utilizing yeast strains. This method may be applicable to other multienzyme systems in yeast. PMID:22740288

  10. Exploring the Saccharomyces cerevisiae Volatile Metabolome: Indigenous versus Commercial Strains

    PubMed Central

    Alves, Zélia; Melo, André; Figueiredo, Ana Raquel; Coimbra, Manuel A.; Gomes, Ana C.; Rocha, Sílvia M.

    2015-01-01

    Winemaking is a highly industrialized process and a number of commercial Saccharomyces cerevisiae strains are used around the world, neglecting the diversity of native yeast strains that are responsible for the production of wines peculiar flavours. The aim of this study was to in-depth establish the S. cerevisiae volatile metabolome and to assess inter-strains variability. To fulfill this objective, two indigenous strains (BT2652 and BT2453 isolated from spontaneous fermentation of grapes collected in Bairrada Appellation, Portugal) and two commercial strains (CSc1 and CSc2) S. cerevisiae were analysed using a methodology based on advanced multidimensional gas chromatography (HS-SPME/GC×GC-ToFMS) tandem with multivariate analysis. A total of 257 volatile metabolites were identified, distributed over the chemical families of acetals, acids, alcohols, aldehydes, ketones, terpenic compounds, esters, ethers, furan-type compounds, hydrocarbons, pyrans, pyrazines and S-compounds. Some of these families are related with metabolic pathways of amino acid, carbohydrate and fatty acid metabolism as well as mono and sesquiterpenic biosynthesis. Principal Component Analysis (PCA) was used with a dataset comprising all variables (257 volatile components), and a distinction was observed between commercial and indigenous strains, which suggests inter-strains variability. In a second step, a subset containing esters and terpenic compounds (C10 and C15), metabolites of particular relevance to wine aroma, was also analysed using PCA. The terpenic and ester profiles express the strains variability and their potential contribution to the wine aromas, specially the BT2453, which produced the higher terpenic content. This research contributes to understand the metabolic diversity of indigenous wine microflora versus commercial strains and achieved knowledge that may be further exploited to produce wines with peculiar aroma properties. PMID:26600152

  11. Exploring the Saccharomyces cerevisiae Volatile Metabolome: Indigenous versus Commercial Strains.

    PubMed

    Alves, Zélia; Melo, André; Figueiredo, Ana Raquel; Coimbra, Manuel A; Gomes, Ana C; Rocha, Sílvia M

    2015-01-01

    Winemaking is a highly industrialized process and a number of commercial Saccharomyces cerevisiae strains are used around the world, neglecting the diversity of native yeast strains that are responsible for the production of wines peculiar flavours. The aim of this study was to in-depth establish the S. cerevisiae volatile metabolome and to assess inter-strains variability. To fulfill this objective, two indigenous strains (BT2652 and BT2453 isolated from spontaneous fermentation of grapes collected in Bairrada Appellation, Portugal) and two commercial strains (CSc1 and CSc2) S. cerevisiae were analysed using a methodology based on advanced multidimensional gas chromatography (HS-SPME/GC×GC-ToFMS) tandem with multivariate analysis. A total of 257 volatile metabolites were identified, distributed over the chemical families of acetals, acids, alcohols, aldehydes, ketones, terpenic compounds, esters, ethers, furan-type compounds, hydrocarbons, pyrans, pyrazines and S-compounds. Some of these families are related with metabolic pathways of amino acid, carbohydrate and fatty acid metabolism as well as mono and sesquiterpenic biosynthesis. Principal Component Analysis (PCA) was used with a dataset comprising all variables (257 volatile components), and a distinction was observed between commercial and indigenous strains, which suggests inter-strains variability. In a second step, a subset containing esters and terpenic compounds (C10 and C15), metabolites of particular relevance to wine aroma, was also analysed using PCA. The terpenic and ester profiles express the strains variability and their potential contribution to the wine aromas, specially the BT2453, which produced the higher terpenic content. This research contributes to understand the metabolic diversity of indigenous wine microflora versus commercial strains and achieved knowledge that may be further exploited to produce wines with peculiar aroma properties. PMID:26600152

  12. Influence of temperature and nutrient strength on the susceptibility of Saccharomyces cerevisiae to heavy metals

    SciTech Connect

    Hsu, T.; Lee, L.W.; Chang, T.H. )

    1992-09-01

    Saccharomyces cerevisiae is not only a key microorganism in brewing or fermentation processes, it has also been employed for monitoring aquatic pollutants. The major advantage of using Saccharomyces cerevisiae as a bioassay system is that this yeast can be easily obtained as dry pellets from commercial sources at low cost. In addition to its economical aspect, Saccharomyces cerevisiae, like other microorganisms, is easy to handle, grows rapidly, and provides a large number of homogeneous individuals for utilization in toxicity tests. Although cell growth, cell viability, electron transport and mitochondrial respiration of Saccharomyces cerevisiaes have all been selected as parameters for toxicity assessment, measuring cell growth by absorbance is by farm the most convenient and rapid method when large amounts of water samples are to be tested. Mochida et al. (1988), however, reported that Saccharomyces cerevisiae was five to ten times less sensitive than cell culture systems to cadmium, mercury and nickel, when cell growth of both systems was monitored. This relative insensitivity to heavy metals might handicap the practical use of this yeast strain for bioassays. Since previous studies indicated that the susceptibility of microorganisms to environmental toxicants can be influenced by incubation temperature and nutrient strength, we attempted to examine the effect of incubation temperature and nutrient strength on the susceptibility of Saccharomyces cerevisiae to heavy metals in order to obtain the optimum bioassay sensitivity. In this study, we used cadmium and mercury as model toxicants. 9 refs., 2 figs., 1 tab.

  13. ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. S. cerevisiae strains engineered for xylose fermentation have been made using constitutive promoters to express the req...

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

    PubMed

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

    2014-04-01

    Saccharomyces cerevisiae, known as baker's yeast, is also used as a probiotic agent to treat gastroenteritis by modulating the endogenous flora and immune system. However, since there have been increasing reports of fungemia due to S.cerevisiae and its subspecies S.boulardii, it is recommended that probiotics should be cautiously used in immunosuppressed patients, people with underlying diseases and low-birth weight babies. To emphasize this phenomenon, in this report, a case of S.cerevisiae fungemia developed in a patient given probiotic treatment for antibiotic-associated diarrhea, was presented. An 88-year-old female patient was admitted to our hospital with left hip pain, hypotension, and confusion. Her medical history included hypertension, chronic renal failure, left knee replacement surgery, and recurrent urinary tract infections due to neurogenic bladder. She was transferred to the intensive care unit with the diagnosis of urosepsis. After obtaining blood and urine samples for culture, empirical meropenem (2 x 500 mg) and linezolid (1 x 600 mg) treatment were administered. A central venous catheter (CVC) was inserted and after one day of inotropic support, her hemodynamic parameters were stabilized. The urine culture obtained on admission yielded extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. Urine culture was repeated after three days and no bacteria were isolated. On the 4th day of admission she developed diarrhea. Toxin A/B tests for Clostridium difficile were negative. To relieve diarrhea, S.boulardii (Reflor 250 mg capsules, Sanofi Aventis, Turkey) was administered twice a day, without opening capsules. Two days later, her C-reactive protein (CRP) level increased from 23.2 mg/L to 100 mg/L without fever. Her blood culture taken from the CVC yielded S.cerevisiae. Linezolid and meropenem therapies were stopped on the 13th and 14th days, respectively, while prophylactic fluconazole therapy was replaced with caspofungin 1 x 50 mg on the fifth day. After seven days of therapy CRP and serum creatinine levels decreased to 9.1 mg/L and 1.2 mg/dl, respectively; and she was discharged from the hospital with improvement. The probiotic capsules were used unopen, thus, it was proposed that S.cerevisiae fungemia originated from translocation from the intestinal mucosa. Since it was not possible to investigate the molecular genetics of the strain isolated from the blood culture and the strain present in the probiotic, a definite conclusion about the origin of the strain could not be reached. It was thought that old age and underlying disease of the patient were the related predisposing factors for S.cerevisiae fungemia. This case emphasized that clinicians should be cautious in case of probiotic application even though in encapsulated form, even in immunocompetent patients with a history of long-term hospital stay and use of broad-spectrum antimicrobials since there may be a risk of S.cerevisiae fungemia development. PMID:24819274

  16. Metabolism of sulfur amino acids in Saccharomyces cerevisiae.

    PubMed Central

    Thomas, D; Surdin-Kerjan, Y

    1997-01-01

    Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region. In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p. The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes. PMID:9409150

  17. Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases

    PubMed Central

    2013-01-01

    Background Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol. Results The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l-1 (0.038 and 0.028 g l-1 h-1), respectively, after 10 days. With a substrate load of 200 g l-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l-1 ethanol (0.58 g l-1 h-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l-1 ethanol (0.36 g l-1 h-1). Conclusions In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases. PMID:24286270

  18. Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.

    PubMed Central

    Perry, J R; Basrai, M A; Steiner, H Y; Naider, F; Becker, J M

    1994-01-01

    We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant. Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid. DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana. Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium. The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S. cerevisiae DNA. PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently. A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene. Images PMID:8264579

  19. Regulation of Saccharomyces cerevisiae FET4 by oxygen and iron.

    PubMed

    Jensen, Laran T; Culotta, Valeria Cizewski

    2002-04-26

    Saccharomyces cerevisiae expresses two distinct iron transport systems under aerobic and anaerobic conditions. The high affinity transporters, Ftr1p and Fet3p, are primarily expressed in oxygenated cultures, whereas anaerobic conditions induce the low affinity iron transporter, Fet4p. The oxygen regulation of FET4 was found to involve the Rox1p transcriptional repressor. The physiological significance of this control by Rox1p is twofold. First, FET4 repression by Rox1p under oxygenated conditions helps minimize metal toxicity. Sensitivity towards cadmium was high in either anaerobically grown wild-type yeast or in oxygenated rox1Delta strains, and in both cases cadmium toxicity was reversed by FET4 mutations. Secondly, the loss of Rox1p repression under anaerobic conditions serves to induce FET4 and facilitate continual accumulation of iron. We noted that fet4 mutants accumulate lower levels of iron under anaerobic conditions. Regulation of FET4 was examined using FET4-lacZ reporters. We found that FET4 contains a complex promoter regulated both by oxygen and iron status. The region surrounding approximately -960 to -490 contains two consensus Rox1p binding sites and mediates Rox1p, but not iron control of FET4. Sequences downstream of -490 harbor a consensus binding site for the iron regulatory factor Aft1p that is essential for iron regulation in wild-type strains. In addition, a secondary mode of iron regulation becomes evident in strains lacking AFT1. The induction by iron limitation in conjunction with low oxygen is more than additive, suggesting that these activities are synergistic. Fet4p is not the only metal transporter that is negatively regulated by oxygen; we find that Rox1p also represses S. cerevisiae SMF3, proposed to function in vacuolar iron transport. This oxygen control of iron transporter gene expression is part of an adaptation response to changes in the redox state of transition metals. PMID:12051835

  20. The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.

    PubMed Central

    van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extensive flux of lipids from these organelles to the plasma membrane is required. Although the pathway of protein traffic to the plasma membrane is similar to that of most of the lipids, the bulk flow of lipids is separate from vesicle-mediated protein transport. Recent advances in the analysis of membrane budding and membrane fusion indicate that the mechanisms of protein transport from the endoplasmic reticulum to the Golgi and from the Golgi to plasma membrane are similar. The majority of plasma membrane proteins transport solutes across the membrane. A number of ATP-dependent export systems have been detected that couple the hydrolysis of ATP to transport of molecules out of the cell. The hydrolysis of ATP by the plasma membrane H(+)-ATPase generates a proton motive force which is used to drive secondary transport processes. In S. cerevisiae, many substrates are transported by more than one system. Transport of monosaccharide is catalyzed by uniport systems, while transport of disaccharides, amino acids, and nucleosides is mediated by proton symport systems. Transport activity can be regulated at the level of transcription, e.g., induction and (catabolite) repression, but transport proteins can also be affected posttranslationally by a process termed catabolite inactivation. Catabolite inactivation is triggered by the addition of fermentable sugars, intracellular acidification, stress conditions, and/or nitrogen starvation. Phosphorylation and/or ubiquitination of the transport proteins has been proposed as an initial step in the controlled inactivation and degradation of the target enzyme. The use of artificial membranes, like secretory vesicles and plasma membranes fused with proteoliposomes, as model systems for studies on the mechanism and regulation of transport is evaluated. PMID:7603412

  1. Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.

    PubMed

    Guimarães, Pedro M R; François, Jean; Parrou, Jean Luc; Teixeira, José A; Domingues, Lucília

    2008-03-01

    The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (beta-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media. PMID:18245248

  2. Modulation of efficiency of translation termination in Saccharomyces cerevisiae

    PubMed Central

    Nizhnikov, Anton A; Antonets, Kirill S; Inge-Vechtomov, Sergey G; Derkatch, Irina L

    2014-01-01

    Nonsense suppression is a readthrough of premature termination codons. It typically occurs either due to the recognition of stop codons by tRNAs with mutant anticodons, or due to a decrease in the fidelity of translation termination. In the latter case, suppressors usually promote the readthrough of different types of nonsense codons and are thus called omnipotent nonsense suppressors. Omnipotent nonsense suppressors were identified in yeast Saccharomyces cerevisiae in 1960s, and most of subsequent studies were performed in this model organism. Initially, omnipotent suppressors were localized by genetic analysis to different protein- and RNA-encoding genes, mostly the components of translational machinery. Later, nonsense suppression was found to be caused not only by genomic mutations, but also by epigenetic elements, prions. Prions are self-perpetuating protein conformations usually manifested by infectious protein aggregates. Modulation of translational accuracy by prions reflects changes in the activity of their structural proteins involved in different aspects of protein synthesis. Overall, nonsense suppression can be seen as a “phenotypic mirror” of events affecting the accuracy of the translational machine. However, the range of proteins participating in the modulation of translation termination fidelity is not fully elucidated. Recently, the list has been expanded significantly by findings that revealed a number of weak genetic and epigenetic nonsense suppressors, the effect of which can be detected only in specific genetic backgrounds. This review summarizes the data on the nonsense suppressors decreasing the fidelity of translation termination in S. cerevisiae, and discusses the functional significance of the modulation of translational accuracy. PMID:25486049

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

    PubMed Central

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

    2013-01-01

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

  4. Distance-Independence of Mitotic Intrachromosomal Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Yuan, L. W.; Keil, R. L.

    1990-01-01

    Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur. We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short. In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences. However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers. The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases. These findings have important implications for the maintenance and evolution of duplicated sequences. PMID:2407612

  5. Coordinated Concentration Changes of Transcripts and Metabolites in Saccharomyces cerevisiae

    PubMed Central

    Bradley, Patrick H.; Brauer, Matthew J.; Rabinowitz, Joshua D.; Troyanskaya, Olga G.

    2009-01-01

    Metabolite concentrations can regulate gene expression, which can in turn regulate metabolic activity. The extent to which functionally related transcripts and metabolites show similar patterns of concentration changes, however, remains unestablished. We measure and analyze the metabolomic and transcriptional responses of Saccharomyces cerevisiae to carbon and nitrogen starvation. Our analysis demonstrates that transcripts and metabolites show coordinated response dynamics. Furthermore, metabolites and gene products whose concentration profiles are alike tend to participate in related biological processes. To identify specific, functionally related genes and metabolites, we develop an approach based on Bayesian integration of the joint metabolomic and transcriptomic data. This algorithm finds interactions by evaluating transcriptmetabolite correlations in light of the experimental context in which they occur and the class of metabolite involved. It effectively predicts known enzymatic and regulatory relationships, including a genemetabolite interaction central to the glycolyticgluconeogenetic switch. This work provides quantitative evidence that functionally related metabolites and transcripts show coherent patterns of behavior on the genome scale and lays the groundwork for building genemetabolite interaction networks directly from systems-level data. PMID:19180179

  6. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae.

    PubMed

    Peters, Theodore W; Miller, Aaron W; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E

    2016-01-01

    Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826

  7. Biosynthesis of diphthamide in the yeast Saccharomyces cerevisiae

    SciTech Connect

    Chen, J.Y.C.

    1985-01-01

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

  8. Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae

    DOE PAGESBeta

    Dar, R. D.; Karig, D. K.; Cooke, J. F.; Cox, C. D.; Simpson, M. L.

    2010-09-01

    Stochasticity is an inherent feature of complex systems with nanoscale structure. In such systems information is represented by small collections of elements (e.g. a few electrons on a quantum dot), and small variations in the populations of these elements may lead to big uncertainties in the information. Unfortunately, little is known about how to work within this inherently noisy environment to design robust functionality into complex nanoscale systems. Here, we look to the biological cell as an intriguing model system where evolution has mediated the trade-offs between fluctuations and function, and in particular we look at the relationships and trade-offsmore » between stochastic and deterministic responses in the gene expression of budding yeast (Saccharomyces cerevisiae). We find gene regulatory arrangements that control the stochastic and deterministic components of expression, and show that genes that have evolved to respond to stimuli (stress) in the most strongly deterministic way exhibit the most noise in the absence of the stimuli. We show that this relationship is consistent with a bursty 2-state model of gene expression, and demonstrate that this regulatory motif generates the most uncertainty in gene expression when there is the greatest uncertainty in the optimal level of gene expression.« less

  9. Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

    PubMed Central

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

    2016-01-01

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

  10. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    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.

  11. Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae

    PubMed Central

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

    1992-01-01

    Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles. PMID:1644271

  12. In vivo Reconstitution of Algal Triacylglycerol Production in Saccharomyces cerevisiae.

    PubMed

    Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

    2016-01-01

    The current fascination with algal biofuel production stems from a high lipid biosynthetic capacity and little conflict with land plant cultivation. However, the mechanisms which enable algae to accumulate massive oil remain elusive. An enzyme for triacylglycerol (TAG) biosynthesis in Chlamydomonas reinhardtii, CrDGTT2, can produce a large amount of TAG when expressed in yeast or higher plants, suggesting a unique ability of CrDGTT2 to enhance oil production in a heterologous system. Here, we performed metabolic engineering in Saccharomyces cerevisiae by taking advantage of CrDGTT2. We suppressed membrane phospholipid biosynthesis at the log phase by mutating OPI3, enhanced TAG biosynthetic pathway at the stationary phase by overexpressing PAH1 and CrDGTT2, and suppressed TAG hydrolysis on growth resumption from the stationary phase by knocking out DGK1. The resulting engineered yeast cells accumulated about 70-fold of TAG compared with wild type cells. Moreover, TAG production was sustainable. Our results demonstrated the enhanced and sustainable TAG production in the yeast synthetic platform. PMID:26913021

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

    PubMed Central

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

    2015-01-01

    Recent experiments established that a culture of Saccharomyces cerevisiae (baker’s yeast) survives sudden high temperatures by specifically duplicating the entire chromosome III and two chromosomal fragments (from IV and XII). Heat shock proteins (HSPs) are not significantly over-abundant in the duplication. In contrast, we suggest a simple algorithm to “ postdict ” the experimental results: Find a small enough chromosome with minimal protein disorder and duplicate this region. This algorithm largely explains all observed duplications. In particular, all regions duplicated in the experiment reduced the overall content of protein disorder. The differential analysis of the functional makeup of the duplication remained inconclusive. Gene Ontology (GO) enrichment suggested over-representation in processes related to reproduction and nutrient uptake. Analyzing the protein-protein interaction network (PPI) revealed that few network-central proteins were duplicated. The predictive hypothesis hinges upon the concept of reducing proteins with long regions of disorder in order to become less sensitive to heat shock attack. PMID:26673203

  14. Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae.

    PubMed Central

    Williams-Hart, Tara; Wu, Xiaolin; Tatchell, Kelly

    2002-01-01

    Protein phosphatase type 1 (PP1) is encoded by the essential gene GLC7 in Saccharomyces cerevisiae. glc7-109 (K259A, R260A) has a dominant, hyperglycogen defect and a recessive, ion and drug sensitivity. Surprisingly, the hyperglycogen phenotype is partially retained in null mutants of GAC1, GIP2, and PIG1, which encode potential glycogen-targeting subunits of Glc7. The R260A substitution in GLC7 is responsible for the dominant and recessive traits of glc7-109. Another mutation at this residue, glc7-R260P, confers only salt sensitivity, indicating that the glycogen and salt traits of glc7-109 are due to defects in distinct physiological pathways. The glc7-109 mutant is sensitive to cations, aminoglycosides, and alkaline pH and exhibits increased rates of l-leucine and 3,3'-dihexyloxacarbocyanine iodide uptake, but it is resistant to molar concentrations of sorbitol or KCl, indicating that it has normal osmoregulation. KCl suppresses the ion and drug sensitivities of the glc7-109 mutant. The CsCl sensitivity of this mutant is suppressed by recessive mutations in PMA1, which encodes the essential plasma membrane H(+)ATPase. Together, these results indicate that Glc7 regulates ion homeostasis by controlling ion transport and/or plasma membrane potential, a new role for Glc7 in budding yeast. PMID:11973298

  15. Mating-type genes and MAT switching in Saccharomyces cerevisiae.

    PubMed

    Haber, James E

    2012-05-01

    Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATα. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATα1, and MATα2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break. PMID:22555442

  16. Tor1 regulates protein solubility in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Rardin, Matthew J.; Czerwieniec, Gregg; Evani, Uday S.; Reis-Rodrigues, Pedro; Lithgow, Gordon J.; Mooney, Sean D.; Gibson, Bradford W.; Hughes, Robert E.

    2012-01-01

    Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase. PMID:23097491

  17. Comparative Genomics of Saccharomyces cerevisiae Natural Isolates for Bioenergy Production

    PubMed Central

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

    2014-01-01

    Lignocellulosic plant material is a viable source of biomass to produce alternative energy including ethanol and other biofuels. However, several factors—including toxic byproducts from biomass pretreatment and poor fermentation of xylose and other pentose sugars—currently limit the efficiency of microbial biofuel production. To begin to understand the genetic basis of desirable traits, we characterized three strains of Saccharomyces cerevisiae with robust growth in a pretreated lignocellulosic hydrolysate or tolerance to stress conditions relevant to industrial biofuel production, through genome and transcriptome sequencing analysis. All stress resistant strains were highly mosaic, suggesting that genetic admixture may contribute to novel allele combinations underlying these phenotypes. Strain-specific gene sets not found in the lab strain were functionally linked to the tolerances of particular strains. Furthermore, genes with signatures of evolutionary selection were enriched for functional categories important for stress resistance and included stress-responsive signaling factors. Comparison of the strains’ transcriptomic responses to heat and ethanol treatment—two stresses relevant to industrial bioethanol production—pointed to physiological processes that were related to particular stress resistance profiles. Many of the genotype-by-environment expression responses occurred at targets of transcription factors with signatures of positive selection, suggesting that these strains have undergone positive selection for stress tolerance. Our results generate new insights into potential mechanisms of tolerance to stresses relevant to biofuel production, including ethanol and heat, present a backdrop for further engineering, and provide glimpses into the natural variation of stress tolerance in wild yeast strains. PMID:25364804

  18. Water-Transfer Slows Aging in Saccharomyces cerevisiae.

    PubMed

    Cohen, Aviv; Weindling, Esther; Rabinovich, Efrat; Nachman, Iftach; Fuchs, Shai; Chuartzman, Silvia; Gal, Lihi; Schuldiner, Maya; Bar-Nun, Shoshana

    2016-01-01

    Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process. PMID:26862897

  19. D-xylulose fermentation to ethanol by Saccharomyces cerevisiae

    SciTech Connect

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

    1981-08-01

    Commercial bakers' yeast (Saccharomyces cerevisiae) was used to study the conversion of D-xylulose to ethanol in the presence of D-xylose. The rate of ethanol production increased with an increase in yeast cell density. The optimal temperature for D-xylulose fermentation was 35 degrees Celcius, and the optimal pH range was 4 to 6. The fermentation of D-xylulose by yeast resulted in the production of ethanol as the major product; small amounts of xylitol and glycerol were also produced. The production of xylitol was influenced by pH as well as temperature. High pH values and low temperatures enhanced xylitol production. The rate of D-xylulose fermentation decreased when the production of ethanol yielded concentrations of 4% or more. The slow conversion rate of D-xylulose to ethanol was increased by increasing the yeast cell density. The overall production of ethanol from D-xylulose by yeast cells under optimal conditions was 90% of the theoretical yield. (Refs. 21).

  20. The chromosomal constitution of wine strains of Saccharomyces cerevisiae.

    PubMed

    Bakalinsky, A T; Snow, R

    1990-01-01

    A general procedure is described for determining the chromosomal constitution of industrial strains of Saccharomyces cerevisiae based on analysis of segregation frequencies for input markers among random spore progeny of industrial-laboratory strain hybrids. The multiply auxotrophic haploid testers used carried a dominant erythromycin-resistance marker, allowing hybrids to be selected in mass matings with spores produced by the wild-type industrial strains. Analysis of a number of independent crosses between the haploid testers and an unselected population of spores of each wine strain distinguished between disomic, trisomic and tetrasomic chromosomal complements in the parents. Possible explanations for a significant class of aberrant segregation frequencies are discussed. Results of the analysis indicate that UCD Enology 522 (Montrachet) is diploid and possibly trisomic for chromosome VII; 522X is diploid; UCD Enology 505 (California Champagne) is disomic for chromosome XVI, trisomic for chromosomes I, II, III, VI, VIII, IX, X, XII, XV, tetrasomic for chromosomes IV, XI, XIII, XIV and either trisomic or tetrasomic for chromosomes V and VII; and that UCD Enology 595 (Pasteur Champagne) is disomic for chromosomes I, II, III, IX, XVI, trisomic for chromosomes IV, VI, X, XII, XIV, XV, tetrasomic for chromosomes V, VIII, XI, XIII and either disomic or tetrasomic for chromosome VII. PMID:2220073

  1. Size control models of Saccharomyces cerevisiae cell proliferation

    SciTech Connect

    Wheals, A.E.

    1982-04-01

    By using time-elapsed photomicroscopy, the individual cycle times and sizes at bud emergence were measured for a population of Saccharomyces cerevisiae cells growing exponentially under balanced growth conditions in a specially constructed filming slide. There was extensive variability in both parameters for daughter and parent cells. The data on 162 pairs of siblings were analyzed for agreement with the predictions of the transition probability hypothesis and the critical-size hypothesis of yeast cell proliferation and also with a model incorporating both of these hypotheses in tandem. None of the models accounted for all of the experimental data, but two models did give good agreement to all of the data. The wobbly tandem model proposes that cells need to attain a critical size, which is very variable, enabling them to enter a start state from which they exit with first-order kinetics. The sloppy size control model suggests that cells have an increasing probability per unit time of traversing start as they increase in size, reaching a high plateau value which is less than one. Both models predict that the kinetics of entry into the cell division sequence will strongly depend on variability in birth size and thus will be quite different for daughters and parents of the asymmetrically dividing yeast cells. Mechanisms underlying these models are discussed.

  2. Novel regulatory properties of Saccharomyces cerevisiae Arp4.

    PubMed

    Steinboeck, Ferdinand; Krupanska, Ludmila; Bogusch, Alexandra; Kaufmann, Alexius; Heidenreich, Erich

    2006-04-01

    ARP4, an essential gene of Saccharomyces cerevisiae, codes for a nuclear actin-related protein. Arp4 is a subunit of several chromatin-modifying complexes and is known to be involved in the transcriptional regulation in yeast. We used a mutant strain with a single amino acid substitution (G161D) in the conserved actin fold domain to investigate the influence of Arp4 on stress and nitrogen catabolite repression genes. The deficiency of functional Arp4 caused a highly increased sensitivity towards nitrogen starvation and to the macrolide antibiotic rapamycin. We show the changes of mRNA levels of selected genes under these conditions. The upregulation of stress genes as a consequence of treatment with rapamycin was largely Msn2p/Msn4p-dependent. The sensitivity towards rapamycin indicates a participation of Arp4 in the regulation of the TOR pathway. Consistently, arp4G161D cells exhibited an affected cell cycle. Long-term cultivation, which leads to a G1 arrest in wild-type cells, provoked arrest in G2/M (more than 60%) in the mutant strain. The same effect was observed upon treatment with rapamycin, indicating an unexpected relationship of Arp4 to TOR-mediated cell cycle arrest. PMID:16672275

  3. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae.

    PubMed

    Conrad, Michaela; Schothorst, Joep; Kankipati, Harish Nag; Van Zeebroeck, Griet; Rubio-Texeira, Marta; Thevelein, Johan M

    2014-03-01

    The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms. Many specific nutrient responses have been elucidated in great detail. This has led to important new concepts and insight into nutrient-controlled cellular regulation. Major highlights include the central role of the Snf1 protein kinase in the glucose repression pathway, galactose induction, the discovery of a G-protein-coupled receptor system, and role of Ras in glucose-induced cAMP signaling, the role of the protein synthesis initiation machinery in general control of nitrogen metabolism, the cyclin-controlled protein kinase Pho85 in phosphate regulation, nitrogen catabolite repression and the nitrogen-sensing target of rapamycin pathway, and the discovery of transporter-like proteins acting as nutrient sensors. In addition, a number of cellular targets, like carbohydrate stores, stress tolerance, and ribosomal gene expression, are controlled by the presence of multiple nutrients. The protein kinase A signaling pathway plays a major role in this general nutrient response. It has led to the discovery of nutrient transceptors (transporter receptors) as nutrient sensors. Major shortcomings in our knowledge are the relationship between rapid and steady-state nutrient signaling, the role of metabolic intermediates in intracellular nutrient sensing, and the identity of the nutrient sensors controlling cellular growth. PMID:24483210

  4. Conversion of Wine Strains of Saccharomyces cerevisiae to Heterothallism

    PubMed Central

    Bakalinsky, Alan T.; Snow, Richard

    1990-01-01

    A general method to convert homothallic strains of the yeast Saccharomyces cerevisiae to heterothallism is described which is applicable to genetically well-behaved diploids, as well as to strains that sporulate poorly or produce few viable and mating-competent spores. The heterothallic (ho) allele was introduced into three widely used wine strains through spore × cell hybridization. The resultant hybrids were sporulated, and heterothallic segregants were isolated for use in successive backcrosses. Heterothallic progeny of opposite mating type and monosomic for chromosome III produced by sixth-backcross hybrids or their progeny were mated together to reconstruct heterothallic derivatives of the wine strain parents. A helpful prerequisite to the introduction of ho was genetic purification of the parental strains based on repeated cycles of sporulation, ascus dissection, and clonal selection. A positive selection to isolate laboratory-wine strain hybrids requiring no prior genetic alteration of the industrial strains, coupled with a partial selection to reduce the number of spore progeny needed to be screened to isolate heterothallic segregants of the proper genotype made the procedure valuable for genetically intractable strains. Trial grape juice fermentations indicated that introduction of ho had no deleterious effect on fermentation behavior. PMID:16348171

  5. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae

    PubMed Central

    Conrad, Michaela; Schothorst, Joep; Kankipati, Harish Nag; Van Zeebroeck, Griet; Rubio-Texeira, Marta; Thevelein, Johan M

    2014-01-01

    The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms. Many specific nutrient responses have been elucidated in great detail. This has led to important new concepts and insight into nutrient-controlled cellular regulation. Major highlights include the central role of the Snf1 protein kinase in the glucose repression pathway, galactose induction, the discovery of a G-protein-coupled receptor system, and role of Ras in glucose-induced cAMP signaling, the role of the protein synthesis initiation machinery in general control of nitrogen metabolism, the cyclin-controlled protein kinase Pho85 in phosphate regulation, nitrogen catabolite repression and the nitrogen-sensing target of rapamycin pathway, and the discovery of transporter-like proteins acting as nutrient sensors. In addition, a number of cellular targets, like carbohydrate stores, stress tolerance, and ribosomal gene expression, are controlled by the presence of multiple nutrients. The protein kinase A signaling pathway plays a major role in this general nutrient response. It has led to the discovery of nutrient transceptors (transporter receptors) as nutrient sensors. Major shortcomings in our knowledge are the relationship between rapid and steady-state nutrient signaling, the role of metabolic intermediates in intracellular nutrient sensing, and the identity of the nutrient sensors controlling cellular growth. PMID:24483210

  6. Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production.

    PubMed

    Feng, Xueyang; Lian, Jiazhang; Zhao, Huimin

    2015-01-01

    Fatty alcohols are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We have engineered Saccharomyces cerevisiae to produce 1-hexadecanol by expressing a fatty acyl-CoA reductase (FAR) from barn owl (Tyto alba). In order to improve fatty alcohol production, we have manipulated both the structural genes and the regulatory genes in yeast lipid metabolism. The acetyl-CoA carboxylase gene (ACC1) was over-expressed, which improved 1-hexadecanol production by 56% (from 45mg/L to 71mg/L). Knocking out the negative regulator of the INO1 gene in phospholipid metabolism, RPD3, further enhanced 1-hexadecanol production by 98% (from 71mg/L to 140mg/L). The cytosolic acetyl-CoA supply was next engineered by expressing a heterologous ATP-dependent citrate lyase, which increased the production of 1-hexadecanol by an additional 136% (from 140mg/L to 330mg/L). Through fed-batch fermentation using resting cells, over 1.1g/L 1-hexadecanol can be produced in glucose minimal medium, which represents the highest titer reported in yeast to date. PMID:25466225

  7. Genome instability in rad54 mutants of Saccharomyces cerevisiae

    PubMed Central

    Schmuckli-Maurer, Jacqueline; Rolfsmeier, Michael; Nguyen, Ho; Heyer, Wolf-Dietrich

    2003-01-01

    The RAD54 gene of Saccharomyces cerevisiae encodes a conserved dsDNA-dependent ATPase of the Swi2/Snf2 family with a specialized function during recombinational DNA repair. Here we analyzed the consequences of the loss of Rad54 function in vegetative (mitotic) cells. Mutants in RAD54 exhibited drastically reduced rates of spontaneous intragenic recombination but were proficient for spontaneous intergenic recombinant formation. The intergenic recombinants likely arose by a RAD54-independent pathway of break-induced replication. Significantly increased rates of spontaneous chromosome loss for diploid rad54/rad54 cells were identified in several independent assays. Inter estingly, the increase in chromosome loss appeared to depend on the presence of a homolog. In addition, the rate of complex genetic events involving chromosome loss were drastically increased in diploid rad54/rad54 cells. Together, these data suggest a role for Rad54 protein in the repair of spontaneous damage, where in the absence of Rad54 protein, homologous recombination is initiated but not properly terminated, leading to misrepair and chromosome loss. PMID:12560498

  8. Dynamics of the Saccharomyces cerevisiae transcriptome during bread dough fermentation.

    PubMed

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

    2013-12-01

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

  9. Nutritional and environmental factors in ethanol fermentation by Saccharomyces cerevisiae

    SciTech Connect

    Wong, H.; Wilke, C.R.; Blanch, H.W.

    1983-05-01

    Using Saccharomyces cerevisiae as a model system, a basic study of the nutritional and environmental factors in ethanol fermentation was carried out to provide fundamental and practical bases for design of fermentation media and culture conditions. The requirements for all active medium components need to be determined in order to establish balanced media, which are important to reduce raw materials costs and to minimize inhibition from buildup of excess feed components in recycle processes with selective ethanol removal. Pulse injection of nutrients into continuous cultures was an effective method for screening active nutrients. In a systematic sensitivity analysis the effect of feed concentration of these individual nutrients was then determined and allowed formulation of media optimal with respect to the major fermentation parameters. Biotin, pantothenate, myo-inositol, potassium and phosphates appeared to stimulate growth preferentially to ethanol production. In contrast, thiamine and pyridoxine appeared to enhance specific ethanol productivity. The effect of ammonium sulfate depended on concentration. A conceptual model was proposed to relate the effects of these nutrients to biochemical pathways and functions. With these data and model the minimum cost combination of raw materials to achieve a medium of well defined components can be determined with a linear program. This computer program shows that many growth factors and minerals can be added to media more economically as pure components than as fractions of complex factors. 225 references, 61 figures, 54 tables.

  10. Nutritional and environmental factors in ethanol fermentation by Saccharomyces cerevisiae

    SciTech Connect

    Wong, H.

    1983-01-01

    Using Saccharomyces cerevisiae as a model system, a basic study of the nutritional and environmental factors in ethanol fermentation was carried out to provide fundamental and practical bases for design of fermentation media and culture conditions. The requirements for all active medium components need to be determined in order to establish balanced media, which are important to reduce raw materials costs and to minimize inhibition from build-up of excess feed components in recycle processes with selective ethanol removal. The effect of feed concentration of individual nutrients was determined and allowed formulation of media optimal with respect to the major fermentation parameters. Biotin, pantothenate, myoinositol, potassium, and phosphates appeared to stimulate growth preferentially to ethanol production. Thiamine and pyridoxine appeared to have the opposite effect. A conceptual model was proposed to relate the effects of these nutrients to biochemical pathways and functions. The minimum cost combination of raw materials to achieve a medium of well defined components can be determined with a linear program. The effect of dissolved oxygen was studied from essentially zero to 346 mm Hg oxygen tension, showing a continuous decline in specific ethanol productivity with increasing oxygen over this range. Long term continuous cultures resulted in decreased media requirements for growth factors and increased tolerance for ethanol inhibition, most probably through adaptation. An ethanol productivity of 5.6 g/l-hr in continuous culture was achieved with a completely synthetic medium with the improved culture.

  11. Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae.

    PubMed

    Hu, Kejin; Guo, Shuguang; Yan, Gonghong; Yuan, Wenjie; Zheng, Yin; Jiang, Yu

    2016-04-01

    In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth-related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12-rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2(W2041R) ) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non-covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization. PMID:26700129

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

    TOXLINE Toxicology Bibliographic Information

    Guan Q; Haroon S; Bravo DG; Will JL; Gasch AP

    2012-10-01

    Cellular memory of past experiences has been observed in several organisms and across a variety of experiences, including bacteria "remembering" prior nutritional status and amoeba "learning" to anticipate future environmental conditions. Here, we show that Saccharomyces cerevisiae maintains a multifaceted memory of prior stress exposure. We previously demonstrated that yeast cells exposed to a mild dose of salt acquire subsequent tolerance to severe doses of H(2)O(2). We set out to characterize the retention of acquired tolerance and in the process uncovered two distinct aspects of cellular memory. First, we found that H(2)O(2) resistance persisted for four to five generations after cells were removed from the prior salt treatment and was transmitted to daughter cells that never directly experienced the pretreatment. Maintenance of this memory did not require nascent protein synthesis after the initial salt pretreatment, but rather required long-lived cytosolic catalase Ctt1p that was synthesized during salt exposure and then distributed to daughter cells during subsequent cell divisions. In addition to and separable from the memory of H(2)O(2) resistance, these cells also displayed a faster gene-expression response to subsequent stress at >1000 genes, representing transcriptional memory. The faster gene-expression response requires the nuclear pore component Nup42p and serves an important function by facilitating faster reacquisition of H(2)O(2) tolerance after a second cycle of salt exposure. Memory of prior stress exposure likely provides a significant advantage to microbial populations living in ever-changing environments.

