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A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein.
Gleave, Emma S; Schmidt, Helgo; Carter, Andrew P
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
Actin from Saccharomyces cerevisiae.
Greer, C; Schekman, R
Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth. Images PMID:6217414
Peptidase activities in Saccharomyces cerevisiae.
Rose, B; Becker, J M; Naider, F
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
Metabolic Engineering of Saccharomyces cerevisiae
Ostergaard, Simon; Olsson, Lisbeth; Nielsen, Jens
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
Cadmium biosorption by Saccharomyces cerevisiae
Volesky, B.; May, H.; Holan, Z.R. )
Cadmium uptake by nonliving and resting cells of Saccharomyces cerevisiae obtained from aerobic or anaerobic cultures from pure cadmium-bearing solutions was examined. The highest cadmium uptake exceeding 70 mg Cd/g was observed with aerobic baker's yeast biomass from the exponential growth phase. Nearly linear sorption isotherms featured by higher sorbing resting cells together with metal deposits localized exclusively in vacuoles indicate the possibility of a different metal-sequestering mechanism when compared to dry nonliving yeasts which did not usually accumulate more than 20 mg Cd/g. The uptake of cadmium was relatively fast, 75% of the sorption completed in less than 5 min.
PET genes of Saccharomyces cerevisiae.
Tzagoloff, A; Dieckmann, C L
We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae. PMID:2215420
Methionine catabolism in Saccharomyces cerevisiae.
Perpète, Philippe; Duthoit, Olivier; De Maeyer, Simon; Imray, Louise; Lawton, Andrew I; Stavropoulos, Konstantinos E; Gitonga, Virginia W; Hewlins, Michael J E; Dickinson, J Richard
The catabolism of methionine to methionol and methanethiol in Saccharomyces cerevisiae was studied using (13)C NMR spectroscopy, GC-MS, enzyme assays and a number of mutants. Methionine is first transaminated to alpha-keto-gamma-(methylthio)butyrate. Methionol is formed by a decarboxylation reaction, which yields methional, followed by reduction. The decarboxylation is effected specifically by Ydr380wp. Methanethiol is formed from both methionine and alpha-keto-gamma-(methylthio)butyrate by a demethiolase activity. In all except one strain examined, demethiolase was induced by the presence of methionine in the growth medium. This pathway results in the production of alpha-ketobutyrate, a carbon skeleton, which can be re-utilized. Hence, methionine catabolism is more complex and economical than the other amino acid catabolic pathways in yeast, which use the Ehrlich pathway and result solely in the formation of a fusel alcohol. PMID:16423070
Glucose repression in Saccharomyces cerevisiae.
Kayikci, Ömur; Nielsen, Jens
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
Postreplication repair in Saccharomyces cerevisiae
Resnick, M.A.; Boyce, J.; Cox, B.
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.
Glucose repression in Saccharomyces cerevisiae
Kayikci, Ömur; Nielsen, Jens
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
Fatal Saccharomyces Cerevisiae Aortic Graft Infection
NASA Technical Reports Server (NTRS)
Meyer, Michael (Technical Monitor); Smith, Davey; Metzgar, David; Wills, Christopher; Fierer, Joshua
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.
Saccharomyces cerevisiae osteomyelitis in an immunocompetent baker.
Seng, Piseth; Cerlier, Alexandre; Cassagne, Carole; Coulange, Mathieu; Legré, Regis; Stein, Andreas
Invasive infection caused by Saccharomyces cerevisiae is rare. We report the first case of osteomyelitis caused by S. cerevisiae (baker's yeast) in a post-traumatic patient. The clinical outcome was favorable after surgical debridement, prolonged antifungal treatment and hyperbaric oxygen therapy. PMID:27347482
Proteomics of Saccharomyces cerevisiae Organelles*
Wiederhold, Elena; Veenhoff, Liesbeth M.; Poolman, Bert; Slotboom, Dirk Jan
Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them with other large scale studies on protein localization and abundance. We evaluate the completeness and reliability of the organelle proteomics studies. Reliability depends on the purity of the organelle preparations, which unavoidably contain (small) amounts of contaminants from different locations. Quantitative proteomics methods can be used to distinguish between true organellar constituents and contaminants. Completeness is compromised when loosely or dynamically associated proteins are lost during organelle preparation and also depends on the sensitivity of the analytical methods for protein detection. There is a clear trend in the data from the 18 organelle proteomics studies showing that proteins of low abundance frequently escape detection. Proteins with unknown function or cellular abundance are also infrequently detected, indicating that these proteins may not be expressed under the conditions used. We discuss that the yeast organelle proteomics studies provide powerful lead data for further detailed studies and that methodological advances in organelle preparation and in protein detection may help to improve the completeness and reliability of the data. PMID:19955081
Local Nanomechanical Motion of the Cell Wall of Saccharomyces cerevisiae
NASA Astrophysics Data System (ADS)
Pelling, Andrew E.; Sehati, Sadaf; Gralla, Edith B.; Valentine, Joan S.; Gimzewski, James K.
We demonstrate that the cell wall of living Saccharomyces cerevisiae (baker's yeast) exhibits local temperature-dependent nanomechanical motion at characteristic frequencies. The periodic motions in the range of 0.8 to 1.6 kHz with amplitudes of ~3 nm were measured using the cantilever of an atomic force microscope (AFM). Exposure of the cells to a metabolic inhibitor causes the periodic motion to cease. From the strong frequency dependence on temperature, we derive an activation energy of 58 kJ/mol, which is consistent with the cell's metabolism involving molecular motors such as kinesin, dynein, and myosin. The magnitude of the forces observed (~10 nN) suggests concerted nanomechanical activity is operative in the cell.
21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.
Code of Federal Regulations, 2012 CFR
... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 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...
21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.
Code of Federal Regulations, 2011 CFR
... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 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...
21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.
Code of Federal Regulations, 2014 CFR
... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 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...
21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.
Code of Federal Regulations, 2013 CFR
... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 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...
Asymmetrical division of Saccharomyces cerevisiae.
Lord, P G; Wheals, A E
The unequal division model proposed for budding yeast (L. H. Hartwell and M. W. Unger, J. Cell Biol. 75:422-435, 1977) was tested by bud scar analyses of steady-state exponential batch cultures of Saccharomyces cerevisiae growing at 30 degrees C at 19 different rates, which were obtained by altering the carbon source. The analyses involved counting the number of bud scars, determining the presence or absence of buds on at least 1,000 cells, and independently measuring the doubling times (gamma) by cell number increase. A number of assumptions in the model were tested and found to be in good agreement with the model. Maximum likelihood estimates of daughter cycle time (D), parent cycle time (P), and the budded phase (B) were obtained, and we concluded that asymmetrical division occurred at all growth rates tested (gamma, 75 to 250 min). D, P, and B are all linearly related to gamma, and D, P, and gamma converge to equality (symmetrical division) at gamma = 65 min. Expressions for the genealogical age distribution for asymmetrically dividing yeast cells were derived. The fraction of daughter cells in steady-state populations is e-alpha P, and the fraction of parent cells of age n (where n is the number of buds that a cell has produced) is (e-alpha P)n-1(1-e-alpha P)2, where alpha = IN2/gamma; thus, the distribution changes with growth rate. The frequency of cells with different numbers of bud scars (i.e., different genealogical ages) was determined for all growth rates, and the observed distribution changed with the growth rate in the manner predicted. In this haploid strain new buds formed adjacent to the previous buds in a regular pattern, but at slower growth rates the pattern was more irregular. The median volume of the cells and the volume at start in the cell cycle both increased at faster growth rates. The implications of these findings for the control of the cell cycle are discussed. PMID:6991494
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...
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.
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…
Preparation of Saccharomyces cerevisiae expression plasmids.
Drew, David; Kim, Hyun
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
[Urinary infection by Saccharomyces cerevisiae: Emerging yeast?].
Elkhihal, B; Elhalimi, M; Ghfir, B; Mostachi, A; Lyagoubi, M; Aoufi, S
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
Sporulation in the Budding Yeast Saccharomyces cerevisiae
Neiman, Aaron M.
In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae. PMID:22084423
Biosorption of heavy metals by Saccharomyces cerevisiae.
Volesky, B; May-Phillips, H A
Abundant and common yeast biomass has been examined for its capacity to sequester heavy metals from dilute aqueous solutions. Live and non-living biomass of Saccharomyces cerevisiae differs in the uptake of uranium, zinc and copper at the optimum pH 4-5. Culture growth conditions can influence the biosorbent metal uptake capacity which normally was: living and non-living brewer's yeast: U > Zn > Cd > Cu; non-living baker's yeast: Zn > (Cd) > U > Cu; living baker's yeast: Zn > Cu approximately (Cd) > U. Non-living brewer's yeast biomass accumulated 0.58 mmol U/g. The best biosorbent of zinc was non-living baker's yeast (approximately 0.56 mmol Zn/g). Dead cells of S. cerevisiae removed approximately 40% more uranium or zinc than the corresponding live cultures. Biosorption of uranium by S. cerevisiae was a rapid process reaching 60% of the final uptake value within the first 15 min of contact. Its deposition differing from that of other heavy metals more associated with the cell wall, uranium was deposited as fine needle-like crystals both on the inside and outside of the S. cerevisiae cells. PMID:7765919
Calcium control of Saccharomyces cerevisiae actin assembly.
Greer, C; Schekman, R
Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo. Images PMID:6757718
Progress in Metabolic Engineering of Saccharomyces cerevisiae
Nevoigt, Elke
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
Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.
Fossati, Elena; Narcross, Lauren; Ekins, Andrew; Falgueyret, Jean-Pierre; Martin, Vincent J J
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
Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae
Fossati, Elena; Narcross, Lauren; Ekins, Andrew; Falgueyret, Jean-Pierre; Martin, Vincent J. J.
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
Myo-inositol transport in Saccharomyces cerevisiae.
Nikawa, J; Nagumo, T; Yamashita, S
myo-Inositol uptake in Saccharomyces cerevisiae was dependent on temperature, time, and substrate concentration. The transport obeyed saturation kinetics with an apparent Km for myo-inositol of 0.1 mM, myo-Inositol analogs, such as scyllo-inositol, 2-inosose, mannitol, and 1,2-cyclohexanediol, had no effect on myo-inositol uptake, myo-Inositol uptake required metabolic energy. Removal of D-glucose resulted in a loss of activity, and azide and cyanide ions were inhibitory. In the presence of D-glucose, myo-inositol was accumulated in the cells against a concentration gradient. A myo-inositol transport mutant was isolated from UV-mutagenized S. cerevisiae cells using the replica-printing technique. The defect in myo-inositol uptake was due to a single nuclear gene mutation. The activities of L-serine and D-glucose transport were not affected by the mutation. Thus it was shown that S. cerevisiae grown under the present culture conditions possessed a single and specific myo-inositol transport system. myo-Inositol transport activity was reduced by the addition of myo-inositol to the culture medium. The activity was reversibly restored by the removal of myo-inositol from the medium. This restoration of activity was completely abolished by cycloheximide. PMID:7040334
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.
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.
Synchronization of the Budding Yeast Saccharomyces cerevisiae.
Foltman, Magdalena; Molist, Iago; Sanchez-Diaz, Alberto
A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle. PMID:26519319
Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae.
Shen, Qing-Ji; Kassim, Hakimi; Huang, Yong; Li, Hui; Zhang, Jing; Li, Guang; Wang, Peng-Ye; Yan, Jun; Ye, Fangfu; Liu, Ji-Long
Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filament-forming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frame-GFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases (Ura7p and Ura8p) and two asparagine synthetases (Asn1p and Asn2p) form filaments both in the cytoplasm and in the nucleus. Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated. PMID:27312010
Killer systems of the yeast Saccharomyces cerevisiae
Nesterova, G.F.
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.
Force Sensitivity in Saccharomyces cerevisiae Flocculins
Chan, Cho X. J.; El-Kirat-Chatel, Sofiane; Joseph, Ivor G.; Jackson, Desmond N.; Ramsook, Caleen B.; Dufrêne, Yves F.
ABSTRACT Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca2+, yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends
Force Sensitivity in Saccharomyces cerevisiae Flocculins.
Chan, Cho X J; El-Kirat-Chatel, Sofiane; Joseph, Ivor G; Jackson, Desmond N; Ramsook, Caleen B; Dufrêne, Yves F; Lipke, Peter N
Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca(2+), yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on
Prediction of Saccharomyces cerevisiae replication origins
Breier, Adam M; Chatterji, Sourav; Cozzarelli, Nicholas R
Background Autonomously replicating sequences (ARSs) function as replication origins in Saccharomyces cerevisiae. ARSs contain the 17 bp ARS consensus sequence (ACS), which binds the origin recognition complex. The yeast genome contains more than 10,000 ACS matches, but there are only a few hundred origins, and little flanking sequence similarity has been found. Thus, identification of origins by sequence alone has not been possible. Results We developed an algorithm, Oriscan, to predict yeast origins using similarity to 26 characterized origins. Oriscan used 268 bp of sequence, including the T-rich ACS and a 3' A-rich region. The predictions identified the exact location of the ACS. A total of 84 of the top 100 Oriscan predictions, and 56% of the top 350, matched known ARSs or replication protein binding sites. The true accuracy was even higher because we tested 25 discrepancies, and 15 were in fact ARSs. Thus, 94% of the top 100 predictions and an estimated 70% of the top 350 were correct. We compared the predictions to corresponding sequences in related Saccharomyces species and found that the ACSs of experimentally supported predictions show significant conservation. Conclusions The high accuracy of the predictions indicates that we have defined near-sufficient conditions for ARS activity, the A-rich region is a recognizable feature of ARS elements with a probable role in replication initiation, and nucleotide sequence is a reliable predictor of yeast origins. Oriscan detected most origins in the genome, demonstrating previously unrecognized generality in yeast replication origins and significant discriminatory power in the algorithm. PMID:15059255
Stationary phase in the yeast Saccharomyces cerevisiae.
