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Sample records for saccharomyces cerevisiae exhibit

  1. Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation.

    PubMed

    Reis, Vanda Renata; Bassi, Ana Paula Guarnieri; da Silva, Jessica Carolina Gomes; Ceccato-Antonini, Sandra Regina

    2013-12-01

    Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains ("52"--rough and "PE-02"--smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone. PMID:24688501

  2. Epigenetics in Saccharomyces cerevisiae

    PubMed Central

    Grunstein, Michael; Gasser, Susan M.

    2013-01-01

    Saccharomyces cerevisiae provides a well-studied model system for heritable silent chromatin, in which a nonhistone protein complex—the SIR complex—represses genes by spreading in a sequence-independent manner, much like heterochromatin in higher eukaryotes. The ability to study mutations in histones and to screen genome-wide for mutations that impair silencing has yielded an unparalleled depth of detail about this system. Recent advances in the biochemistry and structural biology of the SIR-chromatin complex bring us much closer to a molecular understanding of how Sir3 selectively recognizes the deacetylated histone H4 tail and demethylated histone H3 core. The existence of appropriate mutants has also shown how components of the silencing machinery affect physiological processes beyond transcriptional repression. PMID:23818500

  3. Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Ostergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

    2000-01-01

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

  4. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  5. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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 systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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 systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  7. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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 systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  8. The Ras/PKA signaling pathway of Saccharomyces cerevisiae exhibits a functional interaction with the Sin4p complex of the RNA polymerase II holoenzyme.

    PubMed Central

    Howard, S C; Chang, Y W; Budovskaya, Y V; Herman, P K

    2001-01-01

    Saccharomyces cerevisiae cells enter into the G(0)-like resting state, stationary phase, in response to specific types of nutrient limitation. We have initiated a genetic analysis of this resting state and have identified a collection of rye mutants that exhibit a defective transcriptional response to nutrient deprivation. These transcriptional defects appear to disrupt the control of normal growth because the rye mutants are unable to enter into a normal stationary phase upon nutrient deprivation. In this study, we examined the mutants in the rye1 complementation group and found that rye1 mutants were also defective for stationary phase entry. Interestingly, the RYE1 gene was found to be identical to SIN4, a gene that encodes a component of the yeast Mediator complex within the RNA polymerase II holoenzyme. Moreover, mutations that affected proteins within the Sin4p module of the Mediator exhibited specific genetic interactions with the Ras protein signaling pathway. For example, mutations that elevated the levels of Ras signaling, like RAS2(val19), were synthetic lethal with sin4. In all, our data suggest that specific proteins within the RNA polymerase II holoenzyme might be targets of signal transduction pathways that are responsible for coordinating gene expression with cell growth. PMID:11560888

  9. Postreplication repair in Saccharomyces cerevisiae

    SciTech Connect

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

    1981-04-01

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

  10. Microarray Analysis for Saccharomyces cerevisiae

    PubMed Central

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

    2011-01-01

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

  11. Chronological Aging in Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

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

  12. Glucose repression in Saccharomyces cerevisiae

    PubMed Central

    Kayikci, Ömur; Nielsen, Jens

    2015-01-01

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

  13. Glucose repression in Saccharomyces cerevisiae.

    PubMed

    Kayikci, Ömur; Nielsen, Jens

    2015-09-01

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

  14. Choline transport in Saccharomyces cerevisiae.

    PubMed

    Hosaka, K; Yamashita, S

    1980-07-01

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

  15. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  16. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  17. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    ERIC Educational Resources Information Center

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

    2008-01-01

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

  18. 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...

  19. Saccharomyces cerevisiae Sen1 Helicase Domain Exhibits 5'- to 3'-Helicase Activity with a Preference for Translocation on DNA Rather than RNA.

    PubMed

    Martin-Tumasz, Stephen; Brow, David A

    2015-09-18

    In the yeast Saccharomyces cerevisiae, the essential nuclear helicase Sen1 is required for efficient termination of transcription of short noncoding RNA genes by RNA polymerase II. However, the mechanism by which Sen1 promotes transcription termination is not known. Prior biochemical studies on the Sen1 homolog from Schizosaccharomyces pombe showed that it can bind and unwind both DNA and RNA, but the S. pombe protein is not essential and has not been demonstrated to function in transcription. Furthermore, Sen1 from either yeast has not previously been expressed as a recombinant protein, due to its large molecular mass (252 kDa in S. cerevisiae). Here, we report the purification and characterization of the 89-kDa S. cerevisiae Sen1 helicase domain (Sen1-HD) produced in Escherichia coli. Sen1-HD binds single-stranded RNA and DNA with similar affinity in the absence of ATP, but it binds RNA more stably than DNA in the presence of ATP, apparently due to a slower translocation rate on RNA. Translocation occurs in the 5' to 3' direction, as for the S. pombe protein. When purified from E. coli at a moderate salt concentration, Sen1-HD was associated with short RNAs that are enriched for the trinucleotide repeat (CAN)4. We propose that Sen1 binds to RNAs and prevents their stable pairing with DNA, consistent with in vivo studies by others showing increased R-loop (RNA/DNA hybrid) formation when Sen1 activity is impaired by mutations. Our results are consistent with a model in which Sen1 promotes transcription termination by resolving R-loops. PMID:26198638

  20. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  1. Biosynthesis of silver nanoparticles using Saccharomyces cerevisiae.

    PubMed

    Korbekandi, Hassan; Mohseni, Soudabeh; Mardani Jouneghani, Rasoul; Pourhossein, Meraj; Iravani, Siavash

    2016-02-01

    The objectives of this study were the biosynthesis of silver nanoparticles (NPs) by biotransformations using Saccharomyces cerevisiae and analysis of the sizes and shapes of the NPs produced. Dried and freshly cultured S. cerevisiae were used as the biocatalyst. Dried yeast synthesized few NPs, but freshly cultured yeast produced a large amount of them. Silver NPs were spherical, 2-20 nm in diameter, and the NPs with the size of 5.4 nm were the most frequent ones. NPs were seen inside the cells, within the cell membrane, attached to the cell membrane during the exocytosis, and outside of the cells. PMID:25101816

  2. "Malonate uptake and metabolism in Saccharomyces cerevisiae".

    PubMed

    Chen, Wei Ning; Tan, Kee Yang

    2013-09-01

    Malonyl-CoA plays an important role in the synthesis and elongation of fatty acids in yeast Saccharomyces cerevisiae. Malonyl-CoA is at a low concentration inside the cell and is produced mainly from acetyl-CoA through the enzyme acetyl-CoA carboxylase. It would be beneficial to find an alternative source of malonyl-CoA to increase its intracellular concentration and overall synthesis of the fatty acids. MatB gene from the bacteria Rhizobium leguminosarium bv. trifolii encodes for a malonyl-CoA synthetase which catalyzes the formation of the malonyl-CoA directly from malonate and CoA. However, results from high-performance liquid chromatography (HPLC) proved that Saccharomyces cerevisiae itself does not contain enough cytoplasmic malonate within them and is unable to uptake exogenously supplied malonate in the form of malonic acid. A dicarboxylic acid plasma membrane transporter with the ability to uptake exogenous malonic acid was identified from another species of yeast known as Schizosaccharomyces pombe and the gene encoding this transporter is identified as the mae1 gene. From the experiments thus far, the mae1 gene had been successfully cloned and transformed into Saccharomyces cerevisiae. The expression and functional ability of the encoded plasma membrane dicarboxylic acid transporter were also demonstrated and verified using specialized technologies such as RT-PCR, yeast immunofluorescence, HPLC, and LC-MS. PMID:23813405

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

    PubMed

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

    2015-12-01

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

  4. Consolidated bioprocessing for bioethanol production using Saccharomyces cerevisiae.

    PubMed

    van Zyl, Willem H; Lynd, Lee R; den Haan, Riaan; McBride, John E

    2007-01-01

    Consolidated bioprocessing (CBP) of lignocellulose to bioethanol refers to the combining of the four biological events required for this conversion process (production of saccharolytic enzymes, hydrolysis of the polysaccharides present in pretreated biomass, fermentation of hexose sugars, and fermentation of pentose sugars) in one reactor. CBP is gaining increasing recognition as a potential breakthrough for low-cost biomass processing. Although no natural microorganism exhibits all the features desired for CBP, a number of microorganisms, both bacteria and fungi, possess some of the desirable properties. This review focuses on progress made toward the development of baker's yeast (Saccharomyces cerevisiae) for CBP. The current status of saccharolytic enzyme (cellulases and hemicellulases) expression in S. cerevisiae to complement its natural fermentative ability is highlighted. Attention is also devoted to the challenges ahead to integrate all required enzymatic activities in an industrial S. cerevisiae strain(s) and the need for molecular and selection strategies pursuant to developing a yeast capable of CBP. PMID:17846725

  5. Gene duplication in Saccharomyces cerevisiae.

    PubMed

    Hansche, P E; Beres, V; Lange, P

    1978-04-01

    Five independent duplications of the acid-phosphatase (aphtase) structural gene (acp1) were recovered from chemostat populations of S. cerevisiae that were subject to selection for in vivo hyper-aphtase activity. Two of the duplications arose spontaneously. Three of them were induced by UV. All five of the duplication events involved the transpositioning of the aphtase structural gene, acp1, and all known genes distal to acp1 on the right arm of chromosome II, to the terminus of an arm of other unknown chromosomes. One of the five duplicated regions of the right arm of chromosome II was found to be transmitted mitotically and meiotically with very high fidelity. The other four duplicated regions of the right arm of chromosome II were found to be unstable, being lost at a rate of about 2% per mitosis. However, selection for increased fidelity of mitotic transmission was effective in one of these strains. No tandem duplications of the aphtase structural gene were found. PMID:348562

  6. Gene Duplication in SACCHAROMYCES CEREVISIAE

    PubMed Central

    Hansche, P. E.; Beres, V.; Lange, P.

    1978-01-01

    Five indepdendent duplications of the acid-phosphatase (aphtase) structural gene (acp1) were recovered from chemostat populations of S. cerevisiae that were subject to selection for in vivo hyper-aphtase activity. Two of the duplications arose spontaneously. Three of them were induced by UV. All five of the duplication events involved the transpositioning of the aphtase structural gene, acp1, and all known genes distal to acp1 on the right arm of chromosome II, to the terminus of an arm of other unknown chromosomes. One of the five duplicated regions of the right arm of chromosome II was found to be transmitted mitotically and meiotically with very high fidelity. The other four duplicated regions of the right arm of chromosome II were found to be unstable, being lost at a rate of about 2% per mitosis. However, selection for increased fidelity of mitotic transmission was effective in one of these strains. No tandem duplications of the aphtase structural gene were found. PMID:348562

  7. Components of microtubular structures in Saccharomyces cerevisiae.

    PubMed Central

    Pillus, L; Solomon, F

    1986-01-01

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

  8. Assembly of F0 in Saccharomyces cerevisiae

    PubMed Central

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

    2009-01-01

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

  9. Biodiversity study of wine yeasts belonging to the "terroir" of Montepulciano d'Abruzzo "Colline Teramane" revealed Saccharomyces cerevisiae strains exhibiting atypical and unique 5.8S-ITS restriction patterns.

    PubMed

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

    2014-05-01

    The Montepulciano d'Abruzzo "Colline Teramane" premium wine DOCG is produced in the Teramo province (Abruzzo, Italy). This region has a great tradition in winemaking and the wine is produced by a spontaneous fermentation so it could represent a reservoir of wine natural yeasts with important oenological features. The aim of this study was to characterize the yeast community of this wine grape growing region in order to create a Saccharomyces cerevisiae bank, providing data on oenological properties for potential industrial applications. A total of 430 yeasts were isolated at the end of spontaneous fermentation. PCR-RFLP was applied for the identification at the species level and underlined that 14 strains exhibited unusual and characteristic restriction patterns different from those typical of the species S. cerevisiae. This difference was due to the insertion of base C at a position 138 in the ITS1 region that determined an additional cleavage site for the enzyme HaeIII. This insertion could be associated to the fermentative performance and associated to the relationship existing between yeasts and a viticulture region or 'terroir'. PMID:24387846

  10. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae.

    PubMed

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

    2015-01-01

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

  11. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae

    PubMed Central

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

    2015-01-01

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

  12. Interactions between Centromere Complexes in Saccharomyces cerevisiae

    PubMed Central

    Nekrasov, Vladimir S.; Smith, Melanie A.; Peak-Chew, Sew; Kilmartin, John V.

    2003-01-01

    We have purified two new complexes from Saccharomyces cerevisiae, one containing the centromere component Mtw1p together with Nnf1p, Nsl1p, and Dsn1p, which we call the Mtw1p complex, and the other containing Spc105p and Ydr532p, which we call the Spc105p complex. Further purifications using Dsn1p tagged with protein A show, in addition to the other components of the Mtw1p complex, the two components of the Spc105p complex and the four components of the previously described Ndc80p complex, suggesting that all three complexes are closely associated. Fluorescence microscopy and immunoelectron microscopy show that Nnf1p, Nsl1p, Dsn1p, Spc105p, and Ydr532p all localize to the nuclear side of the spindle pole body and along short spindles. Chromatin immunoprecipitation assays show that all five proteins are associated with centromere DNA. Homologues of Nsl1p and Spc105p in Schizosaccharomyces pombe also localize to the centromere. Temperature-sensitive mutations of Nsl1p, Dsn1p, and Spc105p all cause defects in chromosome segregation. Synthetic-lethal interactions are found between temperature-sensitive mutations in proteins from all three complexes, in agreement with their close physical association. These results show an increasingly complex structure for the S. cerevisiae centromere and a probable conservation of structure between parts of the centromeres of S. cerevisiae and S. pombe. PMID:14565975

  13. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Nevoigt, Elke

    2008-01-01

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

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

    PubMed Central

    Erratt, J A; Nasim, A

    1986-01-01

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

  15. Synchronization of the Budding Yeast Saccharomyces cerevisiae.

    PubMed

    Foltman, Magdalena; Molist, Iago; Sanchez-Diaz, Alberto

    2016-01-01

    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

  16. Viruses and prions of Saccharomyces cerevisiae

    PubMed Central

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

    2014-01-01

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

  17. Mitophagy and mitochondrial dynamics in Saccharomyces cerevisiae.

    PubMed

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

    2015-10-01

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

  18. LINE Retrotransposition Assays in Saccharomyces cerevisiae.

    PubMed

    Horn, Axel V; Han, Jeffrey S

    2016-01-01

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

  19. Transcriptional regulatory networks in Saccharomyces cerevisiae.

    PubMed

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

    2002-10-25

    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. PMID:12399584

  20. Transcriptional Regulatory Networks in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

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

    2002-10-01

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

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

    PubMed

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

    2001-10-01

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

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

    PubMed Central

    Jones, Gary E.

    1977-01-01

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

  3. Lipid phosphate phosphatases from Saccharomyces cerevisiae.

    PubMed

    Carman, George M; Wu, Wen-I

    2007-01-01

    DPP1-encoded and LPP1-encoded lipid phosphate phosphatases are integral membrane proteins in the yeast Saccharomyces cerevisiae. They catalyze the Mg(2+)-independent dephosphorylation of bioactive lipid phosphate molecules such as diacylglycerol pyrophosphate and phosphatidate. These enzymes possess a three-domain lipid phosphatase motif that is localized to the hydrophilic surface of the membrane. The lipid phosphate phosphatase activities of DPP1-encoded and LPP1-encoded enzymes are measured by following the release of water-soluble radioactive inorganic phosphate from chloroform-soluble radioactive lipid phosphate substrate following a chloroform/methanol/water phase partition. The DPP1-encoded enzyme, commonly referred to as diacylglycerol pyrophosphate phosphatase, is purified from wild-type S. cerevisiae membranes by detergent solubilization with Triton X-100 followed by chromatography with DEAE-cellulose (DE53), Affi-Gel blue, hydroxylapatite, and Mono Q. The purification scheme yields an essentially homogeneous enzyme preparation that is stable for several years upon storage at -80 degrees . The properties of the DPP1-encoded and LPP1-encoded lipid phosphate phosphatase enzymes are summarized. PMID:17954255

  4. Killer systems of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Nesterova, G.F.

    1989-01-01

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

  5. Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae

    PubMed Central

    Garcia, David E.; Keasling, Jay D.

    2014-01-01

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

  6. Molecular and enological characterization of a natural Saccharomyces uvarum and Saccharomyces cerevisiae hybrid.

    PubMed

    Pérez-Torrado, Roberto; González, Sara Susana; Combina, Mariana; Barrio, Eladio; Querol, Amparo

    2015-07-01

    Saccharomyces cerevisiae plays a main role in the winemaking process, although other species, like Saccharomyces uvarum or Saccharomyces paradoxus, have been associated with must fermentations. It has been reported in recent years, that yeast hybrids of different Saccharomyces species might be responsible for wine productions. Although S. cerevisiae×Saccharomyces kudriavzevii hybrids have been well studied, very little attention has been paid to S. cerevisiae×S. uvarum hybrids. In this work we characterized the genomic composition of S6U, a widely used commercial S. cerevisiae×S. uvarum yeast hybrid isolated in wine fermentations containing one copy of the genome of each parental species, which suggests a relatively recent hybridization event. We also studied its performance under diverse enological conditions. The results show enhanced performance under low temperature enological conditions, increased glycerol production, lower acetic acid production and increased production of interesting aroma compounds. We also examined the transcriptomic response of the S6U hybrid strain compared with the reference species under enological conditions. The results show that although the hybrid strain transcriptome is more similar to S. uvarum than to S. cerevisiae, it presents specifically regulated genes involved in stress response, lipids and amino acid metabolism. The enological performance and aroma profile of this S. cerevisiae×S. uvarum hybrid makes it a good candidate for participating in winemaking, especially at low temperatures. PMID:25867085

  7. The flavoproteome of the yeast Saccharomyces cerevisiae.

    PubMed

    Gudipati, Venugopal; Koch, Karin; Lienhart, Wolf-Dieter; Macheroux, Peter

    2014-03-01

    Genome analysis of the yeast Saccharomyces cerevisiae identified 68 genes encoding flavin-dependent proteins (1.1% of protein encoding genes) to which 47 distinct biochemical functions were assigned. The majority of flavoproteins operate in mitochondria where they participate in redox processes revolving around the transfer of electrons to the electron transport chain. In addition, we found that flavoenzymes play a central role in various aspects of iron metabolism, such as iron uptake, the biogenesis of iron-sulfur clusters and insertion of the heme cofactor into apocytochromes. Another important group of flavoenzymes is directly (Dus1-4p and Mto1p) or indirectly (Tyw1p) involved in reactions leading to tRNA-modifications. Despite the wealth of genetic information available for S. cerevisiae, we were surprised that many flavoproteins are poorly characterized biochemically. For example, the role of the yeast flavodoxins Pst2p, Rfs1p and Ycp4p with regard to their electron donor and acceptor is presently unknown. Similarly, the function of the heterodimeric Aim45p/Cir1p, which is homologous to the electron-transferring flavoproteins of higher eukaryotes, in electron transfer processes occurring in the mitochondrial matrix remains to be elucidated. This lack of information extends to the five membrane proteins involved in riboflavin or FAD transport as well as FMN and FAD homeostasis within the yeast cell. Nevertheless, several yeast flavoproteins, were identified as convenient model systems both in terms of their mechanism of action as well as structurally to improve our understanding of diseases caused by dysfunctional human flavoprotein orthologs. PMID:24373875

  8. The flavoproteome of the yeast Saccharomyces cerevisiae?

    PubMed Central

    Gudipati, Venugopal; Koch, Karin; Lienhart, Wolf-Dieter; Macheroux, Peter

    2014-01-01

    Genome analysis of the yeast Saccharomyces cerevisiae identified 68 genes encoding flavin-dependent proteins (1.1% of protein encoding genes) to which 47 distinct biochemical functions were assigned. The majority of flavoproteins operate in mitochondria where they participate in redox processes revolving around the transfer of electrons to the electron transport chain. In addition, we found that flavoenzymes play a central role in various aspects of iron metabolism, such as iron uptake, the biogenesis of iron–sulfur clusters and insertion of the heme cofactor into apocytochromes. Another important group of flavoenzymes is directly (Dus1-4p and Mto1p) or indirectly (Tyw1p) involved in reactions leading to tRNA-modifications. Despite the wealth of genetic information available for S. cerevisiae, we were surprised that many flavoproteins are poorly characterized biochemically. For example, the role of the yeast flavodoxins Pst2p, Rfs1p and Ycp4p with regard to their electron donor and acceptor is presently unknown. Similarly, the function of the heterodimeric Aim45p/Cir1p, which is homologous to the electron-transferring flavoproteins of higher eukaryotes, in electron transfer processes occurring in the mitochondrial matrix remains to be elucidated. This lack of information extends to the five membrane proteins involved in riboflavin or FAD transport as well as FMN and FAD homeostasis within the yeast cell. Nevertheless, several yeast flavoproteins, were identified as convenient model systems both in terms of their mechanism of action as well as structurally to improve our understanding of diseases caused by dysfunctional human flavoprotein orthologs. PMID:24373875

  9. An assay for functional xylose transporters in Saccharomyces cerevisiae.

    PubMed

    Wang, Chengqiang; Shen, Yu; Hou, Jin; Suo, Fan; Bao, Xiaoming

    2013-11-15

    It has been considered that more efficient uptake of xylose could promote increased xylose metabolic capacity of several microorganisms. In this study, an assay to screen xylose transporters was established in the Saccharomyces cerevisiae strain, which expresses the xylosidase gene of Bacillus pumilus intracellularly. The absorbed xylose analog p-nitrophenyl-?-d-xylopyranoside (pNPX) rapidly hydrolyzed to p-nitrophenol (pNP), which displayed a yellow tint when exposed to xylosidase in vivo. The xylose transporter activities of the strain were computed using the pNP production rate, which was detected extracellularly. This method could be used for both high-throughput screening and smaller scale investigations. AraEp, which is a pentose transporter of Corynebacterium glutamicum, was expressed in S. cerevisiae and exhibited better transport capacity than the endogenous transporters Hxt7p and Gal2p. Moreover, a mutant of AraEp with 103% greater transport capacity was screened out, and the computer simulation suggested that transmembrane domain 5 was an important factor for the transport capacity of AraEp in S. cerevisiae. PMID:23928049

  10. Excision repair genes of Saccharomyces cerevisiae.

    PubMed

    Prakash, L; Prakash, S

    1989-01-01

    In the yeast Saccharomyces cerevisiae, at least ten genes are involved in excision repair of DNA damaged by UV radiation and by other agents that distort the DNA helix. Mutations in the RAD1, RAD2, RAD3, RAD4 and RAD10 genes render cells highly defective in the incision of damaged DNA, whereas mutations in the RAD7, RAD14, RAD16, RAD23 and MMS19 genes reduce the level of damage excision. This review summarizes the evidence for the involvement of these genes in excision repair and highlights the important features in the structures of the proteins encoded by the various RAD genes. The RAD3 protein has been purified and characterized in our laboratory, and it possesses single stranded DNA dependent ATPase and DNA helicase activities. The RAD3 helicase moves along the single-stranded DNA in the 5'----3' direction. We suggest that this activity plays a role in strand displacement synthesis during excision repair and in DNA replication. PMID:2665606

  11. Diacylglycerol pyrophosphate phosphatase in Saccharomyces cerevisiae.

    PubMed

    Oshiro, June; Han, Gil Soo; Carman, George M

    2003-11-30

    Diacylglycerol pyrophosphate (DGPP) phosphatase in the yeast Saccharomyces cerevisiae is a Mg(2+)-independent and N-ethylmaleimide-insensitive 34-kDa vacuolar membrane-associated enzyme. It catalyzes the dephosphorylation of DGPP to form phosphatidate (PA) and then removes the phosphate from PA to form diacylglycerol (DAG). The enzyme is a member of the lipid phosphate phosphatase superfamily that contains a three-domain lipid phosphatase motif required for catalytic activity. Expression of the DPP1 gene, which encodes DGPP phosphatase, is induced by zinc depletion, by inositol supplementation, and when cells enter the stationary phase. Induction by zinc depletion is mediated by the transcription factor Zap1p, which binds to a zinc-responsive element in the DPP1 promoter. Repression of DPP1 expression is mediated by the transcription factor Gis1p, which binds to three post-diauxic shift elements in the promoter. Regulation of DPP1 correlates with the expression of DGPP phosphatase activity and the cellular levels of DGPP and PA. PMID:14642771

  12. Responses of Saccharomyces cerevisiae to thermal stress.

    PubMed

    Guyot, Stéphane; Ferret, Eric; Gervais, Patrick

    2005-11-20

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

  13. Stationary phase in the yeast Saccharomyces cerevisiae.

    PubMed Central

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

    1993-01-01

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

  14. Sugar and Glycerol Transport in Saccharomyces cerevisiae.

    PubMed

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

    2016-01-01

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

  15. 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.

    2004-08-01

    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.

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

    PubMed

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

    2014-04-14

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

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

    PubMed Central

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

    2015-01-01

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

  18. Regulation of Cation Balance in Saccharomyces cerevisiae

    PubMed Central

    Cyert, Martha S.; Philpott, Caroline C.

    2013-01-01

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

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

    PubMed

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

    2014-11-01

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

  20. Complete nucleotide sequence of Saccharomyces cerevisiae chromosome X.

    PubMed Central

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

    1996-01-01

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

  1. Isolation of the ARO1 cluster gene of Saccharomyces cerevisiae.

    PubMed

    Larimer, F W; Morse, C C; Beck, A K; Cole, K W; Gaertner, F H

    1983-09-01

    The AROl cluster gene was isolated by complementation in Saccharomyces cerevisiae after transformation with a comprehensive yeast DNA library of BamHI restriction fragments inserted into the shuttle vector YEp13. Most of the transformants exhibited the expected episomal inheritance of the ARO+ phenotype; however, one stable transformant has been shown to be an integration of the AROl fragment and the vector YEp13 at the arol locus. The insert containing AROl is a 17.2-kilobase pair (kbp) BamHI fragment which complements both nonsense and missense alleles of arol. Subcloning by Sau3AI partial digestion further locates the AROl segment to a 6.2-kbp region. An autonomously replicating sequence (ars) was found on the 17.2-kbp fragment. Yeast arol mutants transformed with the AROl episome express 5 to 12 times the normal level of the five AROl enzyme activities and possess elevated amounts of the AROl protein. The yeast AROl fragment also complemented aroA, aroB, aroD, and aroE mutants of Escherichia coli. The expression of AROl in both S. cerevisiae and E. coli was independent of the orientation of the fragment with respect to the vector. PMID:6355828

  2. Synthesis of ribosomes in Saccharomyces cerevisiae.

    PubMed Central

    Warner, J R

    1989-01-01

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

  3. 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...

  4. The nucleotide sequence of Saccharomyces cerevisiae chromosome XII.

    PubMed

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

    1997-05-29

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

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

    ERIC Educational Resources Information Center

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

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

  6. 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...

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

    PubMed

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

    2015-08-01

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

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

    ERIC Educational Resources Information Center

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

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

  9. Correlation between ethanol stress and cellular fatty acid composition of alcohol producing non-Saccharomyces in comparison with Saccharomyces cerevisiae by multivariate techniques.

    PubMed

    Archana, K M; Ravi, R; Anu-Appaiah, K A

    2015-10-01

    Wine production is a complex process both from biochemical and microbiological point of view in which yeast plays a central role. The use of the wine yeast Saccharomyces cerevisiae and non- Saccharomyces yeasts as mixed starter cultures for wine fermentations is of increasing interest to enhance the quality of wine.The most common stress, yeast cells encounter during wine fermentation is the increase in ethanol concentration.To enhance ethanol tolerance, alteration in the cellular lipid composition is one of its defence mechanism. Ethanol tolerance and cellular fatty acid composition of alcohol producing non Saccharomyces forms were compared with enological strains of Sacccharomyces cerevisiae. Saccharomyces cerevisiae used for the study, tolerated 15 % of ethanol and the non Saccharomyces strains such as, Issatchenkia occidentalis and Issatchenkia orientalis tolerated 10 % of ethanol. On exposure of Saccharomyces cerevisiae to ethanol stress, the proportion of monounsaturated fatty acids increased with concomitant decrease in saturated fatty acids. Decrease in monounsaturated fatty acids, exhibited by non-Saccharomyces yeasts when exposed to ethanol stress, could be one of the reasons for their inability to withstand more than 10 % of alcohol. Multivariate techniques of data analysis - principal component analysis and linear discriminant analysis were employed in order to establish differentiation criteria as function of yeast strains, alcohol stress and their fatty acid profile. Based on the data, Chemometrics, such as principal component analysis and discriminant function analysis, can be successfully applied to fatty acid data to categorize the yeast. PMID:26396428

  10. Potential immobilized Saccharomyces cerevisiae as heavy metal removal

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  11. [Expression of inulinase genes in the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus].

    PubMed

    Sokolenko, G G; Karpechenko, N A

    2015-01-01

    Expression of the genes encoding the enzymes involved in inulin, sucrose, and glucose metabolism in the yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus was studied. The exon-intron structure of the relevant genes was identified and the primers for quantitative PCR were optimized. Expression of the genes was found to depend on the carbon source. Glucose was shown to exhibit a repressive effect on inulinase synthesis by K. marxianus, while in S. cerevisiae glucose and sucrose were inulinase inducer and repressor, respectively. PMID:25916145

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

    PubMed

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

    2014-03-01

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

  13. Purification of Arp2/3 complex from Saccharomyces cerevisiae.

    PubMed

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

    2013-01-01

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

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

    PubMed

    Kelly, Amy C; Wickner, Reed B

    2013-01-01

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

  15. Properties and functional significance of Saccharomyces cerevisiae ADHVI.

    PubMed

    Larroy, Carol; Rosario Fernández, M; González, Eva; Parés, Xavier; Biosca, Josep A

    2003-02-01

    The completion of the Saccharomyces cerevisiae genome project has provided the opportunity to explore for new genes of the medium-chain dehydrogenase/reductase enzyme superfamily. Our group has recently identified a new gene, the YMR318C open reading frame, which coded for a Zn-containing NADP(H)-dependent alcohol dehydrogenase (ADHVI). ADHVI has been purified to homogeneity from over expressing yeast cells, and found to be a homodimer of 40 kDa subunits. The enzyme showed a strict specificity for NADP(H) and high activity with a variety of long chain aliphatic and bulky substrates. Aldehydes exhibited 50-12000 times higher catalytic efficiency than the corresponding alcohols. Substrates with high k(cat)/K(m) were: pentanal, veratraldehyde and cinnamaldehyde. The ADHVI expression was strongly induced when galactose was the sole carbon source in the culture medium. Phylogenetic trees include ADHVI in the cinnamyl alcohol dehydrogenase (CADH) family. In contrast to the plant CADH, involved in lignin biosynthesis, this is not the function for ADHVI, since yeast does not synthesize lignin. ADHVI may be physiologically involved in several steps of the lignin degradation pathway, initiated by other microorganisms, in the synthesis of fusel alcohols, products derived from the aminoacidic metabolism, and in the homeostasis of NADP(H). Disruption of ADH6 was not lethal for the yeast, under laboratory conditions. PMID:12604208

  16. Chromosome mobility during meiotic prophase in Saccharomyces cerevisiae

    PubMed Central

    Scherthan, Harry; Wang, Hailin; Adelfalk, Caroline; White, Eric J.; Cowan, Carrie; Cande, W. Zacheus; Kaback, David B.

    2007-01-01

    In many organisms, a synaptonemal complex (SC) intimately connects each pair of homologous chromosomes during much of the first meiotic prophase and is thought to play a role in regulating recombination. In the yeast Saccharomyces cerevisiae, the central element of each SC contains Zip1, a protein orthologous to mammalian SYCP1. To study the dynamics of SCs in living meiotic cells, a functional ZIP1::GFP fusion was introduced into yeast and analyzed by fluorescence video microscopy. During pachytene, SCs exhibited dramatic and continuous movement throughout the nucleus, traversing relatively large distances while twisting, folding, and unfolding. Chromosomal movements were accompanied by changes in the shape of the nucleus, and all movements were reversibly inhibited by the actin antagonist Latrunculin B. Normal movement required the NDJ1 gene, which encodes a meiosis-specific telomere protein needed for the attachment of telomeres to the nuclear periphery and for normal kinetics of recombination and meiosis. These results show that SC movements involve telomere attachment to the nuclear periphery and are actin-dependent and suggest these movements could facilitate completion of meiotic recombination. PMID:17939997

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

    PubMed

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

    2013-12-01

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

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  20. Genome instability in rad54 mutants of Saccharomyces cerevisiae

    PubMed Central

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

    2003-01-01

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

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

    PubMed

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

    2013-11-01

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

  2. Genotoxicity assessment of amaranth and allura red using Saccharomyces cerevisiae.

    PubMed

    Jabeen, Hafiza Sumara; ur Rahman, Sajjad; Mahmood, Shahid; Anwer, Sadaf

    2013-01-01

    Amaranth (E123) and Allura red (E129), very important food azo dyes used in food, drug, paper, cosmetic and textile industries, were assessed for their genotoxic potential through comet assay in yeast cells. Comet assay was standardized by with different concentration of H(2)O(2). Concentrations of Amaranth and Allura red were maintained in sorbitol buffer starting from 9.76 to 5,000 ?g/mL and 1 × 10(4) cells were incubated at two different incubation temperatures 28 and 37°C. Amaranth (E123) and Allura red (E129) were found to exhibit their genotoxic effect directly in Saccharomyces cerevisiae. No significant genotoxic activity was observed for Amaranth and Allura red at 28°C but at 37°C direct relation of Amaranth concentration with comet tail was significant and no positive relation was seen with time exposure factor. At 37°C the minimum concentration of Amaranth and Allura red at which significant DNA damage observed through comet assay was 1,250 ?g/mL in 2nd h post exposure time. The results indicated that food colors should be carefully used in baking products as heavy concentration of food colors could affect the fermentation process of baking. PMID:23132362

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

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

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

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

    SciTech Connect

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

    2010-01-01

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

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

    SciTech Connect

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

    2010-09-01

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

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

    DOE PAGESBeta

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

    2010-09-01

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

  7. Oligoadenylate is present in the mitochondrial RNA of Saccharomyces cerevisiae

    SciTech Connect

    Yuckenberg, P.D.; Phillips, S.L.