  13. Electroinduced release of recombinant β-galactosidase from Saccharomyces cerevisiae.

    PubMed

    Ganeva, Valentina; Stefanova, Debora; Angelova, Boyana; Galutzov, Bojidar; Velasco, Isabel; Arévalo-Rodríguez, Miguel

    2015-10-10

    Yeasts are one of the most commonly used systems for recombinant protein production. When the protein is intracelullarly expressed the first step comprises a cell lysis, achieved usually by a mechanical disintegration. This leads to non-selective liberation of the cytoplasmic content, which complicates the following downstream process. Here, we present a new approach suitable for more selective and efficient recovery of large intracellular proteins from yeast, based on the combination of electropermeabilisation and subsequent treatment with lytic enzyme. The experiments were performed with Saccharomyces cerevisiae strains expressing LYTAG-β-galactosidase from Escherichia coli. The permeabilzation of plasma membrane was induced by application of rectangular electric pulses, with 1.25ms duration and field intensity of 4.3-5.4kV/cm. In the presence of a reducing agent the cells released approximately 80% of the total protein 4h after electrical treatment. At the same conditions the release of the recombinant protein was very slow, reaching 45% from total activity 20h after pulse application. The great difference in the release kinetics enabled to remove a part of the total protein, without significant loss of β-galactosidase activity, only by substituting the incubation buffer. The subsequent addition of lyticase (1-2U/ml) led to recovery of approximately 70% from the recombinant enzyme, with a factor of purification 2.6, without provoking a significant cell lysis. The applicability of similar protocol for liberation of large recombinant and native proteins from yeast is discussed. PMID:26142064

  14. Error-free DNA-damage tolerance in Saccharomyces cerevisiae.

    PubMed

    Xu, Xin; Blackwell, Susan; Lin, Aiyang; Li, Fangfang; Qin, Zhoushuai; Xiao, Wei

    2015-01-01

    DNA-damage tolerance (DDT) is an important mechanism for living cells to bypass replication blocks on the template strand. In Saccharomyces cerevisiae, DDT is mediated by the RAD6 epistasis group of genes, consisting of two parallel pathways: error-prone translesion DNA synthesis (TLS), and error-free lesion bypass. The two pathways are activated by sequential ubiquitination of PCNA on the Lys164 residue. When a replication fork is stalled at a lesion, PCNA is first monoubiquitinated by Rad6-Rad18, which leads to the TLS pathway. The subsequent ubiquitination by the Mms2-Ubc13-Rad5 complex on the monoubiquitinated PCNA is to form a Lys63-linked polyubiquitin chain that promotes error-free lesion bypass. While the TLS pathway has been extensively characterized, the molecular events leading to error-free lesion bypass by polyubiquitinated PCNA are largely obscure. Furthermore, PCNA can also be sumoylated at the same Lys164 residue, which helps to recruit Srs2, a helicase and anti-recombinase. This review summarizes recent advances in our understanding of error-free DDT and its interplay with Srs2 and homologous recombination. PMID:26041265

  15. Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.

    PubMed Central

    Swedes, J S; Dial, M E; McLaughlin, C S

    1979-01-01

    Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited. PMID:374362

  16. Identification of Saccharomyces cerevisiae spindle pole body remodeling factors.

    PubMed

    Greenland, Kristen B; Ding, Huiming; Costanzo, Michael; Boone, Charles; Davis, Trisha N

    2010-01-01

    The Saccharomyces cerevisiae centrosome or spindle pole body (SPB) is a dynamic structure that is remodeled in a cell cycle dependent manner. The SPB increases in size late in the cell cycle and during most cell cycle arrests and exchanges components during G1/S. We identified proteins involved in the remodeling process using a strain in which SPB remodeling is conditionally induced. This strain was engineered to express a modified SPB component, Spc110, which can be cleaved upon the induction of a protease. Using a synthetic genetic array analysis, we screened for genes required only when Spc110 cleavage is induced. Candidate SPB remodeling factors fell into several functional categories: mitotic regulators, microtubule motors, protein modification enzymes, and nuclear pore proteins. The involvement of candidate genes in SPB assembly was assessed in three ways: by identifying the presence of a synthetic growth defect when combined with an Spc110 assembly defective mutant, quantifying growth of SPBs during metaphase arrest, and comparing distribution of SPB size during asynchronous growth. These secondary screens identified four genes required for SPB remodeling: NUP60, POM152, and NCS2 are required for SPB growth during a mitotic cell cycle arrest, and UBC4 is required to maintain SPB size during the cell cycle. These findings implicate the nuclear pore, urmylation, and ubiquitination in SPB remodeling and represent novel functions for these genes. PMID:21103054

  17. Mutational analysis of capping protein function in Saccharomyces cerevisiae.

    PubMed

    Sizonenko, G I; Karpova, T S; Gattermeir, D J; Cooper, J A

    1996-01-01

    To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis. PMID:8741835

  18. Mutational analysis of capping protein function in Saccharomyces cerevisiae.

    PubMed Central

    Sizonenko, G I; Karpova, T S; Gattermeir, D J; Cooper, J A

    1996-01-01

    To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis. Images PMID:8741835

  19. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Dor, Carole; Deneault, Jean-Sebastien; Rudner, Adam D

    2016-03-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1? cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase. PMID:26715668

  20. Dynamics of the Saccharomyces cerevisiae Transcriptome during Bread Dough Fermentation

    PubMed Central

    Aslankoohi, Elham; Zhu, Bo; Rezaei, Mohammad Naser; Voordeckers, Karin; De Maeyer, Dries; Marchal, Kathleen; Dornez, Emmie

    2013-01-01

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

  1. A novel selection system for chromosome translocations in Saccharomyces cerevisiae.

    PubMed Central

    Tennyson, Rachel B; Ebran, Nathalie; Herrera, Anissa E; Lindsley, Janet E

    2002-01-01

    Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors. While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements. This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae. A translocation YAC was created that contains the breakpoint cluster region from the human MLL gene, a gene frequently involved in translocations in leukemia patients, flanked by positive and negative selection markers. A translocation between the YAC and a yeast chromosome, whose breakpoint falls within the MLL DNA, physically separates the markers and forms the basis for the selection. When RAD52 is deleted, essentially all of the selected and screened cells contain simple translocations. The detectable translocation rates are the same in haploids and diploids, although the mechanisms involved and true translocation rates may be distinct. A unique double-strand break induced within the MLL sequences increases the number of detectable translocation events 100- to 1000-fold. This novel system provides a tractable assay for answering basic mechanistic questions about the development of chromosomal translocations. PMID:11973293

  2. Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

    PubMed Central

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

    2014-01-01

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

  3. Water-Transfer Slows Aging in Saccharomyces cerevisiae

    PubMed Central

    Cohen, Aviv; Weindling, Esther; Rabinovich, Efrat; Nachman, Iftach; Fuchs, Shai; Chuartzman, Silvia; Gal, Lihi; Schuldiner, Maya; Bar-Nun, Shoshana

    2016-01-01

    Transferring Saccharomyces cerevisiae cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on de novo protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process. PMID:26862897

  4. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

    PubMed

    Krogan, Nevan J; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P; Punna, Thanuja; Peregrín-Alvarez, José M; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D; Paccanaro, Alberto; Bray, James E; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J; Gandi, Kiran; Thompson, Natalie J; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H Y; Butland, Gareth; Altaf-Ul, Amin M; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S; Ingles, C James; Hughes, Timothy R; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J; Emili, Andrew; Greenblatt, Jack F

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology. PMID:16554755

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

    PubMed

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

    2007-05-01

    Fermented beverages and foods have played a significant role in most societies worldwide for millennia. To better understand how the yeast species Saccharomyces cerevisiae, the main fermenting agent, evolved along this historical and expansion process, we analysed the genetic diversity among 651 strains from 56 different geographical origins, worldwide. Their genotyping at 12 microsatellite loci revealed 575 distinct genotypes organized in subgroups of yeast types, i.e. bread, beer, wine, sake. Some of these groups presented unexpected relatedness: Bread strains displayed a combination of alleles intermediate between beer and wine strains, and strains used for rice wine and sake were most closely related to beer and bread strains. However, up to 28% of genetic diversity between these technological groups was associated with geographical differences which suggests local domestications. Focusing on wine yeasts, a group of Lebanese strains were basal in an F(ST) tree, suggesting a Mesopotamia-based origin of most wine strains. In Europe, migration of wine strains occurred through the Danube Valley, and around the Mediterranean Sea. An approximate Bayesian computation approach suggested a postglacial divergence (most probable period 10,000-12,000 bp). As our results suggest intimate association between man and wine yeast across centuries, we hypothesize that yeast followed man and vine migrations as a commensal member of grapevine flora. PMID:17498234

  6. Endomitotic effect of a cell cycle mutation of Saccharomyces cerevisiae

    SciTech Connect

    Schild, D.; Ananthaswamy, H.N.; Mortimer, R.K.

    1981-03-01

    A recessive temperature-sensitive mutation of Saccharomyces cerevisiae has been isolated and shown to cause an increase in ploidy in both haploids and diploids. Genetic analysis revealed that the strain carrying the mutation was an aa diploid, although MNNG mutagenesis had been done on an a haploid strain. When the mutant strain was crossed with an ..cap alpha cap alpha.. diploid and the resultant tetraploid sporulated, some of the meiotic progeny of this tetraploid were themselves tetraploid, as shown by both genetic analysis and DNA measurements, instead of diploid as expected of tetraploid meiosis. The ability of these tetraploids to continue to produce tetraploid meiotic progeny was followed for four generations. It was found that tetraploidization was independent of sporulation temperature, but was dependent on the temperature of germination and the growth of the spores. Increase in ploidy occurred when the spores were germinated and grown at 30/sup 0/, but did not occur at 23/sup 0/. Two cycles of sporulation and growth at 23/sup 0/ resulted in haploids, which were shown to diploidize within 24 hr when grown at 30/sup 0/.

  7. Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production

    PubMed Central

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

    2014-01-01

    Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g. tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed. PMID:25133732

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

    PubMed

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

    2016-01-01

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

  9. Proteomic Profiling of Autophagosome Cargo in Saccharomyces cerevisiae

    PubMed Central

    Morimoto, Mayumi; Fujii, Kiyonaga; Noda, Nobuo N.; Inagaki, Fuyuhiko; Ohsumi, Yoshinori

    2014-01-01

    Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy. PMID:24626240

  10. In vivo Reconstitution of Algal Triacylglycerol Production in Saccharomyces cerevisiae

    PubMed Central

    Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

    2016-01-01

    The current fascination with algal biofuel production stems from a high lipid biosynthetic capacity and little conflict with land plant cultivation. However, the mechanisms which enable algae to accumulate massive oil remain elusive. An enzyme for triacylglycerol (TAG) biosynthesis in Chlamydomonas reinhardtii, CrDGTT2, can produce a large amount of TAG when expressed in yeast or higher plants, suggesting a unique ability of CrDGTT2 to enhance oil production in a heterologous system. Here, we performed metabolic engineering in Saccharomyces cerevisiae by taking advantage of CrDGTT2. We suppressed membrane phospholipid biosynthesis at the log phase by mutating OPI3, enhanced TAG biosynthetic pathway at the stationary phase by overexpressing PAH1 and CrDGTT2, and suppressed TAG hydrolysis on growth resumption from the stationary phase by knocking out DGK1. The resulting engineered yeast cells accumulated about 70-fold of TAG compared with wild type cells. Moreover, TAG production was sustainable. Our results demonstrated the enhanced and sustainable TAG production in the yeast synthetic platform. PMID:26913021

  11. Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    PubMed Central

    Gonzalez-Perez, David; Alcalde, Miguel

    2014-01-01

    The ligninolytic enzymatic consortium produced by white-rot fungi is one of the most efficient oxidative systems found in nature, with many potential applications that range from the production of 2nd generation biofuels to chemicals synthesis. In the current study, two high redox potential oxidoreductase fusion genes (laccase -Lac- and versatile peroxidase -Vp-) that had been evolved in the laboratory were re-assembled in Saccharomyces cerevisiae. First, cell viability and secretion were assessed after co-transforming the Lac and Vp genes into yeast. Several expression cassettes were inserted in vivo into episomal bi-directional vectors in order to evaluate inducible promoter and/or terminator pairs of different strengths in an individual and combined manner. The synthetic white-rot yeast model harboring Vp(GAL1/CYC1)-Lac(GAL10/ADH1) displayed up to 1000 and 100 Units per L of peroxidase and laccase activity, respectively, representing a suitable point of departure for future synthetic biology studies. PMID:24830983

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

    PubMed Central

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

    1995-01-01

    The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci [Bang et al. (1992) Nucleic Acids Res. 20, 3925-3931]. More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae [Waters et al. (1993) Mol. Gen. Genet. 239, 28-32]. We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation. Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA. In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced. The basal level of the RAD16 transcript is constant during the mitotic cell cycle. G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation. However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle. Images PMID:7784171

  13. Post-Transcriptional Regulation of Iron Homeostasis in Saccharomyces cerevisiae

    PubMed Central

    Martínez-Pastor, María Teresa; de Llanos, Rosa; Romero, Antonia María; Puig, Sergi

    2013-01-01

    Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in a wide variety of biological processes. Recent studies in Saccharomyces cerevisiae have shown that in response to iron deficiency, an RNA-binding protein denoted Cth2 coordinates a global metabolic rearrangement that aims to optimize iron utilization. The Cth2 protein contains two Cx8Cx5Cx3H tandem zinc fingers (TZFs) that specifically bind to adenosine/uridine-rich elements within the 3′ untranslated region of many mRNAs to promote their degradation. The Cth2 protein shuttles between the nucleus and the cytoplasm. Once inside the nucleus, Cth2 binds target mRNAs and stimulates alternative 3′ end processing. A Cth2/mRNA-containing complex is required for export to the cytoplasm, where the mRNA is degraded by the 5′ to 3′ degradation pathway. This post-transcriptional regulatory mechanism limits iron utilization in nonessential pathways and activates essential iron-dependent enzymes such as ribonucleotide reductase, which is required for DNA synthesis and repair. Recent findings indicate that the TZF-containing tristetraprolin protein also functions in modulating human iron homeostasis. Elevated iron concentrations can also be detrimental for cells. The Rnt1 RNase III exonuclease protects cells from excess iron by promoting the degradation of a subset of the Fe acquisition system when iron levels rise. PMID:23903042

  14. Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae

    SciTech Connect

    Dar, Roy D.; Karig, David K; Cooke, John F; Cox, Chris D.; Simpson, Michael L

    2010-01-01

    Stochasticity is an inherent feature of complex systems with nanoscale structure. In such systems information is represented by small collections of elements (e.g. a few electrons on a quantum dot), and small variations in the populations of these elements may lead to big uncertainties in the information. Unfortunately, little is known about how to work within this inherently noisy environment to design robust functionality into complex nanoscale systems. Here, we look to the biological cell as an intriguing model system where evolution has mediated the trade-offs between fluctuations and function, and in particular we look at the relationships and trade-offs between stochastic and deterministic responses in the gene expression of budding yeast (Saccharomyces cerevisiae). We find gene regulatory arrangements that control the stochastic and deterministic components of expression, and show that genes that have evolved to respond to stimuli (stress) in the most strongly deterministic way exhibit the most noise in the absence of the stimuli. We show that this relationship is consistent with a bursty 2-state model of gene expression, and demonstrate that this regulatory motif generates the most uncertainty in gene expression when there is the greatest uncertainty in the optimal level of gene expression.

  15. Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae

    PubMed Central

    Ramprecht, Claudia; Zellnig, Günther; Leitner, Erich; Hermetter, Albin; Daum, Günther

    2015-01-01

    In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity. PMID:26241051

  16. Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

    PubMed Central

    Orlean, Peter

    2012-01-01

    The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins. PMID:23135325

  17. TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae

    PubMed Central

    Welch, Aaron Z.; Gibney, Patrick A.; Botstein, David; Koshland, Douglas E.

    2013-01-01

    Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance. PMID:23171550

  18. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.

    2015-01-01

    Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826

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

    SciTech Connect

    Visser, W.; Scheffers, W.A.; Batenburg-Van Der Vegte, W.H.; Van Dijken, J.P. )

    1990-12-01

    Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly ({mu}{sub max}, 0.03 and 0.05 h{sup {minus}1}, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.

  20. Continuous ethanol fermentation using immobilized yeast cells. [Saccharomyces cerevisiae

    SciTech Connect

    Nagashima, M.; Azuma, M.; Noguchi, S.; Inuzuka, K.; Samejima, H.

    1984-01-01

    Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidized-bed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and keep cell viability for stable long run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than one-half year. Cell concentration in the carrier was estimated over 250 g dry cell/L gel. A pilot plant with a total column volume of 4kL was constructed and has been operated since 1982. As a result, it was confirmed that 8-10% (v/v) ethanol-containing broth was continuously produced from nonsterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as 0.6 kL ethanol/kL reactor volume day with a 95% conversion yield versus the maximum theoretical yield for the case of 8.5% (v/v) ethanol broth.

  1. Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae

    SciTech Connect

    Dar, R. D.; Karig, D. K.; Cooke, J. F.; Cox, C. D.; Simpson, M. L.

    2010-09-01

    Stochasticity is an inherent feature of complex systems with nanoscale structure. In such systems information is represented by small collections of elements (e.g. a few electrons on a quantum dot), and small variations in the populations of these elements may lead to big uncertainties in the information. Unfortunately, little is known about how to work within this inherently noisy environment to design robust functionality into complex nanoscale systems. Here, we look to the biological cell as an intriguing model system where evolution has mediated the trade-offs between fluctuations and function, and in particular we look at the relationships and trade-offs between stochastic and deterministic responses in the gene expression of budding yeast (Saccharomyces cerevisiae). We find gene regulatory arrangements that control the stochastic and deterministic components of expression, and show that genes that have evolved to respond to stimuli (stress) in the most strongly deterministic way exhibit the most noise in the absence of the stimuli. We show that this relationship is consistent with a bursty 2-state model of gene expression, and demonstrate that this regulatory motif generates the most uncertainty in gene expression when there is the greatest uncertainty in the optimal level of gene expression.

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

    NASA Astrophysics Data System (ADS)

    Ono, Fumihisa; Shibata, Michiko; Torigoe, Motoki; Matsumoto, Yuta; Yamamoto, Shinsuke; Takizawa, Noboru; Hada, Yoshio; Mori, Yoshihisa; Takarabe, Kenichi

    2013-06-01

    In our previous studies on the tolerance of small plants and animals to the high hydrostatic pressure of 7.5 GPa, it was shown that all the living samples could be borne at this high pressure, which is more than one order of magnitude higher than the proteinic denaturation pressure. To make this inconsistency clear, we have extended these studies to a smaller sized fungus, budding yeast Saccharomyces cerevisiae. A several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate (PC72, Sumitomo 3M), and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar (PDA). It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for 12 and 24 h were found dead. The high pressure tolerance of budding yeast is weaker than that of tardigrades.

  3. Transcriptional response of Saccharomyces cerevisiae to desiccation and rehydration.

    PubMed

    Singh, Jatinder; Kumar, Deept; Ramakrishnan, Naren; Singhal, Vibha; Jervis, Jody; Garst, James F; Slaughter, Stephen M; DeSantis, Andrea M; Potts, Malcolm; Helm, Richard F

    2005-12-01

    A transcriptional analysis of the response of Saccharomyces cerevisiae strain BY4743 to controlled air-drying (desiccation) and subsequent rehydration under minimal glucose conditions was performed. Expression of genes involved in fatty acid oxidation and the glyoxylate cycle was observed to increase during drying and remained in this state during the rehydration phase. When the BY4743 expression profile for the dried sample was compared to that of a commercially prepared dry active yeast, strikingly similar expression changes were observed. The fact that these two samples, dried by different means, possessed very similar transcriptional profiles supports the hypothesis that the response to desiccation is a coordinated event independent of the particular conditions involved in water removal. Similarities between "stationary-phase-essential genes" and those upregulated during desiccation were also noted, suggesting commonalities in different routes to reduced metabolic states. Trends in extracellular and intracellular glucose and trehalose levels suggested that the cells were in a "holding pattern" during the rehydration phase, a concept that was reinforced by cell cycle analyses. Application of a "redescription mining" algorithm suggested that sulfur metabolism is important for cell survival during desiccation and rehydration. PMID:16332871

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

    PubMed

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

    2014-01-01

    Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections. PMID:25036027

  5. Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics.

    PubMed Central

    Ernst, J F; Chan, R K

    1985-01-01

    We describe mutants of Saccharomyces cerevisiae that are more sensitive than the wild type to the aminoglycoside antibiotics G418, hygromycin B, destomycin A, and gentamicin X2. In addition, the mutants are sensitive to apramycin, kanamycin B, lividomycin A, neamine, neomycin, paromomycin, and tobramycin--antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is caused by mutations in at least three genes, denoted AGS1, AGS2, and AGS3 (for aminoglycoside antibiotic sensitivity). Mutations in all three genes are required for highest antibiotic sensitivity; ags1 ags2 double mutants have intermediate antibiotic sensitivity. AGS1 was mapped 8 centimorgans distal from LEU2 on chromosome III. Analyses of yeast strains transformed with vectors carrying antibiotic resistance genes revealed that G418, gentamicin X2, kanamycin B, lividomycin A, neamine, and paromomycin are inactivated by the Tn903 phosphotransferase and that destomycin A is inactivated by the hygromycin B phosphotransferase. ags strains are improved host strains for vectors carrying the phosphotransferase genes because a wide spectrum of aminoglycoside antibiotics can be used to select for plasmid maintenance. PMID:2989254

  6. Expression of YAP4 in Saccharomyces cerevisiae under osmotic stress.

    PubMed Central

    Nevitt, Tracy; Pereira, Jorge; Azevedo, Dulce; Guerreiro, Paulo; Rodrigues-Pousada, Claudina

    2004-01-01

    YAP4, a member of the yeast activator protein ( YAP ) gene family, is induced in response to osmotic shock in the yeast Saccharomyces cerevisiae. The null mutant displays mild and moderate growth sensitivity at 0.4 M and 0.8 M NaCl respectively, a fact that led us to analyse YAP4 mRNA levels in the hog1 (high osmolarity glycerol) mutant. The data obtained show a complete abolition of YAP4 gene expression in this mutant, placing YAP4 under the HOG response pathway. YAP4 overexpression not only suppresses the osmosensitivity phenotype of the yap4 mutant but also relieves that of the hog1 mutant. Induction, under the conditions tested so far, requires the presence of the transcription factor Msn2p, but not of Msn4p, as YAP4 mRNA levels are depleted by at least 75% in the msn2 mutant. This result was further substantiated by the fact that full YAP4 induction requires the two more proximal stress response elements. Furthermore we find that GCY1, encoding a putative glycerol dehydrogenase, GPP2, encoding a NAD-dependent glycerol-3-phosphate phosphatase, and DCS2, a homologue to a decapping enzyme, have decreased mRNA levels in the yap4 -deleted strain. Our data point to a possible, as yet not entirely understood, role of the YAP4 in osmotic stress response. PMID:14680476

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

    PubMed

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

    2013-11-01

    Ginsenosides are the primary bioactive components of ginseng, which is a popular medicinal herb and exhibits diverse pharmacological activities. Protopanaxadiol is the aglycon of several dammarane-type ginsenosides, which also has anticancer activity. For microbial production of protopanaxadiol, dammarenediol-II synthase and protopanaxadiol synthase genes of Panax ginseng, together with a NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana, were introduced into Saccharomyces cerevisiae, resulting in production of 0.05 mg/g DCW protopanaxadiol. Increasing squalene and 2,3-oxidosqualene supplies through overexpressing truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, farnesyl diphosphate synthase, squalene synthase and 2,3-oxidosqualene synthase genes, together with increasing protopanaxadiol synthase activity through codon optimization, led to 262-fold increase of protopanaxadiol production. Finally, using two-phase extractive fermentation resulted in production of 8.40 mg/g DCW protopanaxadiol (1189 mg/L), together with 10.94 mg/g DCW dammarenediol-II (1548 mg/L). The yeast strains engineered in this work can serve as the basis for creating an alternative way for production of ginsenosides in place of extraction from plant sources. PMID:24126082

  8. Mechanical double layer model for Saccharomyces cerevisiae cell wall.

    PubMed

    Mercadé-Prieto, Ruben; Thomas, Colin R; Zhang, Zhibing

    2013-08-01

    The elastic modulus of the Baker's yeast (Saccharomyces cerevisiae) cell wall reported in studies using atomic force microscopy (AFM) is two orders of magnitude lower than that obtained using whole cell compression by micromanipulation. Using finite element modelling, it is shown that Hertz-Sneddon analysis cannot be applied to AFM indentation data for single layer core-shell structures. In addition, the Reissner solution for shallow homogeneous spheres is not appropriate for thick walls such as those of yeast cells. In order to explain yeast compression measurements at different length scales, a double layer wall model is presented considering a soft external layer composed of mannoproteins, and a stiff inner layer of β-glucan fibres and chitin. Under this model, previous AFM studies using sharp indenters provide reasonable estimates of the external layer elastic modulus, while micromanipulation provides the total stiffness of the cell wall. Data from both measurements are combined to estimate the mechanical properties of the inner stiff layer. PMID:23653094

  9. The genes for fifteen ribosomal proteins of Saccharomyces cerevisiae.

    PubMed

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

    1981-10-10

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

  10. Rapid identification of chemical genetic interactions in Saccharomyces cerevisiae.

    PubMed

    Dilworth, David; Nelson, Christopher J

    2015-01-01

    Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described. PMID:25867090

  11. Electrophysiology in the eukaryotic model cell Saccharomyces cerevisiae.

    PubMed

    Bertl, A; Bihler, H; Kettner, C; Slayman, C L

    1998-11-01

    Since the mid-1980s, use of the budding yeast, Saccharomyces cerevisiae, for expression of heterologous (foreign) genes and proteins has burgeoned for several major purposes, including facile genetic manipulation, large-scale production of specific proteins, and preliminary functional analysis. Expression of heterologous membrane proteins in yeast has not kept pace with expression of cytoplasmic proteins for two principal reasons: (1) although plant and fungal proteins express and function easily in yeast membranes, animal proteins do not, at least yet; and (2) the yeast plasma membrane is generally regarded as a difficult system to which to apply the standard electrophysiological techniques for detailed functional analysis of membrane proteins. Especially now, since completion of the genome-sequencing project for Saccharomyces, yeast membranes themselves can be seen as an ample source of diverse membrane proteins - including ion channels, pumps, and cotransporters - which lend themselves to electrophysiological analysis, and specifically to patch-clamping. Using some of these native proteins for assay, we report systematic methods to prepare both the yeast plasma membrane and the yeast vacuolar membrane (tonoplast) for patch-clamp experiments. We also describe optimized ambient conditions - such as electrode preparation, buffer solutions, and time regimens - which facilitate efficient patch recording from Saccharomyces membranes. There are two main keys to successful patch-clamping with Saccharomyces. The first is patience; the second is scrupulous cleanliness. Large cells, such as provided by polyploid strains, are also useful in yeast patch recording, especially while the skill required for gigaseal formation is being learned. Cleanliness is aided by (1) osmotic extrusion of protoplasts, after minimal digestion of yeast walls; (2) use of a rather spare suspension of protoplasts in the recording chamber; (3) maintenance of continuous chamber perfusion prior to formation of gigaseals; (4) preparation (pulling and filling) of patch pipettes immediately before use; (5) application of a modest pressure head to the pipette-filling solution before the tip enters the recording bath; (6) optical control for debris at the pipette tip; and (7) discarding of any pipette that does not "work" on the first try at gigaseal formation. Other useful tricks toward gigaseal formation include the making of protoplasts from cells grown aerobically, rather than anaerobically; use of sustained but gentle suction, rather than hard suction; and manipulation of bath temperature and/or osmotic strength. Yeast plasma membranes form gigaseals with difficulty, but these tend to be very stable and allow for long-term cell-attached or whole-cell recording. Yeast tonoplasts form gigaseals with ease, but these tend to be unstable and rarely allow recording for more than 15 min. The difference of stability accrues mainly because of the fact that yeast protoplasts adhere only lightly to the recording chamber and can therefore be lifted away on the patch pipette, whereas yeast vacuoles adhere firmly to the chamber bottom and are subsequently stressed by very slight relative movements of the pipette. With plasma membranes, conversion from cell-attached recording geometry to isolated ISO patch (inside-out) geometry is accomplished by blowing a fine stream of air bubbles across the pipette tip; to whole-cell recording geometry, by combining suction and one high-voltage pulse; and from whole-cell to OSO patch (outside-out) geometry, by sudden acceleration of the bath perfusion stream. With tonoplasts, conversion from the vacuole-attached recording geometry to whole-vacuole geometry is accomplished by application of a large brief voltage pulse; and further conversion to the OSO patch geometry is carried out conventionally, by slow withdrawal of the patch pipette from the vacuole, which usually remains attached to the chamber bottom. PMID:9799419

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

    PubMed Central

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

    2014-01-01

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

  13. [Rab GTPases networks in membrane traffic in Saccharomyces cerevisiae].

    PubMed

    Nagano, Makoto; Toshima, Junko Y; Toshima, Jiro

    2015-01-01

    Intracellular membrane trafficking between membranous compartments is essential for organelle biogenesis, structure, and identity. Rab/Ypt GTPases are well-characterized regulators of intracellular membrane trafficking, functioning as molecular switches that alternate between GTP- and GDP-bound forms. In Saccharomyces cerevisiae, 11 Rab/Ypt GTPases have been identified and their functions are known to be conserved in their mammalian counterparts. In yeast, the secretory pathway is regulated by sequential activation and inactivation (the so-called Rab cascade) of three types of yeast Rab protein -Ypt1p, Ypt31p/32p and Sec4p -via specific guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In addition to these Rabs, we and others have recently demonstrated that Ypt6p is predominantly localized to the early Golgi compartment, and functions as another regulator of anterograde transport for intra-Golgi trafficking in the secretory pathway. On the other hand, the endocytic pathway is known to be regulated by three yeast Rab5s (Vps21p, Ypt52p and Ypt53p) and one Rab7 (Ypt7p). Rab5 and Rab7 are key determinants of endosome identity, and the Rab5-Rab7 cascade is important for the progression from early to late endosome. Our recent study demonstrates that the endocytic pathway branches into two vacuolar targeting pathways, the Rab5-dependent vacuole protein sorting (VPS) pathway and the Rab5-independent pathway. In this review, we focus on recent advances in our understanding of molecular mechanisms that regulate the localization and activity of yeast Rab GTPases in intracellular membrane trafficking. PMID:25759056

  14. Metabolic engineering for enhanced fatty acids synthesis in Saccharomyces cerevisiae.

    PubMed

    Tang, Xiaoling; Feng, Huixing; Chen, Wei Ning

    2013-03-01

    Microbial production of biofuel has attracted significant attention in recent years. The fatty acids are important precursors for the production of fuels and chemicals, and its biosynthesis is initiated by the conversion of acetyl-CoA to malonyl-CoA which requires acetyl-CoA as key substrate. Herein, the yeast Saccharomyces cerevisiae was proposed to be metabolically engineered for cytosol acetyl-CoA enhancement for fatty acid synthesis. By gene disruption strategy, idh1 and idh2 genes involved in citrate turnover in tricarboxylic acid cycle (TCA cycle) were disrupted and the citrate production level was increased to 4- and 5-times in mutant yeast strains. In order to convert accumulated citrate to cytosol acetyl-CoA, a heterologous ATP-citrate lyase (ACL) was overexpressed in yeast wild type and idh1,2 disrupted strains. The wild type strain expressing acl mainly accumulated saturated fatty acids: C14:0, C16:0 and C18:0 at levels about 20%, 14% and 27%, respectively. Additionally, the idh1,2 disrupted strains expressing acl mainly accumulated unsaturated fatty acids. Specifically in Δidh1 strain expressing acl, 80% increase in C16:1 and 60% increase in C18:1 was detected. In Δidh2 strain expressing acl, 60% increase in C16:1 and 45% increase in C18:1 was detected. In Δidh1/2 strain expressing acl, there was 92% increase in C16:1 and 77% increase in C18:1, respectively. The increased fatty acids from our study may well be potential substrates for the production of hydrocarbon molecules as potential biofuels. PMID:23353549

  15. Comparative genomics of Saccharomyces cerevisiae natural isolates for bioenergy production.

    PubMed

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

    2014-09-01

    Lignocellulosic plant material is a viable source of biomass to produce alternative energy including ethanol and other biofuels. However, several factors—including toxic by products from biomass pretreatment and poor fermentation of xylose and other pentose sugars—currently limit the efficiency of microbial biofuel production. To begin to understand the genetic basis of desirable traits, we characterized three strains of Saccharomyces cerevisiae with robust growth in a pretreated lignocellulosic hydrolysate or tolerance to stress conditions relevant to industrial biofuel production, through genome and transcriptome sequencing analysis. All stress resistant strains were highly mosaic, suggesting that genetic admixture may contribute to novel allele combinations underlying these phenotypes. Strain-specific gene sets not found in the lab strain were functionally linked to the tolerances of particular strains. Furthermore,genes with signatures of evolutionary selection were enriched for functional categories important for stress resistance and included stress-responsive signaling factors. Comparison of the strains’ transcriptomic responses to heat and ethanol treatment—two stresses relevant to industrial bioethanol production—pointed to physiological processes that were related to particular stress resistance profiles. Many of the genotype-by-environment expression responses occurred at targets of transcription factors with signatures of positive selection, suggesting that these strains have undergone positive selection for stress tolerance. Our results generate new insights into potential mechanisms of tolerance to stresses relevant to biofuel production, including ethanol and heat, present a backdrop for further engineering, and provide glimpses into the natural variation of stress tolerance in wild yeast strains. PMID:25364804

  16. Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Cid, V J; Durán, A; del Rey, F; Snyder, M P; Nombela, C; Sánchez, M

    1995-01-01

    In fungi and many other organisms, a thick outer cell wall is responsible for determining the shape of the cell and for maintaining its integrity. The budding yeast Saccharomyces cerevisiae has been a useful model organism for the study of cell wall synthesis, and over the past few decades, many aspects of the composition, structure, and enzymology of the cell wall have been elucidated. The cell wall of budding yeasts is a complex and dynamic structure; its arrangement alters as the cell grows, and its composition changes in response to different environmental conditions and at different times during the yeast life cycle. In the past few years, we have witnessed a profilic genetic and molecular characterization of some key aspects of cell wall polymer synthesis and hydrolysis in the budding yeast. Furthermore, this organism has been the target of numerous recent studies on the topic of morphogenesis, which have had an enormous impact on our understanding of the intracellular events that participate in directed cell wall synthesis. A number of components that direct polarized secretion, including those involved in assembly and organization of the actin cytoskeleton, secretory pathways, and a series of novel signal transduction systems and regulatory components have been identified. Analysis of these different components has suggested pathways by which polarized secretion is directed and controlled. Our aim is to offer an overall view of the current understanding of cell wall dynamics and of the complex network that controls polarized growth at particular stages of the budding yeast cell cycle and life cycle. PMID:7565410

  17. Genetic Basis for Saccharomyces cerevisiae Biofilm in Liquid Medium

    PubMed Central

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

    2014-01-01

    Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the Σ1278b background and found 71 genes that were essential for biofilm development. Quantitative northern blots further revealed that AIM1, ASG1, AVT1, DRN1, ELP4, FLO8, FMP10, HMT1, KAR5, MIT1, MRPL32, MSS11, NCP1, NPR1, PEP5, PEX25, RIM8, RIM101, RGT1, SNF8, SPC2, STB6, STP22, TEC1, VID24, VPS20, VTC3, YBL029W, YBL029C-A, YFL054C, YGR161W-C, YIL014C-A, YIR024C, YKL151C, YNL200C, YOR034C-A, and YOR223W controlled biofilm through FLO11 induction. Almost all deletion mutants that were unable to form biofilms in liquid medium also lost the ability to form surface-spreading biofilm colonies (mats) on agar and 69% also lost the ability to grow invasively. The protein kinase A isoform Tpk3p functioned specifically in biofilm and mat formation. In a tpk3 mutant, transcription of FLO11 was induced three-fold compared with wild-type, but biofilm development and cell–cell adhesion was absent, suggesting that Tpk3p regulates FLO11 positive posttranscriptionally and negative transcriptionally. The study provides a resource of biofilm-influencing genes for additional research on biofilm development and suggests that the regulation of FLO11 is more complex than previously anticipated. PMID:25009170

  18. Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Henry, Susan A.; Kohlwein, Sepp D.; Carman, George M.

    2012-01-01

    Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces cerevisiae is a model system of choice for the study of the genetics, biochemistry, and cell biology of eukaryotic lipid metabolism. Glycerolipids (e.g., phospholipids and triacylglycerol) and their precursors are synthesized and metabolized by enzymes associated with the cytosol and membranous organelles, including endoplasmic reticulum, mitochondria, and lipid droplets. Genetic and biochemical analyses have revealed that glycerolipids play important roles in cell signaling, membrane trafficking, and anchoring of membrane proteins in addition to membrane structure. The expression of glycerolipid enzymes is controlled by a variety of conditions including growth stage and nutrient availability. Much of this regulation occurs at the transcriptional level and involves the Ino2–Ino4 activation complex and the Opi1 repressor, which interacts with Ino2 to attenuate transcriptional activation of UASINO-containing glycerolipid biosynthetic genes. Cellular levels of phosphatidic acid, precursor to all membrane phospholipids and the storage lipid triacylglycerol, regulates transcription of UASINO-containing genes by tethering Opi1 to the nuclear/endoplasmic reticulum membrane and controlling its translocation into the nucleus, a mechanism largely controlled by inositol availability. The transcriptional activator Zap1 controls the expression of some phospholipid synthesis genes in response to zinc availability. Regulatory mechanisms also include control of catalytic activity of glycerolipid enzymes by water-soluble precursors, products and lipids, and covalent modification of phosphorylation, while in vivo function of some enzymes is governed by their subcellular location. Genome-wide genetic analysis indicates coordinate regulation between glycerolipid metabolism and a broad spectrum of metabolic pathways. PMID:22345606

  19. Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1

    PubMed Central

    Edlind, Thomas D.