Werner-Washburne, M; Braun, E; Johnston, G C; Singer, R A
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
Regulation of Phosphatidylcholine Biosynthesis in Saccharomyces cerevisiae
Waechter, Charles J.; Lester, Robert L.
Evidence is presented which indicates that the biosynthesis of phosphatidylcholine by the methylation pathway in growing cultures of Saccharomyces cerevisiae is repressed by the presence of choline in the growth medium. This result, obtained previously for glucose-grown cells, was also observed for lactate-grown cells, of which half of the phosphatidylcholine is mitochondrial. A respiration-deficient mutant of the parent wild-type strain has been studied, and its inability to form functional mitochondria cannot be due to an impaired methylation pathway, as it has been shown to incorporate 14C-CH3-methionine into all of the methylated glycerophosphatides. The incorporation rate is depressed by the inclusion of 1 mm choline in the growth medium, suggesting a regulatory effect similar to that demonstrated for the wild-type strain. The effects of choline on the glycerophospholipid composition of lactate and glucose-grown cells is presented. The repressive effects of the two related bases, mono- and dimethylethanolamine, were examined, and reduced levels of 14C-CH3-methionine incorporation were found for cells grown in the presence of these bases. The effect of choline on the methylation rates is reversible and glucosegrown cells regain the nonrepressed level of methylation activity in 60 to 80 min after removal of choline from the growth medium. Images PMID:5547992
Inositol-Requiring Mutants of SACCHAROMYCES CEREVISIAE
Culbertson, Michael R.; Henry, Susan A.
Fifty-two inositol-requiring mutants of Saccharomyces cerevisiae were isolated following mutagenesis with ethyl methanesulfonate. Complementation and tetrad analysis revealed ten major complementation classes, representing ten independently segregating loci (designated ino1 through ino10) which recombined freely with their respective centromeres. Members of any given complementation class segregated as alleles of a single locus. Thirteen complementation subclasses were identified among thirty-six mutants which behaved as alleles of the ino1 locus. The complementation map for these mutants was circular.—Dramatic cell viability losses indicative of unbalanced growth were observed in liquid cultures of representative mutants under conditions of inositol starvation. Investigation of the timing, kinetics, and extent of cell death revealed that losses in cell viability in the range of 2-4 log orders could be prevented by the addition of inositol to the medium or by disruption of protein synthesis with cycloheximide. Mutants defective in nine of the ten loci identified in this study displayed these unusual characteristics. The results suggest an important physiological role for inositol that may be related to its cellular localization and function in membrane phospholipids. The possibility is discussed that inositol deficiency initiates the process of unbalanced growth leading to cell death through the loss of normal assembly, function, or integrity of biomembranes.—Part of this work has been reported in preliminary form (Culbertson and Henry 1974). PMID:1093935
Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning
Stuchell-Brereton, Melissa D.; Siglin, Amanda; Li, Jun; Moore, Jeffrey K.; Ahmed, Shubbir; Williams, John C.; Cooper, John A.
Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function. PMID:21633107
21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.
Code of Federal Regulations, 2010 CFR
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. 866.5785 Section 866.5785 Food and Drugs FOOD AND DRUG ADMINISTRATION... controls). The special control is FDA's "Guidance for Industry and FDA Reviewers: Class II Special...
Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae.
Comitini, Francesca; Gobbi, Mirko; Domizio, Paola; Romani, Cristina; Lencioni, Livio; Mannazzu, Ilaria; Ciani, Maurizio
Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation). PMID:21569929
Regulation of Cation Balance in Saccharomyces cerevisiae
Cyert, Martha S.; Philpott, Caroline C.
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
Analysis of the Saccharomyces cerevisiae proteome with PeptideAtlas
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
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
Invasive Saccharomyces cerevisiae infection: a friend turning foe?
Pillai, Unnikrishnan; Devasahayam, Joe; Kurup, Aparna Narayana; Lacasse, Alexandre
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
A global topology map of the Saccharomyces cerevisiae membrane proteome
NASA Astrophysics Data System (ADS)
Kim, Hyun; Melén, Karin; Österberg, Marie; von Heijne, Gunnar
The yeast Saccharomyces cerevisiae is, arguably, the best understood eukaryotic model organism, yet comparatively little is known about its membrane proteome. Here, we report the cloning and expression of 617 S. cerevisiae membrane proteins as fusions to a C-terminal topology reporter and present experimentally constrained topology models for 546 proteins. By homology, the experimental topology information can be extended to 15,000 membrane proteins from 38 fully sequenced eukaryotic genomes. membrane proteins | membrane proteomics | yeast
[Tolerance of Saccharomyces cerevisiae to monoterpenes--a review].
Liu, Jidong; Zhou, Jingwen; Chen, Jian
Tolerance of Saccharomyces cerevisiae to monoterpenes is important in both metabolic engineering of the yeast to produce these chemicals de novo and efficient use of biomass containing these chemicals. Understanding the mechanisms in the tolerance of S. cerevisiae to monoterpenes could facilitate the construction of yeast strains with enhanced monoterpenes resistance, and therefore improve related bioprocesses. Monoterpenes could disturb the redox balance in S. cerevisiae, therefore increase the accumulation of reactive oxygen species (ROS) and result in cell death. S. cerevisiae has to systematically improve its antioxidative ability to deal with the ROS induced damage. The current review summarized the recent developments in demonstration of the tolerance of S. cerevisiae to different typical monoterpenes mainly from the aspect of the antioxidative mechanisms. Based on the analysis of the previous works, further attempts to demonstrate the mechanisms were proposed. PMID:24028054
Synthesis of ribosomes in Saccharomyces cerevisiae.
Warner, J R
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
Interaction between Hanseniaspora uvarum and Saccharomyces cerevisiae during alcoholic fermentation.
Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio
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
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...
Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"
ERIC Educational Resources Information Center
Deutch, Charles E.; Marshall, Pamela A.
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…
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...
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
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.
Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption.
Scalcinati, Gionata; Otero, José Manuel; Van Vleet, Jennifer R H; Jeffries, Thomas W; Olsson, Lisbeth; Nielsen, Jens
Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S. cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source. PMID:22487265
Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.
Liu, Hong-Zhi; Wang, Qiang; Liu, Xiao-Yong; Tan, Sze-Sze
Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P < 0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (P < 0.05). The spaceflight mutant of S. cerevisiae 2.0016 showed 46.7%, 62.6%, and 146.0% increment in biomass, cell wall thickness and beta-glucan content, respectively, when compared to the ground strain. Moreover, growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production. PMID:18797865
Mutagenesis protocols in Saccharomyces cerevisiae by in vivo overlap extension.
Alcalde, Miguel
A high recombination frequency and its ease of manipulation has made Saccharomyces cerevisiae a unique model eukaryotic organism to study homologous recombination. Indeed, the well-developed recombination machinery in S. cerevisiae facilitates the construction of mutant libraries for directed evolution experiments. In this context, in vivo overlap extension (IVOE) is a particularly attractive protocol that takes advantage of the eukaryotic apparatus to carry out combinatorial saturation mutagenesis, site-directed recombination or site-directed mutagenesis, avoiding ligation steps and additional PCR reactions that are common to standard in vitro protocols. PMID:20676972
The reference genome sequence of Saccharomyces cerevisiae: then and now.
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
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
Saccharomyces cerevisiae: a sexy yeast with a prion problem.
Kelly, Amy C; Wickner, Reed B
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
The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now
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
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
Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.
Rensing, C; Kües, U; Stahl, U; Nies, D H; Friedrich, B
The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity. Images PMID:1735719
The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific.
Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio
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
The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific
Wang, Chunxiao; Mas, Albert; Esteve-Zarzoso, Braulio
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
Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains.
Šuranská, Hana; Vránová, Dana; Omelková, Jiřina
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
Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains
Šuranská, Hana; Vránová, Dana; Omelková, Jiřina
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
... believed to be a strain of Saccharomyces cerevisiae (baker's yeast). Saccharomyces boulardii is used as medicine. Saccharomyces boulardii ... Hansen CBS 5926), Probiotic, Probiotique, Saccharomyces, Saccharomyces boulardii, Saccharomyces Cerevisiae, S. Boulardii.
Saccharomyces cerevisiae: a nomadic yeast with no niche?
Goddard, Matthew R.; Greig, Duncan
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
Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae.
Mormeneo, María; Pastor, Fi Javier; Zueco, Jesús
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
A Saccharomyces cerevisiae Internet protein resource now available.
Latter, G I; Boutell, T; Monardo, P J; Kobayashi, R; Futcher, B; Mclaughlin, C S; Garrels, J I
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
Gains and Losses of Transcription Factor Binding Sites in Saccharomyces cerevisiae and Saccharomyces paradoxus.
Schaefke, Bernhard; Wang, Tzi-Yuan; Wang, Chuen-Yi; Li, Wen-Hsiung
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
Interorganelle signaling is a determinant of longevity in Saccharomyces cerevisiae.
Kirchman, P A; Kim, S; Lai, C Y; Jazwinski, S M
Replicative capacity, which is the number of times an individual cell divides, is the measure of longevity in the yeast Saccharomyces cerevisiae. In this study, a process that involves signaling from the mitochondrion to the nucleus, called retrograde regulation, is shown to determine yeast longevity, and its induction resulted in postponed senescence. Activation of retrograde regulation, by genetic and environmental means, correlated with increased replicative capacity in four different S. cerevisiae strains. Deletion of a gene required for the retrograde response, RTG2, eliminated the increased replicative capacity. RAS2, a gene previously shown to influence longevity in yeast, interacts with retrograde regulation in setting yeast longevity. The molecular mechanism of aging elucidated here parallels the results of genetic studies of aging in nematodes and fruit flies, as well as the caloric restriction paradigm in mammals, and it underscores the importance of metabolic regulation in aging, suggesting a general applicability. PMID:10224252
Advanced biofuel production by the yeast Saccharomyces cerevisiae.
Buijs, Nicolaas A; Siewers, Verena; Nielsen, Jens
Replacement of conventional transportation fuels with biofuels will require production of compounds that can cover the complete fuel spectrum, ranging from gasoline to kerosene. Advanced biofuels are expected to play an important role in replacing fossil fuels because they have improved properties compared with ethanol and some of these may have the energy density required for use in heavy duty vehicles, ships, and aviation. Moreover, advanced biofuels can be used as drop-in fuels in existing internal combustion engines. The yeast cell factory Saccharomyces cerevisiae can be turned into a producer of higher alcohols (1-butanol and isobutanol), sesquiterpenes (farnesene and bisabolene), and fatty acid ethyl esters (biodiesel), and here we discusses progress in metabolic engineering of S. cerevisiae for production of these advanced biofuels. PMID:23628723
Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae
Batt, C.A.; Carvallo, S.; Easson, D.D.; Akedo, M.; Sinskey, A.J.
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.
Purification of fluorescently labeled Saccharomyces cerevisiae Spindle Pole Bodies
Davis, Trisha N.
Centrosomes are components of the mitotic spindle responsible for organizing microtubules and establishing a bipolar spindle for accurate chromosome segregation. In budding yeast, Saccharomyces cerevisiae, the centrosome is called the spindle pole body, a highly organized tri-laminar structure embedded in the nuclear envelope. Here we describe a detailed protocol for the purification of fluorescently labeled spindle pole bodes from S. cerevisiae. Spindle pole bodies are purified from yeast using a TAP-tag purification followed by velocity sedimentation. This highly reproducible TAP-tag purification method improves upon previous techniques and expands the scope of in vitro characterization of yeast spindle pole bodies. The genetic flexibility of this technique allows for the study of spindle pole body mutants as well as the study of spindle pole bodies during different stages of the cell cycle. The ease and reproducibility of the technique makes it possible to study spindle pole bodies using a variety of biochemical, biophysical, and microscopic techniques. PMID:27193850
Energy-dependent effects of resveratrol in Saccharomyces cerevisiae.