    1982-04-01

    The authors examined Saccharomyces cerevisiae mitochondrial RNA for polyadenylate. Using hybridization to (/sup 3/H)polyuridylate as the assay for adenylate sequences, they found adenylate-rich oligonucleotides approximately 8 residues long. Longer polyadenylate was not detected. Most of the adenylate-rich sequence is associated with the large mitochondrial rRNA. The remainder is associated with the 10-12S group of transcripts.

  8. Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

    PubMed Central

    Rensing, C; Kües, U; Stahl, U; Nies, D H; Friedrich, B

    1992-01-01

    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

  9. Efficient screening of environmental isolates for Saccharomyces cerevisiae strains that are suitable for brewing.

    PubMed

    Fujihara, Hidehiko; Hino, Mika; Takashita, Hideharu; Kajiwara, Yasuhiro; Okamoto, Keiko; Furukawa, Kensuke

    2014-01-01

    We developed an efficient screening method for Saccharomyces cerevisiae strains from environmental isolates. MultiPlex PCR was performed targeting four brewing S. cerevisiae genes (SSU1, AWA1, BIO6, and FLO1). At least three genes among the four were amplified from all S. cerevisiae strains. The use of this method allowed us to successfully obtain S. cerevisiae strains. PMID:25036140

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

    PubMed

    Šuranská, Hana; Vránová, Dana; Omelková, Ji?ina

    2016-01-01

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

  11. Growth of non-Saccharomyces yeasts affects nutrient availability for Saccharomyces cerevisiae during wine fermentation.

    PubMed

    Medina, Karina; Boido, Eduardo; Dellacassa, Eduardo; Carrau, Francisco

    2012-07-01

    Yeast produces numerous secondary metabolites during fermentation that impact final wine quality. Although it is widely recognized that growth of diverse non-Saccharomyces (NS) yeast can positively affect flavor complexity during Saccharomyces cerevisiae wine fermentation, the inability to control spontaneous or co-fermentation processes by NS yeast has restricted their use in winemaking. We selected two NS yeasts from our Uruguayan native collection to study NS-S. cerevisiae interactions during wine fermentation. The selected strains of Hanseniaspora vineae and Metschnikowia pulcherrima had different yeast assimilable nitrogen consumption profiles and had different effects on S. cerevisiae fermentation and growth kinetics. Studies in which we varied inoculum size and using either simultaneous or sequential inoculation of NS yeast and S. cerevisiae suggested that competition for nutrients had a significant effect on fermentation kinetics. Sluggish fermentations were more pronounced when S. cerevisiae was inoculated 24h after the initial stage of fermentation with a NS strain compared to co-inoculation. Monitoring strain populations using differential WL nutrient agar medium and fermentation kinetics of mixed cultures allowed for a better understanding of strain interactions and nutrient addition effects. Limitation of nutrient availability for S. cerevisiae was shown to result in stuck fermentations as well as to reduce sensory desirability of the resulting wine. Addition of diammonium phosphate (DAP) and a vitamin mix to a defined medium allowed for a comparison of nutrient competition between strains. Addition of DAP and the vitamin mix was most effective in preventing stuck fermentations. PMID:22687186

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

    PubMed Central

    Guimarães, Pedro MR; Oliveira, Carla

    2010-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

    The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 × 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

  15. Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts

    PubMed Central

    2014-01-01

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

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

    PubMed

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

    2012-03-01

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

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

    PubMed Central

    Goddard, Matthew R.; Greig, Duncan

    2015-01-01

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

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

    PubMed

    Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-02-01

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

  19. Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in Saccharomyces cerevisiae?

    PubMed Central

    Zelle, Rintze M.; Trueheart, Josh; Harrison, Jacob C.; Pronk, Jack T.; van Maris, Antonius J. A.

    2010-01-01

    Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type Saccharomyces cerevisiae. Pyruvate carboxylase-negative (Pyc?) S. cerevisiae strains cannot grow on glucose unless media are supplemented with C4 compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the S. cerevisiae PEPCK encoded by PCK1 is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in S. cerevisiae. Pyc? S. cerevisiae strains constitutively overexpressing the PEPCK either from S. cerevisiae or from Actinobacillus succinogenes did not grow on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h?1, one-half that of the (pyruvate carboxylase-positive) reference strain grown under the same conditions. Growth was dependent on high carbon dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C4 dicarboxylic acids in S. cerevisiae. PMID:20581175

  20. Overproduction of fatty acids in engineered Saccharomyces cerevisiae.

    PubMed

    Li, Xiaowei; Guo, Daoyi; Cheng, Yongbo; Zhu, Fayin; Deng, Zixin; Liu, Tiangang

    2014-09-01

    The long hydrocarbon fatty acyl chain is energy rich, making it an ideal precursor for liquid transportation fuels and high-value oleo chemicals. As Saccharomyces cerevisiae has many advantages for industrial production compared to Escherichia coli. Here, we attempted to engineer Saccharomyces cerevisiae for overproduction of fatty acids. First, disruption of the beta-oxidation pathway, elimination of the acyl-CoA synthetases, overexpression of different thioesterases and acetyl-CoA carboxylase ACC1, and engineering the supply of precursor acetyl-CoA. The engineered strain XL122 produced more than 120 mg/L of fatty acids. In parallel, we inactivated ADH1, the dominant gene for ethanol production, to redirect the metabolic flux to fatty acids synthesis. The engineered strain DG005 produced about 140 mg/L fatty acids. Additionally, Acetyl-CoA carboxylase was identified as a critical bottleneck of fatty acids synthesis in S. cerevisiae with a cell-free system. However, overexpression of ACC1 has little effect on fatty acids biosynthesis. As it has been reported that phosphorylation of ACC1 may influent its activity, so phosphorylation sites of ACC1 were further identified. Although the regulatory mechanisms remain unclear, our results provide rationale for future studies to target this critical step. All these efforts, particularly the discovery of the limiting step are critical for developing a "cell factory" for the overproduction of fatty acids by using type I fatty acids synthase in yeast or other fungi. PMID:24752690

  1. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

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

  2. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  3. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  4. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  5. 40 CFR 180.1246 - Yeast Extract Hydrolysate from Saccharomyces cerevisiae: exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

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

  6. Utilization of D-asparagine by Saccharomyces cerevisiae.

    PubMed

    Dunlop, P C; Roon, R J; Even, H L

    1976-03-01

    Yeast strains sigma1278b and Harden and Young, which synthesize only an internal constitutive form of L-asparaginase, do not grow on D-asparagine, as a sole source of nitrogen, and whole cell suspensions of these strains do not hydrolyze D-asparagine. Strains X2180-A2 and D273-10B, which possess an externally active form of asparaginase, are able to grow slowly on D-asparagine, and nitrogen-starved suspensions of these strains exhibit high activity toward the D-isomer. Nitrogen starvation of strain X218O-A2 results in coordinate increase of D- and L-asparaginase activity; the specific activity observed for the D-isomer is approximately 20% greater than that observed for the L-isomer. It was observed, in studies with cell extracts, that hydrolysis of D-asparagine occurred only with extracts from nitrogen-starved cells of strains that synthesize the external form of asparaginase. Furthermore, the activity of the extracts toward the D-isomer was always higher than that observed with the L-isomer. A 400-fold purified preparation of external asparaginase from Saccharomyces cerevisiae X218U-A2 hydrolyzed D-asparagine with an apparent Km of 0.23 mM and a Vmax of 38.7 mumol/min per mg of protein. D-Asparagine was a competitive inhibitor of L-asparagine hydrolysis and the Ki determined for this inhibition was approximately equal to its Km. These data suggest that D-asparagine is a good substrate for the external yeast asparaginase but is a poor substrate for the internal enzyme. PMID:767332

  7. Characterization and evolution of xylose isomerase screened from the bovine rumen metagenome in Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Shen, Yu; Jiao, Chunlei; Ge, Ruilei; Zhang, Xingjing; Bao, Xiaoming

    2016-02-01

    The conversion of abundant levels of xylose in lignocellulosic materials into viable products would generate economic benefits. The heterologous expression of the xylose isomerase (XI) gene is considered a direct and effective strategy for establishing the xylose metabolic pathway in Saccharomyces cerevisiae. However, only limited sources of xylA are functionally expressed in S. cerevisiae and are capable of driving effective xylose consumption. In this study, Ru-xylA (where Ru represents the rumen), which was screened from the contents of the bovine rumen metagenomic library, was functionally expressed in S. cerevisiae, and the enzyme activity was 1.31 U mg(-1) protein. This is a new source of XI that can exhibit high activity levels in S. cerevisiae. The activity of this enzyme is comparable to those of the Piromyces sp. XI. Then, the Ru-XI activity was further improved through mutagenesis and growth-based screening in a centromeric plasmid. A variant containing two mutations (K11T/D220V) that exhibited a 68% increase in enzyme activity was isolated. Our work identified a new xylose isomerase that can be functionally expressed in S. cerevisiae and results in a higher XI enzyme activity through mutagenesis. PMID:26160406

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    1986-04-01

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

  10. Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

    PubMed Central

    Santangelo, G M; Tornow, J; McLaughlin, C S; Moldave, K

    1988-01-01

    Promoters were isolated at random from the genome of Saccharomyces cerevisiae by using a plasmid that contains a divergently arrayed pair of promoterless reporter genes. A comprehensive library was constructed by inserting random (DNase I-generated) fragments into the intergenic region upstream from the reporter genes. Simple in vivo assays for either reporter gene product (alcohol dehydrogenase or beta-galactosidase) allowed the rapid identification of promoters from among these random fragments. Poly(dA-dT) homopolymer tracts were present in three of five randomly cloned promoters. With two exceptions, each RNA start site detected was 40 to 100 base pairs downstream from a TATA element. All of the randomly cloned promoters were capable of activating reporter gene transcription bidirectionally. Interestingly, one of the promoter fragments originated in a region of the S. cerevisiae rDNA spacer; regulated divergent transcription (presumably by RNA polymerase II) initiated in the same region. Images PMID:2847031

  11. Glucose- and nitrogen sensing and regulatory mechanisms in Saccharomyces cerevisiae.

    PubMed

    Rødkaer, Steven V; Faergeman, Nils J

    2014-08-01

    Pro- and eukaryotic cells are constantly challenged by varying concentrations of nutrients in their environment. Perceiving and adapting to such changes are therefore crucial for cellular viability. Thus, numerous specialized cellular receptors continuously sense and react to the availability of nutrients such as glucose and nitrogen. When stimulated, these receptors initiate various cellular signaling pathways, which in concert constitute a complex regulatory network. To ensure a highly specific response, these pathways and networks cross-communicate with each other and are regulated at several steps and by numerous different regulators. As numerous of these regulating proteins, biochemical mechanisms, and cellular pathways are evolutionary conserved, complex biochemical information relevant to humans can be obtained by studying simple organisms. Thus, the yeast Saccharomyces cerevisiae has been recognized as a powerful model system to study fundamental biochemical processes. In the present review, we highlight central signaling pathways and molecular circuits conferring nitrogen- and glucose sensing in S. cerevisiae. PMID:24738657

  12. Advanced biofuel production by the yeast Saccharomyces cerevisiae.

    PubMed

    Buijs, Nicolaas A; Siewers, Verena; Nielsen, Jens

    2013-06-01

    Replacement of conventional transportation fuels with biofuels will require production of compounds that can cover the complete fuel spectrum, ranging from gasoline to kerosene. Advanced biofuels are expected to play an important role in replacing fossil fuels because they have improved properties compared with ethanol and some of these may have the energy density required for use in heavy duty vehicles, ships, and aviation. Moreover, advanced biofuels can be used as drop-in fuels in existing internal combustion engines. The yeast cell factory Saccharomyces cerevisiae can be turned into a producer of higher alcohols (1-butanol and isobutanol), sesquiterpenes (farnesene and bisabolene), and fatty acid ethyl esters (biodiesel), and here we discusses progress in metabolic engineering of S. cerevisiae for production of these advanced biofuels. PMID:23628723

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

    PubMed

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

    2010-11-01

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

  14. Expression of acylphosphatase in Saccharomyces cerevisiae enhances ethanol fermentation rate

    SciTech Connect

    Raugei, G.; Modesti, A.; Magherini, F.

    1996-06-01

    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.

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

    PubMed

    Gadd, G M; White, C

    1989-01-01

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

  16. Homeostatic and Adaptive Responses to Zinc Deficiency in Saccharomyces cerevisiae*

    PubMed Central

    Eide, David J.

    2009-01-01

    Zinc is an essential nutrient and serves as a structural or catalytic cofactor for many proteins. Thus, cells need mechanisms to maintain zinc homeostasis when available zinc supplies decrease. In addition, cells require other mechanisms to adapt intracellular processes to suboptimal levels of zinc. By exploring the transcriptional responses to zinc deficiency, studies of the yeast Saccharomyces cerevisiae have revealed both homeostatic and adaptive responses to low zinc. The Zap1 zinc-responsive transcription factor regulates several genes in yeast, and the identity of these genes has led to new insights regarding how cells respond to the stress of zinc deficiency. PMID:19363031

  17. Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates.

    PubMed Central

    Ogden, R C; Lee, M C; Knapp, G

    1984-01-01

    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

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

    Diezmann, Stephanie; Dietrich, Fred S.

    2009-01-01

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

  20. Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae?

    PubMed Central

    Brat, Dawid; Boles, Eckhard; Wiedemann, Beate

    2009-01-01

    In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains. PMID:19218403

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

    SciTech Connect

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

    1991-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-08-01

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

  4. Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

    PubMed Central

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

    2015-01-01

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

  5. 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

    2011-06-01

    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.

  6. Copper oxide nanoparticles inhibit the metabolic activity of Saccharomyces cerevisiae.

    PubMed

    Mashock, Michael J; Kappell, Anthony D; Hallaj, Nadia; Hristova, Krassimira R

    2016-01-01

    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. Environ Toxicol Chem 2016;35:134-143. © 2015 SETAC. PMID:26178758

  7. Membrane trafficking in the yeast Saccharomyces cerevisiae model.

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2013-01-01

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

  9. Intracellular analysis of Saccharomyces cerevisiae using CLSM after ultrasonic treatments.

    PubMed

    Kon, Takuo; Nakakura, Shinya; Mitsubayashi, Kohji

    2005-06-01

    The effect of ultrasonic treatments (28, 45, and 100 kHz) as sterilization on Saccharomyces cerevisiae was investigated both by colony-forming ability and by a confocal laser-scanning microscope (CLSM), using a fluorescence staining approach with MDY-64 for endoplasmic reticula and Rhodamine B for mitochondria. The ultrasonic treatments, especially at the lower frequency of 28 kHz, were effective for sterilizing S cerevisiae, thus inducing the remarkable decrease in colony counts of S cerevisiae on YPD plate medium, but with some inactive (dead) cells. The CLSM images of fluorescence-stained organelles in the cell showed the intracellular fracture and the increase in fluorescent intensities of MDY-64 for endoplasmic reticula and Rhodamine B for mitochondria without cell membrane collapse by the ultrasonic treatments, especially at 28 kHz. The effect of the conditions (frequency, power, medium, and so on) of the ultrasonic treatments on cell components such as biological membranes would be different, thus inducing the effective and selective sterilization of some types of microorganisms. PMID:17292073

  10. De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts.

    PubMed

    Carrau, Francisco M; Medina, Karina; Boido, Eduardo; Farina, Laura; Gaggero, Carina; Dellacassa, Eduardo; Versini, Giuseppe; Henschke, Paul A

    2005-02-01

    This paper reports the production of monoterpenes, which elicit a floral aroma in wine, by strains of the yeast Saccharomyces cerevisiae. Terpenes, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of certain wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine they are considered to originate from grape and not fermentation. However, the biosynthesis of monoterpenes by S. cerevisiae in the absence of grape derived precursors is shown here to be of de novo origin in wine yeast strains. Higher concentration of assimilable nitrogen increased accumulation of linalool and citronellol. Microaerobic compared with anaerobic conditions favored terpene accumulation in the ferment. The amount of linalool produced by some strains of S. cerevisiae could be of sensory importance in wine production. These unexpected results are discussed in relation to the known sterol biosynthetic pathway and to an alternative pathway for terpene biosynthesis not previously described in yeast. PMID:15668008

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

    PubMed

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

    2015-07-01

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

  12. Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

    PubMed Central

    2012-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  14. Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  16. Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation

    SciTech Connect

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

    1988-01-01

    An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decease 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.

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

    PubMed

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

    2015-01-01

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

  18. On the Mechanism of Gene Silencing in Saccharomyces cerevisiae

    PubMed Central

    Steakley, David Lee; Rine, Jasper

    2015-01-01

    Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation. This study provided strong support for the steric occlusion mechanism by the discovery that RNA polymerase of bacteriophage T7 could be substantially blocked from transcribing from its cognate promoter when embedded in silenced chromatin. Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin. The effectiveness of the Sir protein–based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing. PMID:26082137

  19. MLH1 mutations differentially affect meiotic functions in Saccharomyces cerevisiae.

    PubMed Central

    Hoffmann, Eva R; Shcherbakova, Polina V; Kunkel, Thomas A; Borts, Rhona H

    2003-01-01

    To test whether missense mutations in the cancer susceptibility gene MLH1 adversely affect meiosis, we examined 14 yeast MLH1 mutations for effects on meiotic DNA transactions and gamete viability in the yeast Saccharomyces cerevisiae. Mutations analogous to those associated with hereditary nonpolyposis colorectal cancer (HNPCC) or those that reduce Mlh1p interactions with ATP or DNA all impair replicative mismatch repair as measured by increased mutation rates. However, their effects on meiotic heteroduplex repair, crossing over, chromosome segregation, and gametogenesis vary from complete loss of meiotic functions to no meiotic defect, and mutants defective in one meiotic process are not necessarily defective in others. DNA binding and ATP binding but not ATP hydrolysis are required for meiotic crossing over. The results reveal clear separation of different Mlh1p functions in mitosis and meiosis, and they suggest that some, but not all, MLH1 mutations may be a source of human infertility. PMID:12618391

  20. Ethylene production by metabolic engineering of the yeast Saccharomyces cerevisiae.

    PubMed

    Pirkov, I; Albers, E; Norbeck, J; Larsson, C

    2008-09-01

    The non-ethylene producing yeast, Saccharomyces cerevisiae, was transformed into an ethylene producer by introducing the ethylene forming enzyme from the plant pathogenic bacterium Pseudomonas syringae. Cultivation of the metabolically engineered strain was performed in well-controlled bioreactors as aerobic batch cultures with an on-line monitoring of ethylene production. The highest productivity was obtained during the respiro-fermentative growth on glucose but there was also a significant rate of formation during the subsequent phase of ethanol respiration. Furthermore, investigations were performed whether substitution of the original nitrogen source, NH(4)(+), for glutamate could improve productivity and yield of ethylene even more. The rationale being that one of the substrates for the enzyme is 2-oxoglutarate and this compound can be formed from glutamate in a single reaction. Indeed, there was a substantial improvement in the rate of production and the final yield of ethylene was almost three times higher when NH(4)(+) was replaced by glutamate. PMID:18640286

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

    PubMed Central

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

    2015-01-01

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

  2. Brazilian propolis protects Saccharomyces cerevisiae cells against oxidative stress.

    PubMed

    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

    2013-01-01

    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 (ctt1?, sod1?, gsh1?, gtt1? and gtt2?) either to 15 mM menadione or to 2 mM hydrogen peroxide during 60 min, we observed that all strains, except the mutant sod1?, acquired tolerance when previously treated with 25 ?g/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

  3. Replication of avocado sunblotch viroid in the yeast Saccharomyces cerevisiae.

    PubMed

    Delan-Forino, Clémentine; Maurel, Marie-Christine; Torchet, Claire

    2011-04-01

    Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, "naked" RNAs, which replicate through RNA-RNA transcription. Viroids of the Avsunviroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, viroids have only been detected in plant cells. Here, we investigate the replication of Avocado sunblotch viroid (ASBVd) of the Avsunviroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrate that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We show that the viroid monomeric RNA is destabilized by the nuclear 3' and the cytoplasmic 5' RNA degradation pathways. For the first time, our results provide evidence that viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd. PMID:21270165

  4. Replication of Avocado Sunblotch Viroid in the Yeast Saccharomyces cerevisiae?

    PubMed Central

    Delan-Forino, Clémentine; Maurel, Marie-Christine; Torchet, Claire

    2011-01-01

    Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, “naked” RNAs, which replicate through RNA-RNA transcription. Viroids of the Avsunviroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, viroids have only been detected in plant cells. Here, we investigate the replication of Avocado sunblotch viroid (ASBVd) of the Avsunviroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrate that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We show that the viroid monomeric RNA is destabilized by the nuclear 3? and the cytoplasmic 5? RNA degradation pathways. For the first time, our results provide evidence that viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd. PMID:21270165

  5. Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues.

    PubMed

    Wiltschi, Birgit; Wenger, Waltraud; Nehring, Sebastian; Budisa, Nediljko

    2008-11-01

    We replaced the single N-terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non-canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae. Our non-canonical amino acid incorporation protocol involves a two-step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of approximately 12% could be elevated to 40% by increasing the non-canonical amino acid concentration in the medium by 10-fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture. PMID:19061186

  6. Brazilian propolis protects Saccharomyces cerevisiae cells against oxidative stress

    PubMed Central

    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.

    2013-01-01

    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 (ctt1?, sod1?, gsh1?, gtt1? and gtt2?) either to 15 mM menadione or to 2 mM hydrogen peroxide during 60 min, we observed that all strains, except the mutant sod1?, acquired tolerance when previously treated with 25 ?g/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

  7. Antioxidant protection of resveratrol and catechin in Saccharomyces cerevisiae.

    PubMed

    Dani, Caroline; Bonatto, Diego; Salvador, Mirian; Pereira, Marcos D; Henriques, João A P; Eleutherio, Elis

    2008-06-11

    Moderate consumption of red wine reduces the risk of heart disease and extends lifespan, but the relative contribution of wine polyphenols to these effects is unclear. In this work, the capacity of resveratrol and catechin to protect the eukaryotic microorganism Saccharomyces cerevisiae against oxidative stress caused by different agents, hydrogen peroxide, carbon tetrachloride, and cadmium, was evaluated. Under all stress conditions, both polyphenols increased tolerance, although their protection was more evident under peroxide exposure. By using mutant strains deficient in specific antioxidant defense systems (superoxide dismutases, catalase, or glutathione), it was observed that increased H2O2 tolerance produced by both polyphenols was associated with catalase, as well as the rise in survival rates caused by resveratrol under CCl4. The acquisition of tolerance was correlated with a reduction in lipid peroxidation, indicating that the antioxidant property of resveratrol and catechin involves protection against membrane oxidation. PMID:18489120

  8. Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae.

    PubMed

    Harris, C L; Kolanko, C J

    1995-07-01

    The size distribution of aminoacyl-tRNA synthetase activity was investigated in cell extracts prepared from Saccharomyces cerevisiae. Bio-Gel A-5M chromatography of 105,000 g supernatants separated isoleucyl-tRNA synthetase activity into three peaks, with apparent molecular masses (Da) of about 100,000, 350,000 and 10(6) or greater. Similar results were obtained with synthetases specific for glutamic acid, serine and tyrosine. Sucrose-density-gradient centrifugation of yeast supernatants also provided evidence for the existence of synthetase complexes. These data provide the first evidence for the existence of a high-molecular-mass aminoacyl-tRNA synthetase complex in yeast, perhaps similar to those reported in higher eukaryotes. PMID:7619074

  9. Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae.

    PubMed Central

    Harris, C L; Kolanko, C J

    1995-01-01

    The size distribution of aminoacyl-tRNA synthetase activity was investigated in cell extracts prepared from Saccharomyces cerevisiae. Bio-Gel A-5M chromatography of 105,000 g supernatants separated isoleucyl-tRNA synthetase activity into three peaks, with apparent molecular masses (Da) of about 100,000, 350,000 and 10(6) or greater. Similar results were obtained with synthetases specific for glutamic acid, serine and tyrosine. Sucrose-density-gradient centrifugation of yeast supernatants also provided evidence for the existence of synthetase complexes. These data provide the first evidence for the existence of a high-molecular-mass aminoacyl-tRNA synthetase complex in yeast, perhaps similar to those reported in higher eukaryotes. PMID:7619074

  10. Modeling growth and telomere dynamics in Saccharomyces cerevisiae

    PubMed Central

    Bertuch, Alison A.

    2013-01-01

    A general branching process is proposed to model a population of cells of the yeast Saccharomyces cerevisiae following loss of telomerase. Previously published experimental data indicate that a population of telomerase-deficient cells regain exponential growth after a period of slowing due to critical telomere shortening. The explanation for this phenomenon is that some cells engage telomerase-independent pathways to maintain telomeres that allow them to become “survivors.” Our model takes into account random variation in individual cell cycle times, telomere length, finite replicative lifespan of mother cells, and survivorship. We identify and estimate crucial parameters such as the probability of an individual cell becoming a survivor, and compare our model predictions to experimental data. PMID:20018194

  11. Regulation of Phospholipid Synthesis in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Carman, George M.; Han, Gil-Soo

    2013-01-01

    The yeast Saccharomyces cerevisiae, with its full complement of organelles, synthesizes membrane phospholipids by pathways that are generally common to those found in higher eukaryotes. Phospholipid synthesis in yeast is regulated in response to a variety of growth conditions (e.g., inositol supplementation, zinc depletion, and growth stage) by a coordination of genetic (e.g., transcriptional activation and repression) and biochemical (e.g., activity modulation and localization) mechanisms. Phosphatidate (PA), whose cellular levels are controlled by the activities of key phospholipid synthesis enzymes, plays a central role in the transcriptional regulation of phospholipid synthesis genes. In addition to the regulation of gene expression, phosphorylation of key phospholipid synthesis catalytic and regulatory proteins controls the metabolism of phospholipid precursors and products. PMID:21275641

  12. Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Melén, Karin; Blomberg, Anders; von Heijne, Gunnar

    2006-07-01

    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

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

    SciTech Connect

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

    2007-11-23

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

  14. Random Segregation of Chromatids at Mitosis in Saccharomyces Cerevisiae

    PubMed Central

    Neff, M. W.; Burke, D. J.

    1991-01-01

    Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges. PMID:2016049

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2015-12-01

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

  17. Global mapping of DNA conformational flexibility on Saccharomyces cerevisiae.

    PubMed

    Menconi, Giulia; Bedini, Andrea; Barale, Roberto; Sbrana, Isabella

    2015-04-01

    In this study we provide the first comprehensive map of DNA conformational flexibility in Saccharomyces cerevisiae complete genome. Flexibility plays a key role in DNA supercoiling and DNA/protein binding, regulating DNA transcription, replication or repair. Specific interest in flexibility analysis concerns its relationship with human genome instability. Enrichment in flexible sequences has been detected in unstable regions of human genome defined fragile sites, where genes map and carry frequent deletions and rearrangements in cancer. Flexible sequences have been suggested to be the determinants of fragile gene proneness to breakage; however, their actual role and properties remain elusive. Our in silico analysis carried out genome-wide via the StabFlex algorithm, shows the conserved presence of highly flexible regions in budding yeast genome as well as in genomes of other Saccharomyces sensu stricto species. Flexibile peaks in S. cerevisiae identify 175 ORFs mapping on their 3'UTR, a region affecting mRNA translation, localization and stability. (TA)n repeats of different extension shape the central structure of peaks and co-localize with polyadenylation efficiency element (EE) signals. ORFs with flexible peaks share common features. Transcripts are characterized by decreased half-life: this is considered peculiar of genes involved in regulatory systems with high turnover; consistently, their function affects biological processes such as cell cycle regulation or stress response. Our findings support the functional importance of flexibility peaks, suggesting that the flexible sequence may be derived by an expansion of canonical TAYRTA polyadenylation efficiency element. The flexible (TA)n repeat amplification could be the outcome of an evolutionary neofunctionalization leading to a differential 3'-end processing and expression regulation in genes with peculiar function. Our study provides a new support to the functional role of flexibility in genomes and a strategy for its characterization inside human fragile sites. PMID:25860149

  18. Global Mapping of DNA Conformational Flexibility on Saccharomyces cerevisiae

    PubMed Central

    Menconi, Giulia; Bedini, Andrea; Barale, Roberto; Sbrana, Isabella

    2015-01-01

    In this study we provide the first comprehensive map of DNA conformational flexibility in Saccharomyces cerevisiae complete genome. Flexibility plays a key role in DNA supercoiling and DNA/protein binding, regulating DNA transcription, replication or repair. Specific interest in flexibility analysis concerns its relationship with human genome instability. Enrichment in flexible sequences has been detected in unstable regions of human genome defined fragile sites, where genes map and carry frequent deletions and rearrangements in cancer. Flexible sequences have been suggested to be the determinants of fragile gene proneness to breakage; however, their actual role and properties remain elusive. Our in silico analysis carried out genome-wide via the StabFlex algorithm, shows the conserved presence of highly flexible regions in budding yeast genome as well as in genomes of other Saccharomyces sensu stricto species. Flexibile peaks in S. cerevisiae identify 175 ORFs mapping on their 3’UTR, a region affecting mRNA translation, localization and stability. (TA)n repeats of different extension shape the central structure of peaks and co-localize with polyadenylation efficiency element (EE) signals. ORFs with flexible peaks share common features. Transcripts are characterized by decreased half-life: this is considered peculiar of genes involved in regulatory systems with high turnover; consistently, their function affects biological processes such as cell cycle regulation or stress response. Our findings support the functional importance of flexibility peaks, suggesting that the flexible sequence may be derived by an expansion of canonical TAYRTA polyadenylation efficiency element. The flexible (TA)n repeat amplification could be the outcome of an evolutionary neofunctionalization leading to a differential 3’-end processing and expression regulation in genes with peculiar function. Our study provides a new support to the functional role of flexibility in genomes and a strategy for its characterization inside human fragile sites. PMID:25860149

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. Effect of GAL4 Gene Dosage on the Level of Galactose Catabolic Enzymes in Saccharomyces cerevisiae

    PubMed Central

    Klar, Amar J. S.; Halvorson, Harlyn O.

    1976-01-01

    Lack of GAL4 gene dosage on the level of uridine diphosphogalactose epimerase (EC 5.1.3.2) activity suggests the positive regulatory role for this locus on the control of galactose catabolic enzymes in Saccharomyces cerevisiae. PMID:1107315

  2. Acquisition of the Ability To Assimilate Mannitol by Saccharomyces cerevisiae through Dysfunction of the General Corepressor Tup1-Cyc8

    PubMed Central

    Chujo, Moeko; Yoshida, Shiori; Ota, Anri; Murata, Kousaku

    2014-01-01

    Saccharomyces cerevisiae normally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. The molecular basis of this inability remains unknown. We found that cells capable of assimilating mannitol arose spontaneously from wild-type S. cerevisiae during prolonged culture in mannitol-containing medium. Based on microarray data, complementation analysis, and cell growth data, we demonstrated that acquisition of mannitol-assimilating ability was due to spontaneous mutations in the genes encoding Tup1 or Cyc8, which constitute a general corepressor complex that regulates many kinds of genes. We also showed that an S. cerevisiae strain carrying a mutant allele of CYC8 exhibited superior salt tolerance relative to other ethanologenic microorganisms; this characteristic would be highly beneficial for the production of bioethanol from marine biomass. Thus, we succeeded in conferring the ability to assimilate mannitol on S. cerevisiae through dysfunction of Tup1-Cyc8, facilitating production of ethanol from mannitol. PMID:25304510

  3. Isolation and characterization of the Saccharomyces cerevisiae DPP1 gene encoding diacylglycerol pyrophosphate phosphatase.

