Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase.

PubMed

Yu, Kyung Ok; Jung, Ju; Kim, Seung Wook; Park, Chul Hwan; Han, Sung Ok

2012-01-01

The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol. Copyright © 2011 Wiley Periodicals, Inc.

Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50▿

PubMed Central

Basanta, Antonio; Herranz, Carmen; Gutiérrez, Jorge; Criado, Raquel; Hernández, Pablo E.; Cintas, Luis M.

2009-01-01

A segregationally stable expression and secretion vector for Saccharomyces cerevisiae, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) into the S. cerevisiae high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from Enterococcus faecium L50 were cloned, separately (entL50A or entL50B) and together (entL50AB), into pYABD01 under the control of the galactose-inducible promoter PGAL1. The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MFα1s-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system. PMID:19218405

Heterologous expression of Trametes versicolor laccase in Saccharomyces cerevisiae.

PubMed

Iimura, Yosuke; Sonoki, Tomonori; Habe, Hiroshi

2018-01-01

Laccase is used in various industrial fields, and it has been the subject of numerous studies. Trametes versicolor laccase has one of the highest redox potentials among the various forms of this enzyme. In this study, we optimized the expression of laccase in Saccharomyces cerevisiae. Optimizing the culture conditions resulted in an improvement in the expression level, and approximately 45 U/L of laccase was functionally secreted in the culture. The recombinant laccase was found to be a heavily hypermannosylated glycoprotein, and the molecular weight of the carbohydrate chain was approximately 60 kDa. These hypermannosylated glycans lowered the substrate affinity, but the optimum pH and thermo-stability were not changed by these hypermannosylated glycans. This functional expression system described here will aid in molecular evolutionary studies conducted to generate new variants of laccase. Copyright © 2017 Elsevier Inc. All rights reserved.

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

2015-06-01

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.

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

Enhanced cell-surface display of a heterologous protein using SED1 anchoring system in SED1-disrupted Saccharomyces cerevisiae strain.

PubMed

Bamba, Takahiro; Inokuma, Kentaro; Hasunuma, Tomohisa; Kondo, Akihiko

2018-03-01

Yeast displaying enzymes on the cell surface are used for developing whole-cell biocatalysts. High enzyme activity on the cell surface is required in certain applications such as direct ethanol production from lignocellulosic materials. However, the cell surface enzyme activity is limited by several factors, one of which is the protein amount of the yeast cell wall. In this study, we attempted to improve the incorporation capacity of a displayed heterologous enzyme by disrupting a native cell-wall protein. β-Glucosidase (BGL1) from Aspergillus aculeatus was fused with Saccharomyces cerevisiae Sed1 and displayed on the cell surface of S. cerevisiae BY4741 strain and its SED1 disruptant. Sed1 is one of the most abundant stationary phase yeast cell wall protein. A time course analysis revealed that BGL1 activity of the control strain reached saturation after 48 h of cultivation. In contrast, the BGL1 activity of the SED1 disruptant increased until 72 h of cultivation and was 22% higher than that of the control strain. We also performed relative quantification of cell wall proteins of these strains by nanoscale ultra pressure liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nano-UPLC-MS E ). The amount of the cell wall-associated BGL1 per unit dry cell-weight of the SED1 disruptant was 19% higher than that of the control strain. These results suggested that the incorporation capacity of the cell wall for BGL1 was increased by disruption of SED1. Disruption of SED1 would be a promising approach for improving display efficiency of heterologous protein fused with Sed1. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Promiscuous activities of heterologous enzymes lead to unintended metabolic rerouting in Saccharomyces cerevisiae engineered to assimilate various sugars from renewable biomass.

PubMed

Yun, Eun Ju; Oh, Eun Joong; Liu, Jing-Jing; Yu, Sora; Kim, Dong Hyun; Kwak, Suryang; Kim, Kyoung Heon; Jin, Yong-Su

2018-01-01

Understanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae . Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites. Unbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively. The heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

Characterization of vacuolar amino acid transporter from Fusarium oxysporum in Saccharomyces cerevisiae.

PubMed

Lunprom, Siriporn; Pongcharoen, Pongsanat; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi; Akiyama, Koichi

2015-01-01

Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

Effects of gene orientation and use of multiple promoters on the expression of XYL1 and XYL2 in Saccharomyces cerevisiae

Treesearch

Ju Yun Bae; Jose Laplaza; Thomas W. Jeffries

2008-01-01

Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We...

Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae.

PubMed

Brain-Isasi, Stephanie; Álvarez-Lueje, Alejandro; Higgins, Thomas Joseph V

2017-06-15

Phaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications. A synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional αAI into the culture supernatants were selected for further analyses. Recombinant αAI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein. This is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.

Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.

PubMed

Robertson, Aaron; Schaltz, Kyle; Neimanis, Karina; Staples, James F; McDonald, Allison E

2016-10-01

Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

In vitro screening of probiotic properties of Saccharomyces cerevisiae var. boulardii and food-borne Saccharomyces cerevisiae strains.

PubMed

van der Aa Kühle, Alis; Skovgaard, Kerstin; Jespersen, Lene

2005-05-01

The probiotic potential of 18 Saccharomyces cerevisiae strains used for production of foods or beverages or isolated from such, and eight strains of Saccharomyces cerevisiae var. boulardii, was investigated. All strains included were able to withstand pH 2.5 and 0.3% Oxgall. Adhesion to the nontumorigenic porcine jejunal epithelial cell line (IPEC-J2) was investigated by incorporation of 3H-methionine into the yeast cells and use of liquid scintillation counting. Only few of the food-borne S. cerevisiae strains exhibited noteworthy adhesiveness with the strongest levels of adhesion (13.6-16.8%) recorded for two isolates from blue veined cheeses. Merely 25% of the S. cerevisiae var. boulardii strains displayed good adhesive properties (16.2-28.0%). The expression of the proinflammatory cytokine IL-1alpha decreased strikingly in IPEC-J2 cells exposed to a Shiga-like toxin 2e producing Escherichia coli strain when the cells were pre- and coincubated with S. cerevisiae var. boulardii even though this yeast strain was low adhesive (5.4%), suggesting that adhesion is not a mandatory prerequisite for such a probiotic effect. A strain of S. cerevisiae isolated from West African sorghum beer exerted similar effects hence indicating that food-borne strains of S. cerevisiae may possess probiotic properties in spite of low adhesiveness.

Expression of Pneumocystis jirovecii Major Surface Glycoprotein in Saccharomyces cerevisiae

PubMed Central

Kutty, Geetha; England, Katherine J.; Kovacs, Joseph A.

2013-01-01

The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis organisms, recombinant Msg localized to the plasma membrane of yeast rather than to the cell wall. Msg expression was targeted to the yeast cell wall by replacing its signal peptide, serine-threonine–rich region, and glycophosphatidylinositol anchor signal region with the signal peptide of cell wall protein α-agglutinin of S. cerevisiae, the serine-threonine–rich region of epithelial adhesin (Epa1) of Candida glabrata, and the carboxyl region of the cell wall protein (Cwp2) of S. cerevisiae, respectively. Immunofluorescence analysis and treatment with β-1,3 glucanase demonstrated that the expressed Msg fusion protein localized to the yeast cell wall. Surface expression of Msg protein resulted in increased adherence of yeast to A549 alveolar epithelial cells. Heterologous expression of Msg in yeast will facilitate studies of the biologic properties of Pneumocystis Msg. PMID:23532098

The Saccharomyces cerevisiae Lipin Homolog is a Mg2+-dependent Phosphatidate Phosphatase Enzyme*

PubMed Central

Han, Gil-Soo; Wu, Wen-I; Carman, George M.

2006-01-01

Mg2+-dependent phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of PA to yield diacylglycerol and Pi. In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene that encodes Mg2+-dependent PA phosphatase using amino acid sequence information derived from a purified preparation of the enzyme (Lin, Y.-P., and Carman, G.M. (1989) J. Biol. Chem. 264, 8641–8645). Overexpression of PAH1 in S. cerevisiae directed elevated levels of Mg2+-dependent PA phosphatase activity, whereas the pah1Δ mutation caused reduced levels of enzyme activity. Heterologous expression of PAH1 in Escherichia coli confirmed that Pah1p is a Mg2+-dependent PA phosphatase enzyme, and showed that its enzymological properties were very similar to those of the enzyme purified from S. cerevisiae. The PAH1-encoded enzyme activity was associated with both the membrane and cytosolic fractions of the cell, and the membrane-bound form of the enzyme was salt-extractable. Lipid analysis showed that mutants lacking PAH1 accumulated PA, and had reduced amounts of diacylglycerol and its derivative triacylglycerol. The PAH1-encoded Mg2+-dependent PA phosphatase shows homology to mammalian lipin, a fat-regulating protein whose molecular function is unknown. Heterologous expression of human LPIN1 in E. coli showed that lipin 1 is also a Mg2+-dependent PA phosphatase enzyme. PMID:16467296

Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt.

PubMed

Zhou, Pingping; Ye, Lidan; Xie, Wenping; Lv, Xiaomei; Yu, Hongwei

2015-10-01

Astaxanthin is a highly valued carotenoid with strong antioxidant activity and has wide applications in aquaculture, food, cosmetic, and pharmaceutical industries. The market demand for natural astaxanthin promotes research in metabolic engineering of heterologous hosts for astaxanthin production. In this study, an astaxanthin-producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis β-carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed β-carotene hyperproducer. Further integration of strategies including codon optimization, gene copy number adjustment, and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in S. cerevisiae reported to date. Besides, the substrate specificity of H. pluvialis CrtZ and BKT and the probable formation route of astaxanthin from β-carotene in S. cerevisiae were figured out by expressing the genes separately and in combination. The yeast strains engineered in this work provide a basis for further improving biotechnological production of astaxanthin and might offer a useful general approach to the construction of heterologous biosynthetic pathways for other natural products.

Progress toward heterologous expression of active G-protein-coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

PubMed Central

O'Malley, Michelle A; Mancini, J Dominic; Young, Carissa L; McCusker, Emily C; Raden, David; Robinson, Anne S

2009-01-01

High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A2a (hA2aR) receptor is active for ligand-binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA2aR, as expected, and partially cleaved for hA2bR, hCCR5R, and hD2LR. Ligand-binding assays conducted on the adenosine family (hA1R, hA2aR, hA2bR, and hA3R) of receptors show that hA2aR and hA2bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae. PMID:19760666

Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must.

PubMed

Cira, Luis Alberto; González, Gloria Angélica; Torres, Juan Carlos; Pelayo, Carlos; Gutiérrez, Melesio; Ramírez, Jesús

2008-03-01

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to alpha-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 muM alpha-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 muM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.

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.

In silico profiling of Escherichia coli and Saccharomyces cerevisiae as terpenoid factories

PubMed Central

2013-01-01

Background Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield. Results Starting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which

Fatal Saccharomyces cerevisiae aortic graft infection.

PubMed

Smith, Davey; Metzgar, David; Wills, Christopher; Fierer, Joshua

2002-07-01

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 (J. N. Aucott, J. Fayan, H. Grossnicklas, A. Morrissey, M. M. Lederman, and R. A. Salata, Rev. Infect. Dis. 12:406-411, 1990). We present a case of S. cerevisiae fungemia and aortic graft infection in an immunocompetent adult. This is the first reported case of S. cerevisiae fungemia where the identity of the pathogen was confirmed by rRNA sequencing.

Saccharomyces cerevisiae vaginitis: transmission from yeast used in baking.

PubMed

Nyirjesy, P; Vazquez, J A; Ufberg, D D; Sobel, J D; Boikov, D A; Buckley, H R

1995-09-01

To determine whether vaginitis due to Saccharomyces cerevisiae can be caused by exposure to exogenous sources of baker's yeast. Eight women with S cerevisiae vaginitis were identified from a cohort of women referred for the evaluation of chronic vaginal symptoms. In those with high-level exposure to exogenous sources of S cerevisiae, isolates from the vagina and those sources were sent in a blinded fashion for contour-clamped homogeneous electric-field electrophoresis. Four women from a cohort of approximately 750 referred patients had high-level exposures to S cerevisiae. In one of these patients, electrophoresis analysis revealed similarities between the strains isolated from her vagina, her husband's fingers, and the yeast he used in his pizza shop. Saccharomyces cerevisiae vaginitis can be the result of the inoculation of this yeast from exogenous sources.

Value-added biotransformation of cellulosic sugars by engineered Saccharomyces cerevisiae.

PubMed

Lane, Stephan; Dong, Jia; Jin, Yong-Su

2018-07-01

The substantial research efforts into lignocellulosic biofuels have generated an abundance of valuable knowledge and technologies for metabolic engineering. In particular, these investments have led to a vast growth in proficiency of engineering the yeast Saccharomyces cerevisiae for consuming lignocellulosic sugars, enabling the simultaneous assimilation of multiple carbon sources, and producing a large variety of value-added products by introduction of heterologous metabolic pathways. While microbial conversion of cellulosic sugars into large-volume low-value biofuels is not currently economically feasible, there may still be opportunities to produce other value-added chemicals as regulation of cellulosic sugar metabolism is quite different from glucose metabolism. This review summarizes these recent advances with an emphasis on employing engineered yeast for the bioconversion of lignocellulosic sugars into a variety of non-ethanol value-added products. Copyright © 2018 Elsevier Ltd. All rights reserved.

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. © 2014 Wiley Periodicals, Inc.

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, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems...

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

PubMed Central

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

2014-01-01

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

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.

Reconstruction and Evaluation of the Synthetic Bacterial MEP Pathway in Saccharomyces cerevisiae

PubMed Central

Partow, Siavash; Siewers, Verena; Daviet, Laurent; Schalk, Michel; Nielsen, Jens

2012-01-01

Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH. PMID:23285068

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

PubMed

Latimer, Luke N; Dueber, John E

2017-06-01

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ∼70% increase in biomass yield and ∼240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae.

PubMed

Solomon, Kevin V; Ovadia, Elisa; Yu, Fujio; Mizunashi, Wataru; O'Malley, Michelle A

2016-12-01

Bio-based isobutantol is a sustainable 'drop in' substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV ), and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1 ) were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae . We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production.

The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by Saccharomyces cerevisiae.

PubMed

Biz, Alessandra; Sugai-Guérios, Maura Harumi; Kuivanen, Joosu; Maaheimo, Hannu; Krieger, Nadia; Mitchell, David Alexander; Richard, Peter

2016-08-18

Pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. One possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. However, the ethanol-producing organism of choice, Saccharomyces cerevisiae, is not able to catabolize D-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, namely, 18 % (w/w) from citrus pulp and 16 % (w/w) sugar beet pulp. In the current work, we describe the construction of a strain of S. cerevisiae in which the five genes of the fungal reductive pathway for D-galacturonic acid catabolism were integrated into the yeast chromosomes: gaaA, gaaC and gaaD from Aspergillus niger and lgd1 from Trichoderma reesei, and the recently described D-galacturonic acid transporter protein, gat1, from Neurospora crassa. This strain metabolized D-galacturonic acid in a medium containing D-fructose as co-substrate. This work is the first demonstration of the expression of a functional heterologous pathway for D-galacturonic acid catabolism in Saccharomyces cerevisiae. It is a preliminary step for engineering a yeast strain for the fermentation of pectin-rich substrates to ethanol.

[Saccharomyces cerevisiae infections].

PubMed

Souza Goebel, Cristine; de Mattos Oliveira, Flávio; Severo, Luiz Carlos

2013-01-01

Saccharomyces cerevisiae is an ubiquitous yeast widely used in industry and it is also a common colonizer of the human mucosae. However, the incidence of invasive infection by these fungi has significantly increased in the last decades. To evaluate the infection by S. cerevisiae in a hospital in southern Brazil during a period of 10 years (2000-2010). Review of medical records of patients infected by this fungus. In this period, 6 patients were found to be infected by S. cerevisiae. The age range of the patients was from 10 years to 84. Urine, blood, ascitic fluid, peritoneal dialysis fluid, and esophageal biopsy samples were analyzed. The predisposing factors were cancer, transplant, surgical procedures, renal failure, use of venous catheters, mechanical ventilation, hospitalization in Intensive Care Unit, diabetes mellitus, chemotherapy, corticosteroid use, and parenteral nutrition. Amphotericin B and fluconazole were the treatments of choice. Three of the patients died and the other 3 were discharged from hospital. We must take special precautions in emerging infections, especially when there are predisposing conditions such as immunosuppression or patients with serious illnesses. The rapid and specific diagnosis of S. cerevisiae infections is important for therapeutic decision. Furthermore, epidemiological and efficacy studies of antifungal agents are necessary for a better therapeutic approach. Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived hydrocarbons.

PubMed

Zhang, Yiming; Nielsen, Jens; Liu, Zihe

2018-06-05

Fatty acid-derived hydrocarbons attract increasing attention as biofuels due to their immiscibility with water, high-energy content, low freezing point, and high compatibility with existing refineries and end-user infrastructures. Yeast Saccharomyces cerevisiae has advantages for production of fatty acid-derived hydrocarbons as its native routes toward fatty acid synthesis involve only a few reactions that allow more efficient conversion of carbon substrates. Here we describe major biosynthetic pathways of fatty acid-derived hydrocarbons in yeast, and summarize key metabolic engineering strategies, including enhancing precursor supply, eliminating competing pathways, and expressing heterologous pathways. With recent advances in yeast production of fatty acid-derived hydrocarbons, our review identifies key research challenges and opportunities for future optimization, and concludes with perspectives and outlooks for further research directions. © 2018 Wiley Periodicals, Inc.

Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals.

PubMed

Yu, Tao; Zhou, Yongjin J; Wenning, Leonie; Liu, Quanli; Krivoruchko, Anastasia; Siewers, Verena; Nielsen, Jens; David, Florian

2017-05-26

Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C 22 H 46 O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l -1 in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.

Analysis of repeat-mediated deletions in the mitochondrial genome of Saccharomyces cerevisiae.

PubMed

Phadnis, Naina; Sia, Rey A; Sia, Elaine A

2005-12-01

Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions.

Analysis of Repeat-Mediated Deletions in the Mitochondrial Genome of Saccharomyces cerevisiae

PubMed Central

Phadnis, Naina; Sia, Rey A.; Sia, Elaine A.

2005-01-01

Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions. PMID:16157666

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

Saccharomyces cerevisiae fungemia: an emerging infectious disease.

PubMed

Muñoz, Patricia; Bouza, Emilio; Cuenca-Estrella, Manuel; Eiros, Jose María; Pérez, María Jesús; Sánchez-Somolinos, Mar; Rincón, Cristina; Hortal, Javier; Peláez, Teresa

2005-06-01

Saccharomyces cerevisiae is well known in the baking and brewing industry and is also used as a probiotic in humans. However, it is a very uncommon cause of infection in humans. During the period of 15-30 April 2003, we found 3 patients with S. cerevisiae fungemia in an intensive care unit (ICU). An epidemiological study was performed, and the medical records for all patients who were in the unit during the second half of April were assessed. The only identified risk factor for S. cerevisiae infection was treatment with a probiotic containing Saccharomyces boulardii (Ultralevura; Bristol-Myers Squibb). This probiotic is used in Europe for the treatment and prevention of Clostridium difficile-associated diarrhea. The 3 patients received the product via nasograstric tube for a mean duration of 8.5 days before the culture result was positive, whereas only 2 of 41 control subjects had received it. Surveillance cultures for the control patients admitted at the same time did not reveal any carriers of the yeast. Strains from the probiotic capsules and the clinical isolates were identified as S. cerevisiae, with identical DNA fingerprinting. Discontinuation of use of the product in the unit stopped the outbreak of infection. A review of the literature identified another 57 cases of S. cerevisiae fungemia. Overall, 60% of these patients were in the ICU, and 71% were receiving enteral or parenteral nutrition. Use of probiotics was detected in 26 patients, and 17 patients died. Use of S. cerevisiae probiotics should be carefully reassessed, particularly in immunosuppressed or critically ill patients.

Improvement of Lead Tolerance of Saccharomyces cerevisiae by Random Mutagenesis of Transcription Regulator SPT3.

PubMed

Zhu, Liying; Gao, Shan; Zhang, Hongman; Huang, He; Jiang, Ling

2018-01-01

Bioremediation of heavy metal pollution with biomaterials such as bacteria and fungi usually suffer from limitations because of microbial sensitivity to high concentration of heavy metals. Herein, we adopted a novel random mutagenesis technique called RAISE to manipulate the transcription regulator SPT3 of Saccharomyces cerevisiae to improve cell lead tolerance. The best strain Mutant VI was selected from the random mutagenesis libraries on account of the growth performance, with higher specific growth rate than the control strain (0.068 vs. 0.040 h -1 ) at lead concentration as high as 1.8 g/L. Combined with the transcriptome analysis of S. cerevisiae, expressing the SPT3 protein was performed to make better sense of the global regulatory effects of SPT3. The data analysis revealed that 57 of S. cerevisiae genes were induced and 113 genes were suppressed, ranging from those for trehalose synthesis, carbon metabolism, and nucleotide synthesis to lead resistance. Especially, the accumulation of intracellular trehalose in S. cerevisiae under certain conditions of stress is considered important to lead resistance. The above results represented that SPT3 was acted as global transcription regulator in the exponential phase of strain and accordingly improved heavy metal tolerance in the heterologous host S. cerevisiae. The present study provides a route to complex phenotypes that are not readily accessible by traditional methods.

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

Heterologously expressed Aspergillus aculeatus β-glucosidase in Saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using Trichoderma reesei cellulases.

PubMed

Treebupachatsakul, Treesukon; Nakazawa, Hikaru; Shinbo, Hideaki; Fujikawa, Hiroki; Nagaiwa, Asami; Ochiai, Nobuhiro; Kawaguchi, Takashi; Nikaido, Mitsuru; Totani, Kazuhide; Shioya, Koki; Shida, Yosuke; Morikawa, Yasushi; Ogasawara, Wataru; Okada, Hirofumi

2016-01-01

Trichoderma reesei is a filamentous organism that secretes enzymes capable of degrading cellulose to cellobiose. The culture supernatant of T. reesei, however, lacks sufficient activity to convert cellobiose to glucose using β-glucosidase (BGL1). In this study, we identified a BGL (Cel3B) from T. reesei (TrCel3B) and compared it with the active β-glucosidases from Aspergillus aculeatus (AaBGL1). AaBGL1 showed higher stability and conversion of sugars to ethanol compared to TrCel3B, and therefore we chose to express this recombinant protein for use in fermentation processes. We expressed the recombinant protein in the yeast Saccharomyces cerevisiae, combined it with the superb T. reesei cellulase machinery and used the combination in a simultaneous saccharification and fermentation (SSF) process, with the hope that the recombinant would supplement the BGL activity. As the sugars were processed, the yeast immediately converted them to ethanol, thereby eliminating the problem posed by end product inhibition. Recombinant AaBGL1 activity was compared with Novozyme 188, a commercially available supplement for BGL activity. Our results show that the recombinant protein is as effective as the commercial supplement and can process sugars with equal efficiency. Expression of AaBGL1 in S. cerevisiae increased ethanol production effectively. Thus, heterologous expression of AaBGL1 in S. cerevisiae is a cost-effective and efficient process for the bioconversion of ethanol from lignocellulosic biomass. Copyright © 2015. Published by Elsevier B.V.

iTRAQ-based proteome profiling of Saccharomyces cerevisiae and cryotolerant species Saccharomyces uvarum and Saccharomyces kudriavzevii during low-temperature wine fermentation.

PubMed

García-Ríos, Estéfani; Querol, Amparo; Guillamón, José Manuel

2016-09-02

Temperature is one of the most important parameters to affect the duration and rate of alcoholic fermentation and final wine quality. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae, which was the case of cryotolerant yeasts Saccharomyces uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the proteomic landscape of these cryotolerant species grown at 12°C and 28°C, which we compared with the proteome of S. cerevisiae (poorly adapted at low temperature). Our results showed that the main differences among the proteomic profiling of the three Saccharomyces strains grown at 12°C and 28°C lay in translation, glycolysis and amino acid metabolism. Our data corroborate previous transcriptomic results, which suggest that S. kudriavzevii is better adapted to grow at low temperature as a result of enhanced more efficient translation. Fitter amino acid biosynthetic pathways can also be mechanisms that better explain biomass yield in cryotolerant strains. Yet even at low temperature, S. cerevisiae is the most fermentative competitive species. A higher concentration of glycolytic and alcoholic fermentation enzymes in the S. cerevisiae strain might explain such greater fermentation activity. Temperature is one of the main relevant environmental variables that microorganisms have to cope with and it is also a key factor in some industrial processes that involve microorganisms. However, we are still far from understanding the molecular and physiological mechanisms of adaptation at low temperatures. The results obtained in this study provided a global atlas of the proteome changes triggered by temperature in three different species of the genus Saccharomyces with different degree of cryotolerance. These results would facilitate a better understanding of mechanisms for how yeast could adapt at the low temperature of growth. Copyright © 2016

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.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

PubMed

Gottardi, Manuela; Knudsen, Jan Dines; Prado, Lydie; Oreb, Mislav; Branduardi, Paola; Boles, Eckhard

2017-06-01

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

Expression of monellin in a food-grade delivery system in Saccharomyces cerevisiae.

PubMed

Liu, Jun; Yan, Da-zhong; Zhao, Sheng-jun

2015-10-01

Genetically modified (GM) foods have caused much controversy. Construction of a food-grade delivery system is a desirable technique with presumptive impact on industrial applications from the perspective of bio-safety. The aim of this study was to construct a food-grade delivery system for Saccharomyces cerevisiae and to study the expression of monellin from the berries of the West African forest plant Dioscoreophyllum cumminsii in this system. A food-grade system for S. cerevisiae was constructed based on ribosomal DNA (rDNA)-mediated homologous recombination to enable high-copy-number integration of the expression cassette inserted into the rDNA locus. A copper resistance gene (CUP1) was used as the selection marker for yeast transformation. Because variants of transformants containing different copy numbers at the CUP1 locus can be readily selected after growth in the presence of elevated copper levels, we suggest that this system would prove useful in the generation of tandemly iterated gene clusters. Using this food-grade system, a single-chain monellin gene was heterologously expressed. The yield of monellin reached a maximum of 675 mg L(-1) . This system harbors exclusively S. cerevisiae DNA with no antibiotic resistance genes, and it should therefore be appropriate for safe use in the food industry. Monellin was shown to be expressed in this food-grade delivery system. To our knowledge, this is the first report so far on expression of monellin in a food-grade expression system in S. cerevisiae. © 2014 Society of Chemical Industry.

Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

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...

Creation of a synthetic xylose-inducible promoter for Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

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...

Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather

Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains withmore » improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. Lastly, the mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min -1•mg -1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation.« less

Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae

DOE PAGES

Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; ...

2016-01-19

Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains withmore » improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. Lastly, the mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min -1•mg -1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation.« less

Genetic Manipulation of Palmitoylethanolamide Production and Inactivation in Saccharomyces cerevisiae

PubMed Central

Muccioli, Giulio G.; Sia, Angela; Muchowski, Paul J.; Stella, Nephi

2009-01-01

Background Lipids can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions. To understand how a newly discovered signaling lipid functions, it is necessary to identify and characterize the enzymes involved in their production and inactivation. The signaling lipid N-palmitoylethanolamine (PEA) is known to activate intracellular and membrane-associated receptors and regulate physiological functions, but little is known about the enzymes involved in its production and inactivation. Principal Findings Here we show that Saccharomyces cerevisiae produce and inactivate PEA, suggesting that genetic manipulations of this lower eukaryote may be used to identify the enzymes involved in PEA metabolism. Accordingly, using single gene deletion mutants, we identified yeast genes that control PEA metabolism, including SPO14 (a yeast homologue of the mammalian phospholipase D) that controls PEA production and YJU3 (a yeast homologue of the mammalian monoacylglycerol lipase) that controls PEA inactivation. We also found that PEA metabolism is affected by heterologous expression of two mammalian proteins involved in neurodegenerative diseases, namely huntingtin and α-synuclein. Significance Together these findings show that forward and reverse genetics in S. cerevisiae can be used to identify proteins involved in PEA production and inactivation, and suggest that mutated proteins causing neurodegenerative diseases might affect the metabolism of this important signaling lipid. PMID:19529773

Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast Saccharomyces cerevisiae.

PubMed

Fišerová, Jindřiška; Richardson, Christine; Goldberg, Martin W

2016-01-01

Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labeling of the yeast Saccharomyces cerevisiae that gives specific and multiple labeling while keeping the finest structural details. We use the protocol to reveal the organization of individual nuclear pore complex proteins and the position of transport factors in the yeast Saccharomyces cerevisiae in relation to actual transport events.

Impact of oxygenation on the performance of three non-Saccharomyces yeasts in co-fermentation with Saccharomyces cerevisiae.