Madrigal-Perez, Luis Alberto; Canizal-Garcia, Melina; González-Hernández, Juan Carlos; Reynoso-Camacho, Rosalia; Nava, Gerardo M; Ramos-Gomez, Minerva
The metabolic effects induced by resveratrol have been associated mainly with the consumption of high-calorie diets; however, its effects with standard or low-calorie diets remain unclear. To better understand the interactions between resveratrol and cellular energy levels, we used Saccharomyces cerevisiae as a model. Herein it is shown that resveratrol: (a) decreased cell viability in an energy-dependent manner; (b) lessening of cell viability occurred specifically when cells were under cellular respiration; and (c) inhibition of oxygen consumption in state 4 occurred at low and standard energy levels, whereas at high energy levels oxygen consumption was promoted. These findings indicate that the effects of resveratrol are dependent on the cellular energy status and linked to metabolic respiration. Importantly, our study also revealed that S. cerevisiae is a suitable and useful model to elucidate the molecular targets of resveratrol under different nutritional statuses. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26945517
ROG1 encodes a monoacylglycerol lipase in Saccharomyces cerevisiae.
Vishnu Varthini, Lakshmanaperumal; Selvaraju, Kandasamy; Srinivasan, Malathi; Nachiappan, Vasanthi
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
Mutants of the Formyltetrahydrofolate Interconversion Pathway of SACCHAROMYCES CEREVISIAE
McKenzie, K. Q.; Jones, Elizabeth W.
Thirteen mutants of Saccharomyces cerevisiae that lack one or more of the three enzyme activities of the pathway for interconversion of tetrahydrofolate coenzymes at the formate level of oxidation have been isolated. They do not require adenine. All fail to complement mutations in the ade3 locus. Mutations that greatly reduce activity for one enzyme also reduce activity for the other two interconversion enzymes. The three enzyme activities cochromatograph on TEAE-cellulose columns. A mutation that eliminates synthetase activity also alters the chromatographic behavior of the remaining cyclohydrolase and dehydrogenase activities. It is suggested that the three activities reside in an enzyme complex encoded by the ade3 locus. PMID:328341
Immobilized cell cross-flow reactor. [Saccharomyces cerevisiae
Chotani, G.K.; Constantinides, A.
A cross-current flow reactor was operated using sodium alginate gel entrapped yeast cells (Saccharomyces cerevisiae) under growth conditions. Micron-sized silica, incorporated into the biocatalyst particles (1 mm mean diameter) improved mechanical strength and internal surface adhesion. The process showed decreased productivity and stability at 35/sup 0/C compared to the normal study done at 30/sup 0/C. The increased number of cross flows diminish the product inhibition effect. The residence time distribution shows that the cross-flow bioreactor system can be approximated to either a train of backmixed fermentors in series or a plug flow fermentor with moderate axial dispersion.
Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates.
Ogden, R C; Lee, M C; Knapp, G
The primary sequences of all the tRNA precursors which contain intervening sequences and which accumulate in the Saccharomyces cerevisiae rnal mutant are presented. A combination of DNA and RNA sequence analysis has led to elucidation of the primary sequence of four hitherto uncharacterized precursors. The location of the intervening sequence has in all cases been unambiguously determined by analysis of the intermediates in the splicing reaction. Secondary structures based upon the tRNA cloverleaf are shown for all the tRNA precursors and discussed with respect to common recognition by the yeast splicing endonuclease. Images PMID:6096826
SACCHAROMYCES CEREVISIAE Recessive Suppressor That Circumvents Phosphatidylserine Deficiency
Atkinson, Katharine D.
Phenotypic reversion of six independent Saccharomyces cerevisiae cho1 mutants was shown to be due predominantly to mutation of an unlinked gene, eam1. The eam1 gene was located very close to ino1 on chromosome X by meiotic tetrad analysis. Recessive eam1 mutations did not correct the primary cho1 defect in phosphatidylserine synthesis but made endogenous ethanolamine available for sustained nitrogenous phospholipid synthesis. A novel biochemical contribution to nitrogenous lipid synthesis is indicated by the eam1 mutants. PMID:17246236
RNAi-Assisted Genome Evolution (RAGE) in Saccharomyces cerevisiae.
Si, Tong; Zhao, Huimin
RNA interference (RNAi)-assisted genome evolution (RAGE) applies directed evolution principles to engineer Saccharomyces cerevisiae genomes. Here, we use acetic acid tolerance as a target trait to describe the key steps of RAGE. Briefly, iterative cycles of RNAi screening are performed to accumulate multiplex knockdown modifications, enabling directed evolution of the yeast genome and continuous improvement of a target phenotype. Detailed protocols are provided on the reconstitution of RNAi machinery, creation of genome-wide RNAi libraries, identification and integration of beneficial knockdown cassettes, and repeated RAGE cycles. PMID:27581294
Isobutanol production from D-xylose by recombinant Saccharomyces cerevisiae.
Brat, Dawid; Boles, Eckhard
Simultaneous overexpression of an optimized, cytosolically localized valine biosynthesis pathway together with overexpression of xylose isomerase XylA from Clostridium phytofermentans, transaldolase Tal1 and xylulokinase Xks1 enabled recombinant Saccharomyces cerevisiae cells to complement the valine auxotrophy of ilv2,3,5 triple deletion mutants for growth on D-xylose as the sole carbon source. Moreover, after additional overexpression of ketoacid decarboxylase Aro10 and alcohol dehydrogenase Adh2, the cells were able to ferment D-xylose directly to isobutanol. PMID:23279585
Differential repair of UV damage in Saccharomyces cerevisiae.
Terleth, C; van Sluis, C A; van de Putte, P
Preferential repair of UV-induced damage is a phenomenon by which mammalian cells might enhance their survival. This paper presents the first evidence that preferential repair occurs in the lower eukaryote Saccharomyces cerevisiae. Moreover an unique approach is reported to compare identical sequences present on the same chromosome and only differing in expression. We determined the removal of pyrimidine dimers from two identical alpha-mating type loci and we were able to show that the active MAT alpha locus is repaired preferentially to the inactive HML alpha locus. In a sir-3 mutant, in which both loci are active this preference is not observed. Images PMID:2664708
Use of bimolecular fluorescence complementation in yeast Saccharomyces cerevisiae.
Skarp, Kari-Pekka; Zhao, Xueqiang; Weber, Marion; Jantti, Jussi
Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. Bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for use of BiFC in the yeast Saccharomyces cerevisiae. PMID:19066026
Expression of acylphosphatase in Saccharomyces cerevisiae enhances ethanol fermentation rate
Raugei, G.; Modesti, A.; Magherini, F.
Previous experiments in vitro have demonstrated the ability of acylphosphatase to increase the rate of glucose fermentation in yeast. To evaluate the possibility of increasing fermentation in vivo also, a chemically synthesized DNA sequence coding for human muscle acylphosphatase was expressed at high level in Saccharomyces cerevisiae. Ethanol production was measured in these engineered strains in comparison with a control. Acylphosphatase expression strongly increased the rate of ethanol production both in aerobic and anaerobic culture. This finding may be potentially important for the development of more efficient industrial fermentation processes. 20 refs., 5 figs.
Sucrose and Saccharomyces cerevisiae: a relationship most sweet.
Marques, Wesley Leoricy; Raghavendran, Vijayendran; Stambuk, Boris Ugarte; Gombert, Andreas Karoly
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
Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae.
Van Hoek, P; Modesti, A; Ramponi, G; Kötter, P; van Dijken, J P; Pron, J T
Human acylphosphatase (h-AP, EC 188.8.131.52) 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
Genetic variation of the repeated MAL loci in natural populations of Saccharomyces cerevisiae and Saccharomyces paradoxus.
Naumov, G I; Naumova, E S; Michels, C A
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
Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.
Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho
Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions. PMID:27221740
A screen for dynein synthetic lethals in Aspergillus nidulans identifies spindle assembly checkpoint genes and other genes involved in mitosis.
Efimov, V P; Morris, N R
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
Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration
DeZwaan, Todd M.; Ellingson, Eric; Pellman, David; Roof, David M.
Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus. PMID:9281581
Membrane trafficking in the yeast Saccharomyces cerevisiae model.
Feyder, Serge; De Craene, Johan-Owen; Bär, Séverine; Bertazzi, Dimitri L; Friant, Sylvie
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
Multiparameter analysis of apoptosis in puromycin-treated Saccharomyces cerevisiae.
Citterio, Barbara; Albertini, Maria Cristina; Ghibelli, Lina; Falcieri, Elisabetta; Battistelli, Michela; Canonico, Barbara; Rocchi, Marco B L; Teodori, Laura; Ciani, Maurizio; Piatti, Elena
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
Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae
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.
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
Biogeographical characterization of Saccharomyces cerevisiae wine yeast by molecular methods
Tofalo, Rosanna; Perpetuini, Giorgia; Schirone, Maria; Fasoli, Giuseppe; Aguzzi, Irene; Corsetti, Aldo; Suzzi, Giovanna
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
Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts.
Makarow, M
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
[Production of β-carotene by metabolically engineered Saccharomyces cerevisiae].
Wang, Beibei; Shi, Mingyu; Wang, Dong; Xu, Jiaoyang; Liu, Yi; Yang, Hongjiang; Dai, Zhubo; Zhang, Xueli
β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production. PMID:25507473
[Production of β-carotene by metabolically engineered Saccharomyces cerevisiae].
Wang, Beibei; Shi, Mingyu; Wang, Dong; Xu, Jiaoyang; Liu, Yi; Yang, Hongjiang; Dai, Zhubo; Zhang, Xueli
β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production. PMID:25423750
Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production.
Chen, Binbin; Lee, Dong-Yup; Chang, Matthew Wook
Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae. PMID:26164646
Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae
Yannai, S.; Berdicevsky, I.; Duek, L. )
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.
Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model
Feyder, Serge; De Craene, Johan-Owen; Bär, Séverine; Bertazzi, Dimitri L.; Friant, Sylvie
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
The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability
NASA Astrophysics Data System (ADS)
Yi, Zong-Chun; Li, Xiao-Fei; Wang, Yan; Wang, Jie; Sun, Yan; Zhuang, Feng-Yuan
The budding yeast Saccharomyces cerevisiae has been proposed as an ideal model organism for clarifying the biological effects caused by spaceflight conditions. The postmitotic S. cerevisiae cells onboard Practice eight recoverable satellite were subjected to spaceflight for 15 days. After recovery, the viability, the glycogen content, the activities of carbohydrate metabolism enzymes, the DNA content and the lipid peroxidation level in yeast cells were analyzed. The viability of the postmitotic yeast cells after spaceflight showed a three-fold increase as compared with that of the ground control cells. Compared to the ground control cells, the lipid peroxidation level in the spaceflight yeast cells markedly decreased. The spaceflight yeast cells also showed an increase in G2/M cell population and a decrease in Sub-G1 cell population. The glycogen content and the activities of hexokinase and succinate dehydrogenase significantly decreased in the yeast cells after spaceflight. In contrast, the activity of malate dehydrogenase showed an obvious increase after spaceflight. These results suggested that microgravity or spaceflight could promote the survival of postmitotic S. cerevisiae cells through regulating carbohydrate metabolism, ROS level and cell cycle progression.
Copper oxide nanoparticles inhibit the metabolic activity of Saccharomyces cerevisiae.
Mashock, Michael J; Kappell, Anthony D; Hallaj, Nadia; Hristova, Krassimira R
Copper oxide nanoparticles (CuO NPs) are used increasingly in industrial applications and consumer products and thus may pose risk to human and environmental health. The interaction of CuO NPs with complex media and the impact on cell metabolism when exposed to sublethal concentrations are largely unknown. In the present study, the short-term effects of 2 different sized manufactured CuO NPs on metabolic activity of Saccharomyces cerevisiae were studied. The role of released Cu(2+) during dissolution of NPs in the growth media and the CuO nanostructure were considered. Characterization showed that the 28 nm and 64 nm CuO NPs used in the present study have different primary diameter, similar hydrodynamic diameter, and significantly different concentrations of dissolved Cu(2+) ions in the growth media released from the same initial NP mass. Exposures to CuO NPs or the released Cu(2+) fraction, at doses that do not have impact on cell viability, showed significant inhibition on S. cerevisiae cellular metabolic activity. A greater CuO NP effect on the metabolic activity of S. cerevisiae growth under respiring conditions was observed. Under the tested conditions the observed metabolic inhibition from the NPs was not explained fully by the released Cu ions from the dissolving NPs. PMID:26178758
Comparative proteomic analysis of Saccharomyces cerevisiae under different nitrogen sources.
Zhao, Shaohui; Zhao, Xinrui; Zou, Huijun; Fu, Jianwei; Du, Guocheng; Zhou, Jingwen; Chen, Jian
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
Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis
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
Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.
D'Amore, T; Panchal, C J; Stewart, G G
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
Mutants of Saccharomyces cerevisiae with defective vacuolar function
Kitamoto, K.; Yoshizawa, K.; Ohsumi, Y.; Anraku, Y.