    PubMed

    Toke, D A; Bennett, W L; Dillon, D A; Wu, W I; Chen, X; Ostrander, D B; Oshiro, J; Cremesti, A; Voelker, D R; Fischl, A S; Carman, G M

    1998-02-01

    Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1Delta mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1Delta mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S. cerevisiae. PMID:9452443

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

    DOEpatents

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

    2002-01-01

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

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

    PubMed

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

    2016-04-01

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

  6. Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin

    PubMed Central

    Badin-Larçon, A. C.; Boscheron, C.; Soleilhac, J. M.; Piel, M.; Mann, C.; Denarier, E.; Fourest-Lieuvin, A.; Lafanechère, L.; Bornens, M.; Job, D.

    2004-01-01

    In most eukaryotic cells, the C-terminal amino acid of ?-tubulin is aromatic (Tyr in mammals and Phe in Saccharomyces cerevisiae) and is preceded by two glutamate residues. In mammals, the C-terminal Tyr of ?-tubulin is subject to cyclic removal from the peptide chain by a carboxypeptidase and readdition to the chain by a tubulin–Tyr ligase. There is evidence that tubulin–Tyr ligase suppression and the resulting accumulation of detyrosinated (Glu) tubulin favor tumor growth, both in animal models and in human cancers. However, the molecular basis for this apparent stimulatory effect of Glu tubulin accumulation on tumor progression is unknown. Here we have developed S. cerevisiae strains expressing only Glu tubulin and used them as a model to assess the consequences of Glu tubulin accumulation in cells. We find that Glu tubulin strains show defects in nuclear oscillations. These defects are linked to a markedly decreased association of the yeast ortholog of CLIP170, Bik1p, with microtubule plus-ends. These results indicate that the accumulation of Glu tubulin in cells affects microtubule tip complexes that are important for microtubule interactions with the cell cortex. PMID:15031428

  7. The nucleotide sequence of Saccharomyces cerevisiae chromosome IV.

    PubMed

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

    1997-05-29

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    1991-03-01

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

  11. Exploring improved endoglucanase expression in Saccharomyces cerevisiae strains.

    PubMed

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

    2010-05-01

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

  12. Quantifying separation and similarity in a Saccharomyces cerevisiae metapopulation

    PubMed Central

    Knight, Sarah; Goddard, Matthew R

    2015-01-01

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

  13. Metabolic engineering of Saccharomyces cerevisiae for itaconic acid production.

    PubMed

    Blazeck, John; Miller, Jarrett; Pan, Anny; Gengler, Jon; Holden, Clinton; Jamoussi, Mariam; Alper, Hal S

    2014-10-01

    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

  14. Structure of the Glycosyltransferase Ktr4p from Saccharomyces cerevisiae

    PubMed Central

    Possner, Dominik D. D.; Claesson, Magnus; Guy, Jodie E.

    2015-01-01

    In the yeast Saccharomyces cerevisiae, members of the Kre2/Mnt1 protein family have been shown to be ?-1,2-mannosyltransferases or ?-1,2-mannosylphosphate transferases, utilising an Mn2+-coordinated GDP-mannose as the sugar donor and a variety of mannose derivatives as acceptors. Enzymes in this family are localised to the Golgi apparatus, and have been shown to be involved in both N- and O-linked glycosylation of newly-synthesised proteins, including cell wall glycoproteins. Our knowledge of the nine proteins in this family is however very incomplete at present. Only one family member, Kre2p/Mnt1p, has been studied by structural methods, and three (Ktr4p, Ktr5p, Ktr7p) are completely uncharacterised and remain classified only as putative glycosyltransferases. Here we use in vitro enzyme activity assays to provide experimental confirmation of the predicted glycosyltransferase activity of Ktr4p. Using GDP-mannose as the donor, we observe activity towards the acceptor methyl-?-mannoside, but little or no activity towards mannose or ?-1,2-mannobiose. We also present the structure of the lumenal catalytic domain of S. cerevisiae Ktr4p, determined by X-ray crystallography to a resolution of 2.2 Å, and the complex of the enzyme with GDP to 1.9 Å resolution. PMID:26296208

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Kavanaugh, Laura A.; Dietrich, Fred S.

    2009-01-01

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

  18. Metabolic engineering of glycerol production in Saccharomyces cerevisiae.

    PubMed

    Overkamp, Karin M; Bakker, Barbara M; Kötter, Peter; Luttik, Marijke A H; Van Dijken, Johannes P; Pronk, Jack T

    2002-06-01

    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 tpi1delta nde1delta nde2delta gut2delta 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

  19. High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

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

  20. Involvement of an Actomyosin Contractile Ring in Saccharomyces cerevisiae Cytokinesis

    PubMed Central

    Bi, Erfei; Maddox, Paul; Lew, Daniel J.; Salmon, E.D.; McMillan, John N.; Yeh, Elaine; Pringle, John R.

    1998-01-01

    In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother–bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother–bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1? strains divide nearly as efficiently as wild type; other myo1? strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell–wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin. PMID:9732290

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2013-04-01

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

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2012-10-01

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

  5. Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae.

    PubMed

    Bakker, B M; Overkamp, K M; van Maris AJ; Kötter, P; Luttik, M A; van Dijken JP; Pronk, J T

    2001-01-01

    In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments. PMID:11152939

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

    SciTech Connect

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

    1992-09-01

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

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

    EPA Science Inventory

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

  8. 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...

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

    PubMed

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

    2014-04-01

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

  10. Effects of ethanol and other alkanols on the general amino acid permease of saccharomyces cerevisiae

    SciTech Connect

    Leao, C.; Van Uden, N.

    1984-01-01

    Among the mechanisms that underlie the inhibition by alkanols of growth and fermentation in Saccharomyces cerevisiae, the noncompetitive exponential inhibition of nutrient transport across the plasma membrane may play a major role. So far this type of inhibition was shown to affect the transport systems of S. cerevisiae for glucose, maltose, and ammonium. The effects of ethanol, isopropanol, propanol, and butanol on the functioning of the general amino acid permease (GAP) of S. cerevisiae are reported.

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

    PubMed Central

    2013-01-01

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

  12. Outcrossing, mitotic recombination, and life-history trade-offs shape genome evolution in Saccharomyces cerevisiae.

    PubMed

    Magwene, Paul M; Kay?kç?, Ömür; Granek, Joshua A; Reininga, Jennifer M; Scholl, Zackary; Murray, Debra

    2011-02-01

    We carried out a population genomic survey of Saccharomyces cerevisiae diploid isolates and find that many budding yeast strains have high levels of genomic heterozygosity, much of which is likely due to outcrossing. We demonstrate that variation in heterozygosity among strains is correlated with a life-history trade-off that involves how readily yeast switch from asexual to sexual reproduction under nutrient stress. This trade-off is reflected in a negative relationship between sporulation efficiency and pseudohyphal development and correlates with variation in the expression of RME1, a transcription factor with pleiotropic effects on meiosis and filamentous growth. Selection for alternate life-history strategies in natural versus human-associated environments likely contributes to differential maintenance of genomic heterozygosity through its effect on the frequency that yeast lineages experience sexual cycles and hence the opportunity for inbreeding. In addition to elevated levels of heterozygosity, many strains exhibit large genomic regions of loss-of-heterozygosity (LOH), suggesting that mitotic recombination has a significant impact on genetic variation in this species. This study provides new insights into the roles that both outcrossing and mitotic recombination play in shaping the genome architecture of Saccharomyces cerevisiae. This study also provides a unique case where stark differences in the genomic distribution of genetic variation among individuals of the same species can be largely explained by a life-history trade-off. PMID:21245305

  13. Comparison of Fermentation and Wines Produced by Inoculation of Hanseniaspora vineae and Saccharomyces cerevisiae

    PubMed Central

    Lleixà, Jessica; Martín, Valentina; Portillo, María del C.; Carrau, Francisco; Beltran, Gemma; Mas, Albert

    2016-01-01

    Interest in the use of non-Saccharomyces yeasts in winemaking has been increasing due to their positive contributions to wine quality. The non-Saccharomyces yeast Hanseniaspora vineae is an apiculate yeast that has been associated with the production of wine with good aromatic properties. However, little is known about the fermentation dynamics of H. vineae in natural must and its interaction with autochthonous yeasts. In the present study, we performed semi industrial fermentations of Macabeo and Merlot musts inoculated with either H. vineae or S. cerevisiae. The yeast population dynamics were monitored by plate culturing, PCR-DGGE and massive sequencing techniques. The results obtained with these techniques show that H. vineae was able dominate the autochthonous microbiota in Macabeo must but not in Merlot must, which exhibited a larger, more diverse yeast population. The presence of H. vineae throughout most of the Macabeo fermentation resulted in more fruity and flowery wine, as indicated by the chemical analysis of the final wines, which demonstrated a strong presence of phenyl ethyl acetate at concentrations higher than the threshold of perception and approximately 50 times more than that produced in wines fermented with S. cerevisiae. This compound is associated with fruity, floral and honey aromas. PMID:27014252

  14. Regulation of allantoate transport in wild-type and mutant strains of Saccharomyces cerevisiae.

    PubMed Central

    Chisholm, V T; Lea, H Z; Rai, R; Cooper, T G

    1987-01-01

    Accumulation of intracellular allantoin and allantoate is mediated by two distinct active transport systems in Saccharomyces cerevisiae. Allantoin transport (DAL4 gene) is inducible, while allantoate uptake is constitutive (it occurs at full levels in the absence of any allantoate-related compounds from the culture medium). Both systems appear to be sensitive to nitrogen catabolite repression, feedback inhibition, and trans-inhibition. Mutants (dal5) that lack allantoate transport have been isolated. These strains also exhibit a 60% loss of allantoin transport capability. Conversely, dal4 mutants previously described are unable to transport allantoin and exhibit a 50% loss of allantoate transport. We interpret the pleiotropic behavior of the dal4 and dal5 mutations as deriving from a functional interaction between elements of the two transport systems. PMID:3549700

  15. Modulation of efficiency of translation termination in Saccharomyces cerevisiae.

    PubMed

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

    2014-01-01

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

  16. Modulation of efficiency of translation termination in Saccharomyces cerevisiae

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1994-01-01

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

  18. Identification and Characterization of Phenylpyruvate Decarboxylase Genes in Saccharomyces cerevisiae

    PubMed Central

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

    2003-01-01

    Catabolism of amino acids via the Ehrlich pathway involves transamination to the corresponding ?-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 Km and Vmax 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 of the Ehrlich pathway. PMID:12902239

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

    Jensen, Laran T; Culotta, Valeria Cizewski

    2002-04-26

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

  1. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. PMID:26801560

  2. Opuntia ficus-indica cladodes as feedstock for ethanol production by Kluyveromyces marxianus and Saccharomyces cerevisiae.

    PubMed

    Kuloyo, Olukayode O; du Preez, James C; García-Aparicio, Maria del Prado; Kilian, Stephanus G; Steyn, Laurinda; Görgens, Johann

    2014-12-01

    The feasibility of ethanol production using an enzymatic hydrolysate of pretreated cladodes of Opuntia ficus-indica (prickly pear cactus) as carbohydrate feedstock was investigated, including a comprehensive chemical analysis of the cladode biomass and the effects of limited aeration on the fermentation profiles and sugar utilization. The low xylose and negligible mannose content of the cladode biomass used in this study suggested that the hemicellulose structure of the O. ficus-indica cladode was atypical of hardwood or softwood hemicelluloses. Separate hydrolysis and fermentation and simultaneous saccharification and fermentation procedures using Kluyveromyces marxianus and Saccharomyces cerevisiae at 40 and 35 °C, respectively, gave similar ethanol yields under non-aerated conditions. In oxygen-limited cultures K. marxianus exhibited almost double the ethanol productivity compared to non-aerated cultures, although after sugar depletion utilization of the produced ethanol was evident. Ethanol concentrations of up to 19.5 and 20.6 g l(-1) were obtained with K. marxianus and S. cerevisiae, respectively, representing 66 and 70 % of the theoretical yield on total sugars in the hydrolysate. Because of the low xylan content of the cladode biomass, a yeast capable of xylose fermentation might not be a prerequisite for ethanol production. K. marxianus, therefore, has potential as an alternative to S. cerevisiae for bioethanol production. However, the relatively low concentration of fermentable sugars in the O. ficus-indica cladode hydrolysate presents a technical constraint for commercial exploitation. PMID:25248867

  3. Introducing a new breed of wine yeast: interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast and Saccharomyces mikatae.

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2002-01-01

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

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

    SciTech Connect

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

    1990-12-01

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

  7. Systematic Identification of Balanced Transposition Polymorphisms in Saccharomyces cerevisiae

    PubMed Central

    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.

    2009-01-01

    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

  8. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae.

    PubMed

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

    2015-01-01

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

  9. D-xylulose fermentation to ethanol by Saccharomyces cerevisiae

    SciTech Connect

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

    1981-08-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

  12. Size control models of Saccharomyces cerevisiae cell proliferation

    SciTech Connect

    Wheals, A.E.

    1982-04-01

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

  13. Towards a genome-wide transcriptogram: the Saccharomyces cerevisiae case.

    PubMed

    Rybarczyk-Filho, José Luiz; Castro, Mauro A A; Dalmolin, Rodrigo J S; Moreira, José C F; Brunnet, Leonardo G; de Almeida, Rita M C

    2011-04-01

    Analysis of genome-wide expression data poses a challenge to extract relevant information. The usual approaches compare cellular expression levels relative to a pre-established control and genes are clustered based on the correlation of their expression levels. This implies that cluster definitions are dependent on the cellular metabolic state, eventually varying from one experiment to another. We present here a computational method that order genes on a line and clusters genes by the probability that their products interact. Protein-protein association information can be obtained from large data bases as STRING. The genome organization obtained this way is independent from specific experiments, and defines functional modules that are associated with gene ontology terms. The starting point is a gene list and a matrix specifying interactions. Considering the Saccharomyces cerevisiae genome, we projected on the ordering gene expression data, producing plots of transcription levels for two different experiments, whose data are available at Gene Expression Omnibus database. These plots discriminate metabolic cellular states, point to additional conclusions, and may be regarded as the first versions of 'transcriptograms'. This method is useful for extracting information from cell stimuli/responses experiments, and may be applied with diagnostic purposes to different organisms. PMID:21169199

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae

    PubMed Central

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

    1992-01-01

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

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

    PubMed

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

    1995-05-25

    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. PMID:7784171

  19. Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae

    PubMed Central

    Michaillat, Lydie; Mayer, Andreas

    2013-01-01

    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

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

    PubMed Central

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

    1990-01-01

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

  1. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae

    PubMed Central

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

    2014-01-01

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

  2. The Network Architecture of the Saccharomyces cerevisiae Genome

    PubMed Central

    Hoang, Stephen A.; Bekiranov, Stefan

    2013-01-01

    We propose a network-based approach for surmising the spatial organization of genomes from high-throughput interaction data. Our strategy is based on methods for inferring architectural features of networks. Specifically, we employ a community detection algorithm to partition networks of genomic interactions. These community partitions represent an intuitive interpretation of genomic organization from interaction data. Furthermore, they are able to recapitulate known aspects of the spatial organization of the Saccharomyces cerevisiae genome, such as the rosette conformation of the genome, the clustering of centromeres, as well as tRNAs, and telomeres. We also demonstrate that simple architectural features of genomic interaction networks, such as cliques, can give meaningful insight into the functional role of the spatial organization of the genome. We show that there is a correlation between inter-chromosomal clique size and replication timing, as well as cohesin enrichment. Together, our network-based approach represents an effective and intuitive framework for interpreting high-throughput genomic interaction data. Importantly, there is a great potential for this strategy, given the rich literature and extensive set of existing tools in the field of network analysis. PMID:24349163

  3. Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion.

    PubMed

    Carman, George M; Han, Gil-Soo

    2007-03-01

    The synthesis of phospholipids in the yeast Saccharomyces cerevisiae is regulated by zinc, an essential mineral required for growth and metabolism. Cells depleted of zinc contain increased levels of phosphatidylinositol and decreased levels of phosphatidylethanolamine. In addition to the major phospholipids, the levels of the minor phospholipids phosphatidate and diacylglycerol pyrophosphate decrease in the vacuole membrane of zinc-depleted cells. Alterations in phosphatidylinositol and phosphatidylethanolamine can be ascribed to an increase in PIS1-encoded phosphatidylinositol synthase activity and to decreases in the activities of CDP-diacylglycerol pathway enzymes including the CHO1-encoded phosphatidylserine synthase, respectively. Alterations in the minor vacuole membrane phospholipids are due to the induction of the DPP1-encoded diacylglycerol pyrophosphate phosphatase. These changes in the activities of phospholipid biosynthetic enzymes result from differential regulation of gene expression at the level of transcription. Under zinc-deplete conditions, the positive transcription factor Zap1p stimulates the expression of the DPP1 and PIS1 genes through the cis-acting element UAS(ZRE). In contrast, the negative regulatory protein Opi1p, which is involved in inositol-mediated regulation of phospholipid synthesis, represses the expression of the CHO1 gene through the cis-acting element UAS(INO). Regulation of phospholipid synthesis may provide an important mechanism by which cells cope with the stress of zinc depletion, given the roles that phospholipids play in the structure and function of cellular membranes. PMID:16807089

  4. Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.

    PubMed

    Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra

    2016-01-01

    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

  5. Bioflavour production from orange peel hydrolysate using immobilized Saccharomyces cerevisiae.

    PubMed

    Lalou, Sofia; Mantzouridou, Fani; Paraskevopoulou, Adamantini; Bugarski, Branko; Levic, Steva; Nedovic, Victor

    2013-11-01

    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, ?-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

  6. Reversal of PCNA Ubiquitylation by Ubp10 in Saccharomyces cerevisiae

    PubMed Central

    Gallego-Sánchez, Alfonso; Andrés, Sonia; Conde, Francisco; San-Segundo, Pedro A.; Bueno, Avelino

    2012-01-01

    Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2BK123 ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in response to the simple presence of ubPCNA. In agreement with this, we show that Ubp10 forms a complex with PCNA in vivo. Interestingly, we also show that deletion of UBP10 alters in different ways the interaction of PCNA with DNA polymerase ?–associated protein Rev1 and with accessory subunit Rev7. While deletion of UBP10 enhances PCNA–Rev1 interaction, it decreases significantly Rev7 binding to the sliding clamp. Finally, we report that Ubp10 counteracts Rad18 E3-ubiquitin ligase activity on PCNA at lysine 164 in such a manner that deregulation of Ubp10 expression causes tolerance impairment and MMS hypersensitivity. PMID:22829782

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

    SciTech Connect

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

    1984-01-01

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

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

    PubMed

    DiCarlo, James E; Norville, Julie E; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M

    2013-04-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast. PMID:23460208

  9. Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae

    PubMed Central

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

    2015-01-01

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

  10. Sulfate Assimilation Mediates Tellurite Reduction and Toxicity in Saccharomyces cerevisiae

    PubMed Central

    Ottosson, Lars-Göran; Logg, Katarina; Ibstedt, Sebastian; Sunnerhagen, Per; Käll, Mikael; Blomberg, Anders; Warringer, Jonas

    2010-01-01

    Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism. PMID:20675578

  11. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  12. Production of resveratrol from tyrosine in metabolically engineered Saccharomyces cerevisiae.

    PubMed

    Shin, So-Yeon; Jung, Sang-Min; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

    2012-09-10

    Resveratrol, a polyphenol compound found in grape skins, has been proposed to account for the beneficial effects of red wine against heart disease. To produce resveratrol in Saccharomyces cerevisiae, four heterologous genes were introduced: the phenylalanine ammonia lyase gene from Rhodosporidium toruloides, the cinnamic acid 4-hydroxylase and 4-coumarate:coenzyme A ligase genes both from Arabidopsis thaliana, and the stilbene synthase gene from Arachis hypogaea. When this recombinant yeast was cultivated by batch fermentation in YP medium containing 2% galactose, it produced 2.6 mg/L p-coumaric acid and 3.3 mg/L resveratrol. In order to increase the pool of malonyl-CoA, a key precursor in resveratrol biosynthesis, the acetyl-CoA carboxylase (ACC1) gene was additionally overexpressed in the yeast by replacing the native promoter of the ACC1 gene with the stronger GAL1 promoter and this resulted in enhanced production of resveratrol (4.3 mg/L). Furthermore, when tyrosine was supplemented in the medium, the concentration of resveratrol increased up to 5.8 mg/L. This result illustrates a possible strategy for developing metabolically engineered yeast strain for the economical production of resveratrol from cheap amino acids. PMID:22883555

  13. Atg36: the Saccharomyces cerevisiae receptor for pexophagy.

    PubMed

    Motley, Alison M; Nuttall, James M; Hettema, Ewald H

    2012-11-01

    Eukaryotic cells adapt their organelle composition and abundance according to environmental conditions. Analysis of the peroxisomal membrane protein Pex3 has revealed that this protein plays a crucial role in peroxisome maintenance as it is required for peroxisome formation, segregation and breakdown. Although its function in peroxisome formation and segregation was known to involve its recruitment to the peroxisomal membrane of factors specific for these processes, the role of Pex3 in peroxisome breakdown was unclear until our recent identification of Atg36 as a novel Saccharomyces cerevisiae Pex3-interacting protein. Atg36 is recruited to peroxisomes by Pex3 and is required specifically for pexophagy. Atg36 is distinct from Atg30, the pexophagy receptor identified in Pichia pastoris. Atg36 interacts with Atg11 in vivo, and to a lesser extent with Atg8. These latter proteins link autophagic cargo receptors to the core autophagy machinery. Like other autophagic cargo receptors, Atg36 is a suicide receptor and is broken down in the vacuole together with its cargo. Unlike other cargo receptors, the interaction between Atg36 and Atg8 does not seem to be direct. Our recent findings suggest that Atg36 is a novel pexophagy receptor that may target peroxisomes for degradation via a noncanonical mechanism. PMID:22874561

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Feng, Xueyang; Lian, Jiazhang; Zhao, Huimin

    2015-01-01

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

  16. A quantitative assay for telomere protection in Saccharomyces cerevisiae.

    PubMed Central

    DuBois, Michelle L; Haimberger, Zara W; McIntosh, Martin W; Gottschling, Daniel E

    2002-01-01

    Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This "chromosomal cap" assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected. PMID:12136006

  17. Identification of Genes Required for ?2 Repression in Saccharomyces Cerevisiae

    PubMed Central

    Wahi, M.; Johnson, A. D.

    1995-01-01

    Transcriptional repression of the a-specific genes in Saccharomyces cerevisiae ? cells involves the concerted action of several proteins. The homeodomain protein ?2, together with MCM1, recruits two general transcriptional repressors, SSN6 and TUP1, to the promoters of a-specific genes. SSN6 and TUP1 then mediate repression of the a-specific genes. SIN4, another general negative regulator, is required for this repression, but unlike tup1 or ssn6 deletions, sin4 deletions cause only partial loss of repression. We have screened for other genes required for a-specific gene repression in ? cells. In addition to recovering multiple alleles of previously identified genes required for this process (referred to as ?2 repression), we have identified four other genes, designated ARE1, ARE2, ARE3, and ARE4 (for alpha2 repression). Recessive mutations in the ARE genes cause partial loss of a-specific gene repression and cause pleiotropic phenotypes similar to those resulting from mutations in SSN6, TUP1, or SIN4, suggesting that the ARE genes are general negative regulators. Based on our initial analysis, we propose that two distinct classes of general negative regulators cooperate to bring about full levels of ?2 repression. The sequence of ARE1 revealed that it encodes a CDC28-related protein kinase, identical to UME5, and thus suggests that protein phosphorylation plays a role in ?2 repression. PMID:7635311

  18. Conversion of Wine Strains of Saccharomyces cerevisiae to Heterothallism

    PubMed Central

    Bakalinsky, Alan T.; Snow, Richard

    1990-01-01

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

  19. Analysis of Meiotic Recombination Pathways in the Yeast Saccharomyces Cerevisiae

    PubMed Central

    Mao-Draayer, Y.; Galbraith, A. M.; Pittman, D. L.; Cool, M.; Malone, R. E.

    1996-01-01

    In the yeast, Saccharomyces cerevisiae, several genes appear to act early in meiotic recombination. HOP1 and RED1 have been classified as such early genes. The data in this paper demonstrate that neither a red1 nor a hop1 mutation can rescue the inviable spores produced by a rad52 spo13 strain; this phenotype helps to distinguish these two genes from other early meiotic recombination genes such as SPO11, REC104, or MEI4. In contrast, either a red1 or a hop1 mutation can rescue a rad50S spo13 strain; this phenotype is similar to that conferred by mutations in the other early recombination genes (e.g., REC104). These two different results can be explained because the data presented here indicate that a rad50S mutation does not diminish meiotic intrachromosomal recombination, similar to the mutant phenotypes conferred by red1 or hop1. Of course, RED1 and HOP1 do act in the normal meiotic interchromosomal recombination pathway; they reduce interchromosomal recombination to ~10% of normal levels. We demonstrate that a mutation in a gene (REC104) required for initiation of exchange is completely epistatic to a mutation in RED1. Finally, mutations in either HOP1 or RED1 reduce the number of double-strand breaks observed at the HIS2 meiotic recombination hotspot. PMID:8878674

  20. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae

    PubMed Central

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

    2015-01-01

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

  1. Water-Transfer Slows Aging in Saccharomyces cerevisiae.

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

  3. Water-Transfer Slows Aging in Saccharomyces cerevisiae

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    TOXLINE Toxicology Bibliographic Information

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

    2012-10-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

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

    PubMed

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

    2007-05-01

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

  8. Isolation and characterization of aminopeptidase mutants of Saccharomyces cerevisiae.

    PubMed Central

    Trumbly, R J; Bradley, G

    1983-01-01

    Mutants of Saccharomyces cerevisiae were isolated which have decreased ability to hydrolyze leucine beta-naphthylamide, a chromogenic substrate for amino-peptidases. The mutations were shown by starch gel electrophoresis to affect one of four different aminopeptidases. Mutations affecting a given enzyme belong to a single complementation group. The four genes were symbolized lap1, lap2, lap3, and lap4, and the corresponding enzymes LAPI, LAPII, LAPIII, and LAPIV. Both lap1 and lap4 were mapped to the left arm of chromosome XI, and lap3 was mapped to the left arm of chromosome XIV. Strains which possessed only one of the four leucine aminopeptidases (LAPs) were constructed. Crude extracts from these strains were used to study the properties of the individual enzymes. Dialysis against EDTA greatly reduced the activity of all the LAPs except for LAPIII. Of the cations tested, Co2+ was the most effective in restoring activity. LAPIV was the only LAP reactivated by Zn2+. LAPI was purified 331-fold and LAPII was purified 126-fold from cell homogenates. Both of the purified enzymes had strong activity on dipeptides and tripeptides. The activity levels of the LAPs are strongly dependent on growth stage in batch culture, with the highest levels in early-stationary phase. Strains lacking all four LAPs have slightly lower growth rates than wild-type strains. The ability of leucine auxotrophs to grow on dipeptides and tripeptides containing leucine is not impaired in strains lacking all four LAPs. Images PMID:6352682

  9. Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae.

    PubMed Central

    Lemire, J M; Willcocks, T; Halvorson, H O; Bostian, K A

    1985-01-01

    We examined the genetic system responsible for transcriptional regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum, EC 3.1.3.2]) in Saccharomyces cerevisiae at the molecular level by analysis of previously isolated and genetically well-defined regulatory gene mutants known to affect APase expression. These mutants identify numerous positive- (PHO4, PHO2, PHO81) and negative-acting (PHO80, PHO85) regulatory loci dispersed throughout the yeast genome. We showed that the interplay of these positive and negative regulatory genes occurs before or during APase gene transcription and that their functions are all indispensible for normal regulation of mRNA synthesis. Biochemical evidence suggests that the regulatory gene products they encode are expressed constitutively. More detailed investigation of APase synthesis is a conditional PHO80(Ts) mutant indicated that neither PHO4 nor any other protein factor necessary for APase mRNA synthesis is transcriptionally regulated by PHO80. Moreover, in the absence of PHO80, the corepressor, presumed to be a metabolite of Pi, did not inhibit their function in the transcriptional activation of APase. Images PMID:3915785

  10. Defects arising from whole-genome duplications in Saccharomyces cerevisiae.

    PubMed

    Andalis, Alex A; Storchova, Zuzana; Styles, Cora; Galitski, Timothy; Pellman, David; Fink, Gerald R

    2004-07-01

    Comparisons among closely related species have led to the proposal that the duplications found in many extant genomes are the remnants of an ancient polyploidization event, rather than a result of successive duplications of individual chromosomal segments. If this interpretation is correct, it would support Ohno's proposal that polyploidization drives evolution by generating the genetic material necessary for the creation of new genes. Paradoxically, analysis of contemporary polyploids suggests that increased ploidy is an inherently unstable state. To shed light on this apparent contradiction and to determine the effects of nascent duplications of the entire genome, we generated isogenic polyploid strains of the budding yeast Saccharomyces cerevisiae. Our data show that an increase in ploidy results in a marked decrease in a cell's ability to survive during stationary phase in growth medium. Tetraploid cells die rapidly, whereas isogenic haploids remain viable for weeks. Unlike haploid cells, which arrest growth as unbudded cells, tetraploid cells continue to bud and form mitotic spindles in stationary phase. The stationary-phase death of tetraploids can be prevented by mutations or conditions that result in growth arrest. These data show that whole-genome duplications are accompanied by defects that affect viability and subsequent survival of the new organism. PMID:15280227

  11. Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

    PubMed Central

    Orlean, Peter

    2012-01-01

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

  12. Ribosome Deficiency Protects Against ER Stress in Saccharomyces cerevisiae

    PubMed Central

    Steffen, Kristan K.; McCormick, Mark A.; Pham, Kim M.; MacKay, Vivian L.; Delaney, Joe R.; Murakami, Christopher J.; Kaeberlein, Matt; Kennedy, Brian K.

    2012-01-01

    In Saccharomyces cerevisiae, 59 of the 78 ribosomal proteins are encoded by duplicated genes that, in most cases, encode identical or very similar protein products. However, different sets of ribosomal protein genes have been identified in screens for various phenotypes, including life span, budding pattern, and drug sensitivities. Due to potential suppressors of growth rate defects among this set of strains in the ORF deletion collection, we regenerated the entire set of haploid ribosomal protein gene deletion strains in a clean genetic background. The new strains were used to create double deletions lacking both paralogs, allowing us to define a set of 14 nonessential ribosomal proteins. Replicative life-span analysis of new strains corresponding to ORF deletion collection strains that likely carried suppressors of growth defects identified 11 new yeast replicative aging genes. Treatment of the collection of ribosomal protein gene deletion strains with tunicamycin revealed a significant correlation between slow growth and resistance to ER stress that was recapitulated by reducing translation of wild-type yeast with cycloheximide. Interestingly, enhanced tunicamycin resistance in ribosomal protein gene deletion mutants was independent of the unfolded protein response transcription factor Hac1. These data support a model in which reduced translation is protective against ER stress by a mechanism distinct from the canonical ER stress response pathway and further add to the diverse yet specific phenotypes associated with ribosomal protein gene deletions. PMID:22377630

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

    PubMed Central

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

    2014-01-01

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

  14. Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    PubMed Central

    Gonzalez-Perez, David; Alcalde, Miguel

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. Genome-wide analysis of the effects of location and number of stress response elements on gene expression in Saccharomyces cerevisiae.

    PubMed

    Yoshikawa, Katsunori; Furusawa, Chikara; Hirasawa, Takashi; Shimizu, Hiroshi

    2008-11-01

    We analyzed the effects of the location and number of stress response elements (STREs) on gene expression in Saccharomyces cerevisiae. Genes containing STRE between 51 and 300 bp upstream from translational start codon tended to be up-regulated and genes with multiple STREs exhibited higher up-regulation under stress conditions. PMID:19111649

  17. Electrophysiology in the eukaryotic model cell Saccharomyces cerevisiae.

    PubMed

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

    1998-11-01

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

  18. Corresponding changes in kynurenine hydroxylase activity, membrane fluidity, and sterol composition in Saccharomyces cerevisiae mitochondria.

    PubMed Central

    McLean-Bowen, C A; Parks, L W

    1981-01-01

    The effect of sterol composition on the properties of the mitochondrial membrane of Saccharomyces cerevisiae was investigated. The physical state of mitochondrial membranes from wild-type strains and sterol mutants was compared, using a fluorescence polarization technique with 1,6-diphenyl-1,3-5-hexatriene. Changes in the rate of depolarization of the probe molecule as a function of temperature suggest the occurrence of a phase transition in the mitochondrial membranes isolated from the sterol mutants but not in the membranes isolated from the wild types. Arrhenius kinetics of the mitochondrial membrane-bound enzyme L-kynurenine-3-hydroxylase exhibited changes in activation energy at temperatures similar to those observed in the fluorescence polarization study. The ratio of mitochondrial sterol to phospholipid and the phospholipid fatty acid composition of the organisms were characterized. PMID:7009578

  19. Molecular Characterization of Two High Affinity Sulfate Transporters in Saccharomyces Cerevisiae

    PubMed Central

    Cherest, H.; Davidian, J. C.; Thomas, D.; Benes, V.; Ansorge, W.; Surdin-Kerjan, Y.