    2012-01-01

    Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncated Saccharomyces cerevisiae Fks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predicted in silico for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2. PMID:22581527

  20. Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae Cells Permeabilized with Ether

    PubMed Central

    Oertel, Wolfgang; Goulian, Mehran

    1979-01-01

    Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With ρ+ strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-1Bρ+) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Density-labeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In ρ0 strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperature-sensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment. PMID:387730

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

    PubMed

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

    2016-01-01

    This study aimed to optimize extracellular glutathione production by a Saccharomyces cerevisiae engineered strain and to concentrate the extracellular glutathione by membrane separation processes, including ultrafiltration (UF) and nanofiltration (NF). Synthetic defined (SD) medium containing 20 g L(-1) glucose was fermented for 48 h; the fermentation liquid was passed through an UF membrane to remove macromolecules. Glutathione in this permeate was concentrated for 48 h to 545.1 ± 33.6 mg L(-1) using the NF membrane; this was a significantly higher concentration than that obtained with yeast extract peptone dextrose (YPD) medium following 96 h NF concentration (217.9 ± 57.4 mg L(-1)). This higher glutathione concentration results from lower cellular growth in SD medium (final OD600 = 6.9 ± 0.1) than in YPD medium (final OD600 = 11.0 ± 0.6) and thus higher production of extracellular glutathione (16.0 ± 1.3 compared to 9.2 ± 2.1 mg L(-1) in YPD medium, respectively). Similar fermentation and membrane processing of sweet sorghum juice containing 20 g L(-1) total sugars provided 240.3 ± 60.6 mg L(-1) glutathione. Increased extracellular production of glutathione by this engineered strain in SD medium and subsequent UF permeation and NF concentration in shortend time may help realize industrial recovery of extracellular glutathione. PMID:26105794

  2. Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.

    PubMed

    Rich, Matthew S; Payen, Celia; Rubin, Alan F; Ong, Giang T; Sanchez, Monica R; Yachie, Nozomu; Dunham, Maitreya J; Fields, Stanley

    2016-05-01

    In the yeast Saccharomyces cerevisiae, beneficial mutations selected during sulfate-limited growth are typically amplifications of the SUL1 gene, which encodes the high-affinity sulfate transporter, resulting in fitness increases of >35% . Cis-regulatory mutations have not been observed at this locus; however, it is not clear whether this absence is due to a low mutation rate such that these mutations do not arise, or they arise but have limited fitness effects relative to those of amplification. To address this question directly, we assayed the fitness effects of nearly all possible point mutations in a 493-base segment of the gene's promoter through mutagenesis and selection. While most mutations were either neutral or detrimental during sulfate-limited growth, eight mutations increased fitness >5% and as much as 9.4%. Combinations of these beneficial mutations increased fitness only up to 11%. Thus, in the case of SUL1, promoter mutations could not induce a fitness increase similar to that of gene amplification. Using these data, we identified functionally important regions of the SUL1 promoter and analyzed three sites that correspond to potential binding sites for the transcription factors Met32 and Cbf1 Mutations that create new Met32- or Cbf1-binding sites also increased fitness. Some mutations in the untranslated region of the SUL1 transcript decreased fitness, likely due to the formation of inhibitory upstream open reading frames. Our methodology-saturation mutagenesis, chemostat selection, and DNA sequencing to track variants-should be a broadly applicable approach. PMID:26936925

  3. Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae.

    PubMed Central

    Brevet, A; Chen, J; Fromant, M; Blanquet, S; Plateau, P

    1991-01-01

    An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988). Images PMID:1653209

  4. Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu.

    PubMed

    Rosenthal, L P; Bodley, J W

    1987-08-15

    Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu. PMID:3301847

  5. Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae.

    PubMed

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

    2010-01-01

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

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

    PubMed

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

    2012-10-01

    Cellular memory of past experiences has been observed in several organisms and across a variety of experiences, including bacteria "remembering" prior nutritional status and amoeba "learning" to anticipate future environmental conditions. Here, we show that Saccharomyces cerevisiae maintains a multifaceted memory of prior stress exposure. We previously demonstrated that yeast cells exposed to a mild dose of salt acquire subsequent tolerance to severe doses of H(2)O(2). We set out to characterize the retention of acquired tolerance and in the process uncovered two distinct aspects of cellular memory. First, we found that H(2)O(2) resistance persisted for four to five generations after cells were removed from the prior salt treatment and was transmitted to daughter cells that never directly experienced the pretreatment. Maintenance of this memory did not require nascent protein synthesis after the initial salt pretreatment, but rather required long-lived cytosolic catalase Ctt1p that was synthesized during salt exposure and then distributed to daughter cells during subsequent cell divisions. In addition to and separable from the memory of H(2)O(2) resistance, these cells also displayed a faster gene-expression response to subsequent stress at >1000 genes, representing transcriptional memory. The faster gene-expression response requires the nuclear pore component Nup42p and serves an important function by facilitating faster reacquisition of H(2)O(2) tolerance after a second cycle of salt exposure. Memory of prior stress exposure likely provides a significant advantage to microbial populations living in ever-changing environments. PMID:22851651

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

    PubMed

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

    2015-01-01

    Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028 mg/l (+)-limonene and 0.060 mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12 mg/l (+)-limonene and 0.49 mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. PMID:25164098

  8. A vector set for systematic metabolic engineering in Saccharomyces cerevisiae

    PubMed Central

    Fang, Fang; Salmon, Kirsty; Shen, Michael W. Y.; Aeling, Kimberly A.; Ito, Elaine; Irwin, Becky; Tran, Uyen Phuong C.; Hatfield, G. Wesley; Da Silva, Nancy A.; Sandmeyer, Suzanne

    2011-01-01

    A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 μ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering. PMID:20936606

  9. Mutational Analysis of NHAoc/NHA2 in Saccharomyces cerevisiae

    PubMed Central

    Huang, Xiaobin; Morse, Leslie R.; Xu, Yan; Zahradka, Jaromir; Sychrová, Hana; Stashenko, Phil; Fan, Feiyue; Battaglino, Ricardo A.

    2010-01-01

    Background NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes. Methods The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth. Results Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 can not grow in 0.4 M NaCl, suggesting that these residues are also essential for antiporter activity. Conclusions Evolutionarily conserved amino acids are required for full antiporter function. General Significance Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo. PMID:20713131

  10. A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae.

    PubMed

    Chu, Daniel B; Burgess, Sean M

    2016-01-01

    Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions. PMID:26747203

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

    PubMed Central

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

    2015-01-01

    How populations that inhabit the same geographical area become genetically differentiated is not clear. To investigate this, we characterized phenotypic and genetic differences between two populations of Saccharomyces cerevisiae that in some cases inhabit the same environment but show relatively little gene flow. We profiled stress sensitivity in a group of vineyard isolates and a group of oak-soil strains and found several niche-related phenotypes that distinguish the populations. We performed bulk-segregant mapping on two of the distinguishing traits: The vineyard-specific ability to grow in grape juice and oak-specific tolerance to the cell wall damaging drug Congo red. To implicate causal genes, we also performed a chemical genomic screen in the lab-strain deletion collection and identified many important genes that fell under quantitative trait loci peaks. One gene important for growth in grape juice and identified by both the mapping and the screen was SSU1, a sulfite-nitrite pump implicated in wine fermentations. The beneficial allele is generated by a known translocation that we reasoned may also serve as a genetic barrier. We found that the translocation is prevalent in vineyard strains, but absent in oak strains, and presents a postzygotic barrier to spore viability. Furthermore, the translocation was associated with a fitness cost to the rapid growth rate seen in oak-soil strains. Our results reveal the translocation as a dual-function locus that enforces ecological differentiation while producing a genetic barrier to gene flow in these sympatric populations. PMID:25953281

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

    PubMed

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

    2010-07-01

    Carob has been widely grown in the Mediterranean region for a long time. It has been regarded as only a forest tree and has been neglected for other economical benefits. However, in recent years, this fruit has gained attention for several applications. As petroleum has become depleted, renewable energy production has started to gain attention all over the world; including the production of ethanol from underutilized agricultural products such as carob. In this project, the optimum extraction conditions were determined for the carob fruit by using the response surface design method. The obtained extract was utilized for production of ethanol by using suspended Saccharomyces cerevisiae fermentation. The effect of various fermentation parameters such as pH, media content and inoculum size were evaluated for ethanol fermentation in carob extract. Also, in order to determine economically appropriate nitrogen sources, four different nitrogen sources were evaluated. The optimum extraction condition for carob extract was determined to be 80 degrees C, 2h in 1:4 dilution rate (fruit: water ratio) according to the result of response surface analysis (115.3g/L). When the fermentation with pH at 5.5 was applied, the final ethanol concentration and production rates were 42.6g/L and 3.37 g/L/h, respectively, which were higher than using an uncontrolled pH. Among inoculum sizes of 1%, 3%, and 5%, 3% was determined as the best inoculum size. The maximum production rate and final ethanol concentration were 3.48 g/L/h and 44.51%, respectively, with an alternative nitrogen source of meat-bone meal. Overall, this study suggested that carob extract can be utilized for production of ethanol in order to meet the demands of renewable energy. PMID:20189805

  13. Biogenesis of the Saccharomyces cerevisiae Mating Pheromone a-Factor

    PubMed Central

    Chen, Peng; Sapperstein, Stephanie K.; Choi, Jonathan D.; Michaelis, Susan

    1997-01-01

    The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification, NH2-terminal proteolysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDSPAGE. We determined the biochemical composition of each by defining their NH2-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH2-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis. PMID:9015298

  14. Experimental bioenergetics of Saccharomyces cerevisiae in respiration and fermentation

    SciTech Connect

    Yerushalmi, L.; Volesky, B.

    1981-10-01

    Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. ''Aerobic fermentation'' and ''aerobic respiration'' were established respectively in the experimental system by performing batch and fed-batch experiments. ''Balanced growth'' batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO2 evolution rate, O2 uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing c, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (delta h) with the fraction of available electrons transferred to oxygen (epsilon O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller epsilon O2 than delta h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficient, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation. (Refs. 19).

  15. Experimental bioenergetics of Saccharomyces cerevisiae in respiration and fermentation

    SciTech Connect

    Yerushalmi, L.; Volesky, B.

    1981-01-01

    Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and EtOH synthesis. Aerobic fermentation and aerobic respiration were established respectively in the experimental system by performing batch and fed-batch experiments. Balanced growth batch cultivations were carried out with initial sugar concentrations of 10-70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as EtOH production rate, CO/sub 2/ evolution rate, O/sub 2/ uptake rate, sp. growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution, and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (delta h) with the fraction of available electrons transferred to O/sub 2/ (epsilon O/sub 2/) indicated equal values of the 2 (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller epsilon O/sub 2/ than delta h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficient, together with the fraction of the available electrons transferred to O/sub 2/, contributed to the estimation of the extent of heat production through oxidative phosphorylation.

  16. Mating-type Gene Switching in Saccharomyces cerevisiae.

    PubMed

    Lee, Cheng-Sheng; Haber, James E

    2015-04-01

    The budding yeast Saccharomyces cerevisiae has two alternative mating types designated MATa and MATα. These are distinguished by about 700 bp of unique sequences, Ya or Yα, including divergent promoter sequences and part of the open reading frames of genes that regulate mating phenotype. Homothallic budding yeast, carrying an active HO endonuclease gene, HO, can switch mating type through a recombination process known as gene conversion, in which a site-specific double-strand break (DSB) created immediately adjacent to the Y region results in replacement of the Y sequences with a copy of the opposite mating type information, which is harbored in one of two heterochromatic donor loci, HMLα or HMRa. HO gene expression is tightly regulated to ensure that only half of the cells in a lineage switch to the opposite MAT allele, thus promoting conjugation and diploid formation. Study of the silencing of these loci has provided a great deal of information about the role of the Sir2 histone deacetylase and its associated Sir3 and Sir4 proteins in creating heterochromatic regions. MAT switching has been examined in great detail to learn about the steps in homologous recombination. MAT switching is remarkably directional, with MATa recombining preferentially with HMLα and MATα using HMRa. Donor preference is controlled by a cis-acting recombination enhancer located near HML. RE is turned off in MATα cells but in MATa binds multiple copies of the Fkh1 transcription factor whose forkhead-associated phosphothreonine binding domain localizes at the DSB, bringing HML into conjunction with MATa. PMID:26104712

  17. Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

    PubMed Central

    Hawes, Catherine S.; Nicholas, D. J. D.

    1973-01-01

    1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at −15°C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP2− complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the Km of each substrate was determined at infinite concentration of the other (Km MgATP2−, 0.07mm; MoO42−, 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5′-[35S]sulphatophosphate and SO42−, 35SO42− and adenosine 5′-sulphatophosphate, occurred only in the presence of either MgATP2− or PPi. This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO32−, S2O32− (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO42− was produced by SO42− (Ki=2.0mm) and non-competitive inhibition by sulphide (Ki=3.4mm). 8. Adenosine 5′-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity–substrate curves with both MgATP2− and MoO42− to sigmoidal forms. PMID:4582048

  18. The anatomy of a hypoxic operator in Saccharomyces cerevisiae.

    PubMed Central

    Deckert, J; Torres, A M; Hwang, S M; Kastaniotis, A J; Zitomer, R S

    1998-01-01

    Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes. PMID:9832521

  19. A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae

    PubMed Central

    Chu, Daniel B.; Burgess, Sean M.

    2016-01-01

    Errors segregating homologous chromosomes during meiosis result in aneuploid gametes and are the largest contributing factor to birth defects and spontaneous abortions in humans. Saccharomyces cerevisiae has long served as a model organism for studying the gene network supporting normal chromosome segregation. Measuring homolog nondisjunction frequencies is laborious, and involves dissecting thousands of tetrads to detect missegregation of individually marked chromosomes. Here we describe a computational method (TetFit) to estimate the relative contributions of meiosis I nondisjunction and random-spore death to spore inviability in wild type and mutant strains. These values are based on finding the best-fit distribution of 4, 3, 2, 1, and 0 viable-spore tetrads to an observed distribution. Using TetFit, we found that meiosis I nondisjunction is an intrinsic component of spore inviability in wild-type strains. We show proof-of-principle that the calculated average meiosis I nondisjunction frequency determined by TetFit closely matches empirically determined values in mutant strains. Using these published data sets, TetFit uncovered two classes of mutants: Class A mutants skew toward increased nondisjunction death, and include those with known defects in establishing pairing, recombination, and/or synapsis of homologous chromosomes. Class B mutants skew toward random spore death, and include those with defects in sister-chromatid cohesion and centromere function. Epistasis analysis using TetFit is facilitated by the low numbers of tetrads (as few as 200) required to compare the contributions to spore death in different mutant backgrounds. TetFit analysis does not require any special strain construction, and can be applied to previously observed tetrad distributions. PMID:26747203

  20. Interaction between lanthanide ions and Saccharomyces cerevisiae cells.

    PubMed

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

    2015-10-01

    Lanthanides are a group of non-essential elements with important imaging and therapeutic applications. Although trivalent lanthanide ions (Ln³⁺) are used as potent blockers of Ca²⁺ channels, the systematic studies correlating Ln³⁺ accumulation and toxicity to Ca²⁺ channel blocking activity are scarce. In this study, we made use of the eukaryotic model Saccharomyces cerevisiae to investigate the correlation between Ln³⁺ accumulation, their toxicity and their capacity to block the exogenous stress-induced Ca²⁺ influx into the cytosol. It was found that the Ln³⁺ blocked the Ca²⁺ entry into the yeast cells only when present at concentration high enough to allow rapid binding to cell surface. At lower concentrations, Ln³⁺ were taken up by the cell, but Ca²⁺ blockage was no longer achieved. At 1 mM concentration, all ions from the Ln³⁺ series could block Ca²⁺ entry into cytosol with the exception of La³⁺, and to a lesser extent, Pr³⁺ and Nd³⁺. The plasma membrane Ca²⁺-channel Cch1/Mid1 contributed to La³⁺ and Gd³⁺ entry into the cells, with a significant preference for La³⁺. The results open the possibility to obtain cells loaded with controlled amounts and ratios of Ln³⁺. PMID:26267167

  1. Initiation of sporulation in Saccharomyces cerevisiae. Mutations preventing initiation.

    PubMed

    Calvert, G R; Dawes, I W

    1984-03-01

    Mutants of Saccharomyces cerevisiae that are unable to initiate sporulation, but can continue vegetative growth under conditions in which the wild-type strain sporulates, have been isolated and characterized. The mutations arose spontaneously as suppressors of the spd1 mutations, restoring the ability of spd1 mutants to grow on glycerol, and also spontaneously in cultures of a wild-type diploid strain undergoing sporulation in continuous culture. The mutations all conferred asporogeny, and were recessive in this respect to the wild-type, but dominant in acting as suppressors of the spd1 mutation. They fell into three complementation groups which corresponded to three unlinked loci, designated spo50, spo51 and spo53. None of these mutations was closely linked to the other initiation mutations defined by the spd1, spd3, spd4, cdc25, cdc28 loci, nor to the cell size control mutations whi1 and whi2. Loose linkage was detected between spd1 and spo53, and spo50, spd3 and spo53 were linked to their respective centromeres. The spo50, spo51 and spo53 mutations are not nonsense suppressors. Mutations in all three genes conferred similar highly pleiotropic phenotypes including: asporogeny; dominant suppression of both spd1 and spd3 mutations; aberrant cell morphology and viability loss on starvation; constitutive ability to reduce tetrazolium (which is subject to carbon source repression in the wild-type); and complete repression of the synthesis of several polypeptides that are subject to carbon source repression in the wild-type strain and derepressed in spd1 mutants derepressed for sporulation. A diploid strain homozygous for the spo50 mutation did not undergo either premeiotic DNA replication or meiotic recombination when transferred to sporulation media. PMID:6374029

  2. Composition of the protoplast membrane from Saccharomyces cerevisiae

    PubMed Central

    Longley, R. P.; Rose, A. H.; Knights, B. A.

    1968-01-01

    1. Protoplasts of Saccharomyces cerevisiae N.C.Y.C. 366 were prepared by incubating washed exponential-phase cells in buffered mannitol (0·8m) containing 10mm-magnesium chloride and snail gut juice (about 8mg. of protein/ml. of reaction mixture). Protoplast membranes were obtained by bursting protoplasts in ice-cold phosphate buffer (pH7·0) containing 10mm-magnesium chloride. 2. Protoplast membranes accounted for 13–20% of the dry weight of the yeast cell. They contained on a weight basis about 39% of lipid, 49% of protein, 6% of sterol (assayed spectrophotometrically) and traces of RNA and carbohydrate (glucan+mannan). 3. The principal fatty acids in membrane lipids were C16:0, C16:1 and C18:1 acids. Whole cells contained a slightly greater proportion of C16:0 and a somewhat smaller proportion of C18:1 acids. Membrane and whole-cell lipids included monoglycerides, diglycerides, triglycerides, sterols, sterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine. Phosphorus analyses on phospholipid fractions from membranes and whole cells showed that membranes contained proportionately more phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine than whole cells, which in turn were richer in phosphatidylcholine. Phospholipid fractions from membranes and whole cells had similar fatty acid compositions. 4. Membranes and whole cells contained two major and three minor sterol components. Gas–liquid chromatography, mass spectrometry and u.v. and i.r. spectra indicated that the major components were probably Δ5,7,22,24(28)-ergostatetraen-3β-ol and zymosterol. The minor sterol components in whole cells were probably episterol (or fecosterol), ergosterol and a C29 di-unsaturated sterol. 5. Defatted whole cells contained slightly more glutamate and ornithine and slightly less leucine and isoleucine than membranes. Otherwise, no major differences were detected in the amino acid compositions of defatted whole cells and membranes. PMID:5667254

  3. Molecular Analysis of Maltotriose Transport and Utilization by Saccharomyces cerevisiae

    PubMed Central

    Day, Rachel E.; Rogers, Peter J.; Dawes, Ian W.; Higgins, Vincent J.

    2002-01-01

    Efficient fermentation of maltotriose is a desired property of Saccharomyces cerevisiae for brewing. In a standard wort, maltotriose is the second most abundant sugar, and slower uptake leads to residual maltotriose in the finished product. The limiting factor of sugar metabolism is its transport, and there are conflicting reports on whether a specific maltotriose permease exists or whether the mechanisms responsible for maltose uptake also carry out maltotriose transport. In this study, radiolabeled maltotriose was used to show that overexpression of the maltose permease gene, MAL61, in an industrial yeast strain resulted in an increase in the rate of transport of maltotriose as well as maltose. A strain derived from W303-1A and lacking any maltose or maltotriose transporter but carrying a functional maltose transport activator (MAL63) was developed. By complementing this strain with permeases encoded by MAL31, MAL61, and AGT1, it was possible to measure their specific transport kinetics by using maltotriose and maltose. All three permeases were capable of high-affinity transport of maltotriose and of allowing growth of the strain on the sugar. Maltotriose utilization from the permease encoded by AGT1 was regulated by the same genetic mechanisms as those involving the maltose transcriptional activator. Competition studies carried out with two industrial strains, one not containing any homologue of AGT1, showed that maltose uptake and maltotriose uptake were competitive and that maltose was the preferred substrate. These results indicate that the presence of residual maltotriose in beer is not due to a genetic or physiological inability of yeast cells to utilize the sugar but rather to the lower affinity for maltotriose uptake in conjunction with deteriorating conditions present at the later stages of fermentation. Here we identify molecular mechanisms regulating the uptake of maltotriose and determine the role of each of the transporter genes in the cells. PMID:12406721

  4. Metabolic engineering of muconic acid production in Saccharomyces cerevisiae.

    PubMed

    Curran, Kathleen A; Leavitt, John M; Karim, Ashty S; Alper, Hal S

    2013-01-01

    The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141mg/L muconic acid in a shake-flask culture, a value nearly 24-fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts. PMID:23164574

  5. Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts

    PubMed Central

    2014-01-01

    Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose. This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase. PMID:24949272

  6. Saccharomyces cerevisiae Genes Involved in Survival of Heat Shock

    PubMed Central

    Jarolim, Stefanie; Ayer, Anita; Pillay, Bethany; Gee, Allison C.; Phrakaysone, Alex; Perrone, Gabriel G.; Breitenbach, Michael; Dawes, Ian W.

    2013-01-01

    The heat-shock response in cells, involving increased transcription of a specific set of genes in response to a sudden increase in temperature, is a highly conserved biological response occurring in all organisms. Despite considerable attention to the processes activated during heat shock, less is known about the role of genes in survival of a sudden temperature increase. Saccharomyces cerevisiae genes involved in the maintenance of heat-shock resistance in exponential and stationary phase were identified by screening the homozygous diploid deletants in nonessential genes and the heterozygous diploid mutants in essential genes for survival after a sudden shift in temperature from 30 to 50°. More than a thousand genes were identified that led to altered sensitivity to heat shock, with little overlap between them and those previously identified to affect thermotolerance. There was also little overlap with genes that are activated or repressed during heat-shock, with only 5% of them regulated by the heat-shock transcription factor. The target of rapamycin and protein kinase A pathways, lipid metabolism, vacuolar H+-ATPase, vacuolar protein sorting, and mitochondrial genome maintenance/translation were critical to maintenance of resistance. Mutants affected in l-tryptophan metabolism were heat-shock resistant in both growth phases; those affected in cytoplasmic ribosome biogenesis and DNA double-strand break repair were resistant in stationary phase, and in mRNA catabolic processes in exponential phase. Mutations affecting mitochondrial genome maintenance were highly represented in sensitive mutants. The cell division transcription factor Swi6p and Hac1p involved in the unfolded protein response also play roles in maintenance of heat-shock resistance. PMID:24142923

  7. From one to many: expanding the Saccharomyces cerevisiae reference genome panel.

    PubMed

    Engel, Stacia R; Weng, Shuai; Binkley, Gail; Paskov, Kelley; Song, Giltae; Cherry, J Michael

    2016-01-01

    In recent years, thousands of Saccharomyces cerevisiae genomes have been sequenced to varying degrees of completion. The Saccharomyces Genome Database (SGD) has long been the keeper of the original eukaryotic reference genome sequence, which was derived primarily from S. cerevisiae strain S288C. Because new technologies are pushing S. cerevisiae annotation past the limits of any system based exclusively on a single reference sequence, SGD is actively working to expand the original S. cerevisiae systematic reference sequence from a single genome to a multi-genome reference panel. We first commissioned the sequencing of additional genomes and their automated analysis using the AGAPE pipeline. Here we describe our curation strategy to produce manually reviewed high-quality genome annotations in order to elevate 11 of these additional genomes to Reference status.Database URL: http://www.yeastgenome.org/. PMID:26989152

  8. From one to many: expanding the Saccharomyces cerevisiae reference genome panel

    PubMed Central

    Engel, Stacia R.; Weng, Shuai; Binkley, Gail; Paskov, Kelley; Song, Giltae; Cherry, J. Michael

    2016-01-01

    In recent years, thousands of Saccharomyces cerevisiae genomes have been sequenced to varying degrees of completion. The Saccharomyces Genome Database (SGD) has long been the keeper of the original eukaryotic reference genome sequence, which was derived primarily from S. cerevisiae strain S288C. Because new technologies are pushing S. cerevisiae annotation past the limits of any system based exclusively on a single reference sequence, SGD is actively working to expand the original S. cerevisiae systematic reference sequence from a single genome to a multi-genome reference panel. We first commissioned the sequencing of additional genomes and their automated analysis using the AGAPE pipeline. Here we describe our curation strategy to produce manually reviewed high-quality genome annotations in order to elevate 11 of these additional genomes to Reference status. Database URL: http://www.yeastgenome.org/ PMID:26989152

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    After accident of Fukushima Daiichi Nuclear Power Plant, the fall-out radiocesium was deposited on the ground. Filamentous fungus is known to accumulate radiocesium in environment, even though many minerals are involved in soil. These facts suggest that fungus affect the migration behavior of radiocesium in the environment. However, accumulation mechanism of radiocesium by fungus is not understood. In the present study, accumulation and chemical states change of Cs by unicellular fungus of Saccharomyces cerevisiae have been studied to elucidate the role of microorganisms in the migration of radiocesium in the environment. Two different experimental conditions were employed; one is the accumulation experiments of radiocesium by S. cerevisiae from the agar medium containing 137Cs and a mineral of zeolite, vermiculite, smectite, mica, or illite. The other is the experiments using stable cesium to examine the chemical states change of Cs. In the former experiment, the cells were grown on membrane filter of 0.45 μm installed on the agar medium. After the grown cells were weighed, radioactivity in the cells was measured by an autoradiography technique. The mineral weight contents were changed from 0.1% to 1% of the medium. In the latter experiment, the cells were grown in the medium containing stable Cs between 1 mM and 10mM. The Cs accumulated cells were analyzed by SEM-EDS and EXAFS. The adsorption experiments of cesium by the cells under resting condition were also conducted to test the effect of cells metabolic activity. Without mineral in the medium, cells of S. cerevisiae accumulated 1.5x103 Bq/g from the medium containing 137Cs of 2.6x102 Bq/g. When mineral was added in the medium, concentration of 137Cs in the cells decreased. The concentration of 137Cs in the cells from the medium containing different minerals were in the following order; smectite, illite, mica > vermiculite > zeolite. This order was nearly the same as the inverse of distribution coefficient of mineral for 137Cs in the medium solution. The concentration of 137Cs in the cells lowered in the medium containing higher mineral content. These results indicate that radiocesium was competively accumulated in the cells with minerals in the soil. Higher concentration of stable Cs was accumulated in the cells in the metabolically active condition than in the resting cells condition. XAFS analyses showed that the k3-weighted extended-XAFS functions and the radial structural function of Cs accumulated by the cells in the metabolically active condition were similar to those in the resting condition, indicating that chemical states of the accumulated Cs were nearly the same between both conditions. These results indicate that the fungus accumulates radiocesium by competitively with minerals in the soils, and performs higher retardation of the migration of Cs in the metabolically active condition than the resting one. A part of this study is the results of "Multidisciplinary investigation on radiocesium fate and transport for safety assessment for interim storage and disposal of heterogeneous waste" carried out under the Initiatives for Atomic Energy Basic and Generic Strategic Research by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

  10. Saccharomyces cerevisiae fungemia, a possible consequence of the treatment of Clostridium difficile colitis with a probioticum.

    PubMed

    Santino, I; Alari, A; Bono, S; Teti, E; Marangi, M; Bernardini, A; Magrini, L; Di Somma, S; Teggi, A

    2014-01-01

    The yeast Saccharomyces boulardii is a biotherapeutic agent used for the prevention and treatment of several gastrointestinal diseases, such as diarrhoea caused by Clostridium difficile, in addition to the antibiotic therapy. In this study we report a case of Saccharomyces cerevisiae fungemia in a patient with Clostridium difficile-associated diarrhoea (CDAD) treated orally with S. boulardii in association with vancomycin. The identification of the S. cerevisiae was confirmed by molecular technique. Fungemia is a rare, but a serious complication to treatment with probiotics. We believe it is important to remind the clinicians of this risk when prescribing probiotics, especially to immunocompromised patients. PMID:24674691

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

    PubMed

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

    2015-01-01

    Formic acid is one of the major inhibitory compounds present in hydrolysates derived from lignocellulosic materials, the presence of which can significantly hamper the efficiency of converting available sugars into bioethanol. This study investigated the potential for screening formic acid tolerance in non-Saccharomyces cerevisiae yeast strains, which could be used for the development of advanced generation bioethanol processes. Spot plate and phenotypic microarray methods were used to screen the formic acid tolerance of 7 non-Saccharomyces cerevisiae yeasts. S. kudriavzeii IFO1802 and S. arboricolus 2.3319 displayed a higher formic acid tolerance when compared to other strains in the study. Strain S. arboricolus 2.3319 was selected for further investigation due to its genetic variability among the Saccharomyces species as related to Saccharomyces cerevisiae and availability of two sibling strains: S. arboricolus 2.3317 and 2.3318 in the lab. The tolerance of S. arboricolus strains (2.3317, 2.3318 and 2.3319) to formic acid was further investigated by lab-scale fermentation analysis, and compared with S. cerevisiae NCYC2592. S. arboricolus 2.3319 demonstrated improved formic acid tolerance and a similar bioethanol synthesis capacity to S. cerevisiae NCYC2592, while S. arboricolus 2.3317 and 2.3318 exhibited an overall inferior performance. Metabolite analysis indicated that S. arboricolus strain 2.3319 accumulated comparatively high concentrations of glycerol and glycogen, which may have contributed to its ability to tolerate high levels of formic acid. PMID:26284784

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

    PubMed Central

    Martorell, P.; Querol, A.; Fernández-Espinar, M. T.

    2005-01-01

    Despite the beneficial role of Saccharomyces cerevisiae in the food industry for food and beverage production, it is able to cause spoilage in wines. We have developed a real-time PCR method to directly detect and quantify this yeast species in wine samples to provide winemakers with a rapid and sensitive method to detect and prevent wine spoilage. Specific primers were designed for S. cerevisiae using the sequence information obtained from a cloned random amplified polymorphic DNA band that differentiated S. cerevisiae from its sibling species Saccharomyces bayanus, Saccharomyces pastorianus, and Saccharomyces paradoxus. The specificity of the primers was demonstrated for typical wine spoilage yeast species. The method was useful for estimating the level of S. cerevisiae directly in sweet wines and red wines without preenrichment when yeast is present in concentrations as low as 3.8 and 5 CFU per ml. This detection limit is in the same order as that obtained from glucose-peptone-yeast growth medium (GPY). Moreover, it was possible to quantify S. cerevisiae in artificially contaminated samples accurately. Limits for accurate quantification in wine were established, from 3.8 × 105 to 3.8 CFU/ml in sweet wine and from 5 × 106 to 50 CFU/ml in red wine. PMID:16269715

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

    PubMed Central

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

    2015-01-01

    Formic acid is one of the major inhibitory compounds present in hydrolysates derived from lignocellulosic materials, the presence of which can significantly hamper the efficiency of converting available sugars into bioethanol. This study investigated the potential for screening formic acid tolerance in non-Saccharomyces cerevisiae yeast strains, which could be used for the development of advanced generation bioethanol processes. Spot plate and phenotypic microarray methods were used to screen the formic acid tolerance of 7 non-Saccharomyces cerevisiae yeasts. S. kudriavzeii IFO1802 and S. arboricolus 2.3319 displayed a higher formic acid tolerance when compared to other strains in the study. Strain S. arboricolus 2.3319 was selected for further investigation due to its genetic variability among the Saccharomyces species as related to Saccharomyces cerevisiae and availability of two sibling strains: S. arboricolus 2.3317 and 2.3318 in the lab. The tolerance of S. arboricolus strains (2.3317, 2.3318 and 2.3319) to formic acid was further investigated by lab-scale fermentation analysis, and compared with S. cerevisiae NCYC2592. S. arboricolus 2.3319 demonstrated improved formic acid tolerance and a similar bioethanol synthesis capacity to S. cerevisiae NCYC2592, while S. arboricolus 2.3317 and 2.3318 exhibited an overall inferior performance. Metabolite analysis indicated that S. arboricolus strain 2.3319 accumulated comparatively high concentrations of glycerol and glycogen, which may have contributed to its ability to tolerate high levels of formic acid. PMID:26284784

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

    SciTech Connect

    Uribe, S.; Rangel, P.; Espinola, G.; Aguirre, G. )

    1990-07-01

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

  15. Quantifying the individual effects of ethanol and temperature on the fitness advantage of Saccharomyces cerevisiae.

    PubMed

    Salvadó, Z; Arroyo-López, F N; Barrio, E; Querol, A; Guillamón, J M

    2011-09-01

    The presence of Saccharomyces cerevisiae in grape berries and fresh musts is usually very low. However, as fermentation progresses, the population levels of this species considerably increase. In this study, we use the concept of fitness advantage to measure how increasing ethanol concentrations (0-25%) and temperature values (4-46 °C) in wine fermentations affects competition between S. cerevisiae and several non-Saccharomyces yeasts (Hanseniaspora uvarum, Torulaspora delbrueckii, Candida zemplinina, Pichia fermentans and Kluyveromyces marxianus). We used a mathematical approach to model the hypothetical time needed for S. cerevisiae to impose itself on a mixed population of the non-Saccharomyces species described above. This approach also took into consideration the influence of environmental factors and the initial population levels of S. cerevisiae (0.1, 1.0 and 10.0%). Our results suggest that Saccharomyces niche construction via ethanol production does not provide a clear ecological advantage (at least not until the ethanol concentration exceeds 9%), whereas a temperature rise (above 15 °C) does give S. cerevisiae a considerable advantage. The initial frequency of S. cerevisiae considerably influences the time it needs to impose itself (until it reaches a final frequency of 99% in the mixed culture), the lowest time values being found at the highest initial frequency. In light of these results, the application of low temperatures in the wine industry could favor the growth and survival of non-Saccharomyces species for a longer period of time. PMID:21645814

  16. Saccharomyces kudriavzevii and Saccharomyces uvarum differ from Saccharomyces cerevisiae during the production of aroma-active higher alcohols and acetate esters using their amino acidic precursors.

    PubMed

    Stribny, Jiri; Gamero, Amparo; Pérez-Torrado, Roberto; Querol, Amparo

    2015-07-16

    Higher alcohols and acetate esters are important flavour and aroma components in the food industry. In alcoholic beverages these compounds are produced by yeast during fermentation. Although Saccharomyces cerevisiae is one of the most extensively used species, other species of the Saccharomyces genus have become common in fermentation processes. This study analyses and compares the production of higher alcohols and acetate esters from their amino acidic precursors in three Saccharomyces species: Saccharomyces kudriavzevii, Saccharomyces uvarum and S. cerevisiae. The global volatile compound analysis revealed that S. kudriavzevii produced large amounts of higher alcohols, whereas S. uvarum excelled in the production of acetate esters. Particularly from phenylalanine, S. uvarum produced the largest amounts of 2-phenylethyl acetate, while S. kudriavzevii obtained the greatest 2-phenylethanol formation from this precursor. The present data indicate differences in the amino acid metabolism and subsequent production of flavour-active higher alcohols and acetate esters among the closely related Saccharomyces species. This knowledge will prove useful for developing new enhanced processes in fragrance, flavour, and food industries. PMID:25886016

  17. Isolation and subcloning analysis of functional centromere DNA(CEN11) from Saccharomyces cerevisiae chromosome XI

    SciTech Connect

    Fitzgerald-Hayes, M.; Buhler, J.M.; Cooper, T.G.; Carbon, J.

    1982-01-01

    The authors have cloned segments of yeast DNA containing the centromere XI-linked MET14 gene. This was done by selecting directly in Saccharomyces cerevisiae for complementation of a met14 mutation after transformation with a hybrid plasmid DNA genomic library. Generic evidence indicates that functional centromere DNA (CEN11) from chromosome XI is also contained on the segment of S. cerevisiae DNA cloned in pYe(MET14)2. This plasmid is maintained stably in budding S. cerevisiae cultures and segregates predominantly 2 + :2- through meiosis. The CEN11 element has been subcloned in vector YRp7' on an S. cerevisiae DNA fragment 900 base pairs in length (pYe(CEN11)10). The mitotic and meiotic behavior of plasmids containing CEN11 plus a DNA replicator (ars) indicates that the centromere DNA sequences enable these plasmids to function as true minichromosomes in S. cerevisiae.

  18. Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae.