Mutants of the yeast Saccharomyces cerevisiae that have a small vacuolar lysine pool were isolated and characterized. Mutant KL97 (lys1 slp1-1) and strain KL197-1A (slp1-1), a prototrophic derivative of KL97, did not grow well in synthetic medium supplemented with 10 mM lysine. Genetic studies indicated that the slp1-1mutation (for small lysine pool) is recessive and is due to a single chromosomal mutation. Mutant KL97 shows the following pleiotropic defects in vacuolar functions. (i) It has small vacuolar pools for lysine, arginine, and histidine. (ii) Its growth is sensitive to lysine, histidine, Ca/sup 2 +/, heavy metal ions, and antibiotics. (iii) It has many small vesicles but no large central vacuole. (iv) It has a normal amount of the vacuolar membrane marker ..cap alpha..-mannosidase but shows reduced activities of the vacuole sap markers proteinase A, proteinase B, and carboxypeptidase Y.
Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.
Williams, J S; Eckdahl, T T; Anderson, J N
Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function. Images PMID:3043195
Identity elements of Saccharomyces cerevisiae tRNA(His).
Nameki, N; Asahara, H; Shimizu, M; Okada, N; Himeno, H
Recognition of tRNA(His) by Saccharomyces cerevisiae histidyl-tRNA synthetase was studied using in vitro transcripts. Histidine tRNA is unique in possessing an extra nucleotide, G-1, at the 5' end. Mutation studies indicate that this irregular secondary structure at the end of the acceptor stem is important for aminoacylation with histidine, while the requirement of either base of this extra base pair is smaller than that in Escherichia coli. The anticodon was also found to be required for histidylation. The regions involved in histidylation are essentially the same as those in E.coli, whereas the proportion of the contributions of the two portions distant from each other, the anticodon and the end of the acceptor stem, makes a substantial difference between the two systems. PMID:7885835
Hormetic Effect of H2O2 in Saccharomyces cerevisiae
Valishkevych, Bohdana V.
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
Bioaccumulation of cadmium by growing Zygosaccharomyces rouxii and Saccharomyces cerevisiae.
Li, Chunsheng; Jiang, Wei; Ma, Ning; Zhu, Yinglian; Dong, Xiaoyan; Wang, Dongfeng; Meng, Xianghong; Xu, Ying
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
MPS3 mediates meiotic bouquet formation in Saccharomyces cerevisiae.
Conrad, Michael N; Lee, Chih-Ying; Wilkerson, Joseph L; Dresser, Michael E
In meiotic prophase, telomeres associate with the nuclear envelope and accumulate adjacent to the centrosome/spindle pole to form the chromosome bouquet, a well conserved event that in Saccharomyces cerevisiae requires the meiotic telomere protein Ndj1p. Ndj1p interacts with Mps3p, a nuclear envelope SUN domain protein that is required for spindle pole body duplication and for sister chromatid cohesion. Removal of the Ndj1p-interaction domain from MPS3 creates an ndj1 Delta-like separation-of-function allele, and Ndj1p and Mps3p are codependent for stable association with the telomeres. SUN domain proteins are found in the nuclear envelope across phyla and are implicated in mediating interactions between the interior of the nucleus and the cytoskeleton. Our observations indicate a general mechanism for meiotic telomere movements. PMID:17495028
The Influence of Microgravity on Invasive Growth in Saccharomyces cerevisiae
NASA Astrophysics Data System (ADS)
Van Mulders, Sebastiaan E.; Stassen, Catherine; Daenen, Luk; Devreese, Bart; Siewers, Verena; van Eijsden, Rudy G. E.; Nielsen, Jens; Delvaux, Freddy R.; Willaert, Ronnie
This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Σ1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Σ1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Σ1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium.
Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae.
Li, C; Li, J; Wang, G; Li, X
Artemisinic acid is a precursor of antimalarial compound artemisinin. The titre of biosynthesis of artemisinic acid using Saccharomyces cerevisiae platform has been achieved up to 25 g l(-1) ; however, the performance of platform cells is still industrial unsatisfied. Many strategies have been proposed to improve the titre of artemisinic acid. The traditional strategies mainly focused on partial target sites, simple up-regulation key genes or repression competing pathways in the total synthesis route. However, this may result in unbalance of carbon fluxes and dysfunction of metabolism. In this review, the recent advances on the promising methods in silico and in vivo for biosynthesis of artemisinic acid have been discussed. The bioinformatics and omics techniques have brought a great prospect for improving production of artemisinin and other pharmacal compounds in heterologous platform. PMID:26743771
Translation initiation factor-dependent extracts from Saccharomyces cerevisiae.
Altmann, M; Blum, S; Pelletier, J; Sonenberg, N; Wilson, T M; Trachsel, H
Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro. Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation. The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA. Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal. PMID:2169890
Availability of substratum enhances ethanol production in Saccharomyces cerevisiae.
Sankh, Santosh N; Arvindekar, Akalpita U
Novel additives that act as substratum for attachment of the yeast cells, increased ethanol production in Saccharomyces cerevisiae. The addition of 2 g rice husk, straw, wood shavings, plastic pieces or silica gel to 100 ml medium enhanced ethanol production by 30-40 (v/v). Six distillery strains showed an average enhancement of 34 from 4.1 (v/v) in control to 5.5 (v/v) on addition of rice husk. The cell wall bound glycogen increased by 40-50 mg g (-1) dry yeast while intracellular glycogen decreased by 10-12 mg g(-1) dry yeast in cells grown in presence of substratum. PMID:15672221
Yap1: a DNA damage responder in Saccharomyces cerevisiae.
Rowe, Lori A; Degtyareva, Natalya; Doetsch, Paul W
Activation of signaling pathways in response to genotoxic stress is crucial for cells to properly repair DNA damage. In response to DNA damage, intracellular levels of reactive oxygen species increase. One important function of such a response could be to initiate signal transduction processes. We have employed the model eukaryote Saccharomyces cerevisiae to delineate DNA damage sensing mechanisms. We report a novel, unanticipated role for the transcription factor Yap1 as a DNA damage responder, providing direct evidence that reactive oxygen species are an important component of the DNA damage signaling process. Our findings reveal an epistatic link between Yap1 and the DNA base excision repair pathway. Corruption of the Yap1-mediated DNA damage response influences cell survival and genomic stability in response to exposure to genotoxic agents. PMID:22433435
Protein disorder reduced in Saccharomyces cerevisiae to survive heat shock.
Vicedo, Esmeralda; Gasik, Zofia; Dong, Yu-An; Goldberg, Tatyana; Rost, Burkhard
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
Higher-order structure of Saccharomyces cerevisiae chromatin
Lowary, P.T.; Widom, J. )
We have developed a method for partially purifying chromatin from Saccharomyces cerevisiae (baker's yeast) to a level suitable for studies of its higher-order folding. This has required the use of yeast strains that are free of the ubiquitous yeast killer virus. Results from dynamic light scattering, electron microscopy, and x-ray diffraction show that the yeast chromatin undergoes a cation-dependent folding into 30-nm filaments that resemble those characteristic of higher-cell chromatin; moreover, the packing of nucleosomes within the yeast 30-nm filaments is similar to that of higher cells. These results imply that yeast has a protein or protein domain that serves the role of the histone H 1 found in higher cells; physical and genetic studies of the yeast activity could help elucidate the structure and function of H 1. Images of the yeast 30-nm filaments can be used to test crossed-linker models for 30-nm filament structure.
Construction of a flocculent Saccharomyces cerevisiae fermenting lactose.
Domingues, L; Teixeira, J A; Lima, N
A flocculent Saccharomyces cerevisiae strain with the ability to express both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus was constructed. This recombinant strain is not only able to grow on lactose, but it can also ferment this substrate. To our knowledge this is the first time that a recombinant S. cervisiae has been found to ferment lactose in a way comparable to that of the existing lactose-fermenting yeast strains. Moreover, the flocculating capacity of the strain used in this work gives the process several advantages. On the one hand, it allows for operation in a continuous mode at high cell concentration, thus increasing the system's overall productivity; on the other hand, the biomass concentration in the effluent is reduced, thus decreasing product separation/purification costs. PMID:10390820
Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae
NASA Astrophysics Data System (ADS)
Melén, Karin; Blomberg, Anders; von Heijne, Gunnar
Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing 600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na+/K+ homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies. caffeine | paraquat | salt tolerance | yeast
Production of natural products through metabolic engineering of Saccharomyces cerevisiae.
Krivoruchko, Anastasia; Nielsen, Jens
Many high-value metabolites are produced in nature by organisms that are not ideal for large-scale production. Therefore, interest exists in expressing the biosynthetic pathways of these compounds in organisms that are more suitable for industrial production. Recent years have seen developments in both the discovery of various biosynthetic pathways, as well as development of metabolic engineering tools that allow reconstruction of complex pathways in microorganisms. In the present review we discuss recent advances in reconstruction of the biosynthetic pathways of various high-value products in the yeast Saccharomyces cerevisiae, a commonly used industrial microorganism. Key achievements in the production of different isoprenoids, aromatics and polyketides are presented and the metabolic engineering strategies underlying these accomplishments are discussed. PMID:25544013
Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae
Hayashi, Naoyuki Kobayashi, Masahiko; Shimizu, Hiroko; Yamamoto, Ken-ichi; Murakami, Seishi; Nishimoto, Takeharu
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.
Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts.
Gallone, Brigida; Steensels, Jan; Prahl, Troels; Soriaga, Leah; Saels, Veerle; Herrera-Malaver, Beatriz; Merlevede, Adriaan; Roncoroni, Miguel; Voordeckers, Karin; Miraglia, Loren; Teiling, Clotilde; Steffy, Brian; Taylor, Maryann; Schwartz, Ariel; Richardson, Toby; White, Christopher; Baele, Guy; Maere, Steven; Verstrepen, Kevin J
Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP. PMID:27610566
Exposure to benzene metabolites causes oxidative damage in Saccharomyces cerevisiae.
Raj, Abhishek; Nachiappan, Vasanthi
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
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.
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.
Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1.
Busygina, Valeria; Gaines, William A; Xu, Yuanyuan; Kwon, Youngho; Williams, Gareth J; Lin, Sheng-Wei; Chang, Hao-Yen; Chi, Peter; Wang, Hong-Wei; Sung, Patrick
The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ∼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase. PMID:23769192
Characterization of Encapsulated Berberine in Yeast Cells of Saccharomyces cerevisiae
Salari, Roshanak; Rajabi, Omid; Khashyarmanesh, Zahra; Fathi Najafi, Mohsen; Fazly Bazzaz, BiBi Sedigheh
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
Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation
Liu, Ling-ling; Jia, Bo; Zhao, Fang; Huang, Wei-dong; Zhan, Ji-cheng
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
Proteomic characterization of a wild-type wine strain of Saccharomyces cerevisiae.
Trabalzini, Lorenza; Paffetti, Alessandro; Ferro, Elisa; Scaloni, Andrea; Talamo, Fabio; Millucci, Lia; Martelli, Paola; Santucci, Annalisa
Saccharomyces cerevisiae is the optimal eukaryotic model system to study mammalian biological responses. At the same time Saccharomyces cerevisiae is also widely utilized as a biotechnological tool in the food industry. Enological Saccharomyces cerevisiae strains have been so far routinely analyzed for their microbiological aspects. Nevertheless, wine yeasts are gaining an increasing interest in the last years since they strongly affect both the vinification process and the organoleptic properties of the final product wine. The protein repertoire is responsible of such features and, consequently, 2D-PAGE can be an useful tool to evaluate and select optimal wine yeast strains. We present here the first proteomic map of a wild-type wine Saccharomyces cerevisiae strain selected for the guided fermentation of very high quality wines. PMID:15141481
Water treatment process and system for metals removal using Saccharomyces cerevisiae
Krauter, Paula A. W.; Krauter, Gordon W.
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.
The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier.
Cameron, D R; Cooper, D G; Neufeld, R J
The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier. PMID:3046488
Transcriptional regulation by ergosterol in the yeast Saccharomyces cerevisiae.
Smith, S J; Crowley, J H; Parks, L W
Sterol biosynthesis in the yeast Saccharomyces cerevisiae is an energy-expensive, aerobic process, requiring heme and molecular oxygen. Heme, also synthesized exclusively during aerobic growth, not only acts as an enzymatic cofactor but also is directly and indirectly responsible for the transcriptional control of several yeast genes. Because of their biosynthetic similarities, we hypothesized that ergosterol, like heme, may have a regulatory function. Sterols are known to play a structural role in membrane integrity, but regulatory roles have not been characterized. To test possible regulatory roles of sterol, the promoter for the ERG3 gene, encoding the sterol C-5 desaturase, was fused to the bacterial lacZ reporter gene. This construct was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as ninefold in erg mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifungal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate for the first time that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae. PMID:8816455
CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.
Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D
This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory. PMID:27250940
Mead production: selection and characterization assays of Saccharomyces cerevisiae strains.