    1997-01-01

    Strains resistant to the toxic analogues of sulfate, selenate and chromate have been isolated. Their genetic analysis allowed us to identify four genes. One, called MET28, encodes a transcriptional factor. The three other genes, called SUL1, SUL2 and SUL3, encode proteins involved in sulfate transport. The sequence of Sul1p and Sul2p indicate that they are integral membrane proteins exhibiting, respectively, 11 and 10 transmembrane domains. Moreover, Sul1p and Sul2p share a high degree of similarity. Sulfate transport kinetic studies made with parental and mutant strains show that, as expected from genetic results, Saccharomyces cerevisiae has two high affinity sulfate transport systems. Sul3p has been shown to be involved in the transcriptional regulation of the SUL2 gene. PMID:9055073

  20. Physical, functional and structural characterization of the cell wall fractions from baker's yeast Saccharomyces cerevisiae.

    PubMed

    Borchani, Chema; Fonteyn, Fabienne; Jamin, Guilhem; Paquot, Michel; Thonart, Philippe; Blecker, Christophe

    2016-03-01

    The yeast cell wall of Saccharomyces cerevisiae is an important source of β-d-glucan, a glucose homopolymer with many functional, nutritional and human health benefits. In the present study, the yeast cell wall fractionation process involving enzymatic treatments (savinase and lipolase enzymes) affected most of the physical and functional characteristics of extracted fractions. Thus, the fractionation process showed that β-d-glucan fraction F4 had significantly higher swelling power and fat binding capacity compared to other fractions (F1, F2 and F3). It also exhibited a viscosity of 652.12mPas and a high degree of brightness of extracted β-d-glucan fraction. Moreover, the fractionation process seemed to have an effect on structural and thermal properties of extracted fractions. Overall, results showed that yeast β-d-glucan had good potential for use as a prebiotic ingredient in food, as well as medicinal and pharmaceutical products. PMID:26471666

  1. Evaluation of industrial Saccharomyces cerevisiae strains as the chassis cell for second-generation bioethanol production

    PubMed Central

    Li, Hongxing; Wu, Meiling; Xu, Lili; Hou, Jin; Guo, Ting; Bao, Xiaoming; Shen, Yu

    2015-01-01

    To develop a suitable Saccharomyces cerevisiae industrial strain as a chassis cell for ethanol production using lignocellulosic materials, 32 wild-type strains were evaluated for their glucose fermenting ability, their tolerance to the stresses they might encounter in lignocellulosic hydrolysate fermentation and their genetic background for pentose metabolism. The strain BSIF, isolated from tropical fruit in Thailand, was selected out of the distinctly different strains studied for its promising characteristics. The maximal specific growth rate of BSIF was as high as 0.65?h?1 in yeast extract peptone dextrose medium, and the ethanol yield was 0.45?g?g?1 consumed glucose. Furthermore, compared with other strains, this strain exhibited superior tolerance to high temperature, hyperosmotic stress and oxidative stress; better growth performance in lignocellulosic hydrolysate; and better xylose utilization capacity when an initial xylose metabolic pathway was introduced. All of these results indicate that this strain is an excellent chassis strain for lignocellulosic ethanol production. PMID:25616171

  2. Asparaginase II of Saccharomyces cerevisiae: positive selection of two mutations that prevent enzyme synthesis.

    PubMed

    Kim, K W; Roon, R J

    1984-03-01

    A positive selection method, D-aspartic acid beta-hydroxamate resistance, was used to isolate Saccharomyces cerevisiae strains lacking the ability to synthesize asparaginase II. Of 100 such mutant strains, 93 exhibited mutations which were allelic with asp3, a previously characterized mutation. The other seven strains carried a new mutation, asp6. The asp6 mutation segregated 2:2 in asp6 X wild-type crosses and assorted from the asp3 mutation in asp6 X asp3 crosses. All seven asp6 mutant isolates reverted at a relatively high frequency, whereas the asp3 mutant isolates did not revert under the same conditions. Various independent asp3 isolates were mated to give heteroallelic diploids, which when sporulated and spread on D-asparagine medium yielded no recombinant strains. PMID:6365897

  3. The DEL1 mutator gene in Saccharomyces cerevisiae does not act in trans.

    PubMed

    Picologlou, S; Liebman, S W

    1985-01-01

    DEL1 strains of the yeast Saccharomyces cerevisiae exhibit a high rate of deletions of the three linked genes, CYC1, OSM1, and RAD7. Classical genetic methods showed that DEL1 segregated as a single Mendelian gene closely linked to CYC1. In addition, genetic evidence suggested that DEL1 was both cis- and trans-dominant (Liebman et al. 1979). Molecular analysis of deletions isolated from a haploid DEL1 strain established that deletion formation was mediated by recombination between yeast transposable elements, Ty's (Liebman et al. 1981). We now report the molecular characterization of deletions isolated from diploids in the trans configuration. This analysis reveals that these deletions probably arose in a two-step process involving mitotic recombination followed by Ty-mediated deletion formation in cis. PMID:2836088

  4. Evaluation of industrial Saccharomyces cerevisiae strains as the chassis cell for second-generation bioethanol production.

    PubMed

    Li, Hongxing; Wu, Meiling; Xu, Lili; Hou, Jin; Guo, Ting; Bao, Xiaoming; Shen, Yu

    2015-03-01

    To develop a suitable Saccharomyces cerevisiae industrial strain as a chassis cell for ethanol production using lignocellulosic materials, 32 wild-type strains were evaluated for their glucose fermenting ability, their tolerance to the stresses they might encounter in lignocellulosic hydrolysate fermentation and their genetic background for pentose metabolism. The strain BSIF, isolated from tropical fruit in Thailand, was selected out of the distinctly different strains studied for its promising characteristics. The maximal specific growth rate of BSIF was as high as 0.65?h(-1) in yeast extract peptone dextrose medium, and the ethanol yield was 0.45?g?g(-1) consumed glucose. Furthermore, compared with other strains, this strain exhibited superior tolerance to high temperature, hyperosmotic stress and oxidative stress; better growth performance in lignocellulosic hydrolysate; and better xylose utilization capacity when an initial xylose metabolic pathway was introduced. All of these results indicate that this strain is an excellent chassis strain for lignocellulosic ethanol production. PMID:25616171

  5. Transporter engineering for improved tolerance against alkane biofuels in Saccharomyces cerevisiae

    PubMed Central

    2013-01-01

    Background Hydrocarbon alkanes, components of major fossil fuels, are considered as next-generation biofuels because their biological production has recently been shown to be possible. However, high-yield alkane production requires robust host cells that are tolerant against alkanes, which exhibit cytotoxicity. In this study, we aimed to improve alkane tolerance in Saccharomyces cerevisiae, a key industrial microbial host, by harnessing heterologous transporters that potentially pump out alkanes. Results To this end, we attempted to exploit ABC transporters in Yarrowia lipolytica based on the observation that it utilizes alkanes as a carbon source. We confirmed the increased transcription of ABC2 and ABC3 transporters upon exposure to a range of alkanes in Y. lipolytica. We then showed that the heterologous expression of ABC2 and ABC3 transporters significantly increased tolerance against decane and undecane in S. cerevisiae through maintaining lower intracellular alkane level. In particular, ABC2 transporter increased the tolerance limit of S. cerevisiae about 80-fold against decane. Furthermore, through site-directed mutagenesis for glutamate (E988 for ABC2, and E989 for ABC3) and histidine (H1020 for ABC2, and H1021 for ABC3), we provided the evidence that glutamate was essential for the activity of ABC2 and ABC3 transporters, with ATP most likely to be hydrolyzed by a catalytic carboxylate mechanism. Conclusions Here, we demonstrated that transporter engineering through expression of heterologous efflux pumps led to significantly improved tolerance against alkane biofuels in S. cerevisiae. We believe that our results laid the groundwork for developing robust alkane-producing yeast cells through transporter engineering, which will greatly aid in next-generation alkane biofuel production and recovery. PMID:23402697

  6. L-Carnosine Affects the Growth of Saccharomyces cerevisiae in a Metabolism-Dependent Manner

    PubMed Central

    Cartwright, Stephanie P.; Bill, Roslyn M.; Hipkiss, Alan R.

    2012-01-01

    The dipeptide L-carnosine (?-alanyl-L-histidine) has been described as enigmatic: it inhibits growth of cancer cells but delays senescence in cultured human fibroblasts and extends the lifespan of male fruit flies. In an attempt to understand these observations, the effects of L-carnosine on the model eukaryote, Saccharomyces cerevisiae, were examined on account of its unique metabolic properties; S. cerevisiae can respire aerobically, but like some tumor cells, it can also exhibit a metabolism in which aerobic respiration is down regulated. L-Carnosine exhibited both inhibitory and stimulatory effects on yeast cells, dependent upon the carbon source in the growth medium. When yeast cells were not reliant on oxidative phosphorylation for energy generation (e.g. when grown on a fermentable carbon source such as 2% glucose), 10–30 mM L-carnosine slowed growth rates in a dose-dependent manner and increased cell death by up to 17%. In contrast, in media containing a non-fermentable carbon source in which yeast are dependent on aerobic respiration (e.g. 2% glycerol), L-carnosine did not provoke cell death. This latter observation was confirmed in the respiratory yeast, Pichia pastoris. Moreover, when deletion strains in the yeast nutrient-sensing pathway were treated with L-carnosine, the cells showed resistance to its inhibitory effects. These findings suggest that L-carnosine affects cells in a metabolism-dependent manner and provide a rationale for its effects on different cell types. PMID:22984600

  7. L-carnosine affects the growth of Saccharomyces cerevisiae in a metabolism-dependent manner.

    PubMed

    Cartwright, Stephanie P; Bill, Roslyn M; Hipkiss, Alan R

    2012-01-01

    The dipeptide L-carnosine (?-alanyl-L-histidine) has been described as enigmatic: it inhibits growth of cancer cells but delays senescence in cultured human fibroblasts and extends the lifespan of male fruit flies. In an attempt to understand these observations, the effects of L-carnosine on the model eukaryote, Saccharomyces cerevisiae, were examined on account of its unique metabolic properties; S. cerevisiae can respire aerobically, but like some tumor cells, it can also exhibit a metabolism in which aerobic respiration is down regulated. L-Carnosine exhibited both inhibitory and stimulatory effects on yeast cells, dependent upon the carbon source in the growth medium. When yeast cells were not reliant on oxidative phosphorylation for energy generation (e.g. when grown on a fermentable carbon source such as 2% glucose), 10-30 mM L-carnosine slowed growth rates in a dose-dependent manner and increased cell death by up to 17%. In contrast, in media containing a non-fermentable carbon source in which yeast are dependent on aerobic respiration (e.g. 2% glycerol), L-carnosine did not provoke cell death. This latter observation was confirmed in the respiratory yeast, Pichia pastoris. Moreover, when deletion strains in the yeast nutrient-sensing pathway were treated with L-carnosine, the cells showed resistance to its inhibitory effects. These findings suggest that L-carnosine affects cells in a metabolism-dependent manner and provide a rationale for its effects on different cell types. PMID:22984600

  8. Saccharomyces cerevisiae Genes Involved in Survival of Heat Shock

    PubMed Central

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

    2013-01-01

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

  9. Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1

    PubMed Central

    Edlind, Thomas D.

    2012-01-01

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

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

    PubMed

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

    2010-07-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  13. Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae.

    PubMed

    Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu

    2009-09-01

    (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter(-1)) rather than GGOH (0.2 mg liter(-1)) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter(-1) GGOH and 6.5 mg liter(-1) squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter(-1) GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering. PMID:19592534

  14. Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae?

    PubMed Central

    Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu

    2009-01-01

    (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter?1) rather than GGOH (0.2 mg liter?1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter?1 GGOH and 6.5 mg liter?1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter?1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering. PMID:19592534

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    2014-09-01

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

  18. Mating-type Gene Switching in Saccharomyces cerevisiae.

    PubMed

    Lee, Cheng-Sheng; Haber, James E

    2015-04-01

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

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

    PubMed Central

    Chu, Daniel B.; Burgess, Sean M.

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  1. Mutational Analysis of NHAoc/NHA2 in Saccharomyces cerevisiae

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    1991-01-01

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

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

    PubMed

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

    2012-10-01

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

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

    PubMed

    Rosenthal, L P; Bodley, J W

    1987-08-15

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

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

    PubMed Central

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

    2015-01-01

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

  6. Saccharomyces boulardii

    MedlinePLUS

    Saccharomyces boulardii is a yeast, which is a type of fungus. Saccharomyces boulardii was previously identified as a unique species of ... a strain of Saccharomyces cerevisiae (baker's yeast). Saccharomyces boulardii is used as medicine. Saccharomyces boulardii is used ...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  9. Idebenone treatment mediates the effect of menadione oxidative stress damage in Saccharomyces cerevisiae.

    PubMed

    Gamondi, Oliver; Chapela, Sebastián; Nievas, Inés; Burgos, Isabel; Alonso, Manuel; Stella, Carlos

    2012-06-01

    We investigated the damage caused by oxidative stress using the yeast Saccharomyces cerevisiae as a model biological system. After inducing oxidative stress with menadione, we were able to evaluate the extent of cellular oxidative stress by utilizing 2',7'-dichlorofluorescein diacetate (DCFH-DA) as a marker of the presence of reactive oxygen species. Cells were grown on different carbon sources in order to compare fermentative and oxidative metabolism. Under these conditions we evaluated the effectiveness of idebenone (2,3-dimethoxy-5-methyl-6-(10- hydroxydecyl)-1,4-benzoquinone) as a molecule that could relieve menadione-induced growth inhibition in Saccharomyces cerevisiae. PMID:22974201

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

    PubMed

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

    2014-01-01

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

  11. Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

    PubMed Central

    Hyma, Katie E; Saerens, Sofie M; Verstrepen, Kevin J; Fay, Justin C

    2011-01-01

    The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea that wine making strains have been domesticated for wine production. In this study, we demonstrate that humans can distinguish between wines produced using wine strains and wild strains of S. cerevisiae as well as its sibling species, Saccharomyces paradoxus. Wine strains produced wine with fruity and floral characteristics, whereas wild strains produced wine with earthy and sulfurous characteristics. The differences that we observe between wine and wild strains provides further evidence that wine strains have evolved phenotypes that are distinct from their wild ancestors and relevant to their use in wine production. PMID:22093681

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

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

    SciTech Connect

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

    1990-07-01

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

  14. Functional Analysis of Free Methionine-R-sulfoxide Reductase from Saccharomyces cerevisiae*S?

    PubMed Central

    Le, Dung Tien; Lee, Byung Cheon; Marino, Stefano M.; Zhang, Yan; Fomenko, Dmitri E.; Kaya, Alaattin; Hacioglu, Elise; Kwak, Geun-Hee; Koc, Ahmet; Kim, Hwa-Young; Gladyshev, Vadim N.

    2009-01-01

    Methionine sulfoxide reductases (Msrs) are oxidoreductases that catalyze thiol-dependent reduction of oxidized methionines. MsrA and MsrB are the best known Msrs that repair methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) residues in proteins, respectively. In addition, an Escherichia coli enzyme specific for free Met-R-SO, designated fRMsr, was recently discovered. In this work, we carried out comparative genomic and experimental analyses to examine occurrence, evolution, and function of fRMsr. This protein is present in single copies and two mutually exclusive subtypes in about half of prokaryotes and unicellular eukaryotes but is missing in higher plants and animals. A Saccharomyces cerevisiae fRMsr homolog was found to reduce free Met-R-SO but not free Met-S-SO or dabsyl-Met-R-SO. fRMsr was responsible for growth of yeast cells on Met-R-SO, and the double fRMsr/MsrA mutant could not grow on a mixture of methionine sulfoxides. However, in the presence of methionine, even the triple fRMsr/MsrA/MsrB mutant was viable. In addition, fRMsr deletion strain showed an increased sensitivity to oxidative stress and a decreased life span, whereas overexpression of fRMsr conferred higher resistance to oxidants. Molecular modeling and cysteine residue targeting by thioredoxin pointed to Cys101 as catalytic and Cys125 as resolving residues in yeast fRMsr. These residues as well as a third Cys, resolving Cys91, clustered in the structure, and each was required for the catalytic activity of the enzyme. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in S. cerevisiae. PMID:19049972

  15. Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Saccharomyces cerevisiae.

    PubMed

    Kurosawa, H; Nomura, N; Tanaka, H

    1989-02-01

    The development of a coimmobilized mixed culture sys tem of aerobic and facultative anaerobic microorganisms in Ca-alginate gel beads and the production of useful metabolites by the system were investigated. A coimmobilized mixed culture system of Aspergillus awamori (obligate aerobe) and Saccharomyces cerevisiae (facultative anaerobe) in Ca-alginate gel beads was used as a model system, and ethanol production from starch by the system was used as a model production. Mold Asp. awamori is an amylolytic microorganism while yeast S. cerevisiae is an ethanol producer. The two microorganisms grew competitively in the oxygen-rich surface area of the gel beads because they had similar oxygen demands in aerobic culture conditions. Neither microorganism exhibited "habitat segregation" in the gel beads and leaked yeast cells grew aerobically without ethanol production in the broth. Ethanol productivity was low under these conditions.A more desirable coimmobilized mixed culture system of Asp. awamori and S. cerevisiae was established by adding Vantocil IB (a biocidal compound) to the production medium. The antimicrobial activity of Vantocil IB was more effective with S. cerevisiae than with Asp. awamori, so that a dense mycelial layer of Asp. awamori formed in the surface of the gel beads While S. cerevisiae grew densely in the more inner areas of the gel beads. Also, yeast cell leakace was repressed and ethanol productivity was improved. The system with Vantocil IB produced ethanol of 4.5 and 12.3 g/L from 16 and 40 g/L starch, respectively. A continuous culture using this system with Vantocil IB was also carried out, and a stable steady state could be maintained for six days without leakage of yeast cells and contamination. The selection of a factor suitable for producing "habitat segregation" enabled the development of a coimmobilized mixed culture system of an aerobe and a facultative anaerobe. In this study, total habitat segregation was used to denote a tendency to exhibit denser growth in different parts of one gel bead. PMID:18587973

  16. Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

    PubMed Central

    Shima, Jun; Hino, Akihiro; Yamada-Iyo, Chie; Suzuki, Yasuo; Nakajima, Ryouichi; Watanabe, Hajime; Mori, Katsumi; Takano, Hiroyuki

    1999-01-01

    Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (?nth1), acid trehalase mutants (?ath1), and double mutants (?nth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The ?nth1 and ?ath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the ?nth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough. PMID:10388673

  17. Novel role for a Saccharomyces cerevisiae nucleoporin, Nup170p, in chromosome segregation.

    PubMed Central

    Kerscher, O; Hieter, P; Winey, M; Basrai, M A

    2001-01-01

    We determined that a mutation in the nucleoporin gene NUP170 leads to defects in chromosome transmission fidelity (ctf) and kinetochore integrity in Saccharomyces cerevisiae. A ctf mutant strain, termed s141, shows a transcription readthrough phenotype and stabilizes a dicentric chromosome fragment in two assays for kinetochore integrity. Previously, these assays led to the identification of two essential kinetochore components, Ctf13p and Ctf14p. Thus, s141 represents another ctf mutant involved in the maintenance of kinetochore integrity. We cloned and mapped the gene complementing the ctf mutation of s141 and showed that it is identical to the S. cerevisiae NUP170 gene. A deletion strain of NUP170 (nup170 Delta::HIS3) has a Ctf(-) phenotype similar to the s141 mutant (nup170-141) and also exhibits a kinetochore integrity defect. We identified a second nucleoporin, NUP157, a homologue of NUP170, as a suppressor of the Ctf(-) phenotype of nup170-141 and nup170 Delta::HIS3 strains. However, a deletion of NUP157 or several other nucleoporins did not affect chromosome segregation. Our data suggest that NUP170 encodes a specialized nucleoporin with a unique role in chromosome segregation and possibly kinetochore function. PMID:11290711

  18. Production of fructanase by a wild strain of Saccharomyces cerevisiae on tequila agave fructan.

    PubMed

    Corona-González, R I; Pelayo-Ortiz, C; Jacques, G; Guatemala, G; Arriola, E; Arias, J A; Toriz, G

    2015-01-01

    A new wild strain of Saccharomyces cerevisiae (CF3) isolated from tequila must was evaluated for production of fructanase on Agave tequilana Weber fructan (FT). Fructanase activity (F) was assessed by a 3(3) factorial design (substrate, temperature and pH). High enzymatic activity (31.1 U/ml) was found at 30 °C, pH 5, using FT (10 g/l) as substrate. The effect of initial substrate concentration on F (FT0, 5.7-66 g/l) was studied and it was found that F was highest (44.8 U/ml) at FT0 25 g/l. A 2(2) factorial experimental design with five central points was utilized to study the effect of stirring and aeration on fructanase activity; stirring exhibited a stronger effect on F. The ratio fructanase to invertase (F/S) was 0.57, which confirms that the enzymes are fructanase. Crude fructanase reached high substrate hydrolysis (48 wt%) in 10 h. It is shown that S. cerevisiae CF3 was able to produce large amounts of fructanase by growing it on fructan from A. tequilana. PMID:25432071

  19. Direct Conversion of Xylan to Ethanol by Recombinant Saccharomyces cerevisiae Strains Displaying an Engineered Minihemicellulosome

    PubMed Central

    Sun, Jie; Wen, Fei; Si, Tong; Xu, Jian-He

    2012-01-01

    Arabinoxylan is a heteropolymeric chain of a β-1,4-linked xylose backbone substituted with arabinose residues, representing a principal component of plant cell walls. Here we developed recombinant Saccharomyces cerevisiae strains as whole-cell biocatalysts capable of combining hemicellulase production, xylan hydrolysis, and hydrolysate fermentation into a single step. These strains displayed a series of uni-, bi-, and trifunctional minihemicellulosomes that consisted of a miniscaffoldin (CipA3/CipA1) and up to three chimeric enzymes. The miniscaffoldin derived from Clostridium thermocellum contained one or three cohesin modules and was tethered to the cell surface through the S. cerevisiae a-agglutinin adhesion receptor. Up to three types of hemicellulases, an endoxylanase (XynII), an arabinofuranosidase (AbfB), and a β-xylosidase (XlnD), each bearing a C-terminal dockerin, were assembled onto the miniscaffoldin by high-affinity cohesin-dockerin interactions. Compared to uni- and bifunctional minihemicellulosomes, the resulting quaternary trifunctional complexes exhibited an enhanced rate of hydrolysis of arabinoxylan. Furthermore, with an integrated d-xylose-utilizing pathway, the recombinant yeast displaying the bifunctional minihemicellulosome CipA3-XynII-XlnD could simultaneously hydrolyze and ferment birchwood xylan to ethanol with a yield of 0.31 g per g of sugar consumed. PMID:22447594

  20. Saccharomyces cerevisiae Expressing Gp43 Protects Mice against Paracoccidioides brasiliensis Infection

    PubMed Central

    Assis-Marques, Mariana Aprigio; Oliveira, Aline Ferreira; Ruas, Luciana Pereira; dos Reis, Thaila Fernanda; Roque-Barreira, Maria Cristina; Coelho, Paulo Sergio Rodrigues

    2015-01-01

    The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM. PMID:25790460

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

    PubMed

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

    2015-07-16

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

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

    SciTech Connect

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

    1982-01-01

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

  3. Growth and fermentation characteristics of Saccharomyces cerevisiae NK28 isolated from kiwi fruit.

    PubMed

    Lee, Jong-Sub; Park, Eun-Hee; Kim, Jung-Wan; Yeo, Soo-Hwan; Kim, Myoung-Dong

    2013-09-28

    The influences of glucose concentration, initial medium acidity (pH), and temperature on the growth and ethanol production of Saccharomyces cerevisiae NK28, which was isolated from kiwi fruit, were examined in shake flask cultures. The optimal glucose concentration, initial medium pH, and temperature for ethanol production were 200 g/l, pH 6.0, and 35oC, respectively. Under this growth condition, S. cerevisiae NK28 produced 98.9 ± 5.67 g/l ethanol in 24 h with a volumetric ethanol production rate of 4.12 ± 0.24 g/l·h. S. cerevisiae NK28 was more tolerant to heat and ethanol than laboratory strain S. cerevisiae BY4742, and its tolerance to ethanol and fermentation inhibitors was comparable to that of an ethanologen, S. cerevisiae D5A. PMID:23893096

  4. Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae.

    PubMed

    Furneisen, J M; Carman, G M

    2000-02-24

    The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-associated enzyme that catalyzes the Mg(2+)-independent dephosphorylation of phosphatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA). The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf-9 insect cells and used to examine the enzymological properties of the enzyme using PA, DGPP, and LPA as substrates. The optimum pH values for PA phosphatase, DGPP phosphatase, and LPA phosphatase activities were 7. 5, 7.0, and 7.0, respectively. Divalent cations (Mn(2+), Co(2+), and Ca(2+)), NaF, heavy metals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA phosphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme. The inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-encoded enzyme were novel properties when compared with other Mg(2+)-independent lipid phosphate phosphatases from S. cerevisiae and mammalian cells. The LPP1-encoded enzyme exhibited saturation kinetics with respect to the surface concentrations of PA (K(m)=0.05 mol%), DGPP (K(m)=0.07 mol%), and LPA (K(m)=0.08 mol%). Based on specificity constants (V(max)/K(m)LPA (1.3 units/mg/mol%). DGPP (K(i)=0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K(i)=0.12 mol%) was a competitive inhibitor with respect to DGPP. This suggested that the binding sites for these substrates were the same. The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S. cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as substrates. PMID:10685032

  5. Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

    PubMed Central

    Romagnoli, Gabriele; Luttik, Marijke A. H.; Kötter, Peter; Pronk, Jack T.

    2012-01-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1? pdc5? pdc6? aro10? thi3? strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols. PMID:22904058

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

    PubMed Central

    2012-01-01

    Background Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules. Result Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations (<5.5 μM). To optimize naringenin titers, a yeast chassis strain was developed. Synthesis of aromatic amino acids was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40-fold increase of extracellular naringenin titers (to approximately 200 μM) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 μM were reached. Conclusion The results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions. PMID:23216753

  7. Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae.

    PubMed

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

    2012-11-01

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

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

    SciTech Connect

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

    1995-12-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

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

    2015-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    EPA Science Inventory

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Berry, D; Volz, P A

    1979-10-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    EPA Science Inventory

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

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

    PubMed

    Darvishi, Farshad

    2012-09-01

    Saccharomyces cerevisiae cannot produce extracellular lipase and utilize low-cost lipid substrates. This study aimed to express extracellular lipase from Yarrowia lipolytica in S.?cerevisiae, construct recombinant oily substrate consumer strains, and compare the roles of native and mutant Y.?lipolytica extracellular lipases in S.?cerevisiae. The LIP2 gene of Y.?lipolytica DSM3286 and its mutant Y.?lipolytica U6 were isolated and cloned by expression vector in S.?cerevisiae. New recombinant S.?cerevisiae strains FDS100 containing the native LIP2 gene, and FDS101 containing the mutant LIP2 gene were produced 10 and 15?U?ml (-1) extracellular lipase respectively, on a production medium containing olive oil. New recombinant S.?cerevisiae strains produce acceptable amount of extracellular lipase in comparison with Y.?lipolytica wild-type strains. These strains can utilize olive oil and lipids as low-cost substrates to produce bioethanol, single cell protein and other biotechnologically valuable products. The recombinant S.?cerevisiae strain with mutant LIP2 produced lipase with 1.5-fold higher activity. The LIP2 gene of Y.?lipolytica was expressed in S.?cerevisiae as a heterologous protein without any modifications. Strong components of the Y.?lipolytica expression/secretion system could be used for high-level production of recombinant proteins in S.?cerevisiae. PMID:22702371

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

    PubMed

    Hyma, Katie E; Fay, Justin C

    2013-06-01

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

  5. High vanillin tolerance of an evolved Saccharomyces cerevisiae strain owing to its enhanced vanillin reduction and antioxidative capacity.

    PubMed

    Shen, Yu; Li, Hongxing; Wang, Xinning; Zhang, Xiaoran; Hou, Jin; Wang, Linfeng; Gao, Nan; Bao, Xiaoming

    2014-11-01

    The phenolic compounds present in hydrolysates pose significant challenges for the sustainable lignocellulosic materials refining industry. Three Saccharomyces cerevisiae strains with high tolerance to lignocellulose hydrolysate were obtained through ethyl methanesulfonate mutation and adaptive evolution. Among them, strain EMV-8 exhibits specific tolerance to vanillin, a phenolic compound common in lignocellulose hydrolysate. The EMV-8 maintains a specific growth rate of 0.104 h(-1) in 2 g L(-1) vanillin, whereas the reference strain cannot grow. Physiological studies revealed that the vanillin reduction rate of EMV-8 is 1.92-fold higher than its parent strain, and the Trolox equivalent antioxidant capacity of EMV-8 is 15 % higher than its parent strain. Transcriptional analysis results confirmed an up-regulated oxidoreductase activity and antioxidant activity in this strain. Our results suggest that enhancing the antioxidant capacity and oxidoreductase activity could be a strategy to engineer S. cerevisiae for improved vanillin tolerance. PMID:25261986

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

    PubMed

    Knight, Sarah J; Goddard, Matthew R

    2016-02-01

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

  7. Interactions between Torulaspora delbrueckii and Saccharomyces cerevisiae in wine fermentation: influence of inoculation and nitrogen content.

    PubMed

    Taillandier, Patricia; Lai, Quoc Phong; Julien-Ortiz, Anne; Brandam, Cédric

    2014-07-01

    Alcoholic fermentation by an oenological strain of Torulaspora delbrueckii in association with an oenological strain of Saccharomyces cerevisiae was studied in mixed and sequential cultures. Experiments were performed in a synthetic grape must medium in a membrane bioreactor, a special tool designed to study indirect interactions between microorganisms. Results showed that the S. cerevisiae strain had a negative impact on the T. delbrueckii strain, leading to a viability decrease as soon as S. cerevisiae was inoculated. Even for high inoculation of T. delbrueckii (more than 20× S. cerevisiae) in mixed cultures, T. delbrueckii growth was inhibited. Substrate competition and cell-to-cell contact mechanism could be eliminated as explanations of the observed interaction, which was probably an inhibition by a metabolite produced by S. cerevisiae. S. cerevisiae should be inoculated 48 h after T. delbrueckii in order to ensure the growth of T. delbrueckii and consequently a decrease of volatile acidity and a higher isoamyl acetate production. In this case, in a medium with a high concentration of assimilable nitrogen (324 mg L(-1)), S. cerevisiae growth was not affected by T. delbrueckii. But in a sequential fermentation in a medium containing 176 mg L(-1) initial assimilable nitrogen, S. cerevisiae was not able to develop because of nitrogen exhaustion by T. delbrueckii growth during the first 48 h, leading to sluggish fermentation. PMID:24500666

  8. Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene

    PubMed Central

    Melo, Sônia C.; Santos, Regineide X.; Melgaço, Ana C.; Pereira, Alanna C. F.; Pungartnik, Cristina; Brendel, Martin

    2015-01-01

    Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae. PMID:26039235

  9. Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.

    PubMed

    Melo, Sônia C; Santos, Regineide X; Melgaço, Ana C; Pereira, Alanna C F; Pungartnik, Cristina; Brendel, Martin

    2015-01-01

    Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet-C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae. PMID:26039235

  10. Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion.

    PubMed

    Turner, Timothy L; Zhang, Guo-Chang; Kim, Soo Rin; Subramaniam, Vijay; Steffen, David; Skory, Christopher D; Jang, Ji Yeon; Yu, Byung Jo; Jin, Yong-Su

    2015-10-01

    Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates. PMID:26043971

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

    PubMed

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

    2011-05-01

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

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

    PubMed

    Zheng, Yan-Lin; Wang, Shi-An

    2015-01-01

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

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

    PubMed Central

    Pérez-Torrado, Roberto; Querol, Amparo

    2016-01-01

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

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

    PubMed Central

    Zheng, Yan-Lin; Wang, Shi-An

    2015-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11.