    PubMed

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

    2012-11-01

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

  19. Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    Background Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media. Results We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to high glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction. Conclusions Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway. PMID:24520859

  20. Network Hubs Buffer Environmental Variation in Saccharomyces cerevisiae

    PubMed Central

    Levy, Sasha F; Siegal, Mark L

    2008-01-01

    Regulatory and developmental systems produce phenotypes that are robust to environmental and genetic variation. A gene product that normally contributes to this robustness is termed a phenotypic capacitor. When a phenotypic capacitor fails, for example when challenged by a harsh environment or mutation, the system becomes less robust and thus produces greater phenotypic variation. A functional phenotypic capacitor provides a mechanism by which hidden polymorphism can accumulate, whereas its failure provides a mechanism by which evolutionary change might be promoted. The primary example to date of a phenotypic capacitor is Hsp90, a molecular chaperone that targets a large set of signal transduction proteins. In both Drosophila and Arabidopsis, compromised Hsp90 function results in pleiotropic phenotypic effects dependent on the underlying genotype. For some traits, Hsp90 also appears to buffer stochastic variation, yet the relationship between environmental and genetic buffering remains an important unresolved question. We previously used simulations of knockout mutations in transcriptional networks to predict that many gene products would act as phenotypic capacitors. To test this prediction, we use high-throughput morphological phenotyping of individual yeast cells from single-gene deletion strains to identify gene products that buffer environmental variation in Saccharomyces cerevisiae. We find more than 300 gene products that, when absent, increase morphological variation. Overrepresented among these capacitors are gene products that control chromosome organization and DNA integrity, RNA elongation, protein modification, cell cycle, and response to stimuli such as stress. Capacitors have a high number of synthetic-lethal interactions but knockouts of these genes do not tend to cause severe decreases in growth rate. Each capacitor can be classified based on whether or not it is encoded by a gene with a paralog in the genome. Capacitors with a duplicate are highly connected in the protein–protein interaction network and show considerable divergence in expression from their paralogs. In contrast, capacitors encoded by singleton genes are part of highly interconnected protein clusters whose other members also tend to affect phenotypic variability or fitness. These results suggest that buffering and release of variation is a widespread phenomenon that is caused by incomplete functional redundancy at multiple levels in the genetic architecture. PMID:18986213

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was designed to determine the effect of supplementing feedlot steers with Saccharomyces cerevisiae CNCM I-1079 (SC) on the acute phase response to a lipopolysaccharide (LPS) challenge. Steers (n = 18; 266 ± 4 kilograms body weight) were separated into three treatment groups (n = 6/treatm...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Phosphate uptake in Saccharomyces cerevisiae Hansen wild type and phenotypes exposed to space flight irradiation.

    PubMed

    Berry, D; Volz, P A

    1979-10-01

    Rates of phosphate uptake were approximately twice as great for Saccharomyces cerevisiae single-cell phenotypic isolates exposed to space parameters as for the wild-type ground control. Quantitative determination of 32P was performed by liquid scintillation spectrometry utilizing Cerenkov radiation counting techniques. PMID:395899

  6. The uptake of different iron salts by the yeast Saccharomyces cerevisiae

    PubMed Central

    Gaensly, Fernanda; Picheth, Geraldo; Brand, Debora; Bonfim, Tania M.B.

    2014-01-01

    Yeasts can be enriched with microelements, including iron; however, special physicochemical conditions are required to formulate a culture media that promotes both yeast growth and iron uptake. Different iron sources do not affect biomass formation; however, considering efficacy, cost, stability, and compatibility with Saccharomyces cerevisiae metabolism, ferrous sulphate is recommended. PMID:25242932

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Oral administration of Saccharomyces cerevisiae boulardii reduces Escherichia coli endotoxin associated mortality in weaned pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of active dry yeast, Saccharomyces cerevisiae boulardii (Scb), on the immune/neuroendocrine response and subsequent mortality to E. coli lipopolysaccharide (LPS) administration were evaluated in newly weaned pigs (26.1 + or - 3.4 d of age). Barrows were assigned to 1 of 2 treatment group...

  9. Engineering Saccharomyces cerevisiae for consolidated bioprocessing in starch and biomass conversion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The conversion of starch or biomass to biofuel is a two-stage process involving enzymatic treatment, followed by yeast fermentation. An alternative route would be to consolidate the process by engineering Saccharomyces cerevisiae capable of both saccharification and fermentation. An approach was d...

  10. PRIMARY STRUCTURE OF THE P450 LANOSTEROL DEMETHYLASE GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have sequenced the structural gene and flanking regions for lanosterol 14oc-demethylase (14DM) from Saccharomyces cerevisiae. n open reading fram of 530 codons encodes a 60.7-kDa protein. hen this gene is disrupted by integrative transformation, the resulting strain requires e...

  11. PRIMARY STRUCTURE OF THE P450 LANOSTEROL DEMETHYLASE GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

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

  12. NDT80, a meiosis-specific gene required for exit from pachytene in Saccharomyces cerevisiae

    SciTech Connect

    Xu, Liuzhong; Ajimura, M.; Padmore, R.; Klein, C.; Kleckner, N.

    1995-12-01

    This report describes the identification of a new meiosis-specific gene of Saccharomyces cerevisiae called NDT80. DNA cloning and molecular analysis revealed that the NDT80 gene maps on the right arm of chromosome 8 and is transcribed during middle meiotic prophase. 82 refs., 6 figs., 3 tabs.

  13. Heat Shock Protein Genes and Newly Integrated Glucose Metabolic Pathways Promote Ethanol Tolerance of Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignocellulose-to-ethanol conversion provides a promising alternative means for production of sustainable and cleaner transportation fuels. Development of stress tolerant ethanologenic Saccharomyces cerevisiae is important for low-cost biobased economy. Tolerance to high levels of ethanol has been...

  14. The uptake of different iron salts by the yeast Saccharomyces cerevisiae.

    PubMed

    Gaensly, Fernanda; Picheth, Geraldo; Brand, Debora; Bonfim, Tania M B

    2014-01-01

    Yeasts can be enriched with microelements, including iron; however, special physicochemical conditions are required to formulate a culture media that promotes both yeast growth and iron uptake. Different iron sources do not affect biomass formation; however, considering efficacy, cost, stability, and compatibility with Saccharomyces cerevisiae metabolism, ferrous sulphate is recommended. PMID:25242932

  15. LACTIC ACID PRODUCTION BY SACCHAROMYCES CEREVISIAE EXPRESSING A RHIZOPUS ORYZAE LACTATE DEHYDROGENASE GENE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work demonstrates the first example of a fungal LDH expressed in yeast. A L(+)-lactate dehydrogenase gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adhl promoter and terminator, then placed in a 2 micron contai...

  16. Benchmark data for identifying N6-methyladenosine sites in the Saccharomyces cerevisiae genome

    PubMed Central

    Chen, Wei; Feng, Pengmian; Ding, Hui; Lin, Hao; Chou, Kuo-Chen

    2015-01-01

    This data article contains the benchmark dataset for training and testing iRNA-Methyl, a web-server predictor for identifying N6-methyladenosine sites in RNA (Chen et al., 2015 [15]). It can also be used to develop other predictors for identifying N6-methyladenosine sites in the Saccharomyces cerevisiae genome.

  17. Null mutants of Saccharomyces cerevisiae Cu,Zn superoxide dismutase: Characterization and spontaneous mutation rates

    SciTech Connect

    Gralla, E.B.; Valentine, J.S. )

    1991-09-01

    Deletion-replacement mutations of the Saccharomyces cerevisiae Cu,Zn superoxide dismutase gene were constructed. They were exquisitely sensitive to redox cycling drugs and showed slight sensitivity to other agents. The aerobic spontaneous mutation rate was three- to fourfold higher in sod1{Delta}1 mutants, while the anaerobic rate was similar to that of the wild type.

  18. Sporulation in soil as an overwinter survival strategy in Saccharomyces cerevisiae.

    PubMed

    Knight, Sarah J; Goddard, Matthew R

    2016-02-01

    Due to its commercial value and status as a research model there is an extensive body of knowledge concerning Saccharomyces cerevisiae's cell biology and genetics. Investigations into S. cerevisiae's ecology are comparatively lacking, and are mostly focused on the behaviour of this species in high sugar, fruit-based environments; however, fruit is ephemeral, and presumably, S. cerevisiae has evolved a strategy to survive when this niche is not available. Among other places, S. cerevisiae has been isolated from soil which, in contrast to fruit, is a permanent habitat. We hypothesize that S. cerevisiae employs a life history strategy targeted at self-preservation rather than growth outside of the fruit niche, and resides in forest niches, such as soil, in a dormant and resistant sporulated state, returning to fruit via vectors such as insects. One crucial aspect of this hypothesis is that S. cerevisiae must be able to sporulate in the 'forest' environment. Here, we provide the first evidence for a natural environment (soil) where S. cerevisiae sporulates. While there are further aspects of this hypothesis that require experimental verification, this is the first step towards an inclusive understanding of the more cryptic aspects of S. cerevisiae's ecology. PMID:26568201

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

    PubMed Central

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

    2012-01-01

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

  20. Cytotoxicity of orthodontic materials assessed by survival tests in Saccharomyces cerevisiae.

    PubMed

    Limberger, Karen M; Westphalen, Graziela H; Menezes, Luciane M; Medina-Silva, Renata

    2011-05-01

    The aim of this study was to assess the cytotoxicity of orthodontic materials (brackets, wires, resin, elastomers and silver solder) using Saccharomyces cerevisiae as a model organism. The induction of cytotoxicity was assessed by two different tests using the wild-type S. cerevisiae strain FF18733: (1) direct exposure to orthodontic materials in YPD broth, and (2) exposure to artificial commercial saliva pre-treated with orthodontic materials. Only the silver solder was tested in mutant S. cerevisiae strains to investigate the origin of the observed cytotoxicity. Colony forming units per mL counts were carried out in all experiments and compared to controls to detect significant survival differences. The results showed that only the silver solder induced significant cytotoxicity, which might have occurred via oxidative stress, although this mechanism is not completely understood. Moreover, S. cerevisiae proved to be a reliable and useful model microorganism for evaluating the cytotoxicity of clinical materials. PMID:21376384

  1. Evaluation of Lactobacillus plantarum and Saccharomyces cerevisiae in the Presence of Bifenthrin.

    PubMed

    Đorđević, Tijana M; Đurović-Pejčev, Rada D

    2016-06-01

    This work describes the effect of insecticide bifenthrin on Lactobacillus plantarum and Saccharomyces cerevisiae. Growths of used microorganisms in growth media supplemented with pesticide were studied. Determination of bacterial and yeast fermentation efficiency in wheat supplemented with bifenthrin was conducted. Additionally, investigation of bifenthrin dissipation during microbiological activity was performed. Experiments applying bifenthrin in different concentrations highlighted a negligible impact of the pesticide on the growth of L. plantarum and S. cerevisiae. This insecticide overall negatively affected the yeast fermentation of wheat, while its presence in wheat had a slight negative impact on lactic acid fermentation. The results of bifenthrin dissipation during lactic acid and yeast fermentations of wheat showed that activities of L. plantarum and S. cerevisiae caused lower pesticide reductions. Average bifenthrin residue reduction within samples fermented with L. plantarum was 5.4 % (maximum ~16 %), while within samples fermented with S. cerevisiae, it was 11.6 % (maximum ~17 %). PMID:26868256

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

    PubMed Central

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

    1981-01-01

    Uranium accumulated extracellularly on the surfaces of Saccharomyces cerevisiae cells. The rate and extent of accumulation were subject to environmental parameters, such as pH, temperature, and interference by certain anions and cations. Uranium accumulation by Pseudomonas aeruginosa occurred intracellularly and was extremely rapid (<10 s), and no response to environmental parameters could be detected. Metabolism was not required for metal uptake by either organism. Cell-bound uranium reached a concentration of 10 to 15% of the dry cell weight, but only 32% of the S. cerevisiae cells and 44% of the P. aeruginosa cells within a given population possessed visible uranium deposits when examined by electron microscopy. Rates of uranium uptake by S. cerevisiae were increased by chemical pretreatment of the cells. Uranium could be removed chemically from S. cerevisiae cells, and the cells could then be reused as a biosorbent. Images PMID:16345691

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

    PubMed Central

    Zheng, Yan-Lin; Wang, Shi-An

    2015-01-01

    The budding yeast Saccharomyces cerevisiae is a platform organism for bioethanol production from various feedstocks and robust strains are desirable for efficient fermentation because yeast cells inevitably encounter stressors during the process. Recently, diverse S. cerevisiae lineages were identified, which provided novel resources for understanding stress tolerance variations and related shaping factors in the yeast. This study characterized the tolerance of diverse S. cerevisiae strains to the stressors of high ethanol concentrations, temperature shocks, and osmotic stress. The results showed that the isolates from human-associated environments overall presented a higher level of stress tolerance compared with those from forests spared anthropogenic influences. Statistical analyses indicated that the variations of stress tolerance were significantly correlated with both ecological sources and geographical locations of the strains. This study provides guidelines for selection of robust S. cerevisiae strains for bioethanol production from nature. PMID:26244846

  4. Opportunistic Strains of Saccharomyces cerevisiae: A Potential Risk Sold in Food Products

    PubMed Central

    Pérez-Torrado, Roberto; Querol, Amparo

    2016-01-01

    In recent decades, fungal infections have emerged as an important health problem associated with more people who present deficiencies in the immune system, such as HIV or transplanted patients. Saccharomyces cerevisiae is one of the emerging fungal pathogens with a unique characteristic: its presence in many food products. S. cerevisiae has an impeccably good food safety record compared to other microorganisms like virus, bacteria and some filamentous fungi. However, humans unknowingly and inadvertently ingest large viable populations of S. cerevisiae (home-brewed beer or dietary supplements that contain yeast). In the last few years, researchers have studied the nature of S. cerevisiae strains and the molecular mechanisms related to infections. Here we review the last advance made in this emerging pathogen and we discuss the implication of using this species in food products. PMID:26779173

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

    Hyma, Katie E.; Fay, Justin C.

    2012-01-01

    Humans have had a significant impact on the distribution and abundance of Saccharomyces cerevisiae through its widespread use in beer, bread and wine production. Yet, similar to other Saccharomyces species, S. cerevisiae has also been isolated from habitats unrelated to fermentations. Strains of S. cerevisiae isolated from grapes, wine must and vineyards worldwide are genetically differentiated from strains isolated from oak-tree bark, exudate and associated soil in North America. However, the causes and consequences of this differentiation have not yet been resolved. Historical differentiation of these two groups may have been influenced by geographic, ecological or human-associated barriers to gene flow. Here, we make use of the relatively recent establishment of vineyards across North America to identify and characterize any active barriers to gene flow between these two groups. We examined S. cerevisiae strains isolated from grapes and oak-trees within three North American vineyards and compared them to those isolated from oak-trees outside of vineyards. Within vineyards we found evidence of migration between grapes and oak-trees and potential gene flow between the divergent oak-tree and vineyard groups. Yet, we found no vineyard genotypes on oak-trees outside of vineyards. In contrast, S. paradoxus isolated from the same sources showed population structure characterized by isolation by distance. The apparent absence of ecological or genetic barriers between sympatric vineyard and oak-tree populations of S. cerevisiae implies that vineyards play an important role in the mixing between these two groups. PMID:23286354

  9. Fumaric Acid Production in Saccharomyces cerevisiae by In Silico Aided Metabolic Engineering

    PubMed Central

    Xu, Guoqiang; Zou, Wei; Chen, Xiulai; Xu, Nan; Liu, Liming; Chen, Jian

    2012-01-01

    Fumaric acid (FA) is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA) revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae. The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610±31 mg L–1 without any apparent change in growth in fed-batch culture. FT-IR and 1H and 13C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134±48 mg L–1 FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675±52 mg L–1 FA in batch culture when the SFC1 gene encoding a succinate–fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering. PMID:23300594

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

    SciTech Connect

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. )

    1993-07-01

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

  11. Next-generation sequencing of Okazaki fragments extracted from Saccharomyces cerevisiae.

    PubMed

    Yanga, Wenchao; Lib, Xinhui

    2013-08-01

    Genome-wide Okazaki fragment distribution can differentiate the discontinuous from the semi-discontinuous DNA replication model. Here, we investigated the genome-wide Okazaki fragment distribution in Saccharomyces cerevisiae S288C. We improved the method based upon lambda exonuclease digestion to purify Okazaki fragments from S288C yeast cells, followed by Illumina sequencing. The distribution of Okazaki fragments around confirmed replication origins, including two highly efficient replication origins, supported the discontinuous DNA replication model. In S. cerevisiae mitochondria, Okazaki fragments were overrepresented in the transcribed regions, indicating the interplay between transcription and DNA replication. PMID:23792162

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

    PubMed Central

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

    1984-01-01

    We have cloned a functional centromeric DNA sequence from Saccharomyces cerevisiae. Using the 2 mu chromosome-loss mapping technique and meiotic tetrad analysis, we have identified this DNA sequence as the centromere of chromosome V (CEN5). The CEN5 sequence has been localized on an 1,100-base-pair BamHI-BglII restriction fragment. Plasmids containing CEN5 and an autonomously replicating sequence are mitotically stable in S. cerevisiae and segregate in a Mendelian fashion during meiosis. Images PMID:6366514

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

    PubMed

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

    2015-08-01

    Ribosomal proteins (RPs), are essential components of the ribosomes, the molecular machines that turn mRNA blueprints into proteins, as they serve to stabilize the structure of the rRNA, thus improving protein biosynthesis. In addition, growing evidence suggests that RPs can function in other cellular roles. In the present review, we summarize several potential extra-ribosomal functions of RPs in ribosomal biogenesis, transcription activity, translation process, DNA repair, replicative life span, adhesive growth, and morphological transformation in Saccharomyces cerevisiae. However, the future in-depth studies are needed to identify these novel secondary functions of RPs in S. cerevisiae. PMID:26211963

  14. Finding of thiosulfate pathway for synthesis of organic sulfur compounds in Saccharomyces cerevisiae and improvement of ethanol production.

    PubMed

    Funahashi, Eri; Saiki, Kyohei; Honda, Kurara; Sugiura, Yuki; Kawano, Yusuke; Ohtsu, Iwao; Watanabe, Daisuke; Wakabayashi, Yukari; Abe, Tetsuya; Nakanishi, Tsuyoshi; Suematsu, Makoto; Takagi, Hiroshi

    2015-12-01

    We found that Saccharomyces cerevisiae utilizes thiosulfate as a sole sulfur source. The energetically-favored thiosulfate rather than sulfate as sulfur sources is also more effective for improving growth and ethanol-production rate in S.cerevisiae due to high levels of intracellular NADPH during thiosulfate utilization. PMID:26188417

  15. Integrated phospholipidomics and transcriptomics analysis of Saccharomyces cerevisiae with enhanced tolerance to a mixture of acetic acid, furfural, and phenol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A mixture of acetic acid, furfural and phenol (AFP), three representative lignocellulose derived inhibitors, significantly inhibited the growth and bioethanol production of Saccharomyces cerevisiae. In order to uncover mechanisms behind the enhanced tolerance of an inhibitor-tolerant S.cerevisiae s...

  16. Saccharomyces cerevisiae boulardii transient fungemia after intravenous self-inoculation

    PubMed Central

    Cohen, Lola; Ranque, Stéphane; Raoult, Didier

    2013-01-01

    We report the case of a young psychotic intravenous drug user injecting herself with Saccharomyces cervisiae (boulardii). She experienced a 24 h fever, resolving spontaneously confirming, quasi experimentally, the inocuity of this yeast in a non-immunocompromised host. PMID:24432219

  17. A comprehensive web resource on RNA helicases from the baker's yeast Saccharomyces cerevisiae.

    PubMed

    Linder, P; Gasteiger, E; Bairoch, A

    2000-04-01

    Members of the RNA helicase protein family are defined by several motifs that have been widely conserved during evolution. They are found in all organisms-from bacteria to humans-and many viruses. The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome. Recent progress in the functional analysis of various family members has confirmed the significance of RNA helicases for most cellular RNA metabolic processes. We have assembled a web resource that focuses on RNA helicases from the budding yeast Saccharomyces cerevisiae. It includes descriptions of RNA helicases and their functions, links to sequence- and yeast-specific databases, an extensive list of references, and links to non-yeast helicase web resources. PMID:10790687

  18. Functional annotations for the Saccharomyces cerevisiae genome: the knowns and the known unknowns

    PubMed Central

    Christie, Karen R.; Hong, Eurie L.; Cherry, J. Michael

    2011-01-01

    The quest to characterize each of the genes of the yeast Saccharomyces cerevisiae has propelled the development and application of novel high-throughput (HTP) experimental techniques. To handle the enormous amount of information generated by these techniques, new bioinformatics tools and resources are needed. Gene Ontology (GO) annotations curated by the Saccharomyces Genome Database (SGD) have facilitated the development of algorithms that analyze HTP data and help predict functions for poorly characterized genes in S. cerevisiae and other organisms. Here, we describe how published results are incorporated into GO annotations at SGD and why researchers can benefit from using these resources wisely to analyze their HTP data and predict gene functions. PMID:19577472

  19. Biodegradation of crude oil by Saccharomyces cerevisiae isolated from fermented zobo (locally fermented beverage in Nigeria).

    PubMed

    Abioye, O P; Akinsola, R O; Aransiola, S A; Damisa, D

    2013-12-15

    The increase in demand for crude oil as a source of energy and as a primary raw material for industries has resulted in an increase in its production, transportation and refining, which in turn has resulted in gross pollution of the environment. In this study, Saccharomyces cerevisiae isolated from a commercially prepared local fermented beverage 'zobo' (prepared from Hibiscus flower) was tested to determine its potential to degrade crude oil for a period of 28 days under aerobic condition. The percentage of oil biodegradation was determined using weight loss method and gas chromatography mass spectroscopy (GC/MS) of the residual crude oil after 28 days. At the end of 28 days 49.29% crude oil degradation was recorded. The result suggests the potential of Saccharomyces cerevisiae for bioremediation of oil polluted sites. PMID:24517030

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

    PubMed

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

    2016-07-01

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

  1. New amylolytic yeast strains for starch and dextrin fermentation. [Schwanniomyces alluvius, Saccharomyces cerevisiae var. diastaticus

    SciTech Connect

    Laluce, C.; Bertolini, M.C.; Ernandes, J.R. ); Martini, A.V.; Martini, A. )

    1988-10-01

    Yeast strains capable of fermenting starch and dextrin to ethanol were isolated from samples collected from Brazilian factories in which cassava flour is produced. Considerable alcohol production was observed for all the strains selected. One strain (DI-10) fermented starch rapidly and secreted 5 times as much amylolytic enzyme than that observed for Schwanniomyces alluvius UCD 54-83. This strain and three other similar isolates were classified as Saccharomyces cerevisiae var. diastaticus by morphological and physiological characteristics and molecular taxonomy.

  2. Mutagenic Inverted Repeats Assisted Genome Engineering (MIRAGE) in Saccharomyces cerevisiae: deletion of gal7.

    PubMed

    Nair, Nikhil U; Zhao, Huimin

    2012-01-01

    MIRAGE is a unique in vivo genome editing technique that exploits the inherent instability of inverted repeats (palindromes) in the Saccharomyces cerevisiae chromosome. As a technique able to quickly create deletions as well as precise point mutations, it is valuable in applications that require creation of designer strains of this yeast. In particular, it has various potential applications in metabolic engineering, systems biology, synthetic biology, and molecular genetics. PMID:22144353

  3. The effect of millimeter waves at the yeast Saccharomyces cerevisiae during heliogeophysical disturbances

    NASA Astrophysics Data System (ADS)

    Rogacheva, Svetlana M.; Babaeva, Milena I.

    2013-02-01

    The isolated and combined effect of heliogeophysical factors and low intensive electromagnetic radiation of millimeter diapason at the metachromasia reaction of the yeast Saccharomyces cerevisiae was studied. It was established that longterm influence of EMR 65 GHz induced changes in the response of cells towards heliogeomagnetic disturbance. On our opinion millimeter waves may reduce the effect of heliogeophysical factors on living organisms because of destabilization of the intracellular water structure.

  4. Analysis of Plasmid Deletion Induced by Ionizing Radiation in Yeast Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Yatsevich, E.; Stepanova, A.; Sprincova, A.; Koltovaya, N.

    2007-11-01

    The article is dedicated to the research of plasmid system YCpL2 with help of quantitative analysis of deletion formation. The cells of yeast Saccharomyces cerevisiae were irradiated by ?-ray with the flux of 0.7 Gy/min and energy of 1.3 MeV as well as heavy ion beam 11B with energy 32 MeV/n. The deletion of plasmid DNA has been analyzed by genetic and restriction analysis.

  5. Saccharomyces cerevisiae Produces a Yeast Substance that Exhibits Estrogenic Activity in Mammalian Systems

    NASA Astrophysics Data System (ADS)

    Feldman, David; Stathis, Peter A.; Hirst, Margaret A.; Price Stover, E.; Do, Yung S.; Kurz, Walter

    1984-06-01

    Partially purified lipid extracts of Saccharomyces cerevisiae contain a substance that displaces tritiated estradiol from rat uterine cytosol estrogen receptors. The yeast product induces estrogenic bioresponses in mammalian systems as measured by induction of progesterone receptors in cultured MCF-7 human breast cancer cells and by a uterotrophic response and progesterone receptor induction after administration to ovariectomized mice. The findings raise the possibility that bakers' yeast may be a source of environmental estrogens.

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

    PubMed Central

    Mendes-Ferreira, A.; del Olmo, M.; García-Martínez, J.; Jiménez-Martí, E.; Leão, C.; Mendes-Faia, A.; Pérez-Ortín, J. E.

    2007-01-01

    Genome-wide analysis of the wine yeast strain Saccharomyces cerevisiae PYCC4072 identified 36 genes highly expressed under conditions of low or absent nitrogen in comparison with a nitrogen-replete condition. Reverse transcription-PCR analysis for four of these transcripts with this strain and its validation with another wine yeast strain underlines the usefulness of these signature genes for predicting nitrogen deficiency and therefore the diagnosis of wine stuck/sluggish fermentations. PMID:17601813

  7. Functional equivalence of translation factor eIF5B from Candida albicans and Saccharomyces cerevisiae.

    PubMed

    Jun, Kyung Ok; Yang, Eun Ji; Lee, Byeong Jeong; Park, Jeong Ro; Lee, Joon H; Choi, Sang Ki

    2008-04-30

    Eukaryotic translation initiation factor 5B (eIF5B) plays a role in recognition of the AUG codon in conjunction with translation factor eIF2, and promotes joining of the 60S ribosomal subunit. To see whether the eIF5B proteins of other organisms function in Saccharomyces cerevisiae, we cloned the corresponding genes from Oryza sativa, Arabidopsis thaliana, Aspergillus nidulans and Candida albican and expressed them under the control of the galactose-inducible GAL promoter in the fun12Delta strain of Saccharomyces cerevisiae. Expression of Candida albicans eIF5B complemented the slow-growth phenotype of the fun12Delta strain, but that of Aspergillus nidulance did not, despite the fact that its protein was expressed better than that of Candida albicans. The Arabidopsis thaliana protein was also not functional in Saccharomyces. These results reveal that the eIF5B in Candida albicans has a close functional relationship with that of Sacharomyces cerevisiae, as also shown by a phylogenetic analysis based on the amino acid sequences of the eIF5Bs. PMID:18414002

  8. The toxic potential of an industrial effluent determined with the Saccharomyces cerevisiae-based assay.

    PubMed

    Schmitt, Marcel; Gellert, Georg; Lichtenberg-Fraté, Hella

    2005-09-01

    Increasing levels of environmental pollution and the continuous monitoring of water quality both request specific and sensitive methods for the detection of detrimental water contents. On a regulatory basis genotoxicity is assessed by the standard umu-test (ISO 13829) that responds to DNA damage induced by chemicals. The focus of this study was the examination of the toxic potential of samples taken from the wastewater treatment plant of a refinery factory to explore the applicability of the Saccharomyces cerevisiae (bakers yeast) test for the detection of bio-available genotoxic activity in complex matrices. The toxic potential of samples without pre-treatment and following centrifugation was determined with the eukaryotic Saccharomyces cerevisiae bioassay based on the transcriptional activation of the green fluorescent protein (gfp) fused to the DNA damage inducible RAD54 promoter and general growth inhibition. Primary effluent samples were taken as qualified sterile spot samples from the final effluent of the purification plant. The Saccharomyces cerevisiae assay yielded geno- and cytotoxic responses in all complex untreated and centrifuged samples with high reproducibility. The obtained results suggest that the yeast assay is suited as a screening tool to monitor genotoxic potential of wastewater. PMID:16002118

  9. [Development of genetically stable recombinant Saccharomyces cerevisiae strains using combinational chromosomal integration].

    PubMed

    Zuo, Qi; Zhao, Xinqing; Liu, Haijun; Hu, Shiyang; Ma, Zhongyi; Bai, Fengwu

    2014-04-01

    Chromosomal integration enables stable phenotype and therefore has become an important strategy for breeding of industrial Saccharomyces cerevisiae strains. pAUR135 is a plasmid that enables recycling use of antibiotic selection marker, and once attached with designated homologous sequences, integration vector for stable expression can be constructed. Development of S. cerevisiae strains by metabolic engineering normally demands overexpression of multiple genes, and employing pAUR135 plasmid, it is possible to construct S. cerevisiae strains by combinational integration of multiple genes in multiple sites, which results in different ratios of expressions of these genes. Xylose utilization pathway was taken as an example, with three pAUR135-based plasmids carrying three xylose assimilation genes constructed in this study. The three genes were sequentially integrated on the chromosome of S. cerevisiae by combinational integration. Xylose utilization rate was improved 24.4%-35.5% in the combinational integration strain comparing with that of the control strain with all the three genes integrated in one location. Strain improvement achieved by combinational integration is a novel method to manipulate multiple genes for genetic engineering of S. cerevisiae, and the recombinant strains are free of foreign sequences and selection markers. In addition, stable phenotype can be maintained, which is important for breeding of industrial strains. Therefore, combinational integration employing pAUR135 is a novel method for metabolic engineering of industrial S. cerevisiae strains. PMID:25195256

  10. Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations.

    PubMed

    Rantsiou, Kalliopi; Dolci, Paola; Giacosa, Simone; Torchio, Fabrizio; Tofalo, Rosanna; Torriani, Sandra; Suzzi, Giovanna; Rolle, Luca; Cocolin, Luca

    2012-03-01

    In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ~0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response. PMID:22247148

  11. Candida zemplinina Can Reduce Acetic Acid Produced by Saccharomyces cerevisiae in Sweet Wine Fermentations

    PubMed Central

    Rantsiou, Kalliopi; Dolci, Paola; Giacosa, Simone; Torchio, Fabrizio; Tofalo, Rosanna; Torriani, Sandra; Suzzi, Giovanna; Rolle, Luca

    2012-01-01

    In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ∼0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response. PMID:22247148

  12. Screening of lactic acid bacteria that can form mixed-species biofilm with Saccharomyces cerevisiae.

    PubMed

    Furukawa, Soichi; Isomae, Ryosuke; Tsuchiya, Noriko; Hirayama, Satoru; Yamagishi, Asuka; Kobayashi, Miho; Suzuki, Chise; Ogihara, Hirokazu; Morinaga, Yasushi

    2015-01-01

    The abilities of lactic acid bacteria (LAB) to form mixed-species biofilm with Saccharomyces cerevisiae in a static co-culture were investigated out of 168 LAB stock cultures, and two Lactobacillus plantarum strains (D71 and E31) and one Leuconostoc mesenteroides strain K01 were found to form mixed-species biofilm with S. cerevisiae BY4741. SEM observation showed that there was no significant difference in morphological properties among these three mixed-species biofilms and they resembled that formed by S. cerevisiae with L. plantarum ML11-11 previously isolated from a brewing sample of Fukuyama pot vinegar. The co-aggregation assays showed that L. plantarum D71 and L. plantarum E31 could co-aggregate with S. cerevisiae similarly to L. plantarum ML11-11, while L. mesenteroides K01 had no ability to co-aggregate with yeast. The above results indicate that aggregation followed by direct cell-to-cell contact is required for mixed-species biofilm formation between these L. plantarum strains and S. cerevisiae, though some different mechanism may be involved in biofilm formation between L. mesenteroides strain and S. cerevisiae. PMID:25514879

  13. Resveratrol increases glycolytic flux in Saccharomyces cerevisiae via a SNF1-dependet mechanism.

    PubMed

    Madrigal-Perez, Luis Alberto; Nava, Gerardo M; González-Hernández, Juan Carlos; Ramos-Gomez, Minerva

    2015-08-01

    Evidence suggests that AMP protein kinase (AMPK) is the main target of the phytochemical resveratrol (RSV) in mammalian cells. Data also indicates that RSV stimulates glucose metabolism; however, the molecular link between RSV and glucose uptake remains unknown. Herein, we provide evidence indicating that RSV stimulates glycolysis via sucrose non-fermenting 1 gene (SNF1, Saccharomyces cerevisiae orthologous of AMPK). S. cerevisiae cultures treated with 30 μM RSV showed an increase in extracellular acidification rate compared to untreated cells, indicating an elevated glycolytic flux. Also, RSV treatment increased transcription levels of two key glycolytic genes, hexokinase 2 (HXK2) and phosphofructokinase 1 (PFK1), as well as production of NADH. Moreover, RSV treatment inhibited mitochondrial respiration when glucose was used as a carbon source. Importantly, the effects of RSV on glycolysis were dependent of SNF1. Taken together, these findings suggest that SNF1 (AMPK in mammalian systems) is the molecular target of RSV in S. cerevisiae. PMID:26091703

  14. Cellular and molecular engineering of yeast Saccharomyces cerevisiae for advanced biobutanol production.

    PubMed

    Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-02-01

    Butanol is an attractive alternative energy fuel owing to several advantages over ethanol. Among the microbial hosts for biobutanol production, yeast Saccharomyces cerevisiae has a great potential as a microbial host due to its powerful genetic tools, a history of successful industrial use, and its inherent tolerance to higher alcohols. Butanol production by S. cerevisiae was first attempted by transferring the 1-butanol-producing metabolic pathway from native microorganisms or using the endogenous Ehrlich pathway for isobutanol synthesis. Utilizing alternative enzymes with higher activity, eliminating competitive pathways, and maintaining cofactor balance achieved significant improvements in butanol production. Meeting future challenges, such as enhancing butanol tolerance and implementing a comprehensive strategy by high-throughput screening, would further elevate the biobutanol-producing ability of S. cerevisiae toward an ideal microbial cell factory exhibiting high productivity of biobutanol. PMID:26712533

  15. Parameter Optimization for Enhancement of Ethanol Yield by Atmospheric Pressure DBD-Treated Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Dong, Xiaoyu; Yuan, Yulian; Tang, Qian; Dou, Shaohua; Di, Lanbo; Zhang, Xiuling

    2014-01-01

    In this study, Saccharomyces cerevisiae (S. cerevisiae) was exposed to dielectric barrier discharge plasma (DBD) to improve its ethanol production capacity during fermentation. Response surface methodology (RSM) was used to optimize the discharge-associated parameters of DBD for the purpose of maximizing the ethanol yield achieved by DBD-treated S. cerevisiae. According to single factor experiments, a mathematical model was established using Box-Behnken central composite experiment design, with plasma exposure time, power supply voltage, and exposed-sample volume as impact factors and ethanol yield as the response. This was followed by response surface analysis. Optimal experimental parameters for plasma discharge-induced enhancement in ethanol yield were plasma exposure time of 1 min, power voltage of 26 V, and an exposed sample volume of 9 mL. Under these conditions, the resulting yield of ethanol was 0.48 g/g, representing an increase of 33% over control.

  16. Toxicity detection using lysosomal enzymes, glycoamylase and thioredoxin fused with fluorescent protein in Saccharomyces cerevisiae.

    PubMed

    Nguyen, Ngoc-Tu; Shin, Hwa-Yoon; Kim, Yang-Hoon; Min, Jiho

    2015-11-20

    Saccharomyces cerevisiae is the simplest and a favorite eukaryotic system that contains lysosome and thus, is a suitable organism for monitoring some toxic effects in environmental pollution. In this study, S. cerevisiae was transformed with two recombinant plasmids. Sporulation-specific glycoamylase (SGA1), which was upregulated in response to arsenic, was fused with the blue fluorescent protein (BFP) for the construction of an oxidative stress-causing chemicals sensor. Additionally, thioredoxin (TRX2), a protein overexpressed exclusively under tetracycline's influence, fused with the cyan fluorescent protein (CFP) to create a detector for this kind of chemical. In summary, we developed two recombinant S. cerevisiae that facilitate the detection of both kinds of toxic chemicals, specifically visualized by different color indicators. PMID:26410455

  17. Two programmed replicative lifespans of Saccharomyces cerevisiae formed by the endogenous molecular-cellular network.