PubMed

Shekhawat, Kirti; Bauer, Florian F; Setati, Mathabatha E

2017-03-01

The sequential or co-inoculation of grape must with non-Saccharomyces yeast species and Saccharomyces cerevisiae wine yeast strains has recently become a common practice in winemaking. The procedure intends to enhance unique aroma and flavor profiles of wine. The extent of the impact of non-Saccharomyces strains depends on their ability to produce biomass and to remain metabolically active for a sufficiently long period. However, mixed-culture wine fermentations tend to become rapidly dominated by S. cerevisiae, reducing or eliminating the non-Saccharomyces yeast contribution. For an efficient application of these yeasts, it is therefore essential to understand the environmental factors that modulate the population dynamics of such ecosystems. Several environmental parameters have been shown to influence population dynamics, but their specific effect remains largely uncharacterized. In this study, the population dynamics in co-fermentations of S. cerevisiae and three non-Saccharomyces yeast species: Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima, was investigated as a function of oxygen availability. In all cases, oxygen availability strongly influenced population dynamics, but clear species-dependent differences were observed. Our data show that L. thermotolerans required the least oxygen, followed by T. delbrueckii and M. pulcherrima. Distinct species-specific chemical volatile profiles correlated in all cases with increased persistence of non-Saccharomyces yeasts, in particular increases in some higher alcohols and medium chain fatty acids. The results highlight the role of oxygen in regulating the succession of yeasts during wine fermentations and suggests that more stringent aeration strategies would be necessary to support the persistence of non-Saccharomyces yeasts in real must fermentations.

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…

The role of tRNA and ribosome competition in coupling the expression of different mRNAs in Saccharomyces cerevisiae

PubMed Central

Chu, Dominique; Barnes, David J.; von der Haar, Tobias

2011-01-01

Protein synthesis translates information from messenger RNAs into functional proteomes. Because of the finite nature of the resources required by the translational machinery, both the overall protein synthesis activity of a cell and activity on individual mRNAs are controlled by the allocation of limiting resources. Upon introduction of heterologous sequences into an organism—for example for the purposes of bioprocessing or synthetic biology—limiting resources may also become overstretched, thus negatively affecting both endogenous and heterologous gene expression. In this study, we present a mean-field model of translation in Saccharomyces cerevisiae for the investigation of two particular translational resources, namely ribosomes and aminoacylated tRNAs. We firstly use comparisons of experiments with heterologous sequences and simulations of the same conditions to calibrate our model, and then analyse the behaviour of the translational system in yeast upon introduction of different types of heterologous sequences. Our main findings are that: competition for ribosomes, rather than tRNAs, limits global translation in this organism; that tRNA aminoacylation levels exert, at most, weak control over translational activity; and that decoding speeds and codon adaptation exert strong control over local (mRNA specific) translation rates. PMID:21558172

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

PubMed

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

2015-07-27

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). © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Saccharomyces cerevisiae Shuttle vectors.

PubMed

Gnügge, Robert; Rudolf, Fabian

2017-05-01

Yeast shuttle vectors are indispensable tools in yeast research. They enable cloning of defined DNA sequences in Escherichia coli and their direct transfer into Saccharomyces cerevisiae cells. There are three types of commonly used yeast shuttle vectors: centromeric plasmids, episomal plasmids and integrating plasmids. In this review, we discuss the different plasmid systems and their characteristic features. We focus on their segregational stability and copy number and indicate how to modify these properties. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Recombinant G protein-coupled receptor expression in Saccharomyces cerevisiae for protein characterization.

PubMed

Blocker, Kory M; Britton, Zachary T; Naranjo, Andrea N; McNeely, Patrick M; Young, Carissa L; Robinson, Anne S

2015-01-01

G protein-coupled receptors (GPCRs) are membrane proteins that mediate signaling across the cellular membrane and facilitate cellular responses to external stimuli. Due to the critical role that GPCRs play in signal transduction, therapeutics have been developed to influence GPCR function without an extensive understanding of the receptors themselves. Closing this knowledge gap is of paramount importance to improving therapeutic efficacy and specificity, where efforts to achieve this end have focused chiefly on improving our knowledge of the structure-function relationship. The purpose of this chapter is to review methods for the heterologous expression of GPCRs in Saccharomyces cerevisiae, including whole-cell assays that enable quantitation of expression, localization, and function in vivo. In addition, we describe methods for the micellular solubilization of the human adenosine A2a receptor and for reconstitution of the receptor in liposomes that have enabled its biophysical characterization. © 2015 Elsevier Inc. All rights reserved.

Glucose-based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae.

PubMed

Germann, Susanne M; Baallal Jacobsen, Simo A; Schneider, Konstantin; Harrison, Scott J; Jensen, Niels B; Chen, Xiao; Stahlhut, Steen G; Borodina, Irina; Luo, Hao; Zhu, Jiangfeng; Maury, Jérôme; Forster, Jochen

2016-05-01

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N-acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L-tryptophan hydroxylase, a 5-hydroxy-L-tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O-methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co-factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L(-1) in a 76h fermentation using simulated fed-batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin. © 2015 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae.

PubMed

Kwak, Suryang; Kim, Soo Rin; Xu, Haiqing; Zhang, Guo-Chang; Lane, Stephan; Kim, Heejin; Jin, Yong-Su

2017-11-01

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc. © 2017 Wiley

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

PubMed

Shirley, Derek; Oppert, Cris; Reynolds, Todd B; Miracle, Bethany; Oppert, Brenda; Klingeman, William E; Jurat-Fuentes, Juan Luis

2014-10-01

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Stoichiometric network constraints on xylose metabolism by recombinant Saccharomyces cerevisiae

Treesearch

Yong-Su Jin; Thomas W. Jeffries

2004-01-01

Metabolic pathway engineering is constrained by the thermodynamic and stoichiometric feasibility of enzymatic activities of introduced genes. Engineering of xylose metabolism in Saccharomyces cerevisiae has focused on introducing genes for the initial xylose assimilation steps from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, a yeast raditionally...

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: Construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

DOE Office of Scientific and Technical Information (OSTI.GOV)

Street, I.P.; Poulter, C.D.

1990-08-14

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinantmore » IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with (4-{sup 3}H)FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.« less

Biosynthesis of cis,cis-muconic acid and its aromatic precursors, catechol and protocatechuic acid, from renewable feedstocks by Saccharomyces cerevisiae.

PubMed

Weber, Christian; Brückner, Christine; Weinreb, Sheila; Lehr, Claudia; Essl, Christine; Boles, Eckhard

2012-12-01

Adipic acid is a high-value compound used primarily as a precursor for the synthesis of nylon, coatings, and plastics. Today it is produced mainly in chemical processes from petrochemicals like benzene. Because of the strong environmental impact of the production processes and the dependence on fossil resources, biotechnological production processes would provide an interesting alternative. Here we describe the first engineered Saccharomyces cerevisiae strain expressing a heterologous biosynthetic pathway converting the intermediate 3-dehydroshikimate of the aromatic amino acid biosynthesis pathway via protocatechuic acid and catechol into cis,cis-muconic acid, which can be chemically dehydrogenated to adipic acid. The pathway consists of three heterologous microbial enzymes, 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase composed of three different subunits, and catechol 1,2-dioxygenase. For each heterologous reaction step, we analyzed several potential candidates for their expression and activity in yeast to compose a functional cis,cis-muconic acid synthesis pathway. Carbon flow into the heterologous pathway was optimized by increasing the flux through selected steps of the common aromatic amino acid biosynthesis pathway and by blocking the conversion of 3-dehydroshikimate into shikimate. The recombinant yeast cells finally produced about 1.56 mg/liter cis,cis-muconic acid.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 formore » 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.« less

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.

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...

[The ABC transporters of Saccharomyces cerevisiae].

PubMed

Wawrzycka, Donata

2011-01-01

The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

PubMed

Barajas, Daniel; Aponte-Ubillus, Juan Jose; Akeefe, Hassibullah; Cinek, Tomas; Peltier, Joseph; Gold, Daniel

2017-01-01

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

Development of a modularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae.

PubMed

Li, Siwei; Ding, Wentao; Zhang, Xueli; Jiang, Huifeng; Bi, Changhao

2016-01-01

Saccharomyces cerevisiae has already been used for heterologous production of fuel chemicals and valuable natural products. The establishment of complicated heterologous biosynthetic pathways in S. cerevisiae became the research focus of Synthetic Biology and Metabolic Engineering. Thus, simple and efficient genomic integration techniques of large number of transcription units are demanded urgently. An efficient DNA assembly and chromosomal integration method was created by combining homologous recombination (HR) in S. cerevisiae and Golden Gate DNA assembly method, designated as modularized two-step (M2S) technique. Two major assembly steps are performed consecutively to integrate multiple transcription units simultaneously. In Step 1, Modularized scaffold containing a head-to-head promoter module and a pair of terminators was assembled with two genes. Thus, two transcription units were assembled with Golden Gate method into one scaffold in one reaction. In Step 2, the two transcription units were mixed with modules of selective markers and integration sites and transformed into S. cerevisiae for assembly and integration. In both steps, universal primers were designed for identification of correct clones. Establishment of a functional β-carotene biosynthetic pathway in S. cerevisiae within 5 days demonstrated high efficiency of this method, and a 10-transcriptional-unit pathway integration illustrated the capacity of this method. Modular design of transcription units and integration elements simplified assembly and integration procedure, and eliminated frequent designing and synthesis of DNA fragments in previous methods. Also, by assembling most parts in Step 1 in vitro, the number of DNA cassettes for homologous integration in Step 2 was significantly reduced. Thus, high assembly efficiency, high integration capacity, and low error rate were achieved.

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

PubMed

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

2012-07-02

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. Copyright © 2012 Elsevier B.V. All rights reserved.

PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications. PMID:23688076

Biosynthesis of levan, a bacterial extracellular polysaccharide, in the yeast Saccharomyces cerevisiae.

PubMed

Franken, Jaco; Brandt, Bianca A; Tai, Siew L; Bauer, Florian F

2013-01-01

Levans are fructose polymers synthesized by a broad range of micro-organisms and a limited number of plant species as non-structural storage carbohydrates. In microbes, these polymers contribute to the formation of the extracellular polysaccharide (EPS) matrix and play a role in microbial biofilm formation. Levans belong to a larger group of commercially important polymers, referred to as fructans, which are used as a source of prebiotic fibre. For levan, specifically, this market remains untapped, since no viable production strategy has been established. Synthesis of levan is catalysed by a group of enzymes, referred to as levansucrases, using sucrose as substrate. Heterologous expression of levansucrases has been notoriously difficult to achieve in Saccharomyces cerevisiae. As a strategy, this study used an invertase (Δsuc2) null mutant and two separate, engineered, sucrose accumulating yeast strains as hosts for the expression of the levansucrase M1FT, previously cloned from Leuconostoc mesenteroides. Intracellular sucrose accumulation was achieved either by expression of a sucrose synthase (Susy) from potato or the spinach sucrose transporter (SUT). The data indicate that in both Δsuc2 and the sucrose accumulating strains, the M1FT was able to catalyse fructose polymerisation. In the absence of the predicted M1FT secretion signal, intracellular levan accumulation was significantly enhanced for both sucrose accumulation strains, when grown on minimal media. Interestingly, co-expression of M1FT and SUT resulted in hyper-production and extracellular build-up of levan when grown in rich medium containing sucrose. This study presents the first report of levan production in S. cerevisiae and opens potential avenues for the production of levan using this well established industrial microbe. Furthermore, the work provides interesting perspectives when considering the heterologous expression of sugar polymerizing enzymes in yeast.

Biotechnology of non-Saccharomyces yeasts--the ascomycetes.

PubMed

Johnson, Eric A

2013-01-01

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

Functional expression of aryl-alcohol oxidase in Saccharomyces cerevisiae and Pichia pastoris by directed evolution.

PubMed

Viña-Gonzalez, Javier; Elbl, Katarina; Ponte, Xavier; Valero, Francisco; Alcalde, Miguel

2018-07-01

Aryl-alcohol oxidase (AAO) plays a fundamental role in the fungal ligninolytic secretome, acting as a supplier of H 2 O 2 . Despite its highly selective mechanism of action, the presence of this flavooxidase in different biotechnological settings has hitherto been hampered by the lack of appropriate heterologous expression systems. We recently described the functional expression of the AAO from Pleurotus eryngii in Saccharomyces cerevisiae by fusing a chimeric signal peptide (preαproK) and applying structure-guided evolution. Here, we have obtained an AAO secretion variant that is readily expressed in S. cerevisiae and overproduced in Pichia pastoris. First, the functional expression of AAO in S. cerevisiae was enhanced through the in vivo shuffling of a panel of secretion variants, followed by the focused evolution of the preαproK peptide. The outcome of this evolutionary campaign-an expression variant that accumulated 4 mutations in the chimeric signal peptide, plus two mutations in the mature protein- showed 350-fold improved secretion (4.5 mg/L) and was stable. This secretion mutant was cloned into P. pastoris and fermented in a fed-batch bioreactor to enhance production to 25 mg/L. While both recombinant AAO from S. cerevisiae and P. pastoris were subjected to the same N-terminal processing and had a similar pH activity profile, they differed in their kinetic parameters and thermostability. The strong glycosylation observed in the evolved AAO from S. cerevisiae underpinned this effect, since when the mutant was produced in the glycosylation-deficient S. cerevisiae strain Δkre2, its kinetic parameters and thermostability were comparable to its poorly glycosylated P. pastoris recombinant counterpart. © 2018 Wiley Periodicals, Inc.

Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

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...

Saccharomyces cerevisiae metabolism in ecological context.

PubMed

Jouhten, Paula; Ponomarova, Olga; Gonzalez, Ramon; Patil, Kiran R

2016-11-01

The architecture and regulation of Saccharomyces cerevisiae metabolic network are among the best studied owing to its widespread use in both basic research and industry. Yet, several recent studies have revealed notable limitations in explaining genotype-metabolic phenotype relations in this yeast, especially when concerning multiple genetic/environmental perturbations. Apparently unexpected genotype-phenotype relations may originate in the evolutionarily shaped cellular operating principles being hidden in common laboratory conditions. Predecessors of laboratory S. cerevisiae strains, the wild and the domesticated yeasts, have been evolutionarily shaped by highly variable environments, very distinct from laboratory conditions, and most interestingly by social life within microbial communities. Here we present a brief review of the genotypic and phenotypic peculiarities of S. cerevisiae in the context of its social lifestyle beyond laboratory environments. Accounting for this ecological context and the origin of the laboratory strains in experimental design and data analysis would be essential in improving the understanding of genotype-environment-phenotype relationships. © FEMS 2016.

Saccharomyces cerevisiae metabolism in ecological context

PubMed Central

Jouhten, Paula; Ponomarova, Olga; Gonzalez, Ramon

2016-01-01

The architecture and regulation of Saccharomyces cerevisiae metabolic network are among the best studied owing to its widespread use in both basic research and industry. Yet, several recent studies have revealed notable limitations in explaining genotype–metabolic phenotype relations in this yeast, especially when concerning multiple genetic/environmental perturbations. Apparently unexpected genotype–phenotype relations may originate in the evolutionarily shaped cellular operating principles being hidden in common laboratory conditions. Predecessors of laboratory S. cerevisiae strains, the wild and the domesticated yeasts, have been evolutionarily shaped by highly variable environments, very distinct from laboratory conditions, and most interestingly by social life within microbial communities. Here we present a brief review of the genotypic and phenotypic peculiarities of S. cerevisiae in the context of its social lifestyle beyond laboratory environments. Accounting for this ecological context and the origin of the laboratory strains in experimental design and data analysis would be essential in improving the understanding of genotype–environment–phenotype relationships. PMID:27634775

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation.

PubMed

Domizio, Paola; Romani, Cristina; Lencioni, Livio; Comitini, Francesca; Gobbi, Mirko; Mannazzu, Ilaria; Ciani, Maurizio

2011-06-30

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol. Copyright © 2011 Elsevier B.V. All rights reserved.

A Member of the Sugar Transporter Family, Stl1p Is the Glycerol/H+ Symporter in Saccharomyces cerevisiae

PubMed Central

Ferreira, Célia; van Voorst, Frank; Martins, António; Neves, Luisa; Oliveira, Rui; Kielland-Brandt, Morten C.; Lucas, Cândida; Brandt, Anders

2005-01-01

Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where glycerol symport is functional. Both the Stl1 protein and the active glycerol transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active glycerol transport system. In S. pombe, STL1 conferred the ability to take up glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the glycerol proton symporter in S. cerevisiae is encoded by STL1. PMID:15703210

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation.

PubMed

Amaya-Delgado, L; Herrera-López, E J; Arrizon, Javier; Arellano-Plaza, M; Gschaedler, A

2013-05-01

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.

Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

PubMed

Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

2016-05-01

Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

The Saccharomyces cerevisiae enolase-related regions encode proteins that are active enolases.

PubMed

Kornblatt, M J; Richard Albert, J; Mattie, S; Zakaib, J; Dayanandan, S; Hanic-Joyce, P J; Joyce, P B M

2013-02-01

In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase-related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2-phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double-null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase-related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes. Copyright © 2012 John Wiley & Sons, Ltd.

Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

PubMed Central

Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.

1998-01-01

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. PMID:9464394

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. Copyright © 2015. Published by Elsevier B.V.

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.

[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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Anti-oxidant effects of pomegranate juice on Saccharomyces cerevisiae cell growth.

PubMed

Aslan, Abdullah; Can, Muhammed İsmail; Boydak, Didem

2014-01-01

Pomegranate juice has a number of positive effects on both human and animal subjects. Four groups were used in this study. i: Control group, ii: H2O2 group, iii: Pomegranate juice (PJ) group and iv: PJ + H2O2 group. Following the sterilization method for pomegranate juice (10%) and H2O2 (6% v/v), Saccharomyces cerevisiae cultures were added and the cultivation incubated at 35°C for 72 hours. Fatty acids and vitamin concentrations were measured using HPLC and GC and the total protein bands profile were determined by SDS-PAGE. According to our results statistically significant differences have been determined among the study groups in terms of fatty acids and vitamin (p<0,05). Fatty acid synthesis, vitamin control and cell density increased in groups to which PJ was given in comparison with the control group (p<0,05). Pomegranate juice increased vitamins, fatty acids and total protein expression in Saccharomyces cerevisiae in comparison with the control. Pomegranate juice has a positive effect on fatty acid, vitamin and protein synthesis by Saccharomyces cerevisiae. Accordingly, we believe that it has significantly decreased oxidative damage thereby making a positive impact on yeast development.

Purification of Arp2/3 complex from Saccharomyces cerevisiae

PubMed Central

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

2014-01-01

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

ALD5, PAD1, ATF1 and ATF2 facilitate the catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid in Saccharomyces cerevisiae

PubMed Central

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

2017-01-01

The ability of Saccharomyces cerevisiae to catabolize phenolic compounds remains to be fully elucidated. Conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid by S. cerevisiae under aerobic conditions was previously reported. A conversion pathway was also proposed. In the present study, possible enzymes involved in the reported conversion were investigated. Aldehyde dehydrogenase Ald5, phenylacrylic acid decarboxylase Pad1, and alcohol acetyltransferases Atf1 and Atf2, were hypothesised to be involved. Corresponding genes for the four enzymes were overexpressed in a S. cerevisiae strain named APT_1. The ability of APT_1 to tolerate and convert the three phenolic compounds was tested. APT_1 was also compared to strains B_CALD heterologously expressing coniferyl aldehyde dehydrogenase from Pseudomonas, and an ald5Δ strain, all previously reported. APT_1 exhibited the fastest conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid. Using the intermediates and conversion products of each compound, the catabolic route of coniferyl aldehyde, ferulic acid and p-coumaric acid in S. cerevisiae was studied in greater detail. PMID:28205618

Direct conversion of starch to ethanol using recombınant Saccharomyces cerevisiae containing glucoamylase gene

NASA Astrophysics Data System (ADS)

Purkan, P.; Baktir, A.; Puspaningsih, N. N. T.; Ni'mah, M.

2017-09-01

Saccharomyces cerevisiae is known for its high fermentative capacity, high ethanol yield and its high ethanol tolerance. The yeast is inability converting starch (relatively inexpensive substrate) into biofuel ethanol. Insertion of glucoamylase gene in yeast cell of Saccharomyces cerevisiae had been done to increase the yeast function in ethanol fermentation from starch. Transformation of yeast of S. cerevisiae with recombinant plasmid yEP-GLO1 carrying gene encoding glucoamylase (GLO1) produced the recombinant yeast which enable to degrade starch. Optimizing of bioconversion process of starch into ethanol by the yeast of recombinant Saccharomyces cerevisiae [yEP-GLO1] had been also done. Starch concentration which could be digested by recombinant yeast of S. cerevisiae [yEP-GLO1] was 10% (w/v). Bioconversion of starch having concentration 10% (b/v) using recombinant yeast of S. cerevisiae BY5207 [yEP-GLO1] could result ethanol as 20% (v/v) to alcoholmeter and 19,5% (v/v) to gas of chromatography. Otherwise, using recombinant yeast S. cerevisiae S. cerevisiae AS3324 [yEP-GLO1] resulted ethanol as 17% (v/v) to alcoholmeter and 17,5% (v/v) to gas of chromatography. The highest ethanol in starch bioconversion using both recombinant yeasts BY5207 and AS3324 could be resulted on 144 hours of fermentation time as well as in pH 5.

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

PubMed

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

2015-01-01

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

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

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

Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women.

PubMed

Papaemmanouil, V; Georgogiannis, N; Plega, M; Lalaki, J; Lydakis, D; Dimitriou, M; Papadimitriou, A

2011-12-01

Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare. The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus. Vaginal samples were collected from a total of 262 (asymptomatic and symptomatic) women with vaginitis attending the centre of family planning of General hospital of Piraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptibility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae. A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker's yeast. Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2017-01-01

ABSTRACT Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection. PMID:27435998

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

PubMed

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

2017-01-02

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

Saccharomyces cerevisiae: a nomadic yeast with no niche?

PubMed

Goddard, Matthew R; Greig, Duncan

2015-05-01

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. © FEMS 2015.

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.

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.

Overlap of copper and iron uptake systems in mitochondria in Saccharomyces cerevisiae

PubMed Central

Wang, Jing; Gammon, Micah G.; Maynard, Margaret K.; White, Olivia L.; Cobine, Jai A.; Mahone, Wilkerson K.

2016-01-01

In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix. PMID:26763345

Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae on alcoholic fermentation behaviour and wine aroma of cherry wines.

PubMed

Sun, Shu Yang; Gong, Han Sheng; Jiang, Xiao Man; Zhao, Yu Ping

2014-12-01

This study examined the effect of mixed fermentation of non-Saccharomyces (Torulaspora delbrueckii ZYMAFLORE Alpha(TD n. Sacch) and Metschnikowia pulcherrima JS22) and Saccharomyces cerevisiae yeasts (D254 and EC1118) on the production of cherry wines, in comparison with commonly used mono-culture. Results obtained during AF demonstrated that negligible inhibitory effect was observed in S. cerevisiae/Alpha pair, whereas a strong antagonistic effect was detected between MJS22 and S. cerevisiae strain, resulting in an early death of MJS22. For volatile components determined, S. cerevisiae/MJS22 couple was found to significantly boost the production of most detected compounds, more particularly in higher alcohols, esters, acids and terpenes; while the characteristic of S. cerevisiae/Alpha pair is an increase in fruity esters, higher alcohols and decrease in acid production. Sensory evaluation revealed that S. cerevisiae/MJS22 pair reinforced sweet, green and fatty notes to the cherry wines, and S. cerevisiae/Alpha trial enhanced the fruity odour and reduced green note. Copyright © 2014 Elsevier Ltd. All rights reserved.

Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae.

PubMed

Liu, Jidong; Zhang, Weiping; Du, Guocheng; Chen, Jian; Zhou, Jingwen

2013-12-01

Monoterpene geraniol, a compound obtained from aromatic plants, has wide applications. In this study, geraniol was synthesized in Saccharomyces cerevisiae through the introduction of geraniol synthase. To increase geraniol production, the mevalonate pathway in S. cerevisiae was genetically manipulated to enhance the supply of geranyl diphosphate, a substrate used for the biosynthesis of geraniol. Identification and optimization of the key regulatory points in the mevalonate pathway in S. cerevisiae increased geraniol production to 36.04 mg L(-1). The results obtained revealed that the IDI1-encoded isopentenyl diphosphate isomerase is a rate-limiting enzyme in the biosynthesis of geraniol in S. cerevisiae, and overexpression of MAF1, a negative regulator in tRNA biosynthesis, is another effective method to increase geraniol production in S. cerevisiae. Copyright © 2013 Elsevier B.V. All rights reserved.

[Invertase Overproduction May Provide for Inulin Fermentation by Selection Strains of Saccharomyces cerevisiae].

PubMed

Naumov, G I; Naumova, E S

2015-01-01

In some recent publications, the ability of selection strains of Saccharomyces cerevisiae to ferment inulin was attributed to inulinase activity. The review summarizes the literature data indicating that overproduction of invertase, an enzyme common to S. cerevisiae, may be responsible for this phenomenon.

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.

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

2μ plasmid in Saccharomyces species and in Saccharomyces cerevisiae.

PubMed

Strope, Pooja K; Kozmin, Stanislav G; Skelly, Daniel A; Magwene, Paul M; Dietrich, Fred S; McCusker, John H

2015-12-01

We determined that extrachromosomal 2μ plasmid was present in 67 of the Saccharomyces cerevisiae 100-genome strains; in addition to variation in the size and copy number of 2μ, we identified three distinct classes of 2μ. We identified 2μ presence/absence and class associations with populations, clinical origin and nuclear genotypes. We also screened genome sequences of S. paradoxus, S. kudriavzevii, S. uvarum, S. eubayanus, S. mikatae, S. arboricolus and S. bayanus strains for both integrated and extrachromosomal 2μ. Similar to S. cerevisiae, we found no integrated 2μ sequences in any S. paradoxus strains. However, we identified part of 2μ integrated into the genomes of some S. uvarum, S. kudriavzevii, S. mikatae and S. bayanus strains, which were distinct from each other and from all extrachromosomal 2μ. We identified extrachromosomal 2μ in one S. paradoxus, one S. eubayanus, two S. bayanus and 13 S. uvarum strains. The extrachromosomal 2μ in S. paradoxus, S. eubayanus and S. cerevisiae were distinct from each other. In contrast, the extrachromosomal 2μ in S. bayanus and S. uvarum strains were identical with each other and with one of the three classes of S. cerevisiae 2μ, consistent with interspecific transfer. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Antioxidant properties and global metabolite screening of the probiotic yeast Saccharomyces cerevisiae var. boulardii.

PubMed

Datta, Suprama; Timson, David J; Annapure, Uday S

2017-07-01

Saccharomyces cerevisiae var. boulardii is the only yeast species with probiotic properties. It is considered to have therapeutic significance in gastrointestinal disorders. In the present study, a comparative physiological study between this yeast and Saccharomyces cerevisiae (BY4742) was performed by evaluating two prominent traits of probiotic species, responses to different stress conditions and antioxidant capacity. A global metabolite profile was also developed aiming to identify which therapeutically important secondary metabolites are produced. Saccharomyces cerevisiae var. boulardii showed no significant difference in growth patterns but greater stress tolerance compared to S. cerevisiae. It also demonstrated a six- to 10-fold greater antioxidant potential (judged by the 1,1-diphenyl-2-picrylhydrazyl assay), with a 70-fold higher total phenolic content and a 20-fold higher total flavonoid content in the extracellular fraction. These features were clearly differentiated by principal component analysis and further indicated by metabolite profiling. The extracellular fraction of the S. cerevisiae var. boulardii cultures was found to be rich in polyphenolic metabolites: vanillic acid, cinnamic acid, phenyl ethyl alcohol (rose oil), erythromycin, amphetamine and vitamin B 6 , which results in the antioxidant capacity of this strain. The present study presents a new perspective for differentiating the two genetically related strains of yeast, S. cerevisiae and S. cerevisiae var. boulardii by assessing their metabolome fingerprints. In addition to the correlation of the phenotypic properties with the secretory metabolites of these two yeasts, the present study also emphasizes the potential to exploit S. cerevisiae var. boulardii in the industrial production of these metabolites. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

PubMed

Oh, Hye Ji; Moon, Hye Yun; Cheon, Seon Ah; Hahn, Yoonsoo; Kang, Hyun Ah

2016-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

Microbially induced separation of quartz from calcite using Saccharomyces cerevisiae.

PubMed

Padukone, S Usha; Natarajan, K A

2011-11-01

Cells of Saccharomyces cerevisiae and their metabolites were successfully utilized to achieve selective separation of quartz and calcite through microbially induced flotation and flocculation. S. cerevisiae was adapted to calcite and quartz minerals. Adsorption studies and electrokinetic investigations were carried out to understand the changes in the surface chemistry of yeast cells and the minerals after mutual interaction. Possible mechanisms in microbially induced flotation and flocculation are outlined. Copyright © 2011 Elsevier B.V. All rights reserved.

The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now

PubMed Central

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

2014-01-01

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

Genetic engineering of industrial strains of Saccharomyces cerevisiae.

PubMed

Le Borgne, Sylvie

2012-01-01

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

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

Complete Sequence of the Intronless Mitochondrial Genome of the Saccharomyces cerevisiae Strain CW252

PubMed Central

2018-01-01

ABSTRACT The mitochondrial genomes of Saccharomyces cerevisiae strains contain up to 13 introns. An intronless recombinant genome introduced into the nuclear background of S. cerevisiae strain W303 gave the S. cerevisiae CW252 strain, which is used to model mitochondrial respiratory pathologies. The complete sequence of this mitochondrial genome was obtained using a hybrid assembling methodology. PMID:29700138

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

Background: The limited xylose utilizing ability of native Saccharomyces cerevisiae has been a major obstacle for efficient cellulosic ethanol production from lignocellulosic materials. Haploid laboratory strains of S. cerevisiae are commonly used for genetic engineering to enable its xylose utiliza...

Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering.

PubMed

Xie, Wenping; Lv, Xiaomei; Ye, Lidan; Zhou, Pingping; Yu, Hongwei

2015-07-01

Improved supply of farnesyl diphosphate (FPP) is often considered as a typical strategy for engineering Saccharomyces cerevisiae towards efficient terpenoid production. However, in the engineered strains with enhanced precursor supply, the production of the target metabolite is often impeded by insufficient capacity of the heterologous terpenoid pathways, which limits further conversion of FPP. Here, we tried to assemble an unimpeded biosynthesis pathway by combining directed evolution and metabolic engineering in S. cerevisiae for lycopene-overproduction. First, the catalytic ability of phytoene syntheses from different sources was investigated based on lycopene accumulation. Particularly, the lycopene cyclase function of the bifunctional enzyme CrtYB from Xanthophyllomyces dendrorhous was inactivated by deletion of functional domain and directed evolution to obtain mutants with solely phytoene synthase function. Coexpression of the resulting CrtYB11M mutant along with the CrtE and CrtI genes from X. dendrorhous, and the tHMG1 gene from S. cerevisiae led to production of 4.47 mg/g DCW (Dry cell weight) of lycopene and 25.66 mg/g DCW of the by-product squalene. To further increase the FPP competitiveness of the lycopene synthesis pathway, we tried to enhance the catalytic performance of CrtE by directed evolution and created a series of pathway variants by varying the copy number of Crt genes. Finally, fed-batch fermentation was conducted for the diploid strain YXWPD-14 resulting in accumulation of 1.61 g/L (24.41 mg/g DCW) of lycopene, meanwhile, the by-production of squalene was reduced to below 1 mg/g DCW. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.

PubMed

Liu, Hong-Zhi; Wang, Qiang; Liu, Xiao-Yong; Tan, Sze-Sze

2008-12-01

Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P < 0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (P < 0.05). The spaceflight mutant of S. cerevisiae 2.0016 showed 46.7%, 62.6%, and 146.0% increment in biomass, cell wall thickness and beta-glucan content, respectively, when compared to the ground strain. Moreover, growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production.

MiYA, an efficient machine-learning workflow in conjunction with the YeastFab assembly strategy for combinatorial optimization of heterologous metabolic pathways in Saccharomyces cerevisiae.

PubMed

Zhou, Yikang; Li, Gang; Dong, Junkai; Xing, Xin-Hui; Dai, Junbiao; Zhang, Chong

2018-05-01

Facing boosting ability to construct combinatorial metabolic pathways, how to search the metabolic sweet spot has become the rate-limiting step. We here reported an efficient Machine-learning workflow in conjunction with YeastFab Assembly strategy (MiYA) for combinatorial optimizing the large biosynthetic genotypic space of heterologous metabolic pathways in Saccharomyces cerevisiae. Using β-carotene biosynthetic pathway as example, we first demonstrated that MiYA has the power to search only a small fraction (2-5%) of combinatorial space to precisely tune the expression level of each gene with a machine-learning algorithm of an artificial neural network (ANN) ensemble to avoid over-fitting problem when dealing with a small number of training samples. We then applied MiYA to improve the biosynthesis of violacein. Feed with initial data from a colorimetric plate-based, pre-screened pool of 24 strains producing violacein, MiYA successfully predicted, and verified experimentally, the existence of a strain that showed a 2.42-fold titer improvement in violacein production among 3125 possible designs. Furthermore, MiYA was able to largely avoid the branch pathway of violacein biosynthesis that makes deoxyviolacein, and produces very pure violacein. Together, MiYA combines the advantages of standardized building blocks and machine learning to accelerate the Design-Build-Test-Learn (DBTL) cycle for combinatorial optimization of metabolic pathways, which could significantly accelerate the development of microbial cell factories. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Invertase SUC2 Is the key hydrolase for inulin degradation in Saccharomyces cerevisiae.

PubMed

Wang, Shi-An; Li, Fu-Li

2013-01-01

Specific Saccharomyces cerevisiae strains were recently found to be capable of efficiently utilizing inulin, but genetic mechanisms of inulin hydrolysis in yeast remain unknown. Here we report functional characteristics of invertase SUC2 from strain JZ1C and demonstrate that SUC2 is the key enzyme responsible for inulin metabolism in S. cerevisiae.

Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

PubMed

Tran, Anh-Minh; Nguyen, Thanh-Thao; Nguyen, Cong-Thuan; Huynh-Thi, Xuan-Mai; Nguyen, Cao-Tri; Trinh, Minh-Thuong; Tran, Linh-Thuoc; Cartwright, Stephanie P; Bill, Roslyn M; Tran-Van, Hieu

2017-04-04

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.

Glucose-free fructose production from Jerusalem artichoke using a recombinant inulinase-secreting Saccharomyces cerevisiae strain.

PubMed

Yu, Jing; Jiang, Jiaxi; Ji, Wangming; Li, Yuyang; Liu, Jianping

2011-01-01

Using inulin (polyfructose) obtained from Jerusalen artichokes, we have produced fructose free of residual glucose using a recombinant inulinase-secreting strain of Saccharomyces cerevisiae in a one-step fermentation of Jerusalem artichoke tubers. For producing fructose from inulin, a recombinant inulinase-producing Saccharomyce cerevisiae strain was constructed with a deficiency in fructose uptake by disruption of two hexokinase genes hxk1 and hxk2. The inulinase gene introduced into S. cerevisiae was cloned from Kluyveromyces cicerisporus. Extracellular inulinase activity of the recombinant hxk-mutated S. cerevisiae strain reached 31 U ml(-1) after 96 h growth. When grown in a medium containing Jerusalem artichoke tubers as the sole component without any additives, the recombinant yeast accumulated fructose up to 9.2% (w/v) in the fermentation broth with only 0.1% (w/v) glucose left after 24 h.

Loss of lager specific genes and subtelomeric regions define two different Saccharomyces cerevisiae lineages for Saccharomyces pastorianus Group I and II strains.

PubMed

Monerawela, Chandre; James, Tharappel C; Wolfe, Kenneth H; Bond, Ursula

2015-03-01

Lager yeasts, Saccharomyces pastorianus, are interspecies hybrids between S. cerevisiae and S. eubayanus and are classified into Group I and Group II clades. The genome of the Group II strain, Weihenstephan 34/70, contains eight so-called 'lager-specific' genes that are located in subtelomeric regions. We evaluated the origins of these genes through bioinformatic and PCR analyses of Saccharomyces genomes. We determined that four are of cerevisiae origin while four originate from S. eubayanus. The Group I yeasts contain all four S. eubayanus genes but individual strains contain only a subset of the cerevisiae genes. We identified S. cerevisiae strains that contain all four cerevisiae 'lager-specific' genes, and distinct patterns of loss of these genes in other strains. Analysis of the subtelomeric regions uncovered patterns of loss in different S. cerevisiae strains. We identify two classes of S. cerevisiae strains: ale yeasts (Foster O) and stout yeasts with patterns of 'lager-specific' genes and subtelomeric regions identical to Group I and II S. pastorianus yeasts, respectively. These findings lead us to propose that Group I and II S. pastorianus strains originate from separate hybridization events involving different S. cerevisiae lineages. Using the combined bioinformatic and PCR data, we describe a potential classification map for industrial yeasts. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

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. © FEMS 2015.

[Thermoresistance in Saccharomyces cerevisiae yeasts].

PubMed

Kaliuzhin, V A

2011-01-01

Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.

Establishing very long-chain fatty alcohol and wax ester biosynthesis in Saccharomyces cerevisiae.

PubMed

Wenning, Leonie; Yu, Tao; David, Florian; Nielsen, Jens; Siewers, Verena

2017-05-01

Wax esters (WEs) are neutral lipids and can be used for a broad range of commercial applications, including personal care products, lubricants, or coatings. They are synthesized by enzymatic reactions catalyzed by a fatty acyl reductase (FAR) and a wax ester synthase (WS). At present, commercially used WEs are mainly isolated from Simmondsia chinensis (jojoba), but the high extraction costs and limited harvest areas constrain their use. The use of FARs in combination with different WSs to achieve a synthesis of jojoba-like WEs in bacteria and yeast has been reported previously, but the products were restricted to C28-C36 WEs. These rather short WEs make up only a very small percentage of the total WEs in natural jojoba oil. The synthesis of longer chain WEs (up to C44) in Saccharomyces cerevisiae has so far only been achieved after substrate feeding. Here we identified new routes for producing very long-chain fatty alcohols (VLCFOHs) up to a chain length of C22 by heterologous expression of a FAR derived from Apis mellifera (AmFAR1) or Marinobacter aquaeolei VT8 (Maqu_2220) in S. cerevisiae and achieved maximum yields of 3.22 ± 0.36 mg/g cell dry weight (CDW) and 7.84 ± 3.09 mg/g CDW, respectively, after 48 h. Moreover, we enabled the synthesis of jojoba-like WEs up to a chain length of C42, catalyzed by a combination of Maqu_2220 together with the WS from S. chinensis (SciWS) and the S. cerevisiae elongase Elo2p, with a maximum yield of 12.24 ± 3.35 mg/g CDW after 48 h. Biotechnol. Bioeng. 2017;114: 1025-1035. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Effect of Temperature on the Prevalence of Saccharomyces Non cerevisiae Species against a S. cerevisiae Wine Strain in Wine Fermentation: Competition, Physiological Fitness, and Influence in Final Wine Composition.

PubMed

Alonso-Del-Real, Javier; Lairón-Peris, María; Barrio, Eladio; Querol, Amparo

2017-01-01

Saccharomyces cerevisiae is the main microorganism responsible for the fermentation of wine. Nevertheless, in the last years wineries are facing new challenges due to current market demands and climate change effects on the wine quality. New yeast starters formed by non-conventional Saccharomyces species (such as S. uvarum or S. kudriavzevii ) or their hybrids ( S. cerevisiae x S. uvarum and S. cerevisiae x S. kudriavzevii ) can contribute to solve some of these challenges. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts. However, S . cerevisiae can competitively displace other yeast species from wine fermentations, therefore the use of these new starters requires an analysis of their behavior during competition with S. cerevisiae during wine fermentation. In the present study we analyzed the survival capacity of non- cerevisiae strains in competition with S. cerevisiae during fermentation of synthetic wine must at different temperatures. First, we developed a new method, based on QPCR, to quantify the proportion of different Saccharomyces yeasts in mixed cultures. This method was used to assess the effect of competition on the growth fitness. In addition, fermentation kinetics parameters and final wine compositions were also analyzed. We observed that some cryotolerant Saccharomyces yeasts, particularly S. uvarum , seriously compromised S. cerevisiae fitness during competences at lower temperatures, which explains why S. uvarum can replace S. cerevisiae during wine fermentations in European regions with oceanic and continental climates. From an enological point of view, mixed co-cultures between S. cerevisiae and S. paradoxus or S. eubayanus , deteriorated fermentation parameters and the final product composition compared to single S. cerevisiae inoculation. However, in co-inoculated synthetic must in which S. kudriavzevii or S. uvarum coexisted with S. cerevisiae , there were

Effect of Temperature on the Prevalence of Saccharomyces Non cerevisiae Species against a S. cerevisiae Wine Strain in Wine Fermentation: Competition, Physiological Fitness, and Influence in Final Wine Composition

PubMed Central

Alonso-del-Real, Javier; Lairón-Peris, María; Barrio, Eladio; Querol, Amparo

2017-01-01

Saccharomyces cerevisiae is the main microorganism responsible for the fermentation of wine. Nevertheless, in the last years wineries are facing new challenges due to current market demands and climate change effects on the wine quality. New yeast starters formed by non-conventional Saccharomyces species (such as S. uvarum or S. kudriavzevii) or their hybrids (S. cerevisiae x S. uvarum and S. cerevisiae x S. kudriavzevii) can contribute to solve some of these challenges. They exhibit good fermentative capabilities at low temperatures, producing wines with lower alcohol and higher glycerol amounts. However, S. cerevisiae can competitively displace other yeast species from wine fermentations, therefore the use of these new starters requires an analysis of their behavior during competition with S. cerevisiae during wine fermentation. In the present study we analyzed the survival capacity of non-cerevisiae strains in competition with S. cerevisiae during fermentation of synthetic wine must at different temperatures. First, we developed a new method, based on QPCR, to quantify the proportion of different Saccharomyces yeasts in mixed cultures. This method was used to assess the effect of competition on the growth fitness. In addition, fermentation kinetics parameters and final wine compositions were also analyzed. We observed that some cryotolerant Saccharomyces yeasts, particularly S. uvarum, seriously compromised S. cerevisiae fitness during competences at lower temperatures, which explains why S. uvarum can replace S. cerevisiae during wine fermentations in European regions with oceanic and continental climates. From an enological point of view, mixed co-cultures between S. cerevisiae and S. paradoxus or S. eubayanus, deteriorated fermentation parameters and the final product composition compared to single S. cerevisiae inoculation. However, in co-inoculated synthetic must in which S. kudriavzevii or S. uvarum coexisted with S. cerevisiae, there were fermentation

Fumaric acid production in Saccharomyces cerevisiae by simultaneous use of oxidative and reductive routes.

PubMed

Xu, Guoqiang; Chen, Xiulai; Liu, Liming; Jiang, Linghuo

2013-11-01

In this study, the simultaneous use of reductive and oxidative routes to produce fumaric acid was explored. The strain FMME003 (Saccharomyces cerevisiae CEN.PK2-1CΔTHI2) exhibited capability to accumulate pyruvate and was used for fumaric acid production. The fum1 mutant FMME004 could produce fumaric acid via oxidative route, but the introduction of reductive route derived from Rhizopus oryzae NRRL 1526 led to lower fumaric acid production. Analysis of the key factors associated with fumaric acid production revealed that pyruvate carboxylase had a low degree of control over the carbon flow to malic acid. The fumaric acid titer was improved dramatically when the heterologous gene RoPYC was overexpressed and 32 μg/L of biotin was added. Furthermore, under the optimal carbon/nitrogen ratio, the engineered strain FMME004-6 could produce up to 5.64 ± 0.16 g/L of fumaric acid. These results demonstrated that the proposed fermentative method is efficient for fumaric acid production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification and characterization of Saccharomyces cerevisiae strains isolated from West African sorghum beer.

PubMed

van der Aa Kühle, A; Jesperen, L; Glover, R L; Diawara, B; Jakobsen, M

2001-08-01

The occurrence and characterization of yeasts isolated from sorghum beer produced in Ghana and Burkina Faso, West Africa, were investigated. The yeasts involved in the fermentations were found to consist of Saccharomyces spp. almost exclusively. Of the isolates investigated, 45% were identified as Saccharomyces cerevisiae, whereas more than half of the isolates (53%) had physiological properties atypical of S. cerevisiae or any other member of the complex sensu strictu, as they were able to assimilate only glucose, maltose and ethanol as carbon sources. Both ITS-PCR RFLP and PFGE strongly indicated that these isolates were related to S. cerevisiae, regardless of their phenotypic characteristics. Sequencing of the D1/D2 domain of the 26S rDNA confirmed the close relatedness to S. cerevisiae with 0.5% nucleotide differences. The MAL1 and MAL3 loci were found for all isolates as the only recognized MAL loci. Besides, for 40% of the isolates the MAL61 probe hybridized to a position of about 950 kbp, which has not formerly been described as a MAL locus. The results showed that the spontaneous fermentation of West African sorghum beer is dominated by a variety of strains of S.cerevisiae not previously described, among which starter cultures should be selected. Copyright 2001 John Wiley & Sons, Ltd.

Microaerobic glycerol formation in Saccharomyces cerevisiae.

PubMed

Costenoble, R; Valadi, H; Gustafsson, L; Niklasson, C; Franzén, C J

2000-12-01

The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD(+)-dependent glycerol-3-phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol-3-phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae. Copyright 2000 John Wiley & Sons, Ltd.

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. © 2013 John Wiley & Sons Ltd.

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.

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

PubMed

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

2008-04-30

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

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…

Response of Saccharomyces cerevisiae to D-limonene-induced oxidative stress.

PubMed

Liu, Jidong; Zhu, Yibo; Du, Guocheng; Zhou, Jingwen; Chen, Jian

2013-07-01

In the present study, we investigated the mode of cell response induced by D-limonene in Saccharomyces cerevisiae. D-limonene treatment was found to be accompanied by intracellular accumulation of reactive oxygen species (ROS). Since ROS impair cell membranes, an engineered strain with enhanced membrane biosynthesis exhibited a higher tolerance to D-limonene. Subsequent addition of an ROS scavenger significantly reduced the ROS level and alleviated cell growth inhibition. Thus, D-limonene-induced ROS accumulation plays an important role in cell death in S. cerevisiae. In D-limonene-treated S. cerevisiae strains, higher levels of antioxidants, antioxidant enzymes, and nicotinamide adenine dinucleotide phosphate (NADPH) were synthesized. Quantitative real-time PCR results also verified that D-limonene treatment triggered upregulation of genes involved in the antioxidant system and the regeneration of NADPH at the transcription level in S. cerevisiae. These data indicate that D-limonene treatment results in intracellular ROS accumulation, an important factor in cell death, and several antioxidant mechanisms in S. cerevisiae were enhanced in response to D-limonene treatment.

Omics analysis of acetic acid tolerance in Saccharomyces cerevisiae.

PubMed

Geng, Peng; Zhang, Liang; Shi, Gui Yang

2017-05-01

Acetic acid is an inhibitor in industrial processes such as wine making and bioethanol production from cellulosic hydrolysate. It causes energy depletion, inhibition of metabolic enzyme activity, growth arrest and ethanol productivity losses in Saccharomyces cerevisiae. Therefore, understanding the mechanisms of the yeast responses to acetic acid stress is essential for improving acetic acid tolerance and ethanol production. Although 329 genes associated with acetic acid tolerance have been identified in the Saccharomyces genome and included in the database ( http://www.yeastgenome.org/observable/resistance_to_acetic_acid/overview ), the cellular mechanistic responses to acetic acid remain unclear in this organism. Post-genomic approaches such as transcriptomics, proteomics, metabolomics and chemogenomics are being applied to yeast and are providing insight into the mechanisms and interactions of genes, proteins and other components that together determine complex quantitative phenotypic traits such as acetic acid tolerance. This review focuses on these omics approaches in the response to acetic acid in S. cerevisiae. Additionally, several novel strains with improved acetic acid tolerance have been engineered by modifying key genes, and the application of these strains and recently acquired knowledge to industrial processes is also discussed.

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

PubMed Central

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

2012-01-01

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

Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae.

PubMed

Chardwiriyapreecha, Soracom; Manabe, Kunio; Iwaki, Tomoko; Kawano-Kawada, Miyuki; Sekito, Takayuki; Lunprom, Siriporn; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2015-01-01

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.

Clinical Saccharomyces cerevisiae isolates cannot cross the epithelial barrier in vitro.

PubMed

Pérez-Torrado, Roberto; Llopis, Silvia; Jespersen, Lene; Fernández-Espinar, Teresa; Querol, Amparo

2012-06-15

Saccharomyces cerevisiae is generally considered to be a safe organism and is essential to produce many different kinds of foods as well as being widely used as a dietary supplement. However, several isolates, which are genetically related to brewing and baking yeasts, have shown virulent traits, being able to produce human infections in immunodeficient patients. Previously it has been shown that the administration of S. cerevisiae clinical isolates can lead to systemic infections, reaching several organs in murine systems. In this work, we studied S. cerevisiae clinical isolates in an in vitro intestinal epithelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. The results showed that, in contrast to C. glabrata and C. albicans, S. cerevisiae was not able to cross the intestinal barrier. We concluded that S. cerevisiae can only perform opportunistic or passive crossings when epithelial barrier integrity is previously compromised. Copyright © 2012 Elsevier B.V. All rights reserved.

Oral administration of myostatin-specific recombinant Saccharomyces cerevisiae vaccine in rabbit.

PubMed

Liu, Zhongtian; Zhou, Gang; Ren, Chonghua; Xu, Kun; Yan, Qiang; Li, Xinyi; Zhang, Tingting; Zhang, Zhiying

2016-04-29

Yeast is considered as a simple and cost-effective host for protein expression, and our previous studies have proved that Saccharomyces cerevisiae can deliver recombinant protein and DNA into mouse dendritic cells and can further induce immune responses as novel vaccines. In order to know whether similar immune responses can be induced in rabbit by oral administration of such recombinant S. cerevisiae vaccine, we orally fed the rabbits with heat-inactivated myostatin-recombinant S. cerevisiae for 5 weeks, and then myostatin-specific antibody in serum was detected successfully by western blotting and ELISA assay. The rabbits treated with myostatin-recombinant S. cerevisiae vaccine grew faster and their muscles were much heavier than that of the control group. As a common experimental animal and a meat livestock with great economic value, rabbit was proved to be the second animal species that have been successfully orally immunized by recombinant S. cerevisiae vaccine after mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Repurposing the Saccharomyces cerevisiae peroxisome for compartmentalizing multi-enzyme pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeLoache, William; Russ, Zachary; Samson, Jennifer

The peroxisome of Saccharomyces cerevisiae was targeted for repurposing in order to create a synthetic organelle that provides a generalizable compartment for engineered metabolic pathways. Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk, improving pathway efficiency, and ultimately modifying the chemical environment to be distinct from that of the cytoplasm. We focused on the Saccharomyces cerevisiae peroxisome, as this organelle is not required for viability when grown on conventional media. We identified an enhanced peroxisomal targeting signal type 1 (PTS1) for rapidly importing non-native cargo proteins. Additionally, we performed the first systematic in vivo measurementsmore » of nonspecific metabolite permeability across the peroxisomal membrane using a polymer exclusion assay and characterized the size dependency of metabolite trafficking. Finally, we applied these new insights to compartmentalize a two-enzyme pathway in the peroxisome and characterize the expression regimes where compartmentalization leads to improved product titer. This work builds a foundation for using the peroxisome as a synthetic organelle, highlighting both promise and future challenges on the way to realizing this goal.« less

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.

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks.

PubMed

d'Espaux, Leo; Ghosh, Amit; Runguphan, Weerawat; Wehrs, Maren; Xu, Feng; Konzock, Oliver; Dev, Ishaan; Nhan, Melissa; Gin, Jennifer; Reider Apel, Amanda; Petzold, Christopher J; Singh, Seema; Simmons, Blake A; Mukhopadhyay, Aindrila; García Martín, Héctor; Keasling, Jay D

2017-07-01

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2g/L fatty alcohols in shake flasks, and 6.0g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products. Published by Elsevier Inc.

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. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Kluyveromyces marxianus as a host for heterologous protein synthesis.

PubMed

Gombert, Andreas K; Madeira, José Valdo; Cerdán, María-Esperanza; González-Siso, María-Isabel

2016-07-01

The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

Metabolic Engineering of Probiotic Saccharomyces boulardii

PubMed Central

Liu, Jing-Jing; Kong, In Iok; Zhang, Guo-Chang; Jayakody, Lahiru N.; Kim, Heejin; Xia, Peng-Fei; Kwak, Suryang; Sung, Bong Hyun; Sohn, Jung-Hoon; Walukiewicz, Hanna E.; Rao, Christopher V.

2016-01-01

Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae. Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2, ura3, his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools for S. cerevisiae. We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome of S. boulardii. We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii. Our results suggest that more sophisticated genetic perturbations to improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii. PMID:26850302

Metabolic Engineering of Probiotic Saccharomyces boulardii.

PubMed

Liu, Jing-Jing; Kong, In Iok; Zhang, Guo-Chang; Jayakody, Lahiru N; Kim, Heejin; Xia, Peng-Fei; Kwak, Suryang; Sung, Bong Hyun; Sohn, Jung-Hoon; Walukiewicz, Hanna E; Rao, Christopher V; Jin, Yong-Su

2016-04-01

Saccharomyces boulardiiis a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae Therefore, S. boulardiiis an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2,ura3,his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools forS. cerevisiae We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome ofS. boulardii We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii Our results suggest that more sophisticated genetic perturbations to improveS. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineeredS. boulardii. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Sporulation in the Budding Yeast Saccharomyces cerevisiae

PubMed Central

Neiman, Aaron M.

2011-01-01

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae. PMID:22084423

K2 killer toxin-induced physiological changes in the yeast Saccharomyces cerevisiae.

PubMed

Orentaite, Irma; Poranen, Minna M; Oksanen, Hanna M; Daugelavicius, Rimantas; Bamford, Dennis H

2016-03-01

Saccharomyces cerevisiae cells produce killer toxins, such as K1, K2 and K28, that can modulate the growth of other yeasts giving advantage for the killer strains. Here we focused on the physiological changes induced by K2 toxin on a non-toxin-producing yeast strain as well as K1, K2 and K28 killer strains. Potentiometric measurements were adjusted to observe that K2 toxin immediately acts on the sensitive cells leading to membrane permeability. This correlated with reduced respiration activity, lowered intracellular ATP content and decrease in cell viability. However, we did not detect any significant ATP leakage from the cells treated by killer toxin K2. Strains producing heterologous toxins K1 and K28 were less sensitive to K2 than the non-toxin producing one suggesting partial cross-protection between the different killer systems. This phenomenon may be connected to the observed differences in respiratory activities of the killer strains and the non-toxin-producing strain at low pH. This might also have practical consequences in wine industry; both as beneficial ones in controlling contaminating yeasts and non-beneficial ones causing sluggish fermentation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

High hydrostatic pressure and the cell membrane: stress response of Saccharomyces cerevisiae.

PubMed

Bravim, Fernanda; de Freitas, Jéssica M; Fernandes, A Alberto R; Fernandes, Patricia M B

2010-02-01

The brewing and baking yeast Saccharomyces cerevisiae is a useful eukaryotic model of stress response systems whose study could lead to the understanding of stress response mechanisms in other organisms. High hydrostatic pressure (HHP) exerts broad effects upon yeast cells, interfering with cell membranes, cellular architecture, and the processes of polymerization and denaturation of proteins. In this review, we focus on the effect of HHP on the S. cerevisiae cell membrane and describe the main signaling pathways involved in the pressure response.

[Saccharomyces cerevisiae invasive infection: The first reported case in Morocco].

PubMed

Maleb, A; Sebbar, E; Frikh, M; Boubker, S; Moussaoui, A; El Mekkaoui, A; Khannoussi, W; Kharrasse, G; Belefquih, B; Lemnouer, A; Ismaili, Z; Elouennass, M

2017-06-01

Saccharomyces cerevisiae is a cosmopolitan yeast, widely used in agro-alimentary and pharmaceutical industry. Its impact in human pathology is rare, but maybe still underestimated compared to the real situation. This yeast is currently considered as an emerging and opportunistic pathogen. Risk factors are immunosuppression and intravascular device carrying. Fungemias are the most frequent clinical forms. We report the first case of S. cerevisiae invasive infection described in Morocco, and to propose a review of the literature cases of S. cerevisiae infections described worldwide. A 77-year-old patient, with no notable medical history, who was hospitalized for a upper gastrointestinal stenosis secondary to impassable metastatic gastric tumor. Its history was marked by the onset of septic shock, with S. cerevisiae in his urine and in his blood, with arguments for confirmation of invasion: the presence of several risk factors in the patient, positive direct microbiological examination, abundant and exclusive culture of S. cerevisiae from clinical samples. Species identification was confirmed by the study of biochemical characteristics of the isolated yeast. Confirmation of S. cerevisiae infection requires a clinical suspicion in patients with risk factors, but also a correct microbiological diagnosis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein*

PubMed Central

Vest, Katherine E.; Leary, Scot C.; Winge, Dennis R.; Cobine, Paul A.

2013-01-01

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria. PMID:23846699

Copper import into the mitochondrial matrix in Saccharomyces cerevisiae is mediated by Pic2, a mitochondrial carrier family protein.

PubMed

Vest, Katherine E; Leary, Scot C; Winge, Dennis R; Cobine, Paul A

2013-08-16

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.

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.

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae.

PubMed

Madhavan, Anjali; Srivastava, Aradhana; Kondo, Akihiko; Bisaria, Virendra S

2012-03-01

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.

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

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...

Enological characterization of Spanish Saccharomyces kudriavzevii strains, one of the closest relatives to parental strains of winemaking and brewing Saccharomyces cerevisiae × S. kudriavzevii hybrids.