    PubMed Central

    Bressan, D A; Olivares, H A; Nelms, B E; Petrini, J H

    1998-01-01

    Saccharomyces cerevisiae Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous recombination. Null mutants of MRE11, RAD50, and XRS2 are characterized by ionizing radiation sensitivity and mitotic interhomologue hyperrecombination. We mutagenized the four highly conserved phosphoesterase signature motifs of Mre11 to create mre11-11, mre11-2, mre11-3, and mre11-4 and assessed the functional consequences of these mutant alleles with respect to mitotic interhomologue recombination, chromosome loss, ionizing radiation sensitivity, double-strand break repair, and protein interaction. We found that mre11 mutants that behaved as the null were sensitive to ionizing radiation and deficient in double-strand break repair. We also observed that these null mutants exhibited a hyperrecombination phenotype in mitotic cells, consistent with previous reports, but did not exhibit an increased frequency of chromosome loss. Differential ionizing radiation sensitivities among the hypomorphic mre11 alleles correlated with the trends observed in the other phenotypes examined. Two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the Mre11-Rad50 interaction. Mutagenesis of the phosphoesterase signatures in Mre11 thus demonstrated the importance of these conserved motifs for recombinational DNA repair. PMID:9755192

  19. Nucleosome alterations caused by mutations at modifiable histone residues in Saccharomyces cerevisiae

    PubMed Central

    Liu, Hongde; Wang, Pingyan; Liu, Lingjie; Min, Zhu; Luo, Kun; Wan, Yakun

    2015-01-01

    Nucleosome organization exhibits dynamic properties depending on the cell state and environment. Histone proteins, fundamental components of nucleosomes, are subject to chemical modifications on particular residues. We examined the effect of substituting modifiable residues of four core histones with the non-modifiable residue alanine on nucleosome dynamics. We mapped the genome-wide nucleosomes in 22 histone mutants of Saccharomyces cerevisiae and compared the nucleosome alterations relative to the wild-type strain. Our results indicated that different types of histone mutation resulted in different phenotypes and a distinct reorganization of nucleosomes. Nucleosome occupancy was altered at telomeres, but not at centromeres. The first nucleosomes upstream (?1) and downstream (+1) of the transcription start site (TSS) were more dynamic than other nucleosomes. Mutations in histones affected the nucleosome array downstream of the TSS. Highly expressed genes, such as ribosome genes and genes involved in glycolysis, showed increased nucleosome occupancy in many types of histone mutant. In particular, the H3K56A mutant exhibited a high percentage of dynamic genomic regions, decreased nucleosome occupancy at telomeres, increased occupancy at the +1 and ?1 nucleosomes, and a slow growth phenotype under stress conditions. Our findings provide insight into the influence of histone mutations on nucleosome dynamics. PMID:26498326

  20. The actin-related protein Act3p of Saccharomyces cerevisiae is located in the nucleus.

    PubMed Central

    Weber, V; Harata, M; Hauser, H; Wintersberger, U

    1995-01-01

    Actin-related proteins, a group of protein families that exhibit about 50% sequence identity among each other and to conventional actin, have been found in a variety of eukaryotic organisms. In the budding yeast Saccharomyces cerevisiae, genes for one conventional actin (ACT1) and for three actin-related proteins (ACT2, ACT3, and ACT5) are known. ACT3, which we recently discovered, is an essential gene coding for a polypeptide of 489 amino acids (Act3p), with a calculated molecular mass of 54.8 kDa. Besides its homology to conventional actin, Act3p possesses a domain exhibiting weak similarity to the chromosomal protein HMG-14 as well as a potential nuclear localization signal. An antiserum prepared against a specific segment of the ACT3 gene product recognizes a polypeptide band of approximately 55 kDa in yeast extract. Indirect immunofluorescence experiments with this antiserum revealed that Act3p is located in the nucleus. Nuclear staining was observed in all cells regardless of the stage of the cell cycle. Independently, immunoblotting experiments with subcellular fractions showed that Act3p is indeed highly enriched in the nuclear fraction. We suggest that Act3p is an essential constituent of yeast chromatin. Images PMID:8573785

  1. RSC Chromatin-Remodeling Complex Is Important for Mitochondrial Function in Saccharomyces cerevisiae

    PubMed Central

    Imamura, Yuko; Yu, Feifei; Nakamura, Misaki; Chihara, Yuhki; Okane, Kyo; Sato, Masahiro; Kanai, Muneyoshi; Hamada, Ryoko; Ueno, Masaru; Yukawa, Masashi; Tsuchiya, Eiko

    2015-01-01

    RSC (Remodel the Structure of Chromatin) is an ATP-dependent chromatin remodeling complex essential for the growth of Saccharomyces cerevisiae. RSC exists as two distinct isoforms that share core subunits including the ATPase subunit Nps1/Sth1 but contain either Rsc1or Rsc2. Using the synthetic genetic array (SGA) of the non-essential null mutation method, we screened for mutations exhibiting synthetic growth defects in combination with the temperature-sensitive mutant, nps1-105, and found connections between mitochondrial function and RSC. rsc mutants, including rsc1?, rsc2?, and nps1-13, another temperature-sensitive nps1 mutant, exhibited defective respiratory growth; in addition, rsc2? and nps1-13 contained aggregated mitochondria. The rsc2? phenotypes were relieved by RSC1 overexpression, indicating that the isoforms play a redundant role in respiratory growth. Genome-wide expression analysis in nps1-13 under respiratory conditions suggested that RSC regulates the transcription of some target genes of the HAP complex, a transcriptional activator of respiratory gene expression. Nps1 physically interacted with Hap4, the transcriptional activator moiety of the HAP complex, and overexpression of HAP4 alleviated respiratory defects in nps1-13, suggesting that RSC plays pivotal roles in mitochondrial gene expression and shares a set of target genes with the HAP complex. PMID:26086550

  2. Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11.

    PubMed

    Bressan, D A; Olivares, H A; Nelms, B E; Petrini, J H

    1998-10-01

    Saccharomyces cerevisiae Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous recombination. Null mutants of MRE11, RAD50, and XRS2 are characterized by ionizing radiation sensitivity and mitotic interhomologue hyperrecombination. We mutagenized the four highly conserved phosphoesterase signature motifs of Mre11 to create mre11-11, mre11-2, mre11-3, and mre11-4 and assessed the functional consequences of these mutant alleles with respect to mitotic interhomologue recombination, chromosome loss, ionizing radiation sensitivity, double-strand break repair, and protein interaction. We found that mre11 mutants that behaved as the null were sensitive to ionizing radiation and deficient in double-strand break repair. We also observed that these null mutants exhibited a hyperrecombination phenotype in mitotic cells, consistent with previous reports, but did not exhibit an increased frequency of chromosome loss. Differential ionizing radiation sensitivities among the hypomorphic mre11 alleles correlated with the trends observed in the other phenotypes examined. Two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the Mre11-Rad50 interaction. Mutagenesis of the phosphoesterase signatures in Mre11 thus demonstrated the importance of these conserved motifs for recombinational DNA repair. PMID:9755192

  3. "A comparison between sugar consumption and ethanol production in wort by immobilized Saccharomyces Cerevisiae, Saccharomyces Ludwigii and Saccharomyces Rouxii on Brewer'S Spent Grain".

    PubMed

    Mohammadi, Aniseh; Razavi, Seyyed Hadi; Mousavi, Seyyed Mohammad; Rezaei, Karamatollah

    2011-04-01

    The immobilization of Saccharomyces cerevisiae DSM 70424, Saccharomyces ludwigii DSM 3447 and Saccharomyces rouxii DSM 2531 on brewer's spent grain and then ethanol production and sugar consumption of these immobilized yeasts were investigated. The aim of this study was to investigate the abilities of these three immobilized yeasts for producing alcohol for brewing at two temperatures (7 and 12 °C) using two different sugar levels (one at original level supplied in the brewery and one with 2.5% (w/v), added glucose to the wort). Increasing both parameters resulted in higher alcohol production by all the yeasts studied. At 7 °C and with original wort density the ethanol content at the end of fermentation was 2.7% (v/v) for S. cerevisiae, 1.7% for S. ludwigii and 2.0% for S. rouxii. After the addition of 2.5% (w/v) glucose at the same temperature (7 °C), the alcohol production was increased to 4.1, 2.8 and 4.1%, respectively. Similar improvements were observed when the fermentation was carried out at 12 °C with/without the addition of glucose to the wort. However, temperature indicated greater influence on S. ludwigii than did on S. rouxii and S. cerevisiae. The immobilization as carried out in this study impacted both S. ludwigii and S. rouxii in a way that they could consume maltose under certain conditions. PMID:24031672

  4. “A comparison between sugar consumption and ethanol production in wort by immobilized Saccharomyces Cerevisiae, Saccharomyces Ludwigii and Saccharomyces Rouxii on Brewer’S Spent Grain”

    PubMed Central

    Mohammadi, Aniseh; Razavi, Seyyed Hadi; Mousavi, Seyyed Mohammad; Rezaei, Karamatollah

    2011-01-01

    The immobilization of Saccharomyces cerevisiae DSM 70424, Saccharomyces ludwigii DSM 3447 and Saccharomyces rouxii DSM 2531 on brewer’s spent grain and then ethanol production and sugar consumption of these immobilized yeasts were investigated. The aim of this study was to investigate the abilities of these three immobilized yeasts for producing alcohol for brewing at two temperatures (7 and 12 °C) using two different sugar levels (one at original level supplied in the brewery and one with 2.5% (w/v), added glucose to the wort). Increasing both parameters resulted in higher alcohol production by all the yeasts studied. At 7 °C and with original wort density the ethanol content at the end of fermentation was 2.7% (v/v) for S. cerevisiae, 1.7% for S. ludwigii and 2.0% for S. rouxii. After the addition of 2.5% (w/v) glucose at the same temperature (7 °C), the alcohol production was increased to 4.1, 2.8 and 4.1%, respectively. Similar improvements were observed when the fermentation was carried out at 12 °C with/without the addition of glucose to the wort. However, temperature indicated greater influence on S. ludwigii than did on S. rouxii and S. cerevisiae. The immobilization as carried out in this study impacted both S. ludwigii and S. rouxii in a way that they could consume maltose under certain conditions. PMID:24031672

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

    PubMed Central

    Hyma, Katie E.; Fay, Justin C.

    2012-01-01

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

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

    SciTech Connect

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

    1993-07-01

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

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

    PubMed

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

    2015-08-01

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

  8. Modeling mutant phenotypes and oscillatory dynamics in the Saccharomyces cerevisiae cAMP-PKA pathway

    PubMed Central

    2013-01-01

    Background The cyclic AMP-Protein Kinase A (cAMP-PKA) pathway is an evolutionarily conserved signal transduction mechanism that regulates cellular growth and differentiation in animals and fungi. We present a mathematical model that recapitulates the short-term and long-term dynamics of this pathway in the budding yeast, Saccharomyces cerevisiae. Our model is aimed at recapitulating the dynamics of cAMP signaling for wild-type cells as well as single (pde1? and pde2?) and double (pde1?pde2?) phosphodiesterase mutants. Results Our model focuses on PKA-mediated negative feedback on the activity of phosphodiesterases and the Ras branch of the cAMP-PKA pathway. We show that both of these types of negative feedback are required to reproduce the wild-type signaling behavior that occurs on both short and long time scales, as well as the the observed responses of phosphodiesterase mutants. A novel feature of our model is that, for a wide range of parameters, it predicts that intracellular cAMP concentrations should exhibit decaying oscillatory dynamics in their approach to steady state following glucose stimulation. Experimental measurements of cAMP levels in two genetic backgrounds of S. cerevisiae confirmed the presence of decaying cAMP oscillations as predicted by the model. Conclusions Our model of the cAMP-PKA pathway provides new insights into how yeast respond to alterations in their nutrient environment. Because the model has both predictive and explanatory power it will serve as a foundation for future mathematical and experimental studies of this important signaling network. PMID:23680078

  9. Mathematical model for the aerobic growth of saccharomyces cerevisiae with a saturated respiratory capacity

    SciTech Connect

    Barford, J.P.; Hall, R.J.

    1981-08-01

    A mathematical model for the aerobic growth of Saccharomyces cerevisiae in both batch and continuous culture is described. It was based on the experimental observation that the respiratory capacity of this organism may become saturated and exhibit a maximum specific oxygen uptake rate after suitable adaptation. This experimental observation led to the possibility that transport into and out of the mitochondrion was of major importance in the overall metabolism of S. cerevisiae and was subject to long-term adaptation. Consistent with this observation a distributed model was proposed which, as its basis, assumed the control of respiration and fermentation to be the result of saturation of respiration without any specific repression or inhibition of the uptake rates of other substrates. No other regulation of fermentation and respiration was assumed. The model provided a suitable structure allowing precise quantification of the changes in rate and stoichiometry of energy production. The model clearly indicated that growth under the wide range of experimental conditions reported could not be predicted using constant values for the maximum specific respiratory rate or constant values of Yatp (g biomass/mol ATP) and PO ratio of (mol ATP/atom oxygen). The causes of the variation in the respiratory rate were not determined and it was concluded that a more detailed analysis (reported subsequently) was required. The variation of Y atp and PO ratio with specific growth rate implied that the efficiency of ATP generation or ATP utilization decreased with increasing specific growth rate. It was concluded that it was not possible to quantify the individual effect of Yatp and PO ratio until independent means for their reliable estimation is available. (Refs. 84).

  10. Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents.

    PubMed

    Kim, Sun-Ki; Jin, Yong-Su; Choi, In-Geol; Park, Yong-Cheol; Seo, Jin-Ho

    2015-05-01

    Fermentation inhibitors present in lignocellulose hydrolysates are inevitable obstacles for achieving economic production of biofuels and biochemicals by industrial microorganisms. Here we show that spermidine (SPD) functions as a chemical elicitor for enhanced tolerance of Saccharomyces cerevisiae against major fermentation inhibitors. In addition, the feasibility of constructing an engineered S. cerevisiae strain capable of tolerating toxic levels of the major inhibitors without exogenous addition of SPD was explored. Specifically, we altered expression levels of the genes in the SPD biosynthetic pathway. Also, OAZ1 coding for ornithine decarboxylase (ODC) antizyme and TPO1 coding for the polyamine transport protein were disrupted to increase intracellular SPD levels through alleviation of feedback inhibition on ODC and prevention of SPD excretion, respectively. Especially, the strain with combination of OAZ1 and TPO1 double disruption and overexpression of SPE3 not only contained spermidine content of 1.1mg SPD/g cell, which was 171% higher than that of the control strain, but also exhibited 60% and 33% shorter lag-phase period than that of the control strain under the medium containing furan derivatives and acetic acid, respectively. While we observed a positive correlation between intracellular SPD contents and tolerance phenotypes among the engineered strains accumulating different amounts of intracellular SPD, too much SPD accumulation is likely to cause metabolic burden. Therefore, genetic perturbations for intracellular SPD levels should be optimized in terms of metabolic burden and SPD contents to construct inhibitor tolerant yeast strains. We also found that the genes involved in purine biosynthesis and cell wall and chromatin stability were related to the enhanced tolerance phenotypes to furfural. The robust strains constructed in this study can be applied for producing chemicals and advanced biofuels from cellulosic hydrolysates. PMID:25724339

  11. The Saccharomyces cerevisiae Actin Patch Protein App1p Is a Phosphatidate Phosphatase Enzyme*?

    PubMed Central

    Chae, Minjung; Han, Gil-Soo; Carman, George M.

    2012-01-01

    Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1? dpp1? lpp1? triple mutant indicated another gene(s) encoding the enzyme. We purified PAP from the pah1? dpp1? lpp1? triple mutant by salt extraction of mitochondria followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, we found that APP1, a gene whose molecular function has been unknown, confers ?30% PAP activity of wild type cells. The overexpression of APP1 in the pah1? dpp1? lpp1? mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1? mutation into the pah1? dpp1? lpp1? triple mutant resulted in a complete loss of PAP activity, indicating that distinct PAP enzymes in S. cerevisiae are encoded by APP1, PAH1, DPP1, and LPP1. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity. PMID:23071111

  12. Development of a cellulolytic Saccharomyces cerevisiae strain with enhanced cellobiohydrolase activity.

    PubMed

    Hong, Jiefang; Yang, Huajun; Zhang, Kun; Liu, Cheng; Zou, Shaolan; Zhang, Minhua

    2014-11-01

    Consolidated bioprocessing (CBP) is a promising technology for lignocellulosic ethanol production, and the key is the engineering of a microorganism that can efficiently utilize cellulose. Development of Saccharomyces cerevisiae for CBP requires high level expression of cellulases, particularly cellobiohydrolases (CBH). In this study, to construct a CBP-enabling yeast with enhanced CBH activity, three cassettes containing constitutively expressed CBH-encoding genes (cbh1 from Aspergillus aculeatus, cbh1 and cbh2 from Trichoderma reesei) were constructed. T. reesei eg2, A. aculeatus bgl1, and the three CBH-encoding genes were then sequentially integrated into the S. cerevisiae W303-1A chromosome via δ-sequence-mediated integration. The resultant strains W1, W2, and W3, expressing uni-, bi-, and trifunctional cellulases, respectively, exhibited corresponding cellulase activities. Furthermore, both the activities and glucose producing activity ascended. The growth test on cellulose containing plates indicated that CBH was a necessary component for successful utilization of crystalline cellulose. The three recombinant strains and the control strains W303-1A and AADY were evaluated in acid- and alkali-pretreated corncob containing media with 5 FPU exogenous cellulase/g biomass loading. The highest ethanol titer (g/l) within 7 days was 5.92 ± 0.51, 18.60 ± 0.81, 28.20 ± 0.84, 1.40 ± 0.12, and 2.12 ± 0.35, respectively. Compared with the control strains, W3 efficiently fermented pretreated corncob to ethanol. To our knowledge, this is the first study aimed at creating cellulolytic yeast with enhanced CBH activity by integrating three types of CBH-encoding gene with a strong constitutive promoter Ptpi. PMID:25164958

  13. Isolation and characterization of the Saccharomyces cerevisiae LPP1 gene encoding a Mg2+-independent phosphatidate phosphatase.

    PubMed

    Toke, D A; Bennett, W L; Oshiro, J; Wu, W I; Voelker, D R; Carman, G M

    1998-06-01

    The DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase enzyme accounts for half of the Mg2+-independent phosphatidate (PA) phosphatase activity in Saccharomyces cerevisiae. The LPP1 (lipid phosphate phosphatase) gene encodes a protein that contains a novel phosphatase sequence motif found in DGPP phosphatase and in the mouse Mg2+-independent PA phosphatase. A genomic copy of the S. cerevisiae LPP1 gene was isolated and was used to construct lpp1Delta and lpp1Delta dpp1Delta mutants. A multicopy plasmid containing the LPP1 gene directed a 12.9-fold overexpression of Mg2+-independent PA phosphatase activity in the S. cerevisiae lpp1Delta dpp1Delta double mutant. The heterologous expression of the S. cerevisiae LPP1 gene in Sf-9 insect cells resulted in a 715-fold overexpression of Mg2+-independent PA phosphatase activity relative to control insect cells. The Mg2+-independent PA phosphatase activity encoded by the LPP1 gene was associated with the membrane fraction of the cell. The LPP1 gene product also exhibited lyso-PA phosphatase and DGPP phosphatase activities. The order of substrate preference was PA > lyso-PA > DGPP. Like the dpp1Delta mutant, the lpp1Delta mutant and the lpp1Delta dpp1Delta double mutant were viable and did not exhibit obvious growth defects. Biochemical analyses of lpp1Delta, dpp1Delta, and lpp1Delta dpp1Delta mutants showed that the LPP1 and DPP1 gene products encoded nearly all of the Mg2+-independent PA phosphatase and lyso-PA phosphatase activities and all of the DGPP phosphatase activity in S. cerevisiae. Moreover, the analyses of the mutants showed that the LPP1 and DPP1 gene products played a role in the regulation of phospholipid metabolism and the cellular levels of phosphatidylinositol and PA. PMID:9603941

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2015-12-01

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

  16. Comparative characterization of endo-polygalacturonase (Pgu1) from Saccharomyces cerevisiae and Saccharomyces paradoxus under winemaking conditions.

    PubMed

    Eschstruth, Alexis; Divol, Benoit

    2011-08-01

    Wine strains of Saccharomyces cerevisiae have no to weak natural pectinase activity, despite their genetic ability to secrete an endo-polygalacturonase. The addition of external pectinase of fungal origin has therefore become a common step of winemaking in order to enhance the extraction of compounds located in the grape berry skins during maceration and to ease wine clarification after maturation. Recently, the strong pectinase activity of a wine strain of Saccharomyces paradoxus has been reported. In this study, the endo-polygalacturonase-encoding gene of S. paradoxus was sequenced and its activity was characterised, compared with that of S. cerevisiae and tested under winemaking conditions through overexpression of both genes individually in S. cerevisiae. A few differences in the amino acids sequences between the two proteins were revealed and the activity of the Pgu1 enzyme of S. paradoxus was shown to be weaker under winemaking conditions. Clear indicators of extracellular activity were observed in the wines made with both recombinant strains (i.e. enzyme activity in cell-free wine, higher methanol concentration and higher free-run wine), but the actual composition of the wines fermented with the mutants was only sparingly altered. Although unexpectedly found in lower concentrations in the latter wines, phenolic compounds were shown to be the most discriminatory components. Overexpressing the PGU1 gene of S. paradoxus or that of S. cerevisiae did not make much difference, showing that the higher activity of S. paradoxus strains under laboratory conditions could be due to a different regulation mechanism rather than to a different sequence of PGU1. PMID:21479715

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

    PubMed

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

    2013-12-15

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

  18. Crystallization and Preliminary X-ray Diffraction Analysis of motif N from Saccharomyces cerevisiae Dbf4

    SciTech Connect

    Matthews, L.; Duong, A; Prasad, A; Duncker, B; Guarne, A

    2009-01-01

    The Cdc7-Dbf4 complex plays an instrumental role in the initiation of DNA replication and is a target of replication-checkpoint responses in Saccharomyces cerevisiae. Cdc7 is a conserved serine/threonine kinase whose activity depends on association with its regulatory subunit, Dbf4. A conserved sequence near the N-terminus of Dbf4 (motif N) is necessary for the interaction of Cdc7-Dbf4 with the checkpoint kinase Rad53. To understand the role of the Cdc7-Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. A complete native data set was collected at 100 K from crystals that diffracted X-rays to 2.75 {angstrom} resolution and structure determination is currently under way.

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

    PubMed Central

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

    2011-01-01

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

  20. Isolation of cobalt hyper-resistant mutants of Saccharomyces cerevisiae by in vivo evolutionary engineering approach.

    PubMed

    Cakar, Z Petek; Alkim, Ceren; Turanli, Burcu; Tokman, Nilgün; Akman, Süleyman; Sarikaya, Mehmet; Tamerler, Candan; Benbadis, Laurent; François, Jean M

    2009-08-20

    Cobalt is an important element with magnetic properties used in various industrial applications, but is also needed for biological activity. Very little is known about the cellular response of living systems to cobalt stress. Towards investigating this mechanism, we isolated individual Saccharomyces cerevisiae cells resistant to high cobalt concentrations up to 8 mmoll(-1), by employing four different 'in vivo' evolutionary engineering strategies: selection under constant or gradually increasing stress levels, and selection under continuous or pulse exposure to cobalt stress. Selection under continuous exposure to gradually increasing cobalt stress levels yielded the most resistant cell population to cobalt. However, the resistance was highly heterogeneous within the mutant populations ranging from 3- to 3700-fold survival rate of isolated individuals to 8 mmoll(-1) CoCl2 in the most resistant population. Moreover, cobalt-resistant individual colonies were associated with 2-4-times lower intracellular cobalt contents as compared to wild-type, and with cross-resistance to metals such as nickel, zinc, manganese, but not to copper and chromium ions. Contrary to mutants evolved under continuous exposure to cobalt, those isolated by pulse exposure strategy also exhibited resistance to heat shock and hydrogen peroxide stress. Taken together, this study reinforced the fact that evolutionary engineering is useful in selecting strains with very specific phenotypes, and further illustrated the importance of the strategy chosen to isolate the best evolved strain. PMID:19577596

  1. A Colony Color Assay for Saccharomyces Cerevisiae Mutants Defective in Kinetochore Structure and Function

    PubMed Central

    Perier, F.; Carbon, J.

    1992-01-01

    We have designed a colony color assay for monitoring centromere DNA-protein interactions in yeast (Saccharomyces cerevisiae). The assay is based on the ability of centromere DNA sequences to block (in cis) transcription initiated from a hybrid CEN-GAL1 promoter. Using a IacZ reporter gene under control of the CEN-GAL1 promoter, we screened colonies derived from mutagenized cells for a blue color phenotype indicative of derepression of the hybrid construct. A limited screen in which a 61-bp CEN11 DNA fragment containing an intact CDEIII subregion plus flanking sequences was used as the ``pseudo-operator'' led to the identification of mutations (blu) in three complementation groups. The blu1 mutants exhibited a decrease in activity of the major CEN DNA-binding proteins in vitro. The BLU1 gene was shown to be identical to the previously isolated SPT3 gene, known to be involved in the transcriptional regulation of a subset of yeast genes. Our results indicate that the BLU1/SPT3 gene product may also be required to maintain optimal levels of functional centromere DNA-binding proteins. PMID:1398062

  2. Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae

    PubMed Central

    Thota, Swarna Gowri; Unnikannan, C. P.; Thampatty, Sitalakshmi R.; Manorama, R.; Bhandari, Rashna

    2014-01-01

    Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1? yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1? yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a ?-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1? yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription. PMID:25423617

  3. Evidence that the antiproliferative effects of auranofin in Saccharomyces cerevisiae arise from inhibition of mitochondrial respiration.

    PubMed

    Gamberi, Tania; Fiaschi, Tania; Modesti, Alessandra; Massai, Lara; Messori, Luigi; Balzi, Manuela; Magherini, Francesca

    2015-08-01

    Auranofin is a gold based drug in clinical use since 1985 for the treatment of rheumatoid arthritis. Beyond its antinflammatory properties, auranofin exhibits other attractive biological and pharmacological actions such as a potent in vitro cytotoxicity and relevant antimicrobial and antiparasitic effects that make it amenable for new therapeutic indications. For instance, auranofin is currently tested as an anticancer agent in four independent clinical trials; yet, its mode of action is highly controversial. With the present study, we explore the effects of auranofin in Saccharomyces cerevisiae and its likely mechanism. Notably, auranofin is reported to induce remarkable yeast growth inhibition. Solid evidence is provided that growth inhibition is the consequence of a direct cytotoxic insult occurring at the mitochondrial level; a profound depression of cell respiration is indeed clearly documented as the main cause of cell death while induction of ROS plays only a secondary role. More in detail, the mitochondrial NADH kinase Pos5 is identified as a primary target for auranofin. The implications of these results are discussed in the frame of current mechanistic knowledge on the cellular effects of auranofin and of its role as a prospective anticancer drug. PMID:26024642

  4. Enhanced Bioconversion of Cellobiose by Industrial Saccharomyces cerevisiae Used for Cellulose Utilization

    PubMed Central

    Hu, Meng-Long; Zha, Jian; He, Lin-Wei; Lv, Ya-Jin; Shen, Ming-Hua; Zhong, Cheng; Li, Bing-Zhi; Yuan, Ying-Jin

    2016-01-01

    Cellobiose accumulation and the compromised temperature for yeast fermentation are the main limiting factors of enzymatic hydrolysis process during simultaneous saccharification and fermentation (SSF). In this study, genes encoding cellobiose transporter and β-glucosidase were introduced into an industrial Saccharomyces cerevisiae strain, and evolution engineering was carried out to improve the cellobiose utilization of the engineered yeast strain. The evolved strain exhibited significantly higher cellobiose consumption rate (2.8-fold) and ethanol productivity (4.9-fold) compared with its parent strain. Besides, the evolved strain showed a high cellobiose consumption rate of 3.67 g/L/h at 34°C and 3.04 g/L/h at 38°C. Moreover, little cellobiose was accumulated during SSF of Avicel using the evolved strain at 38°C, and the ethanol yield from Avicel increased by 23% from 0.34 to 0.42 g ethanol/g cellulose. Overexpression of the genes encoding cellobiose transporter and β-glucosidase accelerated cellobiose utilization, and the improvement depended on the strain background. The results proved that fast cellobiose utilization enhanced ethanol production by reducing cellobiose accumulation during SSF at high temperature. PMID:26973619

  5. A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements.

    PubMed

    Moskvina, E; Schüller, C; Maurer, C T; Mager, W H; Ruis, H

    1998-08-01

    Stress response elements (STREs, core consensus AG4 or C4T) have been demonstrated previously to occur in the upstream region of a number of genes responsive to induction by a variety of stress signals. This stress response is mediated by the homologous transcription factors Msn2p and Msn4p, which bind specifically to STREs. Double mutants (msn2 msn4) deficient in these transcription factors have been shown to be hypersensitive to severe stress conditions. To obtain a more representative overview of the set of yeast genes controlled via this regulon, a computer search of the Saccharomyces cerevisiae genome was carried out for genes, which, similar to most known STRE-controlled genes, exhibit at least two STREs in their upstream region. In addition to the great majority of genes previously known to be controlled via STREs, 69 open reading-frames were detected. Expression patterns of a set of these were examined by grid filter hybridization, and 14 genes were examined by Northern analysis. Comparison of the expression patterns of these genes demonstrates that they are all STRE-controlled although their detailed expression patterns differ considerably. PMID:9730283

  6. SPL1-1, a Saccharomyces cerevisiae mutation affecting tRNA splicing.

    PubMed Central

    Kolman, C; Söll, D

    1993-01-01

    A genetic approach was used to isolate and characterize Saccharomyces cerevisiae genes affecting tRNA processing. Three mutants were isolated which were able to process and utilize splicing-deficient transcripts from inactivated Schizosaccharomyces pombe suppressor tRNA genes. Extragenic recovery of suppressibility was verified by the suppression of nonsense mutations in LEU2, HIS4, and ADE1. One mutant, SPL1-1, was chosen for detailed analysis on the basis of its increased synthesis of mature suppressor tRNA over wild-type cell levels as determined by Northern (RNA) analysis. This mutant exhibited strong suppression exclusively with the defective tRNA gene used in the mutant selection. Genetic analysis revealed that a single, dominant, haplo-lethal mutation was responsible for the suppression phenotype. The mutation mapped on chromosome III to an essential 1.5-kb open reading frame (L. S. Symington and T. D. Petes, Mol. Cell. Biol. 8:595-604, 1988), recently named NFS1 (S. G. Oliver et al., Nature [London] 357:38-46, 1992), located adjacent (centromere proximal) to LEU2. Images PMID:8444805

  7. Enhanced xylose fermentation and ethanol production by engineered Saccharomyces cerevisiae strain.

    PubMed

    Vilela, Leonardo de Figueiredo; de Araujo, Verônica Parente Gomes; Paredes, Raquel de Sousa; Bon, Elba Pinto da Silva; Torres, Fernando Araripe Gonçalves; Neves, Bianca Cruz; Eleutherio, Elis Cristina Araújo

    2015-01-01

    We have recently demonstrated that heterologous expression of a bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia enabled a laboratorial Saccharomyces cerevisiae strain to ferment xylose anaerobically, without xylitol accumulation. However, the recombinant yeast fermented xylose slowly. In this study, an evolutionary engineering strategy was applied to improve xylose fermentation by the xylA-expressing yeast strain, which involved sequential batch cultivation on xylose. The resulting yeast strain co-fermented glucose and xylose rapidly and almost simultaneously, exhibiting improved ethanol production and productivity. It was also observed that when cells were grown in a medium containing higher glucose concentrations before being transferred to fermentation medium, higher rates of xylose consumption and ethanol production were obtained, demonstrating that xylose utilization was not regulated by catabolic repression. Results obtained by qPCR demonstrate that the efficiency in xylose fermentation showed by the evolved strain is associated, to the increase in the expression of genes HXT2 and TAL1, which code for a low-affinity hexose transporter and transaldolase, respectively. The ethanol productivity obtained after the introduction of only one genetic modification and the submission to a one-stage process of evolutionary engineering was equivalent to those of strains submitted to extensive metabolic and evolutionary engineering, providing solid basis for future applications of this strategy in industrial strains. PMID:25852993

  8. Soluble Moringa oleifera leaf extract reduces intracellular cadmium accumulation and oxidative stress in Saccharomyces cerevisiae.

    PubMed

    Kerdsomboon, Kittikhun; Tatip, Supinda; Kosasih, Sattawat; Auesukaree, Choowong

    2016-05-01

    Moringa oleifera leaves are a well-known source of antioxidants and traditionally used for medicinal applications. In the present study, the protective action of soluble M. oleifera leaf extract (MOLE) against cadmium toxicity was investigated in the model eukaryote Saccharomyces cerevisiae. The results showed that this extract exhibited a protective effect against oxidative stress induced by cadmium and H2O2 through the reduction of intracellular reactive oxygen species. Interestingly, not only the co-exposure of soluble MOLE with cadmium but also pretreatment of this extract prior to cadmium exposure significantly reduced the cadmium uptake through an inhibition of Fet4p, a low-affinity iron(II) transporter. In addition, the supplementation of soluble MOLE significantly reduced intracellular iron accumulation in a Fet4p-independent manner. Our findings suggest the potential use of soluble extract from M. oleifera leaves as a dietary supplement for protection against cadmium accumulation and oxidative stress. PMID:26675819

  9. Thiol Peroxidase Deficiency Leads to Increased Mutational Load and Decreased Fitness in Saccharomyces cerevisiae

    PubMed Central

    Kaya, Alaattin; Lobanov, Alexei V.; Gerashchenko, Maxim V.; Koren, Amnon; Fomenko, Dmitri E.; Koc, Ahmet; Gladyshev, Vadim N.