    PubMed

    Hu, Jie; Zhu, Xiaomei; Wang, Xinan; Yuan, Ruoshi; Zheng, Wei; Xu, Minjuan; Ao, Ping

    2014-12-01

    Cellular replicative capacity is a therapeutic target for regenerative medicine as well as cancer treatment. The mechanism of replicative senescence and cell immortality is still unclear. We investigated the diauxic growth of Saccharomyces cerevisiae and demonstrate that the replicative capacity revealed by the yeast growth curve can be understood by using the dynamical property of the molecular-cellular network regulating S. cerevisiae. The endogenous network we proposed has a limit cycle when pheromone signaling is disabled, consistent with the exponential growth phase with an infinite replicative capacity. In the post-diauxic phase, the cooperative effect of the pheromone activated mitogen-activated protein kinase (MAPK) signaling pathway with the cell cycle leads to a fixed point attractor instead of the limit cycle. The cells stop dividing after several generations counting from the beginning of the post-diauxic growth. By tuning the MAPK pathway, S. cerevisiae therefore programs the number of offsprings it replicates. PMID:24447585

  18. A new biological test of water toxicity-yeast Saccharomyces cerevisiae conductometric test.

    PubMed

    Dolezalova, Jaroslava; Rumlova, Lubomira

    2014-11-01

    This new biological test of water toxicity is based on monitoring of specific conductivity changes of yeast Saccharomyces cerevisiae suspension as a result of yeast fermentation activity inhibition in toxic conditions. The test was verified on ten substances with various mechanisms of toxic effect and the results were compared with two standard toxicity tests based on Daphnia magna mobility inhibition (EN ISO 6341) and Vibrio fischeri bioluminescence inhibition (EN ISO 11348-2) and with the results of the S. cerevisiae lethal test (Rumlova and Dolezalova, 2012). The new biological test - S. cerevisiae conductometric test - is an express method developed primarily for field conditions. It is applicable in case of need of immediate information about water toxicity. Fast completion is an advantage of this test (time necessary for test completion is about 60min), the test is simple and the test organism - dried instant yeast - belongs among its biggest advantages because of its long-term storage life and broad availability. PMID:25461558

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

    PubMed Central

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

    2016-01-01

    The natural biology of Saccharomyces cerevisiae, the best known unicellular model eukaryote, remains poorly documented and understood although recent progress has started to change this situation. Studies carried out recently in the Northern Hemisphere revealed the existence of wild populations associated with oak trees in North America, Asia, and in the Mediterranean region. However, in spite of these advances, the global distribution of natural populations of S. cerevisiae, especially in regions were oaks and other members of the Fagaceae are absent, is not well understood. Here we investigate the occurrence of S. cerevisiae in Brazil, a tropical region where oaks and other Fagaceae are absent. We report a candidate natural habitat of S. cerevisiae in South America and, using whole-genome data, we uncover new lineages that appear to have as closest relatives the wild populations found in North America and Japan. A population structure analysis revealed the penetration of the wine genotype into the wild Brazilian population, a first observation of the impact of domesticated microbe lineages on the genetic structure of wild populations. Unexpectedly, the Brazilian population shows conspicuous evidence of hybridization with an American population of Saccharomyces paradoxus. Introgressions from S. paradoxus were significantly enriched in genes encoding secondary active transmembrane transporters. We hypothesize that hybridization in tropical wild lineages may have facilitated the habitat transition accompanying the colonization of the tropical ecosystem. PMID:26782936

  20. A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism.

    PubMed

    Solis-Escalante, Daniel; Kuijpers, Niels G A; Barrajon-Simancas, Nuria; van den Broek, Marcel; Pronk, Jack T; Daran, Jean-Marc; Daran-Lapujade, Pascale

    2015-08-01

    As a result of ancestral whole-genome and small-scale duplication events, the genomes of Saccharomyces cerevisiae and many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae's current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae strains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the "minimal glycolysis" [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic of S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community. PMID:26071034

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

    PubMed

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

    2016-01-01

    The natural biology of Saccharomyces cerevisiae, the best known unicellular model eukaryote, remains poorly documented and understood although recent progress has started to change this situation. Studies carried out recently in the Northern Hemisphere revealed the existence of wild populations associated with oak trees in North America, Asia, and in the Mediterranean region. However, in spite of these advances, the global distribution of natural populations of S. cerevisiae, especially in regions were oaks and other members of the Fagaceae are absent, is not well understood. Here we investigate the occurrence of S. cerevisiae in Brazil, a tropical region where oaks and other Fagaceae are absent. We report a candidate natural habitat of S. cerevisiae in South America and, using whole-genome data, we uncover new lineages that appear to have as closest relatives the wild populations found in North America and Japan. A population structure analysis revealed the penetration of the wine genotype into the wild Brazilian population, a first observation of the impact of domesticated microbe lineages on the genetic structure of wild populations. Unexpectedly, the Brazilian population shows conspicuous evidence of hybridization with an American population of Saccharomyces paradoxus. Introgressions from S. paradoxus were significantly enriched in genes encoding secondary active transmembrane transporters. We hypothesize that hybridization in tropical wild lineages may have facilitated the habitat transition accompanying the colonization of the tropical ecosystem. PMID:26782936

  2. A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism

    PubMed Central

    Solis-Escalante, Daniel; Kuijpers, Niels G. A.; Barrajon-Simancas, Nuria; van den Broek, Marcel; Pronk, Jack T.; Daran, Jean-Marc

    2015-01-01

    As a result of ancestral whole-genome and small-scale duplication events, the genomes of Saccharomyces cerevisiae and many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae's current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae strains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the “minimal glycolysis” [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic of S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community. PMID:26071034

  3. Immunology and Microbiology Devices; Classification of Anti-Saccharomyces cerevisiae (S. cerevisiae) Antibody (ASCA) Test Systems. Food and Drug Administration, HHS. Final rule.

    PubMed

    2000-11-22

    The Food and Drug Administration (FDA) is classifying the Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system into class II (special controls). The special control that will apply to this device is a guidance document entitled "Guidance for Industry and FDA Reviewers: Class II Special Control Guidance Document for Anti-Saccharomyces cerevisiae (S. cerevisiae) Antibody (ASCA) Premarket Notifications." Elsewhere in this issue of the Federal Register. FDA is announcing the availability of this guidance document. The agency is taking this action in response to a petition submitted under the Federal Food, Drug, and Cosmetic Act (the act) as amended by the Medical Device Amendments of 1976, the Safe Medical Devices Act of 1990, and the Food and Drug Administration Modernization Act of 1997. The agency is classifying these devices into class II (special controls) in order to provide a reasonable assurance of the safety and effectiveness of the devices. PMID:11503713

  4. High-Throughput Microscopy-Based Screening in Saccharomyces cerevisiae.

    PubMed

    Styles, Erin B; Friesen, Helena; Boone, Charles; Andrews, Brenda J

    2016-01-01

    The budding yeastSaccharomyces cerevisiaehas served as the pioneer model organism for virtually all genome-scale methods, including genome sequencing, DNA microarrays, gene deletion collections, and a variety of proteomic platforms. Yeast has also provided a test-bed for the development of systematic fluorescence-based imaging screens to enable the analysis of protein localization and abundance in vivo. Especially important has been the integration of high-throughput microscopy with automated image-processing methods, which has allowed researchers to overcome issues associated with manual image analysis and acquire unbiased, quantitative data. Here we provide an introduction to automated imaging in budding yeast. PMID:27037080

  5. "Active" one-carbon generation in Saccharomyces cerevisiae.

    PubMed Central

    Ogur, M; Liu, T N; Cheung, I; Paulavicius, I; Wales, W; Mehnert, D; Blaise, D

    1977-01-01

    A new mutation introducing a one-carbon requirement (e.g., formate) for the glycine-supplemented growth of a serine-glycine auxotroph (ser1) was correlated with a lack of glycine decarboxylase activity. The presence of oxalate decarboxylase activity or glyoxylate decarboxylase activity did not overcome the one-carbon requirement. Another mutation characterized by the absence of oxalate decarboxylase activity did not introduce a one-carbon requirement. The presence and physiological significance of glycine decarboxylase activity in Saccharomyces are thus inferred. PMID:320197

  6. Screening for hydrolytic enzymes reveals Ayr1p as a novel triacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Ploier, Birgit; Scharwey, Melanie; Koch, Barbara; Schmidt, Claudia; Schatte, Jessica; Rechberger, Gerald; Kollroser, Manfred; Hermetter, Albin; Daum, Günther

    2013-12-13

    Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317-23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301-37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source. PMID:24187129

  7. Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

    PubMed Central

    Guerrini, A M; Ascenzioni, F; Tribioli, C; Donini, P

    1985-01-01

    Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids. Images Fig. 1. Fig. 2. Fig. 4. PMID:3896773

  8. Effect of fermented sea tangle on the alcohol dehydrogenase and acetaldehyde dehydrogenase in Saccharomyces cerevisiae.

    PubMed

    Cha, Jae-Young; Jeong, Jae-Jun; Yang, Hyun-Ju; Lee, Bae-Jin; Cho, Young-Su

    2011-08-01

    Sea tangle, a kind of brown seaweed, was fermented with Lactobacillus brevis BJ-20. The gamma-aminobutyric acid (GABA) content in fermented sea tangle (FST) was 5.56% (w/w) and GABA in total free amino acid of FST was 49.5%. The effect of FST on the enzyme activities and mRNA protein expression of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) involved in alcohol metabolism in Saccharomyces cerevisiae was investigated. Yeast was cultured in YPD medium supplemented with different concentrations of FST powder [0, 0.4, 0.8, and 1.0% (w/v)] for 18 h. FST had no cytotoxic effect on the yeast growth. The highest activities and protein expressions of ADH and ALDH from the cell-free extracts of S. cerevisiae were evident with the 0.4% and 0.8% (w/v) FST-supplemented concentrations, respectively. The highest concentrations of GABA as well as minerals (Zn, Ca, and Mg) were found in the cell-free extracts of S. cerevisiae cultured in medium supplemented with 0.4% (w/v) FST. The levels of GABA, Zn, Ca, and Mg in S. cerevisiae were strongly correlated with the enzyme activities of ADH and ALDH in yeast. These results indicate that FST can enhance the enzyme activities and protein expression of ADH and ALDH in S. cerevisiae. PMID:21876367

  9. Label-Free Proteomic Analysis of Flavohemoglobin Deleted Strain of Saccharomyces cerevisiae

    PubMed Central

    Panja, Chiranjit; Setty, Rakesh K. S.; Vaidyanathan, Gopal; Ghosh, Sanjay

    2016-01-01

    Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in the nitrosative stress responses in Saccharomyces cerevisiae. It is still unclear how S. cerevisiae can withstand this NO level in the absence of flavohemoglobin. To better understand the physiological function of flavohemoglobin in yeast, in the present study a label-free differential proteomics study has been carried out in wild-type and YHB1 deleted strains of S. cerevisiae grown under fermentative conditions. From the analysis, 417 proteins in Y190 and 392 proteins in ΔYHB1 were identified with high confidence. Interestingly, among the differentially expressed identified proteins, 40 proteins were found to be downregulated whereas 41 were found to be upregulated in ΔYHB1 strain of S. cerevisiae (p value < 0.05). The differentially expressed proteins were also classified according to gene ontology (GO) terms. The most enriched and significant GO terms included nitrogen compound biosynthesis, amino acid biosynthesis, translational regulation, and protein folding. Interactions of differentially expressed proteins were generated using Search Tool for the Retrieval of Interacting Genes (STRING) database. This is the first report which offers a more complete view of the proteome changes in S. cerevisiae in the absence of flavohemoglobin. PMID:26881076

  10. Effects of sequential mixed cultures of Wickerhamomyces anomalus and Saccharomyces cerevisiae on apple cider fermentation.

    PubMed

    Ye, Mengqi; Yue, Tianli; Yuan, Yahong

    2014-09-01

    The fermentation of cider by mixed cultures of Wickerhamomyces anomalus and Saccharomyces cerevisiae was carried out to study their effect on the cider quality. The results showed that growth of W. anomalus and S. cerevisiae was affected by each other during co-fermentation process. All the mixed cultures produced statistically the same level of ethanol as S. cerevisiae monoculture. The mixed fermentation could produce more variety and higher amounts of acetate esters, ethyl esters, higher alcohols, aldehydes, and ketones. Sensory evaluation demonstrated that ciders obtained from co-fermentation with W. anomalus gained higher scores than ciders fermented by pure S. cerevisiae, especially the co-fermentation cultures WS3, WS4, WS6, and WS8. Only 3 days of fermentation with W. anomalus in sequential mixtures were enough to improve the quality of cider. Wickerhamomyces anomalus could be used in association with S. cerevisiae to improve the quality of cider. The modulation of inoculation time may provide an effective means of manipulating cider aroma for different characteristics. PMID:24931623

  11. Dominance of Saccharomyces cerevisiae in alcoholic fermentation processes: role of physiological fitness and microbial interactions.

    PubMed

    Albergaria, Helena; Arneborg, Nils

    2016-03-01

    Winemaking, brewing and baking are some of the oldest biotechnological processes. In all of them, alcoholic fermentation is the main biotransformation and Saccharomyces cerevisiae the primary microorganism. Although a wide variety of microbial species may participate in alcoholic fermentation and contribute to the sensory properties of end-products, the yeast S. cerevisiae invariably dominates the final stages of fermentation. The ability of S. cerevisiae to outcompete other microbial species during alcoholic fermentation processes, such as winemaking, has traditionally been ascribed to its high fermentative power and capacity to withstand the harsh environmental conditions, i.e. high levels of ethanol and organic acids, low pH values, scarce oxygen availability and depletion of certain nutrients. However, in recent years, several studies have raised evidence that S. cerevisiae, beyond its remarkable fitness for alcoholic fermentation, also uses defensive strategies mediated by different mechanisms, such as cell-to-cell contact and secretion of antimicrobial peptides, to combat other microorganisms. In this paper, we review the main physiological features underlying the special aptitude of S. cerevisiae for alcoholic fermentation and discuss the role of microbial interactions in its dominance during alcoholic fermentation, as well as its relevance for winemaking. PMID:26728020

  12. Intracellular pH in Schizosaccharomyces pombe--comparison with Saccharomyces cerevisiae.

    PubMed

    Haworth, R S; Fliegel, L

    1993-07-21

    We examined cytoplasmic pH regulation in Schizosaccharomyces pombe and Saccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 from S. pombe was much slower than leakage from C. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 for S. pombe and 6.6 for S. cerevisiae. We found that internal pH in S. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater in S. cerevisiae than in S. pombe. The plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC50 of approximately 35 microM in both species. Amiloride also inhibited internal alkalinisation with IC50's of 745 microM for S. cerevisiae and 490 microM for S. pombe. PMID:8232284

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

    PubMed Central

    2013-01-01

    Efficient degradation of cellulosic biomass requires the synergistic action of the cellulolytic enzymes endoglucanase, cellobiohydrolase, and ?-glucosidase. Although there are many reports describing consolidation of hydrolysis and fermentation steps using recombinant Saccharomyces cerevisiae that express cellulolytic enzymes, the efficiency of cellulose degradation has not been sufficiently improved. Although the yeast S. cerevisiae cannot take up cellooligosaccharide, some fungi can take up and assimilate cellooligosaccharide through a cellodextrin transporter. In this study, a S. cerevisiae strain co-expressing genes for several cell surface display cellulases and the cellodextrin transporter was constructed for the purpose of improving the efficiency of direct ethanol fermentation from phosphoric acid swollen cellulose (PASC). The cellulase/cellodextrin transporter-coexpressing strain produced 1.7-fold more ethanol (4.3g/L) from PASC during a 72-h fermentation than did a strain expressing cellulase only (2.5g/L). Direct ethanol production from PASC by the recombinant S. cerevisiae strain was improved by co-expression of cellulase display and cellodextrin transporter genes. These results suggest that cellulase- and cellodextrin transporter-co-expressing S. cerevisiae could be a promising technology for efficient direct ethanol production from cellulose. PMID:23800294

  14. Saccharomyces cerevisiae: Population Divergence and Resistance to Oxidative Stress in Clinical, Domesticated and Wild Isolates

    PubMed Central

    Diezmann, Stephanie; Dietrich, Fred S.

    2009-01-01

    Background Saccharomyces cerevisiae has been associated with human life for millennia in the brewery and bakery. Recently it has been recognized as an emerging opportunistic pathogen. To study the evolutionary history of S. cerevisiae, the origin of clinical isolates and the importance of a virulence-associated trait, population genetics and phenotypic assays have been applied to an ecologically diverse set of 103 strains isolated from clinics, breweries, vineyards, fruits, soil, commercial supplements and insect guts. Methodology/Principal Findings DNA sequence data from five nuclear DNA loci were analyzed for population structure and haplotype distribution. Additionally, all strains were tested for survival of oxidative stress, a trait associated with microbial pathogenicity. DNA sequence analyses identified three genetic subgroups within the recombining S. cerevisiae strains that are associated with ecology, geography and virulence. Shared alleles suggest that the clinical isolates contain genetic contribution from the fruit isolates. Clinical and fruit isolates exhibit high levels of recombination, unlike the genetically homogenous soil isolates in which no recombination was detected. However, clinical and soil isolates were more resistant to oxidative stress than any other population, suggesting a correlation between survival in oxidative stress and yeast pathogenicity. Conclusions/Significance Population genetic analyses of S. cerevisiae delineated three distinct groups, comprising primarily the (i) human-associated brewery and vineyard strains, (ii) clinical and fruit isolates (iii) and wild soil isolates from eastern U.S. The interactions between S. cerevisiae and humans potentiate yeast evolution and the development of genetically, ecologically and geographically divergent groups. PMID:19390633

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

    PubMed

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

    2015-07-01

    In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae. PMID:26037463

  16. Fermentation profile of Saccharomyces cerevisiae and Candida tropicalis as starter cultures on barley malt medium.

    PubMed

    Alloue-Boraud, Wazé Aimée Mireille; N'Guessan, Kouadio Florent; Djeni, N'Dédé Théodore; Hiligsmann, Serge; Djè, Koffi Marcellin; Delvigne, Franck

    2015-08-01

    Saccharomyces cerevisiae C8-5 and Candida tropicalis F0-5 isolated from traditional sorghum beer were tested for kinetic parameters on barley malt extract, YPD (863 medium) and for alcohol production. The results showed that C. tropicalis has the highest maximum growth rate and the lowest doubling time. Values were 0.22 and 0.32 h(-1) for maximum growth rate, 3 h 09 min and 2 h 09 min for doubling time respectively on barley malt extract and YPD. On contrary, glucose consumption was the fastest with S. cerevisiae (-0.36 and -0.722 g/l/h respectively on barley malt extract and YPD). When these two yeasts were used as starters in pure culture and co-culture at proportion of 1:1 and 2:1 (cell/cell) for barley malt extract fermentation, we noticed that maltose content increased first from 12.12 g/l to 13.62-16.46 g/l and then decreased. The highest increase was obtained with starter C. tropicalis + S. cerevisiae 2:1. On contrary, glucose content decreased throughout all the fermentation process. For all the starters used, the major part of the ethanol was produced at 16 h of fermentation. Values obtained in the final beers were 11.4, 11.6, 10.4 and 10.9 g/l for fermentation conducted with S. cerevisiae, C. tropicalis, C. tropicalis + S. cerevisiae 1:1 and C. tropicalis + S. cerevisiae 2:1. Cell viability measurement during the fermentation by using flow cytometry revealed that the lowest mean channel fluorescence for FL3 (yeast rate of death) was obtained with C. tropicalis + S. cerevisiae 2:1 after 48 h of fermentation. PMID:26243947

  17. Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris.

    PubMed Central

    Soares-Silva, Isabel; Schuller, Dorit; Andrade, Raquel P; Baltazar, Fátima; Cássio, Fernanda; Casal, Margarida

    2003-01-01

    In Saccharomyces cerevisiae the activity for the lactate-proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a V (max) of 2.1 nmol x s(-1) x mg of dry weight(-1). Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest V (max) (0.84 nmol x s(-1) x mg of dry weight(-1)) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels. PMID:12962538

  18. Quercetin increases oxidative stress resistance and longevity in Saccharomyces cerevisiae.

    PubMed

    Belinha, Iracema; Amorim, Maria Amélia; Rodrigues, Pedro; de Freitas, Victor; Moradas-Ferreira, Pedro; Mateus, Nuno; Costa, Vítor

    2007-03-21

    Quercetin, the major flavonol found in several fruits and vegetables, is a natural antioxidant with potential anticancer and antiaging activities. In this paper, the effect of quercetin in Sacharomyces cerevisiae cells submitted to oxidative stress was studied. Hydrogen peroxide resistance increased in cells pretreated with quercetin. Cellular protection was correlated with a decrease in oxidative stress markers, namely, levels of reactive oxygen species, glutathione oxidation, protein carbonylation, and lipid peroxidation. The acquisition of H2O2 resistance was not associated with the induction of antioxidant defenses or with iron chelation. Oxidative stress is a limiting factor for longevity. In agreement, quercetin also increased 60% chronological life span. These results support the utilization of yeast as a useful model to screen in vivo for natural antioxidants with putative health beneficial effects. PMID:17323973

  19. Histone H1 of Saccharomyces cerevisiae Inhibits Transcriptional Silencing

    PubMed Central

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

    2006-01-01

    Eukaryotic genomes contain euchromatic regions, which are transcriptionally active, and heterochromatic regions, which are repressed. These domains are separated by “barrier elements”: DNA sequences that protect euchromatic regions from encroachment by neighboring heterochromatin. To identify proteins that play a role in the function of barrier elements we have carried out a screen in S. cerevisiae. We recovered the gene HHO1, which encodes the yeast ortholog of histone H1, as a high-copy modifier of barrier activity. Histone H1 is a linker histone that binds the outside of nucleosomes and modifies chromatin dynamics. Here we show that Hho1p reinforces the action of several types of barrier elements, and also inhibits silencing on its own. PMID:16582449

  20. Microencapsulation of Saccharomyces cerevisiae and its evaluation to protect in simulated gastric conditions

    PubMed Central

    Ghorbani-Choboghlo, Hassan; Zahraei-Salehi, Taghi; Ashrafi-Helan, Javad; Yahyaraeyat, Ramak; Pourjafar, Hadi; Nikaein, Donya; Balal, Asad; Khosravi, Ali-Reza

    2015-01-01

    Background and Objectives: Probiotic yeasts are used in production of functional foods and pharmaceutical products. They play an important role in promoting and maintaining human health. Until now, little work has been published on improving the survival of Saccharomyces in stimulated gastrointestinal condition. Material and Methods: In this study the exposure of the yeast in the capsulate and free forms to artificial gastrointestinal conditions was assessed and the number of viable Saccharomyces cerevisiae cells during 0 to 120 mines in these conditions was evaluated by a pour plate method using sabouraud dextrose agar. Results: Results showed the shape of the beads was generally spherical, sometimes elliptical with a mean diameter of about 50–90 μm. Also count of viable probiotic cells obtained for all the microcapsules were above the recommended levels for a probiotic food. Also decrease of approximately 4 logs was noted in the number of free cells after 2 h of incubation at pH 2 and 8, when compared to decreases of about 2 logs in the all microencapsulated S. cerevisiae under similar conditions. Conclusion: It is concluded that microencapsulation process was significantly able to increase the survival rate of Saccharomyces in a simulated gastrointestinal condition (p<0.05).. PMID:26885335

  1. High hydrostatic pressure activates transcription factors involved in Saccharomyces cerevisiae stress tolerance

    PubMed Central

    Bravim, Fernanda; da Silva, Lucas F.; Souza, Diego T.; Lippman, Soyeon I.; Broach, James R.; Fernandes, A. Alberto R.; Fernandes, Patricia M, B.

    2016-01-01

    A number of transcriptional control elements are activated when Saccharomyces cerevisiae cells are submitted to various stress conditions, including high hydrostatic pressure (HHP). Exposure of Saccharomyces cerevisiae cells to HHP results in global transcriptional reprogramming, similar to that observed under other industrial stresses, such as temperature, ethanol and oxidative stresses. Moreover, treatment with a mild hydrostatic pressure renders yeast cells multi-stress tolerant. In order to identify transcriptional factors involved in coordinating response to high hydrostatic pressure, we performed a time series microarray expression analysis on a wild S. cerevisiae strain exposed to 50 MPa for 30 min followed by recovery at atmospheric pressure (0.1 MPa) for 5, 10 and 15 min. We identified transcription factors and corresponding DNA and RNA motifs targeted in response to hydrostatic pressure. Moreover, we observed that different motif elements are present in the promoters of induced or repressed genes during HHP treatment. Overall, as we have already published, mild HHP treatment to wild yeast cells provides multiple protection mechanisms, and this study suggests that the TFs and motifs identified as responding to HHP may be informative for a wide range of other biotechnological and industrial applications, such as fermentation, that may utilize HHP treatment. PMID:23072392

  2. CHARACTERIZATION OF THE PYROGENICITY OF CANDIDA ALBICANS, SACCHAROMYCES CEREVISIAE, AND CRYPTOCOCCUS NEOFORMANS.

    PubMed

    KOBAYASHI, G S; FRIEDMAN, L

    1964-09-01

    Kobayashi, George S. (Tulane University, New Orleans, La.), and Lorraine Friedman. Characterization of the pyrogenicity of Candida albicans, Saccharomyces cerevisiae, and Cryptococcus neoformans. J. Bacteriol. 88:660-666. 1964.-The intravenous injection into rabbits of 10(9) yeast cells of Candida albicans, Saccharomyces cerevisiae, or Cryptococcus neoformans (both slightly and heavily encapsulated forms) induced a febrile response indistinguishable from that elicited by gram-negative bacterial endotoxin. There was a brisk rise in body temperature which began as early as 30 min after injection, peaked once or twice, and then returned to normal after about 10 hr. With viable C. albicans, the febrile response did not return to normal but remained elevated for several days and terminated at death of the animal. Of three extraction procedures employed in attempts to isolate the endotoxin-like pyrogenically active substances from C. albicans, only one, the phenol extraction method, was successful. Pyrogenic substances were more easily extractable from S. cerevisiae, but extracted cells of both species were still highly pyrogenic. It was concluded that the particulate nature of the yeast cell did not contribute to the induction of fever, for latex particles of a similar size were nonpyrogenic. Viable or heat-killed C. albicans, phenol extract of C. albicans, zymosan, and polystyrene latex particles all failed to induce in rabbits increased dermal reactivity to epinephrine. PMID:14208504

  3. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  4. Nitrogen and carbon assimilation by Saccharomyces cerevisiae during Sauvignon blanc juice fermentation.

    PubMed

    Pinu, Farhana R; Edwards, Patrick J B; Gardner, Richard C; Villas-Boas, Silas G

    2014-12-01

    To investigate the assimilation and production of juice metabolites by Saccharomyces cerevisiae during winemaking, we compared the metabolite profiles of 63 Sauvignon blanc (SB) grape juices collected over five harvesting seasons from different locations of New Zealand before and after fermentation by the commercial wine yeast strain EC1118 at 15 °C. Metabolite profiles were obtained using gas chromatography-mass spectrometry and nuclear magnetic resonance and the oenological parameters were determined by Fourier transform infrared spectroscopy. Our results revealed that the amino acids threonine and serine were the most consumed organic nitrogen sources, while proline and gamma-aminobutyric acid were the least consumed amino acids during SB juice fermentation. Saccharomyces cerevisiae metabolised some uncommon nitrogen sources (e.g. norleucine, norvaline and pyroglutamic acid) and several organic acids, including some fatty acids, most likely after fermenting the main juice sugars (glucose, fructose and mannose). However, consumption showed large variation between juices and in some cases between seasons. Our study clearly shows that preferred nitrogen and carbon sources were consumed by S. cerevisiae EC1118 independent of the juice fine composition, whilst the consumption of other nutrient sources mainly depended on the concentration of other juice metabolites, which explains the uniqueness of each barrel of wine. PMID:25345561

  5. Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

    PubMed

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  6. The yeast dynein Dyn2-Pac11 complex is a dynein dimerization/processivity factor: structural and single-molecule characterization

    PubMed Central

    Rao, Lu; Romes, Erin M.; Nicholas, Matthew P.; Brenner, Sibylle; Tripathy, Ashutosh; Gennerich, Arne; Slep, Kevin C.

    2013-01-01

    Cytoplasmic dynein is the major microtubule minus end–directed motor. Although studies have probed the mechanism of the C-terminal motor domain, if and how dynein's N-terminal tail and the accessory chains it binds regulate motor activity remain to be determined. Here, we investigate the structure and function of the Saccharomyces cerevisiae dynein light (Dyn2) and intermediate (Pac11) chains in dynein heavy chain (Dyn1) movement. We present the crystal structure of a Dyn2-Pac11 complex, showing Dyn2-mediated Pac11 dimerization. To determine the molecular effects of Dyn2 and Pac11 on Dyn1 function, we generated dyn2Δ and dyn2Δpac11Δ strains and analyzed Dyn1 single-molecule motor activity. We find that the Dyn2-Pac11 complex promotes Dyn1 homodimerization and potentiates processivity. The absence of Dyn2 and Pac11 yields motors with decreased velocity, dramatically reduced processivity, increased monomerization, aggregation, and immobility as determined by single-molecule measurements. Deleting dyn2 significantly reduces Pac11-Dyn1 complex formation, yielding Dyn1 motors with activity similar to Dyn1 from the dyn2Δpac11Δ strain. Of interest, motor phenotypes resulting from Dyn2-Pac11 complex depletion bear similarity to a point mutation in the mammalian dynein N-terminal tail (Loa), highlighting this region as a conserved, regulatory motor element. PMID:23761070

  7. Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive. Results In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH) and fumarase (RoFUM1) were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2) was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1) than the control strain FMME-001 empty vector. Conclusions The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner. PMID:22335940

  8. Functional interactions between potassium and phosphate homeostasis in Saccharomyces cerevisiae.

    PubMed

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

    2015-02-01

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

  9. Determination of ethanol in whey-sugar solutions by freezing. [Kluyveromyces fragilis and Saccharomyces cerevisiae

    SciTech Connect

    Demott, B.J.

    1982-01-01

    The composition of solutions undergoing yeast fermentation was stimulated by using direct-acid-set cottage cheese whey containing increasing amounts of EtOH (0 to 5.4%) with decreasing amounts of sucrose (10 to 0%). Each decrease of 0.54 g EtOH decreased specific gravity by 0.0046 unit and lowered the freezing point by 0.159 H (the Hortvet unit). Whey containing 10% added sucrose was treated as follows: (a) inoculated with Kluyveromyces fragilis, (b) carbohydrate splitting enzymes added and inoculated with Kluyveromyces fragilis and (c) carbohydrate splitting enzymes added and inoculated with Saccharomyces cerevisiae. All mixtures were incubated 48 h at 32 degrees during which 6 samples from each treatment were analyzed for total solids, specific gravity and freezing point. No difference was noted between samples treated with enzymes or those treated with the 2 yeasts cultured as related to decrease in total solids concentration or specific gravity. Each 0.001 H decrease in freezing point was accompanied by a total solids decrease of 0.006 g/100 g whey in the nonenzyme treated sample, and 0.008 g and 0.010 g/100 g whey in the enzyme-treated samples inoculated with Kluyveromyces fragilis and Saccharomyces cerevisiae, respectively. Each 0.001 H change in freezing point was equivalent to a change of 0.00003 specific gravity unit in the nonenzyme-treated sample and 0.000043 and 0.000048 specific gravity unit in the enzyme-treated samples inoculated with Kluyveromyces fragilis and Saccharomyces cerevisiae, respectively. The precision with which freezing point can be determined suggests its use in evaluating the amount of EtOH produced during fermentation.

  10. Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

    PubMed

    Haworth, R S; Lemire, B D; Crandall, D; Cragoe, E J; Fliegel, L

    1991-12-01

    We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase. PMID:1661160

  11. Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

    TOXLINE Toxicology Bibliographic Information

    Haworth RS; Lemire BD; Crandall D; Cragoe EJ Jr; Fliegel L

    1991-12-03

    We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.

  12. Genomic diversity of Saccharomyces cerevisiae yeasts associated with alcoholic fermentation of bacanora produced by artisanal methods.

    PubMed

    Álvarez-Ainza, M L; Zamora-Quiñonez, K A; Moreno-Ibarra, G M; Acedo-Félix, E

    2015-03-01

    Bacanora is a spirituous beverage elaborated with Agave angustifolia Haw in an artisanal process. Natural fermentation is mostly performed with native yeasts and bacteria. In this study, 228 strains of yeast like Saccharomyces were isolated from the natural alcoholic fermentation on the production of bacanora. Restriction analysis of the amplified region ITS1-5.8S-ITS2 of the ribosomal DNA genes (RFLPr) were used to confirm the genus, and 182 strains were identified as Saccharomyces cerevisiae. These strains displayed high genomic variability in their chromosomes profiles by karyotyping. Electrophoretic profiles of the strains evaluated showed a large number of chromosomes the size of which ranged between 225 and 2200 kpb approximately. PMID:25561061

  13. Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae

    PubMed Central

    Pâques, Frédéric; Haber, James E.

    1999-01-01

    The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination. PMID:10357855

  14. Genomic exploration of the hemiascomycetous yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae.

    PubMed

    Llorente, B; Durrens, P; Malpertuy, A; Aigle, M; Artiguenave, F; Blandin, G; Bolotin-Fukuhara, M; Bon, E; Brottier, P; Casaregola, S; Dujon, B; de Montigny, J; Lépingle, A; Neuvéglise, C; Ozier-Kalogeropoulos, O; Potier, S; Saurin, W; Tekaia, F; Toffano-Nioche, C; Wésolowski-Louvel, M; Wincker, P; Weissenbach, J; Souciet, J; Gaillardin, C

    2000-12-22

    We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others. PMID:11152895

  15. Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid

    PubMed Central

    Giannattasio, Sergio; Guaragnella, Nicoletta; Ždralević, Maša; Marra, Ersilia

    2013-01-01

    Beyond its classical biotechnological applications such as food and beverage production or as a cell factory, the yeast Saccharomyces cerevisiae is a valuable model organism to study fundamental mechanisms of cell response to stressful environmental changes. Acetic acid is a physiological product of yeast fermentation and it is a well-known food preservative due to its antimicrobial action. Acetic acid has recently been shown to cause yeast cell death and aging. Here we shall focus on the molecular mechanisms of S. cerevisiae stress adaptation and programmed cell death in response to acetic acid. We shall elaborate on the intracellular signaling pathways involved in the cross-talk of pro-survival and pro-death pathways underlying the importance of understanding fundamental aspects of yeast cell homeostasis to improve the performance of a given yeast strain in biotechnological applications. PMID:23430312

  16. Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Klein, H. P.; Jahnke, L.

    1979-01-01

    Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

  17. Improvement of Phytase Activity by a New Saccharomyces cerevisiae Strain Using Statistical Optimization

    PubMed Central

    Ries, Edi Franciele; Alves Macedo, Gabriela

    2011-01-01

    Using statistical optimization, we enhanced the activity of phytase by a new Saccharomyces cerevisiae strain cultured in mineral medium. Concentrations of carbon source and inducer of phytase production were optimized using a 22 full factorial CCD and response surface methodology (RSM). Urea was fixed as nitrogen source in culture medium (0.15%, w/v). The culture medium consisting of 2.5% sucrose and 0.5% sodium phytate optimally supported the maximum phytase activity. In addition, we found that culture of the yeast at 35°C with shaking at 150 rpm supports maximum phytase production. The validity of this model was verified by culturing the organisms in flasks on a shaker. Using the optimized media and growth conditions, we obtained a 10-fold improvement in the production of phytase by S. cerevisiae. PMID:21837273

  18. Effect of acetaldehyde on Saccharomyces cerevisiae and Zymomonas mobilis subjected to environmental shocks

    SciTech Connect

    Stanley, G.A.; Hobley, T.J.; Pamment, N.B.

    1997-01-05

    The lag phase of Saccharomyces cerevisiae subjected to a step increase in temperature or ethanol concentration was reduced by as much as 60% when acetaldehyde was added to the medium at concentrations less than 0.1 g/L. Maximum specific growth rates were also substantially increased. Even greater proportional reductions in lag time due to acetaldehyde addition were observed for ethanol-shocked cultures of Zymomonas mobilis. Acetaldehyde had no effect on S. cerevisiae cultures started from stationary phase inocula in the absence of environmental shock and its lag-reducing effects were greater in complex medium than in a defined synthetic medium. Acetaldehyde reacted strongly with the ingredients of complex culture media. It is proposed that the effect of added acetaldehyde may be to compensate for the inability of cells to maintain transmembrane acetaldehyde gradients following an environmental shock.

  19. Genome-wide construction of a series of designed segmental aneuploids in Saccharomyces cerevisiae

    PubMed Central

    Natesuntorn, Waranya; Iwami, Kotaro; Matsubara, Yuki; Sasano, Yu; Sugiyama, Minetaka; Kaneko, Yoshinobu; Harashima, Satoshi

    2015-01-01

    Segmental aneuploidy can play an important role in environmental adaptation. However, study of segmental aneuploids is severely hampered by the difficulty of creating them in a designed fashion. Here, we describe a PCR-mediated chromosome duplication (PCDup) technology that enables the generation of segmental aneuploidy at any desired chromosomal region in Saccharomyces cerevisiae. We constructed multiple strains harboring 100 kb to 200 kb segmental duplications covering the whole of the S. cerevisiae genome. Interestingly, some segmental aneuploidies confer stress tolerance, such as to high temperature, ethanol and strong acids, while others induce cell lethality and stress sensitivity, presumably as result of the simultaneous increases in dosages of multiple genes. We suggest that our PCDup technology will accelerate studies into the phenotypic changes resulting from alteration of gene dosage balance of multiple genes and will provide new insights into the adaptive molecular mechanisms in the genome in segmental aneuploidy-derived human diseases. PMID:26224198

  20. Physiological response of Saccharomyces cerevisiae to 15-azasterol-mediated growth inhibition.

    PubMed Central

    Rodriguez, R J; Parks, L W

    1981-01-01

    We studied 15-aza-24-methylene-8,14-cholestadiene-3 beta-ol (15-azasterol) inhibition of Saccharomyces cerevisiae growth. Exposure to sublethal concentrations of this drug caused S. cerevisiae cells to undergo a transient period of inhibition at midlog phase. During growth inhibition the turbidity of each culture remained constant, as did the total cell number. Although the proportion of viable cells in cultures decreased from 90 to 12% during inhibition, methylene blue staining showed that less than 40% of the cells underwent metabolic inactivation. We monitored adenosine triphosphate levels throughout the inhibition cycle, and these levels followed kinetics identical to cell growth kinetics. After overcoming inhibition, cellular lipid extracts revealed the presence of a modified form of 15-azasterol. It appeared that the yeast cells were able to overcome 15-azasterol inhibition by an inactivating transmethylation reaction involving S-adenosylmethionine. PMID:7025753

  1. Action of antifungal peptidolipids from Bacillus subtilis on the cell membrane of Saccharomyces cerevisiae.

    PubMed

    Besson, F; Peypoux, F; Quentin, M J; Michel, G

    1984-02-01

    Iturin A and bacillomycin L, antibiotics of the iturin group inhibit the growth of Saccharomyces cerevisiae and the lethal doses were respectively 10 and 60 micrograms/ml. Both antibiotics had an effect on the incorporation of radioactive precursors into macromolecules which decreased with increasing concentrations of antibiotics. However, no specificity was observed on the various macromolecules, proteins, ribonucleic acids and polysaccharides. The site of action on yeast cells was demonstrated to be the cytoplasmic membrane: both antibiotics of iturin group lysed spheroplasts of S. cerevisiae. Moreover, a rapid leakage of potassium ions occurred in the presence of the antibiotics; this leakage was directly associated to the killing effect. These results are consistent with a disruption of the structural integrity of the cytoplasmic membrane correlated to the loss of viability of the yeast cells. PMID:6423598

  2. Production of Volatile and Sulfur Compounds by 10 Saccharomyces cerevisiae Strains Inoculated in Trebbiano Must.

    PubMed

    Patrignani, Francesca; Chinnici, Fabio; Serrazanetti, Diana I; Vernocchi, Pamela; Ndagijimana, Maurice; Riponi, Claudio; Lanciotti, Rosalba

    2016-01-01

    In wines, the presence of sulfur compounds is the resulting of several contributions among which yeast metabolism. The characterization of the starter Saccharomyces cerevisiae needs to be performed also taking into account this ability even if evaluated together with the overall metabolic profile. In this perspective, principal aim of this experimental research was the evaluation of the volatile profiles, throughout GC/MS technique coupled with solid phase micro extraction, of wines obtained throughout the fermentation of 10 strains of S. cerevisiae. In addition, the production of sulfur compounds was further evaluated by using a gas-chromatograph coupled with a Flame Photometric Detector. Specifically, the 10 strains were inoculated in Trebbiano musts and the fermentations were monitored for 19 days. In the produced wines, volatile and sulfur compounds as well as amino acid concentrations were investigated. Also the physico-chemical characteristics of the wines and their electronic nose profiles were evaluated. PMID:26973621

  3. Comparative transcriptome analysis between original and evolved recombinant lactose-consuming Saccharomyces cerevisiae strains.