PubMed

Peris, D; Pérez-Través, L; Belloch, C; Querol, A

2016-02-01

Wine fermentation and innovation have focused mostly on Saccharomyces cerevisiae strains. However, recent studies have shown that other Saccharomyces species can also be involved in wine fermentation or are useful for wine bouquet, such as Saccharomyces uvarum and Saccharomyces paradoxus. Many interspecies hybrids have also been isolated from wine fermentation, such as S. cerevisiae × Saccharomyces kudriavzevii hybrids. In this study, we explored the genetic diversity and fermentation performance of Spanish S. kudriavzevii strains, which we compared to other S. kudriavzevii strains. Fermentations of red and white grape musts were performed, and the phenotypic differences between Spanish S. kudriavzevii strains under different temperature conditions were examined. An ANOVA analysis suggested striking similarity between strains for glycerol and ethanol production, although a high diversity of aromatic profiles among fermentations was found. The sources of these phenotypic differences are not well understood and require further investigation. Although the Spanish S. kudriavzevii strains showed desirable properties, particularly must fermentations, the quality of their wines was no better than those produced with a commercial S. cerevisiae. We suggest hybridization or directed evolution as methods to improve and innovate wine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Induction of a Mitosis Delay and Cell Lysis by High-Level Secretion of Mouse α-Amylase from Saccharomyces cerevisiae

PubMed Central

Wang, Bi-Dar; Kuo, Tsong-Teh

2001-01-01

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse α-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in α-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of α-amylase. Our data also suggest that high levels of α-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous α-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis. PMID:11472949

Investigation of the Best Saccharomyces cerevisiae Growth Condition.

PubMed

Salari, Roshanak; Salari, Rosita

2017-01-01

Saccharomyces cerevisiae is known as one of the useful yeasts which are utilized in baking and other industries. It can be easily cultured at an economic price. Today the introduction of safe and efficient carriers is being considered. Due to its generally round shape, and the volume that is enclosed by its membrane and cell wall, it is used to encapsulate active materials to protect them from degradation or to introduce a sustained release drug delivery system. Providing the best conditions in order to achieve the best morphological properties of Saccharomyces cerevisiae as a carrier. In this research, the most suitable growth condition of yeast cells which provides the best size for use as drug carriers was found by a bioreactor in a synthetic culture medium. Yeast cell reproduction and growth curves were obtained, based on pour plate colony counting data and UV/Visible sample absorption at 600 nm. Yeast cell growth patterns and growth rates were determined by Matlab mathematical software. Results showed that pH=4 and dissolving oxygen (DO) 5% was the best condition for yeast cells to grow and reproduce. This condition also provided the largest size (2 × 3 μ) yeast cells. Owing to the yeast cells' low-cost production and their structural characteristics, they could be used as potent drug carriers. This work was supported by a grant from the Vice Chancellor of Research of Mashhad University of Medical Sciences.

The new modern era of yeast genomics: community sequencing and the resulting annotation of multiple Saccharomyces cerevisiae strains at the Saccharomyces Genome Database

PubMed Central

Engel, Stacia R.; Cherry, J. Michael

2013-01-01

The first completed eukaryotic genome sequence was that of the yeast Saccharomyces cerevisiae, and the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) is the original model organism database. SGD remains the authoritative community resource for the S. cerevisiae reference genome sequence and its annotation, and continues to provide comprehensive biological information correlated with S. cerevisiae genes and their products. A diverse set of yeast strains have been sequenced to explore commercial and laboratory applications, and a brief history of those strains is provided. The publication of these new genomes has motivated the creation of new tools, and SGD will annotate and provide comparative analyses of these sequences, correlating changes with variations in strain phenotypes and protein function. We are entering a new era at SGD, as we incorporate these new sequences and make them accessible to the scientific community, all in an effort to continue in our mission of educating researchers and facilitating discovery. Database URL: http://www.yeastgenome.org/ PMID:23487186

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation.

PubMed

Yamaoka, Chizuru; Kurita, Osamu; Kubo, Tomoko

2014-12-01

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool. Copyright © 2014 Elsevier GmbH. All rights reserved.

Preparation of cell-free splicing extracts from Saccharomyces cerevisiae.

PubMed

Ares, Manuel

2013-10-01

Much of our understanding of the mechanism of splicing comes from the analysis of cell extracts able to carry out splicing complex formation and splicing reactions in vitro using exogenously added synthetic model pre-mRNA transcripts. This protocol describes the preparation of whole-cell extracts from the budding yeast Saccharomyces cerevisiae. These extracts can be used to dissect the biochemical steps of the splicing reaction and to determine the macromolecules, cofactors, and substrate features necessary for successful splicing.

The Oenological Potential of Hanseniaspora uvarum in Simultaneous and Sequential Co-fermentation with Saccharomyces cerevisiae for Industrial Wine Production

PubMed Central

Tristezza, Mariana; Tufariello, Maria; Capozzi, Vittorio; Spano, Giuseppe; Mita, Giovanni; Grieco, Francesco

2016-01-01

In oenology, the utilization of mixed starter cultures composed by Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers in order to enhance sensory quality and complexity of the final product without compromising the general quality and safety of the oenological products. In fact, several non-Saccharomyces yeasts are already commercialized as oenological starter cultures to be used in combination with Saccharomyces cerevisiae, while several others are the subject of various studies to evaluate their application. Our aim, in this study was to assess, for the first time, the oenological potential of H. uvarum in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for industrial wine production. Three previously characterized H. uvarum strains were separately used as multi-starter together with an autochthonous S. cerevisiae starter culture in lab-scale micro-vinification trials. On the basis of microbial development, fermentation kinetics and secondary compounds formation, the strain H. uvarum ITEM8795 was further selected and it was co- and sequentially inoculated, jointly with the S. cerevisiae starter, in a pilot scale wine production. The fermentation course and the quality of final product indicated that the co-inoculation was the better performing modality of inoculum. The above results were finally validated by performing an industrial scale vinification The mixed starter was able to successfully dominate the different stages of the fermentation process and the H. uvarum strain ITEM8795 contributed to increasing the wine organoleptic quality and to simultaneously reduce the volatile acidity. At the best of our knowledge, the present report is the first study regarding the utilization of a selected H. uvarum strain in multi-starter inoculation with S. cerevisiae for the industrial production of a wine. In addition, we demonstrated, at an industrial scale, the importance of non-Saccharomyces in

The Oenological Potential of Hanseniaspora uvarum in Simultaneous and Sequential Co-fermentation with Saccharomyces cerevisiae for Industrial Wine Production.

PubMed

Tristezza, Mariana; Tufariello, Maria; Capozzi, Vittorio; Spano, Giuseppe; Mita, Giovanni; Grieco, Francesco

2016-01-01

In oenology, the utilization of mixed starter cultures composed by Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers in order to enhance sensory quality and complexity of the final product without compromising the general quality and safety of the oenological products. In fact, several non-Saccharomyces yeasts are already commercialized as oenological starter cultures to be used in combination with Saccharomyces cerevisiae, while several others are the subject of various studies to evaluate their application. Our aim, in this study was to assess, for the first time, the oenological potential of H. uvarum in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for industrial wine production. Three previously characterized H. uvarum strains were separately used as multi-starter together with an autochthonous S. cerevisiae starter culture in lab-scale micro-vinification trials. On the basis of microbial development, fermentation kinetics and secondary compounds formation, the strain H. uvarum ITEM8795 was further selected and it was co- and sequentially inoculated, jointly with the S. cerevisiae starter, in a pilot scale wine production. The fermentation course and the quality of final product indicated that the co-inoculation was the better performing modality of inoculum. The above results were finally validated by performing an industrial scale vinification The mixed starter was able to successfully dominate the different stages of the fermentation process and the H. uvarum strain ITEM8795 contributed to increasing the wine organoleptic quality and to simultaneously reduce the volatile acidity. At the best of our knowledge, the present report is the first study regarding the utilization of a selected H. uvarum strain in multi-starter inoculation with S. cerevisiae for the industrial production of a wine. In addition, we demonstrated, at an industrial scale, the importance of non-Saccharomyces in

Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP.

PubMed

McIntosh, E M; Kunz, B A; Haynes, R H

1986-01-01

Cytosine arabinoside (araC), a potent inhibitor of DNA replication in mammalian cells, was found to be completely ineffective in Saccharomyces cerevisiae. The 5' monophosphate derivative, araCMP, is toxic and effectively inhibits both nuclear and mitochondrial DNA synthesis in this organism. Although wild-type strains can be inhibited by araCMP, dTMP permeable (tup-) strains were found to be much more sensitive to the analogue. In vivo labelling experiments indicate that araC enters yeast cells; however, it is extensively catabolized by deamination and breakage of the glycosidic bond. In addition, the analogue is not efficiently phosphorylated in S. cerevisiae owing to an apparent lack of deoxynucleoside kinase activity. These results provide further evidence that deoxyribonucleotides can be synthesized only through de novo pathways in this organism. Finally, araCMP was found to be recombinagenic in S. cerevisiae which suggests, together with other previous studies, that, in general, inhibition of DNA synthesis in yeast promotes mitotic recombination events.

Ethanol production by Saccharomyces cerevisiae using lignocellulosic hydrolysate from Chrysanthemum waste degradation.

PubMed

Quevedo-Hidalgo, Balkys; Monsalve-Marín, Felipe; Narváez-Rincón, Paulo César; Pedroza-Rodríguez, Aura Marina; Velásquez-Lozano, Mario Enrique

2013-03-01

Ethanol production derived from Saccharomyces cerevisiae fermentation of a hydrolysate from floriculture waste degradation was studied. The hydrolysate was produced from Chrysanthemum (Dendranthema grandiflora) waste degradation by Pleurotus ostreatus and characterized to determine the presence of compounds that may inhibit fermentation. The products of hydrolysis confirmed by HPLC were cellobiose, glucose, xylose and mannose. The hydrolysate was fermented by S. cerevisiae, and concentrations of biomass, ethanol, and glucose were determined as a function of time. Results were compared to YGC modified medium (yeast extract, glucose and chloramphenicol) fermentation. Ethanol yield was 0.45 g g(-1), 88 % of the maximal theoretical value. Crysanthemum waste hydrolysate was suitable for ethanol production, containing glucose and mannose with adequate nutrients for S. cerevisiae fermentation and low fermentation inhibitor levels.

Radioprotective effect of orally administered beta-d-glucan derived from Saccharomyces cerevisiae.

PubMed

Liu, Fang; Wang, Zhuanzi; Liu, Jia; Li, Wenjian

2018-04-21

The present study was to evaluate the in vivo radioprotective effect of oral administration of Saccharomyces cerevisiae-derived-beta-d-glucan (S. cerevisiae-BG) and to investigate the protective mechanism. The results demonstrated that oral pretreatment with 350 mg/kg S. cerevisiae-BG once daily for 14 consecutive days significantly increased the survival rate of mice from 6 Gy X-rays irradiation. At the 30th day after irradiation, cellularity and the percentage of hematopoietic stem/progenitor cells in bone marrow (BM) of surviving mice were increased by S. cerevisiae-BG. Further studies showed that S. cerevisiae-BG decreased BM cell DNA damage and improved BM cell cycle progress in irradiated mice. And the reactive oxygen species (ROS) levels in BM cells of irradiated mice were also decreased by S. cerevisiae-BG. These results indicated that oral S. cerevisiae-BG exhibited obviously radioprotective effect in mice and the protective effect may be attributed to the polysaccharide's hematopoiesis-modulating action and free radical scavenging property. S. cerevisiae-BG protects BM cells from radiation damage through scavenging BM cell ROS, mitigating BM cell DNA damage and improving cell cycle progress, and thus mitigated myelosuppression induced by irradiation and stimulated hematopoiesis, ultimately increased the survival of radiated mice. Copyright © 2018. Published by Elsevier B.V.

Screening and evolution of a novel protist xylose isomerase from the termite Reticulitermes speratus for efficient xylose fermentation in Saccharomyces cerevisiae.

PubMed

Katahira, Satoshi; Muramoto, Nobuhiko; Moriya, Shigeharu; Nagura, Risa; Tada, Nobuki; Yasutani, Noriko; Ohkuma, Moriya; Onishi, Toru; Tokuhiro, Kenro

2017-01-01

The yeast Saccharomyces cerevisiae , a promising host for lignocellulosic bioethanol production, is unable to metabolize xylose. In attempts to confer xylose utilization ability in S. cerevisiae , a number of xylose isomerase (XI) genes have been expressed heterologously in this yeast. Although several of these XI encoding genes were functionally expressed in S. cerevisiae , the need still exists for a S. cerevisiae strain with improved xylose utilization ability for use in the commercial production of bioethanol. Although currently much effort has been devoted to achieve the objective, one of the solutions is to search for a new XI gene that would confer superior xylose utilization in S. cerevisiae . Here, we searched for novel XI genes from the protists residing in the hindgut of the termite Reticulitermes speratus . Eight novel XI genes were obtained from a cDNA library, prepared from the protists of the R. speratus hindgut, by PCR amplification using degenerated primers based on highly conserved regions of amino acid sequences of different XIs. Phylogenetic analysis classified these cloned XIs into two groups, one showed relatively high similarities to Bacteroidetes and the other was comparatively similar to Firmicutes . The growth rate and the xylose consumption rate of the S. cerevisiae strain expressing the novel XI, which exhibited highest XI activity among the eight XIs, were superior to those exhibited by the strain expressing the XI gene from Piromyces sp. E2. Substitution of the asparagine residue at position 337 of the novel XI with a cysteine further improved the xylose utilization ability of the yeast strain. Interestingly, introducing point mutations in the corresponding asparagine residues in XIs originated from other organisms, such as Piromyces sp. E2 or Clostridium phytofermentans , similarly improved xylose utilization in S. cerevisiae . A novel XI gene conferring superior xylose utilization in S. cerevisiae was successfully isolated from the

Interactions between Lactobacillus kefiranofaciens and Saccharomyces cerevisiae in mixed culture for kefiran production.

PubMed

Cheirsilp, Benjamas; Shoji, Hirofumi; Shimizu, Hiroshi; Shioya, Suteaki

2003-01-01

Since a positive effect on the growth and kefiran production of Lactobacillus kefiranofaciens was observed in a mixed culture with Saccharomyces cerevisiae, the elucidation of the interactions between L. kefiranofaciens and S. cerevisiae may lead to higher productivity. Hence, the microbial interaction of each strain was investigated. Apart from the positive effect of a reduction in the amount of lactic acid by S. cerevisiae, a positive effect of S. cerevisiae on the growth and kefiran production of L. kefiranofaciens in a mixed culture was observed. Various experiments were carried out to study this effect. In this study, the observed increase in capsular kefiran in a mixed culture with inactivated S. cerevisiae correlated well to that in an anaerobic mixed culture. Differences in capsular kefiran production were observed for different initial S. cerevisiae concentrations under anaerobic conditions. From these fermentation results, it was concluded that the physical contact with S. cerevisiae mainly enhanced the capsular kefiran production of L. kefiranofaciens in a mixed culture. Therefore, in an anaerobic mixed culture, this direct contact resulted in higher capsular kefiran production than that in pure culture.

Secretory Overexpression of Bacillus thermocatenulatus Lipase in Saccharomyces cerevisiae Using Combinatorial Library Strategy.

PubMed

Kajiwara, Shota; Yamada, Ryosuke; Ogino, Hiroyasu

2018-04-10

Simple and cost-effective lipase expression host microorganisms are highly desirable. A combinatorial library strategy is used to improve the secretory expression of lipase from Bacillus thermocatenulatus (BTL2) in the culture supernatant of Saccharomyces cerevisiae. A plasmid library including expression cassettes composed of sequences encoding one of each 15 promoters, 15 secretion signals, and 15 terminators derived from yeast species, S. cerevisiae, Pichia pastoris, and Hansenula polymorpha, is constructed. The S. cerevisiae transformant YPH499/D4, comprising H. polymorpha GAP promoter, S. cerevisiae SAG1 secretion signal, and P. pastoris AOX1 terminator, is selected by high-throughput screening. This transformant expresses BTL2 extra-cellularly with a 130-fold higher than the control strain, comprising S. cerevisiae PGK1 promoter, S. cerevisiae α-factor secretion signal, and S. cerevisiae PGK1 terminator, after cultivation for 72 h. This combinatorial library strategy holds promising potential for application in the optimization of the secretory expression of proteins in yeast. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reconstruction of the High-Osmolarity Glycerol (HOG) Signaling Pathway from the Halophilic Fungus Wallemia ichthyophaga in Saccharomyces cerevisiae.

PubMed

Konte, Tilen; Terpitz, Ulrich; Plemenitaš, Ana

2016-01-01

The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like other fungi, W. ichthyophaga detects changes in environmental salinity mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with protein modeling data, our study reveals conserved and non-conserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.

Division of labour in the yeast: Saccharomyces cerevisiae.

PubMed

Wloch-Salamon, Dominika M; Fisher, Roberta M; Regenberg, Birgitte

2017-10-01

Division of labour between different specialized cell types is a central part of how we describe complexity in multicellular organisms. However, it is increasingly being recognized that division of labour also plays an important role in the lives of predominantly unicellular organisms. Saccharomyces cerevisiae displays several phenotypes that could be considered a division of labour, including quiescence, apoptosis and biofilm formation, but they have not been explicitly treated as such. We discuss each of these examples, using a definition of division of labour that involves phenotypic variation between cells within a population, cooperation between cells performing different tasks and maximization of the inclusive fitness of all cells involved. We then propose future research directions and possible experimental tests using S. cerevisiae as a model organism for understanding the genetic mechanisms and selective pressures that can lead to the evolution of the very first stages of a division of labour. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Energy-dependent effects of resveratrol in Saccharomyces cerevisiae.

PubMed

Madrigal-Perez, Luis Alberto; Canizal-Garcia, Melina; González-Hernández, Juan Carlos; Reynoso-Camacho, Rosalia; Nava, Gerardo M; Ramos-Gomez, Minerva

2016-06-01

The metabolic effects induced by resveratrol have been associated mainly with the consumption of high-calorie diets; however, its effects with standard or low-calorie diets remain unclear. To better understand the interactions between resveratrol and cellular energy levels, we used Saccharomyces cerevisiae as a model. Herein it is shown that resveratrol: (a) decreased cell viability in an energy-dependent manner; (b) lessening of cell viability occurred specifically when cells were under cellular respiration; and (c) inhibition of oxygen consumption in state 4 occurred at low and standard energy levels, whereas at high energy levels oxygen consumption was promoted. These findings indicate that the effects of resveratrol are dependent on the cellular energy status and linked to metabolic respiration. Importantly, our study also revealed that S. cerevisiae is a suitable and useful model to elucidate the molecular targets of resveratrol under different nutritional statuses. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Habitat Predicts Levels of Genetic Admixture in Saccharomyces cerevisiae.

PubMed

Tilakaratna, Viranga; Bensasson, Douda

2017-09-07

Genetic admixture can provide material for populations to adapt to local environments, and this process has played a crucial role in the domestication of plants and animals. The model yeast, Saccharomyces cerevisiae , has been domesticated multiple times for the production of wine, sake, beer, and bread, but the high rate of admixture between yeast lineages has so far been treated as a complication for population genomic analysis. Here, we make use of the low recombination rate at centromeres to investigate admixture in yeast using a classic Bayesian approach and a locus-by-locus phylogenetic approach. Using both approaches, we find that S. cerevisiae from stable oak woodland habitats are less likely to show recent genetic admixture compared with those isolated from transient habitats such as fruits, wine, or human infections. When woodland yeast strains do show recent genetic admixture, the degree of admixture is lower than in strains from other habitats. Furthermore, S. cerevisiae populations from oak woodlands are genetically isolated from each other, with only occasional migration between woodlands and local fruit habitats. Application of the phylogenetic approach suggests that there is a previously undetected population in North Africa that is the closest outgroup to the European S. cerevisiae , including the domesticated Wine population. Careful testing for admixture in S. cerevisiae leads to a better understanding of the underlying population structure of the species and will be important for understanding the selective processes underlying domestication in this economically important species. Copyright © 2017 Tilakaratna and Bensasson.

Chromosome Duplication in Saccharomyces cerevisiae

PubMed Central

Bell, Stephen P.; Labib, Karim

2016-01-01

The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. PMID:27384026

Saccharomyces cerevisiae × Saccharomyces uvarum hybrids generated under different conditions share similar winemaking features.

PubMed

Origone, Andrea Cecilia; Rodríguez, María Eugenia; Oteiza, Juan Martín; Querol, Amparo; Lopes, Christian Ariel

2018-01-01

Interspecific hybrids among species in the Saccharomyces genus are frequently detected in anthropic habitats and can also be obtained easily in the laboratory. This occurs because the most important genetic barriers among Saccharomyces species are post-zygotic. Depending on several factors, including the involved strains, the hybridization mechanism and stabilization conditions, hybrids that bear differential genomic constitutions, and hence phenotypic variability, can be obtained. In the present study, Saccharomyces cerevisiae × Saccharomyces uvarum hybrids were constructed using genetically and physiologically different S. uvarum parents at distinct temperatures (13 and 20°C). The effect of those variables on the main oenological features of the wines obtained with these hybrids was evaluated. Hybrids were successfully obtained in all cases. However, genetic stabilization based on successive fermentations in white wine at 13°C was significantly longer than that at 20°C. Our results demonstrated that, irrespective of the S. uvarum parent and temperature used for hybrid generation and stabilization, similar physicochemical and aromatic features were found in wines. The hybrids generated herein were characterized by low ethanol production, high glycerol synthesis and the capacity to grow at low temperature and to produce malic acid with particular aroma profiles. These features make these hybrids useful for the new winemaking industry within the climate change era frame. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Deletion of JJJ1 improves acetic acid tolerance and bioethanol fermentation performance of Saccharomyces cerevisiae strains.

PubMed

Wu, Xuechang; Zhang, Lijie; Jin, Xinna; Fang, Yahong; Zhang, Ke; Qi, Lei; Zheng, Daoqiong

2016-07-01

To improve tolerance to acetic acid that is present in lignocellulosic hydrolysates and affects bioethanol production by Saccharomyces cerevisiae. Saccharomyces cerevisiae strains with improved tolerance to acetic acid were obtained through deletion of the JJJ1 gene. The lag phase of the JJJ1 deletion mutant BYΔJJJ1 was ~16 h shorter than that of the parent strain, BY4741, when the fermentation medium contained 4.5 g acetic acid/l. Additionally, the specific ethanol production rate of BYΔJJJ1 was increased (0.057 g/g h) compared to that of the parent strain (0.051 g/g h). Comparative transcription and physiological analyses revealed higher long chain fatty acid, trehalose, and catalase contents might be critical factors responsible for the acetic acid resistance of JJJ1 knockout strains. JJJ1 deletion improves acetic acid tolerance and ethanol fermentation performance of S. cerevisiae.

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

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae.

PubMed

Schoeman, H; Vivier, M A; Du Toit, M; Dicks, L M; Pretorius, I S

1999-06-15

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end-products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast-based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1.0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA-1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA-1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA-1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1P-MFa1S-pedA-ADH1T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1P) and terminator (ADH1T). Secretion of the PED1-encoded pediocin PA-1 was directed by the yeast mating pheromone alpha-factor's secretion signal (MFa1S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4.6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the

Sucrose fermentation by Saccharomyces cerevisiae lacking hexose transport.

PubMed

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

2004-01-01

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

Optimization of air-blast drying process for manufacturing Saccharomyces cerevisiae and non-Saccharomyces yeast as industrial wine starters.

PubMed

Lee, Sae-Byuk; Choi, Won-Seok; Jo, Hyun-Jung; Yeo, Soo-Hwan; Park, Heui-Dong

2016-12-01

Wine yeast (Saccharomyces cerevisiae D8) and non-Saccharomyces wine yeasts (Hanseniaspora uvarum S6 and Issatchenkia orientalis KMBL5774) were studied using air-blast drying instead of the conventional drying methods (such as freeze and spray drying). Skim milk-a widely used protective agent-was used and in all strains, the highest viabilities following air-blast drying were obtained using 10% skim milk. Four excipients (wheat flour, nuruk, artichoke powder, and lactomil) were evaluated as protective agents for yeast strains during air-blast drying. Our results showed that 7 g lactomil was the best excipient in terms of drying time, powder form, and the survival rate of the yeast in the final product. Finally, 7 types of sugars were investigated to improve the survival rate of air-blast dried yeast cells: 10% trehalose, 10% sucrose, and 10% glucose had the highest survival rate of 97.54, 92.59, and 79.49% for S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respectively. After 3 months of storage, S. cerevisiae D8 and H. uvarum S6 demonstrated good survival rates (making them suitable for use as starters), whereas the survival rate of I. orientalis KMBL5774 decreased considerably compared to the other strains. Air-blast dried S. cerevisiae D8 and H. uvarum S6 showed metabolic activities similar to those of non-dried yeast cells, regardless of the storage period. Air-blast dried I. orientalis KMBL5774 showed a noticeable decrease in its ability to decompose malic acid after 3 months of storage at 4 °C.

Genome Sequence of Saccharomyces cerevisiae Double-Stranded RNA Virus L-A-28.

PubMed

Konovalovas, Aleksandras; Serviené, Elena; Serva, Saulius

2016-06-16

We cloned and sequenced the complete genome of the L-A-28 virus from the Saccharomyces cerevisiae K28 killer strain. This sequence completes the set of currently identified L-A helper viruses required for expression of double-stranded RNA-originated killer phenotypes in baking yeast. Copyright © 2016 Konovalovas et al.

Exploring the northern limit of the distribution of Saccharomyces cerevisiae and Saccharomyces paradoxus in North America.

PubMed

Charron, Guillaume; Leducq, Jean-Baptiste; Bertin, Chloé; Dubé, Alexandre K; Landry, Christian R

2014-03-01

We examined the northern limit of Saccharomyces cerevisiae and Saccharomyces paradoxus in northeast America. We collected 876 natural samples at 29 sites and applied enrichment methods for the isolation of mesophilic yeasts. We uncovered a large diversity of yeasts, in some cases, associated with specific substrates. Sequencing of the ITS1, 5.8S and ITS2 loci allowed to assign 226 yeast strains at the species level, including 41 S. paradoxus strains. Our intensive sampling suggests that if present, S. cerevisiae is rare at these northern latitudes. Our sampling efforts spread across several months of the year revealed that successful sampling increases throughout the summer and diminishes significantly at the beginning of the fall. The data obtained on the ecological context of yeasts corroborate what was previously reported on Pichiaceae, Saccharomycodaceae, Debaryomycetaceae and Phaffomycetaceae yeast families. We identified 24 yeast isolates that could not be assigned to any known species and that may be of taxonomic, medical, or biotechnological importance. Our study reports new data on the taxonomic diversity of yeasts and new resources for studying the evolution and ecology of S. paradoxus. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Development of a Saccharomyces cerevisiae Strain with Enhanced Resistance to Phenolic Fermentation Inhibitors in Lignocellulose Hydrolysates by Heterologous Expression of Laccase

PubMed Central

Larsson, Simona; Cassland, Pierre; Jönsson, Leif J.

2001-01-01

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose. PMID:11229906

Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase.

PubMed

Larsson, S; Cassland, P; Jönsson, L J

2001-03-01

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.

Saccharomyces cerevisiae show low levels of traversal across human endothelial barrier in vitro.

PubMed

Pérez-Torrado, Roberto; Querol, Amparo

2017-01-01

Background :   Saccharomyces cerevisiae is generally considered safe, and is involved in the production of many types of foods and dietary supplements. However, some isolates, which are genetically related to strains used in brewing and baking, have shown virulent traits, being able to produce infections in humans, mainly in immunodeficient patients. This can lead to systemic infections in humans. Methods : In this work, we studied S. cerevisiae isolates in an in vitro human endothelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans . Results : The results showed that this food related yeast is able to cross the endothelial barrier in vitro . However, in contrast to C. glabrata and C. albicans , S. cerevisiae showed very low levels of traversal. Conclusions : We conclude that using an in vitro human endothelial barrier model with S. cerevisiae can be useful to evaluate the safety of S. cerevisiae strains isolated from foods.

Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p

PubMed Central

Li, Li; Lipke, Peter N.; Dranginis, Anne M.

2016-01-01

ABSTRACT FLO11 encodes a yeast cell wall flocculin that mediates a variety of adhesive phenotypes in Saccharomyces cerevisiae. Flo11p is implicated in many developmental processes, including flocculation, formation of pseudohyphae, agar invasion, and formation of microbial mats and biofilms. However, Flo11p mediates different processes in different yeast strains. To investigate the mechanisms by which FLO11 determines these differences in colony morphology, flocculation, and invasion, we studied gene structure, function, and expression levels. Nonflocculent Saccharomyces cerevisiae Σ1278b cells exhibited significantly higher FLO11 mRNA expression, especially in the stationary phase, than highly flocculent S. cerevisiae var. diastaticus. The two strains varied in cell surface hydrophobicity, and Flo11p contributed significantly to surface hydrophobicity in S. cerevisiae var. diastaticus but not in strain Σ1278b. Sequencing of the FLO11 gene in S. cerevisiae var. diastaticus revealed strain-specific differences, including a 15-amino-acid insertion in the adhesion domain. Flo11p adhesion domains from strain Σ1278b and S. cerevisiae var. diastaticus were expressed and used to coat magnetic beads. The adhesion domain from each strain bound preferentially to homologous cells, and the preferences were independent of the cells in which the adhesion domains were produced. These results are consistent with the idea that strain-specific variations in the amino acid sequences in the adhesion domains cause different Flo11p flocculation activities. The results also imply that strain-specific differences in expression levels, posttranslational modifications, and allelic differences outside the adhesion domains have little effect on flocculation. IMPORTANCE As a nonmotile organism, Saccharomyces cerevisiae employs the cell surface flocculin Flo11/Muc1 as an important means of adapting to environmental change. However, there is a great deal of strain variation in the

Enhancing Fatty Acid Production of Saccharomyces cerevisiae as an Animal Feed Supplement.