    2014-01-01

    Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (?8) is viable. In this study, we employed two independent ?8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ?8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ?8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ?8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ?8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ?8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness. PMID:25173844

  10. REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis

    SciTech Connect

    Larimer, F.W.; Perry, J.R.; Hardigree, A.A.

    1989-01-01

    The REV1 gene of Saccharomyces cerevisiae is required for normal induction of mutations by physical and chemical agents. We have determined the sequence of a 3,485-base-pair segment of DNA that complements the rev1-1 mutant. Gene disruption was used to confirm that this DNA contained the REV1 gene. The sequenced segment contains a single long open reading frame, which can encode a polypeptide of 985 amino acid residues. The REV1 transcript is 3.1 kilobase pairs in length. Frameshift mutations introduced into the open reading frame yielded a Rev-phenotype. A base substitution, encoding Gly-193 to Arg-193, was found in this open reading frame in rev1-1. Deletion mutants, lacking segments of the 5' region of REV1, had intermediate mutability relative to REV1 and rev1-1; a complete deletion exhibited lower mutability than rev1-1. REV1 is not an essential gene. An in-frame fusion of the 5' end of the REV1 open reading frame to the lacZ gene produced beta-galactosidase activity constitutively. The predicted REV1 protein is hydrophilic, with a predicted pI of 9.82. No homologies to RAD1, RAD2, RAD3, RAD7, or RAD10 proteins were noted. A 152-residue internal segment displayed 25% identity with UMUC protein.

  11. The REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis.

    PubMed

    Larimer, F W; Perry, J R; Hardigree, A A

    1989-01-01

    The REV1 gene of Saccharomyces cerevisiae is required for normal induction of mutations by physical and chemical agents. We have determined the sequence of a 3,485-base-pair segment of DNA that complements the rev1-1 mutant. Gene disruption was used to confirm that this DNA contained the REV1 gene. The sequenced segment contains a single long open reading frame, which can encode a polypeptide of 985 amino acid residues. The REV1 transcript is 3.1 kilobase pairs in length. Frameshift mutations introduced into the open reading frame yielded a Rev-phenotype. A base substitution, encoding Gly-193 to Arg-193, was found in this open reading frame in rev1-1. Deletion mutants, lacking segments of the 5' region of REV1, had intermediate mutability relative to REV1 and rev1-1; a complete deletion exhibited lower mutability than rev1-1. REV1 is not an essential gene. An in-frame fusion of the 5' end of the REV1 open reading frame to the lacZ gene produced beta-galactosidase activity constitutively. The predicted REV1 protein is hydrophilic, with a predicted pI of 9.82. No homologies to RAD1, RAD2, RAD3, RAD7, or RAD10 proteins were noted. A 152-residue internal segment displayed 25% identity with UMUC protein. PMID:2492497

  12. Senescence Mutants of Saccharomyces Cerevisiae with a Defect in Telomere Replication Identify Three Additional Est Genes

    PubMed Central

    Lendvay, T. S.; Morris, D. K.; Sah, J.; Balasubramanian, B.; Lundblad, V.

    1996-01-01

    The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere. The only component of this enzyme that has been identified in Saccharomyces cerevisiae is the TLC1 gene, encoding the telomerase RNA subunit. However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function. We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains. These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4. Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function. Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity. PMID:8978029

  13. Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle

    PubMed Central

    1995-01-01

    The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer- aided reconstruction from electron micrographs of serially cross- sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 microns have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 micron, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3- 1.6 microns long, and we suggest that the approximately 32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 microns), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast. PMID:7790357

  14. The Ubp15 deubiquitinase promotes timely entry into S phase in Saccharomyces cerevisiae

    PubMed Central

    Ostapenko, Denis; Burton, Janet L.; Solomon, Mark J.

    2015-01-01

    The anaphase-promoting complex in partnership with its activator, Cdh1, is an E3 ubiquitin ligase responsible for targeting cell cycle proteins during G1 phase. In the budding yeast Saccharomyces cerevisiae, Cdh1 associates with the deubiquitinating enzyme Ubp15, but the significance of this interaction is unclear. To better understand the physiological role(s) of Ubp15, we examined cell cycle phenotypes of cells lacking Ubp15. We found that ubp15∆ cells exhibited delayed progression from G1 into S phase and increased sensitivity to the DNA synthesis inhibitor hydroxyurea. Both phenotypes of ubp15∆ cells were rescued by additional copies of the S-phase cyclin gene CLB5. Clb5 is an unstable protein targeted for proteasome-mediated degradation by several pathways. We found that during G1 phase, the APCCdh1-mediated degradation of Clb5 was accelerated in ubp15∆ cells. Ubp15 interacted with Clb5 independent of Cdh1 and deubiquitinated Clb5 in a reconstituted system. Thus deubiquitination by Ubp15 counteracts APC activity toward cyclin Clb5 to allow Clb5 accumulation and a timely entry into S phase. PMID:25877870

  15. Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae.

    PubMed

    Peng, Bingyin; Shen, Yu; Li, Xiaowei; Chen, Xiao; Hou, Jin; Bao, Xiaoming

    2012-01-01

    Effective conversion of xylose in lignocelluloses is expected to reduce the production cost of second-generation biofuels significantly. The factors affecting xylose fermentation in Saccharomyces cerevisiae that express xylose reductase-xylitol dehydrogenase (XR-XDH) are studied. Although overproduction of non-oxidative pentose phosphate pathway significantly increased the aerobic-specific growth rate on xylose and slightly improved conversion of xylose to ethanol under oxygen-limited conditions, the elimination of respiration by deleting cytochrome C oxidase subunit IV gene impeded aerobic growth on xylose. However, the adaptive evolution of the respiratory-deficient strain with an NADP(+)-preferring XDH mutant in xylose media dramatically improved its xylose-fermenting ability. The specific growth rate, ethanol yield, and xylitol yield of the evolved strain on xylose were 0.06h(-1), 0.39gg(-1), and 0.13gg(-1) consumed xylose, respectively. Similar to anaerobic fermentation, the evolved strain exhibited accumulated ethanol rather than recycled it under aerobic conditions. PMID:22178745

  16. Strain-specific nuclear genetic background differentially affects mitochondria-related phenotypes in Saccharomyces cerevisiae.

    PubMed

    Montanari, Arianna; Francisci, Silvia; Fazzi D'Orsi, Mario; Bianchi, Michele Maria

    2014-06-01

    In the course of our studies on mitochondrial defects, we have observed important phenotypic variations in Saccharomyces cerevisiae strains suggesting that a better characterization of the genetic variability will be essential to define the relationship between the mitochondrial efficiency and the presence of different nuclear backgrounds. In this manuscript, we have extended the study of such relations by comparing phenotypic assays related to mitochondrial functions of three wild-type laboratory strains. In addition to the phenotypic variability among the wild-type strains, important differences have been observed among strains bearing identical mitochondrial tRNA mutations that could be related only to the different nuclear background of the cells. Results showed that strains exhibited an intrinsic variability in the severity of the effects of the mitochondrial mutations and that specific strains might be used preferentially to evaluate the phenotypic effect of mitochondrial mutations on carbon metabolism, stress responses, and mitochondrial DNA stability. In particular, while W303-1B and MCC123 strains should be used to study the effect of severe mitochondrial tRNA mutations, D273-10B/A1 strain is rather suitable for studying the effects of milder mutations. PMID:24700775

  17. Spore germination in Saccharomyces cerevisiae: global gene expression patterns and cell cycle landmarks

    PubMed Central

    Joseph-Strauss, Daphna; Zenvirth, Drora; Simchen, Giora; Barkai, Naama

    2007-01-01

    Background Spore germination in the yeast Saccharomyces cerevisiae is a process in which non-dividing haploid spores re-enter the mitotic cell cycle and resume vegetative growth. To study the signals and pathways underlying spore germination we examined the global changes in gene expression and followed cell-cycle and germination markers during this process. Results We find that the germination process can be divided into two distinct stages. During the first stage, the induced spores respond only to glucose. The transcription program during this stage recapitulates the general transcription response of yeast cells to glucose. Only during the second phase are the cells able to sense and respond to other nutritional components in the environment. Components of the mitotic machinery are involved in spore germination but in a distinct pattern. In contrast to the mitotic cell cycle, growth-related events during germination are not coordinated with nuclear events and are separately regulated. Thus, genes that are co-induced during G1/S of the mitotic cell cycle, the dynamics of the septin Cdc10 and the kinetics of accumulation of the cyclin Clb2 all exhibit distinct patterns of regulation during spore germination, which allow the separation of cell growth from nuclear events. Conclusion Taken together, genome-wide expression profiling enables us to follow the progression of spore germination, thus dividing this process into two major stages, and to identify germination-specific regulation of components of the mitotic cell cycle machinery. PMID:17999778

  18. Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

    SciTech Connect

    Hegemann, J.H.; Shero, J.H.; Cottarel, G.; Philippsen, P.; Hieter, P.

    1988-06-01

    Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three different centromere DNA sequence elements (CDEI, CDEII, and CDEIII). The authors generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10/sup -4/) to nonfunctional (>10/sup -1/). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance to mitotic CEN function.

  19. Dimeric Coiled-coil Structure of Saccharomyces cerevisiae Atg16 and Its Functional Significance in Autophagy*

    PubMed Central

    Fujioka, Yuko; Noda, Nobuo N.; Nakatogawa, Hitoshi; Ohsumi, Yoshinori; Inagaki, Fuyuhiko

    2010-01-01

    Atg16 interacts with the Atg12-Atg5 protein conjugate through its N-terminal domain and self-assembles through its coiled-coil domain (CCD). Formation of the Atg12-Atg5·Atg16 complex is essential for autophagy, the bulk degradation process conserved among most eukaryotes. Here, we report the crystal structures of full-length Saccharomyces cerevisiae Atg16 at 2.8 ? resolution and its CCD at 2.5 ? resolution. The CCD and full-length Atg16 each exhibit an extended ?-helix, 90 and 130 ?, respectively, and form a parallel coiled-coil dimer in the crystals. Although the apparent molecular weight of Atg16 observed by gel-filtration chromatography suggests that Atg16 is tetrameric, an analytical ultracentrifugation study showed Atg16 as a dimer in solution, consistent with the crystal structure. Evolutionary conserved surface residues clustered at the C-terminal half of Atg16 CCD were shown to be crucial for autophagy. These results will give a structural basis for understanding the molecular functions and significance of Atg16 in autophagy. PMID:19889643

  20. Thiol peroxidase deficiency leads to increased mutational load and decreased fitness in Saccharomyces cerevisiae.

    PubMed

    Kaya, Alaattin; Lobanov, Alexei V; Gerashchenko, Maxim V; Koren, Amnon; Fomenko, Dmitri E; Koc, Ahmet; Gladyshev, Vadim N

    2014-11-01

    Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (?8) is viable. In this study, we employed two independent ?8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ?8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ?8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ?8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ?8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ?8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness. PMID:25173844

  1. The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity.

    PubMed

    Megarity, Clare F; Looi, Hong Keat; Timson, David J

    2014-08-01

    Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p. PMID:24866129

  2. Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl, Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiae

    PubMed Central

    Eudes, Aymerick; Teixeira Benites, Veronica; Wang, George; Baidoo, Edward E. K.; Lee, Taek Soon; Keasling, Jay D.; Loqué, Dominique

    2015-01-01

    Biological synthesis of pharmaceuticals and biochemicals offers an environmentally friendly alternative to conventional chemical synthesis. These alternative methods require the design of metabolic pathways and the identification of enzymes exhibiting adequate activities. Cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates are natural metabolites which possess beneficial activities for human health, and the search is expanding for novel derivatives that might have enhanced biological activity. For example, biosynthesis in Dianthus caryophyllus is catalyzed by hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT), which couples hydroxycinnamoyl-CoAs and benzoyl-CoAs to anthranilate. We recently demonstrated the potential of using yeast (Saccharomyces cerevisiae) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from Arabidopsis thaliana (4CL5) and HCBT. Here we report that, by exploiting the substrate flexibility of both 4CL5 and HCBT, we achieved rapid biosynthesis of more than 160 cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates in yeast upon feeding with both natural and non-natural cinnamates, dihydrocinnamates, benzoates, and anthranilates. Our results demonstrate the use of enzyme promiscuity in biological synthesis to achieve high chemical diversity within a defined class of molecules. This work also points to the potential for the combinatorial biosynthesis of diverse and valuable cinnamoylated, dihydrocinnamoylated, and benzoylated products by using the versatile biological enzyme 4CL5 along with characterized cinnamoyl-CoA- and benzoyl-CoA-utilizing transferases. PMID:26430899

  3. Pronounced and extensive microtubule defects in a Saccharomyces cerevisiae DIS3 mutant.

    PubMed

    Smith, Sarah B; Kiss, Daniel L; Turk, Edward; Tartakoff, Alan M; Andrulis, Erik D

    2011-11-01

    Subunits of the RNA processing exosome assemble into structurally distinct protein complexes that function in disparate cellular compartments and RNA metabolic pathways. Here, in a genetic, cell biological and transcriptomic analysis, we examined the role of Dis3, an essential polypeptide with endo- and 3'?5' exo-ribonuclease activity, in cell cycle progression. We present several lines of evidence that perturbation of DIS3 affects microtubule (MT) localization and structure in Saccharomyces cerevisiae. Cells with a DIS3 mutant: (a) accumulate anaphase and pre-anaphase mitotic spindles; (b) exhibit spindles that are misorientated and displaced from the bud neck; (c) harbour elongated spindle-associated astral MTs; (d) have an increased G1 astral MT length and number; and (e) are hypersensitive to MT poisons. Mutations in the core exosome genes RRP4 and MTR3 and the exosome cofactor gene MTR4, but not other exosome subunit gene mutants, also elicit MT phenotypes. RNA deep sequencing analysis (RNA-seq) shows broad changes in the levels of cell cycle- and MT-related transcripts in mutant strains. Collectively, the data presented in this study suggest an evolutionarily conserved role for Dis3 in linking RNA metabolism, MTs and cell cycle progression. PMID:21919057

  4. SNG1--a new gene involved in nitrosoguanidine resistance in Saccharomyces cerevisiae.

    PubMed

    Grey, M; Pich, C T; Haase, E; Brendel, M

    1995-04-01

    We have molecularly characterized the SNG1 gene that confers hyper-resistance to the mutagen N-methyl-N'nitro-N-nitrosoguanidine (MNNG) in Saccharomyces cerevisiae when overexpressed on a multi-copy plasmid. This hyper-resistance to MNNG is not due to depletion of glutathione pools since multi-copy SNG1 containing yeast transformants contain at least wild type levels of glutathione; DNA repair seems unaffected in these transformants as the multi-copy SNG1-mediated MNNG hyper-resistance is also seen in DNA repair mutants belonging to each of the three epistasis groups of yeast repair mutants. It could be shown that SNG1 is not under control of the YAP1 encoded transcription activator that controls expression of at least two genes involved in MNNG metabolism in yeast. sng1 null mutants are viable but exhibit only slight sensitivity to MNNG, indicating that SNG1 does not encode a protein involved in a major detoxification step of this mutagen. Sequencing of the HYR-mediating passenger DNA revealed that SNG1 encodes a 547 a polypeptide containing seven transmembrane-spanning regions that may be membrane-bound. Comparison of the DNA sequence with established gene databanks revealed that SNG1 is a novel yeast gene. PMID:7753113

  5. SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae

    PubMed Central

    Gale, Cheryl A.; Leonard, Michelle D.; Finley, Kenneth R.; Christensen, Leah; McClellan, Mark; Abbey, Darren; Kurischko, Cornelia; Bensen, Eric; Tzafrir, Iris; Kauffman, Sarah; Becker, Jeff; Berman, Judith

    2009-01-01

    The early endocytic patch protein Sla2 is important for morphogenesis and growth rates in Saccharomyces cerevisiae and Candida albicans, but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2 or ScSLA2 are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other components of early endocytic patches (Sla1 and Abp1) with those in strains lacking Sla2. Only sla2 strains had defects in actin cables, a known trigger of the morphogenesis checkpoint, yet all three strains exhibited Swe1-dependent phenotypes. Thus, Swe1 appears to monitor actin patch in addition to actin cable function. Furthermore, Swe1 contributed to virulence in a mouse model of disseminated candidiasis, implying a role for the morphogenesis checkpoint during the pathogenesis of C. albicans infections. PMID:19778960

  6. CDC19 encoding pyruvate kinase is important for high-temperature tolerance in Saccharomyces cerevisiae.

    PubMed

    Benjaphokee, Suthee; Koedrith, Preeyaporn; Auesukaree, Choowong; Asvarak, Thipa; Sugiyama, Minetaka; Kaneko, Yoshinobu; Boonchird, Chuenchit; Harashima, Satoshi

    2012-01-15

    Use of thermotolerant strains is a promising way to reduce the cost of maintaining optimum temperatures in the fermentation process. Here we investigated genetically a Saccharomyces cerevisiae strain showing a high-temperature (41°C) growth (Htg(+)) phenotype and the result suggested that the Htg(+) phenotype of this Htg(+) strain is dominant and under the control of most probably six genes, designated HTG1 to HTG6. As compared with a Htg(-) strain, the Htg(+) strain showed a higher survival rate after exposure to heat shock at 48°C. Moreover, the Htg(+) strain exhibited a significantly high content of trehalose when cultured at high temperature and stronger resistance to Congo Red, an agent that interferes with cell wall construction. These results suggest that a strengthened cell wall in combination with increased trehalose accumulation can support growth at high temperature. The gene CDC19, encoding pyruvate kinase, was cloned as the HTG2 gene. The CDC19 allele from the Htg(+) strain possessed five base changes in its upstream region, and two base changes resulting in silent mutations in its coding region. Interestingly, the latter base changes are probably responsible for the increased pyruvate kinase activity of the Htg(+) strain. The possible mechanism leading to this increased activity and to the Htg(+) phenotype, which may lead to the activation of energy metabolism to maintain cellular homeostasis, is discussed. PMID:21459167

  7. Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics.

    PubMed

    Walther, Thomas; Mtimet, Narjes; Alkim, Ceren; Vax, Amélie; Loret, Marie-Odile; Ullah, Azmat; Gancedo, Carlos; Smits, Gertien J; François, Jean Marie

    2013-09-01

    In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H?-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H?-ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism. PMID:23763276

  8. Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl, Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiae.

    PubMed

    Eudes, Aymerick; Teixeira Benites, Veronica; Wang, George; Baidoo, Edward E K; Lee, Taek Soon; Keasling, Jay D; Loqué, Dominique

    2015-01-01

    Biological synthesis of pharmaceuticals and biochemicals offers an environmentally friendly alternative to conventional chemical synthesis. These alternative methods require the design of metabolic pathways and the identification of enzymes exhibiting adequate activities. Cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates are natural metabolites which possess beneficial activities for human health, and the search is expanding for novel derivatives that might have enhanced biological activity. For example, biosynthesis in Dianthus caryophyllus is catalyzed by hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT), which couples hydroxycinnamoyl-CoAs and benzoyl-CoAs to anthranilate. We recently demonstrated the potential of using yeast (Saccharomyces cerevisiae) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from Arabidopsis thaliana (4CL5) and HCBT. Here we report that, by exploiting the substrate flexibility of both 4CL5 and HCBT, we achieved rapid biosynthesis of more than 160 cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates in yeast upon feeding with both natural and non-natural cinnamates, dihydrocinnamates, benzoates, and anthranilates. Our results demonstrate the use of enzyme promiscuity in biological synthesis to achieve high chemical diversity within a defined class of molecules. This work also points to the potential for the combinatorial biosynthesis of diverse and valuable cinnamoylated, dihydrocinnamoylated, and benzoylated products by using the versatile biological enzyme 4CL5 along with characterized cinnamoyl-CoA- and benzoyl-CoA-utilizing transferases. PMID:26430899

  9. A Novel Allele of Hap1 Causes Uninducible Expression of Hem13 in Saccharomyces Cerevisiae

    PubMed Central

    Ushinsky, S. C.; Keng, T.

    1994-01-01

    Transcription of HEM13 in Saccharomyces cerevisiae is repressed by heme and oxygen. We have isolated two mutants in which expression of HEM13 is aberrant. The mutant alleles in these strains represent two different alleles of HAP1. HAP1 encodes an activator protein whose DNA binding activity is stimulated by heme, and is required for the transcription of CYC1, ROX1 and a number of other heme-dependent genes. One of our mutant alleles confers a phenotype much like that of the hap1::LEU2 allele. Expression of HEM13 in a strain with this mutation is elevated under repressing conditions and not fully inducible in the absence of heme. The other mutant allele of HAP1 we uncovered confers a novel phenotype. A strain containing this allele exhibits heme-independent expression of CYC1 and ROX1 and uninducible expression of HEM13 and ANB1. The mutation associated with this novel allele of HAP1 was localized to a glycine to aspartate change in amino acid 235 of HAP1, between the DNA binding and heme responsive domains. DNA binding assays demonstrated that the protein made from this HAP1 allele retains the ability to bind DNA, but that unlike wild-type HAP1 protein, this binding is not stimulated by heme. PMID:8005437

  10. Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

    PubMed Central

    Shiroma, Shodai; Jayakody, Lahiru Niroshan; Horie, Kenta; Okamoto, Koji

    2014-01-01

    Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO2 production and final ethanol concentration generated by the atg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts. PMID:24271183

  11. Cardiolipin and Mitochondrial Phosphatidylethanolamine Have Overlapping Functions in Mitochondrial Fusion in Saccharomyces cerevisiae*

    PubMed Central

    Joshi, Amit S.; Thompson, Morgan N.; Fei, Naomi; Hüttemann, Maik; Greenberg, Miriam L.

    2012-01-01

    The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have shown previously that CL and PE have overlapping functions, and the loss of both is synthetically lethal. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, we hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional mutant crd1?psd1? containing null alleles of CRD1 (CL synthase) and PSD1 (mitochondrial phosphatidylserine decarboxylase), in which the wild type CRD1 gene is expressed on a plasmid under control of the TETOFF promoter. In the presence of tetracycline, the mutant exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate for the first time in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion. PMID:22433850

  12. Investigation of the Highly Active Manganese Superoxide Dismutase from Saccharomyces cerevisiae

    SciTech Connect

    Cabelli, D.E.; Barnese, K.; Sheng, Y.; Stich, T.A.; Gralla, E.B.; Britt, R.D.; Valentine, J.S.

    2010-09-15

    Manganese superoxide dismutase (MnSOD) from different species differs in its efficiency in removing high concentrations of superoxide (O{sub 2}{sup -}), due to different levels of product inhibition. Human MnSOD exhibits a substantially higher level of product inhibition than the MnSODs from bacteria. In order to investigate the mechanism of product inhibition and whether it is a feature common to eukaryotic MnSODs, we purified MnSOD from Saccharomyces cerevisiae (ScMnSOD). It was a tetramer with 0.6 equiv of Mn per monomer. The catalytic activity of ScMnSOD was investigated by pulse radiolysis and compared with human and two bacterial (Escherichia coli and Deinococcus radiodurans) MnSODs. To our surprise, ScMnSOD most efficiently facilitates removal of high concentrations of O{sub 2}{sup -} among these MnSODs. The gating value k{sub 2}/k{sub 3} that characterizes the level of product inhibition scales as ScMnSOD > D. radiodurans MnSOD > E. coli MnSOD > human MnSOD. While most MnSODs rest as the oxidized form, ScMnSOD was isolated in the Mn{sup 2+} oxidation state as revealed by its optical and electron paramagnetic resonance spectra. This finding poses the possibility of elucidating the origin of product inhibition by comparing human MnSOD with ScMnSOD.

  13. Quantitative study of interactions between Saccharomyces cerevisiae and Oenococcus oeni strains.

    PubMed

    Nehme, Nancy; Mathieu, Florence; Taillandier, Patricia

    2008-07-01

    This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained. PMID:18317828

  14. Secretion can proceed uncoupled from net plasma membrane expansion in inositol-starved Saccharomyces cerevisiae.

    PubMed Central

    Atkinson, K D; Ramirez, R M

    1984-01-01

    Secretion of acid phosphatase and invertase was examined in an inositol-requiring ino1 mutant of the yeast Saccharomyces cerevisiae. Inositol starvation is known to block plasma membrane expansion, presumably due to restricted membrane phospholipid synthesis. If membrane expansion and extracellular protein secretion are accomplished by the same intracellular transport process, one would expect secretion to fail coordinately with cessation of plasma membrane growth in inositol-starved cells. In glucose-grown, inositol-starved cells, plasma membrane expansion and acid phosphatase secretion stopped coordinately, and intracellular acid phosphatase accumulated. In sucrose-grown, inositol-starved cells, plasma membrane growth halted, but secretion of both acid phosphatase and invertase continued until the onset of inositol-less death. Although glucose-grown and sucrose-grown cells differ in their ability to secrete when deprived of inositol, they exhibited the same disturbances in phospholipid synthesis. Phosphatidylinositol synthesis failed, and its precursors phosphatidic acid and CDP-diglyceride accumulated equally in both cultures. Sucrose-grown yeast cells appear to accomplish normal levels of extracellular protein secretion by an inositol-independent mechanism. In glucose-grown yeasts, both plasma membrane expansion and secretion are inositol dependent. PMID:6384202

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

    PubMed Central

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

    2007-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2007-11-01

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

  17. Glutamine synthesis is a regulatory signal controlling glucose catabolism in Saccharomyces cerevisiae.

    PubMed Central

    Flores-Samaniego, B; Olivera, H; González, A

    1993-01-01

    The effect of glutamine biosynthesis and degradation on glucose catabolism in Saccharomyces cerevisiae was studied. A wild-type strain and mutants altered in glutamine biosynthesis and degradation were analyzed. Cells having low levels of glutamine synthetase activity showed high ATP/ADP ratios and a diminished rate of glucose metabolism. It is proposed that glutamine biosynthesis plays a role in the regulation of glucose catabolism. PMID:7902349

  18. Seven-Base-Pair Inverted Repeats in DNA Form Stable Hairpins in Vivo in Saccharomyces Cerevisiae

    PubMed Central

    Nag, D. K.; Petes, T. D.

    1991-01-01

    Palindromic sequences in single-stranded DNA and RNA have the potential for intrastrand base pairing, resulting in formation of ``hairpin'' structures. We previously reported a genetic method for detecting such structures in vivo in the yeast Saccharomyces cerevisiae. Below, we describe evidence indicating that a 14-base-pair palindrome (7 bp per inverted repeat) is sufficient for formation of a hairpin in vivo. PMID:1752412

  19. Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: excision of dimers in cell extracts.

    PubMed

    Reynolds, R J; Love, J D; Friedberg, E C

    1981-08-01

    Cell-free extracts prepared from rad1-19, rd2-2, rad3-1, rad4-3, rad7-1, rad10-1, rd14-1, rad16-1, and cyc1-1 (rad7) mutants of Saccharomyces cerevisiae all catalyze the preferential excision of thymine-containing pyrimidine dimers from ultraviolet-irradiated DNA specifically incised with M. luteus ultraviolet deoxyribonucleic acid incising activity. PMID:7021538

  20. Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: excision of dimers in cell extracts

    SciTech Connect

    Reynolds, R.J.; Love, J.D.; Friedberg, E.C.

    1981-08-01

    Cell-free extracts prepared from rad1-19, rad2-2, rad3-1, rad4-3, rad7-1, rad10-1, rad14-1, rad16-1, and cycl-1 (rad7) mutants of Saccharomyces cerevisiae all catalyze the preferential excision of thymine-containing pyrimidine dimers from ultraviolet-irradiated DNA specifically incised with M. luteus ultraviolet deoxyribonucleic acid incising activity.

  1. Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae.

    PubMed

    Covello, P S; Reed, D W

    1996-05-01

    The functional expression in yeast of the Arabidopsis thaliana FAD2 gene, encoding the extraplastidial oleate desaturase (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase) is reported. Dienoic fatty acids constituted up to 11% (w/w) of the total fatty acids in transformed Saccharomyces cerevisiae cells and were confirmed to be linoleic acid and delta 9, delta 12-hexadecadienoic acid by gas chromatography-mass spectrometry. PMID:8685264

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

    PubMed

    Nair, Nikhil U; Zhao, Huimin

    2012-01-01

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

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

    SciTech Connect

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

    1988-10-01

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

  4. Biosorption of water-soluble dyes on magnetically modified Saccharomyces cerevisiae subsp. uvarum cells.

    PubMed

    Safaríková, M; Ptácková, L; Kibriková, I; Safarík, I

    2005-05-01

    Brewer's yeast (bottom yeast, Saccharomyces cerevisiae subsp. uvarum) cells were magnetically modified using water based magnetic fluid stabilized with perchloric acid. Magnetically modified yeast cells efficiently adsorbed various water soluble dyes. The dyes adsorption can be described by the Langmuir adsorption model. The maximum adsorption capacity of the magnetic cells differed substantially for individual dyes; the highest value was found for aniline blue (approx. 220 mg per g of dried magnetic adsorbent). PMID:15811411

  5. Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families.

    PubMed

    de la Cruz, J; Kressler, D; Linder, P

    1999-05-01

    Members of the RNA-helicase family are defined by several evolutionary conserved motifs. They are found in all organisms - from bacteria to humans - and many viruses. The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome. Recent progress in the functional analysis of various family members has given new insights into, and confirmed the significance of these proteins for, most cellular RNA metabolic processes. PMID:10322435

  6. Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae

    SciTech Connect

    Field, J.; Xu, Haopeng; Michaeli, T.; Ballester, R.; Sass, P.; Wigler, M.; Colicelli, J. )

    1990-01-26

    The interaction between RAS proteins and adenylyl cyclase was studied by using dominant interfering mutations of adenylyl cyclase from the yeast Saccharomyces cerevisiae. RAS proteins active adenylyl cyclase in this organism. A plasmid expressing a catalytically inactive adenylyl cyclase was found to interfere dominantly with this activation. The interfering region mapped to the leucine-rich repeat region of adenylyl cyclase, which is homologous to domains present in several other proteins and is thought to participate in protein-protein interactions.

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

    NASA Astrophysics Data System (ADS)

    Rogacheva, Svetlana M.; Babaeva, Milena I.

    2013-02-01

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

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

    SciTech Connect

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

    2007-11-26

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

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

    PubMed Central

    de Bruijn, F; Greer, H

    1981-01-01

    A Saccharomyces cerevisiae transposable element which carries the his4C structural gene and which is capable of transposition, excision, and mutator activity is described. Physical evidence is presented for transposition of the his4C deoxyribonucleic acid sequences to a new location in the genome and for precise excision of these transposed deoxyribonucleic acid sequences in spontaneous his4C- segregants. Images PMID:6100963

  10. Assessment of the Biological Pathways Targeted by Isocyanate Using N-Succinimidyl N-Methylcarbamate in Budding Yeast Saccharomyces cerevisiae

    PubMed Central

    Azad, Gajendra Kumar; Singh, Vikash; Tomar, Raghuvir S.

    2014-01-01

    Isocyanates, a group of low molecular weight aromatic and aliphatic compounds possesses the functional isocyanate group. They are highly toxic in nature hence; we used N-succinimidyl N-methylcarbamate (NSNM), a surrogate chemical containing a functional isocyanate group to understand the mode of action of this class of compounds. We employed budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by NSNM. Our screening with yeast mutants revealed that it affects chromatin, DNA damage response, protein-ubiquitylation and chaperones, oxidative stress, TOR pathway and DNA repair processes. We also show that NSNM acts as an epigenetic modifier as its treatment causes reduction in global histone acetylation and formation of histone adducts. Cells treated with NSNM exhibited increase in mitochondrial membrane potential as well as intracellular ROS levels and the effects were rescued by addition of reduced glutathione to the medium. We also report that deletion of SOD1 and SOD2, the superoxide dismutase in Saccharomyces cerevisiae displayed hypersensitivity to NSNM. Furthermore, NSNM treatment causes rapid depletion of total glutathione and reduced glutathione. We also demonstrated that NSNM induces degradation of Sml1, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. In summary, we define the various biological pathways targeted by isocyanates. PMID:24664350

  11. Molecular Characterization of New Natural Hybrids of Saccharomyces cerevisiae and S. kudriavzevii in Brewing? †

    PubMed Central

    González, Sara S.; Barrio, Eladio; Querol, Amparo

    2008-01-01

    We analyzed 24 beer strains from different origins by using PCR-restriction fragment length polymorphism analysis of different gene regions, and six new Saccharomyces cerevisiae × Saccharomyces kudriavzevii hybrid strains were found. This is the first time that the presence in brewing of this new type of hybrid has been demonstrated. From the comparative molecular analysis of these natural hybrids with respect to those described in wines, it can be concluded that these originated from at least two hybridization events and that some brewing hybrids share a common origin with wine hybrids. Finally, a reduction of the S. kudriavzevii fraction of the hybrid genomes was observed, but this reduction was found to vary among hybrids regardless of the source of isolation. The fact that 25% of the strains analyzed were discovered to be S. cerevisiae × S. kudriavzevii hybrids suggests that an important fraction of brewing strains classified as S. cerevisiae may correspond to hybrids, contributing to the complexity of Saccharomyces diversity in brewing environments. The present study raises new questions about the prevalence of these new hybrids in brewing as well as their contribution to the properties of the final product. PMID:18296532

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

    PubMed

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

    2005-09-01

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

  13. Anti-Saccharomyces cerevisiae autoantibodies in autoimmune diseases: from bread baking to autoimmunity.

    PubMed

    Rinaldi, Maurizio; Perricone, Roberto; Blank, Miri; Perricone, Carlo; Shoenfeld, Yehuda

    2013-10-01

    Saccharomyces cerevisiae is best known as the baker's and brewer's yeast, but its residual traces are also frequent excipients in some vaccines. Although anti-S. cerevisiae autoantibodies (ASCAs) are considered specific for Crohn's disease, a growing number of studies have detected high levels of ASCAs in patients affected with autoimmune diseases as compared with healthy controls, including antiphospholipid syndrome, systemic lupus erythematosus, type 1 diabetes mellitus, and rheumatoid arthritis. Commensal microorganisms such as Saccharomyces are required for nutrition, proper development of Peyer's aggregated lymphoid tissue, and tissue healing. However, even the commensal nonclassically pathogenic microbiota can trigger autoimmunity when fine regulation of immune tolerance does not work properly. For our purposes, the protein database of the National Center for Biotechnology Information (NCBI) was consulted, comparing Saccharomyces mannan to several molecules with a pathogenetic role in autoimmune diseases. Thanks to the NCBI bioinformation technology tool, several overlaps in molecular structures (50-100 %) were identified when yeast mannan, and the most common autoantigens were compared. The autoantigen U2 snRNP B? was found to conserve a superfamily protein domain that shares 83 % of the S. cerevisiae mannan sequence. Furthermore, ASCAs may be present years before the diagnosis of some associated autoimmune diseases as they were retrospectively found in the preserved blood samples of soldiers who became affected by Crohn's disease years later. Our results strongly suggest that ASCAs' role in clinical practice should be better addressed in order to evaluate their predictive or prognostic relevance. PMID:23292495

  14. Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation.