    PubMed

    Guimarães, Pedro M R; Le Berre, Véronique; Sokol, Serguei; François, Jean; Teixeira, José A; Domingues, Lucília

    2008-12-01

    The engineering of Saccharomyces cerevisiae strains for lactose utilization has been attempted with the intent of developing high productivity processes for alcoholic fermentation of cheese whey. A recombinant S. cerevisiae flocculent strain that efficiently ferments lactose to ethanol was previously obtained by evolutionary engineering of an original recombinant that displayed poor lactose fermentation performance. We compared the transcriptomes of the original and the evolved recombinant strains growing in lactose, using cDNA microarrays. Microarray data revealed 173 genes whose expression levels differed more than 1.5-fold. About half of these genes were related to RNA-mediated transposition. We also found genes involved in DNA repair and recombination mechanisms, response to stress, chromatin remodeling, cell cycle control, mitosis regulation, glycolysis and alcoholic fermentation. These transcriptomic data are in agreement with some of the previously identified physiological and molecular differences between the recombinants, and point to further hypotheses to explain those differences. PMID:19039778

  4. Production of Volatile and Sulfur Compounds by 10 Saccharomyces cerevisiae Strains Inoculated in Trebbiano Must

    PubMed Central

    Patrignani, Francesca; Chinnici, Fabio; Serrazanetti, Diana I.; Vernocchi, Pamela; Ndagijimana, Maurice; Riponi, Claudio; Lanciotti, Rosalba

    2016-01-01

    In wines, the presence of sulfur compounds is the resulting of several contributions among which yeast metabolism. The characterization of the starter Saccharomyces cerevisiae needs to be performed also taking into account this ability even if evaluated together with the overall metabolic profile. In this perspective, principal aim of this experimental research was the evaluation of the volatile profiles, throughout GC/MS technique coupled with solid phase micro extraction, of wines obtained throughout the fermentation of 10 strains of S. cerevisiae. In addition, the production of sulfur compounds was further evaluated by using a gas-chromatograph coupled with a Flame Photometric Detector. Specifically, the 10 strains were inoculated in Trebbiano musts and the fermentations were monitored for 19 days. In the produced wines, volatile and sulfur compounds as well as amino acid concentrations were investigated. Also the physico-chemical characteristics of the wines and their electronic nose profiles were evaluated. PMID:26973621

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

    PubMed Central

    Choi, Hak Joo; Kim, Eun A; Kim, Dong Hee

    2014-01-01

    A ?-glucosidase producing yeast strain was isolated from Korean traditional rice wine. Based on the sequence of the YCL008c gene and analysis of the fatty acid composition, the isolate was identified as Saccharomyces cerevisiae strain HJ-014. S. cerevisiae HJ-014 produced ginsenoside Rd, F2, and compound K from the ethanol extract of red ginseng. The production was increased by shaking culture, where the bioconversion efficiency was increased 2-fold compared to standing culture. The production of ginsenoside F2 and compound K was time-dependent and thought to proceed by the transformation pathway of: red ginseng extract?Rd?F2?compound K. The optimum incubation time and concentration of red ginseng extract for the production of compound K was 96 hr and 4.5% (w/v), respectively. PMID:25346602

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

    PubMed

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

    2014-09-01

    A β-glucosidase producing yeast strain was isolated from Korean traditional rice wine. Based on the sequence of the YCL008c gene and analysis of the fatty acid composition, the isolate was identified as Saccharomyces cerevisiae strain HJ-014. S. cerevisiae HJ-014 produced ginsenoside Rd, F2, and compound K from the ethanol extract of red ginseng. The production was increased by shaking culture, where the bioconversion efficiency was increased 2-fold compared to standing culture. The production of ginsenoside F2 and compound K was time-dependent and thought to proceed by the transformation pathway of: red ginseng extract→Rd→F2→compound K. The optimum incubation time and concentration of red ginseng extract for the production of compound K was 96 hr and 4.5% (w/v), respectively. PMID:25346602

  7. Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters.

    PubMed

    Ha, Suk-Jin; Galazka, Jonathan M; Joong Oh, Eun; Kordić, Vesna; Kim, Heejin; Jin, Yong-Su; Cate, Jamie H D

    2013-01-01

    Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular β-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6kJmol(-1)). A thermodynamic "push" from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae. PMID:23178501

  8. Lead sulfide nanoparticles increase cell wall chitin content and induce apoptosis in Saccharomyces cerevisiae.

    PubMed

    Sun, Meiqing; Yu, Qilin; Hu, Mengyuan; Hao, Zhenwei; Zhang, Chengdong; Li, Mingchun

    2014-05-30

    Although there have been numerous studies on bacterial toxicity, the cytotoxicity of nanoparticles toward fungi remains poorly understood. We investigated the toxicity of various sizes of lead sulfide particles against the important model fungus, Saccharomyces cerevisiae. The smallest particle exerted the highest toxicity, inhibiting cell growth and decreasing cell viability, likely reflecting reduced sedimentation and persistent cell wall attack. In response to cell wall stress, S. cerevisiae showed an increase in the cell wall chitin content and the overexpression of FKS2 and PRM5, two genes of the cell wall integrity signaling pathway. Cell wall stress increased the concentration of intracellular reactive oxygen species, leading to mitochondrial dysfunction and cell apoptosis. The contribution of dissolved lead ions to the overall toxicity was negligible. These findings provide the first demonstration of the physiological protective response of a fungus toward nanoparticles, thereby contributing useful information to the assessment of the environmental impact of metal nanoparticles. PMID:24704549

  9. Budding Yeast for Budding Geneticists: A Primer on the Saccharomyces cerevisiae Model System

    PubMed Central

    Duina, Andrea A.; Miller, Mary E.; Keeney, Jill B.

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans. PMID:24807111

  10. A Novel Saccharomyces cerevisiae FG Nucleoporin Mutant Collection for Use in Nuclear Pore Complex Functional Experiments

    PubMed Central

    Adams, Rebecca L.; Terry, Laura J.; Wente, Susan R.

    2015-01-01

    FG nucleoporins (Nups) are the class of proteins that both generate the permeability barrier and mediate selective transport through the nuclear pore complex (NPC). The FG Nup family has 11 members in Saccharomyces cerevisiae, and the study of mutants lacking different FG domains has been instrumental in testing transport models. To continue analyzing the distinct functional roles of FG Nups in vivo, additional robust genetic tools are required. Here, we describe a novel collection of S. cerevisiae mutant strains in which the FG domains of different groups of Nups are absent (Δ) in the greatest number documented to date. Using this plasmid-based ΔFG strategy, we find that a GLFG domain-only pore is sufficient for viability. The resulting extensive plasmid and strain resources are available to the scientific community for future in-depth in vivo studies of NPC transport. PMID:26530420

  11. Biogenic amine accumulation in silver carp sausage inoculated with Lactobacillus plantarum plus Saccharomyces cerevisiae.

    PubMed

    Nie, Xiaohua; Zhang, Qilin; Lin, Shengli

    2014-06-15

    The effect of an amine-negative mixed starter culture (Lactobacillus plantarum ZY40 plus Saccharomyces cerevisiae JM19) on biogenic amine accumulation in fermented silver carp sausage was studied. Microbial counts, pH, titratable acid and free amino acids were also determined. Putrescine, cadaverine and tyramine were the main amines formed during sausage fermentation. The contents of putrescine and cadaverine were greatly reduced by the addition of L. plantarum ZY40 plus S. cerevisiae JM19, whereas tyramine accumulation was enhanced as compared to the control batch. Histamine and spermidine were not affected by the mixed starter culture, and their levels varied slightly throughout the fermentation. Besides, no positive correction between pH, free amino acid content and biogenic amine accumulation were found. PMID:24491750

  12. Metabolic engineering of Saccharomyces cerevisiae ethanol strains PE-2 and CAT-1 for efficient lignocellulosic fermentation.

    PubMed

    Romaní, Aloia; Pereira, Filipa; Johansson, Björn; Domingues, Lucília

    2015-03-01

    In this work, Saccharomyces cerevisiae strains PE-2 and CAT-1, commonly used in the Brazilian fuel ethanol industry, were engineered for xylose fermentation, where the first fermented xylose faster than the latter, but also produced considerable amounts of xylitol. An engineered PE-2 strain (MEC1121) efficiently consumed xylose in presence of inhibitors both in synthetic and corn-cob hydrolysates. Interestingly, the S. cerevisiae MEC1121 consumed xylose and glucose simultaneously, while a CEN.PK based strain consumed glucose and xylose sequentially. Deletion of the aldose reductase GRE3 lowered xylitol production to undetectable levels and increased xylose consumption rate which led to higher final ethanol concentrations. Fermentation of corn-cob hydrolysate using this strain, MEC1133, resulted in an ethanol yield of 0.47 g/g of total sugars which is 92% of the theoretical yield. PMID:25536512

  13. Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae.

    PubMed Central

    Walker-Caprioglio, H M; Parks, L W

    1987-01-01

    Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition. This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde. The effect is duplicated precisely in fresh medium by the addition of acetaldehyde. Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism. Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde. PMID:3548591

  14. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    PubMed

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

    2015-09-01

    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. PMID:26091838

  15. Oxygen requirements for formation and activity of the squalene expoxidase in Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Jahnke, L.; Klein, H. P.

    1983-01-01

    The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the 'mitochondrial' fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the 'microsomal' fraction. At 0.03 percent (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent K sub m for oxygen of 0.38 percent (vol/vol) was determined from a crude particulate preparation for the epoxidase.

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

    PubMed Central

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

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

  17. Cleavage of cruciform DNA structures by an activity from Saccharomyces cerevisiae.

    PubMed Central

    West, S C; Körner, A

    1985-01-01

    Protein extracts from Saccharomyces cerevisiae have been fractionated to reveal a nuclease activity that cleaves cruciform structures in DNA. Negatively supercoiled plasmids that contain inverted repeats that are extruded into cruciform structures have been used as DNA substrates. The sites of cleavage of pColIR215 DNA are located within the extruded cruciform stems and are symmetrically opposed to each other across the cruciform junction. Neither relaxed duplex DNA nor single-stranded DNA serve as substrates. The native molecular weight of the activity was estimated to be approximately equal to 200,000 by gel filtration. Images PMID:3901001

  18. Saccharomyces cerevisiae mutants resistant to catabolite repression: use in cheese whey hydrolysate fermentation

    SciTech Connect

    Bailey, R.B.; Benitez, T.; Woodward, A.

    1982-09-01

    Mutants of an industrial-type strain of Saccharomyces cerevisiae which rapidly and completely fermented equimolar mixtures of glucose and galactose to ethanol were isolated. These mutants fell into two general phenotypic classes based upon their fermentation kinetics and enzyme induction patterns. One class apparently specifically effects the utilization of galactose and allows sequential utilization of first glucose and then galactose in an anaerobic fermentation. The second class of mutants was resistant to general catabolite repression and produced maltase, invertase, and galactokinase in the presence of repressive levels of glucose. These mutants were completely dominant and appear to represent an as yet undescribed class of mutant. (Refs. 23).

  19. Saccharomyces cerevisiae Mutants Resistant to Catabolite Repression: Use in Cheese Whey Hydrolysate Fermentation

    PubMed Central

    Bailey, Richard B.; Benitez, Tahia; Woodward, Anne

    1982-01-01

    Mutants of an industrial-type strain of Saccharomyces cerevisiae which rapidly and completely fermented equimolar mixtures of glucose and galactose to ethanol were isolated. These mutants fell into two general phenotypic classes based upon their fermentation kinetics and enzyme induction patterns. One class apparently specifically effects the utilization of galactose and allows sequential utilization of first glucose and then galactose in an anaerobic fermentation. The second class of mutants was resistant to general catabolite repression and produced maltase, invertase, and galactokinase in the presence of repressive levels of glucose. These mutants were completely dominant and appear to represent an as yet undescribed class of mutant. PMID:16346092

  20. Ordered Restriction Maps of Saccharomyces cerevisiae Chromosomes Constructed by Optical Mapping

    NASA Astrophysics Data System (ADS)

    Schwartz, David C.; Li, Xiaojun; Hernandez, Luis I.; Ramnarain, Satyadarshan P.; Huff, Edward J.; Wang, Yu-Ker

    1993-10-01

    A light microscope-based technique for rapidly constructing ordered physical maps of chromosomes has been developed. Restriction enzyme digestion of elongated individual DNA molecules (about 0.2 to 1.0 megabases in size) was imaged by fluorescence microscopy after fixation in agarose gel. The size of the resulting individual restriction fragments was determined by relative fluorescence intensity and apparent molecular contour length. Ordered restriction maps were then created from genomic DNA without reliance on cloned or amplified sequences for hybridization or analytical gel electrophoresis. Initial application of optical mapping is described for Saccharomyces cerevisiae chromosomes.

  1. Improvement of a Wine Saccharomyces cerevisiae Strain by a Breeding Program

    PubMed Central

    Romano, Patrizia; Soli, M. Grazia; Suzzi, Giovanna; Grazia, Luigi; Zambonelli, Carlo

    1985-01-01

    Hybridization by spore conjugation was used to develop new and improved wine yeasts of Saccharomyces cerevisiae. The procedure was achieved with diploid, homothallic strains with high sporulation frequency and high spore viability. The method was verified by crossing flocculent and non-H2S-forming strains. Single-spore descendants of the hybrids were studied by tetrad analysis with regard to the aforementioned characters and the other two winemaking traits, i.e., ethanol production and fermentation rate. A highly flocculent, non-H2S-forming wine yeast strain with a high fermentation rate and high ethanol production was obtained. PMID:16346903

  2. The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains.

    PubMed Central

    Duncan, K; Edwards, R M; Coggins, J R

    1987-01-01

    The nucleotide sequence of the Saccharomyces cerevisiae ARO1 gene which encodes the arom multifunctional enzyme has been determined. The protein sequence deduced for the pentafunctional arom polypeptide is 1588 amino acids in length and has a calculated Mr of 174555. Functional regions within the polypeptide chain have been identified by comparison with the sequences of the five monofunctional Escherichia coli enzymes whose activities correspond with those of the arom multifunctional enzyme. The observed homologies demonstrate that the arom polypeptide is a mosaic of functional domains and are consistent with the hypothesis that the ARO1 gene evolved by the linking of ancestral E. coli-like genes. PMID:2825635

  3. Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Yamashita, I; Fukui, S

    1985-01-01

    In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores. Images PMID:3939312

  4. Evidence for control of nitrogen metabolism by a START-dependent mechanism in Saccharomyces cerevisiae.

    PubMed

    Bryan, B A; McGrew, E; Lu, Y; Polymenis, M

    2004-02-01

    It is generally thought that cell growth and metabolism regulate cell division and not vice versa. Here, we examined Saccharomyces cerevisiae cells growing under conditions of continuous culture in a chemostat. We found that loss of G1 cyclins, or inactivation of the cyclin-dependent kinase Cdc28p, reduced the activity of glutamate synthase (Glt1p), a key enzyme in nitrogen assimilation. We also present evidence indicating that the G1 cyclin-dependent control of Glt1p may involve Jem1p, a DnaJ-type chaperone. Our results suggest that completion of START may be linked to nitrogen metabolism. PMID:14648201

  5. High-resolution analysis of condition-specific regulatory modules in Saccharomyces cerevisiae

    PubMed Central

    Lee, Hun-Goo; Lee, Hyo-Soo; Jeon, Sang-Hoon; Chung, Tae-Hoon; Lim, Young-Sung; Huh, Won-Ki

    2008-01-01

    We present an approach for identifying condition-specific regulatory modules by using separate units of gene expression profiles along with ChIP-chip and motif data from Saccharomyces cerevisiae. By investigating the unique and common features of the obtained condition-specific modules, we detected several important properties of transcriptional network reorganization. Our approach reveals the functionally distinct coregulated submodules embedded in a coexpressed gene module and provides an effective method for identifying various condition-specific regulatory events at high resolution. PMID:18171483

  6. Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Mortimer, R.K.; Contopoulou, R.; Schild, D.

    1981-09-01

    Cells of Saccharomyces cerevisiae with mutations in the RAD52 gene have previously been shown to be defective in meiotic and mitotic recombination, in sporulation, and in repair of radiation-induced damage to DNA. In this study we show that diploid cells homozygous for rad52 lose chromosomes at high frequencies and that these frequencies of loss can be increased dramatically by exposure of these cells to x-rays. Genetic analyses of survivors of x-ray treatment demonstrate that chromosome loss events result in the conversion of diploid cells to cells with near haploid chromosome numbers.

  7. X-Ray Absorption Spectroscopy of Cuprous-Thiolate Clusters in Saccharomyces Cerevisiae Metallothionein

    SciTech Connect

    Zhang, L.; Pickering, I.J.; Winge, D.R.; George, G.N.

    2009-05-28

    Copper (Cu) metallothioneins are cuprous-thiolate proteins that contain multimetallic clusters, and are thought to have dual functions of Cu storage and Cu detoxification. We have used a combination of X-ray absorption spectroscopy (XAS) and density-functional theory (DFT) to investigate the nature of Cu binding to Saccharomyces cerevisiae metallothionein. We found that the XAS of metallothionein prepared, containing a full complement of Cu, was quantitatively consistent with the crystal structure, and that reconstitution of the apo-metallothionein with stoichiometric Cu results in the formation of a tetracopper cluster, indicating cooperative binding of the Cu ions by the metallothionein.

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

    NASA Astrophysics Data System (ADS)

    Safarik, Ivo; Sabatkova, Zdenka; Safarikova, Mirka

    2009-05-01

    Invert sugar (an equimolar mixture of glucose and fructose prepared by sucrose hydrolysis) is a very important food component. We have prepared magnetically responsive alginate microbeads containing entrapped Saccharomyces cerevisiae cells and magnetite microparticles which can be easily separated in an appropriate magnetic separator. The microbeads (typical diameter between 50 and 100 μm) were prepared using the water-in-oil emulsification process. The prepared microbeads containing yeast cells with invertase activity enabled efficient sucrose conversion. The biocatalyst was quite stable; the same catalytic activity was observed after one month storage at 4 °C and the microbeads could be used at least six times.

  9. Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

    PubMed Central

    Montiel, J F; Norbury, C J; Tuite, M F; Dobson, M J; Mills, J S; Kingsman, A J; Kingsman, S M

    1984-01-01

    We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence. PMID:6320114

  10. A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids.

    PubMed Central

    Kikuchi, Y; Toh-e, A

    1986-01-01

    We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication. Images PMID:3025627

  11. Induction of "General Control" and thermotolerance in cdc mutants of Saccharomyces cerevisiae.

    PubMed

    Messenguy, F; Scherens, B

    1990-11-01

    In Saccharomyces cerevisiae starvation for a single amino acid activates the transcription of a set of genes belonging to different amino acid biosynthetic pathways (General Control, GC). We show that mutants affected in GC regulation are also affected in their response to thermal stress. Moreover, growth conditions that are known to induce heat shock proteins induce the GC response. However, unlike heat shock proteins, the transcriptional activator of GC, GCN4, is not induced after a short exposure to heat, and in gcn mutant strains induction of heat resistance is normal. PMID:2277643

  12. Oxygen requirements for formation and activity of the squalene epoxidase in Saccharomyces cerevisiae.

    PubMed Central

    Jahnke, L; Klein, H P

    1983-01-01

    The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the "mitochondrial" fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the "microsomal" fraction. At 0.03% (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent Km for oxygen of 0.38% (vol/vol) was determined from a crude particulate preparation for the epoxidase. PMID:6348021

  13. Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae

    SciTech Connect

    Walker-Caprioglio, H.M.; Parks, L.W.

    1987-01-01

    Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition. This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde. The effect is duplicated precisely in fresh medium by the addition of acetaldehyde. Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism. Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde.

  14. Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae.

    PubMed

    Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

    2015-01-01

    Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved. PMID:26588105

  15. Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae

    PubMed Central

    Chemler, Joseph A; Yan, Yajun; Koffas, Mattheos AG

    2006-01-01

    Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties. PMID:16719921

  16. Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae.

    PubMed Central

    Trabalzini, Lorenza; Paffetti, Alessandro; Scaloni, Andrea; Talamo, Fabio; Ferro, Elisa; Coratza, Grazietta; Bovalini, Lucia; Lusini, Paola; Martelli, Paola; Santucci, Annalisa

    2003-01-01

    We report a study on the adaptive response of a wild-type wine Saccharomyces cerevisiae strain, isolated from natural spontaneous grape must, to mild and progressive physiological stresses due to fermentation. We observed by two-dimensional electrophoresis how the yeast proteome changes during glucose exhaustion, before the cell enters its complete stationary phase. On the basis of their identification, the proteins representing the S. cerevisiae proteomic response to fermentation stresses were divided into three classes: repressed proteins, induced proteins and autoproteolysed proteins. In an overall view, the proteome adaptation of S. cerevisiae at the time of glucose exhaustion seems to be directed mainly against the effects of ethanol, causing both hyperosmolarity and oxidative responses. Stress-induced autoproteolysis is directed mainly towards specific isoforms of glycolytic enzymes. Through the use of a wild-type S. cerevisiae strain and PMSF, a specific inhibitor of vacuolar proteinase B, we could also distinguish the specific contributions of the vacuole and the proteasome to the autoproteolytic process. PMID:12401115

  17. Effects of Saccharomyces cerevisiae on survival rate and growth performance of Convict Cichlid (Amatitlania nigrofasciata)

    PubMed Central

    Mohammadi, F; Mousavi, S. M.; Ahmadmoradi, E.; Zakeri, M.; Jahedi, A.

    2015-01-01

    Using probiotics can control pathogens by a variety of mechanisms. Probiotics can promote growth performance and have, therefore, become increasingly important in the aquaculture industry. Convict Cichlid belongs to the family of Cichlidae and is known for its rapid development in laboratory conditions and is suitable for behavioral examinations. The aim of this study was to evaluate the effects of Saccharomyces cerevisiae on growth performance, survival rate and body composition of Convict Cichlids (Amatitlania nigrofasciata). One hundred sixty eight Convict Cichlids (mean weight: 2.1 ± 0.12 g and mean length: 2.2 ± 0.05 cm) were fed by commercial diets with different concentrations of S. cerevisiae (0, 0.5%, 1%, 2%). At the end of the experiment, survival rate and growth indices were measured. Based on the results, growth performance significantly increased with probiotic, S. cerevisiae, specially, at the 2% probiotic level of concentration. In the present study, the best FCR (feed conversion rate), SGR (specific growth rate), CF (condition factor) and BWG (body weight gain) values were observed in a 2% concentration of S. cerevisiae. The results suggest that this yeast could improve feed utilization in this fish species.

  18. Proteomic Evaluation of Cellular Responses of Saccharomyces cerevisiae to Formic Acid Stress

    PubMed Central

    Lee, Sung-Eun; Park, Byeoung-Soo

    2010-01-01

    Formic acid is a representative carboxylic acid that inhibits bacterial cell growth, and thus it is generally considered to constitute an obstacle to the reuse of renewable biomass. In this study, Saccharomyces cerevisiae was used to elucidate changes in protein levels in response to formic acid. Fifty-seven differentially expressed proteins in response to formic acid toxicity in S. cerevisiae were identified by 1D-PAGE and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analyses. Among the 28 proteins increased in expression, four were involved in the MAP kinase signal transduction pathway and one in the oxidative stress-induced pathway. A dramatic increase was observed in the number of ion transporters related to maintenance of acid-base balance. Regarding the 29 proteins decreased in expression, they were found to participate in transcription during cell division. Heat shock protein 70, glutathione reductase, and cytochrome c oxidase were measured by LC-MS/MS analysis. Taken together, the inhibitory action of formic acid on S. cerevisiae cells might disrupt the acid-base balance across the cell membrane and generate oxidative stress, leading to repressed cell division and death. S. cerevisiae also induced expression of ion transporters, which may be required to maintain the acid-base balance when yeast cells are exposed to high concentrations of formic acid in growth medium. PMID:23956670

  19. Genomic structural variation contributes to phenotypic change of industrial bioethanol yeast Saccharomyces cerevisiae.

    PubMed

    Zhang, Ke; Zhang, Li-Jie; Fang, Ya-Hong; Jin, Xin-Na; Qi, Lei; Wu, Xue-Chang; Zheng, Dao-Qiong

    2016-03-01

    Genomic structural variation (GSV) is a ubiquitous phenomenon observed in the genomes of Saccharomyces cerevisiae strains with different genetic backgrounds; however, the physiological and phenotypic effects of GSV are not well understood. Here, we first revealed the genetic characteristics of a widely used industrial S. cerevisiae strain, ZTW1, by whole genome sequencing. ZTW1 was identified as an aneuploidy strain and a large-scale GSV was observed in the ZTW1 genome compared with the genome of a diploid strain YJS329. These GSV events led to copy number variations (CNVs) in many chromosomal segments as well as one whole chromosome in the ZTW1 genome. Changes in the DNA dosage of certain functional genes directly affected their expression levels and the resultant ZTW1 phenotypes. Moreover, CNVs of large chromosomal regions triggered an aneuploidy stress in ZTW1. This stress decreased the proliferation ability and tolerance of ZTW1 to various stresses, while aneuploidy response stress may also provide some benefits to the fermentation performance of the yeast, including increased fermentation rates and decreased byproduct generation. This work reveals genomic characters of the bioethanol S. cerevisiae strain ZTW1 and suggests that GSV is an important kind of mutation that changes the traits of industrial S. cerevisiae strains. PMID:26733503

  20. [Surface display of phytase on Saccharomyces cerevisiae for efficient bioethanol production from corn starch].

    PubMed

    Xiao, Yan; Chen, Xianzhong; Shen, Wei; Yang, Haiquan; Fan, You

    2015-12-01

    Production of bioethanol using starch as raw material has become a very prominent technology. However, phytate in the raw material not only decreases ethanol production efficiency, but also increases phosphorus discharge. In this study, to decrease phytate content in an ethanol fermentationprocess, Saccharomyces cerevisiae was engineered forheterologous expression of phytase on the cell surface. The phy gene encoding phytase gene was fused with the C-terminal-half region of α-agglutinin and then inserted downstream of the secretion signal gene, to produce a yeast surface-display expression vector pMGK-AG-phy, which was then transformed into S. cerevisiae. The recombinant yeast strain, PHY, successfully displayed phytase on the surface of cells producing 6.4 U/g wet cells and its properties were further characterized. The growthrate and ethanol production of the PHY strain were faster than the parent S. cerevisiae strain in the fermentation medium by simultaneous saccharification and fermentation. Moreover, the phytate concentration decreased by 91% in dry vinasse compared to the control. In summary, we constructed recombinant S. cerevisiae strain displaying phytase on the cell surface, which could effectively reduce the content of phytate, improve the utilization value of vinasse and reduce the discharge of phosphorus. The strain reported here represents a useful novel engineering platform for developing an environment-friendly system for bioethanol production from a corn substrate. PMID:27093833

  1. Identification of Novel Knockout Targets for Improving Terpenoids Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Li, Jing; Wang, Jianfeng; Li, Qian; Wang, Yong; Zhang, Yansheng

    2014-01-01

    Many terpenoids have important pharmacological activity and commercial value; however, application of these terpenoids is often limited by problems associated with the production of sufficient amounts of these molecules. The use of Saccharomyces cerevisiae (S. cerevisiae) for the production of heterologous terpenoids has achieved some success. The objective of this study was to identify S. cerevisiae knockout targets for improving the synthesis of heterologous terpeniods. On the basis of computational analysis of the S. cerevisiae metabolic network, we identified the knockout sites with the potential to promote terpenoid production and the corresponding single mutant was constructed by molecular manipulations. The growth rates of these strains were measured and the results indicated that the gene deletion had no adverse effects. Using the expression of amorphadiene biosynthesis as a testing model, the gene deletion was assessed for its effect on the production of exogenous terpenoids. The results showed that the dysfunction of most genes led to increased production of amorphadiene. The yield of amorphadiene produced by most single mutants was 8–10-fold greater compared to the wild type, indicating that the knockout sites can be engineered to promote the synthesis of exogenous terpenoids. PMID:25386654

  2. Probiotic Saccharomyces cerevisiae strains as biotherapeutic tools: is there room for improvement?

    PubMed

    Palma, Mariana L; Zamith-Miranda, Daniel; Martins, Flaviano S; Bozza, Fernando A; Nimrichter, Leonardo; Montero-Lomeli, Mônica; Marques, Ernesto T A; Douradinha, Bruno

    2015-08-01

    The probiotic yeast Saccharomyces cerevisiae var boulardii is widely used as a low cost and efficient adjuvant against gastrointestinal tract disorders such as inflammatory bowel disease and treatment of several types of diarrhea, both in humans and animals. S. boulardii exerts its protective mechanisms by binding and neutralizing enteric pathogens or their toxins, by reducing inflammation and by inducing the secretion of sIgA. Although several S. cerevisiae strains have proven probiotic potential in both humans and animals, only S. boulardii is currently licensed for use in humans. Recently, some researchers started using S. boulardii as heterologous protein expression systems. Combined with their probiotic activity, the use of these strains as prophylactic and therapeutic proteins carriers might result in a positive combined effort to fight specific diseases. Here, we provide an overview of the current use of S. cerevisiae strains as probiotics and their mechanisms of action. We also discuss their potential to produce molecules with biotherapeutic application and the advantages and hurdles of this approach. Finally, we suggest future directions and alternatives for which the combined effort of specific immunomodulatory effects of probiotic S. cerevisiae strains and ability to express desired foreign genes would find a practical application. PMID:26142388

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-03-01

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

  5. Novel image cytometric method for detection of physiological and metabolic changes in Saccharomyces cerevisiae.

    PubMed

    Chan, Leo L; Kury, Alexandria; Wilkinson, Alisha; Berkes, Charlotte; Pirani, Alnoor

    2012-11-01

    The studying and monitoring of physiological and metabolic changes in Saccharomyces cerevisiae (S. cerevisiae) has been a key research area for the brewing, baking, and biofuels industries, which rely on these economically important yeasts to produce their products. Specifically for breweries, physiological and metabolic parameters such as viability, vitality, glycogen, neutral lipid, and trehalose content can be measured to better understand the status of S. cerevisiae during fermentation. Traditionally, these physiological and metabolic changes can be qualitatively observed using fluorescence microscopy or flow cytometry for quantitative fluorescence analysis of fluorescently labeled cellular components associated with each parameter. However, both methods pose known challenges to the end-users. Specifically, conventional fluorescent microscopes lack automation and fluorescence analysis capabilities to quantitatively analyze large numbers of cells. Although flow cytometry is suitable for quantitative analysis of tens of thousands of fluorescently labeled cells, the instruments require a considerable amount of maintenance, highly trained technicians, and the system is relatively expensive to both purchase and maintain. In this work, we demonstrate the first use of Cellometer Vision for the kinetic detection and analysis of vitality, glycogen, neutral lipid, and trehalose content of S. cerevisiae. This method provides an important research tool for large and small breweries to study and monitor these physiological behaviors during production, which can improve fermentation conditions to produce consistent and higher-quality products. PMID:22864608

  6. Phenotypic Landscape of Saccharomyces cerevisiae during Wine Fermentation: Evidence for Origin-Dependent Metabolic Traits

    PubMed Central

    Camarasa, Carole; Sanchez, Isabelle; Brial, Pascale; Bigey, Frédéric; Dequin, Sylvie

    2011-01-01

    The species Saccharomyces cerevisiae includes natural strains, clinical isolates, and a large number of strains used in human activities. The aim of this work was to investigate how the adaptation to a broad range of ecological niches may have selectively shaped the yeast metabolic network to generate specific phenotypes. Using 72 S. cerevisiae strains collected from various sources, we provide, for the first time, a population-scale picture of the fermentative metabolic traits found in the S. cerevisiae species under wine making conditions. Considerable phenotypic variation was found suggesting that this yeast employs diverse metabolic strategies to face environmental constraints. Several groups of strains can be distinguished from the entire population on the basis of specific traits. Strains accustomed to growing in the presence of high sugar concentrations, such as wine yeasts and strains obtained from fruits, were able to achieve fermentation, whereas natural yeasts isolated from “poor-sugar” environments, such as oak trees or plants, were not. Commercial wine yeasts clearly appeared as a subset of vineyard isolates, and were mainly differentiated by their fermentative performances as well as their low acetate production. Overall, the emergence of the origin-dependent properties of the strains provides evidence for a phenotypic evolution driven by environmental constraints and/or human selection within S. cerevisiae. PMID:21949874

  7. Chromosomal rearrangements as a major mechanism in the onset of reproductive isolation in Saccharomyces cerevisiae.

    PubMed

    Hou, Jing; Friedrich, Anne; de Montigny, Jacky; Schacherer, Joseph

    2014-05-19

    Understanding the molecular basis of how reproductive isolation evolves between individuals from the same species offers valuable insight into patterns of genetic differentiation as well as the onset of speciation [1, 2]. The yeast Saccharomyces cerevisiae constitutes an ideal model partly due to its vast ecological range, high level of genetic diversity [3-6], and laboratory-amendable sexual reproduction. Between S. cerevisiae and its sibling species in the Saccharomyces sensu stricto complex, reproductive isolation acts postzygotically and could be attributed to chromosomal rearrangements [7], cytonuclear incompatibility [8, 9], and antirecombination [10, 11], although the implication of these mechanisms at the incipient stage of speciation remains unclear due to further divergence in the nascent species. Recently, several studies assessed the onset of intraspecific reproductive isolation in S. cerevisiae by evaluating the effect of the mismatch repair system [12-14] or by fostering incipient speciation using the same initial genetic background [15-18]. Nevertheless, the overall genetic diversity within this species was largely overlooked, and no systematic evaluation has been performed. Here, we carried out the first species-wide survey for postzygotic reproductive isolation in S. cerevisiae. We crossed 60 natural isolates sampled from diverse niches with the reference strain S288c and identified 16 cases of reproductive isolation with reduced offspring viabilities ranging from 44% to 86%. Using different mapping strategies, we identified reciprocal translocations in a large fraction of all isolates surveyed, indicating that large-scale chromosomal rearrangements might play a major role in the onset of reproductive isolation in this species. PMID:24814147

  8. Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae

    SciTech Connect

    Kobayashi, N.; McEntee, K. )

    1993-01-01

    In Saccharomyces cerevisiae, the stress-responsive gene DDR2 is transcribed at elevated levels following heat shock or DNA damage. A pentanucleotide, CCCCT, has been identified as an essential component of the stress response sequence. This research demonstrates the ability of oligonucleotides containing CCCCT sequence to confer heat shock inducibility on the report gene and show that the presence of two such elements produces more than additive effects on induction. It also identify key cis- and transacting components of a novel heat shock stress response pathway in Saccharomyces cerevisiae.

  9. Impact of Acute Metal Stress in Saccharomyces cerevisiae

    PubMed Central

    Lichtenberg-Frat, Hella; Glaser, Walter; Schller, Christoph; Klipp, Edda

    2014-01-01

    Although considered as essential cofactors for a variety of enzymatic reactions and for important structural and functional roles in cell metabolism, metals at high concentrations are potent toxic pollutants and pose complex biochemical problems for cells. We report results of single dose acute toxicity testing in the model organism S. cerevisiae. The effects of moderate toxic concentrations of 10 different human health relevant metals, Ag+, Al3+, As3+, Cd2+, Co2+, Hg2+, Mn2+, Ni2+, V3+, and Zn2+, following short-term exposure were analyzed by transcription profiling to provide the identification of early-on target genes or pathways. In contrast to common acute toxicity tests where defined endpoints are monitored we focused on the entire genomic response. We provide evidence that the induction of central elements of the oxidative stress response by the majority of investigated metals is the basic detoxification process against short-term metal exposure. General detoxification mechanisms also comprised the induction of genes coding for chaperones and those for chelation of metal ions via siderophores and amino acids. Hierarchical clustering, transcription factor analyses, and gene ontology data further revealed activation of genes involved in metal-specific protein catabolism along with repression of growth-related processes such as protein synthesis. Metal ion group specific differences in the expression responses with shared transcriptional regulators for both, up-regulation and repression were also observed. Additionally, some processes unique for individual metals were evident as well. In view of current concerns regarding environmental pollution our results may support ongoing attempts to develop methods to monitor potentially hazardous areas or liquids and to establish standardized tests using suitable eukaryotic a model organism. PMID:24416162

  10. Screening of optimal cellulases from symbiotic protists of termites through expression in the secretory pathway of Saccharomyces cerevisiae.

    PubMed

    Todaka, Nemuri; Nakamura, Risa; Moriya, Sigeharu; Ohkuma, Moriya; Kudo, Toshiaki; Takahashi, Haruo; Ishida, Nobuhiro

    2011-01-01

    For direct and efficient ethanol production from cellulosic materials, we screened optimal cellulases from symbiotic protists of termites through heterologous expression with Saccharomyces cerevisiae. 11 cellulases, belonging to glycoside hydrolase families 5, 7, and 45 endoglucanases (EGs), were confirmed to produce with S. cerevisiae for the first time. A recombinant yeast expressing SM2042B24 EG I was more efficient at degrading carboxylmethyl cellulose than was Trichoderma reesei EG I, a major EG with high cellulolytic activity. PMID:22056456

  11. Mediated electrochemical measurement of the inhibitory effects of furfural and acetic acid on Saccharomyces cerevisiae and Candida shehatae.