Pereira, Ana Paula; Dias, Teresa; Andrade, João; Ramalhosa, Elsa; Estevinho, Letícia M
Mead is a traditional drink, which results from the alcoholic fermentation of diluted honey carried out by yeasts. However, when it is produced in a homemade way, mead producers find several problems, namely, the lack of uniformity in the final product, delayed and arrested fermentations, and the production of "off-flavours" by the yeasts. These problems are usually associated with the inability of yeast strains to respond and adapt to unfavourable and stressful growth conditions. The main objectives of this work were to evaluate the capacity of Saccharomyces cerevisiae strains, isolated from honey of the Trás-os-Montes (Northeast Portugal), to produce mead. Five strains from honey, as well as one laboratory strain and one commercial wine strain, were evaluated in terms of their fermentation performance under ethanol, sulphur dioxide and osmotic stress. All the strains showed similar behaviour in these conditions. Two yeasts strains isolated from honey and the commercial wine strain were further tested for mead production, using two different honey (a dark and a light honey), enriched with two supplements (one commercial and one developed by the research team), as fermentation media. The results obtained in this work show that S. cerevisiae strains isolated from honey, are appropriate for mead production. However it is of extreme importance to take into account the characteristics of the honey, and supplements used in the fermentation medium formulation, in order to achieve the best results in mead production. PMID:19481129
Metabolic engineering of Saccharomyces cerevisiae for itaconic acid production.
Blazeck, John; Miller, Jarrett; Pan, Anny; Gengler, Jon; Holden, Clinton; Jamoussi, Mariam; Alper, Hal S
Renewable alternatives for petroleum-derived chemicals are achievable through biosynthetic production. Here, we utilize Saccharomyces cerevisiae to enable the synthesis of itaconic acid, a molecule with diverse applications as a petrochemical replacement. We first optimize pathway expression within S. cerevisiae through the use of a hybrid promoter. Next, we utilize sequential, in silico computational genome-scanning to identify beneficial genetic perturbations that are metabolically distant from the itaconic acid synthesis pathway. In this manner, we successfully identify three non-obvious genetic targets (∆ade3 ∆bna2 ∆tes1) that successively improve itaconic acid titer. We establish that focused manipulations of upstream pathway enzymes (localized refactoring) and enzyme re-localization to both mitochondria and cytosol fail to improve itaconic acid titers. Finally, we establish a higher cell density fermentation that ultimately achieves itaconic acid titer of 168 mg/L, a sevenfold improvement over initial conditions. This work represents an attempt to increase itaconic acid production in yeast and demonstrates the successful utilization of computationally guided genetic manipulation to increase metabolic capacity. PMID:24997118
Lactose fermentation by engineered Saccharomyces cerevisiae capable of fermenting cellobiose.
Liu, Jing-Jing; Zhang, Guo-Chang; Oh, Eun Joong; Pathanibul, Panchalee; Turner, Timothy L; Jin, Yong-Su
Lactose is an inevitable byproduct of the dairy industry. In addition to cheese manufacturing, the growing Greek yogurt industry generates excess acid whey, which contains lactose. Therefore, rapid and efficient conversion of lactose to fuels and chemicals would be useful for recycling the otherwise harmful acid whey. Saccharomyces cerevisiae, a popular metabolic engineering host, cannot natively utilize lactose. However, we discovered that an engineered S. cerevisiae strain (EJ2) capable of fermenting cellobiose can also ferment lactose. This finding suggests that a cellobiose transporter (CDT-1) can transport lactose and a β-glucosidase (GH1-1) can hydrolyze lactose by acting as a β-galactosidase. While the lactose fermentation by the EJ2 strain was much slower than the cellobiose fermentation, a faster lactose-fermenting strain (EJ2e8) was obtained through serial subcultures on lactose. The EJ2e8 strain fermented lactose with a consumption rate of 2.16g/Lh. The improved lactose fermentation by the EJ2e8 strain was due to the increased copy number of cdt-1 and gh1-1 genes. Looking ahead, the EJ2e8 strain could be exploited for the production of other non-ethanol fuels and chemicals from lactose through further metabolic engineering. PMID:27457698
Data on dynamic study of cytoophidia in Saccharomyces cerevisiae.
Li, Hui; Huang, Yong; Wang, Peng-Ye; Ye, Fangfu; Liu, Ji-Long
The data in this paper are related to the research article entitled "Filamentation of metabolic enzymes in Saccharomyces cerevisiae" Q.J. Shen et al. (2016) [1]. Cytoophidia are filamentous structures discovered in fruit flies (doi:10.1016/S1673-8527(09)60046-1) J.L. Liu (2010) [2], bacteria (doi:10.1038/ncb2087) M. Ingerson-Mahar et al. (2010) [3], yeast (doi:10.1083/jcb.201003001; doi:10.1242/bio.20149613) C. Noree et al. (2010) and J. Zhang, L. Hulme, J.L. Liu (2014) [4], [5] and human cells (doi:10.1371/journal.pone.0029690; doi:10.1016/j.jgg.2011.08.004) K. Chen et al. (2011) and W.C. Carcamo et al. (2011) ( [6], [7]. However, there is little research on the motility of the cytoophidia. Here we selected cytoophidia formed by 6 filament-forming proteins in the budding yeast S. cerevisiae, and performed living-cell imaging of cells expressing the proteins fused with GFP. The dynamic features of the six types of cytoophidia were analyzed. In the data, both raw movies and analysed results of the dynamics of cytoophidia are presented. PMID:27274529
Genetic determinants for enhanced glycerol growth of Saccharomyces cerevisiae.
Swinnen, Steve; Ho, Ping-Wei; Klein, Mathias; Nevoigt, Elke
The yeast Saccharomyces cerevisiae generally shows a low natural capability to utilize glycerol as the sole source of carbon, particularly when synthetic medium is used and complex supplements are omitted. Nevertheless, wild type isolates have been identified that show a moderate growth under these conditions. In the current study we made use of intraspecies diversity to identify targets suitable for reverse metabolic engineering of the non-growing laboratory strain CEN.PK113-1A. A genome-wide genetic mapping experiment using pooled-segregant whole-genome sequence analysis was conducted, and one major and several minor genetic loci were identified responsible for the superior glycerol growth phenotype of the previously selected S. cerevisiae strain CBS 6412-13A. Downscaling of the major locus by fine-mapping and reciprocal hemizygosity analysis allowed the parallel identification of two superior alleles (UBR2CBS 6412-13A and SSK1CBS 6412-13A). These alleles together with the previously identified GUT1CBS 6412-13A allele were used to replace the corresponding alleles in the strain CEN.PK113-1A. In this way, glycerol growth could be established reaching a maximum specific growth rate of 0.08h(-1). Further improvement to a maximum specific growth rate of 0.11h(-1) could be achieved by heterologous expression of the glycerol facilitator FPS1 from Cyberlindnera jadinii. PMID:26971668
Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.
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
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
Long-chain alkane production by the yeast Saccharomyces cerevisiae.
Buijs, Nicolaas A; Zhou, Yongjin J; Siewers, Verena; Nielsen, Jens
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
Quantifying separation and similarity in a Saccharomyces cerevisiae metapopulation
Knight, Sarah; Goddard, Matthew R
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
Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining.
Yu, Xin; Gabriel, Abram
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
Metabolomic approach for improving ethanol stress tolerance in Saccharomyces cerevisiae.
Ohta, Erika; Nakayama, Yasumune; Mukai, Yukio; Bamba, Takeshi; Fukusaki, Eiichiro
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
Systems biology of GAL regulon in Saccharomyces cerevisiae.
Pannala, Venkat Reddy; Bhat, Paike Jayadeva; Bhartiya, Sharad; Venkatesh, K V
Evolutionary success of an organism depends on its ability to express or adapt to constantly changing environmental conditions. Saccharomyces cerevisiae has evolved an elaborate genetic circuit to regulate the expression of galactose-metabolizing enzymes in the presence of galactose but in the absence of glucose. The circuit possesses molecular mechanisms such as multiple binding sites, cooperativity, autoregulation, nucleocytoplasmic shuttling, and substrate sensing mechanism. Furthermore, the GAL system consists of two positive (activating) feedback and one negative (repressing) feedback loops. These individual mechanisms, elucidated through experimental approach, can be integrated to obtain a system-wide behavior. Mathematical models in conjunction with guided experiments have demonstrated system-level properties such as ultrasensitivity, memory, noise attenuation, rapid response, and sensitive response arising out of the molecular interactions. These system-level properties allow S. cerevisiae to adapt and grow in a galactose medium under noisy and changing environments. This review focuses on system-level models and properties of the GAL regulon. PMID:20836013
Genomic Evolution of Saccharomyces cerevisiae under Chinese Rice Wine Fermentation
Li, Yudong; Zhang, Weiping; Zheng, Daoqiong; Zhou, Zhan; Yu, Wenwen; Zhang, Lei; Feng, Lifang; Liang, Xinle; Guan, Wenjun; Zhou, Jingwen; Chen, Jian; Lin, Zhenguo
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
Human G protein-coupled receptor studies in Saccharomyces cerevisiae.
Liu, Rongfang; Wong, Winsy; IJzerman, Adriaan P
G protein-coupled receptors (GPCRs) are one of the largest families of membrane proteins, with approximately 800 different GPCRs in the human genome. Signaling via GPCRs regulates many biological processes, such as cell proliferation, differentiation, and development. In addition, many receptors have a pivotal role in immunophysiology. Many hormones and neurotransmitters are ligands for these receptors, and hence it is not surprising that many drugs, either mimicking or blocking the action of the bodily substances, have been developed. It is estimated that 30-40% of current drugs on the market target GPCRs. Further identifying and elucidating the functions of GPCRs will provide opportunities for novel drug discovery, including for immunotherapy. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a very important and useful platform in this respect. There are many advantages of using a yeast assay system, as it is cheap, safe and stable; it is also convenient for rapid feasibility and optimization studies. Moreover, it offers a "null" background when studying human GPCRs. New developments regarding human GPCRs expressed in a yeast platform are providing insight into GPCR activation and signaling, and facilitate agonist and antagonist identification. In this review we summarize the latest findings regarding human G-protein-coupled receptors in studies using S. cerevisiae, ever since the year 2005 when we last published a review on this topic. We describe 11 families of GPCRs in detail, while including the principles and developments of each yeast system applied to these different GPCRs and highlight and generalize the experimental findings of GPCR function in these systems. PMID:26920251
Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae.
Hou, Jin; Osterlund, Tobias; Liu, Zihe; Petranovic, Dina; Nielsen, Jens
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
Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.
Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber
Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development. PMID:27470141
Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae
Overkamp, Karin M.; Bakker, Barbara M.; Kötter, Peter; Luttik, Marijke A. H.; van Dijken, Johannes P.; Pronk, Jack T.
Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1Δ nde1Δ nde2Δ gut2Δ quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h−1 at 100 g of glucose · liter−1). In aerated batch cultures grown on 400 g of glucose · liter−1, this engineered S. cerevisiae strain produced over 200 g of glycerol · liter−1, corresponding to a molar yield of glycerol on glucose close to unity. PMID:12039737
Exploring the Saccharomyces cerevisiae Volatile Metabolome: Indigenous versus Commercial Strains
Alves, Zélia; Melo, André; Figueiredo, Ana Raquel; Coimbra, Manuel A.; Gomes, Ana C.; Rocha, Sílvia M.
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
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...
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...
Metabolism of sulfur amino acids in Saccharomyces cerevisiae.
Thomas, D; Surdin-Kerjan, Y
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
Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases
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
The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.
van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N
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
Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae
Pablo-Hernando, M Evangelina; Arnaiz-Pita, Yolanda; Tachikawa, Hiroyuki; del Rey, Francisco; Neiman, Aaron M; Vázquez de Aldana, Carlos R
Background In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. Results Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. Conclusion Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources. PMID:18826657
Engineering the monomer composition of polyhydroxyalkanoates synthesized in Saccharomyces cerevisiae.
Zhang, Bo; Carlson, Ross; Srienc, Friedrich
Polyhydroxyalkanoates (PHAs) have received considerable interest as renewable-resource-based, biodegradable, and biocompatible plastics with a wide range of potential applications. We have engineered the synthesis of PHA polymers composed of monomers ranging from 4 to 14 carbon atoms in either the cytosol or the peroxisome of Saccharomyces cerevisiae by harnessing intermediates of fatty acid metabolism. Cytosolic PHA production was supported by establishing in the cytosol critical beta-oxidation chemistries which are found natively in peroxisomes. This platform was utilized to supply medium-chain (C6 to C14) PHA precursors from both fatty acid degradation and synthesis to a cytosolically expressed medium-chain-length (mcl) polymerase from Pseudomonas oleovorans. Synthesis of short-chain-length PHAs (scl-PHAs) was established in the peroxisome of a wild-type yeast strain by targeting the Ralstonia eutropha scl polymerase to the peroxisome. This strain, harboring a peroxisomally targeted scl-PHA synthase, accumulated PHA up to approximately 7% of its cell dry weight. These results indicate (i) that S. cerevisiae expressing a cytosolic mcl-PHA polymerase or a peroxisomal scl-PHA synthase can use the 3-hydroxyacyl coenzyme A intermediates from fatty acid metabolism to synthesize PHAs and (ii) that fatty acid degradation is also possible in the cytosol as beta-oxidation might not be confined only to the peroxisomes. Polymers of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers can be controlled by feeding the appropriate substrates. This ability should permit the rational design and synthesis of polymers with desired material properties. PMID:16391089
Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons.