    PubMed

    Lopez, Claudia Lorena Fernandez; Beaufort, Sandra; Brandam, Cédric; Taillandier, Patricia

    2014-08-01

    Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces. PMID:24677041

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

    PubMed

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

    2012-03-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-08-01

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

  18. Development of a system for multicopy gene integration in Saccharomyces cerevisiae.

    PubMed

    Semkiv, Marta V; Dmytruk, Kostyantyn V; Sibirny, Andriy A

    2016-01-01

    In this study we describe construction and evaluation of a vector for multicopy integration in yeast Saccharomyces cerevisiae. In this vector a modified selective marker and a reporter gene PHO8 (encoding alkaline phosphatase) were flanked with delta sequences of the Ty1 transposon. Modified by error-prone PCR version of selection marker kanMX4 was obtained from Escherichia coli clone with impaired geneticin (G418) resistance. The attenuation of kanMX4 gene provides an opportunity to select for explicitly multicopy integration of the module in S. cerevisiae using moderate (200mgL(-1)) antibiotic concentrations. The developed system provided integration of 3-10 copies of the module in the genome of S. cerevisiae. High copy integration events were confirmed by qRT-PCR, Southern hybridization and reporter enzyme activity measurements. PMID:26529647

  19. Anthranilic acid release in adenosine-inhibited cultures of Saccharomyces cerevisiae and its inhibition by thiamin.

    PubMed

    Iwashima, A; Kawasaki, Y; Kimura, Y; Hasegawa, T

    1992-10-01

    Adenosine, at 1 mM concentrations or above, was found to have a fungistatic effect on Saccharomyces cerevisiae. A substance with amethyst fluorescence was detected in the medium of adenosine-inhibited cultures of S. cerevisiae. This compound was isolated and physicochemically identified as anthranilic acid. Both the inhibition of growth and release of anthranilic acid induced by adenosine were abrogated by thiamin or by the pyrimidine portion of thiamin, 2-methyl-4-amino-5-hdroxymethyl-pyrimidine (hydroxymethyl-pyrimidine); the latter was found to restore intracellular thiamin content that had been reduced by adenosine. It was demonstrated that effects of thiamin and hydroxymethylpyrimidine on S. cerevisiae cultured with adenosine resulted from their inhibition of adenosine uptake by growing yeast cells. PMID:1426996

  20. Improve carbon metabolic flux in Saccharomyces cerevisiae at high temperature by overexpressed TSL1 gene.

    PubMed

    Ge, Xiang-Yang; Xu, Yan; Chen, Xiang

    2013-04-01

    This study describes a novel strategy to improve the glycolysis flux of Saccharomyces cerevisiae at high temperature. The TSL1 gene-encoding regulatory subunit of the trehalose synthase complex was overexpressed in S. cerevisiae Z-06, which increased levels of trehalose synthase activity in extracts, enhanced stress tolerance and glucose consuming rate of the yeast cells. As a consequence, the final ethanol concentration of 185.5 g/L was obtained at 38 °C for 36 h (with productivity up to 5.2 g/L/h) in 7-L fermentor, and the ethanol productivity was 92.7 % higher than that of the parent strain. The results presented here provide a novel way to enhance the carbon metabolic flux at high temperature, which will be available for the purposes of producing other primary metabolites of commercial interest using S. cerevisiae as a host. PMID:23377879

  1. Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae.

    PubMed Central

    Abouzied, M M; Reddy, C A

    1986-01-01

    Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae. PMID:3539016

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

    PubMed

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

    2014-12-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  7. Saccharomyces cerevisiae pol30 (Proliferating Cell Nuclear Antigen) Mutations Impair Replication Fidelity and Mismatch Repair

    PubMed Central

    Chen, Clark; Merrill, Bradley J.; Lau, Patrick J.; Holm, Connie; Kolodner, Richard D.

    1999-01-01

    To understand the role of POL30 in mutation suppression, 11 Saccharomyces cerevisiae pol30 mutator mutants were characterized. These mutants were grouped based on their mutagenic defects. Many pol30 mutants harbor multiple mutagenic defects and were placed in more than one group. Group A mutations (pol30-52, -104, -108, and -126) caused defects in mismatch repair (MMR). These mutants exhibited mutation rates and spectra reminiscent of MMR-defective mutants and were defective in an in vivo MMR assay. The mutation rates of group A mutants were enhanced by a msh2 or a msh6 mutation, indicating that MMR deficiency is not the only mutagenic defect present. Group B mutants (pol30-45, -103, -105, -126, and -114) exhibited increased accumulation of either deletions alone or a combination of deletions and duplications (4 to 60 bp). All deletion and duplication breakpoints were flanked by 3 to 7 bp of imperfect direct repeats. Genetic analysis of one representative group B mutant, pol30-126, suggested polymerase slippage as the likely mutagenic mechanism. Group C mutants (pol30-100, -103, -105, -108, and -114) accumulated base substitutions and exhibited synergistic increases in mutation rate when combined with msh6 mutations, suggesting increased DNA polymerase misincorporation as a mutagenic defect. The synthetic lethality between a group A mutant, pol30-104, and rad52 was almost completely suppressed by the inactivation of MSH2. Moreover, pol30-104 caused a hyperrecombination phenotype that was partially suppressed by a msh2 mutation. These results suggest that pol30-104 strains accumulate DNA breaks in a MSH2-dependent manner. PMID:10523669

  8. Improvement of ethanol production by electrochemical redox coupling of Zymomonas mobilis and Saccharomyces cerevisiae.

    PubMed

    Jeon, Bo Young; Park, Doo Hyun

    2010-01-01

    Zymomonas mobilis was immobilized in a modified graphite felt cathode with neutral red (NR-graphite cathode) and Saccharomyces cerevisiae was cultivated on a platinum plate anode to electrochemically activate ethanol fermentation. Electrochemical redox reaction was induced by 3 approximately 4 volt of electric potential charged to a cathode and an anode. Z. mobilis produced 1.3 approximately 1.5 M of ethanol in the cathode compartment and S. cerevisiae did 1.7 approximately 1.9 M in the anode compartment for 96 hr. The ethanol production by Z. mobilis immobilized in the NR-graphite cathode and S. cerevisiae cultivated on the platinum plate was 1.5 approximately 1.6 times higher than those cultivated in the conventional condition. The electrochemical oxidation potential greatly inhibited ethanol fermentation of Z. mobilis but did not S. cerevisiae. Total soluble protein pattern of Z. mobilis cultivated in the electrochemical oxidation condition was getting simplified in proportion to potential intensity based on SDS-PAGE pattern; however the SDS-PAGE pattern of protein extracted from S. cerevisiae cultivated in both oxidation and reduction condition was not changed. When Z. mobilis culture incubated in the cathode compartment for 24 hr was transferred to S. cerevisiae culture in the anode compartment, 0.8 approximately 0.9 M of ethanol was additionally produced by S. cerevisiae for another 24 hr. Conclusively, total 2.0 approximately 2.1 M of ethanol was produced by the electrochemical redox coupling of Z. mobilis and S. cerevisiae for 48 hr. PMID:20134239

  9. Comparative toxicities of putative phagocyte-generated oxidizing radicals toward a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae).

    PubMed

    King, David A; Sheafor, Mark W; Hurst, James K

    2006-09-01

    Toxicities of the radiolytically generated oxidizing radicals HO(*), CO(3)(-)(*), and NO(2)(*) toward suspension cultures of a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae) were examined. As demonstrated by the absence of protection from the membrane-impermeable HO(*) scavenger polyethylene glycol (PEG), externally generated HO(*) was not bactericidal under these conditions; however, partial protection by PEG was observed for S. cerevisiae, indicating the presence of a fungicidal pathway involving external HO(*). For both organisms, conversion of external HO(*) to the secondary radical, CO(3)(-)(*), by reaction with HCO(3)(-) increases their susceptibility to radiolytic killing. In contrast, externally generated NO(2)(*) exhibited toxicity comparable to that of CO(3)(-)(*) toward E. coli, but completely blocked the extracellular toxicity of HO(*) toward S. cerevisiae. Cogeneration of equal fluxes of NO(2)(-)(*) and either HO(*) or CO(3)(-)(*) also essentially eliminated the extracellular microbicidal reactions. This behavior is consistent with expectations based upon relative rates of radical-radical self-coupling and cross-coupling reactions. The different patterns of toxicity observed imply fundamentally different microbicidal mechanisms for the two organisms, wherein the bacterium is susceptible to killing by oxidation of highly reactive targets on its cellular envelope but, despite undergoing similar oxidative insult, the fungus is not. PMID:16895797

  10. Understanding the Mechanism of Thermotolerance Distinct From Heat Shock Response Through Proteomic Analysis of Industrial Strains of Saccharomyces cerevisiae.

    PubMed

    Shui, Wenqing; Xiong, Yun; Xiao, Weidi; Qi, Xianni; Zhang, Yong; Lin, Yuping; Guo, Yufeng; Zhang, Zhidan; Wang, Qinhong; Ma, Yanhe

    2015-07-01

    Saccharomyces cerevisiae has been intensively studied in responses to different environmental stresses such as heat shock through global omic analysis. However, the S. cerevisiae industrial strains with superior thermotolerance have not been explored in any proteomic studies for elucidating the tolerance mechanism. Recently a new diploid strain was obtained through evolutionary engineering of a parental industrial strain, and it exhibited even higher resistance to prolonged thermal stress. Herein, we performed iTRAQ-based quantitative proteomic analysis on both the parental and evolved industrial strains to further understand the mechanism of thermotolerant adaptation. Out of ∼ 2600 quantifiable proteins from biological quadruplicates, 193 and 204 proteins were differentially regulated in the parental and evolved strains respectively during heat-stressed growth. The proteomic response of the industrial strains cultivated under prolonged thermal stress turned out to be substantially different from that of the laboratory strain exposed to sudden heat shock. Further analysis of transcription factors underlying the proteomic perturbation also indicated the distinct regulatory mechanism of thermotolerance. Finally, a cochaperone Mdj1 and a metabolic enzyme Adh1 were selected to investigate their roles in mediating heat-stressed growth and ethanol production of yeasts. Our proteomic characterization of the industrial strain led to comprehensive understanding of the molecular basis of thermotolerance, which would facilitate future improvement in the industrially important trait of S. cerevisiae by rational engineering. PMID:25926660

  11. Genetic approaches for identifying kinetochore components in Saccharomyces cerevisiae

    SciTech Connect

    Doheny, K.F.; Puziss, J.; Spencer, F.; Hieter, P.

    1993-12-31

    A fundamental aspect of the cell division cycle is the chromosome cycle in which each of the chromosomal DNA molecules undergoes a series of morphological changes and complex movements to ensure faithful distribution at mitosis. The gene products responsible for execution of the chromosome cycle include structural components, such as those that assemble into the mitotic spindle apparatus, and regulatory components, such as those that coordinate the ordered series of events leading to chromosome segregation within the cell cycle. We have been taking several genetic approaches to identify genes encoding determinants critical to the chromosome cycle in the budding yeast, S. cerevisiae.

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

    PubMed

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

    2013-12-13

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

  13. Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface

    PubMed Central

    Bae, Jungu; Kuroda, Kouichi

    2014-01-01

    Proximity effect is a form of synergistic effect exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other), and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25-fold or 2.22-fold higher activity than MIX-yeast did at a yeast concentration equal to the yeast cell number in 1 ml of yeast suspension with an optical density (OD) at 600 nm of 10 (OD10) or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22-fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. A proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming yeast technology can also be applied to examine proximity effects in other diverse fields. PMID:25304511

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

    PubMed Central

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

    2012-01-01

    Genetic variation for plastic phenotypes potentially contributes phenotypic variation to populations that can be selected during adaptation to novel ecological contexts. However, the basis and extent of plastic variation that manifests in diverse environments remains elusive. Here, we characterize copper reaction norms for mRNA abundance among five Saccharomyces cerevisiae strains to 1) describe population variation across the full range of ecologically relevant copper concentrations, from starvation to toxicity, and 2) to test the hypothesis that plastic networks exhibit increased population variation for gene expression. We find that although the vast majority of the variation is small in magnitude (considerably <2-fold), not just some, but most genes demonstrate variable expression across environments, across genetic backgrounds, or both. Plastically expressed genes included both genes regulated directly by copper-binding transcription factors Mac1 and Ace1 and genes indirectly responding to the downstream metabolic consequences of the copper gradient, particularly genes involved in copper, iron, and sulfur homeostasis. Copper-regulated gene networks exhibited more similar behavior within the population in environments where those networks have a large impact on fitness. Nevertheless, expression variation in genes like Cup1, important to surviving copper stress, was linked with variation in mitotic fitness and in the breadth of differential expression across the genome. By revealing a broader and deeper range of population variation, our results provide further evidence for the interconnectedness of genome-wide mRNA levels, their dependence on environmental context and genetic background, and the abundance of variation in gene expression that can contribute to future evolution. PMID:23019066

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

    PubMed

    Ye, Mengqi; Yue, Tianli; Yuan, Yahong

    2014-09-01

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

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

    PubMed

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

    2011-08-01

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

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

    PubMed

    Albergaria, Helena; Arneborg, Nils

    2016-03-01

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

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    1985-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2003-01-01

    In Saccharomyces cerevisiae the activity for the lactate-proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a V (max) of 2.1 nmol x s(-1) x mg of dry weight(-1). Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest V (max) (0.84 nmol x s(-1) x mg of dry weight(-1)) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels. PMID:12962538

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

    PubMed

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

    2015-08-01

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

  4. Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive. Results In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH) and fumarase (RoFUM1) were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2) was up-regulated. The resultant yeast strain, FMME-001 ?PYC2 + ?RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1) than the control strain FMME-001 empty vector. Conclusions The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner. PMID:22335940

  5. Saccharomyces cerevisiae-based system for studying clustered DNA damages

    SciTech Connect

    Moscariello, M.M.; Sutherland, B.

    2010-08-01

    DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs) on the same or opposing DNA strands. In the latter, attempts to repair MDS can generate closely opposed single-strand break intermediates that may convert non-lethal or mutagenic base damage into double-strand breaks (DSBs). We constructed a diploid S. cerevisiae yeast strain with a chromosomal context targeted by integrative DNA fragments carrying different damages to determine whether closely opposed base damages are converted to DSBs following the outcomes of the homologous recombination repair pathway. As a model of MDS, we studied clustered uracil DNA damages with a known location and a defined distance separating the lesions. The system we describe might well be extended to assessing the repair of MDSs with different compositions, and to most of the complex DNA lesions induced by physical and chemical agents.

  6. Histone H1 of Saccharomyces cerevisiae Inhibits Transcriptional Silencing

    PubMed Central

    Veron, Marie; Zou, Yanfei; Yu, Qun; Bi, Xin; Selmi, Abdelkader; Gilson, Eric; Defossez, Pierre-Antoine

    2006-01-01

    Eukaryotic genomes contain euchromatic regions, which are transcriptionally active, and heterochromatic regions, which are repressed. These domains are separated by “barrier elements”: DNA sequences that protect euchromatic regions from encroachment by neighboring heterochromatin. To identify proteins that play a role in the function of barrier elements we have carried out a screen in S. cerevisiae. We recovered the gene HHO1, which encodes the yeast ortholog of histone H1, as a high-copy modifier of barrier activity. Histone H1 is a linker histone that binds the outside of nucleosomes and modifies chromatin dynamics. Here we show that Hho1p reinforces the action of several types of barrier elements, and also inhibits silencing on its own. PMID:16582449

  7. The effect of citric acid and pH on growth and metabolism of anaerobic Saccharomyces cerevisiae and Zygosaccharomyces bailii cultures.

    PubMed

    Nielsen, Marina Karelina; Arneborg, Nils

    2007-02-01

    The effects of citric acid at pH values of 3.0, 4.0, and 4.5 on growth and metabolism of anaerobic Saccharomyces cerevisiae and Zygosaccharomyces bailii cultures were investigated. S. cerevisiae and Z. bailii exhibited similar tolerances to citric acid, as determined by growth measurements, at all three pH values investigated. The citric-acid-induced growth inhibition of both yeast species increased with increasing pH values, indicating that the antimicrobial mechanism of citric acid differs from that of classical weak-acid preservatives. In S. cerevisiae, citric acid shifted the primary energy metabolism towards lower ethanol production and higher glycerol production, thus resulting in lower ATP production. These metabolic changes in S. cerevisiae were pH-dependent; i.e. the higher the pH, the lower the ATP production, and they may explain why growth of S. cerevisiae is more inhibited by citric acid at higher pH values. In Z. bailii, citric acid also caused an increased glycerol production, although to a lesser extent than in S. cerevisiae, but it caused virtually no changes in ethanol and ATP production. PMID:16943101

  8. Microencapsulation of Saccharomyces cerevisiae and its evaluation to protect in simulated gastric conditions

    PubMed Central

    Ghorbani-Choboghlo, Hassan; Zahraei-Salehi, Taghi; Ashrafi-Helan, Javad; Yahyaraeyat, Ramak; Pourjafar, Hadi; Nikaein, Donya; Balal, Asad; Khosravi, Ali-Reza

    2015-01-01

    Background and Objectives: Probiotic yeasts are used in production of functional foods and pharmaceutical products. They play an important role in promoting and maintaining human health. Until now, little work has been published on improving the survival of Saccharomyces in stimulated gastrointestinal condition. Material and Methods: In this study the exposure of the yeast in the capsulate and free forms to artificial gastrointestinal conditions was assessed and the number of viable Saccharomyces cerevisiae cells during 0 to 120 mines in these conditions was evaluated by a pour plate method using sabouraud dextrose agar. Results: Results showed the shape of the beads was generally spherical, sometimes elliptical with a mean diameter of about 50–90 μm. Also count of viable probiotic cells obtained for all the microcapsules were above the recommended levels for a probiotic food. Also decrease of approximately 4 logs was noted in the number of free cells after 2 h of incubation at pH 2 and 8, when compared to decreases of about 2 logs in the all microencapsulated S. cerevisiae under similar conditions. Conclusion: It is concluded that microencapsulation process was significantly able to increase the survival rate of Saccharomyces in a simulated gastrointestinal condition (p<0.05).. PMID:26885335

  9. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  10. Nitrogen and carbon assimilation by Saccharomyces cerevisiae during Sauvignon blanc juice fermentation.

    PubMed

    Pinu, Farhana R; Edwards, Patrick J B; Gardner, Richard C; Villas-Boas, Silas G

    2014-12-01

    To investigate the assimilation and production of juice metabolites by Saccharomyces cerevisiae during winemaking, we compared the metabolite profiles of 63 Sauvignon blanc (SB) grape juices collected over five harvesting seasons from different locations of New Zealand before and after fermentation by the commercial wine yeast strain EC1118 at 15 °C. Metabolite profiles were obtained using gas chromatography-mass spectrometry and nuclear magnetic resonance and the oenological parameters were determined by Fourier transform infrared spectroscopy. Our results revealed that the amino acids threonine and serine were the most consumed organic nitrogen sources, while proline and gamma-aminobutyric acid were the least consumed amino acids during SB juice fermentation. Saccharomyces cerevisiae metabolised some uncommon nitrogen sources (e.g. norleucine, norvaline and pyroglutamic acid) and several organic acids, including some fatty acids, most likely after fermenting the main juice sugars (glucose, fructose and mannose). However, consumption showed large variation between juices and in some cases between seasons. Our study clearly shows that preferred nitrogen and carbon sources were consumed by S. cerevisiae EC1118 independent of the juice fine composition, whilst the consumption of other nutrient sources mainly depended on the concentration of other juice metabolites, which explains the uniqueness of each barrel of wine. PMID:25345561

  11. High hydrostatic pressure activates transcription factors involved in Saccharomyces cerevisiae stress tolerance

    PubMed Central

    Bravim, Fernanda; da Silva, Lucas F.; Souza, Diego T.; Lippman, Soyeon I.; Broach, James R.; Fernandes, A. Alberto R.; Fernandes, Patricia M, B.

    2016-01-01

    A number of transcriptional control elements are activated when Saccharomyces cerevisiae cells are submitted to various stress conditions, including high hydrostatic pressure (HHP). Exposure of Saccharomyces cerevisiae cells to HHP results in global transcriptional reprogramming, similar to that observed under other industrial stresses, such as temperature, ethanol and oxidative stresses. Moreover, treatment with a mild hydrostatic pressure renders yeast cells multi-stress tolerant. In order to identify transcriptional factors involved in coordinating response to high hydrostatic pressure, we performed a time series microarray expression analysis on a wild S. cerevisiae strain exposed to 50 MPa for 30 min followed by recovery at atmospheric pressure (0.1 MPa) for 5, 10 and 15 min. We identified transcription factors and corresponding DNA and RNA motifs targeted in response to hydrostatic pressure. Moreover, we observed that different motif elements are present in the promoters of induced or repressed genes during HHP treatment. Overall, as we have already published, mild HHP treatment to wild yeast cells provides multiple protection mechanisms, and this study suggests that the TFs and motifs identified as responding to HHP may be informative for a wide range of other biotechnological and industrial applications, such as fermentation, that may utilize HHP treatment. PMID:23072392

  12. Candida albicans SSD1 can suppress multiple mutations in Saccharomyces cerevisiae.

    PubMed

    Chen, C Y; Rosamond, J

    1998-11-01

    The SSD1 gene of Saccharomyces encodes a 160 kDa cytoplasmic protein that can suppress mutations in a number of other genes. A functional homologue of SSD1 from the human pathogen Candida albicans was isolated on the basis of its ability to restore viability at the restrictive temperature in a Saccharomyces cerevisiae swi4 ssd1-d strain. The C. albicans gene, designated CaSSD1, encodes a 1262 aa protein which has 47% identity overall to S. cerevisiae SSD1 as well as significant identity to Schizosaccharomyces pombe dis3 and sts5 products. It is shown that CaSSD1 expression is constitutive through the mitotic cell cycle, which is consistent with a role for the protein in cell growth. CaSSD1 rescues the swi4ts defect in an ssd1-d background when expressed from its own promoter on a single-copy plasmid and under the same conditions can rescue mutations in genes encoding protein phosphatase type 2A catalytic subunits. These data suggest that CaSSD1, like its S. cerevisiae homologue, can limit the effect of mutations on a variety of cellular processes. PMID:9846729

  13. Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

    PubMed

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  14. PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae.

    PubMed

    Van Dyck, L; Pearce, D A; Sherman, F

    1994-01-01

    The PIM1 nuclear gene in the yeast Saccharomyces cerevisiae encodes a mitochondrial ATP-dependent protease that exhibits over 30% identity with ATP-dependent protease La from Escherichia coli, Lon from Bacillus brevis, and one from Myxococcus xanthus. In addition, Pim1 is 1133 amino acids long and has a putative mitochondrial import signal in the N-terminal region. Enzymatic comparisons of normal PIM1+ and deficient pim1-delta strains revealed that the ATP-dependent protease is located within the mitochondrial matrix. The pim1-delta strains are unable to utilize nonfermentable substrates as the sole carbon source and are unable to maintain functional mitochondrial DNA, indicating that the Pim1 protease is required for mitochondrial function. PIM1 mRNA is constitutively expressed but is increased after thermal stress, suggesting that Pim1 may play a role in the heat shock response. PMID:8276800

  15. Role of Saccharomyces cerevisiae ISA1 and ISA2 in Iron Homeostasis

    PubMed Central

    Jensen, Laran T.; Culotta, Valeria Cizewski

    2000-01-01

    The budding yeast Saccharomyces cerevisiae contains two homologues of bacterial IscA proteins, designated Isa1p and Isa2p. Bacterial IscA is a product of the isc (iron-sulfur cluster) operon and has been suggested to participate in Fe-S cluster formation or repair. To test the function of yeast Isa1p and Isa2p, single or combinatorial disruptions were introduced in ISA1 and ISA2. The resultant isa? mutants were viable but exhibited a dependency on lysine and glutamate for growth and a respiratory deficiency due to an accumulation of mutations in mitochondrial DNA. As with other yeast genes proposed to function in Fe-S cluster assembly, mitochondrial iron concentration was significantly elevated in the isa mutants, and the activities of the Fe-S cluster-containing enzymes aconitase and succinate dehydrogenase were dramatically reduced. An inspection of Isa-like proteins from bacteria to mammals revealed three invariant cysteine residues, which in the case of Isa1p and Isa2p are essential for function and may be involved in iron binding. As predicted, Isa1p is targeted to the mitochondrial matrix. However, Isa2p is present within the intermembrane space of the mitochondria. Our deletion analyses revealed that Isa2p harbors a bipartite N-terminal leader sequence containing a mitochondrial import signal linked to a second sequence that targets Isa2p to the intermembrane space. Both signals are needed for Isa2p function. A model for the nonredundant roles of Isa1p and Isa2p in delivering iron to sites of the Fe-S cluster assembly is discussed. PMID:10805735

  16. Functional Implications and Ubiquitin-Dependent Degradation of the Peptide Transporter Ptr2 in Saccharomyces cerevisiae

    PubMed Central

    Kawai, Ken; Moriya, Atsuto; Uemura, Satoshi

    2014-01-01

    The peptide transporter Ptr2 plays a central role in di- or tripeptide import in Saccharomyces cerevisiae. Although PTR2 transcription has been extensively analyzed in terms of upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this transporter is limited. Here we identified 14 amino acid residues required for peptide import through Ptr2 based on the crystallographic information of Streptococcus thermophilus peptide transporter PepTst and based on the conservation of primary sequences among the proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 mutations of which the corresponding residues of PepTst are involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enable ptr2?trp1 cell growth in alanyl-tryptophan (Ala-Trp) medium. We observed that Ptr2 underwent rapid degradation after cycloheximide treatment (half-life, approximately 1 h), and this degradation depended on Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, W362L, and E480D) exhibited severe defects in peptide import and were subjected to Rsp5-dependent degradation when cells were moved to Ala-Trp medium, whereas, similar to what occurs in the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results suggest that Ptr2 undergoes quality control and the defects in peptide binding and the concomitant conformational change render Ptr2 subject to efficient ubiquitination and subsequent degradation. PMID:25172766

  17. The Cdc34/SCF ubiquitination complex mediates Saccharomyces cerevisiae cell wall integrity.

    PubMed

    Varelas, Xaralabos; Stuart, David; Ellison, Michael J; Ptak, Christopher

    2006-12-01

    To identify novel functions for the Cdc34/SCF ubiquitination complex, we analyzed genomewide transcriptional profiles of cdc53-1 and cdc34-2 Saccharomyces cerevisiae mutants. This analysis revealed altered expression for several gene families, including genes involved in the regulation of cell wall organization and biosynthesis. This led us to uncover a role for the Cdc34/SCF complex in the regulation of cell wall integrity. In support of this, cdc53-1 and cdc34-2 mutants exhibit phenotypes characteristic of cell wall integrity mutants, such as SDS sensitivity and temperature-sensitive suppression by osmotic stabilizers. Examination of these mutants revealed defects in their induction of Slt2 phosphorylation, indicating defects in Pkc1-Slt2 MAPK signaling. Consistent with this, synthetic genetic interactions were observed between the genes encoding the Cdc34/SCF complex and key components of the Pck1-Slt2 MAPK pathway. Further analysis revealed that Cdc34/SCF mutants have reduced levels of active Rho1, suggesting that these defects stem from the deregulated activity of the Rho1 GTPase. Altering the activity of Rho1 via manipulation of the Rho1-GAPs LRG1 or SAC7 affected Cdc34/SCF mutant growth. Strikingly, however, deletion of LRG1 rescued the growth defects associated with Cdc34/SCF mutants, whereas deletion of SAC7 enhanced these defects. Given the differential roles that these GAPs play in the regulation of Rho1, these observations indicate the importance of coordinating Cdc34/SCF activity with specific Rho1 functions. PMID:17028344

  18. Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae.

    PubMed

    Deng, Xu; Petitjean, Marjorie; Teste, Marie-Ange; Kooli, Wafa; Tranier, Samuel; François, Jean Marie; Parrou, Jean-Luc

    2014-01-01

    The yeast Saccharomyces cerevisiae IMA multigene family encodes four isomaltases sharing high sequence identity from 65% to 99%. Here, we explore their functional diversity, with exhaustive in-vitro characterization of their enzymological and biochemical properties. The four isoenzymes exhibited a preference for the ?-(1,6) disaccharides isomaltose and palatinose, with Michaëlis-Menten kinetics and inhibition at high substrates concentration. They were also able to hydrolyze trisaccharides bearing an ?-(1,6) linkage, but also ?-(1,2), ?-(1,3) and ?-(1,5) disaccharides including sucrose, highlighting their substrate ambiguity. While Ima1p and Ima2p presented almost identical characteristics, our results nevertheless showed many singularities within this protein family. In particular, Ima3p presented lower activities and thermostability than Ima2p despite only three different amino acids between the sequences of these two isoforms. The Ima3p_R279Q variant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240 significantly increased the stability of Ima3p and supported the role of prolines in thermostability. The most distant protein, Ima5p, presented the lowest optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specific amino acids for determining the specificity for ?-(1,6) substrates. We finally found a mixed inhibition by maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitive at very low isomaltose concentrations and uncompetitive as the substrate concentration increased. Altogether, this work illustrates that a gene family encoding proteins with strong sequence similarities can lead to enzyme with notable differences in biochemical and enzymological properties. PMID:24649402

  19. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

    PubMed Central

    1996-01-01

    M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity. PMID:8567717

  20. Acid Black 48 dye biosorption using Saccharomyces cerevisiae immobilized with treated sugarcane bagasse.

    PubMed

    Mitter, E K; Corso, C R

    2012-01-01

    The textile industry consumes large quantities of water and chemicals, especially in dyeing and finishing processes. Textile dye adsorption can be accomplished with natural or synthetic compounds. Cell immobilization using biomaterials allows the reduction of toxicity and mechanical resistance and opens spaces within the matrix for cell growth. The use of natural materials, such as sugarcane bagasse, is promising due to the low costs involved. The aim of the present study was to evaluate the use of sugarcane bagasse treated with either polyethyleneimine (PEI), NaOH or distilled water in the cell immobilization of Saccharomyces cerevisiae for textile dye removal. Three different adsorption tests were conducted: treated sugarcane bagasse alone, free yeast cells and bagasse-immobilized yeast cells. Yeast immobilization was 31.34% with PEI-treated bagasse, 8.56% with distilled water and 22.54% with NaOH. PEI-treated bagasse exhibited the best removal rates of the dye at all pH values studied (2.50, 4.50 and 6.50). The best Acid Black 48 adsorption rates were obtained with use of free yeast cells. At pH 2.50, 1 mg of free yeast cells was able to remove 5488.49 g of the dye. The lowest adsorption capacity rates were obtained using treated bagasse alone. However, the use of bagasse-immobilized cells increased adsorption efficiency from 20 to 40%. The use of immobilized cells in textile dye removal is very attractive due to adsorbed dye precipitation, which eliminates the industrial need for centrifugation processes. Dye adsorption using only yeast cells or sugarcane bagasse requires separation methods. PMID:22864427

  1. Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production.