    PubMed

    Zhao, Jinsheng; Wang, Min; Yang, Zhenyu; Gong, Qintao; Lu, Yao; Yang, Zhengyu

    2005-02-01

    The toxic effects of furfural and acetic acid on two yeasts, Saccharomyces cerevisiae and Candida shehatae, were evaluated using an electrochemical method. Intracellular redox activities were lowered by 40% and 78% for S. cerevisiae and C. shehatae, respectively, by 8 g furfural l(-1), and by 46% and 67%, respectively, by 8 g acetic acid l(-1). The proposed method can accurately measure the effects of inhibitors on cell cultures. PMID:15717131

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

    PubMed

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

    2009-09-22

    Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations. PMID:19805302

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

    PubMed Central

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

    2009-01-01

    Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations. PMID:19805302

  14. The peroxisomal lumen in Saccharomyces cerevisiae is alkaline.

    PubMed

    van Roermund, Carlo W T; de Jong, Mark; IJlst, Lodewijk; van Marle, Jan; Dansen, Tobias B; Wanders, Ronald J A; Waterham, Hans R

    2004-08-15

    Peroxisomes have a central function in lipid metabolism, including the beta-oxidation of various fatty acids. The products and substrates involved in the beta-oxidation have to cross the peroxisomal membrane, which previously has been demonstrated to constitute a closed barrier, implying the existence of specific transport mechanisms. Fatty acid transport across the yeast peroxisomal membrane may follow two routes: one for activated fatty acids, dependent on the peroxisomal ABC half transporter proteins Pxa1p and Pxa2p, and one for free fatty acids, which depends on the peroxisomal acyl-CoA synthetase Faa2p and the ATP transporter Ant1p. A proton gradient across the peroxisomal membrane as part of a proton motive force has been proposed to be required for proper peroxisomal function, but the nature of the peroxisomal pH has remained inconclusive and little is known about its generation. To determine the pH of Sacharomyces cerevisiae peroxisomes in vivo, we have used two different pH-sensitive yellow fluorescent proteins targeted to the peroxisome by virtue of a C-terminal SKL and found the peroxisomal matrix in wild-type cells to be alkaline (pH(per) 8.2), while the cytosolic pH was neutral (pH(cyt) 7.0). No Delta pH was present in ant1 Delta cells, indicating that the peroxisomal pH is regulated in an ATP-dependent way and suggesting that Ant1p activity is directly involved in maintenance of the peroxisomal pH. Moreover, we found a high peroxisomal pH of >8.6 in faa2 Delta cells, while the peroxisomal pH remained 8.1+/-0.2 in pxa2 Delta cells. Our combined results suggest that the proton gradient across the peroxisomal membrane is dependent on Ant1p activity and required for the beta-oxidation of medium chain fatty acids. PMID:15316083

  15. Metabolomic analysis of antimicrobial mechanisms of ε-poly-L-lysine on Saccharomyces cerevisiae.

    PubMed

    Bo, Tao; Liu, Miao; Zhong, Cheng; Zhang, Qian; Su, Qin-Zhi; Tan, Zhi-Lei; Han, Pei-Pei; Jia, Shi-Ru

    2014-05-14

    ε-Poly-L-lysine (ε-PL), a naturally occurring amino acid homopolymer, has been widely used as a food preservative. However, its antimicrobial mechanism has not been fully understood. This study investigated the antimicrobial mode of action of ε-PL on a yeast, Saccharomyces cerevisiae. When treated with ε-PL at the concentration of 500 μg/mL, cell mortality was close to 100% and the phospholipid bilayer curvature, pores, and micelles on the surface of S. cerevisiae were clearly observed by scanning electron microscopy (SEM). At the level of 200 μg/mL, ε-PL significantly inhibited the cell growth of S. cerevisiae. When treated with 50 μg/mL ε-PL, the yeast cell was able to grow but the cell cycle was prolonged. A significant increase in cell membrane permeability was induced by ε-PL at higher concentrations. Metabolomics analysis revealed that the ε-PL stress led to the inhibition of primary metabolic pathways through the suppression of the tricarboxylic acid cycle and glycolysis. It is therefore proposed that the microbiostatic effect of ε-PL at lower levels on S. cerevisiae is achieved by inducing intracellular metabolic imbalance via disruption of cell membrane functions. Moreover, the results suggested that the antimicrobial mechanism of ε-PL on S. cerevisiae can in fact change from microbiostatic to microbicidal when the concentration of ε-PL increased, and the mechanisms of these two modes of action were completely different. PMID:24735012

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

    PubMed Central

    Salma, Mohammad; Rousseaux, Sandrine; Sequeira-Le Grand, Anabelle; Divol, Benoit; Alexandre, Hervé

    2013-01-01

    The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to “resuscitate”. The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO2-induced VBNC state in S. cerevisiae and that the stress removal allows the “resuscitation” of VBNC cells during the VBNC state. PMID:24204887

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

    PubMed

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

    2014-10-01

    Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments. PMID:24318365

  18. Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation.

    PubMed

    Dong, Shi-Jun; Yi, Chen-Feng; Li, Hao

    2015-12-01

    During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C18:1) but lower levels of hexadecanoic (C16:0) and palmitelaidic (C16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance. PMID:26515124

  19. DNA sequence and functional analysis of homologous ARS elements of Saccharomyces cerevisiae and S. carlsbergensis.

    PubMed Central

    Theis, J F; Yang, C; Schaefer, C B; Newlon, C S

    1999-01-01

    ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication. Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation. We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S. cerevisiae and S. carlsbergensis. In all but one case, the ARS308-ARS308(carl) pair, significant blocks of homology were detected. In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S. carlsbergensis homologs. Mutation of the conserved sequences in the S. carlsbergensis ARS elements revealed that the homologous sequences are required for function. These observations suggested that the sequences important for ARS function would be conserved in other ARS elements. Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310(carl), though not of ARS310. PMID:10388814

  20. Determination of biological activity from fluorescence-lifetime measurements in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Rudek, F.; Baselt, T.; Lempe, B.; Taudt, C.; Hartmann, P.

    2015-03-01

    The importance of fluorescence lifetime measurement as an optical analysis tool is growing. Many applications already exist in order to determine the fluorescence lifetime, but the majority of these require the addition of fluorescence-active substances to enable measurements. Every usage of such foreign materials has an associated risk. This paper investigates the use of auto-fluorescing substances in Saccharomyces cerevisiae (Baker's yeast) as a risk free alternative to fluorescence-active substance enabled measurements. The experimental setup uses a nitrogen laser with a pulse length of 350 ps and a wavelength of 337 nm. The excited sample emits light due to fluorescence of NADH/NADPH and collagen. A fast photodiode collects the light at the output of an appropriate high-pass edge-filter at 400 nm. Fluorescence lifetimes can be determined from the decay of the measurement signals, which in turn characterizes the individual materials and their surrounding environment. Information about the quantity of the fluorescence active substances can also be measured based on the received signal intensity. The correlation between the fluorescence lifetime and the metabolic state of Saccharomyces cerevisiae was investigated and is presented here.

  1. Comparison of Fermentation and Wines Produced by Inoculation of Hanseniaspora vineae and Saccharomyces cerevisiae.

    PubMed

    Lleixà, Jessica; Martín, Valentina; Portillo, María Del C; Carrau, Francisco; Beltran, Gemma; Mas, Albert

    2016-01-01

    Interest in the use of non-Saccharomyces yeasts in winemaking has been increasing due to their positive contributions to wine quality. The non-Saccharomyces yeast Hanseniaspora vineae is an apiculate yeast that has been associated with the production of wine with good aromatic properties. However, little is known about the fermentation dynamics of H. vineae in natural must and its interaction with autochthonous yeasts. In the present study, we performed semi industrial fermentations of Macabeo and Merlot musts inoculated with either H. vineae or S. cerevisiae. The yeast population dynamics were monitored by plate culturing, PCR-DGGE and massive sequencing techniques. The results obtained with these techniques show that H. vineae was able dominate the autochthonous microbiota in Macabeo must but not in Merlot must, which exhibited a larger, more diverse yeast population. The presence of H. vineae throughout most of the Macabeo fermentation resulted in more fruity and flowery wine, as indicated by the chemical analysis of the final wines, which demonstrated a strong presence of phenyl ethyl acetate at concentrations higher than the threshold of perception and approximately 50 times more than that produced in wines fermented with S. cerevisiae. This compound is associated with fruity, floral and honey aromas. PMID:27014252

  2. New aspects of Saccharomyces cerevisiae as a novel carrier for berberine

    PubMed Central

    2013-01-01

    Background Berberine was encapsulated in yeast cells of Saccharomyces cerevisiae as novel carriers to be used in different food and drug industries. The microcapsules were characterized by differential scanning calorimetry (DSC), fourier transform infra red spectroscopy (FT-IR) and fluorescence microscopy. The encapsulation factors such as plasmolysis of yeast cells which affects the % encapsulation yield were studied. Results Fluorescence microscopy showed the yeast cells became fluorescent after encapsulation process. DSC diagram was representing of new peak for microcapsule which was not the same as berberine and the empty yeast cells peaks, separately. FTIR spectrums of microcapsules and yeast cells were almost the same. The plasmolysed and non plasmolysed microcapsules were loaded with berberine up to about 40.2 ± 0.2% w/w. Conclusion Analytical methods proved that berberine was encapsulated in the yeast cells. Fluorescence microscopy and FTIR results showed the entrance of berberine inside the yeasts. DSC diagram indicated the appearance of new peak which is due to the synthesis of new product. Although plasmolysis caused changes in yeast cell structure and properties, it did not enhance berberine loading in the cells. The results confirmed that Saccharomyces cerevisiae could be an efficient and safe carrier for active materials. PMID:24359687

  3. RNAi-assisted genome evolution in Saccharomyces cerevisiae for complex phenotype engineering.

    PubMed

    Si, Tong; Luo, Yunzi; Bao, Zehua; Zhao, Huimin

    2015-03-20

    A fundamental challenge in basic and applied biology is to reprogram cells with improved or novel traits on a genomic scale. However, the current ability to reprogram a cell on the genome scale is limited to bacterial cells. Here, we report RNA interference (RNAi)-assisted genome evolution (RAGE) as a generally applicable method for genome-scale engineering in the yeast Saccharomyces cerevisiae. Through iterative cycles of creating a library of RNAi induced reduction-of-function mutants coupled with high throughput screening or selection, RAGE can continuously improve target trait(s) by accumulating multiplex beneficial genetic modifications in an evolving yeast genome. To validate the RNAi library constructed with yeast genomic DNA and convergent-promoter expression cassette, we demonstrated RNAi screening in Saccharomyces cerevisiae for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation yku70Δ. We then showed the application of RAGE for improved acetic acid tolerance, a key trait for microbial production of chemicals and fuels. Three rounds of iterative RNAi screening led to the identification of three gene knockdown targets that acted synergistically to confer an engineered yeast strain with substantially improved acetic acid tolerance. RAGE should greatly accelerate the design and evolution of organisms with desired traits and provide new insights on genome structure, function, and evolution. PMID:24758359

  4. Glucose uptake kinetics of Saccharomyces cerevisiae monitored with a newly developed FIA.

    PubMed

    Rothen, S A; Saner, M; Meenakshisundaram, S; Sonnleitner, B; Fiechter, A

    1996-09-13

    The glucose content of the culture liquid during shift experiments and synchronized cultures of Saccharomyces cerevisiae H1022 (ATCC 32167) was monitored using a greatly improved and highly precise FIA. During shift-up experiments on the dilution rate, an overshoot of the glucose-concentration was observed. The amplitude of the overshoot showed a dependency on the duration of undisturbed cultivation before application of the shift. Mutarotational non-equilibrium was excluded as the cause of the observed overshoot. For the first time glucose measurements of oscillating cultures of Saccharomyces cerevisiae are demonstrated with high accuracy and reproducibility. The data strongly support the proposals by Münch et al. (1992a, b) that faint oscillations in glucose concentration are responsible for the persistence of the synchronization. Analytical subsystems prove to be a powerful tool for investigation of the dynamics of metabolic pathways of microbial organisms. Accurate glucose measurements at low concentrations point out the limits and allow refinements of commonly used models. PMID:8987846

  5. High-level production of animal-free recombinant transferrin from saccharomyces cerevisiae

    PubMed Central

    2010-01-01

    Background Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin. Results The first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 μm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin. Conclusions A Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics. PMID:21083917

  6. Comparison of Fermentation and Wines Produced by Inoculation of Hanseniaspora vineae and Saccharomyces cerevisiae

    PubMed Central

    Lleixà, Jessica; Martín, Valentina; Portillo, María del C.; Carrau, Francisco; Beltran, Gemma; Mas, Albert

    2016-01-01

    Interest in the use of non-Saccharomyces yeasts in winemaking has been increasing due to their positive contributions to wine quality. The non-Saccharomyces yeast Hanseniaspora vineae is an apiculate yeast that has been associated with the production of wine with good aromatic properties. However, little is known about the fermentation dynamics of H. vineae in natural must and its interaction with autochthonous yeasts. In the present study, we performed semi industrial fermentations of Macabeo and Merlot musts inoculated with either H. vineae or S. cerevisiae. The yeast population dynamics were monitored by plate culturing, PCR-DGGE and massive sequencing techniques. The results obtained with these techniques show that H. vineae was able dominate the autochthonous microbiota in Macabeo must but not in Merlot must, which exhibited a larger, more diverse yeast population. The presence of H. vineae throughout most of the Macabeo fermentation resulted in more fruity and flowery wine, as indicated by the chemical analysis of the final wines, which demonstrated a strong presence of phenyl ethyl acetate at concentrations higher than the threshold of perception and approximately 50 times more than that produced in wines fermented with S. cerevisiae. This compound is associated with fruity, floral and honey aromas. PMID:27014252

  7. Response of blood platelets to beta-glucan from Saccharomyces cerevisiae.

    PubMed

    Saluk-Juszczak, Joanna; Krlewska, Karolina; Wachowicz, Barbara

    2010-01-01

    The effects of the beta-D-glucan, a polysaccharide derived from the yeast cell walls of species such as Saccharomyces cerevisiae, on blood platelets activation induced by physiological agonists (thrombin, ADP, collagen) in vitro were studied. The aim of our study was to assess in vitro if beta-glucan, a naturally strong biological response modifier, may modify platelet activation, i.e. platelet aggregation and degranulation (release of proteins and adenine nucleotides) induced by thrombin, ADP and collagen. Cytochrome c reduction method was used to test the ability of beta-glucan to change superoxide anion generation in platelets. Moreover, we determined also its effect on enzymatic arachidonic acid cascade. The obtained results indicate that beta-glucan has the inhibitory effects on platelet aggregation and secretion. beta-glucan distinctly reduced the arachidonic acid pathway and superoxide anion radical generation in platelets stimulated by biological agonists. The results of the present study suggest that beta-glucan from Saccharomyces cerevisiae has antiplatelet and antioxidative activities, and therefore may be beneficial in the prevention of the excessive blood platelet activation-related diseases, such as cardiovascular or inflammatory diseases. PMID:19891527

  8. Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production.

    PubMed

    Meersman, Esther; Steensels, Jan; Struyf, Nore; Paulus, Tinneke; Saels, Veerle; Mathawan, Melissa; Allegaert, Leen; Vrancken, Gino; Verstrepen, Kevin J

    2016-01-01

    Microbial starter cultures have extensively been used to enhance the consistency and efficiency of industrial fermentations. Despite the advantages of such controlled fermentations, the fermentation involved in the production of chocolate is still a spontaneous process that relies on the natural microbiota at cocoa farms. However, recent studies indicate that certain thermotolerant Saccharomyces cerevisiae cultures can be used as starter cultures for cocoa pulp fermentation. In this study, we investigate the potential of specifically developed starter cultures to modulate chocolate aroma. Specifically, we developed several new S. cerevisiae hybrids that combine thermotolerance and efficient cocoa pulp fermentation with a high production of volatile flavor-active esters. In addition, we investigated the potential of two strains of two non-Saccharomyces species that produce very large amounts of fruity esters (Pichia kluyveri and Cyberlindnera fabianii) to modulate chocolate aroma. Gas chromatography-mass spectrometry (GC-MS) analysis of the cocoa liquor revealed an increased concentration of various flavor-active esters and a decrease in spoilage-related off-flavors in batches inoculated with S. cerevisiae starter cultures and, to a lesser extent, in batches inoculated with P. kluyveri and Cyb. fabianii. Additionally, GC-MS analysis of chocolate samples revealed that while most short-chain esters evaporated during conching, longer and more-fat-soluble ethyl and acetate esters, such as ethyl octanoate, phenylethyl acetate, ethyl phenylacetate, ethyl decanoate, and ethyl dodecanoate, remained almost unaffected. Sensory analysis by an expert panel confirmed significant differences in the aromas of chocolates produced with different starter cultures. Together, these results show that the selection of different yeast cultures opens novel avenues for modulating chocolate flavor. PMID:26590272

  9. Hurdle technology applied to prickly pear beverages for inhibiting Saccharomyces cerevisiae and Escherichia coli.

    PubMed

    García-García, R; Escobedo-Avellaneda, Z; Tejada-Ortigoza, V; Martín-Belloso, O; Valdez-Fragoso, A; Welti-Chanes, J

    2015-06-01

    The effect of pH reduction (from 6·30-6·45 to 4·22-4·46) and the addition of antimicrobial compounds (sodium benzoate and potassium sorbate) on the inhibition of Saccharomyces cerevisiae and Escherichia coli in prickly pear beverages formulated with the pulp and peel of Villanueva (V, Opuntia albicarpa) and Rojo Vigor (RV, Opuntia ficus-indica) varieties during 14 days of storage at 25°C, was evaluated. RV variety presented the highest microbial inhibition. By combining pH reduction and preservatives, reductions of 6·2-log10 and 2·3-log10 for E. coli and S. cerevisiae were achieved respectively. Due to the low reduction of S. cerevisiae, pulsed electric fields (PEF) (11-15 μs/25-50 Hz/27-36 kV cm(-1)) was applied as another preservation factor. The combination of preservatives, pH reduction and PEF at 13-15 μs/25-50 Hz for V variety, and 11 μs/50 Hz, 13-15 μs/25-50 Hz for RV, had a synergistic effect on S. cerevisiae inhibition, achieving at least 3·4-log10 of microbial reduction immediately after processing, and more than 5-log10 at fourth day of storage at 25°C maintained this reduction during 21 days of storage (P > 0·05). Hurdle technology using PEF in combination with other factors is adequate to maintain stable prickly pear beverages during 21 days/25°C. Significance and impact of the study: Prickly pear is a fruit with functional value, with high content of nutraceuticals and antioxidant activity. Functional beverages formulated with the pulp and peel of this fruit represent an alternative for its consumption. Escherichia coli and Saccharomyces cerevisiae are micro-organisms that typically affect fruit beverage quality and safety. The food industry is looking for processing technologies that maintain quality without compromising safety. Hurdle technology, including pulsed electric fields (PEF) could be an option to achieve this. The combination of PEF, pH reduction and preservatives is an alternative to obtain safe and minimally processed prickly pear beverages with convenient shelf-life. PMID:25702901

  10. Improving the performance of the Granulosis virus of Codling moth (Lepidoptera: Tortricideae) by adding the yeast Saccharomyces cerevisiae with sugar

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies evaluated the effectiveness of adding Saccharomyces cerevisiae with brown cane sugar (sugar) to the codling moth granulosis virus, CpGV, to improve larval control of Cydia pomonella (L.), on apple. Neither the use of the yeast or sugar alone caused larval mortality greater than the water con...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Production of Yarrowia lipolytica Nha2 Na+/H+ antiporter improves the salt tolerance of Saccharomyces cerevisiae.

    PubMed

    Papousková, K; Sychrová, H

    2007-01-01

    Yarrowia lipolytica plasma-membrane Na+/H+ antiporter, encoded by the YlNHA2 gene, is a very efficient exporter of surplus sodium from the cytosol. Its heterologous expression in Saccharomyces cerevisiae wild-type laboratory strains increased their sodium tolerance more efficiently than the expression of ZrSod2-22 antiporter from the osmotolerant yeast Zygosaccharomvces rouxii. PMID:18450222

  13. Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a differ...

  14. Saccharomyces cerevisiae mass transformed with FLEXGenes results in strain capable of anaerobic fermentation of pentose and hexose sugars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Advanced automated high-throughput mass transformation of yeast full-genome libraries into Saccharomyces cerevisiae and screening for growth on xylose produced a yeast strain that is capable of fully utilizing pentose as well as hexose sugars anaerobically. This is the first yeast strain capable of...

  15. Draft Genome Sequence of Saccharomyces cerevisiae IR-2, a Useful Industrial Strain for Highly Efficient Production of Bioethanol

    PubMed Central

    Fujimori, Kazuhiro E.; Nezuo, Maiko; Tsukahara, Masatoshi; Tochigi, Yuki; Ohgiya, Satoru; Kamagata, Yoichi

    2014-01-01

    We sequenced the genome of Saccharomyces cerevisiae IR-2, which is a diploid industrial strain with flocculation activity and the ability to efficiently produce bioethanol. The approximately 11.4-Mb draft genome information provides useful insights into metabolic engineering for the production of bioethanol from biomass. PMID:24435865

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

    PubMed

    Sravanthi Goud, Burragoni; Ulaganathan, Kandasamy

    2015-01-01

    Here, we report the draft genome sequence of Saccharomyces cerevisiae strain NCIM3186 used in bioethanol production from sweet sorghum. The size of the genome is approximately 11.9 Mb and contains 5,347 protein-coding genes. PMID:26227595

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

    PubMed Central

    Sravanthi Goud, Burragoni

    2015-01-01

    Here, we report the draft genome sequence of Saccharomyces cerevisiae strain NCIM3186 used in bioethanol production from sweet sorghum. The size of the genome is approximately 11.9 Mb and contains 5,347 protein-coding genes. PMID:26227595

  18. Effects of supplementing a Saccharomyces cerevisiae fermentation product in sow diets on performance of sows and nursing piglets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty-two sows (Camborough-22, PIC) were used to determine the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; Diamond V Original XPC) in gestation and lactation diets on performance of sows and their progeny. On 5 d before breeding, sows were allotted to 2 dietary tr...

  19. Effects of supplementing Saccharomyces cerevisiae fermentation product in sow diets on performance of sows and nursing piglets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Forty-two sows were used to determine the effects of adding Saccharomyces cerevisiae fermentation product to gestation and lactation diets on performance of sows and their progeny. On 5 d before breeding, sows were allotted to 2 dietary treatments representing: (1) sows fed a diet with 12.0 g fermen...

  20. GMAX-L Saccharomyces Cerevisiae Strains for Profitable Sustainable Cellulosic Ethanol and Biodiesel Production Concurrently using Engineered Workcell

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A stable GMAX-L strain of Saccharomyces cerevisiae is being constructed using pSUMO expression cassettes that are extremely high expression level plasmids designed for use on automated workcell. This strain expresses xylose isomerase, xylulokinase, XIB1, and XIG1 for anaerobic cellulosic ethanol pr...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. ISOLATION OF THE CANDIDA TROPICALIS GENE FOR P450 LANOSTEROL DEMETHYLASE AND ITS EXPRESSION IN SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have isolated the gene for cytochrome P450 lanosterol 14a-demethylase (14DM) from the yeast Candida tropicalis. his was accomplished by screening genomic libraries of strain ATCC750 in E. coli using a DNA fragment containing the yeast Saccharomyces cerevisiae 14DM gene. dentit...

  3. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONANZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-Cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14a-demethylase. esistance is restored through complementation by the plasmid-born...

  4. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  5. Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by the yeast, particularly when the carbon source is acid-treated lignocell...

  6. Evaluation of engineered xylose-fermenting industrial strains of Saccharomyces cerevisiae for improved ethanol production from lignocellulosic feedstocks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. Hemicellulose can make up to 20-30% of biomass and is primarily composed of xylose. Enzymes from native xylose-assimi...

  7. Different effects of sodium chloride preincubation on cadmium tolerance of Pichia kudriavzevii and Saccharomyces cerevisiae.

    PubMed

    Ma, Ning; Li, Chunsheng; Dong, Xiaoyan; Wang, Dongfeng; Xu, Ying

    2015-08-01

    Application of growing microorganisms for cadmium removal is restricted by high cadmium toxicity. The effects of sodium chloride (NaCl) preincubation on the cadmium tolerance and removal ability of Pichia kudriavzevii and Saccharomyces cerevisiae were investigated in this study. NaCl preincubation improved the biomass of P. kudriavzevii under cadmium stress, while no obvious effect was observed in S. cerevisiae. The improved activities of peroxidase (POD) and catalase (CAT) after NaCl preincubation might be an important reason for the decrease of the reactive oxygen species (ROS) accumulation, cell death, and oxidative damage of proteins and lipids induced by cadmium, contributing to the improvement of the yeast growth. The cadmium bioaccumulation capacity of P. kudriavzevii decreased significantly after NaCl preincubation, which played an important role in mitigating the cadmium toxicity to the yeast. The cadmium removal rate of P. kudriavzevii was obviously higher than S. cerevisiae and was significantly enhanced after NaCl preincubation. The results suggested that NaCl preincubation improved the cadmium tolerance and removal ability of P. kudriavzevii. PMID:25721585

  8. Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.

    PubMed

    Hu, Yanhui; Rolfs, Andreas; Bhullar, Bhupinder; Murthy, Tellamraju V S; Zhu, Cong; Berger, Michael F; Camargo, Anamaria A; Kelley, Fontina; McCarron, Seamus; Jepson, Daniel; Richardson, Aaron; Raphael, Jacob; Moreira, Donna; Taycher, Elena; Zuo, Dongmei; Mohr, Stephanie; Kane, Michael F; Williamson, Janice; Simpson, Andrew; Bulyk, Martha L; Harlow, Edward; Marsischky, Gerald; Kolodner, Richard D; LaBaer, Joshua

    2007-04-01

    The availability of an annotated genome sequence for the yeast Saccharomyces cerevisiae has made possible the proteome-scale study of protein function and protein-protein interactions. These studies rely on availability of cloned open reading frame (ORF) collections that can be used for cell-free or cell-based protein expression. Several yeast ORF collections are available, but their use and data interpretation can be hindered by reliance on now out-of-date annotations, the inflexible presence of N- or C-terminal tags, and/or the unknown presence of mutations introduced during the cloning process. High-throughput biochemical and genetic analyses would benefit from a "gold standard" (fully sequence-verified, high-quality) ORF collection, which allows for high confidence in and reproducibility of experimental results. Here, we describe Yeast FLEXGene, a S. cerevisiae protein-coding clone collection that covers over 5000 predicted protein-coding sequences. The clone set covers 87% of the current S. cerevisiae genome annotation and includes full sequencing of each ORF insert. Availability of this collection makes possible a wide variety of studies from purified proteins to mutation suppression analysis, which should contribute to a global understanding of yeast protein function. PMID:17322287

  9. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae.

    PubMed

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D; Mukhopadhyay, Aindrila

    2016-01-01

    Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min(-1)•mg(-1)) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation. PMID:26781725

  10. A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair.

    PubMed

    Shivange, Gururaj; Kodipelli, Naveena; Monisha, Mohan; Anindya, Roy

    2014-12-26

    Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity. PMID:25381260

  11. A Role for Saccharomyces cerevisiae Tpa1 Protein in Direct Alkylation Repair*

    PubMed Central

    Shivange, Gururaj; Kodipelli, Naveena; Monisha, Mohan; Anindya, Roy

    2014-01-01

    Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity. PMID:25381260

  12. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

    PubMed Central

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-01-01

    Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min−1•mg−1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation. PMID:26781725

  13. Genome-wide screening of Saccharomyces cerevisiae genes regulated by vanillin.

    PubMed

    Park, Eun-Hee; Kim, Myoung-Dong

    2015-01-01

    During pretreatment of lignocellulosic biomass, a variety of fermentation inhibitors, including acetic acid and vanillin, are released. Using DNA microarray analysis, this study explored genes of the budding yeast Saccharomyces cerevisiae that respond to vanillin-induced stress. The expression of 273 genes was upregulated and that of 205 genes was downregulated under vanillin stress. Significantly induced genes included MCH2, SNG1, GPH1, and TMA10, whereas NOP2, UTP18, FUR1, and SPR1 were down regulated. Sequence analysis of the 5'-flanking region of upregulated genes suggested that vanillin might regulate gene expression in a stress response element (STRE)-dependent manner, in addition to a pathway that involved the transcription factor Yap1p. Retardation in the cell growth of mutant strains indicated that MCH2, SNG1, and GPH1 are intimately involved in vanillin stress response. Deletion of the genes whose expression levels were decreased under vanillin stress did not result in a notable change in S. cerevisiae growth under vanillin stress. This study will provide the basis for a better understanding of the stress response of the yeast S. cerevisiae to fermentation inhibitors. PMID:25269814

  14. Secretory pathway engineering enhances secretion of cellobiohydrolase I from Trichoderma reesei in Saccharomyces cerevisiae.

    PubMed

    Xu, Lili; Shen, Yu; Hou, Jin; Peng, Bingyin; Tang, Hongting; Bao, Xiaoming

    2014-01-01

    Improving the cellulase secretion is beneficial for Saccharomyces cerevisiae used in consolidated bioprocessing (CBP) of cellulosic ethanol. In this study, protein secretory pathway, including protein folding, disulfide bond formation, and protein trafficking and sorting, was modified in S. cerevisiae. The effects of these modifications on the secretion of cellobiohydrolase I (Tr-Cel7A) with its native signal peptide from Trichoderma reesei were investigated. The results showed that overexpression of the protein disulfide isomerase Sc-PDI1 and the plasma membrane targeting soluble N-ethylmaleimide-sensitive factor attachment protein receptor Sc-SSO1, and disruption of the sorting receptor Sc-VPS10 and a Ca(2+)/Mn(2+) ATPase Sc-PMR1, improved respectively the extracellular Tr-Cel7A activities. Among them, disruption of Sc-PMR1 showed better improvement of 162% in the extracellular activity and decreased the glycosylation of Tr-Cel7A. Multiple modifications generally resulted in higher activities. The extracellular activities of the quadruple-modified strain (vps10Δ/pmr1Δ/SSO1/PDI1/cel7AF) using p-nitrophenyl-β-d-cellobioside (pNPC) and phosphoric acid swollen cellulose (PASC) as the substrates, respectively, were 3.9-fold and 1.3-fold higher than that of the reference strain cel7AF. The results indicated that engineering of the protein secretory pathway is an effective approach to improve the Tr-Cel7A secretion in S. cerevisiae. PMID:23890871

  15. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.

    PubMed

    Runguphan, Weerawat; Keasling, Jay D

    2014-01-01

    As the serious effects of global climate change become apparent and access to fossil fuels becomes more limited, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. In recent years, microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fatty acid-derived biofuels and chemicals from simple sugars. Specifically, we overexpressed all three fatty acid biosynthesis genes, namely acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1) and fatty acid synthase 2 (FAS2), in S. cerevisiae. When coupled to triacylglycerol (TAG) production, the engineered strain accumulated lipid to more than 17% of its dry cell weight, a four-fold improvement over the control strain. Understanding that TAG cannot be used directly as fuels, we also engineered S. cerevisiae to produce drop-in fuels and chemicals. Altering the terminal "converting enzyme" in the engineered strain led to the production of free fatty acids at a titer of approximately 400 mg/L, fatty alcohols at approximately 100mg/L and fatty acid ethyl esters (biodiesel) at approximately 5 mg/L directly from simple sugars. We envision that our approach will provide a scalable, controllable and economic route to this important class of chemicals. PMID:23899824

  16. Saccharomyces cerevisiae: A novel and efficient biological control agent for Colletotrichum acutatum during pre-harvest.

    PubMed

    Lopes, Marcos Roberto; Klein, Mariana Nadjara; Ferraz, Luriany Pompeo; da Silva, Aline Caroline; Kupper, Katia Cristina

    2015-06-01

    In this study, we evaluated the efficiency of six isolates of Saccharomyces cerevisiae in controlling Colletotrichum acutatum, the causal agent of postbloom fruit drop that occur in pre-harvest citrus. We analyzed the mechanisms of action involved in biological control such as: production of antifungal compounds, nutrient competition, detection of killer activity, and production of hydrolytic enzymes of the isolates of S. cerevisiae on C. acutatum and their efficiency in controlling postbloom fruit drop on detached citrus flowers. Our results showed that all six S. cerevisiae isolates produced antifungal compounds, competed for nutrients, inhibited pathogen germination, and produced killer activity and hydrolytic enzymes when in contact with the fungus wall. The isolates were able to control the disease when detached flowers were artificially inoculated, both preventively and curatively. In this work we identified a novel potential biological control agent for C. acutatum during pre-harvest. This is the first report of yeast efficiency for the biocontrol of postbloom fruit drop, which represents an important contribution to the field of biocontrol of diseases affecting citrus populations worldwide. PMID:25960430

  17. Identification of a gene, FMP21, whose expression levels are involved in thermotolerance in Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    Elucidation of the mechanism of high temperature tolerance in yeasts is important for the molecular breeding of high temperature-tolerant yeasts that can be used in bioethanol production. We identified genes whose expression is correlated with the degree of thermotolerance in Saccharomyces cerevisiae by DNA microarray analysis. Gene expression profiles of three S. cerevisiae strains showing different levels of thermotolerance were compared, and we chose three of them as candidate genes. Among these genes, FMP21 was investigated as a thermotolerance-related gene in S. cerevisiae by comparing the growth at high temperature with the gene expression in eight strains. The expression ratio of FMP21 at 37°C was correlated with the doubling time ratio at a coefficient of determination of 0.787. The potential involvement of the Fmp21 in the thermotolerance of yeasts was evaluated. The FMP21 deletion variant showed a decreased respiratory growth rate and increased thermosensitivity. Furthermore, the overexpression of FMP21 improved thermotolerance in yeasts. In conclusion, the function of Fmp21 is important for thermotolerance in yeasts. PMID:25177541

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

    SciTech Connect

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

    2008-11-25

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

  19. Biosorptive recovery of platinum from platinum group metal refining wastewaters by immobilised Saccharomyces cerevisiae.

    PubMed

    Mack, C L; Wilhelmi, B; Duncan, J R; Burgess, J E

    2011-01-01

    The process of platinum group metal (PGM) refining can be up to 99.99% efficient at best, and although it may seem small, the amount of valuable metal lost to waste streams is appreciable enough to warrant recovery. The method currently used to remove entrained metal ions from refinery wastewaters, chemical precipitation, is not effective for selective recovery of PGMs. The yeast Saccharomyces cerevisiae has been found capable of sorbing numerous precious and base metals, and is a cheap and abundant source of biomass. In this investigation, S. cerevisiae was immobilised using polyethyleneimine and glutaraldehyde to produce a suitable sorbent, capable of high platinum uptake (150-170 mg/g) at low pH (<2). The sorption mechanism was found to be a chemical reaction, which made effective desorption impossible. When applied to PGM refinery wastewater, two key wastewater characteristics limited the success of the sorption process; high inorganic ion content and complex speciation of the platinum ions. The results proved the concept principle of platinum recovery by immobilised yeast biosorption and indicated that a more detailed understanding of the platinum speciation within the wastewater is required before biosorption can be applied. Overall, the sorption of platinum by the S. cerevisiae sorbent was demonstrated to be highly effective in principle, but the complexity of the wastewater requires that pretreatment steps be taken before the successful application of this process to industrial wastewater. PMID:21245567

  20. Adhesion-dependent rupturing of Saccharomyces cerevisiae on biological antimicrobial nanostructured surfaces

    PubMed Central

    Nowlin, Kyle; Boseman, Adam; Covell, Alan; LaJeunesse, Dennis

    2015-01-01

    Recent studies have shown that some nanostructured surfaces (NSS), many of which are derived from surfaces found on insect cuticles, rupture and kill adhered prokaryotic microbes. Most important, the nanoscale topography is directly responsible for this effect. Although parameters such as cell adhesion and cell wall rigidity have been suggested to play significant roles in this process, there is little experimental evidence regarding the underlying mechanisms involving NSS-induced microbial rupture. In this work, we report the NSS-induced rupturing of a eukaryotic microorganism, Saccharomyces cerevisiae. We show that the amount of NSS-induced rupture of S. cerevisiae is dependent on both the adhesive qualities of the yeast cell and the nanostructure geometry of the NSS. Thus, we are providing the first empirical evidence that these parameters play a direct role in the rupturing of microbes on NSS. Our observations of this phenomenon with S. cerevisiae, particularly the morphological changes, are strikingly similar to that reported for bacteria despite the differences in the yeast cell wall structure. Consequently, NSS provide a novel approach for the control of microbial growth and development of broad-spectrum microbicidal surfaces. PMID:25551144

  1. Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae

    PubMed Central

    Hu, Yanhui; Rolfs, Andreas; Bhullar, Bhupinder; Murthy, Tellamraju V. S.; Zhu, Cong; Berger, Michael F.; Camargo, Anamaria A.; Kelley, Fontina; McCarron, Seamus; Jepson, Daniel; Richardson, Aaron; Raphael, Jacob; Moreira, Donna; Taycher, Elena; Zuo, Dongmei; Mohr, Stephanie; Kane, Michael F.; Williamson, Janice; Simpson, Andrew; Bulyk, Martha L.; Harlow, Edward; Marsischky, Gerald; Kolodner, Richard D.; LaBaer, Joshua

    2007-01-01

    The availability of an annotated genome sequence for the yeast Saccharomyces cerevisiae has made possible the proteome-scale study of protein function and protein–protein interactions. These studies rely on availability of cloned open reading frame (ORF) collections that can be used for cell-free or cell-based protein expression. Several yeast ORF collections are available, but their use and data interpretation can be hindered by reliance on now out-of-date annotations, the inflexible presence of N- or C-terminal tags, and/or the unknown presence of mutations introduced during the cloning process. High-throughput biochemical and genetic analyses would benefit from a “gold standard” (fully sequence-verified, high-quality) ORF collection, which allows for high confidence in and reproducibility of experimental results. Here, we describe Yeast FLEXGene, a S. cerevisiae protein-coding clone collection that covers over 5000 predicted protein-coding sequences. The clone set covers 87% of the current S. cerevisiae genome annotation and includes full sequencing of each ORF insert. Availability of this collection makes possible a wide variety of studies from purified proteins to mutation suppression analysis, which should contribute to a global understanding of yeast protein function. PMID:17322287

  2. Terminal acidic shock inhibits sour beer bottle conditioning by Saccharomyces cerevisiae.

    PubMed

    Rogers, Cody M; Veatch, Devon; Covey, Adam; Staton, Caleb; Bochman, Matthew L

    2016-08-01

    During beer fermentation, the brewer's yeast Saccharomyces cerevisiae experiences a variety of shifting growth conditions, culminating in a low-oxygen, low-nutrient, high-ethanol, acidic environment. In beers that are bottle conditioned (i.e., carbonated in the bottle by supplying yeast with a small amount of sugar to metabolize into CO2), the S. cerevisiae cells must overcome these stressors to perform the ultimate act in beer production. However, medium shock caused by any of these variables can slow, stall, or even kill the yeast, resulting in production delays and economic losses. Here, we describe a medium shock caused by high lactic acid levels in an American sour beer, which we refer to as "terminal acidic shock". Yeast exposed to this shock failed to bottle condition the beer, though they remained viable. The effects of low pH/high [lactic acid] conditions on the growth of six different brewing strains of S. cerevisiae were characterized, and we developed a method to adapt the yeast to growth in acidic beer, enabling proper bottle conditioning. Our findings will aid in the production of sour-style beers, a trending category in the American craft beer scene. PMID:27052714

  3. Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance.