Zitomer, R S; Walthall, D A; Rymond, B C; Hollenberg, C P
A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results. PMID:6390186
Modulation of efficiency of translation termination in Saccharomyces cerevisiae
Nizhnikov, Anton A; Antonets, Kirill S; Inge-Vechtomov, Sergey G; Derkatch, Irina L
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
Identification and characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae.
Vuralhan, Zeynep; Morais, Marcos A; Tai, Siew-Leng; Piper, Matthew D W; Pronk, Jack T
Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding alpha-keto acids, followed by decarboxylation to an aldehyde and then reduction to an alcohol. Alternatively, the aldehyde may be oxidized to an acid. This pathway is functional in Saccharomyces cerevisiae, since during growth in glucose-limited chemostat cultures with phenylalanine as the sole nitrogen source, phenylethanol and phenylacetate were produced in quantities that accounted for all of the phenylalanine consumed. Our objective was to identify the structural gene(s) required for the decarboxylation of phenylpyruvate to phenylacetaldehyde, the first specific step in the Ehrlich pathway. S. cerevisiae possesses five candidate genes with sequence similarity to genes encoding thiamine diphosphate-dependent decarboxylases that could encode this activity: YDR380w/ARO10, YDL080C/THI3, PDC1, PDC5, and PDC6. Phenylpyruvate decarboxylase activity was present in cultures grown with phenylalanine as the sole nitrogen source but was absent from ammonia-grown cultures. Furthermore, the transcript level of one candidate gene (ARO10) increased 30-fold when phenylalanine replaced ammonia as the sole nitrogen source. Analyses of phenylalanine catabolite production and phenylpyruvate decarboxylase enzyme assays indicated that ARO10 was sufficient to encode phenylpyruvate decarboxylase activity in the absence of the four other candidate genes. There was also an alternative activity with a higher capacity but lower affinity for phenylpyruvate. The candidate gene THI3 did not itself encode an active phenylpyruvate decarboxylase but was required along with one or more pyruvate decarboxylase genes (PDC1, PDC5, and PDC6) for the alternative activity. The K(m) and V(max) values of the two activities differed, showing that Aro10p is the physiologically relevant phenylpyruvate decarboxylase in wild-type cells. Modifications to this gene could therefore be important for metabolic engineering
Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.
Perry, J R; Basrai, M A; Steiner, H Y; Naider, F; Becker, J M
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
The plasma membrane of Saccharomyces cerevisiae: structure, function, and biogenesis.
van der Rest, M E; Kamminga, A H; Nakano, A; Anraku, Y; Poolman, B; Konings, W N
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
Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.
Guimarães, Pedro M R; François, Jean; Parrou, Jean Luc; Teixeira, José A; Domingues, Lucília
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
Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae
Bellon, Jennifer R.; Schmid, Frank; Capone, Dimitra L.; Dunn, Barbara L.; Chambers, Paul J.
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
Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae.
Buchanan, Bryce W; Lloyd, Michael E; Engle, Sarah M; Rubenstein, Eric M
Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation. PMID:27167179
TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae
Welch, Aaron Z.; Gibney, Patrick A.; Botstein, David; Koshland, Douglas E.
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
Chromatin remodeling during Saccharomyces cerevisiae ADH2 gene activation.
Verdone, L; Camilloni, G; Di Mauro, E; Caserta, M
We have analyzed at both low and high resolution the distribution of nucleosomes over the Saccharomyces cerevisiae ADH2 promoter region in its chromosomal location, both under repressing (high-glucose) conditions and during derepression. Enzymatic treatments (micrococcal nuclease and restriction endonucleases) were used to probe the in vivo chromatin structure during ADH2 gene activation. Under glucose-repressed conditions, the ADH2 promoter was bound by a precise array of nucleosomes, the principal ones positioned at the RNA initiation sites (nucleosome +1), at the TATA box (nucleosome -1), and upstream of the ADR1-binding site (UAS1) (nucleosome -2). The UAS1 sequence and the adjacent UAS2 sequence constituted a nucleosome-free region. Nucleosomes -1 and +1 were destabilized soon after depletion of glucose and had become so before the appearance of ADH2 mRNA. When the transcription rate was high, nucleosomes -2 and +2 also underwent rearrangement. When spheroplasts were prepared from cells grown in minimal medium, detection of this chromatin remodeling required the addition of a small amount of glucose. Cells lacking the ADR1 protein did not display any of these chromatin modifications upon glucose depletion. Since the UAS1 sequence to which Adr1p binds is located immediately upstream of nucleosome -1, Adr1p is presumably required for destabilization of this nucleosome and for aiding the TATA-box accessibility to the transcription machinery. PMID:8628264
Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae.
Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian
Natural products with anti-aging property have drawn great attention recently but examples of such compounds are exceedingly scarce. By applying a high-throughput assay based on yeast chronological lifespan measurement, we screened the anti-aging activity of 144 botanical materials and found that dried roots of Salvia miltiorrhiza Bunge have significant anti-aging activity. Tanshinones isolated from the plant including cryptotanshione, tanshinone I, and tanshinone IIa, are the active components. Among them, cryptotanshinone can greatly extend the budding yeast Saccharomyces cerevisiae chronological lifespan (up to 2.5 times) in a dose- and the-time-of-addition-dependent manner at nanomolar concentrations without disruption of cell growth. We demonstrate that cryptotanshinone prolong chronological lifespan via a nutrient-dependent regime, especially essential amino acid sensing, and three conserved protein kinases Tor1, Sch9, and Gcn2 are required for cryptotanshinone-induced lifespan extension. In addition, cryptotanshinone significantly increases the lifespan of SOD2-deleted mutants. Altogether, those data suggest that cryptotanshinone might be involved in the regulation of, Tor1, Sch9, Gcn2, and Sod2, these highly conserved longevity proteins modulated by nutrients from yeast to humans. PMID:24970458
Tor1 regulates protein solubility in Saccharomyces cerevisiae
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.
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
mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.
Jerome, J F; Jaehning, J A
We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II. Images PMID:3537708
Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae
Gonzalez-Perez, David; Alcalde, Miguel
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
Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16.
Bang, D D; Timmermans, V; Verhage, R; Zeeman, A M; van de Putte, P; Brouwer, J
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
Dynamics of the Saccharomyces cerevisiae Transcriptome during Bread Dough Fermentation
Aslankoohi, Elham; Zhu, Bo; Rezaei, Mohammad Naser; Voordeckers, Karin; De Maeyer, Dries; Marchal, Kathleen; Dornez, Emmie
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
Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics.
Ernst, J F; Chan, R K
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
Characterization of the Biotin Transport System in Saccharomyces cerevisiae1
Rogers, Thomas O.; Lichstein, Herman C.
The characteristics of the biotin transport mechanism of Saccharomyces cerevisiae were investigated in nonproliferating cells. Microbiological and radioisotope assays were employed to measure biotin uptake. The vitamin existed intracellularly in both free and bound forms. Free biotin was extracted by boiling water. Chromatography of the free extract showed it to consist entirely of d-biotin. Cellular bound biotin was released by treating cells with 6 n H2SO4. The rate of biotin uptake was linear with time for 10 min, reaching a maximum at about 20 min followed by a gradual loss of accumulated free vitamin from the cells. Biotin was not degraded or converted to vitamers during uptake. Transport was temperature- and pH-dependent, optimum conditions for uptake being 30 C and pH 4.0. Glucose markedly stimulated biotin transport. In its presence, large intracellular free-biotin concentration gradients were established. Iodoacetate inhibited the glucose stimulation of biotin uptake. The rate of vitamin transport increased in a linear fashion with increasing cell mass. The transport system was saturated with increasing concentrations of the vitamin. The apparent Km for uptake was 3.23 × 10−7m. Uptake of radioactive biotin was inhibited by unlabeled biotin and a number of analogues including homobiotin, desthiobiotin, oxybiotin, norbiotin, and biotin sulfone. Proline, hydroxyproline, and 7,8-diaminopelargonic acid did not inhibit uptake. Unlabeled biotin and desthiobiotin exchanged with accumulated intracellular 14C-biotin, whereas hydroxyproline did not. PMID:5354931
Protective Effects of Arginine on Saccharomyces cerevisiae Against Ethanol Stress
Cheng, Yanfei; Du, Zhaoli; Zhu, Hui; Guo, Xuena; He, Xiuping
Yeast cells are challenged by various environmental stresses in the process of industrial fermentation. As the currently main organism for bio-ethanol production, Saccharomyces cerevisiae suffers from ethanol stress. Some amino acids have been reported to be related to yeast tolerance to stresses. Here the relationship between arginine and yeast response to ethanol stress was investigated. Marked inhibitions of ethanol on cell growth, expression of genes involved in arginine biosynthesis and intracellular accumulation of arginine were observed. Furthermore, extracellular addition of arginine can abate the ethanol damage largely. To further confirm the protective effects of arginine on yeast cells, yeast strains with different levels of arginine content were constructed by overexpression of ARG4 involved in arginine biosynthesis or CAR1 encoding arginase. Intracellular arginine was increased by 18.9% or 13.1% respectively by overexpression of ARG4 or disruption of CAR1, which enhanced yeast tolerance to ethanol stress. Moreover, a 41.1% decrease of intracellular arginine was observed in CAR1 overexpressing strain, which made yeast cells keenly sensitive to ethanol. Further investigations indicated that arginine protected yeast cells from ethanol damage by maintaining the integrity of cell wall and cytoplasma membrane, stabilizing the morphology and function of organellae due to low ROS generation. PMID:27507154
Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae
Dar, R. D.; Karig, D. K.; Cooke, J. F.; Cox, C. D.; Simpson, M. L.
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.
Distribution and regulation of stochasticity and plasticity in Saccharomyces cerevisiae
Dar, R. D.; Karig, D. K.; Cooke, J. F.; Cox, C. D.; Simpson, M. L.
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
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)
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.
Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production.
Feng, Xueyang; Lian, Jiazhang; Zhao, Huimin
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
MAP Kinase Pathways in the Yeast Saccharomyces cerevisiae
Gustin, Michael C.; Albertyn, Jacobus; Alexander, Matthew; Davenport, Kenneth
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. PMID:9841672
Post-Transcriptional Regulation of Iron Homeostasis in Saccharomyces cerevisiae
Martínez-Pastor, María Teresa; de Llanos, Rosa; Romero, Antonia María; Puig, Sergi
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
Water-Transfer Slows Aging in Saccharomyces cerevisiae
Cohen, Aviv; Weindling, Esther; Rabinovich, Efrat; Nachman, Iftach; Fuchs, Shai; Chuartzman, Silvia; Gal, Lihi; Schuldiner, Maya; Bar-Nun, Shoshana
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
A novel selection system for chromosome translocations in Saccharomyces cerevisiae.
Tennyson, Rachel B; Ebran, Nathalie; Herrera, Anissa E; Lindsley, Janet E
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
Water-Transfer Slows Aging in Saccharomyces cerevisiae.
Cohen, Aviv; Weindling, Esther; Rabinovich, Efrat; Nachman, Iftach; Fuchs, Shai; Chuartzman, Silvia; Gal, Lihi; Schuldiner, Maya; Bar-Nun, Shoshana
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
Biochemical basis of mitochondrial acetaldehyde dismutation in Saccharomyces cerevisiae.
Thielen, J; Ciriacy, M
As reported previously, Saccharomyces cerevisiae cells deficient in all four known genes coding for alcohol dehydrogenases (ADH1 through ADH4) produce considerable amounts of ethanol during aerobic growth on glucose. It has been suggested that ethanol production in such adh0 cells is a corollary of acetaldehyde dismutation in mitochondria. This could be substantiated further by showing that mitochondrial ethanol formation requires functional electron transport, while the proton gradient or oxidative phosphorylation does not interfere with reduction of acetaldehyde in isolated mitochondria. This acetaldehyde-reducing activity is different from classical alcohol dehydrogenases in that it is associated with the inner mitochondrial membrane and also is unable to carry out ethanol oxidation. The putative cofactor is NADH + H+ generated by a soluble, matrix-located aldehyde dehydrogenase upon acetaldehyde oxidation to acetate. This enzyme has been purified from mitochondria of glucose-grown cells. It is clearly different from the known mitochondrial aldehyde dehydrogenase, which is absent in glucose-grown cells. Both acetaldehyde-reducing and acetaldehyde-oxidizing activities are also present in the mitochondrial fraction of fermentation-proficient (ADH+) cells. Mitochondrial acetaldehyde dismutation may have some significance in the removal of surplus acetaldehyde and in the formation of acetate in mitochondria during aerobic glucose fermentation. Images FIG. 4 PMID:1938903
Electroinduced release of recombinant β-galactosidase from Saccharomyces cerevisiae.
Ganeva, Valentina; Stefanova, Debora; Angelova, Boyana; Galutzov, Bojidar; Velasco, Isabel; Arévalo-Rodríguez, Miguel
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
Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall
Orlean, Peter
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
Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae.