PubMed

You, Seung Kyou; Joo, Young-Chul; Kang, Dae Hee; Shin, Sang Kyu; Hyeon, Jeong Eun; Woo, Han Min; Um, Youngsoon; Park, Chulhwan; Han, Sung Ok

2017-12-20

Saccharomyces cerevisiae is used for edible purposes, such as human food or as an animal feed supplement. Fatty acids are also beneficial as feed supplements, but S. cerevisiae produces small amounts of fatty acids. In this study, we enhanced fatty acid production of S. cerevisiae by overexpressing acetyl-CoA carboxylase, thioesterase, and malic enzyme associated with fatty acid metabolism. The enhanced strain pAMT showed 2.4-fold higher fatty acids than the wild-type strain. To further increase the fatty acids, various nitrogen sources were analyzed and calcium nitrate was selected as an optimal nitrogen source for fatty acid production. By concentration optimization, 672 mg/L of fatty acids was produced, which was 4.7-fold higher than wild-type strain. These results complement the low level fatty acid production and make it possible to obtain the benefits of fatty acids as an animal feed supplement while, simultaneously, maintaining the advantages of S. cerevisiae.

Determination of biological activity from fluorescence-lifetime measurements in Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Rudek, F.; Baselt, T.; Lempe, B.; Taudt, C.; Hartmann, P.

2015-03-01

The importance of fluorescence lifetime measurement as an optical analysis tool is growing. Many applications already exist in order to determine the fluorescence lifetime, but the majority of these require the addition of fluorescence-active substances to enable measurements. Every usage of such foreign materials has an associated risk. This paper investigates the use of auto-fluorescing substances in Saccharomyces cerevisiae (Baker's yeast) as a risk free alternative to fluorescence-active substance enabled measurements. The experimental setup uses a nitrogen laser with a pulse length of 350 ps and a wavelength of 337 nm. The excited sample emits light due to fluorescence of NADH/NADPH and collagen. A fast photodiode collects the light at the output of an appropriate high-pass edge-filter at 400 nm. Fluorescence lifetimes can be determined from the decay of the measurement signals, which in turn characterizes the individual materials and their surrounding environment. Information about the quantity of the fluorescence active substances can also be measured based on the received signal intensity. The correlation between the fluorescence lifetime and the metabolic state of Saccharomyces cerevisiae was investigated and is presented here.

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae

PubMed Central

Feng, Quanzhou; Weber, Scott A.; Li, Shizhong

2018-01-01

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production. PMID:29621349

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae.

PubMed

Feng, Quanzhou; Liu, Z Lewis; Weber, Scott A; Li, Shizhong

2018-01-01

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production.

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

USDA-ARS?s Scientific Manuscript database

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...

Cell Surface Display of Four Types of Solanum nigrum Metallothionein on Saccharomyces cerevisiae for Biosorption of Cadmium.

PubMed

Wei, Qinguo; Zhang, Honghai; Guo, Dongge; Ma, Shisheng

2016-05-28

We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd(2+) in this study. The transformed yeast strains showed much higher resistance ability to Cd(2+) compared with the control. Strikingly, their Cd(2+) accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd(2+) under a wide range of pH levels, from 3 to 7, without disturbing the Cu(2+) and Hg(2+). Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd(2+)-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants.

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.

Production and Purification of the Native Saccharomyces cerevisiae Hsp12 in Escherichia coli.

PubMed

Léger, Antoine; Hocquellet, Agnès; Dieryck, Wilfrid; Moine, Virginie; Marchal, Axel; Marullo, Philippe; Josseaume, Annabelle; Cabanne, Charlotte

2017-09-20

Hsp12 is a small heat shock protein produced in many organisms, including the yeast Saccharomyces cerevisiae. It has been described as an indicator of yeast stress rate and has also been linked to the sweetness sensation of wine. To obtain a sufficient amount of protein, we produced and purified Hsp12 without tag in Escherichia coli. A simple fast two-step process was developed using a microplate approach and a design of experiments. A capture step on an anion-exchange salt-tolerant resin was followed by size exclusion chromatography for polishing, leading to a purity of 97%. Thereafter, specific anti-Hsp12 antibodies were obtained by rabbit immunization. An ELISA was developed to quantify Hsp12 in various strains of Saccharomyces cerevisiae. The antibodies showed high specificity and allowed the quantitation of Hsp12 in the yeast. The quantities of Hsp12 measured in the strains differed in direct proportion to the level of expression found in previous studies.

Saccharomyces cerevisiae KTR4, KTR5 and KTR7 encode mannosyltransferases differentially involved in the N- and O-linked glycosylation pathways.

PubMed

Hernández, Nahúm V; López-Ramírez, Luz A; Díaz-Jiménez, Diana F; Mellado-Mojica, Erika; Martínez-Duncker, Iván; López, Mercedes G; Mora-Montes, Héctor M

2017-10-01

Saccharomyces cerevisiae is a model to understand basic aspects of protein glycosylation pathways. Although these metabolic routes have been thoroughly studied, there are still knowledge gaps; among them, the role of the MNT1/KRE2 gene family. This family is composed of nine members, with only six functionally characterized. The enzymes Ktr1, Ktr3, and Mnt1/Kre2 have overlapping activities in both O-linked and N-linked glycan synthesis; while Ktr2 and Yur1 participate exclusively in the elongation of the N-linked glycan outer chain. KTR6 encodes for a phosphomannosyltransferase that synthesizes the cell wall phosphomannan. Here, we aimed to establish the functional role of KTR4, KTR5 and KTR7 in the protein glycosylation pathways, by using heterologous complementation in Candida albicans null mutants lacking members of the MNT1/KRE2 gene family. The three S. cerevisiae genes restored defects in the C. albicans N-linked glycosylation pathway. KTR5 and KTR7 partially complemented a C. albicans null mutant with defects in the synthesis of O-linked glycans, and only KTR4 fully elongated the O-linked glycans like wild-type cells. Therefore, our results suggest that the three genes have a redundant activity in the S. cerevisiae N-linked glycosylation pathway, but KTR4 plays a major role in O-linked glycan synthesis. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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.

Immunoproteomic profiling of Saccharomyces cerevisiae systemic infection in a murine model.

PubMed

Hernández-Haro, Carolina; Llopis, Silvia; Molina, María; Monteoliva, Lucía; Gil, Concha

2015-01-01

Saccharomyces cerevisiae is considered a safe microorganism widely used as a dietary supplement. However, in the latest decades several cases of S. cerevisiae infections have been reported. Recent studies in a murine model of systemic infection have also revealed the virulence of some S. cerevisiae dietary strains. Here we use an immunoproteomic approach based on protein separation by 2D-PAGE followed by Western-blotting to compare the serological response against a virulent dietary and a non-virulent laboratory strains leading to the identification of highly different patterns of antigenic proteins. Thirty-six proteins that elicit a serological response in mice have been identified. Most of them are involved in stress responses and metabolic pathways. Their selectivity as putative biomarkers for S. cerevisiae infections was assessed by testing sera from S. cerevisiae-infected mice against Candida albicans and C. glabrata proteins. Some chaperones and metabolic proteins showed cross-reactivity. We also compare the S. cerevisiae immunodetected proteins with previously described C. albicans antigens. The results point to the stress-related proteins Ahp1, Yhb1 and Oye2, as well as the glutamine synthetase Gln1 and the oxysosterol binding protein Kes1 as putative candidates for being evaluated as biomarkers for diagnostic assays of S. cerevisiae infections. S. cerevisiae can cause opportunistic infections, and therefore, a precise diagnosis of fungal infections is necessary. This immunoproteomic analysis of sera from a model murine infection with a virulent dietary S. cerevisiae strain has been shown to be a source of candidate proteins for being evaluated as biomarkers to develop assays for diagnosis of S. cerevisiae infections. To our knowledge, this is the first study devoted to the identification of S. cerevisiae immunogenic proteins and the results allowed the proposal of five antigens to be further investigated. Copyright © 2014 Elsevier B.V. All rights reserved.

Monoterpenoid biosynthesis in Saccharomyces cerevisiae.

PubMed

Oswald, Marilyne; Fischer, Marc; Dirninger, Nicole; Karst, Francis

2007-05-01

Plant monoterpenoids belong to a large family of plant secondary metabolites with valuable applications in cosmetics and medicine. Their usual low levels and difficult purification justify the need for alternative fermentative processes for large-scale production. Geranyl diphosphate is the universal precursor of monoterpenoids. In yeast it occurs exclusively as an intermediate of farnesyl diphosphate synthesis. In the present study we investigated the potential use of Saccharomyces cerevisiae as an alternative engineering tool. The expression of geraniol synthase of Ocimum basilicum in yeast allowed a strong and specific excretion of geraniol to the growth medium, in contrast to mutants defective in farnesyl diphosphate synthase which excreted geraniol and linalool in similar amounts. A further increase of geraniol synthesis was obtained using yeast mutants defective in farnesyl diphosphate synthase. We also showed that geraniol synthase expression affects the general ergosterol pathway, but in a manner dependent on the genetic background of the strain.

Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

PubMed Central

Otterstedt, Karin; Larsson, Christer; Bill, Roslyn M; Ståhlberg, Anders; Boles, Eckhard; Hohmann, Stefan; Gustafsson, Lena

2004-01-01

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction. PMID:15071495

Mitochondrial genomic dysfunction causes dephosphorylation of Sch9 in the yeast Saccharomyces cerevisiae.

PubMed

Kawai, Shigeyuki; Urban, Jörg; Piccolis, Manuele; Panchaud, Nicolas; De Virgilio, Claudio; Loewith, Robbie

2011-10-01

TORC1-dependent phosphorylation of Saccharomyces cerevisiae Sch9 was dramatically reduced upon exposure to a protonophore or in respiration-incompetent ρ(0) cells but not in respiration-incompetent pet mutants, providing important insight into the molecular mechanisms governing interorganellar signaling in general and retrograde signaling in particular.

Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host.

PubMed

Santos, João H P M; Costa, Iris M; Molino, João V D; Leite, Mariana S M; Pimenta, Marcela V; Coutinho, João A P; Pessoa, Adalberto; Ventura, Sónia P M; Lopes, André M; Monteiro, Gisele

2017-03-01

l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His) 6 -tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni 2+ -charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg -1 ) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17-fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017. © 2016 American Institute of Chemical Engineers.

Functional relevance of water and glycerol channels in Saccharomyces cerevisiae.

PubMed

Sabir, Farzana; Loureiro-Dias, Maria C; Soveral, Graça; Prista, Catarina

2017-05-01

Our understanding of the functional relevance of orthodox aquaporins and aquaglyceroporins in Saccharomyces cerevisiae is essentially based on phenotypic variations obtained by expression/overexpression/deletion of these major intrinsic proteins in selected strains. These water/glycerol channels are considered crucial during various life-cycle phases, such as sporulation and mating and in some life processes such as rapid freeze-thaw tolerance, osmoregulation and phenomena associated with cell surface. Despite their putative functional roles not only as channels but also as sensors, their underlying mechanisms and their regulation are still poorly understood. In the present review, we summarize and discuss the physiological relevance of S. cerevisiae aquaporins (Aqy1 and Aqy2) and aquaglyceroporins (Fps1 and Yfl054c). In particular, the fact that most S. cerevisiae laboratory strains harbor genes coding for non-functional aquaporins, while wild and industrial strains possess at least one functional aquaporin, suggests that aquaporin activity is required for cell survival under more harsh conditions. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Early transcriptional response to biotic stress in mixed starter fermentations involving Saccharomyces cerevisiae and Torulaspora delbrueckii.

PubMed

Tronchoni, Jordi; Curiel, Jose Antonio; Morales, Pilar; Torres-Pérez, Rafael; Gonzalez, Ramon

2017-01-16

Advances in microbial wine biotechnology have led to the recent commercialization of several non-Saccharomyces starter cultures. These are intended to be used in either simultaneous or sequential inoculation with Saccharomyces cerevisiae. The different types of microbial interactions that can be stablished during wine fermentation acquire an increased relevance in the context of these mixed-starter fermentations. We analysed the transcriptional response to co-cultivation of S. cerevisiae and Torulaspora delbrueckii. The study focused in the initial stages of wine fermentation, before S. cerevisiae completely dominates the mixed cultures. Both species showed a clear response to the presence of each other, even though the portion of the genome showing altered transcription levels was relatively small. Changes in the transcription pattern suggested a stimulation of metabolic activity and growth, as a consequence of the presence of competitors in the same medium. The response of S. cerevisiae seems to take place earlier, as compared to T. delbrueckii. Enhanced glycolytic activity of the mixed culture was confirmed by the CO 2 production profile during these early stages of fermentation. Interestingly, HSP12 expression appeared induced by co-cultivation for both of S. cerevisiae and Torulaspora delbrueckii in the two time points studied. This might be related with a recently described role of Hsp12 in intercellular communication in yeast. Expression of S. cerevisiae PAU genes was also stimulated in mixed cultures. Copyright © 2016. Published by Elsevier B.V.

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.

Can Zymomonas mobilis Substitute Saccharomyces cerevisiae in Cereal Dough Leavening?

PubMed Central

Musatti, Alida; Mapelli, Chiara

2018-01-01

Baker’s yeast intolerance is rising among Western populations, where Saccharomyces cerevisiae is spread in fermented food and food components. Zymomonas mobilis is a bacterium commonly used in tropical areas to produce alcoholic beverages, and it has only rarely been considered for dough leavening probably because it only ferments glucose, fructose and sucrose, which are scarcely present in flour. However, through alcoholic fermentation, similarly to S. cerevisiae, it provides an equimolar mixture of ethanol and CO2 that can rise a dough. Here, we propose Z. mobilis as a new leavening agent, as an alternative to S. cerevisiae, overcoming its technological limit with different strategies: (1) adding glucose to the dough formulation; and (2) exploiting the maltose hydrolytic activity of Lactobacillus sanfranciscensis associated with Z. mobilis. CO2 production, dough volume increase, pH value, microbial counts, sugars consumption and ethanol production were monitored. Results suggest that glucose addition to the dough lets Z. mobilis efficiently leaven a dough, while glucose released by L. sanfranciscensis is not so well fermented by Z. mobilis, probably due to the strong acidification. Nevertheless, the use of Z. mobilis as a leavening agent could contribute to increasing the variety of baked goods alternative to those leavened by S. cerevisiae. PMID:29659515

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

... Hydrolysate from Saccharomyces cerevisiae on all food commodities when applied/used for the management of... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1246 Yeast Extract Hydrolysate from...

Alkaline pH enhances farnesol production by Saccharomyces cerevisiae.

PubMed

Muramatsu, Masayoshi; Ohto, Chikara; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2009-07-01

External environments affect prenyl alcohol production by squalene synthetase-deficient mutant Saccharomyces cerevisiae ATCC 64031. Cultivation of the yeast in medium with an initial pH ranging from 7.0 to 8.0 increased the amount of secreted farnesol (FOH). In contrast, acidic medium with a pH below 4.0 increased the intracellular FOH and its isomer nerolidol. These effects of alkaline pH were also observed on constant pH cultivation in a jar fermenter. On cultivation for 133 h, the FOH production reached 102.8 mg/l.

Induction of polypeptides in Saccharomyces cerevisiae after ultraviolet irradiation.

PubMed

Angulo, J F; Schwencke, J; Fernandez, I; Moustacchi, E

1986-07-31

Alterations in the synthesis of proteins following exposure of Saccharomyces cerevisiae to UV light were investigated using radioactive labelling and two dimensional electrophoresis. UV-irradiation induced the synthesis of various proteins. Among them the analogue of the RecA protein of Escherichia coli (Angulo et al. 1985) and two other polypeptides (34 Kd and 35 Kd, pI 5.8) were observed in all four strains analyzed namely two DNA-repair deficient (rad-) strains: (rad6-1 and pso2-1) and their isogenic wild type RAD+ strains.

YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

PubMed Central

Guo, Yakun; Dong, Junkai; Zhou, Tong; Auxillos, Jamie; Li, Tianyi; Zhang, Weimin; Wang, Lihui; Shen, Yue; Luo, Yisha; Zheng, Yijing; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Cai, Yizhi; Dai, Junbiao

2015-01-01

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts. PMID:25956650

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

USDA-ARS?s Scientific Manuscript database

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...

Ethanol-independent biofilm formation by a flor wine yeast strain of Saccharomyces cerevisiae.

PubMed

Zara, Severino; Gross, Michael K; Zara, Giacomo; Budroni, Marilena; Bakalinsky, Alan T

2010-06-01

Flor strains of Saccharomyces cerevisiae form a biofilm on the surface of wine at the end of fermentation, when sugar is depleted and growth on ethanol becomes dependent on oxygen. Here, we report greater biofilm formation on glycerol and ethyl acetate and inconsistent formation on succinic, lactic, and acetic acids.

The heterologous expression strategies of antimicrobial peptides in microbial systems.

PubMed

Deng, Ting; Ge, Haoran; He, Huahua; Liu, Yao; Zhai, Chao; Feng, Liang; Yi, Li

2017-12-01

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid by Saccharomyces cerevisiae yields less toxic products.

PubMed

Adeboye, Peter Temitope; Bettiga, Maurizio; Aldaeus, Fredrik; Larsson, Per Tomas; Olsson, Lisbeth

2015-09-21

Lignocellulosic substrates and pulping process streams are of increasing relevance to biorefineries for second generation biofuels and biochemical production. They are known to be rich in sugars and inhibitors such as phenolic compounds, organic acids and furaldehydes. Phenolic compounds are a group of aromatic compounds known to be inhibitory to fermentative organisms. It is known that inhibition of Sacchromyces cerevisiae varies among phenolic compounds and the yeast is capable of in situ catabolic conversion and metabolism of some phenolic compounds. In an approach to engineer a S. cerevisiae strain with higher tolerance to phenolic inhibitors, we selectively investigated the metabolic conversion and physiological effects of coniferyl aldehyde, ferulic acid, and p-coumaric acid in Saccharomyces cerevisiae. Aerobic batch cultivations were separately performed with each of the three phenolic compounds. Conversion of each of the phenolic compounds was observed on time-based qualitative analysis of the culture broth to monitor various intermediate and final metabolites. Coniferyl aldehyde was rapidly converted within the first 24 h, while ferulic acid and p-coumaric acid were more slowly converted over a period of 72 h. The conversion of the three phenolic compounds was observed to involved several transient intermediates that were concurrently formed and converted to other phenolic products. Although there were several conversion products formed from coniferyl aldehyde, ferulic acid and p-coumaric acid, the conversion products profile from the three compounds were similar. On the physiology of Saccharomyces cerevisiae, the maximum specific growth rates of the yeast was not affected in the presence of coniferyl aldehyde or ferulic acid, but it was significantly reduced in the presence of p-coumaric acid. The biomass yields on glucose were reduced to 73 and 54 % of the control in the presence of coniferyl aldehyde and ferulic acid, respectively, biomass yield

Acidic Calcium Stores of Saccharomyces cerevisiae

PubMed Central

Cunningham, Kyle W.

2011-01-01

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

Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae.

PubMed

Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

2012-08-01

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

Zinc oxide and silver nanoparticles toxicity in the baker's yeast, Saccharomyces cerevisiae.

PubMed

Galván Márquez, Imelda; Ghiyasvand, Mergan; Massarsky, Andrey; Babu, Mohan; Samanfar, Bahram; Omidi, Katayoun; Moon, Thomas W; Smith, Myron L; Golshani, Ashkan

2018-01-01

Engineered nanomaterials (ENMs) are increasingly incorporated into a variety of commercial applications and consumer products; however, ENMs may possess cytotoxic properties due to their small size. This study assessed the effects of two commonly used ENMs, zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs), in the model eukaryote Saccharomyces cerevisiae. A collection of ≈4600 S. cerevisiae deletion mutant strains was used to deduce the genes, whose absence makes S. cerevisiae more prone to the cytotoxic effects of ZnONPs or AgNPs. We demonstrate that S. cerevisiae strains that lack genes involved in transmembrane and membrane transport, cellular ion homeostasis, and cell wall organization or biogenesis exhibited the highest sensitivity to ZnONPs. In contrast, strains that lack genes involved in transcription and RNA processing, cellular respiration, and endocytosis and vesicular transport exhibited the highest sensitivity to AgNPs. Secondary assays confirmed that ZnONPs affected cell wall function and integrity, whereas AgNPs exposure decreased transcription, reduced endocytosis, and led to a dysfunctional electron transport system. This study supports the use of S. cerevisiae Gene Deletion Array as an effective high-throughput technique to determine cellular targets of ENM toxicity.

Disrupted short chain specific β-oxidation and improved synthase expression increase synthesis of short chain fatty acids in Saccharomyces cerevisiae.

PubMed

Leber, Christopher; Choi, Jin Wook; Polson, Brian; Da Silva, Nancy A

2016-04-01

Biologically derived fatty acids have gained tremendous interest as an alternative to petroleum-derived fuels and chemical precursors. We previously demonstrated the synthesis of short chain fatty acids in Saccharomyces cerevisiae by introduction of the Homo sapiens fatty acid synthase (hFAS) with heterologous phosphopantetheine transferases and heterologous thioesterases. In this study, short chain fatty acid production was improved by combining a variety of novel enzyme and metabolic engineering strategies. The use of a H. sapiens-derived thioesterase and phosphopantetheine transferase were evaluated. In addition, strains were engineered to disrupt either the full β-oxidation (by deleting FAA2, PXA1, and POX1) or short chain-specific β-oxidation (by deleting FAA2, ANT1, and PEX11) pathways. Prohibiting full β-oxidation increased hexanoic and octanoic acid levels by 8- and 79-fold relative to the parent strain expressing hFAS. However, by targeting only short chain β-oxidation, hexanoic and octanoic acid levels increased further to 31- and 140-fold over the parent. In addition, an optimized hFAS gene increased hexanoic, octanoic, decanoic and total short chain fatty acid levels by 2.9-, 2.0-, 2.3-, and 2.2-fold, respectively, relative to the non-optimized counterpart. By combining these unique enzyme and metabolic engineering strategies, octanoic acid was increased more than 181-fold over the parent strain expressing hFAS. © 2015 Wiley Periodicals, Inc.

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

USDA-ARS?s Scientific Manuscript database

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...

Engineering and Evolution of Saccharomyces cerevisiae to Produce Biofuels and Chemicals.

PubMed

Turner, Timothy L; Kim, Heejin; Kong, In Iok; Liu, Jing-Jing; Zhang, Guo-Chang; Jin, Yong-Su

To mitigate global climate change caused partly by the use of fossil fuels, the production of fuels and chemicals from renewable biomass has been attempted. The conversion of various sugars from renewable biomass into biofuels by engineered baker's yeast (Saccharomyces cerevisiae) is one major direction which has grown dramatically in recent years. As well as shifting away from fossil fuels, the production of commodity chemicals by engineered S. cerevisiae has also increased significantly. The traditional approaches of biochemical and metabolic engineering to develop economic bioconversion processes in laboratory and industrial settings have been accelerated by rapid advancements in the areas of yeast genomics, synthetic biology, and systems biology. Together, these innovations have resulted in rapid and efficient manipulation of S. cerevisiae to expand fermentable substrates and diversify value-added products. Here, we discuss recent and major advances in rational (relying on prior experimentally-derived knowledge) and combinatorial (relying on high-throughput screening and genomics) approaches to engineer S. cerevisiae for producing ethanol, butanol, 2,3-butanediol, fatty acid ethyl esters, isoprenoids, organic acids, rare sugars, antioxidants, and sugar alcohols from glucose, xylose, cellobiose, galactose, acetate, alginate, mannitol, arabinose, and lactose.

Engineering of Saccharomyces cerevisiae for the synthesis of short chain fatty acids.

PubMed

Leber, Christopher; Da Silva, Nancy A

2014-02-01

Carbon feedstocks from fossilized sources are being rapidly depleted due to rising demand for industrial and commercial applications. Many petroleum-derived chemicals can be directly or functionally substituted with chemicals derived from renewable feedstocks. Several short chain organic acids may fulfill this role using their functional groups as a target for chemical catalysis. Saccharomyces cerevisiae was engineered to produce short chain carboxylic acids (C6 to C10 ) from glucose using the heterologous Homo sapiens type I fatty acid synthase (hFAS). This synthase was activated by phosphopantetheine transfereases AcpS and Sfp from Escherichia coli and Bacillus subtilis, respectively, both in vitro and in vivo. hFAS was produced in the holo-form and produced carboxylic acids in vitro, confirmed by NADPH and ADIFAB assays. Overexpression of hFAS in a yeast FAS2 knockout strain, deficient in de novo fatty acid synthesis, demonstrated the full functional replacement of the native fungal FAS by hFAS. Two active heterologous short chain thioesterases (TEs) from Cuphea palustris (CpFatB1) and Rattus norvegicus (TEII) were evaluated for short chain fatty acid (SCFA) synthesis in vitro and in vivo. Three hFAS mutants were constructed: a mutant deficient in the native TE domain, a mutant with a linked CpFatB1 TE and a mutant with a linked TEII TE. Using the native yeast fatty acid synthase for growth, the overexpression of the hFAS mutants and the short-chain TEs (linked or plasmid-based) increased in vivo caprylic acid and total SCFA production up to 64-fold (63 mg/L) and 52-fold (68 mg/L), respectively, over the native yeast levels. Combined over-expression of the phosphopantetheine transferase with the hFAS mutant resulted in C8 titers of up to 82 mg/L and total SCFA titers of up to 111 mg/L. © 2013 Wiley Periodicals, Inc.

Effects of Saccharomyces cerevisiae or boulardii yeasts on acute stress induced intestinal dysmotility.

PubMed

West, Christine; Stanisz, Andrew M; Wong, Annette; Kunze, Wolfgang A

2016-12-28

To investigate the capacity of Saccharomyces cerevisiae ( S. cerevisiae ) and Saccharomyces boulardii ( S. boulardii ) yeasts to reverse or to treat acute stress-related intestinal dysmotility. Adult Swiss Webster mice were stressed for 1 h in a wire-mesh restraint to induce symptoms of intestinal dysmotility and were subsequently killed by cervical dislocation. Jejunal and colon tissue were excised and placed within a tissue perfusion bath in which S. cerevisiae , S. boulardii , or their supernatants were administered into the lumen. Video recordings of contractility and gut diameter changes were converted to spatiotemporal maps and the velocity, frequency, and amplitude of propagating contractile clusters (PCC) were measured. Motility pre- and post-treatment was compared between stressed animals and unstressed controls. S. boulardii and S. cerevisiae helped to mediate the effects of stress on the small and large intestine. Restraint stress reduced jejunal transit velocity (mm/s) from 2.635 ± 0.316 to 1.644 ± 0.238, P < 0.001 and jejunal transit frequency (Hz) from 0.032 ± 0.008 to 0.016 ± 0.005, P < 0.001. Restraint stress increased colonic transit velocity (mm/s) from 0.864 ± 0.183 to 1.432 ± 0.329, P < 0.001 and frequency to a lesser degree. Luminal application of S. boulardii helped to restore jejunal and colonic velocity towards the unstressed controls; 1.833 ± 0.688 to 2.627 ± 0.664, P < 0.001 and 1.516 ± 0.263 to 1.036 ± 0.21, P < 0.001, respectively. S. cerevisiae also had therapeutic effects on the stressed gut, but was most apparent in the jejunum. S. cerevisiae increased PCC velocity in the stressed jejunum from 1.763 ± 0.397 to 2.017 ± 0.48, P = 0.0031 and PCC frequency from 0.016 ± 0.009 to 0.027 ± 0.007, P < 0.001. S. cerevisiae decreased colon PCC velocity from 1.647 ± 0.187 to 1.038 ± 0.222, P < 0.001. Addition of S. boulardii or S. cerevisiae supernatants also helped to restore motility to unstressed values in similar capacity

Effects of Saccharomyces cerevisiae or boulardii yeasts on acute stress induced intestinal dysmotility

PubMed Central

West, Christine; Stanisz, Andrew M; Wong, Annette; Kunze, Wolfgang A

2016-01-01

AIM To investigate the capacity of Saccharomyces cerevisiae (S. cerevisiae) and Saccharomyces boulardii (S. boulardii) yeasts to reverse or to treat acute stress-related intestinal dysmotility. METHODS Adult Swiss Webster mice were stressed for 1 h in a wire-mesh restraint to induce symptoms of intestinal dysmotility and were subsequently killed by cervical dislocation. Jejunal and colon tissue were excised and placed within a tissue perfusion bath in which S. cerevisiae, S. boulardii, or their supernatants were administered into the lumen. Video recordings of contractility and gut diameter changes were converted to spatiotemporal maps and the velocity, frequency, and amplitude of propagating contractile clusters (PCC) were measured. Motility pre- and post-treatment was compared between stressed animals and unstressed controls. RESULTS S. boulardii and S. cerevisiae helped to mediate the effects of stress on the small and large intestine. Restraint stress reduced jejunal transit velocity (mm/s) from 2.635 ± 0.316 to 1.644 ± 0.238, P < 0.001 and jejunal transit frequency (Hz) from 0.032 ± 0.008 to 0.016 ± 0.005, P < 0.001. Restraint stress increased colonic transit velocity (mm/s) from 0.864 ± 0.183 to 1.432 ± 0.329, P < 0.001 and frequency to a lesser degree. Luminal application of S. boulardii helped to restore jejunal and colonic velocity towards the unstressed controls; 1.833 ± 0.688 to 2.627 ± 0.664, P < 0.001 and 1.516 ± 0.263 to 1.036 ± 0.21, P < 0.001, respectively. S. cerevisiae also had therapeutic effects on the stressed gut, but was most apparent in the jejunum. S. cerevisiae increased PCC velocity in the stressed jejunum from 1.763 ± 0.397 to 2.017 ± 0.48, P = 0.0031 and PCC frequency from 0.016 ± 0.009 to 0.027 ± 0.007, P < 0.001. S. cerevisiae decreased colon PCC velocity from 1.647 ± 0.187 to 1.038 ± 0.222, P < 0.001. Addition of S. boulardii or S. cerevisiae supernatants also helped to restore motility to unstressed values in similar

Thiamin biosynthesis in Saccharomyces cerevisiae. Origin of carbon-2 of the thiazole moiety.