    PubMed

    Garcia Sanchez, Rosa; Solodovnikova, Natalia; Wendland, Jürgen

    2012-08-01

    Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. PMID:22887121

  4. Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance.

    TOXLINE Toxicology Bibliographic Information

    Garcia Sanchez R; Solodovnikova N; Wendland J

    2012-08-01

    Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent.

  5. Effects of microcystin-LR on Saccharomyces cerevisiae growth, oxidative stress and apoptosis.

    PubMed

    Valério, Elisabete; Vilares, Arminda; Campos, Alexandre; Pereira, Paulo; Vasconcelos, Vitor

    2014-11-01

    Microcystins (MC) are cyanotoxins occurring globally, known for causing acute hepatotoxicity in humans/animals, tumor promotion in animals and potential carcinogenicity. The mechanism of MC toxicity is considered a multi-pathway process involving the inhibition of protein phosphatases PP1/PP2A and the production of reactive oxygen species (ROS). However, their mechanism of action is not fully characterized, thus hampering the complete hazard identification. In this study, we evaluated the effect of several microcystin-LR concentrations on the growth, ROS levels, antioxidant system response and apoptosis induction on Saccharomyces cerevisiae. Our results showed that the growth of S. cerevisiae was not inhibited when compared to control cells. However, the staining of cells with DHR123 and DHE revealed an intracellular increase of the ROS levels. This ROS increase resulted in an augment of catalase activity and inhibition of SOD. All these facts suggest that hydrogen peroxide was the main ROS induced by MCLR. Signs of apoptosis were also detected in the cells exposed to toxin. Our results show that S. cerevisiae VL3 displays MCLR toxicity effects known to occur in higher eukaryotes and confirmed that it can be a simple and good model to help further in the elucidation of MCLR molecular mechanisms of toxicity. PMID:25157802

  6. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. PMID:26801560

  7. Heterologous expression of human membrane receptors in the yeast Saccharomyces cerevisiae.

    PubMed

    Joubert, Olivier; Nehmé, Rony; Bidet, Michel; Mus-Veteau, Isabelle

    2010-01-01

    Due to their implication in numerous diseases like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension, and Alzheimer disease, membrane proteins (MPs) represent around 50% of drug targets. However, only 204 crystal structures of MPs have been solved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. The yeast Saccharomyces cerevisiae is a convenient host for the production of mammalian MPs for functional and structural studies. Like bacteria, they are straightforward to manipulate genetically, are well characterized, can be easily cultured, and can be grown inexpensively in large quantities. The advantage of yeast compared to bacteria is that they have protein-processing and posttranslational modification mechanisms related to those found in mammalian cells. The recombinant rabbit muscle Ca(2+)-ATPase (adenosine triphosphatase), the first heterologously expressed mammalian MP for which the crystal structure was resolved, has been produced in S. cerevisiae. In this chapter, the focus is on expression of recombinant human integral MPs in a functional state at the plasma membrane of the yeast S. cerevisiae. Optimization of yeast culture and of MP preparations is detailed for two human receptors of the Hedgehog pathway: Patched and Smoothened. PMID:20099141

  8. L-Histidine Inhibits Biofilm Formation and FLO11-Associated Phenotypes in Saccharomyces cerevisiae Flor Yeasts

    PubMed Central

    Bou Zeidan, Marc; Zara, Giacomo; Viti, Carlo; Decorosi, Francesca; Mannazzu, Ilaria; Budroni, Marilena; Giovannetti, Luciana; Zara, Severino

    2014-01-01

    Flor yeasts of Saccharomyces cerevisiae have an innate diversity of FLO11 which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling FLO11 alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce FLO11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to FLO11 expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air–liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the FLO11 gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts. PMID:25369456

  9. Saccharomyces cerevisiae KNU5377 Stress Response during High-Temperature Ethanol Fermentation

    PubMed Central

    Kim, Il-Sup; Kim, Young-Saeng; Kim, Hyun; Jin, Ingnyol; Yoon, Ho-Sung

    2013-01-01

    Fuel ethanol production is far more costly to produce than fossil fuels. There are a number of approaches to cost-effective fuel ethanol production from biomass. We characterized stress response of thermotolerant Saccharomyces cerevisiae KNU5377 during glucose-based batch fermentation at high temperature (40°C). S. cerevisiae KNU5377 (KNU5377) transcription factors (Hsf1, Msn2/4, and Yap1), metabolic enzymes (hexokinase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, and alcohol dehydrogenase), antioxidant enzymes (thioredoxin 3, thioredoxin reductase, and porin), and molecular chaperones and its cofactors (Hsp104, Hsp82, Hsp60, Hsp42, Hsp30, Hsp26, Cpr1, Sti1, and Zpr1) are upregulated during fermentation, in comparison to S. cerevisiae S288C (S288C). Expression of glyceraldehyde-3-phosphate dehydrogenase increased significantly in KNU5377 cells. In addition, cellular hydroperoxide and protein oxidation, particularly lipid peroxidation of triosephosphate isomerase, was lower in KNU5377 than in S288C. Thus, KNU5377 activates various cell rescue proteins through transcription activators, improving tolerance and increasing alcohol yield by rapidly responding to fermentation stress through redox homeostasis and proteostasis. PMID:23512334

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

    PubMed

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

    2015-05-01

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

  11. Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae

    PubMed Central

    Da Silva, S.M.; Vang, L.K.; Olson, N.D.; Lund, S.P.; Downey, A.S.; Kelman, Z.; Salit, M.L.; Lin, N.J.; Morrow, J.B.

    2016-01-01

    Aims We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR). Methods and results S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. cerevisiae BY4739 to convey selectivity. For the interlaboratory study, a binomial regression model was used to select three cell concentrations, high (4 × 107 cells ml−1), intermediate (4 × 105 cells ml−1) and low (4 × 103 cells ml−1), and the number of samples per concentration. Seven participants, including potential end users, had combined rates of positive qPCR detection (quantification cycle <37) of 100%, 40%, and 0% for high, intermediate, and low concentrations, respectively. Conclusions The NE095 strain was successfully detected by all participants, with the high concentration indicating a potential target concentration for a reference material. Significance and impact of the study The engineered yeast has potential to support measurement assurance for the analytical process of qPCR, encompassing the method, equipment, and operator, to increase confidence in results and better inform decision-making in areas of applied microbiology. This material can also support process assessment for other DNA-based detection technologies. PMID:27077050

  12. Improvement of lactic acid production in Saccharomyces cerevisiae by a deletion of ssb1.

    PubMed

    Lee, Jinsuk J; Crook, Nathan; Sun, Jie; Alper, Hal S

    2016-01-01

    Polylactic acid (PLA) is an important renewable polymer, but current processes for producing its precursor, lactic acid, suffer from process inefficiencies related to the use of bacterial hosts. Therefore, improving the capacity of Saccharomyces cerevisiae to produce lactic acid is a promising approach to improve industrial production of lactic acid. As one such improvement required, the lactic acid tolerance of yeast must be significantly increased. To enable improved tolerance, we employed an RNAi-mediated genome-wide expression knockdown approach as a means to rapidly identify potential genetic targets. In this approach, several gene knockdown targets were identified which confer increased acid tolerance to S. cerevisiae BY4741, of which knockdown of the ribosome-associated chaperone SSB1 conferred the highest increase (52%). This target was then transferred into a lactic acid-overproducing strain of S. cerevisiae CEN.PK in the form of a knockout and the resulting strain demonstrated up to 33% increased cell growth, 58% increased glucose consumption, and 60% increased L-lactic acid production. As SSB1 contains a close functional homolog SSB2 in yeast, this result was counterintuitive and may point to as-yet-undefined functional differences between SSB1 and SSB2 related to lactic acid production. The final strain produced over 50 g/L of lactic acid in under 60 h of fermentation. PMID:26660479

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

    PubMed

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

    2016-01-01

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

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

    PubMed

    Matsushika, Akinori; Hoshino, Tamotsu

    2015-12-01

    The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes. PMID:26438430

  15. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

    PubMed

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

    2015-07-24

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. PMID:26071590

  16. Characterization of vacuolar amino acid transporter from Fusarium oxysporum in Saccharomyces cerevisiae.

    PubMed

    Lunprom, Siriporn; Pongcharoen, Pongsanat; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi; Akiyama, Koichi

    2015-01-01

    Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter. PMID:26083447

  17. The Chromatin and Transcriptional Landscape of Native Saccharomyces cerevisiae Telomeres and Subtelomeric Domains.

    PubMed

    Ellahi, Aisha; Thurtle, Deborah M; Rine, Jasper

    2015-06-01

    Saccharomyces cerevisiae telomeres have been a paradigm for studying telomere position effects on gene expression. Telomere position effect was first described in yeast by its effect on the expression of reporter genes inserted adjacent to truncated telomeres. The reporter genes showed variable silencing that depended on the Sir2/3/4 complex. Later studies examining subtelomeric reporter genes inserted at natural telomeres hinted that telomere position effects were less pervasive than previously thought. Additionally, more recent data using the sensitive technology of chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq) revealed a discrete and noncontinuous pattern of coenrichment for all three Sir proteins at a few telomeres, calling the generality of these conclusions into question. Here we combined the ChIP-Seq of the Sir proteins with RNA sequencing (RNA-Seq) of messenger RNAs (mRNAs) in wild-type and in SIR2, SIR3, and SIR4 deletion mutants to characterize the chromatin and transcriptional landscape of all native S. cerevisiae telomeres at the highest achievable resolution. Most S. cerevisiae chromosomes had subtelomeric genes that were expressed, with only ∼6% of subtelomeric genes silenced in a SIR-dependent manner. In addition, we uncovered 29 genes with previously unknown cell-type-specific patterns of expression. These detailed data provided a comprehensive assessment of the chromatin and transcriptional landscape of the subtelomeric domains of a eukaryotic genome. PMID:25823445

  18. Genome-wide transposon mutagenesis in Saccharomyces cerevisiae and Candida albicans.

    PubMed

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2011-01-01

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

  19. ¹³C-based metabolic flux analysis of Saccharomyces cerevisiae with a reduced Crabtree effect.

    PubMed

    Kajihata, Shuichi; Matsuda, Fumio; Yoshimi, Mika; Hayakawa, Kenshi; Furusawa, Chikara; Kanda, Akihisa; Shimizu, Hiroshi

    2015-08-01

    Saccharomyces cerevisiae shows a Crabtree effect that produces ethanol in a high glucose concentration even under fully aerobic condition. For efficient production of cake yeast or compressed yeast for baking, ethanol by-production is not desired since glucose limited chemostat or fed-batch cultivations are performed to suppress the Crabtree effect. In this study, the (13)C-based metabolic flux analysis ((13)C-MFA) was performed for the S288C derived S. cerevisiae strain to characterize a metabolic state under the reduced Crabtree effect. S. cerevisiae cells were cultured at a low dilution rate (0.1 h(-1)) under the glucose-limited chemostat condition. The estimated metabolic flux distribution showed that the acetyl-CoA in mitochondria was mainly produced from pyruvate by pyruvate dehydrogenase (PDH) reaction and that the level of the metabolic flux through the pentose phosphate pathway was much higher than that of the Embden-Meyerhof-Parnas pathway, which contributes to high biomass yield at low dilution rate by supplying NADPH required for cell growth. PMID:25634548

  20. Opuntia ficus-indica cladodes as feedstock for ethanol production by Kluyveromyces marxianus and Saccharomyces cerevisiae.

    PubMed

    Kuloyo, Olukayode O; du Preez, James C; Garca-Aparicio, Maria del Prado; Kilian, Stephanus G; Steyn, Laurinda; Grgens, Johann

    2014-12-01

    The feasibility of ethanol production using an enzymatic hydrolysate of pretreated cladodes of Opuntia ficus-indica (prickly pear cactus) as carbohydrate feedstock was investigated, including a comprehensive chemical analysis of the cladode biomass and the effects of limited aeration on the fermentation profiles and sugar utilization. The low xylose and negligible mannose content of the cladode biomass used in this study suggested that the hemicellulose structure of the O. ficus-indica cladode was atypical of hardwood or softwood hemicelluloses. Separate hydrolysis and fermentation and simultaneous saccharification and fermentation procedures using Kluyveromyces marxianus and Saccharomyces cerevisiae at 40 and 35C, respectively, gave similar ethanol yields under non-aerated conditions. In oxygen-limited cultures K. marxianus exhibited almost double the ethanol productivity compared to non-aerated cultures, although after sugar depletion utilization of the produced ethanol was evident. Ethanol concentrations of up to 19.5 and 20.6gl(-1) were obtained with K. marxianus and S. cerevisiae, respectively, representing 66 and 70% of the theoretical yield on total sugars in the hydrolysate. Because of the low xylan content of the cladode biomass, a yeast capable of xylose fermentation might not be a prerequisite for ethanol production. K. marxianus, therefore, has potential as an alternative to S. cerevisiae for bioethanol production. However, the relatively low concentration of fermentable sugars in the O. ficus-indica cladode hydrolysate presents a technical constraint for commercial exploitation. PMID:25248867

  1. A noncoding RNA in Saccharomyces cerevisiae is an RNase P substrate

    PubMed Central

    Yang, Li; Altman, Sidney

    2007-01-01

    Ribonuclease P (RNase P) is involved in regulation of noncoding RNA (ncRNA) expression in Saccharomyces cerevisiae. A hidden-in-reading-frame antisense-1 (HRA1) RNA in S. cerevisiae, which belongs to a class of ncRNAs located in the antisense strand to verified protein coding regions, was cloned for further use in RNase P assays. Escherichia coli RNase P assays in vitro of HRA1 RNA show two cleavage sites, one major and one minor in terms of rates. The same result was observed with a partially purified S. cerevisiae RNase P activity, both at 30°C and 37°C. These latter cells are normally grown at 30°C. Predictions of the secondary structure of HRA1 RNA in silico show the cleavage sites are canonical RNase P recognition sites. A relatively small amount of endogenous HRA1 RNA was identified by RT-PCR in yeast cells. The endogenous HRA1 RNA is increased in amount in strains that are deficient in RNase P activity. A deletion of 10 nucleotides in the HRA1 gene that does not overlap with the gene coding for a protein (DRS2) in the sense strand shows no defective growth in galactose or glucose. These data indicate that HRA1 RNA is a substrate for RNase P and does not appear as a direct consequence of separate regulatory effects of the enzyme on ncRNAs. PMID:17379814

  2. GENOME-WIDE ASSOCIATION ANALYSIS OF CLINICAL VERSUS NON-CLINICAL ORIGIN PROVIDES INSIGHTS INTO SACCHAROMYCES CEREVISIAE PATHOGENESIS

    PubMed Central

    Muller, Ludo A. H.; Lucas, Joseph E.; Georgianna, D. Ryan; McCusker, John H.

    2011-01-01

    Because domesticated Saccharomyces cerevisiae strains have been used to produce fermented food and beverages for centuries without apparent health implications, S. cerevisiae has always been considered a Generally Recognized As Safe (GRAS) microorganism. However, the number of reported mucosal and systemic S. cerevisiae infections in the human population has increased and fatal infections have occured even in relatively healthy individuals. In order to gain insight into the pathogenesis of S. cerevisiae and improve our understanding of the emergence of fungal pathogens, we performed a population-based genome-wide environmental association analysis of clinical versus non-clinical origin in S. cerevisiae. Using tiling array-based, high density genotypes of 44 clinical and 44 non-clinical S. cerevisiae strains from diverse geographical origins and source substrates, we identified several genetic loci associated with clinical background in S. cerevisiae. Associated polymorphisms within the coding sequences of VRP1, KIC1, SBE22 and PDR5, and the 5′ upstream region of YGR146C indicate the importance of pseudohyphal formation, robust cell wall maintenance and cellular detoxification for S. cerevisiae pathogenesis, and constitute good candidates for follow-up verification of virulence and virulence-related factors underlying the pathogenicity of S. cerevisiae. PMID:21880084

  3. Xylose Isomerase Improves Growth and Ethanol Production Rates from Biomass Sugars for Both Saccharomyces Pastorianus and Saccharomyces Cerevisiae

    PubMed Central

    Miller, Kristen P.; Gowtham, Yogender Kumar; Henson, J. Michael; Harcum, Sarah W.

    2013-01-01

    The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion. PMID:22866331

  4. Selection of indigenous Saccharomyces cerevisiae strains in Shanshan County (Xinjiang, China) for winemaking and their aroma-producing characteristics.

    PubMed

    Liu, Ning; Qin, Yi; Song, Yuyang; Ye, Dongqin; Yuan, Wei; Pei, Yingfang; Xue, Bo; Liu, Yanlin

    2015-11-01

    In order to select potential indigenous Saccharomyces strains, diversity of indigenous Saccharomyces strains in Shanshan County (Xinjiang, China) was preliminarily analyzed. Twenty-one genotypes were found through interdelta fingerprinting analysis. According to this result, representatives of each genotype were chosen to test the enological criteria. After tests of fermentation characteristics and growth ability, eight strains were finally selected as starters to further fermentation of Merlot must for aroma analysis and sensory evaluation at the same testing conditions, with one commercial strain F15 as control. Each strain of Saccharomyces cerevisiae produced individual volatiles in different concentrations and combinations which significantly influenced resulting wine flavour. Except of LFP522, all indigenous isolates produced more concentration of esters than F15. Higher concentrations of linalool, β-damascenone and citral, associated with S. cerevisiae LFE1809, considerably distinguished this strain from the others. Sensory evaluation present the Merlot wine fermented by LFE1225 isolated from Merlot, had the highest sensory score. PMID:26323948

  5. The Oenological Potential of Hanseniaspora uvarum in Simultaneous and Sequential Co-fermentation with Saccharomyces cerevisiae for Industrial Wine Production

    PubMed Central

    Tristezza, Mariana; Tufariello, Maria; Capozzi, Vittorio; Spano, Giuseppe; Mita, Giovanni; Grieco, Francesco

    2016-01-01

    In oenology, the utilization of mixed starter cultures composed by Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers in order to enhance sensory quality and complexity of the final product without compromising the general quality and safety of the oenological products. In fact, several non-Saccharomyces yeasts are already commercialized as oenological starter cultures to be used in combination with Saccharomyces cerevisiae, while several others are the subject of various studies to evaluate their application. Our aim, in this study was to assess, for the first time, the oenological potential of H. uvarum in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for industrial wine production. Three previously characterized H. uvarum strains were separately used as multi-starter together with an autochthonous S. cerevisiae starter culture in lab-scale micro-vinification trials. On the basis of microbial development, fermentation kinetics and secondary compounds formation, the strain H. uvarum ITEM8795 was further selected and it was co- and sequentially inoculated, jointly with the S. cerevisiae starter, in a pilot scale wine production. The fermentation course and the quality of final product indicated that the co-inoculation was the better performing modality of inoculum. The above results were finally validated by performing an industrial scale vinification The mixed starter was able to successfully dominate the different stages of the fermentation process and the H. uvarum strain ITEM8795 contributed to increasing the wine organoleptic quality and to simultaneously reduce the volatile acidity. At the best of our knowledge, the present report is the first study regarding the utilization of a selected H. uvarum strain in multi-starter inoculation with S. cerevisiae for the industrial production of a wine. In addition, we demonstrated, at an industrial scale, the importance of non-Saccharomyces in the design of tailored starter cultures for typical wines.

  6. Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis

    PubMed Central

    2013-01-01

    Background Glycolic acid is a C2 hydroxy acid that is a widely used chemical compound. It can be polymerised to produce biodegradable polymers with excellent gas barrier properties. Currently, glycolic acid is produced in a chemical process using fossil resources and toxic chemicals. Biotechnological production of glycolic acid using renewable resources is a desirable alternative. Results The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are suitable organisms for glycolic acid production since they are acid tolerant and can grow in the presence of up to 50 g l-1 glycolic acid. We engineered S. cerevisiae and K. lactis for glycolic acid production using the reactions of the glyoxylate cycle to produce glyoxylic acid and then reducing it to glycolic acid. The expression of a high affinity glyoxylate reductase alone already led to glycolic acid production. The production was further improved by deleting genes encoding malate synthase and the cytosolic form of isocitrate dehydrogenase. The engineered S. cerevisiae strain produced up to about 1 g l-1 of glycolic acid in a medium containing d-xylose and ethanol. Similar modifications in K. lactis resulted in a much higher glycolic acid titer. In a bioreactor cultivation with d-xylose and ethanol up to 15 g l-1 of glycolic acid was obtained. Conclusions This is the first demonstration of engineering yeast to produce glycolic acid. Prior to this work glycolic acid production through the glyoxylate cycle has only been reported in bacteria. The benefit of a yeast host is the possibility for glycolic acid production also at low pH, which was demonstrated in flask cultivations. Production of glycolic acid was first shown in S. cerevisiae. To test whether a Crabtree negative yeast would be better suited for glycolic acid production we engineered K. lactis in the same way and demonstrated it to be a better host for glycolic acid production. PMID:24053654

  7. Lipid Raft-Based Membrane Compartmentation of a Plant Transport Protein Expressed in Saccharomyces cerevisiae

    PubMed Central

    Grossmann, Guido; Opekarova, Miroslava; Novakova, Linda; Stolz, Jürgen; Tanner, Widmar

    2006-01-01

    The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H+ symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H+/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms. PMID:16757742

  8. Characterization of Schizosaccharomyces pombe Malate Permease by Expression in Saccharomyces cerevisiae

    PubMed Central

    Camarasa, Carole; Bidard, Frédérique; Bony, Muriel; Barre, Pierre; Dequin, Sylvie

    2001-01-01

    In Saccharomyces cerevisiae, l-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression in S. cerevisiae of the MAE1 gene, encoding Schizosaccharomyces pombe malate permease, markedly increased l-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes), l-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters: Vmax = 8.7 nmol/mg/min; Km = 1.6 mM) and some simple diffusion of the undissociated l-malic acid (Kd = 0.057 min−1). As total l-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain. l-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the ΔpH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related to l-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibited l-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. In S. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediated l-malic acid import in S. cerevisiae, and intracellular pH remained constant. This suggests that, in S. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport. PMID:11526017

  9. Transporter engineering for improved tolerance against alkane biofuels in Saccharomyces cerevisiae

    PubMed Central

    2013-01-01

    Background Hydrocarbon alkanes, components of major fossil fuels, are considered as next-generation biofuels because their biological production has recently been shown to be possible. However, high-yield alkane production requires robust host cells that are tolerant against alkanes, which exhibit cytotoxicity. In this study, we aimed to improve alkane tolerance in Saccharomyces cerevisiae, a key industrial microbial host, by harnessing heterologous transporters that potentially pump out alkanes. Results To this end, we attempted to exploit ABC transporters in Yarrowia lipolytica based on the observation that it utilizes alkanes as a carbon source. We confirmed the increased transcription of ABC2 and ABC3 transporters upon exposure to a range of alkanes in Y. lipolytica. We then showed that the heterologous expression of ABC2 and ABC3 transporters significantly increased tolerance against decane and undecane in S. cerevisiae through maintaining lower intracellular alkane level. In particular, ABC2 transporter increased the tolerance limit of S. cerevisiae about 80-fold against decane. Furthermore, through site-directed mutagenesis for glutamate (E988 for ABC2, and E989 for ABC3) and histidine (H1020 for ABC2, and H1021 for ABC3), we provided the evidence that glutamate was essential for the activity of ABC2 and ABC3 transporters, with ATP most likely to be hydrolyzed by a catalytic carboxylate mechanism. Conclusions Here, we demonstrated that transporter engineering through expression of heterologous efflux pumps led to significantly improved tolerance against alkane biofuels in S. cerevisiae. We believe that our results laid the groundwork for developing robust alkane-producing yeast cells through transporter engineering, which will greatly aid in next-generation alkane biofuel production and recovery. PMID:23402697

  10. Local ancestry corrects for population structure in Saccharomyces cerevisiae genome-wide association studies.

    PubMed

    Diao, Liyang; Chen, Kevin C

    2012-12-01

    Genome-wide association studies (GWAS) have become an important method for mapping the genetic loci underlying complex phenotypic traits in many species. A crucial issue when performing GWAS is to control for the underlying population structure because not doing so can lead to spurious associations. Population structure is a particularly important issue in nonhuman species since it is often difficult to control for population structure during the study design phase, requiring population structure to be corrected statistically after the data have been collected. It has not yet been established if GWAS is a feasible approach in Saccharomyces cerevisiae, an important model organism and agricultural species. We thus performed an empirical study of statistical methods for controlling for population structure in GWAS using a set of 201 phenotypic traits measured in multiple resequenced strains of S. cerevisiae. We complemented our analysis of real data with an extensive set of simulations. Our main result is that a mixed linear model using the local ancestry of the strain as a covariate is effective at controlling for population structure, consistent with the mosaic structure of many S. cerevisiae strains. We further studied the evolutionary forces acting on the GWAS SNPs and found that SNPs associated with variation in phenotypic traits are enriched for low minor allele frequencies, consistent with the action of negative selection on these SNPs. Despite the effectiveness of local ancestry correction, GWAS remains challenging in highly structured populations, such as S. cerevisiae. Nonetheless, we found that, even after correcting for population structure, there is still sufficient statistical power to recover biologically meaningful associations. PMID:23023004

  11. AGAPE (Automated Genome Analysis PipelinE) for Pan-Genome Analysis of Saccharomyces cerevisiae

    PubMed Central

    Song, Giltae; Dickins, Benjamin J. A.; Demeter, Janos; Engel, Stacia; Dunn, Barbara; Cherry, J. Michael

    2015-01-01

    The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which integrates the steps of assembly, annotation, and variation calling. To assign strain-specific functional annotations, we identified genes that were not present in the reference genome. We classified these according to their presence or absence across strains and characterized each group of genes with known functional and phenotypic features. The functional roles of novel genes not found in the reference genome and associated with strains or groups of strains appear to be consistent with anticipated adaptations in specific lineages. As more S. cerevisiae strain genomes are released, our analysis can be used to collate genome data and relate it to lineage-specific patterns of genome evolution. Our new tool set will enhance our understanding of genomic and functional evolution in S. cerevisiae, and will be available to the yeast genetics and molecular biology community. PMID:25781462

  12. Utilization of waste products of dehydrated onion industry for production of fodder yeast by Saccharomyces cerevisiae.

    PubMed

    Ghonaim, S A; Abou-Zeid, A A; Abd El-Fattah, A F; Farid, M A

    1980-01-01

    One strain of Saccharomyces cerevisiae was selected from different yeasts, isolated from black strap molasses. This microorganism was cultivated on seven fermentation media for the production of protein. Medium I exhibited the highest potentiality for formation of protein. Therefore strain 1 of S. cerevisiae and medium I were used for further studies in the formation of protein. Factors controlling production of protein were explored. The required incubation period for the fermentation process was 72 hrs, while the initial pH value of the medium was 6.0. Sucrose supported the microorganism for higher production of protein (40.96%), while the best concentration of sucrose was shown to be 10.0 g/l. The best inorganic and organic nitrogen sources for protein formation were (NH4)2HPO4, (NH4)3PO4 and yeast extract, respectively. The best concentrations of (NH4)2HPO4 and yeast extract, supporting protein formation, were 5.0 g/l and 10.0 g/l, respectively. Addition of MgSO4, ZnSO4, ferrous ammonium sulphate, copper sulphate, biotin, Ca-pantothenate, thiamine, pyridoxine, and inositol to the synthetic medium did not markedly influence high level of protein formation. Glutamic acid was the best amino acid, supporting protein formation by S. cerevisiae. Onion juice was found to be a good medium, after deletion of inhibitory volatile sulphur organic compounds, for the production of protein by S. cerevisiae. Addition of (NH4)2HPO4 to the best concentration of onion juice assisted the onion medium in production of fodder yeast, containing high level of protein. Addition of MgSO4 to onion juice and (NH4)2HPO4 did not increase the total nitrogen of the biomass. Fodder yeast, produced by onion juice medium, contained more valuable ingredients than fodder yeast, produced by synthetic medium. PMID:6990654

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

    PubMed

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

    2016-05-01

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

  14. Investigation of the dominance behavior of Saccharomyces cerevisiae strains during wine fermentation.

    PubMed

    Perrone, Benedetta; Giacosa, Simone; Rolle, Luca; Cocolin, Luca; Rantsiou, Kalliopi

    2013-07-15

    During wine fermentation, different strains of Saccharomyces cerevisiae compete in the same fermenting must and dominance takes place when one strain overcomes all the others. The purpose of this study was to investigate this phenomenon by identifying S. cerevisiae strains endowed with this feature and to test them in laboratory fermentations. First, autochthonous S. cerevisiae from Nebbiolo fermentations were isolated, molecularly identified and characterized. Genetically diverse S. cerevisiae strains were subsequently subjected to physiological characterization and to micro-scale fermentation, the weight loss kinetics was measured and HPLC analysis was performed at the end of the fermentation. Then, the strains that presented good fermentation characteristics were chosen for further analysis and to determine the dominance feature. For this purpose, couples of strains were co-inoculated in Nebbiolo must and the fermentations were monitored by microbiological and chemical analysis. Two different inoculation approaches were used: co-fermentations in flasks with mixed cells and reactor co-fermentations, in which the cells from the two different strains were kept separate by means of a 0.45 μm filter membrane, which allowed the fermenting must to move freely between the two compartments. During the flask co-fermentations, a minisatellite PCR protocol was applied, in order to differentiate the two strains and determine which one was able to dominate. The protocol included a culture-dependent approach and an independent one. In the first case, DNA extraction was performed on all the colonies scraped off the plates after sampling. In the second case, DNA extraction was performed directly on the fermenting must. The strains that were able to dominate were tested against several S. cerevisiae in order to confirm this dominance behavior. Dominance was observed in the early stages of fermentation, as early as 3days. Combinations of dominant and not-dominant strains were subjected to further tests in a co-fermentation reactor system, in order to perform single-strain analysis so as to obtain a better understanding of the dominance behavior. Surprisingly, the results obtained in the flask co-fermentations were not confirmed. In fact, the two strains, one which was hypothesized to be dominant and the other not-dominant, coexisted throughout the fermentation period. The results of this study suggest that the dominant behavior of S. cerevisiae is only expressed when they sense other yeasts in the same environment. PMID:23728432

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

    PubMed Central

    Hall, Charles; Dietrich, Fred S.

    2007-01-01

    The synthesis of biotin, a vitamin required for many carboxylation reactions, is a variable trait in Saccharomyces cerevisiae. Many S. cerevisiae strains, including common laboratory strains, contain only a partial biotin synthesis pathway. We here report the identification of the first step necessary for the biotin synthesis pathway in S. cerevisiae. The biotin auxotroph strain S288c was able to grow on media lacking biotin when BIO1 and the known biotin synthesis gene BIO6 were introduced together on a plasmid vector. BIO1 is a paralog of YJR154W, a gene of unknown function and adjacent to BIO6. The nature of BIO1 illuminates the remarkable evolutionary history of the biotin biosynthesis pathway in S. cerevisiae. This pathway appears to have been lost in an ancestor of S. cerevisiae and subsequently rebuilt by a combination of horizontal gene transfer and gene duplication followed by neofunctionalization. Unusually, for S. cerevisiae, most of the genes required for biotin synthesis in S. cerevisiae are grouped in two subtelomeric gene clusters. The BIO1–BIO6 functional cluster is an example of a cluster of genes of “dispensable function,” one of the few categories of genes in S. cerevisiae that are positionally clustered. PMID:18073433

  16. SO2 protects the amino nitrogen metabolism of Saccharomyces cerevisiae under thermal stress

    PubMed Central

    Ancn?Azpilicueta, Carmen; Barriuso?Esteban, Blanca; Nieto?Rojo, Rodrigo; Aristizbal?Lpez, Nerea

    2012-01-01

    Summary Thermal stress conditions during alcoholic fermentation modify yeasts' plasma membrane since they become more hyperfluid, which results in a loss of bilayer integrity. In this study, the influence of elevated temperatures on nitrogen metabolism of a Saccharomyces cerevisiae strain was studied, as well as the effect of different concentrations of SO2 on nitrogen metabolism under thermal stress conditions. The results obtained revealed that amino nitrogen consumption was lower in the fermentation sample subjected to thermal stress than in the control, and differences in amino acid consumption preferences were also detected, especially at the beginning of the fermentation. Under thermal stress conditions, among the three doses of SO2 studied (0, 35, 70?mg?l?1 SO2), the highest dose was observed to favour amino acid utilization during the fermentative process, whereas sugar consumption presented higher rates at medium doses. PMID:22452834

  17. Rpb4 and Rpb9 mediate subpathways of transcription-coupled DNA repair in Saccharomyces cerevisiae

    PubMed Central

    Li, Shisheng; Smerdon, Michael J.

    2002-01-01

    Rpb9, a non-essential subunit of RNA polymerase II, mediates a transcription-coupled repair (TCR) subpathway in Saccharomyces cerevisiae. This subpathway initiates at the same upstream site as the previously identified Rad26 subpathway. However, the Rpb9 subpathway operates more effectively in the coding region than in the region upstream of the transcription start site, whereas the Rad26 subpathway operates equally in the two regions. Rpb4, another non-essential subunit of RNA polymerase II, plays a dual role in regulating the two subpathways, suppressing the Rpb9 subpathway and facilitating the Rad26 subpathway. Simultaneous deletion of RPB9 and RAD26 genes completely abolishes TCR in both the coding and upstream regions, indicating that no other TCR subpathway exists in RNA polymerase II-transcribed genes. PMID:12411509

  18. Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens.

    PubMed Central

    Huang, Z; Dostal, L; Rosazza, J P

    1993-01-01

    Saccharomyces cerevisiae (dry baker's yeast) and Pseudomonas fluorescens were used to convert trans-ferulic acid into 4-hydroxy-3-methoxystyrene in 96 and 89% yields, respectively. The metabolites were isolated by solid-phase extraction and analyzed by thin-layer chromatography and high-performance liquid chromatography. The identities of the metabolites were determined by 1H- and 13C-nuclear magnetic resonance spectroscopy and by mass spectrometry. The mechanism of the decarboxylation of ferulic acid was investigated by measuring the degree and position of deuterium incorporated into the styrene derivative from D2O by mass spectrometry and by both proton and deuterium nuclear magnetic resonance spectroscopies. Resting cells of baker's yeast reduced ferulic acid to 4-hydroxy-3-methoxyphenylpropionic acid in 54% yield when incubations were under an argon atmosphere. PMID:8395165

  19. Effects of ubiquinone derivatives on the mitochondrial unselective channel of Saccharomyces cerevisiae.

    PubMed

    Gutiérrez-Aguilar, Manuel; López-Carbajal, Helga M; Uribe-Alvarez, Cristina; Espinoza-Simón, Emilio; Rosas-Lemus, Mónica; Chiquete-Félix, Natalia; Uribe-Carvajal, Salvador

    2014-12-01

    Ubiquinone derivatives modulate the mammalian mitochondrial Permeability Transition Pore (PTP). Yeast mitochondria harbor a similar structure: the respiration- and ATP-induced Saccharomyces cerevisiae Mitochondrial Unselective Channel ( Sc MUC). Here we show that decylubiquinone, a well-characterized inhibitor of the PTP, suppresses Sc MUC opening in diverse strains and independently of respiratory chain modulation or redox-state. We also found that naturally occurring derivatives such as hexaprenyl and decaprenyl ubiquinones lacked effects on the Sc MUC. The PTP-inactive ubiquinone 5 (Ub5) promoted the Sc MUC-independent activation of the respiratory chain in most strains tested. In an industrial strain however, Ub5 blocked the protection elicited by dUb. The results indicate the presence of a ubiquinone-binding site in the Sc MUC. PMID:25465614

  20. Metabolic response of Danaüs archippus and Saccharomyces cerevisiae to weak oscillatory magnetic fields

    NASA Astrophysics Data System (ADS)

    Russell, D. N.; Webb, S. J.

    1981-09-01

    Respiration of the insect larva, Danaüs archippus, and the yeast, Saccharomyces cerevisiae, in log phase has been monitored before and after an oscillatory magnetic insult of 0.005 Gauss rms amplitude and 40 50 min duration. Frequencies used were 10 16 Hz for the insect and 100 200 Hz for the yeast. Depression of as much as 30% in metabolic rate has been found to occur immediately after the field is both imposed and eliminated with a general recovery over the 30-min period thereafter both in and out of the imposed field, although complete recovery to original levels may take much longer. Evidence is given that the response may depend on the frequency pattern used. This data is used to formulate an hypothesis whereby changes in the geomagnetic field variability pattern may act as a biochronometric zeitgeber.