Gonzalez-Perez, David; Alcalde, Miguel
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
Functional studies of aldo-keto reductases in Saccharomyces cerevisiae*
Chang, Qing; Griest, Terry A.; Harter, Theresa M.; Petrash, J. Mark
SUMMARY We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678
Systematic Identification of Balanced Transposition Polymorphisms in Saccharomyces cerevisiae
Faddah, Dina A.; Ganko, Eric W.; McCoach, Caroline; Pickrell, Joseph K.; Hanlon, Sean E.; Mann, Frederick G.; Mieczkowska, Joanna O.; Jones, Corbin D.; Lieb, Jason D.; Vision, Todd J.
High-throughput techniques for detecting DNA polymorphisms generally do not identify changes in which the genomic position of a sequence, but not its copy number, varies among individuals. To explore such balanced structural polymorphisms, we used array-based Comparative Genomic Hybridization (aCGH) to conduct a genome-wide screen for single-copy genomic segments that occupy different genomic positions in the standard laboratory strain of Saccharomyces cerevisiae (S90) and a polymorphic wild isolate (Y101) through analysis of six tetrads from a cross of these two strains. Paired-end high-throughput sequencing of Y101 validated four of the predicted rearrangements. The transposed segments contained one to four annotated genes each, yet crosses between S90 and Y101 yielded mostly viable tetrads. The longest segment comprised 13.5 kb near the telomere of chromosome XV in the S288C reference strain and Southern blotting confirmed its predicted location on chromosome IX in Y101. Interestingly, inter-locus crossover events between copies of this segment occurred at a detectable rate. The presence of low-copy repetitive sequences at the junctions of this segment suggests that it may have arisen through ectopic recombination. Our methodology and findings provide a starting point for exploring the origins, phenotypic consequences, and evolutionary fate of this largely unexplored form of genomic polymorphism. PMID:19503594
Adaptation of a Saccharomyces cerevisiae strain to high copper concentrations.
Sarais, I; Manzano, M; De Bertoldi, M; Romandini, P; Beltramini, M; Salvato, B; Rocco, G P
A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mM). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent. PMID:8043987
Protective Effects of Arginine on Saccharomyces cerevisiae Against Ethanol Stress.
Cheng, Yanfei; Du, Zhaoli; Zhu, Hui; Guo, Xuena; He, Xiuping
Yeast cells are challenged by various environmental stresses in the process of industrial fermentation. As the currently main organism for bio-ethanol production, Saccharomyces cerevisiae suffers from ethanol stress. Some amino acids have been reported to be related to yeast tolerance to stresses. Here the relationship between arginine and yeast response to ethanol stress was investigated. Marked inhibitions of ethanol on cell growth, expression of genes involved in arginine biosynthesis and intracellular accumulation of arginine were observed. Furthermore, extracellular addition of arginine can abate the ethanol damage largely. To further confirm the protective effects of arginine on yeast cells, yeast strains with different levels of arginine content were constructed by overexpression of ARG4 involved in arginine biosynthesis or CAR1 encoding arginase. Intracellular arginine was increased by 18.9% or 13.1% respectively by overexpression of ARG4 or disruption of CAR1, which enhanced yeast tolerance to ethanol stress. Moreover, a 41.1% decrease of intracellular arginine was observed in CAR1 overexpressing strain, which made yeast cells keenly sensitive to ethanol. Further investigations indicated that arginine protected yeast cells from ethanol damage by maintaining the integrity of cell wall and cytoplasma membrane, stabilizing the morphology and function of organellae due to low ROS generation. PMID:27507154
Genetic dissection of acetic acid tolerance in Saccharomyces cerevisiae.
Geng, Peng; Xiao, Yin; Hu, Yun; Sun, Haiye; Xue, Wei; Zhang, Liang; Shi, Gui-Yang
Dissection of the hereditary architecture underlying Saccharomyces cerevisiae tolerance to acetic acid is essential for ethanol fermentation. In this work, a genomics approach was used to dissect hereditary variations in acetic acid tolerance between two phenotypically different strains. A total of 160 segregants derived from these two strains were obtained. Phenotypic analysis indicated that the acetic acid tolerance displayed a normal distribution in these segregants, and suggested that the acetic acid tolerant traits were controlled by multiple quantitative trait loci (QTLs). Thus, 220 SSR markers covering the whole genome were used to detect QTLs of acetic acid tolerant traits. As a result, three QTLs were located on chromosomes 9, 12, and 16, respectively, which explained 38.8-65.9 % of the range of phenotypic variation. Furthermore, twelve genes of the candidates fell into the three QTL regions by integrating the QTL analysis with candidates of acetic acid tolerant genes. These results provided a novel avenue to obtain more robust strains. PMID:27430512
Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history.
Legras, Jean-Luc; Merdinoglu, Didier; Cornuet, Jean-Marie; Karst, Francis
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
Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production
Jin, Lu; Bhuiya, Mohammad Wadud; Li, Mengmeng; Liu, XiangQi; Han, Jixiang; Deng, WeiWei; Wang, Min; Yu, Oliver; Zhang, Zhengzhu
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
Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae.
Flis, Vid V; Fankl, Ariane; Ramprecht, Claudia; Zellnig, Günther; Leitner, Erich; Hermetter, Albin; Daum, Günther
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
Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae
Ramprecht, Claudia; Zellnig, Günther; Leitner, Erich; Hermetter, Albin; Daum, Günther
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
Endomitotic effect of a cell cycle mutation of Saccharomyces cerevisiae
Schild, D.; Ananthaswamy, H.N.; Mortimer, R.K.
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/.
Transcriptional regulatory network shapes the genome structure of Saccharomyces cerevisiae
Li, Songling; Heermann, Dieter W.
Among cellular processes gene transcription is central. More and more evidence is mounting that transcription is tightly connected with the spatial organization of the chromosomes. Spatial proximity of genes sharing transcriptional machinery is one of the consequences of this organization. Motivated by information on the physical relationship among genes identified via chromosomal conformation capture methods, we complement the spatial organization with the idea that genes under similar transcription factor control, but possible scattered throughout the genome, might be in physically proximity to facilitate the access of their commonly used transcription factors. Unlike the transcription factory model, "interacting" genes in our "Gene Proximity Model" are not necessarily immediate physical neighbors but are in spatial proximity. Considering the stochastic nature of TF-promoter binding, this local condensation mechanism could serve as a tie to recruit co-regulated genes to guarantee the swiftness of biological reactions. We tested this idea with a simple eukaryotic organism, Saccharomyces cerevisiae. Chromosomal interaction patterns and folding behavior generated by our model re-construct those obtained from experiments. We show that the transcriptional regulatory network has a close linkage with the genome organization in budding yeast, which is fundamental and instrumental to later studies on other more complex eukaryotes. PMID:23674068
Characterization of Alcohol-induced Filamentous Growth in Saccharomyces cerevisiae
Lorenz, Michael C.; Cutler, N. Shane; Heitman, Joseph
Diploid cells of the budding yeast Saccharomyces cerevisiae starved for nitrogen differentiate into a filamentous growth form. Poor carbon sources such as starches can also stimulate filamentation, whereas haploid cells undergo a similar invasive growth response in rich medium. Previous work has demonstrated a role for various alcohols, by-products of amino acid metabolism, in altering cellular morphology. We found that several alcohols, notably isoamyl alcohol and 1-butanol, stimulate filamentous growth in haploid cells in which this differentiation is normally repressed. Butanol also induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology, even in liquid medium. The filamentous colony morphology and cell elongation require elements of the pheromone-responsive MAPK cascade and TEC1, whereas components of the nutrient-sensing machinery, such as MEP2, GPA2, and GPR1, do not affect this phenomenon. A screen for 1-butanol–insensitive mutants identified additional proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG1), mitochondrial function (MSM1, MRP21, and HMI1), and a transcriptional regulator (CHD1). Furthermore, we have also found that ethanol stimulates hyperfilamentation in diploid cells, again in a MAPK-dependent manner. Together, these results suggest that yeast may sense a combination of nutrient limitation and metabolic by-products to regulate differentiation. PMID:10637301
In vivo Reconstitution of Algal Triacylglycerol Production in Saccharomyces cerevisiae
Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki
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
Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.
Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D
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
Investigation of Batten disease with the yeast Saccharomyces cerevisiae.
Pearce, D A; Sherman, F
The CLN3 gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the human CLN3 gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance. Severity of Batten disease due to mutations in CLN3 and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease. PMID:10191120
Brazilian propolis protects Saccharomyces cerevisiae cells against oxidative stress
de Sá, Rafael A.; de Castro, Frederico A.V.; Eleutherio, Elis C.A.; de Souza, Raquel M.; da Silva, Joaquim F.M.; Pereira, Marcos D.
Propolis is a natural product widely used for humans. Due to its complex composition, a number of applications (antimicrobial, antiinflammatory, anesthetic, cytostatic and antioxidant) have been attributed to this substance. Using Saccharomyces cerevisiae as a eukaryotic model we investigated the mechanisms underlying the antioxidant effect of propolis from Guarapari against oxidative stress. Submitting a wild type (BY4741) and antioxidant deficient strains (ctt1Δ, sod1Δ, gsh1Δ, gtt1Δ and gtt2Δ) either to 15 mM menadione or to 2 mM hydrogen peroxide during 60 min, we observed that all strains, except the mutant sod1Δ, acquired tolerance when previously treated with 25 μg/mL of alcoholic propolis extract. Such a treatment reduced the levels of ROS generation and of lipid peroxidation, after oxidative stress. The increase in Cu/Zn-Sod activity by propolis suggests that the protection might be acting synergistically with Cu/Zn-Sod. PMID:24516431
Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.
Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra
This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jen1, Ady2, Fps1 and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jen1 and Ady2 can mediate the uptake of the anionic form of these substrates by a H(+)-symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fps1 channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions. PMID:26721276
Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.
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
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
Bioflavour production from orange peel hydrolysate using immobilized Saccharomyces cerevisiae.
Lalou, Sofia; Mantzouridou, Fani; Paraskevopoulou, Adamantini; Bugarski, Branko; Levic, Steva; Nedovic, Victor
The rising trend of bioflavour synthesis by microorganisms is hindered by the high manufacturing costs, partially attributed to the cost of the starting material. To overcome this limitation, in the present study, dilute-acid hydrolysate of orange peel was employed as a low-cost, rich in fermentable sugars substrate for the production of flavour-active compounds by Saccharomyces cerevisiae. With this purpose, the use of immobilized cell technology to protect cells against the various inhibitory compounds present in the hydrolysate was evaluated with regard to yeast viability, carbon and nitrogen consumption and cell ability to produce flavour active compounds. For cell immobilization the encapsulation in Ca alginate beads was used. The results were compared with those obtained using free-cell system. Based on the data obtained immobilized cells showed better growth performance and increased ability for de novo synthesis of volatile esters of "fruity" aroma (phenylethyl acetate, ethyl hexanoate, octanoate, decanoate and dodecanoate) than those of free cells. The potential for in situ production of new formulations containing flavour-active compounds derive from yeast cells and also from essential oil of orange peel (limonene, α-terpineol) was demonstrated by the fact that bioflavour mixture was found to accumulate within the beads. Furthermore, the ability of the immobilized yeast to perform efficiently repeated batch fermentations of orange peel hydrolysate for bioflavour production was successfully maintained after six consecutive cycles of a total period of 240 h. PMID:23995224
Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae.
Lundin, M; Deopujari, S W; Lichko, L; da Silva, L P; Baltscheffsky, H
We have studied a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae. The uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the ionophores valinomycin and nigericin stimulate the PPase activity of repeatedly washed yeast mitochondria 2-3-fold. We have previously cloned a yeast gene, PPA2, encoding the catalytic subunit of a mitochondrial PPase. Uncouplers stimulate the PPase activity several-fold in mitochondria from both cells that overexpress PPA2 from a high copy number plasmid and cells with normal expression. These results indicate that the PPA2 polypeptide functions as an energy linked and membrane associated PPase. The stimulation of mitochondrial PPase activity by FCCP, but not by valinomycin and nigericin, was greatly enhanced by the presence of DTT. The antibiotics Dio-9, equisetin and the F0F1-ATPase inhibitor oligomycin also increase mitochondrial PPase activity several fold. This stimulation is much higher, whereas basal PPase activity is lower, in isotonic than in hypotonic solution, which indicates that intact membranes are a prerequisite for maximal effects. PMID:1309654
D-xylulose fermentation to ethanol by Saccharomyces cerevisiae
Chiang, L.C.; Gong, C.S.; Chen, L.F.; Tsao, G.T.
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).
Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.
Kron, S J; Styles, C A; Fink, G R
Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape. Images PMID:7841518
Oxygen requirements of yeasts. [Saccharomyces cerevisiae; Candida tropicalis
Visser, W.; Scheffers, W.A.; Batenburg-Van Der Vegte, W.H.; Van Dijken, J.P. )
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.
Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects
Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves
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
Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.
Swedes, J S; Dial, M E; McLaughlin, C S
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
Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae
Conrad, Michaela; Schothorst, Joep; Kankipati, Harish Nag; Van Zeebroeck, Griet; Rubio-Texeira, Marta; Thevelein, Johan M
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
Nutritional and environmental factors in ethanol fermentation by Saccharomyces cerevisiae
Wong, H.; Wilke, C.R.; Blanch, H.W.