    PubMed

    Argueso, Juan Lucas; Carazzolle, Marcelo F; Mieczkowski, Piotr A; Duarte, Fabiana M; Netto, Osmar V C; Missawa, Silvia K; Galzerani, Felipe; Costa, Gustavo G L; Vidal, Ramon O; Noronha, Melline F; Dominska, Margaret; Andrietta, Maria G S; Andrietta, Sílvio R; Cunha, Anderson F; Gomes, Luiz H; Tavares, Flavio C A; Alcarde, André R; Dietrich, Fred S; McCusker, John H; Petes, Thomas D; Pereira, Gonçalo A G

    2009-12-01

    Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (approximately 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies. PMID:19812109

  2. Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae.

    PubMed Central

    Araki, H; Hamatake, R K; Morrison, A; Johnson, A L; Johnston, L H; Sugino, A

    1991-01-01

    DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptides with apparent molecular masses of greater than 200, 80, 34, 30 and 29 kDa, the two largest of which (subunits A and B) are encoded by the essential genes POL2 and DPB2. By probing a lambda gt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C'). The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product. The predicted amino acid sequence contained a possible NTP-binding motif and a glutamate-rich region. NTP-binding motif and a glutamate-rich region. A dpb3 deletion mutant (dpb3 delta) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides. dpb3 delta strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication. Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C') and D. The 5' nontranscribed region of DPB3 contained the MulI-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary. The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes. DPB3 was mapped to chromosome II, 30 cM distal to his7. Images PMID:1923754

  3. Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase.

    PubMed

    Tani, Motohiro; Toume, Moeko

    2015-12-01

    In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group and five types of ceramide; however, the physiological significance of the structural diversity is not fully understood. Here, we report that deletion of vacuolar H+-ATPase (V-ATPase) in yeast causes dramatic alteration of the complex sphingolipid composition, which includes decreases in hydroxylation at the C-4 position of long-chain bases and the C-2 position of fatty acids in the ceramide moiety, decreases in inositol phosphorylceramide (IPC) levels, and increases in mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)2C] levels. V-ATPase-deleted cells exhibited slow growth at pH 7.2, whereas the increase in MIPC levels was significantly enhanced when V-ATPase-deleted cells were incubated at pH 7.2. The protein expression levels of MIPC and M(IP)2C synthases were significantly increased in V-ATPase-deleted cells incubated at pH 7.2. Loss of MIPC synthesis or an increase in the hydroxylation level of the ceramide moiety of sphingolipids on overexpression of Scs7 and Sur2 sphingolipid hydroxylases enhanced the growth defect of V-ATPase-deleted cells at pH 7.2. On the contrary, the growth rate of V-ATPase-deleted cells was moderately increased on the deletion of SCS7 and SUR2. In addition, supersensitivities to Ca2+, Zn2+ and H2O2, which are typical phenotypes of V-ATPase-deleted cells, were enhanced by the loss of MIPC synthesis. These results indicate the possibility that alteration of the complex sphingolipid composition is an adaptation mechanism for a defect of V-ATPase. PMID:26404656

  4. PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae.

    PubMed

    Baccarini, Leticia; Martínez-Montañés, Fernando; Rossi, Silvia; Proft, Markus; Portela, Paula

    2015-11-01

    Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B, and RPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in catalytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different β-karyopherin pathway. Moreover, β-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic subunits is required for its association to target genes and properly gene expression. PMID:26403272

  5. Pheromones and Pheromone Receptors Are the Primary Determinants of Mating Specificity in the Yeast Saccharomyces Cerevisiae

    PubMed Central

    Bender, A.; Sprague-Jr, G. F.

    1989-01-01

    Saccharomyces cerevisiae has two haploid cell types, a and ?, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize ?-factor pheromone and a-factor receptor is sufficient to allow mating by mat?1 mutants, mutants that normally do not express any ?- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and ?-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and ?-factor pheromone constitute the minimum set of ?-specific proteins that must be produced to allow mating as an ? cell. Furthermore, the production of these two proteins dictates the mating specificity exhibited by that cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat?2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat?2 background. These constructions reveal that mat?2 mutants can be made to mate as either a cells or as ? cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat?2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to ? factor by the a-factor receptor. PMID:2653961

  6. Adjustment of trehalose metabolism in wine Saccharomyces cerevisiae strains to modify ethanol yields.

    PubMed

    Rossouw, D; Heyns, E H; Setati, M E; Bosch, S; Bauer, F F

    2013-09-01

    The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1? and tdh3?), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines. PMID:23793638

  7. Enhanced hexose fermentation by Saccharomyces cerevisiae through integration of stoichiometric modeling and genetic screening.

    PubMed

    Quarterman, Josh; Kim, Soo Rin; Kim, Pan-Jun; Jin, Yong-Su

    2015-01-20

    In order to determine beneficial gene deletions for ethanol production by the yeast Saccharomyces cerevisiae, we performed an in silico gene deletion experiment based on a genome-scale metabolic model. Genes coding for two oxidative phosphorylation reactions (cytochrome c oxidase and ubiquinol cytochrome c reductase) were identified by the model-based simulation as potential deletion targets for enhancing ethanol production and maintaining acceptable overall growth rate in oxygen-limited conditions. Since the two target enzymes are composed of multiple subunits, we conducted a genetic screening study to evaluate the in silico results and compare the effect of deleting various portions of the respiratory enzyme complexes. Over two-thirds of the knockout mutants identified by the in silico study did exhibit experimental behavior in qualitative agreement with model predictions, but the exceptions illustrate the limitation of using a purely stoichiometric model-based approach. Furthermore, there was a substantial quantitative variation in phenotype among the various respiration-deficient mutants that were screened in this study, and three genes encoding respiratory enzyme subunits were identified as the best knockout targets for improving hexose fermentation in microaerobic conditions. Specifically, deletion of either COX9 or QCR9 resulted in higher ethanol production rates than the parental strain by 37% and 27%, respectively, with slight growth disadvantages. Also, deletion of QCR6 led to improved ethanol production rate by 24% with no growth disadvantage. The beneficial effects of these gene deletions were consistently demonstrated in different strain backgrounds and with four common hexoses. The combination of stoichiometric modeling and genetic screening using a systematic knockout collection was useful for narrowing a large set of gene targets and identifying targets of interest. PMID:25435378

  8. Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37.

    PubMed

    Li, Yun-Cheng; Mitsumasu, Kanako; Gou, Zi-Xi; Gou, Min; Tang, Yue-Qin; Li, Guo-Ying; Wu, Xiao-Lei; Akamatsu, Takashi; Taguchi, Hisataka; Kida, Kenji

    2016-02-01

    Industrial yeast strains with good xylose fermentation ability and inhibitor tolerance are important for economical lignocellulosic bioethanol production. The flocculating industrial Saccharomyces cerevisiae strain NAPX37, harboring the xylose reductase-xylitol dehydrogenase (XR-XDH)-based xylose metabolic pathway, displayed efficient xylose fermentation during batch and continuous fermentation. During batch fermentation, the xylose consumption rates at the first 36 h were similar (1.37 g/L/h) when the initial xylose concentrations were 50 and 75 g/L, indicating that xylose fermentation was not inhibited even when the xylose concentration was as high as 75 g/L. The presence of glucose, at concentrations of up to 25 g/L, did not affect xylose consumption rate at the first 36 h. Strain NAPX37 showed stable xylose fermentation capacity during continuous ethanol fermentation using xylose as the sole sugar, for almost 1 year. Fermentation remained stable at a dilution rate of 0.05/h, even though the xylose concentration in the feed was as high as 100 g/L. Aeration rate, xylose concentration, and MgSO4 concentration were found to affect xylose consumption and ethanol yield. When the xylose concentration in the feed was 75 g/L, a high xylose consumption rate of 6.62 g/L/h and an ethanol yield of 0.394 were achieved under an aeration rate of 0.1 vvm, dilution rate of 0.1/h, and 5 mM MgSO4. In addition, strain NAPX37 exhibited good tolerance to inhibitors such as weak acids, furans, and phenolics during xylose fermentation. These findings indicate that strain NAPX37 is a promising candidate for application in the industrial production of lignocellulosic bioethanol. PMID:26603762

  9. Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

    PubMed Central

    Rossouw, D.; Heyns, E. H.; Setati, M. E.; Bosch, S.

    2013-01-01

    The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1? and tdh3?), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines. PMID:23793638

  10. Determination of ethanol in whey-sugar solutions by freezing. [Kluyveromyces fragilis and Saccharomyces cerevisiae

    SciTech Connect

    Demott, B.J.

    1982-01-01

    The composition of solutions undergoing yeast fermentation was stimulated by using direct-acid-set cottage cheese whey containing increasing amounts of EtOH (0 to 5.4%) with decreasing amounts of sucrose (10 to 0%). Each decrease of 0.54 g EtOH decreased specific gravity by 0.0046 unit and lowered the freezing point by 0.159 H (the Hortvet unit). Whey containing 10% added sucrose was treated as follows: (a) inoculated with Kluyveromyces fragilis, (b) carbohydrate splitting enzymes added and inoculated with Kluyveromyces fragilis and (c) carbohydrate splitting enzymes added and inoculated with Saccharomyces cerevisiae. All mixtures were incubated 48 h at 32 degrees during which 6 samples from each treatment were analyzed for total solids, specific gravity and freezing point. No difference was noted between samples treated with enzymes or those treated with the 2 yeasts cultured as related to decrease in total solids concentration or specific gravity. Each 0.001 H decrease in freezing point was accompanied by a total solids decrease of 0.006 g/100 g whey in the nonenzyme treated sample, and 0.008 g and 0.010 g/100 g whey in the enzyme-treated samples inoculated with Kluyveromyces fragilis and Saccharomyces cerevisiae, respectively. Each 0.001 H change in freezing point was equivalent to a change of 0.00003 specific gravity unit in the nonenzyme-treated sample and 0.000043 and 0.000048 specific gravity unit in the enzyme-treated samples inoculated with Kluyveromyces fragilis and Saccharomyces cerevisiae, respectively. The precision with which freezing point can be determined suggests its use in evaluating the amount of EtOH produced during fermentation.

  11. Heritable capture of heterochromatin dynamics in Saccharomyces cerevisiae

    PubMed Central

    Dodson, Anne E; Rine, Jasper

    2015-01-01

    Heterochromatin exerts a heritable form of eukaryotic gene repression and contributes to chromosome segregation fidelity and genome stability. However, to date there has been no quantitative evaluation of the stability of heterochromatic gene repression. We designed a genetic strategy to capture transient losses of gene silencing in Saccharomyces as permanent, heritable changes in genotype and phenotype. This approach revealed rare transcription within heterochromatin that occurred in approximately 1/1000 cell divisions. In concordance with multiple lines of evidence suggesting these events were rare and transient, single-molecule RNA FISH showed that transcription was limited. The ability to monitor fluctuations in heterochromatic repression uncovered previously unappreciated roles for Sir1, a silencing establishment factor, in the maintenance and/or inheritance of silencing. In addition, we identified the sirtuin Hst3 and its histone target as contributors to the stability of the silenced state. These approaches revealed dynamics of a heterochromatin function that have been heretofore inaccessible. DOI: http://dx.doi.org/10.7554/eLife.05007.001 PMID:25581000

  12. D-xylulose fermentation to ethanol by Saccharomyces cerevisiae

    SciTech Connect

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

    1981-01-01

    Commercial bakers' yeast (S. 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, and the optimal pH range was 4-6. 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 by 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 greater than 4%. The slow conversion rate of C-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.

  13. Measurement of inorganic pyrophosphate levels in Saccharomyces cerevisiae

    SciTech Connect

    Ahmad, N.; Cooperman, B.S.

    1987-05-01

    Inorganic pyrophosphate (PPi) in microorganisms has been shown to reach quite high levels, with profound implications for many aspects of cellular metabolism. They have modified the method of Heinonen et al., developed for studies on E. coli and based on selective precipitation of PPi by added Ca/sup 2 +/ and F/sup -/, to measure PPi levels in S. cerevisiae. Cells were lysed in acidic medium containing known amounts of added /sup 32/PPi by vortexing with glass beads. Yields of isolated PPi were calculated on the basis of recovered radioactivity. Measurement of the protein content of the lysate allowed a correction to be made for variability in the efficiency of cell lysis. The results show a remarkable variation in PPi levels along the growth curve. For cells grown with aeration on 2% glucose, the peak value, corresponding to an internal concentration of at least 5mM, is attained in mid- to late-log phase. Concentrations of PPi in early-log phase are at least 10-fold lower.

  14. Saccharomyces cerevisiae GTPase complex: Gtr1p-Gtr2p regulates cell-proliferation through Saccharomyces cerevisiae Ran-binding protein, Yrb2p

    SciTech Connect

    Wang Yonggang; Nakashima, Nobutaka; Sekiguchi, Takeshi . E-mail: sekigu@molbiol.med.kyushu-u.ac.jp; Nishimoto, Takeharu

    2005-10-21

    A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2{delta} gtr1{delta} gtr2{delta} was lethal, while a double mutant: gtr1{delta} gtr2{delta} survived well, indicating that Yrb2p protected cells from the killing effect of gtr1{delta} gtr2{delta}. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtr1p-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtr1p-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p.

  15. Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae

    PubMed Central

    Pâques, Frédéric; Haber, James E.

    1999-01-01

    The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination. PMID:10357855

  16. Metabolic engineering of Saccharomyces cerevisiae ethanol strains PE-2 and CAT-1 for efficient lignocellulosic fermentation.

    PubMed

    Romaní, Aloia; Pereira, Filipa; Johansson, Björn; Domingues, Lucília

    2015-03-01

    In this work, Saccharomyces cerevisiae strains PE-2 and CAT-1, commonly used in the Brazilian fuel ethanol industry, were engineered for xylose fermentation, where the first fermented xylose faster than the latter, but also produced considerable amounts of xylitol. An engineered PE-2 strain (MEC1121) efficiently consumed xylose in presence of inhibitors both in synthetic and corn-cob hydrolysates. Interestingly, the S. cerevisiae MEC1121 consumed xylose and glucose simultaneously, while a CEN.PK based strain consumed glucose and xylose sequentially. Deletion of the aldose reductase GRE3 lowered xylitol production to undetectable levels and increased xylose consumption rate which led to higher final ethanol concentrations. Fermentation of corn-cob hydrolysate using this strain, MEC1133, resulted in an ethanol yield of 0.47 g/g of total sugars which is 92% of the theoretical yield. PMID:25536512

  17. Production of Volatile and Sulfur Compounds by 10 Saccharomyces cerevisiae Strains Inoculated in Trebbiano Must.

    PubMed

    Patrignani, Francesca; Chinnici, Fabio; Serrazanetti, Diana I; Vernocchi, Pamela; Ndagijimana, Maurice; Riponi, Claudio; Lanciotti, Rosalba

    2016-01-01

    In wines, the presence of sulfur compounds is the resulting of several contributions among which yeast metabolism. The characterization of the starter Saccharomyces cerevisiae needs to be performed also taking into account this ability even if evaluated together with the overall metabolic profile. In this perspective, principal aim of this experimental research was the evaluation of the volatile profiles, throughout GC/MS technique coupled with solid phase micro extraction, of wines obtained throughout the fermentation of 10 strains of S. cerevisiae. In addition, the production of sulfur compounds was further evaluated by using a gas-chromatograph coupled with a Flame Photometric Detector. Specifically, the 10 strains were inoculated in Trebbiano musts and the fermentations were monitored for 19 days. In the produced wines, volatile and sulfur compounds as well as amino acid concentrations were investigated. Also the physico-chemical characteristics of the wines and their electronic nose profiles were evaluated. PMID:26973621

  18. A Novel Saccharomyces cerevisiae FG Nucleoporin Mutant Collection for Use in Nuclear Pore Complex Functional Experiments

    PubMed Central

    Adams, Rebecca L.; Terry, Laura J.; Wente, Susan R.

    2015-01-01

    FG nucleoporins (Nups) are the class of proteins that both generate the permeability barrier and mediate selective transport through the nuclear pore complex (NPC). The FG Nup family has 11 members in Saccharomyces cerevisiae, and the study of mutants lacking different FG domains has been instrumental in testing transport models. To continue analyzing the distinct functional roles of FG Nups in vivo, additional robust genetic tools are required. Here, we describe a novel collection of S. cerevisiae mutant strains in which the FG domains of different groups of Nups are absent (?) in the greatest number documented to date. Using this plasmid-based ?FG strategy, we find that a GLFG domain-only pore is sufficient for viability. The resulting extensive plasmid and strain resources are available to the scientific community for future in-depth in vivo studies of NPC transport. PMID:26530420

  19. Budding Yeast for Budding Geneticists: A Primer on the Saccharomyces cerevisiae Model System

    PubMed Central

    Duina, Andrea A.; Miller, Mary E.; Keeney, Jill B.

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans. PMID:24807111

  20. Genomic exploration of the hemiascomycetous yeasts: 20. Evolution of gene redundancy compared to Saccharomyces cerevisiae.

    PubMed

    Llorente, B; Durrens, P; Malpertuy, A; Aigle, M; Artiguenave, F; Blandin, G; Bolotin-Fukuhara, M; Bon, E; Brottier, P; Casaregola, S; Dujon, B; de Montigny, J; Lépingle, A; Neuvéglise, C; Ozier-Kalogeropoulos, O; Potier, S; Saurin, W; Tekaia, F; Toffano-Nioche, C; Wésolowski-Louvel, M; Wincker, P; Weissenbach, J; Souciet, J; Gaillardin, C

    2000-12-22

    We have evaluated the degree of gene redundancy in the nuclear genomes of 13 hemiascomycetous yeast species. Saccharomyces cerevisiae singletons and gene families appear generally conserved in these species as singletons and families of similar size, respectively. Variations of the number of homologues with respect to that expected affect from 7 to less than 24% of each genome. Since S. cerevisiae homologues represent the majority of the genes identified in the genomes studied, the overall degree of gene redundancy seems conserved across all species. This is best explained by a dynamic equilibrium resulting from numerous events of gene duplication and deletion rather than by a massive duplication event occurring in some lineages and not in others. PMID:11152895

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

    PubMed

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

    2014-09-01

    A β-glucosidase producing yeast strain was isolated from Korean traditional rice wine. Based on the sequence of the YCL008c gene and analysis of the fatty acid composition, the isolate was identified as Saccharomyces cerevisiae strain HJ-014. S. cerevisiae HJ-014 produced ginsenoside Rd, F2, and compound K from the ethanol extract of red ginseng. The production was increased by shaking culture, where the bioconversion efficiency was increased 2-fold compared to standing culture. The production of ginsenoside F2 and compound K was time-dependent and thought to proceed by the transformation pathway of: red ginseng extract→Rd→F2→compound K. The optimum incubation time and concentration of red ginseng extract for the production of compound K was 96 hr and 4.5% (w/v), respectively. PMID:25346602

  2. Improvement of Phytase Activity by a New Saccharomyces cerevisiae Strain Using Statistical Optimization

    PubMed Central

    Ries, Edi Franciele; Alves Macedo, Gabriela

    2011-01-01

    Using statistical optimization, we enhanced the activity of phytase by a new Saccharomyces cerevisiae strain cultured in mineral medium. Concentrations of carbon source and inducer of phytase production were optimized using a 22 full factorial CCD and response surface methodology (RSM). Urea was fixed as nitrogen source in culture medium (0.15%, w/v). The culture medium consisting of 2.5% sucrose and 0.5% sodium phytate optimally supported the maximum phytase activity. In addition, we found that culture of the yeast at 35°C with shaking at 150 rpm supports maximum phytase production. The validity of this model was verified by culturing the organisms in flasks on a shaker. Using the optimized media and growth conditions, we obtained a 10-fold improvement in the production of phytase by S. cerevisiae. PMID:21837273

  3. Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae

    SciTech Connect

    Barry, K.; Stiles, J.I.; Pietras, D.F.; Melnick, L.; Sherman, F.

    1987-02-01

    Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae. The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures. The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively. There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene. The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S. cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes.

  4. Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae.

    PubMed

    Barry, K; Stiles, J I; Pietras, D F; Melnick, L; Sherman, F

    1987-02-01

    Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae. The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures. The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively. There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene. The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S. cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes. PMID:3029562

  5. Genome-wide construction of a series of designed segmental aneuploids in Saccharomyces cerevisiae

    PubMed Central

    Natesuntorn, Waranya; Iwami, Kotaro; Matsubara, Yuki; Sasano, Yu; Sugiyama, Minetaka; Kaneko, Yoshinobu; Harashima, Satoshi

    2015-01-01

    Segmental aneuploidy can play an important role in environmental adaptation. However, study of segmental aneuploids is severely hampered by the difficulty of creating them in a designed fashion. Here, we describe a PCR-mediated chromosome duplication (PCDup) technology that enables the generation of segmental aneuploidy at any desired chromosomal region in Saccharomyces cerevisiae. We constructed multiple strains harboring 100 kb to 200 kb segmental duplications covering the whole of the S. cerevisiae genome. Interestingly, some segmental aneuploidies confer stress tolerance, such as to high temperature, ethanol and strong acids, while others induce cell lethality and stress sensitivity, presumably as result of the simultaneous increases in dosages of multiple genes. We suggest that our PCDup technology will accelerate studies into the phenotypic changes resulting from alteration of gene dosage balance of multiple genes and will provide new insights into the adaptive molecular mechanisms in the genome in segmental aneuploidy-derived human diseases. PMID:26224198

  6. Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans.

    PubMed

    Hollis, R P; Lagido, C; Pettitt, J; Porter, A J; Killham, K; Paton, G I; Glover, L A

    2001-10-01

    This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n-decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms. PMID:11591388

  7. Production of Volatile and Sulfur Compounds by 10 Saccharomyces cerevisiae Strains Inoculated in Trebbiano Must

    PubMed Central

    Patrignani, Francesca; Chinnici, Fabio; Serrazanetti, Diana I.; Vernocchi, Pamela; Ndagijimana, Maurice; Riponi, Claudio; Lanciotti, Rosalba

    2016-01-01

    In wines, the presence of sulfur compounds is the resulting of several contributions among which yeast metabolism. The characterization of the starter Saccharomyces cerevisiae needs to be performed also taking into account this ability even if evaluated together with the overall metabolic profile. In this perspective, principal aim of this experimental research was the evaluation of the volatile profiles, throughout GC/MS technique coupled with solid phase micro extraction, of wines obtained throughout the fermentation of 10 strains of S. cerevisiae. In addition, the production of sulfur compounds was further evaluated by using a gas-chromatograph coupled with a Flame Photometric Detector. Specifically, the 10 strains were inoculated in Trebbiano musts and the fermentations were monitored for 19 days. In the produced wines, volatile and sulfur compounds as well as amino acid concentrations were investigated. Also the physico-chemical characteristics of the wines and their electronic nose profiles were evaluated. PMID:26973621

  8. Purification, crystallization and preliminary X-ray analysis of Hsp33 from Saccharomyces cerevisiae

    SciTech Connect

    Liu, Wei; Zhou, Ye-Yun; Teng, Mai-Kun; Zhou, Cong-Zhao

    2007-02-01

    Heat-shock protein Hsp33 from S. cerevisiae was crystallized and diffraction data were collected to 2.7 Å resolution. The heat-shock protein Hsp33 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. A crystal was obtained using the hanging-drop vapour-diffusion method and a data set was collected to 2.7 Å resolution. The crystal belongs to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 96.43, c = 132.22 Å, ? = ? = ? = 90°. The asymmetric unit is assumed to contain two subunits of Hsp33, with a V{sub M} value of 2.96 Å{sup 3} Da{sup ?1} and a solvent content of 58.41%.

  9. Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters.

    TOXLINE Toxicology Bibliographic Information

    Ha SJ; Galazka JM; Joong Oh E; Kordić V; Kim H; Jin YS; Cate JH

    2013-01-01

    Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular ?-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (?G°=+3.6kJmol(-1)). A thermodynamic "push" from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae.

  10. Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters.

    PubMed

    Ha, Suk-Jin; Galazka, Jonathan M; Joong Oh, Eun; Kordi?, Vesna; Kim, Heejin; Jin, Yong-Su; Cate, Jamie H D

    2013-01-01

    Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular ?-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (?G°=+3.6kJmol(-1)). A thermodynamic "push" from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae. PMID:23178501

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

    PubMed Central

    Choi, Hak Joo; Kim, Eun A; Kim, Dong Hee

    2014-01-01

    A ?-glucosidase producing yeast strain was isolated from Korean traditional rice wine. Based on the sequence of the YCL008c gene and analysis of the fatty acid composition, the isolate was identified as Saccharomyces cerevisiae strain HJ-014. S. cerevisiae HJ-014 produced ginsenoside Rd, F2, and compound K from the ethanol extract of red ginseng. The production was increased by shaking culture, where the bioconversion efficiency was increased 2-fold compared to standing culture. The production of ginsenoside F2 and compound K was time-dependent and thought to proceed by the transformation pathway of: red ginseng extract?Rd?F2?compound K. The optimum incubation time and concentration of red ginseng extract for the production of compound K was 96 hr and 4.5% (w/v), respectively. PMID:25346602

  12. Effect of acetaldehyde on Saccharomyces cerevisiae and Zymomonas mobilis subjected to environmental shocks

    SciTech Connect

    Stanley, G.A.; Hobley, T.J.; Pamment, N.B.

    1997-01-05

    The lag phase of Saccharomyces cerevisiae subjected to a step increase in temperature or ethanol concentration was reduced by as much as 60% when acetaldehyde was added to the medium at concentrations less than 0.1 g/L. Maximum specific growth rates were also substantially increased. Even greater proportional reductions in lag time due to acetaldehyde addition were observed for ethanol-shocked cultures of Zymomonas mobilis. Acetaldehyde had no effect on S. cerevisiae cultures started from stationary phase inocula in the absence of environmental shock and its lag-reducing effects were greater in complex medium than in a defined synthetic medium. Acetaldehyde reacted strongly with the ingredients of complex culture media. It is proposed that the effect of added acetaldehyde may be to compensate for the inability of cells to maintain transmembrane acetaldehyde gradients following an environmental shock.

  13. Crystallization and preliminary X-ray analysis of CTP:phosphoethanolamine cytidylyltransferase (ECT) from Saccharomyces cerevisiae

    SciTech Connect

    Ohtsuka, Jun; Nagata, Koji; Lee, Woo Cheol; Ono, Yusuke; Fukuda, Ryouichi; Ohta, Akinori; Tanokura, Masaru

    2006-10-01

    CTP:phosphoethanolamine cytidylyltransferase from S. cerevisiae has been expressed, purified and crystallized. CTP:phosphoethanolamine cytidylyltransferase (ECT) is the enzyme that catalyzes the conversion of phosphoethanolamine to CDP-ethanolamine in the phosphatidylethanolamine-biosynthetic pathway (Kennedy pathway). ECT from Saccharomyces cerevisiae was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 as precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.88 Å resolution. The space group was assigned as primitive tetragonal, P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 66.3, c = 150.8 Å. The crystals contain one ECT molecule in the asymmetric unit (V{sub M} = 2.2 Å{sup 3} Da{sup ?1}), with a solvent content of 43%.

  14. Assembly of bacteriophage Qbeta virus-like particles in yeast Saccharomyces cerevisiae and Pichia pastoris.

    PubMed

    Freivalds, Janis; Dislers, Andris; Ose, Velta; Skrastina, Dace; Cielens, Indulis; Pumpens, Paul; Sasnauskas, Kestutis; Kazaks, Andris

    2006-05-29

    Recombinant bacteriophage Qbeta coat protein (CP), which has been proposed as a promising carrier of foreign epitopes via their incorporation either by gene engineering techniques or by chemical coupling, efficiently self-assembles into virus-like particles (VLPs) when expressed in Escherichia coli. Here, we demonstrate expression and self-assembly of Qbeta CP in yeast Saccharomyces cerevisiae and Pichia pastoris. Production reached 3-4 mg/1g of wet cells for S. cerevisiae and 4-6 mg for P. pastoris, which was about 15-20% and 20-30% of the E. coli expression level, respectively. Qbeta VLPs were easily purified by size-exclusion chromatography in both cases and contained nucleic acid, shown by native agarose gel electrophoresis. The obtained particles were highly immunogenic in mice and the resulting sera recognized both E. coli- and yeast-derived Qbeta VLPs equally well. PMID:16406160

  15. Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Klein, H. P.; Jahnke, L.

    1979-01-01

    Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

  16. Crystal structure of Cdc11, a septin subunit from Saccharomyces cerevisiae.

    PubMed

    Brausemann, Anton; Gerhardt, Stefan; Schott, Anne-Kathrin; Einsle, Oliver; Große-Berkenbusch, Andreas; Johnsson, Nils; Gronemeyer, Thomas

    2016-03-01

    Septins are a conserved family of GTP-binding proteins that assemble into a highly ordered array of filaments at the mother bud neck in Saccharomyces cerevisiae cells. Many molecular functions and mechanisms of the septins in S. cerevisiae were already uncovered. However, structural information is only available from modeling the crystallized subunits of the human septins into the EM cryomicroscopy data of the yeast hetero-octameric septin rod. Octameric rods are the building block of septin filaments in yeast. We present here the first crystal structure of Cdc11, the terminal subunit of the octameric rod and discuss its structure in relation to its human homologues. Size exclusion chromatography analysis revealed that Cdc11 forms homodimers through its C-terminal coiled coil tail. PMID:26780475

  17. Lysine inhibition of Saccharomyces cerevisiae: role of repressible L-lysine ?-aminotransferase.

    PubMed

    Thomas, K C; Ingledew, W M

    1994-09-01

    Lysine added to grain mashes under nitrogen-limiting conditions (as in most industrial fermentations) inhibited growth of Saccharomyces cerevisiae. This inhibition was relieved by raising the assimilable nitrogen content. Lysine-induced inhibition is not mediated through accumulation of ?-oxoadipic acid, an intermediate of lysine metabolism which accumulates by a back up of intermediates in de novo synthesis. Lysine degradation is regulated by the synthesis of L-lysine ?-aminotransferase, an enzyme that catalyses the first step in one of three possible routes of lysine degradation (not previously reported in S. cerevisiae). Synthesis is repressed under nitrogenlimiting conditions, but derepressed when excess assimilable nitrogen is available. Derepression results in degradation of lysine and decreases inhibitory effects on growth. The toxic compound appears to be lysine itself. PMID:24421137

  18. Molecular mechanisms of Saccharomyces cerevisiae stress adaptation and programmed cell death in response to acetic acid

    PubMed Central

    Giannattasio, Sergio; Guaragnella, Nicoletta; Ždralevi?, Maša; Marra, Ersilia

    2013-01-01

    Beyond its classical biotechnological applications such as food and beverage production or as a cell factory, the yeast Saccharomyces cerevisiae is a valuable model organism to study fundamental mechanisms of cell response to stressful environmental changes. Acetic acid is a physiological product of yeast fermentation and it is a well-known food preservative due to its antimicrobial action. Acetic acid has recently been shown to cause yeast cell death and aging. Here we shall focus on the molecular mechanisms of S. cerevisiae stress adaptation and programmed cell death in response to acetic acid. We shall elaborate on the intracellular signaling pathways involved in the cross-talk of pro-survival and pro-death pathways underlying the importance of understanding fundamental aspects of yeast cell homeostasis to improve the performance of a given yeast strain in biotechnological applications. PMID:23430312

  19. Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae.

    PubMed

    Avery, S V; Tobin, J M

    1992-12-01

    Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain. PMID:1335718

  20. Changes and roles of membrane compositions in the adaptation of Saccharomyces cerevisiae to ethanol.

    PubMed

    Wang, Yanfeng; Zhang, Shuxian; Liu, Huaqing; Zhang, Lei; Yi, Chenfeng; Li, Hao

    2015-12-01

    Bioethanol fermentation by Saccharomyces cerevisiae is often stressed by the accumulation of ethanol. Cell membrane is the first assaulting target of ethanol. Ethanol-adapted S. cerevisiae strains provide opportunity to shed light on membrane functions in the ethanol tolerance. This study aimed at clarifying the roles of cell membrane in the ethanol tolerance of S. cerevisiae through comparing membrane components between S. cerevisiae parental strain and ethanol-adapted strains. A directed evolutionary engineering was performed to obtain the ethanol-adapted S. cerevisiae strains. The parental, ethanol-adapted M5 and M10 strains were selected to be compared the percentage of viable cells after exposing to ethanol stress and cell membrane compositions (i.e., ergosterol, trehalose, and fatty acids). Compared with the parental strain, M5 or M10 strain had higher survival rate in the presence of 10% v/v ethanol. Compared with that in the parental strain, contents of trehalose, ergosterol, and fatty acids increased about 15.7, 12.1, and 29.3%, respectively, in M5 strain, and about 47.5, 107.8, and 61.5%, respectively, in M10 strain. Moreover, expression differences of genes involved in fatty acids metabolisms among the parental, M5 and M10 strains were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and results demonstrated that M5 or M10 strain had higher expression of ACC1 and OLE1 than the parental strain. These results indicated that although being exposed to step-wise increased ethanol, S. cerevisiae cells might remodel membrane components or structure to adapt to the ethanol stress. PMID:26265555