PubMed Central

White, R L; Spenser, I D

1979-01-01

Radioactivity from [2-14C]glycine enters C-2 of the thiazole moiety of thiamin and no other site, in Saccharomyces cerevisiae (strains A.T.C.C. 24903 and 39916, H.J. Bunker). Radioactivity from L-[Me-14C]methionine or from DL-[2-14C]tyrosine does not enter thiamin. PMID:384994

Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae.

PubMed

Kim, Sujin; Bae, Sang-Jeong; Hahn, Ji-Sook

2016-04-07

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine.

PubMed

Gobbi, Mirko; Comitini, Francesca; Domizio, Paola; Romani, Cristina; Lencioni, Livio; Mannazzu, Ilaria; Ciani, Maurizio

2013-04-01

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine. In this specific pairing of yeast strains in mixed fermentations (S. cerevisiae EC1118 and L. thermotolerans 101), this non-Saccharomyces yeast showed a high level of competitiveness. Nevertheless the S. cerevisiae strain dominated the fermentation over the spontaneous S. cerevisiae strains also under the industrial fermentation conditions. The different condition tested (modalities of inoculum, temperature of fermentation, different grape juice) influenced the specific interactions and the fermentation behaviour of the co-culture of S. cerevisiae and L. thermotolerans. However, some metabolic behaviours such as pH reduction and enhancement of 2-phenylethanol and glycerol, were shown here under all of the conditions tested. The specific chemical profiles of these wines were confirmed by the sensory analysis test, which expressed these results at the tasting level as significant increases in the spicy notes and in terms of total acidity increases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation and characterization of a resident tolerant Saccharomyces cerevisiae strain from a spent sulfite liquor fermentation plant

PubMed Central

2012-01-01

Spent Sulfite Liquor (SSL) from wood pulping facilities is a sugar rich effluent that can be used as feedstock for ethanol production. However, depending on the pulping process conditions, the release of monosaccharides also generates a range of compounds that negatively affect microbial fermentation. In the present study, we investigated whether endogenous yeasts in SSL-based ethanol plant could represent a source of Saccharomyces cerevisiae strains with a naturally acquired tolerance towards this inhibitory environment. Two isolation processes were performed, before and after the re-inoculation of the plant with a commercial baker’s yeast strain. The isolates were clustered by DNA fingerprinting and a recurrent Saccharomyces cerevisiae strain, different from the inoculated commercial baker’s yeast strain, was isolated. The strain, named TMB3720, flocculated heavily and presented high furaldehyde reductase activity. During fermentation of undiluted SSL, TMB3720 displayed a 4-fold higher ethanol production rate and 1.8-fold higher ethanol yield as compared to the commercial baker’s yeast. Another non-Saccharomyces cerevisiae species, identified as the pentose utilizing Pichia galeiformis, was also recovered in the last tanks of the process where the hexose to pentose sugar ratio and the inhibitory pressure are expected to be the lowest. PMID:23237549

Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.

PubMed

Rodríguez-Escudero, María; Cid, Víctor J; Molina, María; Schulze-Luehrmann, Jan; Lührmann, Anja; Rodríguez-Escudero, Isabel

2016-01-01

Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.

Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation

PubMed Central

Rodríguez-Escudero, María; Cid, Víctor J.; Molina, María; Schulze-Luehrmann, Jan; Lührmann, Anja; Rodríguez-Escudero, Isabel

2016-01-01

Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors

Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae.

PubMed

Dai, Zhongxue; Gu, Honglian; Zhang, Shangjie; Xin, Fengxue; Zhang, Wenming; Dong, Weiliang; Ma, Jiangfeng; Jia, Honghua; Jiang, Min

2017-12-01

The aim of this study was to metabolically construct Saccharomyces cerevisiae for achievement of direct methanol utilization and value added product (mainly pyruvate) production. After successful integration of methanol oxidation pathway originated from Pichia pastoris into the chromosome of S. cerevisiae, the recombinant showed 1.04g/L consumption of methanol and 3.13% increase of cell growth (OD 600 ) when using methanol as the sole carbon source. Moreover, 0.26g/L of pyruvate was detected in the fermentation broth. The supplementation of 1g/L yeast extract could further improve cell growth with increase of 11.70% and methanol consumption to 2.35g/L. This represents the first genetically modified non-methylotrophic eukaryotic microbe for direct methanol utilization and would be of great value concerning the development of biotechnological processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters.

PubMed

Boswell-Casteel, Rebba C; Johnson, Jennifer M; Roe-Žurž, Zygy; Duggan, Kelli D; Schmitz, Hannah; Hays, Franklin A

2018-02-01

Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Improved bread-baking process using Saccharomyces cerevisiae displayed with engineered cyclodextrin glucanotransferase.

PubMed

Shim, Jae-Hoon; Seo, Nam-Seok; Roh, Sun-Ah; Kim, Jung-Wan; Cha, Hyunju; Park, Kwan-Hwa

2007-06-13

A bread-baking process was developed using a potential novel enzyme, cyclodextrin glucanotransferase[3-18] (CGTase[3-18]), that had previously been engineered to have enhanced hydrolyzing activity with little cyclodextrin (CD) formation activity toward starch. CGTase[3-18] was primarily manipulated to be displayed on the cell surface of Saccharomyces cerevisiae. S. cerevisiae carrying pdeltaCGT integrated into the chromosome exhibited starch-hydrolyzing activity at the same optimal pH and temperature as the free enzyme. Volumes of the bread loaves and rice cakes prepared using S. cerevisiae/pdeltaCGT increased by 20% and 45%, respectively, with no detectable CD. Retrogradation rates of the bread and rice cakes decreased significantly during storage. In comparison to the wild type, S. cerevisiae/pdeltaCGT showed improved viability during four freeze-thaw cycles. The results indicated that CGTase[3-18] displayed on the surface of yeast hydrolyzed starch to glucose and maltose that can be used more efficiently for yeast fermentation. Therefore, display of an antistaling enzyme on the cell surface of yeast has potential for enhancing the baking process.

ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

DOE PAGES

Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.; ...

2016-03-03

With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsicmore » physiological constraints—in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. In conclusion, by combining the push/pull/block strategies, we significantly

ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez, Sarah; Denby, Charles M.; Van Vu, T.

With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsicmore » physiological constraints—in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints. We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate. In conclusion, by combining the push/pull/block strategies, we significantly

Feasibility of brewing makgeolli using Pichia anomala Y197-13, a non-Saccharomyces cerevisiae.

PubMed

Kim, Hye Ryun; Kim, Jae-Ho; Bai, Dong-Hoon; Ahn, ByungHak

2012-12-01

Makgeolli is a traditional rice wine favored by the general public in Korea. This study investigated the fermentation and sensory characteristics of using wild yeast strains for brewing makgeolli. A non-Saccharomyces cerevisiae strain was isolated from nuruk and termed Y197-13. It showed 98% similarity to Pichia anomala and had an optimal growth temperature of 25 degrees C. Makgeolli was manufactured using koji, jinju nuruk, and improved nuruk as fermentation agents. Y197-13 makgeolli brewed with koji had alcohol and solids contents of 11.1% and 13.9%, respectively. Sweet sensory characteristics were attributed to residual sugars in makgeolli with 6% alcohol. The makgeolli had a fresh sour taste and carbonated taste. Volatile component analysis showed the isoamyl alcohol, phenylethyl alcohol, isoamyl acetate, and fatty acid, including ethyl oleate and ethyl linoleate, relative peak area was higher in Y197-13 makgeolli than in makgeolli with Saccharomyces cerevisiae. These results suggest the wild yeast, Y197-13, as a candidate for brewing makgeolli.

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains.

PubMed

Chigira, Yuko; Oka, Takuji; Okajima, Tetsuya; Jigami, Yoshifumi

2008-04-01

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

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. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Screening for Hydrolytic Enzymes Reveals Ayr1p as a Novel Triacylglycerol Lipase in Saccharomyces cerevisiae*

PubMed Central

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

2013-01-01

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

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

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.

Detection of yeast Saccharomyces cerevisiae with ionic liquid mediated carbon dots.

PubMed

Wang, Jia-Li; Teng, Ji-Yuan; Jia, Te; Shu, Yang

2018-02-01

Hydrophobic nitrogen-doped carbon dots are prepared with energetic ionic liquid (1,3-dibutylimidazolium dicyandiamide, BbimDCN) as carbon source. A yield of as high as 58% is obtained for the carbon dots, shortly termed as BbimDCN-OCDs, due to the presence of thermal-instable N(CN) 2 - moiety. BbimDCN-OCDs exhibit favorable biocompability and excellent imaging capacity for fluorescence labelling of yeast cell Saccharomyces cerevisiae. In addition, chitosan-modified Dy 3+ -doped magnetic nanoparticles (shortly as Chitosan@Fe 2.75 Dy 0.25 O 4 ) with superparamagnetism are prepared. The electrostatic attraction between positively charged magnetic nanoparticles and negatively charged yeast cells facilitates exclusive recognition/isolation of S. cerevisiae. In practice, S. cerevisiae is labelled by BbimDCN-OCDs and adhered onto the Chitosan@Fe 2.75 Dy 0.25 O 4 . The yeast/ BbimDCN-OCDs/Chitosan@Fe 2.75 Dy 0.25 O 4 composite is then isolated with an external magnet and the fluorescence from BbimDCN-OCDs incorporated in S. cerevisiae is monitored. The fluorescence intensity is linearly correlated with the content of yeast cell, showing a calibration graph of F = 3.01log[C]+11.7, offering a detection limit of 5×10 2 CFU/mL. S. cerevisiae content in various real sample matrixes are quantified by using this protocol. Copyright © 2017 Elsevier B.V. All rights reserved.

Advances in metabolic engineering of yeast Saccharomyces cerevisiae for production of chemicals.

PubMed

Borodina, Irina; Nielsen, Jens

2014-05-01

Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up the development of yeast cell factories. We also present an overview of metabolic engineering strategies for developing yeast strains for production of polymer monomers: lactic, succinic, and cis,cis-muconic acids. S. cerevisiae has already firmly established itself as a cell factory in industrial biotechnology and the advances in yeast strain engineering will stimulate development of novel yeast-based processes for chemicals production. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

PubMed

Nijland, Jeroen G; Shin, Hyun Yong; Boender, Leonie G M; de Waal, Paul P; Klaassen, Paul; Driessen, Arnold J M

2017-06-01

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8 , or SSN6 , which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10 , HXT13 , HXT15 , and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism ( V max ) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell. IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased

Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes.

PubMed Central

Pérez-Gonzalez, J A; De Graaff, L H; Visser, J; Ramón, D

1996-01-01

Two Aspergillus nidulans genes, xlnA and xlnB, encoding the X22 and X24 xylanases from this fungus, respectively, have been cloned and sequenced. Their cDNAs have been expressed in a laboratory Saccharomyces cerevisiae strain under the control of a constitutive yeast promoter, resulting in the construction of recombinant xylanolytic yeast strains. PMID:8787417

Enhanced ethanol fermentation by engineered Saccharomyces cerevisiae strains with high spermidine contents.

PubMed

Kim, Sun-Ki; Jo, Jung-Hyun; Jin, Yong-Su; Seo, Jin-Ho

2017-05-01

Construction of robust and efficient yeast strains is a prerequisite for commercializing a biofuel production process. We have demonstrated that high intracellular spermidine (SPD) contents in Saccharomyces cerevisiae can lead to improved tolerance against various fermentation inhibitors, including furan derivatives and acetic acid. In this study, we examined the potential applicability of the S. cerevisiae strains with high SPD contents under two cases of ethanol fermentation: glucose fermentation in repeated-batch fermentations and xylose fermentation in the presence of fermentation inhibitors. During the sixteen times of repeated-batch fermentations using glucose as a sole carbon source, the S. cerevisiae strains with high SPD contents maintained higher cell viability and ethanol productivities than a control strain with lower SPD contents. Specifically, at the sixteenth fermentation, the ethanol productivity of a S. cerevisiae strain with twofold higher SPD content was 31% higher than that of the control strain. When the SPD content was elevated in an engineered S. cerevisiae capable of fermenting xylose, the resulting S. cerevisiae strain exhibited much 40-50% higher ethanol productivities than the control strain during the fermentations of synthetic hydrolysate containing high concentrations of fermentation inhibitors. These results suggest that the strain engineering strategy to increase SPD content is broadly applicable for engineering yeast strains for robust and efficient production of ethanol.

Biosorption of the strontium ion by irradiated Saccharomyces cerevisiae under culture conditions.

PubMed

Qiu, Liang; Feng, Jundong; Dai, Yaodong; Chang, Shuquan

2017-06-01

As a new-emerging method for strontium disposal, biosorption has shown advantages such as high sorption capacity; low cost. In this study, we investigated the potential of Saccharomyces cerevisiae (S. cerevisiae) in strontium disposal under culture conditions and the effects of irradiation on their biosorption capabilities. We found that S. cerevisiae can survive irradiation and grow. Pre-exposure to irradiation rendered S. cerevisiae resistant to further irradiation. Surprisingly, the pre-exposure to irradiation can increase the biosorption capability of S. cerevisiae. We further investigated the factors that influenced the biosorption efficiency, which were (strongest to weakest): pH > strontium concentration > time > temperature. In our orthogonal experiment, the optimal conditions for strontium biosorption by irradiated S. cerevisiae were: pH 7, 150 mg L -1 strontium at the temperature of 32 °C with 30 h. The equilibrium of strontium biosorption was analyzed by Langmuir and Freundlich models, from which the formal model is found to provide a better fit for the experimental results. The kinetics of strontium biosorption by living irradiated S. cerevisiae was found to be comprised of three phases: dramatically increased during 0-9 h, decreased during 12-24 h, and increased during 30-50 h. These results provide a systematic understanding of the biosorption capabilities of irradiated S. cerevisiae, which can contribute to the development of remediating nuclear waste water. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pathways and Mechanisms that Prevent Genome Instability in Saccharomyces cerevisiae

PubMed Central

Putnam, Christopher D.; Kolodner, Richard D.

2017-01-01

Genome rearrangements result in mutations that underlie many human diseases, and ongoing genome instability likely contributes to the development of many cancers. The tools for studying genome instability in mammalian cells are limited, whereas model organisms such as Saccharomyces cerevisiae are more amenable to these studies. Here, we discuss the many genetic assays developed to measure the rate of occurrence of Gross Chromosomal Rearrangements (called GCRs) in S. cerevisiae. These genetic assays have been used to identify many types of GCRs, including translocations, interstitial deletions, and broken chromosomes healed by de novo telomere addition, and have identified genes that act in the suppression and formation of GCRs. Insights from these studies have contributed to the understanding of pathways and mechanisms that suppress genome instability and how these pathways cooperate with each other. Integrated models for the formation and suppression of GCRs are discussed. PMID:28684602

Saccharomyces cerevisiae variety diastaticus friend or foe?-spoilage potential and brewing ability of different Saccharomyces cerevisiae variety diastaticus yeast isolates by genetic, phenotypic and physiological characterization.

PubMed

Meier-Dörnberg, Tim; Kory, Oliver Ingo; Jacob, Fritz; Michel, Maximilian; Hutzler, Mathias

2018-06-01

Saccharomyces cerevisiae variety diastaticus is generally considered to be an obligatory spoilage microorganism and spoilage yeast in beer and beer-mixed beverages. Their super-attenuating ability causes increased carbon dioxide concentrations, beer gushing and potential bottle explosion along with changes in flavor, sedimentation and increased turbidity. This research shows clear differences in the super-attenuating properties of S. cerevisiae var. diastaticus yeast strains and their potential for industrial brewing applications. Nineteen unknown spoilage yeast cultures were obtained as isolates and characterized using a broad spectrum of genetic and phenotypic methods. Results indicated that all isolates represent genetically different S. cerevisiae var. diastaticus strains except for strain TUM PI BA 124. Yeast strains were screened for their super-attenuating ability and sporulation. Even if the STA1 gene responsible for super-attenuation by encoding for the enzyme glucoamylase could be verified by real-time polymerase chain reaction, no correlation to the spoilage potential could be demonstrated. Seven strains were further characterized focusing on brewing and sensory properties according to the yeast characterization platform developed by Meier-Dörnberg. Yeast strain TUM 3-H-2 cannot metabolize dextrin and soluble starch and showed no spoilage potential or super-attenuating ability even when the strain belongs to the species S. cerevisiae var. diastaticus. Overall, the beer produced with S. cerevisiae var. diastaticus has a dry and winey body with noticeable phenolic off-flavors desirable in German wheat beers.

Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae.

PubMed Central

Erdmann, R; Veenhuis, M; Mertens, D; Kunau, W H

1989-01-01

Two mutants of Saccharomyces cerevisiae affected in peroxisomal assembly (pas mutants) have been isolated and characterized. Each strain contains a single mutation that results in (i) the inability to grow on oleic acid, (ii) accumulation of peroxisomal matrix enzymes in the cytosol, and (iii) absence of detectable peroxisomes at the ultrastructural level. These lesions (pas1-1 and pas2) are shown to be nonallelic and recessive. Crossing of pas1-1 and pas2 strains resulted in diploid cells that had regained the ability to grow on oleic acid as sole carbon source and to form peroxisomes. These pas mutants may provide useful tools for future studies on the molecular mechanisms involved in peroxisomal assembly. Images PMID:2568633

Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae.

PubMed

Mishra, C; Semino, C E; McCreath, K J; de la Vega, H; Jones, B J; Specht, C A; Robbins, P W

1997-03-30

Chitin deacetylase (EC 3.5.1.41), which hydrolyses the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, has been demonstrated in crude extracts from sporulating Saccharomyces cerevisiae. Two S. cerevisiae open reading frames (ORFs), identified by the Yeast Genome Project, have protein sequence homology to a chitin deacetylase from Mucor rouxii. Northern blot hybridizations show each ORF was transcribed in diploid cells after transfer to sporulation medium and prior to formation of asci. Each ORF was cloned in a vector under transcriptional control of the GAL 1, 10 promoter and introduced back into haploid strains of S. cerevisiae. Chitin deacetylase activity was detected by in vitro assays from vegetative cells grown in galactose. Chemical analysis of these cells also demonstrated the synthesis of chitosam in vivo. Both recombinant chitin deacetylases showed similar qualitative and quantitative activities toward chitooligosaccharides in vitro. A diploid strain deleted to both ORFs, when sporulated, did not show deacetylase activity. The mutant spores were hypersensitive to lytic enzymes (Glusulase or Zymolyase).

Biosynthesis and engineering of kaempferol in Saccharomyces cerevisiae.

PubMed

Duan, Lijin; Ding, Wentao; Liu, Xiaonan; Cheng, Xiaozhi; Cai, Jing; Hua, Erbing; Jiang, Huifeng

2017-09-26

Kaempferol is a flavonol with broad bioactivity of anti-oxidant, anti-cancer, anti-diabetic, anti-microbial, cardio-protective and anti-asthma. Microbial synthesis of kaempferol is a promising strategy because of the low content in primary plant source. In this study, the biosynthesis pathway of kaempferol was constructed in the budding yeast Saccharomyces cerevisiae to produce kaempferol de novo, and several biological measures were taken for high production. Firstly, a high efficient flavonol synthases (FLS) from Populus deltoides was introduced into the biosynthetic pathway of kaempferol. Secondly, a S. cerevisiae recombinant was constructed for de novo synthesis of kaempferol, which generated about 6.97 mg/L kaempferol from glucose. To further promote kaempferol production, the acetyl-CoA biosynthetic pathway was overexpressed and p-coumarate was supplied as substrate, which improved kaempferol titer by about 23 and 120%, respectively. Finally, a fed-batch process was developed for better kaempferol fermentation performance, and the production reached 66.29 mg/L in 40 h. The titer of kaempferol in our engineered yeast is 2.5 times of the highest reported titer. Our study provides a possible strategy to produce kaempferol using microbial cell factory.

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

USDA-ARS?s Scientific Manuscript database

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...

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

Truncation of Gal4p explains the inactivation of the GAL/MEL regulon in both Saccharomyces bayanus and some Saccharomyces cerevisiae wine strains.

PubMed

Dulermo, Rémi; Legras, Jean-Luc; Brunel, François; Devillers, Hugo; Sarilar, Véronique; Neuvéglise, Cécile; Nguyen, Huu-Vang

2016-09-01

In the past, the galactose-negative (Gal(-)) phenotype was a key physiological character used to distinguish Saccharomyces bayanus from S. cerevisiae In this work, we investigated the inactivation of GAL gene networks in S. bayanus, which is an S. uvarum/S. eubayanus hybrid, and in S. cerevisiae wine strains erroneously labelled 'S. bayanus'. We made an inventory of their GAL genes using genomes that were either available publicly, re-sequenced by us, or assembled from public data and completed with targeted sequencing. In the S. eubayanus/S. uvarum CBS 380(T) hybrid, the GAL/MEL network is composed of genes from both parents: from S. uvarum, an otherwise complete set that lacks GAL4, and from S. eubayanus, a truncated version of GAL4 and an additional copy of GAL3 and GAL80 Similarly, two different truncated GAL4 alleles were found in S. cerevisiae wine strains EC1118 and LalvinQA23. The lack of GAL4 activity in these strains was corrected by introducing a full-length copy of S. cerevisiae GAL4 on a CEN4/ARS plasmid. Transformation with this plasmid restored galactose utilisation in Gal(-) strains, and melibiose fermentation in strain CBS 380(T) The melibiose fermentation phenotype, formerly regarded as characteristic of S. uvarum, turned out to be widespread among Saccharomyces species. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

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

Transposon mutagenesis to improve the growth of recombinant Saccharomyces cerevisiae on D-xylose

Treesearch

Haiying Ni; Jose M. Laplaza; Thomas W. Jeffries

2007-01-01

Saccharomyces cerevisiae L2612 transformed with genes for xylose reductase and xylitol dehydrogenase (XYL1 and XYL2) grows well on glucose but very poorly on D-xylose. When a gene for D-xylulokinase (XYL3 or XKS1) is overexpressed, growth on glucose is unaffected, but growth on xylose is blocked. Spontaneous or chemically induced mutants of this engineered yeast that...

[Molecular evolution of the sulphite efflux gene SSU1 in Saccharomyces cerevisiae].

PubMed

Peng, Li-Xin; Sun, Fei-Fei; Huang, Yan-Yan; Li, Zhen-Chong

2013-11-01

The SSU1 gene encoding a membrane sulfite pump is a main facilitator invovled in sulfite efflux. In Saccharomyce cerevisiae, various range of resistance to sulfite was observed among strains. To explore the evolution traits of SSU1 gene, the population data of S. cerevisiae were collected and analyzed. The phylogenetic analysis indicated that S. cerevisiae population can be classified into three sub-populations, and the positive selection was detected in population by McDonald-Kreitman test. The anaylsis of Ka/Ks ratios further showed that S. cerevisiae sub-population was undergoing positive selection. This finding was also supported by PAML branch model. Nine potential positive selection sites were predicted by branch-site model, and four sites exclusively belong to the sub-population under positive seletion. The data from ssulp protein structure demonstrated that three sites are substitutions between polar and hydrophobic amino acids, and only one site of substitutaion from basic amino acid to basic amino acid (345R/K). Because amino acid pKa values are crucial for sulfite pump to maintain their routine function, positive selection of these amino acid substitutions might affect sulfite efflux efficient.

Tentacle-type immobilized metal affinity cryogel for invertase purification from Saccharomyces cerevisiae.

PubMed

Çetin, Kemal; Perçin, Işık; Denizli, Fatma; Denizli, Adil

2017-11-01

The aim of this study is to investigate the usability of cryogel columns for the purification of invertase from Saccharomyces cerevisiae. Poly(2-hydroxyethyl methacrylate) monolithic columns were produced via cryogelation. Ester groups of the poly(2-hydroxyethyl methacrylate) structure were then converted to imine groups by the reaction with poly(ethylene imine) in the presence of NaHCO 3 . Transition metal ions, Cu(II), Co(II), and Ni(II), were chelated on the PEI-modified cryogel columns. Purification of invertase from natural source namely S. cerevisiae was also studied, and the purification fold values were obtained as 41.350, 44.714, and 30.302 for Cu(II)-chelated, Co(II)-chelated, and Ni(II)-chelated PHEMA/PEI columns, respectively.

Trichoderma virens β-glucosidase I (BGLI) gene; expression in Saccharomyces cerevisiae including docking and molecular dynamics studies.

PubMed

Wickramasinghe, Gammadde Hewa Ishan Maduka; Rathnayake, Pilimathalawe Panditharathna Attanayake Mudiyanselage Samith Indika; Chandrasekharan, Naduviladath Vishvanath; Weerasinghe, Mahindagoda Siril Samantha; Wijesundera, Ravindra Lakshman Chundananda; Wijesundera, Wijepurage Sandhya Sulochana

2017-06-21

Cellulose, a linear polymer of β 1-4, linked glucose, is the most abundant renewable fraction of plant biomass (lignocellulose). It is synergistically converted to glucose by endoglucanase (EG) cellobiohydrolase (CBH) and β-glucosidase (BGL) of the cellulase complex. BGL plays a major role in the conversion of randomly cleaved cellooligosaccharides into glucose. As it is well known, Saccharomyces cerevisiae can efficiently convert glucose into ethanol under anaerobic conditions. Therefore, S.cerevisiae was genetically modified with the objective of heterologous extracellular expression of the BGLI gene of Trichoderma virens making it capable of utilizing cellobiose to produce ethanol. The cDNA and a genomic sequence of the BGLI gene of Trichoderma virens was cloned in the yeast expression vector pGAPZα and separately transformed to Saccharomyces cerevisiae. The size of the BGLI cDNA clone was 1363 bp and the genomic DNA clone contained an additional 76 bp single intron following the first exon. The gene was 90% similar to the DNA sequence and 99% similar to the deduced amino acid sequence of 1,4-β-D-glucosidase of T. atroviride (AC237343.1). The BGLI activity expressed by the recombinant genomic clone was 3.4 times greater (1.7 x 10 -3  IU ml -1 ) than that observed for the cDNA clone (5 x 10 -4  IU ml -1 ). Furthermore, the activity was similar to the activity of locally isolated Trichoderma virens (1.5 x 10 -3  IU ml -1 ). The estimated size of the protein was 52 kDA. In fermentation studies, the maximum ethanol production by the genomic and the cDNA clones were 0.36 g and 0.06 g /g of cellobiose respectively. Molecular docking results indicated that the bare protein and cellobiose-protein complex behave in a similar manner with considerable stability in aqueous medium. The deduced binding site and the binding affinity of the constructed homology model appeared to be reasonable. Moreover, it was identified that the five hydrogen bonds formed

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

DOE PAGES

Tran, Tam N. T.; Breuer, Rebecca J.; Avanasi Narasimhan, Ragothaman; ...

2017-03-20

Background: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storagemore » lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts. Results: In order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol

The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier.

PubMed Central

Cameron, D R; Cooper, D G; Neufeld, R J

1988-01-01

The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier. PMID:3046488

Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

PubMed

Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

2015-09-01

The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae.

PubMed

Zhou, Pingping; Xie, Wenping; Li, Aipeng; Wang, Fan; Yao, Zhen; Bian, Qi; Zhu, Yongqiang; Yu, Hongwei; Ye, Lidan

2017-05-01

Highly efficient biosynthesis of the commercially valuable carotenoid astaxanthin by microbial cells is an attractive alternative to chemical synthesis and microalgae extraction. With the goal of enhancing heterologous astaxanthin production in Saccharomyces cerevisiae, metabolic engineering and protein engineering were integrated to improve both the expression and activity of rate-limiting enzymes. Firstly, to increase the supply of β-carotene as a key precursor for astaxanthin, a positive mutant of GGPP synthase (CrtE03M) was overexpressed together with three other rate-limiting enzymes tHMG1, CrtI and CrtYB. Subsequently, to accelerate the conversion of β-carotene to astaxanthin, a color screening system was developed and adopted for directed evolution of β-carotene ketolase (OBKT), generating a triple mutant OBKTM (H165R/V264D/F298Y) with 2.4-fold improved activity. After adjusting copy numbers of the above-mentioned rate-limiting enzymes to further balance the metabolic flux, a diploid strain YastD-01 was generated by mating two astaxanthin-producing haploid strains carrying the same carotenogenic pathway. Finally, further overexpression of OCrtZ and OBKTM in YastD-01 resulted in accumulation of 8.10mg/g DCW (47.18mg/l) of (3S, 3'S)-astaxanthin in shake-flask cultures. This combinatorial strategy might be also applicable for alleviation of metabolic bottleneck in biosynthesis of other value-added products, especially colored metabolites. Copyright © 2017 Elsevier Inc. All rights reserved.

Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl, Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiae

DOE PAGES

Eudes, Aymerick; Teixeira Benites, Veronica; Wang, George; ...

2015-10-02

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 anthranilatesmore » 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. Finally, 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.« less

Altering the Rate of Mitosis by Introducing Low-Gigahertz Radiation to Saccharomyces cerevisiae Cells

NASA Astrophysics Data System (ADS)

Garg, S.; Ashby, C.

2017-12-01

This experiment aims to assess the impact of low-frequency radiation (from common technological tools such as cell phones, scanners, and wifi) on the mitotic rates of cells. In particular, the focus of the study was on the growth and development of Saccharomyces cerevisiae cultures that were exposed to radio waves from a wifi router, which were then compared to a cohort of the same species without exposure. Though routers emit a low gigahertz frequency, they are categorized as Group 2B radiation (possibly carcinogenic) by the International Agency for Research on Cancer of the World Health Organization, signifying that constant exposure poses a potential risk to humans. Twelve agar dishes of active Saccharomyces cerevisiae solution were prepared, with six dishes acting as the control under no added radiation and six acting as the experimental group under 2.4 GHz of radiation due to their proximity to the router. Data on how many cultures proliferated in each dish was collected every three days, with the experiment running for a total of twelve days. All subjects experienced growth curves until day 9 when the experimental group's growth peaked with an average of 62 colonies/dish. Three of the six dishes in this group lost colonies in the following three days, leaving the experimental group with an average of 61 colonies/dish on day 12, while the control group was still increasing by day 12 with an average of 48 colonies/dish, with only one dish undergoing a loss of colonies. Exposing the Saccharomyces cerevisiae cells to low grade radiation resulted in accelerated mitosis, and though the experimental group faced colony death after nine days, the loss was likely due to overpopulation in the dish.

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress▿

PubMed Central

Rodríguez-Vargas, Sonia; Sánchez-García, Alicia; Martínez-Rivas, Jose Manuel; Prieto, Jose Antonio; Randez-Gil, Francisca

2007-01-01

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ9 position. We expressed two sunflower (Helianthus annuus) oleate Δ12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp+ or Trp− strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains. PMID:17071783

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

Purification and Characterization of Put1p from Saccharomyces cerevisiae

PubMed Central

Wanduragala, Srimevan; Sanyal, Nikhilesh; Liang, Xinwen; Becker, Donald F.

2010-01-01

In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH)1 enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A Km value of 36 mM proline and a kcat = 27 s−1 were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ1) as an electron acceptor with a kcat = 9.6 s−1 and a Km of 33 µM for CoQ1. In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast. PMID:20450881

Statistics-based model for prediction of chemical biosynthesis yield from Saccharomyces cerevisiae

PubMed Central

2011-01-01

Background The robustness of Saccharomyces cerevisiae in facilitating industrial-scale production of ethanol extends its utilization as a platform to synthesize other metabolites. Metabolic engineering strategies, typically via pathway overexpression and deletion, continue to play a key role for optimizing the conversion efficiency of substrates into the desired products. However, chemical production titer or yield remains difficult to predict based on reaction stoichiometry and mass balance. We sampled a large space of data of chemical production from S. cerevisiae, and developed a statistics-based model to calculate production yield using input variables that represent the number of enzymatic steps in the key biosynthetic pathway of interest, metabolic modifications, cultivation modes, nutrition and oxygen availability. Results Based on the production data of about 40 chemicals produced from S. cerevisiae, metabolic engineering methods, nutrient supplementation, and fermentation conditions described therein, we generated mathematical models with numerical and categorical variables to predict production yield. Statistically, the models showed that: 1. Chemical production from central metabolic precursors decreased exponentially with increasing number of enzymatic steps for biosynthesis (>30% loss of yield per enzymatic step, P-value = 0); 2. Categorical variables of gene overexpression and knockout improved product yield by 2~4 folds (P-value < 0.1); 3. Addition of notable amount of intermediate precursors or nutrients improved product yield by over five folds (P-value < 0.05); 4. Performing the cultivation in a well-controlled bioreactor enhanced the yield of product by three folds (P-value < 0.05); 5. Contribution of oxygen to product yield was not statistically significant. Yield calculations for various chemicals using the linear model were in fairly good agreement with the experimental values. The model generally underestimated the ethanol production as

The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae.

PubMed

García-Ríos, Estéfani; Morard, Miguel; Parts, Leopold; Liti, Gianni; Guillamón, José M

2017-02-14

Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci. In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions' adaptive nature. The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.

Metabolomic Comparison of Saccharomyces cerevisiae and the Cryotolerant Species S. bayanus var. uvarum and S. kudriavzevii during Wine Fermentation at Low Temperature

PubMed Central

López-Malo, María; Querol, Amparo; Guillamon, José Manuel

2013-01-01

Temperature is one of the most important parameters affecting the length and rate of alcoholic fermentation and final wine quality. Wine produced at low temperature is often considered to have improved sensory qualities. However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase, and sluggish or stuck fermentations. To investigate the effects of temperature on commercial wine yeast, we compared its metabolome growing at 12°C and 28°C in a synthetic must. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae. This is the case of the cryotolerant yeasts Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the metabolome of these species growing at 12°C, which we compared with the metabolome of S. cerevisiae (not well adapted at low temperature) at the same temperature. Our results show that the main differences between the metabolic profiling of S. cerevisiae growing at 12°C and 28°C were observed in lipid metabolism and redox homeostasis. Moreover, the global metabolic comparison among the three species revealed that the main differences between the two cryotolerant species and S. cerevisiae were in carbohydrate metabolism, mainly fructose metabolism. However, these two species have developed different strategies for cold resistance. S. bayanus var. uvarum presented elevated shikimate pathway activity, while S. kudriavzevii displayed increased NAD+ synthesis. PMID:23527304

Schizosaccharomyces pombe and Saccharomyces cerevisiae yeasts in sequential fermentations: Effect on phenolic acids of fermented Kei-apple (Dovyalis caffra L.) juice.

PubMed

Minnaar, P P; Jolly, N P; Paulsen, V; Du Plessis, H W; Van Der Rijst, M

2017-09-18

Kei-apple (Dovyalis caffra) is an evergreen tree indigenous to Southern Africa. The fruit contains high concentrations of l-malic acid, ascorbic acid, and phenolic acids. Kei-apple juice was sequentially inoculated with Schizosaccharomyces pombe and Saccharomyces cerevisiae yeasts. A reference fermentation using only S. cerevisiae was included. The fermentation was monitored by recording mass loss. At the end of fermentation, twelve untrained judges conducted free choice aroma profiling on the fruit wines. The Kei-apple juice and wines were analysed for total titratable acidity, total soluble solids, pH, alcohol, l-malic acid, and phenolic acids. Total titratable acidity was ca. 70% lower in Kei-apple wines produced with S. pombe+S. cerevisiae than in Kei-apple juice. Kei-apple wines produced with S. pombe+S. cerevisiae showed substantially lower concentrations of l-malic acid than Kei-apple wines produced with S. cerevisiae only. Wines produced with S. cerevisiae only proved higher in phenolic acid concentrations than wines produced with S. pombe+S. cerevisiae. Chlorogenic acid was the most abundant phenolic acid measured in the Kei-apple wines, followed by protocatechuic acid. Judges described the Kei-apple wines produced with S. pombe+S. cerevisiae as having noticeable off-odours, while wines produced with S. cerevisiae were described as fresh and fruity. Kei-apple wines (S. pombe+S. cerevisiae and S. cerevisiae) were of comparable vegetative and organic character. Saccharomyces cerevisiae produced Kei-apple wine with increased caffeic, chlorogenic, protocatechuic, and sinapic acids, whereas S. pombe+S. cerevisiae produced Kei-apple wines with increased ferulic, and p-coumaric acids and low l-malic acid. Copyright © 2017 Elsevier B.V. All rights reserved.

Epidemiological Investigation of Vaginal Saccharomyces cerevisiae Isolates by a Genotypic Method

PubMed Central

McCullough, Michael J.; Clemons, Karl V.; Farina, Claudio; McCusker, John H.; Stevens, David A.

1998-01-01

Saccharomyces cerevisiae is a ubiquitous, ascomycetous yeast, and vaginitis caused by this organism has been reported only very rarely. The aim of the present investigation was to assess the epidemiological relatedness of a group of vaginal and commercial S. cerevisiae isolates by a previously reported genetic typing method, which divided the isolates into two broad groups with numerous subtypes. Nineteen S. cerevisiae isolates obtained from patients suffering from vaginitis and four isolates from commercial products in the same city were analyzed. The cellular DNA from each isolate was digested with the restriction endonuclease EcoRI, and restriction fragment length polymorphisms were generated by horizontal gel electrophoresis. The results showed that although vaginal isolates did not cluster in any particular genetic subtype, multiple patients were infected with indistinguishable strains (there were nine distinct strains among 23 isolates). For two of three patients, all three with two episodes of S. cerevisiae vaginitis, different strains were isolated during the recurrence of this disease. Three other patients with indistinguishable isolates were epidemiologically related in that two were practitioners in the same clinic and the third was a patient at this clinic. We also found that one commercial strain was indistinguishable from the strain isolated from three different women at the time that they were suffering from vaginitis. The findings of the present study suggest that some S. cerevisiae strains may possess properties permitting persistence in the human host. Furthermore, person-to-person contact and the proliferation of the use of S. cerevisiae as a health-food product, in home baking, and in home brewing may be a contributing factor in human colonization and infection with this organism. PMID:9466776

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

Force Sensitivity in Saccharomyces cerevisiae Flocculins.

PubMed

Chan, Cho X J; El-Kirat-Chatel, Sofiane; Joseph, Ivor G; Jackson, Desmond N; Ramsook, Caleen B; Dufrêne, Yves F; Lipke, Peter N

2016-01-01

Many fungal adhesins have short, β-aggregation-prone sequences that play important functional roles, and in the Candida albicans adhesin Als5p, these sequences cluster the adhesins after exposure to shear force. Here, we report that Saccharomyces cerevisiae flocculins Flo11p and Flo1p have similar β-aggregation-prone sequences and are similarly stimulated by shear force, despite being nonhomologous. Shear from vortex mixing induced the formation of small flocs in cells expressing either adhesin. After the addition of Ca(2+), yeast cells from vortex-sheared populations showed greatly enhanced flocculation and displayed more pronounced thioflavin-bright surface nanodomains. At high concentrations, amyloidophilic dyes inhibited Flo1p- and Flo11p-mediated agar invasion and the shear-induced increase in flocculation. Consistent with these results, atomic force microscopy of Flo11p showed successive force-distance peaks characteristic of sequentially unfolding tandem repeat domains, like Flo1p and Als5p. Flo11p-expressing cells bound together through homophilic interactions with adhesion forces of up to 700 pN and rupture lengths of up to 600 nm. These results are consistent with the potentiation of yeast flocculation by shear-induced formation of high-avidity domains of clustered adhesins at the cell surface, similar to the activation of Candida albicans adhesin Als5p. Thus, yeast adhesins from three independent gene families use similar force-dependent interactions to drive cell adhesion. IMPORTANCE The Saccharomyces cerevisiae flocculins mediate the formation of cellular aggregates and biofilm-like mats, useful in clearing yeast from fermentations. An important property of fungal adhesion proteins, including flocculins, is the ability to form catch bonds, i.e., bonds that strengthen under tension. This strengthening is based, at least in part, on increased avidity of binding due to clustering of adhesins in cell surface nanodomains. This clustering depends on

Maltotriose fermentation by Saccharomyces cerevisiae.

PubMed

Zastrow, C R; Hollatz, C; de Araujo, P S; Stambuk, B U

2001-07-01

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l(-1) glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific alpha-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells.

Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins.

PubMed Central

Van der Vaart, J M; te Biesebeke, R; Chapman, J W; Toschka, H Y; Klis, F M; Verrips, C T

1997-01-01

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production. alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell. PMID:9023939

Bioethanol strains of Saccharomyces cerevisiae characterised by microsatellite and stress resistance.

PubMed

Reis, Vanda Renata; Antonangelo, Ana Teresa Burlamaqui Faraco; Bassi, Ana Paula Guarnieri; Colombi, Débora; Ceccato-Antonini, Sandra Regina

Strains of Saccharomyces cerevisiae may display characteristics that are typical of rough-type colonies, made up of cells clustered in pseudohyphal structures and comprised of daughter buds that do not separate from the mother cell post-mitosis. These strains are known to occur frequently in fermentation tanks with significant lower ethanol yield when compared to fermentations carried out by smooth strains of S. cerevisiae that are composed of dispersed cells. In an attempt to delineate genetic and phenotypic differences underlying the two phenotypes, this study analysed 10 microsatellite loci of 22 S. cerevisiae strains as well as stress resistance towards high concentrations of ethanol and glucose, low pH and cell sedimentation rates. The results obtained from the phenotypic tests by Principal-Component Analysis revealed that unlike the smooth colonies, the rough colonies of S. cerevisiae exhibit an enhanced resistance to stressful conditions resulting from the presence of excessive glucose and ethanol and high sedimentation rate. The microsatellite analysis was not successful to distinguish between the colony phenotypes as phenotypic assays. The relevant industrial strain PE-2 was observed in close genetic proximity to rough-colony although it does not display this colony morphology. A unique genetic pattern specific to a particular phenotype remains elusive. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

USDA-ARS?s Scientific Manuscript database

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a differ...

Draft Genome Sequence of Saccharomyces cerevisiae Barra Grande (BG-1), a Brazilian Industrial Bioethanol-Producing Strain

PubMed Central

Coutouné, Natalia; Mulato, Aline Tieppo Nogueira

2017-01-01

ABSTRACT Here, we present the draft genome sequence of Saccharomyces cerevisiae BG-1, a Brazilian industrial strain widely used for bioethanol production from sugarcane. The 11.7-Mb genome sequence consists of 216 scaffolds and harbors 5,607 predicted protein-coding genes. PMID:28360170

Invasive Saccharomyces cerevisiae in a liver transplant patient: case report and review of infection in transplant recipients.

PubMed

Popiel, K Y; Wong, P; Lee, M J; Langelier, M; Sheppard, D C; Vinh, D C

2015-06-01

Saccharomyces cerevisiae, an ascosporogenous yeast commonly used in the production of food, is an emerging infection in immunocompromised patients. We report the case of a 60-year-old man whose orthotopic liver transplant was complicated by S. cerevisiae fungemia and peritoneal abscess, successfully treated with caspofungin and drainage. We also review the literature of invasive saccharomycoses in recipients of hematologic and solid organ transplants. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Saccharomyces cerevisiae KNU5377 stress response during high-temperature ethanol fermentation.

PubMed

Kim, Il-Sup; Kim, Young-Saeng; Kim, Hyun; Jin, Ingnyol; Yoon, Ho-Sung

2013-03-01

Fuel ethanol production is far more costly to produce than fossil fuels. There are a number of approaches to cost-effective fuel ethanol production from biomass. We characterized stress response of thermotolerant Saccharomyces cerevisiae KNU5377 during glucose-based batch fermentation at high temperature (40°C). S. cerevisiae KNU5377 (KNU5377) transcription factors (Hsf1, Msn2/4, and Yap1), metabolic enzymes (hexokinase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, and alcohol dehydrogenase), antioxidant enzymes (thioredoxin 3, thioredoxin reductase, and porin), and molecular chaperones and its cofactors (Hsp104, Hsp82, Hsp60, Hsp42, Hsp30, Hsp26, Cpr1, Sti1, and Zpr1) are upregulated during fermentation, in comparison to S. cerevisiae S288C (S288C). Expression of glyceraldehyde-3-phosphate dehydrogenase increased significantly in KNU5377 cells. In addition, cellular hydroperoxide and protein oxidation, particularly lipid peroxidation of triosephosphate isomerase, was lower in KNU5377 than in S288C. Thus, KNU5377 activates various cell rescue proteins through transcription activators, improving tolerance and increasing alcohol yield by rapidly responding to fermentation stress through redox homeostasis and proteostasis.

The Response to Heat Shock and Oxidative Stress in Saccharomyces cerevisiae

PubMed Central

Morano, Kevin A.; Grant, Chris M.; Moye-Rowley, W. Scott

2012-01-01

A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review the progress in understanding the response of the yeast Saccharomyces cerevisiae to two important environmental stresses: heat shock and oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of these environmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding of eukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperature and levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stress response is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, much remains to be learned about the mechanisms dedicated to dealing with these environmental parameters. PMID:22209905

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

USDA-ARS?s Scientific Manuscript database

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...

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

PubMed

Dolezalova, Jaroslava; Rumlova, Lubomira

2014-11-01

This new biological test of water toxicity is based on monitoring of specific conductivity changes of yeast Saccharomyces cerevisiae suspension as a result of yeast fermentation activity inhibition in toxic conditions. The test was verified on ten substances with various mechanisms of toxic effect and the results were compared with two standard toxicity tests based on Daphnia magna mobility inhibition (EN ISO 6341) and Vibrio fischeri bioluminescence inhibition (EN ISO 11348-2) and with the results of the S. cerevisiae lethal test (Rumlova and Dolezalova, 2012). The new biological test - S. cerevisiae conductometric test - is an express method developed primarily for field conditions. It is applicable in case of need of immediate information about water toxicity. Fast completion is an advantage of this test (time necessary for test completion is about 60min), the test is simple and the test organism - dried instant yeast - belongs among its biggest advantages because of its long-term storage life and broad availability. Copyright © 2014 Elsevier B.V. All rights reserved.

Glycerol positive promoters for tailored metabolic engineering of the yeast Saccharomyces cerevisiae.

PubMed

Ho, Ping-Wei; Klein, Mathias; Futschik, Matthias; Nevoigt, Elke

2018-05-01

Glycerol offers several advantages as a substrate for biotechnological applications. An important step toward using the popular production host Saccharomyces cerevisiae for glycerol-based bioprocesses has been the fact that in recent studies commonly used S. cerevisiae strains were engineered to grow in synthetic medium containing glycerol as the sole carbon source. For metabolic engineering projects of S. cerevisiae growing on glycerol, characterized promoters are missing. In the current study, we used transcriptome analysis and a yECitrine-based fluorescence reporter assay to select and characterize 25 useful promoters. The promoters of the genes ALD4 and ADH2 showed 4.2-fold and 3-fold higher activities compared to the well-known strong TEF1 promoter. Moreover, the collection contains promoters with graded activities in synthetic glycerol medium and different degrees of glucose repression. To demonstrate the general applicability of the promoter collection, we successfully used a subset of the characterized promoters with graded activities in order to optimize growth on glycerol in an engineered derivative of CEN.PK, in which glycerol catabolism exclusively occurs via a non-native DHA pathway.

Chromium (VI) biosorption by Saccharomyces cerevisiae subjected to chemical and thermal treatments.

PubMed

De Rossi, Andrea; Rigon, Magali Rejane; Zaparoli, Munise; Braido, Rafael Dalmas; Colla, Luciane Maria; Dotto, Guilherme Luiz; Piccin, Jeferson Steffanello

2018-05-28

The potential of chemically and thermally treated Saccharomyces cerevisiae as biosorbents for chromium (VI) was investigated in this work. The presence of this toxic metal in industrial effluents is harmful to the environment, so, it is important to develop environmental friendly methods for Cr(VI) removal from these effluents. Biosorption using microorganisms such as S. cerevisiae is a viable treatment option because this biomass is easily available as a residue of fermentation industries. In this study, the affecting variables on Cr(VI) biosorption were studied by constructing biosorption isotherms, using lyophilized yeast subjected to chemical and thermal treatments. S. cerevisiae was able to remove 99.66% of Cr(VI) from effluents by biosorption. The significant variables affecting biosorption were pH, initial Cr(VI) concentration, and contact time. The biosorption isotherms were represented by the Freundlich model for the untreated biomass, BET model for the chemically treated biomass, and Langmuir model for the heat-treated biomass. Thermal treatment increased the biosorption affinity of the biomass for chromium, while the chemical treatment facilitated the formation of a multilayer.

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation.

PubMed

Satomura, Atsushi; Katsuyama, Yoshiaki; Miura, Natsuko; Kuroda, Kouichi; Tomio, Ayako; Bamba, Takeshi; Fukusaki, Eiichiro; Ueda, Mitsuyoshi

2013-01-01

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60-1, was bred from a parental strain, MT8-1, via stepwise adaptation. YK60-1 grew at 40°C, a temperature at which MT8-1 could not grow at all. YK60-1 exhibited faster growth than MT8-1 at 30°C. To investigate the mechanisms how MT8-1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress-responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60-1. Furthermore, nontargeting metabolome analysis showed that YK60-1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8-1. In conclusion, S. cerevisiae MT8-1 acquired thermotolerance by induction of specific stress-responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers.

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

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

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

Genomic Sequence of Saccharomyces cerevisiae BAW-6, a Yeast Strain Optimal for Brewing Barley Shochu

PubMed Central

Mori, Kazuki; Tashiro, Kosuke; Higuchi, Yujiro; Takashita, Hideharu

2018-01-01

ABSTRACT Here, we report the draft genome sequence of Saccharomyces cerevisiae strain BAW-6, which is used for the production of barley shochu, a traditional Japanese spirit. This genomic information can be used to elucidate the genetic basis underlying the high alcohol production capacity and citric acid tolerance of shochu yeast. PMID:29622617

Torulaspora delbrueckii contribution in mixed brewing fermentations with different Saccharomyces cerevisiae strains.

PubMed

Canonico, Laura; Comitini, Francesca; Ciani, Maurizio

2017-10-16

In recent years, there has been growing demand for distinctive high quality beer. Fermentation management has a fundamental role in beer quality and the levels of aroma compounds. Use of non-conventional yeast has been proposed to enhance beer bioflavor. In the present work we investigated mixed fermentations using three commercial Saccharomyces cerevisiae strains, without and with addition of a selected Torulaspora delbrueckii strain evaluating their interactions, as well as the aroma profiles. At the S. cerevisiae/T. delbrueckii co-inoculation ratio of 1:20, viable cell counts indicated that T. delbrueckii dominated all of the three combinations. In the mixed fermentations, T. delbrueckii provided higher levels of higher alcohols (excepting of β-phenyl ethanol), in contrast to data obtained in winemaking, where higher alcohols had lower levels. Moreover, mixed fermentations showed significantly higher ethyl acetate (from 5 to 16mg/L) and isoamyl acetate (from 0.019 to 0.128mg/L), and were generally lower in ethyl hexanoate and ethyl octanoate. Therefore, irrespective of S. cerevisiae strain, T. delbrueckii influenced on all mixed fermentations. On the other hand, the mixed fermentations were also affected by each of the three S. cerevisiae strains, which resulted in beers with distinctive flavors. Copyright © 2017 Elsevier B.V. All rights reserved.

Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification

USDA-ARS?s Scientific Manuscript database

Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by the yeast, particularly when the carbon source is acid-treated lignocell...

DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

EPA Science Inventory

Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

PubMed Central

Dever, Thomas E.; Kinzy, Terri Goss; Pavitt, Graham D.

2016-01-01

In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs. PMID:27183566

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa.

PubMed

Carú, M; Cifuentes, V; Pincheira, G; Jiménez, A

1989-10-01

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suco and N. crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa, although S. cerevisiae suc+ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.

Advancing Metabolic Engineering of Saccharomyces cerevisiae Using the CRISPR/Cas System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lian, Jiazhang; HamediRad, Mohammad; Zhao, Huimin

Thanks to its ease of use, modularity, and scalability, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been increasingly used in the design and engineering of Saccharomyces cerevisiae, one of the most popular hosts for industrial biotechnology. This review summarizes the recent development of this disruptive technology for metabolic engineering applications, including CRISPR-mediated gene knock-out and knock-in as well as transcriptional activation and interference. More importantly, multi-functional CRISPR systems that combine both gain- and loss-of-function modulations for combinatorial metabolic engineering are highlighted.

Advancing Metabolic Engineering of Saccharomyces cerevisiae Using the CRISPR/Cas System

DOE PAGES

Lian, Jiazhang; HamediRad, Mohammad; Zhao, Huimin

2018-04-18

Thanks to its ease of use, modularity, and scalability, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been increasingly used in the design and engineering of Saccharomyces cerevisiae, one of the most popular hosts for industrial biotechnology. This review summarizes the recent development of this disruptive technology for metabolic engineering applications, including CRISPR-mediated gene knock-out and knock-in as well as transcriptional activation and interference. More importantly, multi-functional CRISPR systems that combine both gain- and loss-of-function modulations for combinatorial metabolic engineering are highlighted.

Genomic reconstruction to improve bioethanol and ergosterol production of industrial yeast Saccharomyces cerevisiae.

PubMed

Zhang, Ke; Tong, Mengmeng; Gao, Kehui; Di, Yanan; Wang, Pinmei; Zhang, Chunfang; Wu, Xuechang; Zheng, Daoqiong

2015-02-01

Baker's yeast (Saccharomyces cerevisiae) is the common yeast used in the fields of bread making, brewing, and bioethanol production. Growth rate, stress tolerance, ethanol titer, and byproducts yields are some of the most important agronomic traits of S. cerevisiae for industrial applications. Here, we developed a novel method of constructing S. cerevisiae strains for co-producing bioethanol and ergosterol. The genome of an industrial S. cerevisiae strain, ZTW1, was first reconstructed through treatment with an antimitotic drug followed by sporulation and hybridization. A total of 140 mutants were selected for ethanol fermentation testing, and a significant positive correlation between ergosterol content and ethanol production was observed. The highest performing mutant, ZG27, produced 7.9 % more ethanol and 43.2 % more ergosterol than ZTW1 at the end of fermentation. Chromosomal karyotyping and proteome analysis of ZG27 and ZTW1 suggested that this breeding strategy caused large-scale genome structural variations and global gene expression diversities in the mutants. Genetic manipulation further demonstrated that the altered expression activity of some genes (such as ERG1, ERG9, and ERG11) involved in ergosterol synthesis partly explained the trait improvement in ZG27.

Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae.

PubMed

Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

2015-01-01

Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.

Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis.

PubMed

Werner, Sean R; Morgan, John A

2009-07-15

Glycosyltransferases are promising biocatalysts for the synthesis of small molecule glycosides. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase (GT) from Dianthus caryophyllus (carnation) was investigated as a whole-cell biocatalyst. Two yeast expression systems were compared using the flavonoid naringenin as a model substrate. Under in vitro conditions, naringenin-7-O-glucoside was formed and a higher specific glucosyl transfer activity was found using a galactose inducible expression system compared to a constitutive expression system. However, S. cerevisiae expressing the GT constitutively was significantly more productive than the galactose inducible system under in vivo conditions. Interestingly, the glycosides were recovered directly from the culture broth and did not accumulate intracellularly. A previously uncharacterized naringenin glycoside formed using the D. caryophyllus GT was identified as naringenin-4'-O-glucoside. It was found that S. cerevisiae cells hydrolyze naringenin-7-O-glucoside during whole-cell biocatalysis, resulting in a low final glycoside titer. When phloretin was added as a substrate to the yeast strain expressing the GT constitutively, the natural product phlorizin was formed. This study demonstrates S. cerevisiae is a promising whole-cell biocatalyst host for the production of valuable glycosides.

Nonhomologous Synapsis and Reduced Crossing over in a Heterozygous Paracentric Inversion in Saccharomyces Cerevisiae

PubMed Central

Dresser, M. E.; Ewing, D. J.; Harwell, S. N.; Coody, D.; Conrad, M. N.

1994-01-01

Homologous chromosome synapsis (``homosynapsis'') and crossing over are well-conserved aspects of meiotic chromosome behavior. The long-standing assumption that these two processes are causally related has been challenged recently by observations in Saccharomyces cerevisiae of significant levels of crossing over (1) between small sequences at nonhomologous locations and (2) in mutants where synapsis is abnormal or absent. In order to avoid problems of local sequence effects and of mutation pleiotropy, we have perturbed synapsis by making a set of isogenic strains that are heterozygous and homozygous for a large chromosomal paracentric inversion covering a well marked genetic interval and then measured recombination. We find that reciprocal recombination in the marked interval in heterozygotes is reduced variably across the interval, on average to ~55% of that in the homozygotes, and that positive interference still modulates crossing over. Cytologically, stable synapsis across the interval is apparently heterologous rather than homologous, consistent with the interpretation that stable homosynapsis is required to initiate or consummate a large fraction of the crossing over observed in wild-type strains. When crossing over does occur in heterozygotes, dicentric and acentric chromosomes are formed and can be visualized and quantitated on blots though not demonstrated in viable spores. We find that there is no loss of dicentric chromosomes during the two meiotic divisions and that the acentric chromosome is recovered at only 1/3 to 1/2 of the expected level. PMID:7851761

Efficient ethanol production from beetle-killed lodgepole pine using SPORL technology and Saccharomyces cerevisiae without detoxification

Treesearch

Junyong Zhu; Xiaolin Luo; Shen Tian; Roland Gleisner; Jose Negron; Eric Horn

2011-01-01

This study applied Sulfite Pretreatment to Overcome Recalcitrance of Lignocelluloses (SPORL) to evaluate the potential of mountain pine beetle-killed lodgepole pine for ethanol production using conventional Saccharomyces cerevisiae without hydrolysate detoxification. The results indicate that the beetle-killed trees are more susceptible to SPORL pretreatment than live...