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.
Nutritional and environmental factors in ethanol fermentation by Saccharomyces cerevisiae
Wong, H.
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.
Transcriptional Response of Saccharomyces cerevisiae to Desiccation and Rehydration†
Singh, Jatinder; Kumar, Deept; Ramakrishnan, Naren; Singhal, Vibha; Jervis, Jody; Garst, James F.; Slaughter, Stephen M.; DeSantis, Andrea M.; Potts, Malcolm; Helm, Richard F.
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
Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae
Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.
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
Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae
Michaillat, Lydie; Mayer, Andreas
The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property. PMID:23383298
Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects.
Moysés, Danuza Nogueira; Reis, Viviane Castelo Branco; de Almeida, João Ricardo Moreira; de Moraes, Lidia Maria Pepe; Torres, Fernando Araripe Gonçalves
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
Rapid identification of chemical genetic interactions in Saccharomyces cerevisiae.
Dilworth, David; Nelson, Christopher J
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
Biotransformation of malachite green by Saccharomyces cerevisiae MTCC 463.
Jadhav, J P; Govindwar, S P
In recent years, use of microbial biomass for decolourization of textile industry wastewater is becoming a promising alternative in which some bacteria and fungi are used to replace present treatment processes. Saccharomyces cerevisiae MTCC 463 decolourized the triphenylmethane dyes (malachite green, cotton blue, methyl violet and crystal violet) by biosorption, showing different decolourization patterns. However, malachite green decolourized by biosorption at the initial stage and further biodegradation occurred, about 85% in plain distilled water within 7 h, and about 95.5% in 5% glucose medium within 4 h, under aerobic conditions and at room temperature. Decolourization of malachite green depends on various conditions, such as concentration of dye, concentration of cells, composition of medium and agitation. HPLC, UV-VIS, FTIR and TLC analysis of samples extracted with ethyl acetate from decolourized culture flasks confirmed the biodegradation of malachite green into several metabolites. A study of the enzymes responsible for the biodegradation of malachite green in the control and cells obtained after decolourization showed the activities of laccase, lignin peroxidase, NADH-DCIP reductase, malachite green reductase and aminopyrine N-demethylase in control cells. A significant increase in the activities of NADH-DCIP reductase and MG reductase was observed in the cells obtained after decolourization, indicating a major involvement of reductases in malachite green degradation. PMID:16544273
Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae
Haber, James E.
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
Pressure treatment of Saccharomyces cerevisiae in low-moisture environments.
Moussa, Marwen; Espinasse, Vincent; Perrier-Cornet, Jean-Marie; Gervais, Patrick
We investigated the influence of cell hydration on the ability of Saccharomyces cerevisiae CBS 1171 to withstand extreme hydrostatic pressure in order to determine the mechanisms involved in cell resistance. Hydration conditions were modified in two different ways. We first modulated the chemical potential of water by adding glycerol in cell suspensions. Another procedure consisted in dehydrating cells aerobically and immersing them in perfluorooctane, an innocuous hydrophobic liquid used as a pressure-transmitting medium, prior to pressure treatments. This original method made it possible to transmit isostatic pressure to yeast powders without changing the initial water activity (aw) level at which cells had been equilibrated. The aw ranged between 0.11 and 0.99. Pressure treatments were applied at levels of up to 600 MPa for 10 min, 24 h, and 6 days. The dehydration of cells was found to strongly limit, or even prevent, cell inactivation under pressure. Notably, cells suspended in a water-glycerol mixture with aw levels of 0.71 or below were completely protected against all pressure treatments. Moreover, cells dehydrated aerobically survived for 6 days at 600 MPa even when aw levels were relatively high (up to 0.94). We highlighted the crucial role of water content in determining cellular damage under pressure. When water is available in a sufficient amount, high pressure induces membrane permeabilization, causing uncontrolled mass transfers that could lead to death during a prolonged holding under pressure. Possible mechanisms of membrane permeabilization are discussed. PMID:19633838
Electrophysiology in the eukaryotic model cell Saccharomyces cerevisiae.
Bertl, A; Bihler, H; Kettner, C; Slayman, C L
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
Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.
Quarterman, Josh; Skerker, Jeffrey M; Feng, Xueyang; Liu, Ian Y; Zhao, Huimin; Arkin, Adam P; Jin, Yong-Su
In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without
Ethanol fermentation from Jerusalem artichoke powder using Saccharomyces cerevisiae KCCM50549 without pretreatment for inulin hydrolysis.
Lim, Seok-Hwan; Ryu, Ji-Myoung; Lee, Hongweon; Jeon, Jae Heung; Sok, Dai-Eun; Choi, Eui-Sung
A strain of Saccharomyces cerevisiae, KCCM50549, was found to efficiently ferment the inulin-containing carbohydrates in Jerusalem artichoke without acidic or enzymatic pretreatment prior to fermentation. S. cerevisiae KCCM50549 could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke (up to degree of polymerization (DP) of 15), in contrast to the other S. cerevisiae strain such as NCYC625 that fermented the fructo-oligosaccharides with DP of up to around six. Inulin-fermenting S. cerevisiae KCCM50549 produced c.a. 1.6 times more ethanol from Jerusalem artichoke compared with S. cerevisiae NCYC625. Direct ethanol fermentation of Jerusalem artichoke flour at 180 g/L without any supplements or pretreatments by S. cerevisiae KCCM50549 in a 5 L jar fermentor yielded 36.2 g/L of ethanol within 36 h. The conversion efficiency of inulin-type sugars to ethanol was 70% of the theoretical ethanol yield. PMID:20833540
Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley.
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
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 × 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production. PMID:25242923
Nanofiltration concentration of extracellular glutathione produced by engineered Saccharomyces cerevisiae.
Sasaki, Kengo; Hara, Kiyotaka Y; Kawaguchi, Hideo; Sazuka, Takashi; Ogino, Chiaki; Kondo, Akihiko
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
Saccharomyces cerevisiae Genes Involved in Survival of Heat Shock
Jarolim, Stefanie; Ayer, Anita; Pillay, Bethany; Gee, Allison C.; Phrakaysone, Alex; Perrone, Gabriel G.; Breitenbach, Michael; Dawes, Ian W.
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
Mating-type Gene Switching in Saccharomyces cerevisiae.
Lee, Cheng-Sheng; Haber, James E
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
Ergosterol production from molasses by genetically modified Saccharomyces cerevisiae.
He, Xiuping; Guo, Xuena; Liu, Nan; Zhang, Borun
Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation. PMID:17225097
A vector set for systematic metabolic engineering in Saccharomyces cerevisiae
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
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
[Construction of Saccharomyces cerevisiae cell factories for lycopene production].
Shi, Ming-Yu; Liu Yi; Wang, Dong; Lu, Fu-Ping; Huang, Lu-Qi; Dai, Zhu-Bo; Zhang, Xue-Li
For microbial production of lycopene, the lycopene synthetic genes from Pantoea agglomerans were integrated into Saccharomyces cerevisiae strain BY4742, to obtain strain ZD-L-000 for production of 0.17 mg · L(-1) lycopene. Improving supplies of isoprenoid precursors was then investigated for increasing lycopene production. Four key genes were chosen to be overexpressed, inclu- ding truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMG1), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, a mutated global regulatory factor gene (upc2.1), a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1), which is a key enzyme in the diterpenoid synthetic pathway, and GGPP synthase gene (SaGGPS) from Sulfolobus acidocaldarius. Over-expression of upc2.1 could not improve lycopene production, while over-expression of tHMGI , BTS1-ERG20 and SaGGPS genes led to 2-, 16. 9- and20. 5-fold increase of lycopene production, respectively. In addition, three effective genes, tHMG1, BTS1-ERG20 and SaGGPS, were integrated into rDNA sites of ZD-L-000, resulting in strain ZD-L-201 for production of 13.23 mg · L(-1) lycopene, which was 77-fold higher than that of the parent strain. Finally, two-phase extractive fermentation was performed. The titer of lycopene increased 10-fold to 135.21 mg · L(-1). The engineered yeast strains obtained in this work provided the basis for fermentative production of lycopene. PMID:25751950
Comprehensive Analysis of the SUL1 Promoter of Saccharomyces cerevisiae.
Rich, Matthew S; Payen, Celia; Rubin, Alan F; Ong, Giang T; Sanchez, Monica R; Yachie, Nozomu; Dunham, Maitreya J; Fields, Stanley
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
Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae.
Cid, V J; Durán, A; del Rey, F; Snyder, M P; Nombela, C; Sánchez, M
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
In vivo rearrangement of mitochondrial DNA in Saccharomyces cerevisiae.
Clark-Walker, G D
A revertant (SPR1) from a high-frequency petite strain of Saccharomyces cerevisiae has been shown by mapping and sequence analysis to have a rearranged mitochondrial genome. In vivo rearrangement has occurred through a subgenomic-recombination pathway involving the initial formation of subgenomic molecules in nascent petite mutants, recombination between these molecules to form an intermediate with direct repeats, and subsequent excision of the resident or symposed duplication to yield a molecule with three novel junctions and a changed gene order. Sequencing of the novel junctions shows that intramolecular recombination in each case occurs by means of G + C-rich short direct repeats of 40-51 base pairs. Mapping and sequence analysis also reveal that the SPR1 mitochondrial genome lacks three sectors of the wild-type molecule of 4.4, 1.7, and 0.5 kilobases. Each of these sectors occurs in nontemplate, base-biased DNA, that is over 90% A + T. Absence of these sectors together with a rearranged gene order does not appear to affect the phenotype of SPR1, as colony morphology and growth rate on a number of different substrates are not detectably different from the wild type. Lack of phenotypic change suggests that mitochondrial gene expression has not been noticeably disrupted in SPR1 despite deletion of the consensus nonomer promoter upstream from the glutamic acid tRNA gene. Dispensability of DNA sectors and the presence of recombinogenic short, direct repeats are mandatory features of the subgenomic-recombination pathway for creating rearrangements in baker's yeast mtDNA. It is proposed that, in other organisms, organelle genomes containing these elements may undergo rearrangement by the same steps. Images PMID:2682661
MET17 and Hydrogen Sulfide Formation in Saccharomyces cerevisiae
Spiropoulos, Apostolos; Bisson, Linda F.
Commercial isolates of Saccharomyces cerevisiae differ in the production of hydrogen sulfide (H2S) during fermentation, which has been attributed to variation in the ability to incorporate reduced sulfur into organic compounds. We transformed two commercial strains (UCD522 and UCD713) with a plasmid overexpressing the MET17 gene, which encodes the bifunctional O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase), to test the hypothesis that the level of activity of this enzyme limits reduced sulfur incorporation, leading to H2S release. Overexpression of MET17 resulted in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H2S produced. In contrast, OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced H2S formation. Overexpression of OAS/OAH SHLase activity was greater in UCD713 when grown under low-nitrogen conditions, but the impact on reduction of H2S was greater under high-nitrogen conditions. Thus, there was not a good correlation between the level of enzyme activity and H2S production. We measured cellular levels of cysteine to determine the impact of overexpression of OAS/OAH SHLase activity on sulfur incorporation. While Met17p activity was not correlated with increased cysteine production, conditions that led to elevated cytoplasmic levels of cysteine also reduced H2S formation. Our data do not support the simple hypothesis that variation in OAS/OAH SHLase activity is correlated with H2S production and release. PMID:11010893
Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae.
Edlich, Astrid; Magdanz, Veronika; Rasch, Detlev; Demming, Stefanie; Aliasghar Zadeh, Shobeir; Segura, Rodrigo; Kähler, Christian; Radespiel, Rolf; Büttgenbach, Stephanus; Franco-Lara, Ezequiel; Krull, Rainer
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
A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae
Chu, Daniel B.; Burgess, Sean M.
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
A Computational Approach to Estimating Nondisjunction Frequency in Saccharomyces cerevisiae.
Chu, Daniel B; Burgess, Sean M
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
Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae
Alani, E.; Reenan, RAG.; Kolodner, R. D.
The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA. PMID:8056309
Ecological and Genetic Barriers Differentiate Natural Populations of Saccharomyces cerevisiae.
Clowers, Katie J; Heilberger, Justin; Piotrowski, Jeff S; Will, Jessica L; Gasch, Audrey P
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
Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response.
Hoffman, Kyle S; Duennwald, Martin L; Karagiannis, Jim; Genereaux, Julie; McCarton, Alexander S; Brandl, Christopher J
The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2 The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly. PMID:27172216
Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response
Hoffman, Kyle S.; Duennwald, Martin L.; Karagiannis, Jim; Genereaux, Julie; McCarton, Alexander S.; Brandl, Christopher J.
The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2. The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae. We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly. PMID:27172216
Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts
Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine 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
Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae
Henry, Susan A.; Kohlwein, Sepp D.; Carman, George M.
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
Ecological and Genetic Barriers Differentiate Natural Populations of Saccharomyces cerevisiae
Clowers, Katie J.; Heilberger, Justin; Piotrowski, Jeff S.; Will, Jessica L.; Gasch, Audrey P.
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