RNAseq-based transcriptome comparison of Saccharomyces cerevisiae strains isolated from diverse fermentative environments.

PubMed

Ibáñez, Clara; Pérez-Torrado, Roberto; Morard, Miguel; Toft, Christina; Barrio, Eladio; Querol, Amparo

2017-09-18

Transcriptome analyses play a central role in unraveling the complexity of gene expression regulation in Saccharomyces cerevisiae. This species, one of the most important microorganisms for humans given its industrial applications, shows an astonishing degree of genetic and phenotypic variability among different strains adapted to specific environments. In order to gain novel insights into the Saccharomyces cerevisiae biology of strains adapted to different fermentative environments, we analyzed the whole transcriptome of three strains isolated from wine, flor wine or mezcal fermentations. An RNA-seq transcriptome comparison of the different yeasts in the samples obtained during synthetic must fermentation highlighted the differences observed in the genes that encode mannoproteins, and in those involved in aroma, sugar transport, glycerol and alcohol metabolism, which are important under alcoholic fermentation conditions. These differences were also observed in the physiology of the strains after mannoprotein and aroma determinations. This study offers an essential foundation for understanding how gene expression variations contribute to the fermentation differences of the strains adapted to unequal fermentative environments. Such knowledge is crucial to make improvements in fermentation processes and to define targets for the genetic improvement or selection of wine yeasts. Copyright © 2017 Elsevier B.V. All rights reserved.

Hybridization and Polyploidization of Saccharomyces cerevisiae Strains by Transformation-Associated Cell Fusion

PubMed Central

Takagi, Atsuko; Harashima, Satoshi; Oshima, Yasuji

1985-01-01

Hybrid or polyploid clones of Saccharomyces cerevisiae produced by protoplast fusion were easily isolated by selecting transformants with the plasmid phenotype because the transformation was directly associated with cell fusion. When haploid cells were used as the original strain, the transformants were mostly diploids with a significant fraction of polyploids (triploids or tetraploids). Repeated transformation after curing the plasmid gave rise to clones with higher ploidy, but the frequency of cell fusion was severely reduced as ploidy increased. Images PMID:16346702

Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 wasmore » 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.« less

Induction and characterization of morphologic mutants in a natural Saccharomyces cerevisiae strain.

PubMed

Barberio, Claudia; Bianchi, Lucia; Pinzauti, Francesca; Lodi, Tiziana; Ferrero, Iliana; Polsinelli, Mario; Casalone, Enrico

2007-02-01

Saccharomyces cerevisiae is a good model with which to study the effects of morphologic differentiation on the ecological behaviour of fungi. In this work, 33 morphologic mutants of a natural strain of S. cerevisiae, obtained with UV mutagenesis, were selected for their streak shape and cell shape on rich medium. Two of them, showing both high sporulation proficiency and constitutive pseudohyphal growth, were analysed from a genetic and physiologic point of view. Each mutant carries a recessive monogenic mutation, and the two mutations reside in unlinked genes. Flocculation ability and responsiveness to different stimuli distinguished the two mutants. Growth at 37 degrees C affected the cell but not the colony morphology, suggesting that these two phenotypes are regulated differently. The effect of ethidium bromide, which affects mitochondrial DNA replication, suggested a possible "retrograde action" of mitochondria in pseudohyphal growth.

Genomic Sequence of Saccharomyces cerevisiae BAW-6, a Yeast Strain Optimal for Brewing Barley Shochu.

PubMed

Kajiwara, Yasuhiro; Mori, Kazuki; Tashiro, Kosuke; Higuchi, Yujiro; Takegawa, Kaoru; Takashita, Hideharu

2018-04-05

Here, we report the draft genome sequence of Saccharomyces cerevisiae strain BAW-6, which is used for the production of barley shochu, a traditional Japanese spirit. This genomic information can be used to elucidate the genetic basis underlying the high alcohol production capacity and citric acid tolerance of shochu yeast. Copyright © 2018 Kajiwara et al.

Energetic and metabolic transient response of Saccharomyces cerevisiae to benzoic acid.

PubMed

Kresnowati, M T A P; van Winden, W A; van Gulik, W M; Heijnen, J J

2008-11-01

Saccharomyces cerevisiae is known to be able to adapt to the presence of the commonly used food preservative benzoic acid with a large energy expenditure. Some mechanisms for the adaptation process have been suggested, but its quantitative energetic and metabolic aspects have rarely been discussed. This study discusses use of the stimulus response approach to quantitatively study the energetic and metabolic aspects of the transient adaptation of S. cerevisiae to a shift in benzoic acid concentration, from 0 to 0.8 mM. The information obtained also serves as the basis for further utilization of benzoic acid as a tool for targeted perturbation of the energy system, which is important in studying the kinetics and regulation of central carbon metabolism in S. cerevisiae. Using this experimental set-up, we found significant fast-transient (< 3000 s) increases in O(2) consumption and CO(2) production rates, of approximately 50%, which reflect a high energy requirement for the adaptation process. We also found that with a longer exposure time to benzoic acid, S. cerevisiae decreases the cell membrane permeability for this weak acid by a factor of 10 and decreases the cell size to approximately 80% of the initial value. The intracellular metabolite profile in the new steady-state indicates increases in the glycolytic and tricarboxylic acid cycle fluxes, which are in agreement with the observed increases in specific glucose and O(2) uptake rates.

Single Cell Protein Production by Saccharomyces cerevisiae Using an Optimized Culture Medium Composition in a Batch Submerged Bioprocess.

PubMed

Hezarjaribi, Mehrnoosh; Ardestani, Fatemeh; Ghorbani, Hamid Reza

2016-08-01

Saccharomyces cerevisiae PTCC5269 growth was evaluated to specify an optimum culture medium to reach the highest protein production. Experiment design was conducted using a fraction of the full factorial methodology, and signal to noise ratio was used for results analysis. Maximum cell of 8.84 log (CFU/mL) was resulted using optimized culture composed of 0.3, 0.15, 1, and 50 g L(-1) of ammonium sulfate, iron sulfate, glycine, and glucose, respectively at 300 rpm and 35 °C. Glycine concentration (39.32 % contribution) and glucose concentration (36.15 % contribution) were determined as the most effective factors on the biomass production, while Saccharomyces cerevisiae growth had showed the least dependence on ammonium sulfate (5.2 % contribution) and iron sulfate (19.28 % contribution). The most interaction was diagnosed between ammonium sulfate and iron sulfate concentrations with interaction severity index of 50.71 %, while the less one recorded for glycine and glucose concentration was equal to 8.12 %. An acceptable consistency of 84.26 % was obtained between optimum theoretical cell numbers determined by software of 8.91 log (CFU/mL), and experimentally measured one at optimal condition confirms the suitability of the applied method. High protein content of 44.6 % using optimum culture suggests that Saccharomyces cerevisiae is a good commercial case for single cell protein production.

From Saccharomyces cerevisiae to human: The important gene co-expression modules.

PubMed

Liu, Wei; Li, Li; Ye, Hua; Chen, Haiwei; Shen, Weibiao; Zhong, Yuexian; Tian, Tian; He, Huaqin

2017-08-01

Network-based systems biology has become an important method for analyzing high-throughput gene expression data and gene function mining. Yeast has long been a popular model organism for biomedical research. In the current study, a weighted gene co-expression network analysis algorithm was applied to construct a gene co-expression network in Saccharomyces cerevisiae . Seventeen stable gene co-expression modules were detected from 2,814 S. cerevisiae microarray data. Further characterization of these modules with the Database for Annotation, Visualization and Integrated Discovery tool indicated that these modules were associated with certain biological processes, such as heat response, cell cycle, translational regulation, mitochondrion oxidative phosphorylation, amino acid metabolism and autophagy. Hub genes were also screened by intra-modular connectivity. Finally, the module conservation was evaluated in a human disease microarray dataset. Functional modules were identified in budding yeast, some of which are associated with patient survival. The current study provided a paradigm for single cell microorganisms and potentially other organisms.

Electron microscopy for ultrastructural analysis and protein localization in Saccharomyces cerevisiae

PubMed Central

Frankl, Andri; Mari, Muriel; Reggiori, Fulvio

2015-01-01

The yeast Saccharomyces cerevisiae is a key model system for studying of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. Ultrastructural examinations of this organism, though, are traditionally difficult because of the presence of a thick cell wall and the high density of cytoplasmic proteins. A series of recent methodological and technical developments, however, has revived interest in morphological analyses of yeast (e.g. 123). Here we present a review of established and new methods, from sample preparation to imaging, for the ultrastructural analysis of S. cerevisiae. We include information for the use of different fixation methods, embedding procedures, approaches for contrast enhancement, and sample visualization techniques, with references to successful examples. The goal of this review is to guide researchers that want to investigate a particular process at the ultrastructural level in yeast by aiding in the selection of the most appropriate approach to visualize a specific structure or subcellular compartment. PMID:28357267

Enhanced pathway efficiency of Saccharomyces cerevisiae by introducing thermo-tolerant devices.

PubMed

Liu, Yueqin; Zhang, Genli; Sun, Huan; Sun, Xiangying; Jiang, Nisi; Rasool, Aamir; Lin, Zhanglin; Li, Chun

2014-10-01

In this study, thermo-tolerant devices consisting of heat shock genes from thermophiles were designed and introduced into Saccharomyces cerevisiae for improving its thermo-tolerance. Among ten engineered thermo-tolerant yeasts, T.te-TTE2469, T.te-GroS2 and T.te-IbpA displayed over 25% increased cell density and 1.5-4-fold cell viability compared with the control. Physiological characteristics of thermo-tolerant strains revealed that better cell wall integrity, higher trehalose content and enhanced metabolic energy were preserved by thermo-tolerant devices. Engineered thermo-tolerant strain was used to investigate the impact of thermo-tolerant device on pathway efficiency by introducing β-amyrin synthesis pathway, showed 28.1% increased β-amyrin titer, 28-35°C broadened growth temperature range and 72h shortened fermentation period. The results indicated that implanting heat shock proteins from thermophiles to S. cerevisiae would be an efficient approach to improve its thermo-tolerance. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structure of the tandem PX-PH domains of Bem3 from Saccharomyces cerevisiae.

PubMed

Ali, Imtiaz; Eu, Sungmin; Koch, Daniel; Bleimling, Nathalie; Goody, Roger S; Müller, Matthias P

2018-05-01

The structure of the tandem lipid-binding PX and pleckstrin-homology (PH) domains of the Cdc42 GTPase-activating protein Bem3 from Saccharomyces cerevisiae (strain S288c) has been determined to a resolution of 2.2 Å (R work = 21.1%, R free = 23.4%). It shows that the domains adopt a relative orientation that enables them to simultaneously bind to a membrane and suggests possible cooperativity in membrane binding. open access.

Structure of the tandem PX-PH domains of Bem3 from Saccharomyces cerevisiae

PubMed Central

Ali, Imtiaz; Eu, Sungmin; Bleimling, Nathalie

2018-01-01

The structure of the tandem lipid-binding PX and pleckstrin-homology (PH) domains of the Cdc42 GTPase-activating protein Bem3 from Saccharomyces cerevisiae (strain S288c) has been determined to a resolution of 2.2 Å (R work = 21.1%, R free = 23.4%). It shows that the domains adopt a relative orientation that enables them to simultaneously bind to a membrane and suggests possible cooperativity in membrane binding. PMID:29718000

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

NASA Astrophysics Data System (ADS)

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

1984-06-01

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

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

PubMed Central

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

1981-01-01

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

ATP-dependent export of neutral amino acids by vacuolar membrane vesicles of Saccharomyces cerevisiae.

PubMed

Ishimoto, Masaya; Sugimoto, Naoko; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2012-01-01

Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H(+)-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.

Saccharomyces cerevisiae populations and other yeasts associated with indigenous beers (chicha) of Ecuador.

PubMed

Piló, Fernanda Barbosa; Carvajal-Barriga, Enrique Javier; Guamán-Burneo, Maria Cristina; Portero-Barahona, Patricia; Dias, Arthur Matoso Morato; Freitas, Larissa Falabella Daher de; Gomes, Fátima de Cássia Oliveira; Rosa, Carlos Augusto

2018-03-01

Chicha, a type of beer made mainly with maize or cassava, is a traditional fermented beverage of the Andean region. There have only been a few studies on yeasts associated with chicha fermentation, and the species diversity occurring during the production of this beverage is not known. The objective of this study was to determine the biodiversity of yeasts in chicha, and to characterize the Saccharomyces cerevisiae populations associated with the production of chicha de jora, seven-grain chicha, chicha de yuca, and chicha de morocho in Ecuador. The molecular diversity of S. cerevisiae populations was determined by restriction polymorphism mitochondrial profiles. The beverages were characterized based on their physicochemical parameters. Twenty-six species were identified, and the most prevalent species were S. cerevisiae and Torulaspora delbrueckii. Other yeast species were isolated at low frequencies. Among 121 isolates of S. cerevisiae, 68 different mtDNA molecular profiles were identified. These results showed that chichas are fermented by a high number of different strains of S. cerevisiae. Some other species provided a minor contribution to the fermentation process. The chicha presented generally similar physicochemical parameters to those observed for other traditional fermented beverages, and can be considered as an acid fermented beverage. Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

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

PubMed

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

2014-10-06

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

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.

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.

Comprehensive quantitative analysis of central carbon and amino-acid metabolism in Saccharomyces cerevisiae under multiple conditions by targeted proteomics

PubMed Central

Costenoble, Roeland; Picotti, Paola; Reiter, Lukas; Stallmach, Robert; Heinemann, Matthias; Sauer, Uwe; Aebersold, Ruedi

2011-01-01

Decades of biochemical research have identified most of the enzymes that catalyze metabolic reactions in the yeast Saccharomyces cerevisiae. The adaptation of metabolism to changing nutritional conditions, in contrast, is much less well understood. As an important stepping stone toward such understanding, we exploit the power of proteomics assays based on selected reaction monitoring (SRM) mass spectrometry to quantify abundance changes of the 228 proteins that constitute the central carbon and amino-acid metabolic network in the yeast Saccharomyces cerevisiae, at five different metabolic steady states. Overall, 90% of the targeted proteins, including families of isoenzymes, were consistently detected and quantified in each sample, generating a proteomic data set that represents a nutritionally perturbed biological system at high reproducibility. The data set is near comprehensive because we detect 95–99% of all proteins that are required under a given condition. Interpreted through flux balance modeling, the data indicate that S. cerevisiae retains proteins not necessarily used in a particular environment. Further, the data suggest differential functionality for several metabolic isoenzymes. PMID:21283140

Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains.

PubMed

Kong, In Iok; Turner, Timothy Lee; Kim, Heejin; Kim, Soo Rin; Jin, Yong-Su

2018-02-01

Microorganisms have been studied and used extensively to produce value-added fuels and chemicals. Yeasts, specifically Saccharomyces cerevisiae, receive industrial attention because of their well-known ability to ferment glucose and produce ethanol. Thousands of natural or genetically modified S. cerevisiae have been found in industrial environments for various purposes. These industrial strains are isolated from industrial fermentation sites, and they are considered as potential host strains for superior fermentation processes. In many cases, industrial yeast strains have higher thermotolerance, increased resistances towards fermentation inhibitors and increased glucose fermentation rates under anaerobic conditions when compared with laboratory yeast strains. Despite the advantages of industrial strains, they are often not well characterized. Through screening and phenotypic characterization of commercially available industrial yeast strains, industrial fermentation processes requiring specific environmental conditions may be able to select an ideal starting yeast strain to be further engineered. Here, we have characterized and compared 21 industrial S. cerevisiae strains under multiple conditions, including their tolerance to varying pH conditions, resistance to fermentation inhibitors, sporulation efficiency and ability to ferment lignocellulosic sugars. These data may be useful for the selection of a parental strain for specific biotechnological applications of engineered yeast. © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Terminal acidic shock inhibits sour beer bottle conditioning by Saccharomyces cerevisiae.

PubMed

Rogers, Cody M; Veatch, Devon; Covey, Adam; Staton, Caleb; Bochman, Matthew L

2016-08-01

During beer fermentation, the brewer's yeast Saccharomyces cerevisiae experiences a variety of shifting growth conditions, culminating in a low-oxygen, low-nutrient, high-ethanol, acidic environment. In beers that are bottle conditioned (i.e., carbonated in the bottle by supplying yeast with a small amount of sugar to metabolize into CO2), the S. cerevisiae cells must overcome these stressors to perform the ultimate act in beer production. However, medium shock caused by any of these variables can slow, stall, or even kill the yeast, resulting in production delays and economic losses. Here, we describe a medium shock caused by high lactic acid levels in an American sour beer, which we refer to as "terminal acidic shock". Yeast exposed to this shock failed to bottle condition the beer, though they remained viable. The effects of low pH/high [lactic acid] conditions on the growth of six different brewing strains of S. cerevisiae were characterized, and we developed a method to adapt the yeast to growth in acidic beer, enabling proper bottle conditioning. Our findings will aid in the production of sour-style beers, a trending category in the American craft beer scene. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression of three Trichoderma reesei cellulase genes in Saccharomyces pastorianus for the development of a two-step process of hydrolysis and fermentation of cellulose.

PubMed

Fitzpatrick, J; Kricka, W; James, T C; Bond, U

2014-07-01

To compare the production of recombinant cellulase enzymes in two Saccharomyces species so as to ascertain the most suitable heterologous host for the degradation of cellulose-based biomass and its conversion into bioethanol. cDNA copies of genes representing the three major classes of cellulases (Endoglucanases, Cellobiohydrolases and β-glucosidases) from Trichoderma reesei were expressed in Saccharomyces pastorianus and Saccharomyces cerevisiae. The recombinant enzymes were secreted by the yeast hosts into the medium and were shown to act in synergy to hydrolyse cellulose. The conditions required to achieve maximum release of glucose from cellulose by the recombinant enzymes were defined and the activity of the recombinant enzymes was compared to a commercial cocktail of T. reesei cellulases. We demonstrate that significantly higher levels of cellulase activity were achieved by expression of the genes in S. pastorianus compared to S. cerevisiae. Hydrolysis of cellulose by the combined activity of the recombinant enzymes was significantly better at 50°C than at 30°C, the temperature used for mesophilic yeast fermentations, reflecting the known temperature profiles of the native enzymes. The results demonstrate that host choice is important for the heterologous production of cellulases. On the basis of the low activity of the T. reesei recombinant enzymes at fermentation temperatures, we propose a two-step process for the hydrolysis of cellulose and its fermentation into alcohol using cellulases produced in situ. © 2014 The Society for Applied Microbiology.

Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols.

PubMed

Ferreira, Raphael; Teixeira, Paulo Gonçalves; Gossing, Michael; David, Florian; Siewers, Verena; Nielsen, Jens

2018-06-01

Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although Saccharomyces cerevisiae is the favored microbial cell factory for industrial production of biochemicals, it does not produce large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered S. cerevisiae to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy by overexpressing genes encoding a deregulated acetyl-CoA carboxylase, ACC1 S659A/S1157A (ACC1**) , as well as the last two steps of TAG formation: phosphatidic phosphatase ( PAH1 ) and diacylglycerol acyltransferase ( DGA1 ), ultimately leading to 129 mg∙gCDW -1 of TAGs. Disruption of TAG lipase genes TGL3 , TGL4 , TGL5 and sterol acyltransferase gene ARE1 increased the TAG content to 218 mg∙gCDW -1 . Further disruption of the beta-oxidation by deletion of POX1 , as well as glycerol-3-phosphate utilization through deletion of GUT2 , did not affect TAGs levels. Finally, disruption of the peroxisomal fatty acyl-CoA transporter PXA1 led to accumulation of 254 mg∙gCDW -1 . The TAG levels achieved here are the highest titer reported in S. cerevisiae , reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species for high level lipid production.

Review of current methods for characterizing virulence and pathogenicity potential of industrial Saccharomyces cerevisiae strains towards humans.

PubMed

Anoop, Valar; Rotaru, Sever; Shwed, Philip S; Tayabali, Azam F; Arvanitakis, George

2015-09-01

Most industrial Saccharomyces cerevisiae strains used in food or biotechnology processes are benign. However, reports of S. cerevisiae infections have emerged and novel strains continue to be developed. In order to develop recommendations for the human health risk assessment of S. cerevisiae strains, we conducted a literature review of current methods used to characterize their pathogenic potential and evaluated their relevance towards risk assessment. These studies revealed that expression of virulence traits in S. cerevisiae is complex and depends on many factors. Given the opportunistic nature of this organism, an approach using multiple lines of evidence is likely necessary for the reasonable prediction of the pathogenic potential of a particular strain. Risk assessment of S. cerevisiae strains would benefit from more research towards the comparison of virulent and non-virulent strains in order to better understand those genotypic and phenotypic traits most likely to be associated with pathogenicity. © Her Majesty the Queen in Right of Canada 2015. Reproduced with the permission of the Minister of Health.

Transcriptomic analysis of Saccharomyces cerevisiae upon honokiol treatment.

PubMed

Zhu, Xiaolong; Zou, Shenshen; Li, Youbin; Liang, Yongheng

2017-09-01

Honokiol (HNK), one of the main medicinal components in Magnolia officinalis, possesses antimicrobial activity against a variety of pathogenic bacteria and fungi. However, little is known of the molecular mechanisms underpinning the antimicrobial activity. To explore the molecular mechanism of its antifungal activity, we determined the effects of HNK on the mRNA expression profile of Saccharomyces cerevisiae using a DNA microarray approach. HNK markedly induced the expression of genes related to iron uptake and homeostasis. Conversely, genes associated with respiratory electron transport were downregulated, mirroring the effects of iron starvation. Meanwhile, HNK-induced growth deficiency was partly rescued by iron supplementation and HNK reacted with iron, producing iron complexes that depleted iron. These results suggest that HNK treatment induced iron starvation. Additionally, HNK treatment resulted in the upregulation of genes involved in protein synthesis and drug resistance networks. Furthermore, the deletion of PDR5, a gene encoding the plasma membrane ATP binding cassette (ABC) transporter, conferred sensitivity to HNK. Overexpression of PDR5 enhanced resistance of WT and pdr5Δ strains to HNK. Taken together, these findings suggest that HNK, which can be excluded by overexpression of Pdr5, functions in multiple cellular processes in S. cerevisiae, particularly in inducing iron starvation to inhibit cell growth. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Identification of the gene for β-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background β-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries. Results We identified a β-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family. Heterologous expression of the CmINV gene in Saccharomyces cerevisiae BY4742∆suc2 provided further evidence that CmINV indeed functions as a β-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the ∆suc2 deletion mutant strain of S. cerevisiae to grow on sucrose as sole carbohydrate source. Secondly, the recombinant protein was capable of producing short-chain fructooligosaccharides (scFOS) when incubated in the presence of 10% sucrose. Purified deglycosylated CmINV protein showed a molecular weight of ca. 66 kDa and a Km and Vmax on sucrose of 7.50 mM and 986 μmol/min/mg protein, respectively. Its optimal pH and temperature conditions were determined to be 6.0 and 62.5°C, respectively. The addition of 50 mM LiCl led to a 186% increase in CmINV activity. Another striking feature was the relatively high volumetric production of this protein in S. cerevisiae as one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme. Conclusion The properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry. PMID:24225070

Natural and modified promoters for tailored metabolic engineering of the yeast Saccharomyces cerevisiae.

PubMed

Hubmann, Georg; Thevelein, Johan M; Nevoigt, Elke

2014-01-01

The ease of highly sophisticated genetic manipulations in the yeast Saccharomyces cerevisiae has initiated numerous initiatives towards development of metabolically engineered strains for novel applications beyond its traditional use in brewing, baking, and wine making. In fact, baker's yeast has become a key cell factory for the production of various bulk and fine chemicals. Successful metabolic engineering requires fine-tuned adjustments of metabolic fluxes and coordination of multiple pathways within the cell. This has mostly been achieved by controlling gene expression at the transcriptional level, i.e., by using promoters with appropriate strengths and regulatory properties. Here we present an overview of natural and modified promoters, which have been used in metabolic pathway engineering of S. cerevisiae. Recent developments in creating promoters with tailor-made properties are also discussed.

Genetic Approaches to Study Meiosis and Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

PubMed

Kassir, Yona; Stuart, David T

2017-01-01

The budding yeast Saccharomyces cerevisiae has a long history as a model organism for studies of meiosis and the cell cycle. The popularity of this yeast as a model is in large part due to the variety of genetic and cytological approaches that can be effectively performed with the cells. Cultures of the cells can be induced to synchronously progress through meiosis and sporulation allowing large-scale gene expression and biochemical studies to be performed. Additionally, the spore tetrads resulting from meiosis make it possible to characterize the haploid products of meiosis allowing investigation of meiotic recombination and chromosome segregation. Here we describe genetic methods for analysis progression of S. cerevisiae through meiosis and sporulation with an emphasis on strategies for the genetic analysis of regulators of meiosis-specific genes.

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.

Functional analysis of Paracoccidioides brasiliensis 14-3-3 adhesin expressed in Saccharomyces cerevisiae.

PubMed

Assato, Patricia Akemi; da Silva, Julhiany de Fátima; de Oliveira, Haroldo Cesar; Marcos, Caroline Maria; Rossi, Danuza; Valentini, Sandro Roberto; Mendes-Giannini, Maria José Soares; Zanelli, Cleslei Fernando; Fusco-Almeida, Ana Marisa

2015-11-04

14-3-3 proteins comprise a family of eukaryotic multifunctional proteins involved in several cellular processes. The Pb14-3-3 of Paracoccidioides brasiliensis seems to play an important role in the Paracoccidioides-host interaction. Paracoccidioides brasiliensis is an etiological agent of paracoccidioidomycosis, which is a systemic mycosis that is endemic in Latin America. In the initial steps of the infection, Paracoccidioides spp. synthetizes adhesins that allow it to adhere and invade host cells. Therefore, the aim of this work was to perform a functional analysis of Pb14-3-3 using Saccharomyces cerevisiae as a model. The functional analysis of Pb14-3-3 was performed in S. cerevisiae, and it was found that Pb14-3-3 partially complemented S. cerevisiae proteins Bmh1p and Bmh2p, which are recognized as two yeast 14-3-3 homologues. When we evaluated the adhesion profile of S. cerevisiae transformants, Pb14-3-3 acted as an adhesin in S. cerevisiae; however, Bmh1p did not show this function. The influence of Pb14-3-3 in S. cerevisiae ergosterol pathway was also evaluated and our results showed that Pb14-3-3 up-regulates genes involved in ergosterol biosynthesis. Our data showed that Pb14-3-3 was able to partially complement Bmh1p and Bmh2p proteins in S. cerevisiae; however, we suggest that Pb14-3-3 has a differential role as an adhesin. In addition, Pb-14-3-3 may be involved in Paracoccidioides spp. ergosterol biosynthesis which makes it an interest as a therapeutic target.

Copper/Zinc-Superoxide Dismutase Is Required for Oxytetracycline Resistance of Saccharomyces cerevisiae

PubMed Central

Avery, Simon V.; Malkapuram, Srividya; Mateus, Carolina; Babb, Kimberly S.

2000-01-01

Saccharomyces cerevisiae, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S. cerevisiae hypersensitive to oxytetracycline (OTC): a sod1Δ mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 μg ml−1, whereas concentrations of up to 1,000 μg ml−1 had no effect on the growth of the wild type. OTC resistance was restored in the sod1Δ mutant by complementation with wild-type SOD1. The effect of OTC appeared to be cytotoxic and was not evident in a ctt1Δ (cytosolic catalase) mutant or in the presence of tetracycline. SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and sod1Δ strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1Δ mutant, but not the wild type, to OTC. We propose that Sod1p protects S. cerevisiae against a mode of OTC action that is dependent on oxidative damage. PMID:10613865

Hxt-carrier-mediated glucose efflux upon exposure of Saccharomyces cerevisiae to excess maltose.

PubMed

Jansen, Mickel L A; De Winde, Johannes H; Pronk, Jack T

2002-09-01

When wild-type Saccharomyces cerevisiae strains pregrown in maltose-limited chemostat cultures were exposed to excess maltose, release of glucose into the external medium was observed. Control experiments confirmed that glucose release was not caused by cell lysis or extracellular maltose hydrolysis. To test the hypothesis that glucose efflux involved plasma membrane glucose transporters, experiments were performed with an S. cerevisiae strain in which all members of the hexose transporter (HXT) gene family had been eliminated and with an isogenic reference strain. Glucose efflux was virtually eliminated in the hexose-transport-deficient strain. This constitutes experimental proof that Hxt transporters facilitate export of glucose from S. cerevisiae cells. After exposure of the hexose-transport-deficient strain to excess maltose, an increase in the intracellular glucose level was observed, while the concentrations of glucose 6-phosphate and ATP remained relatively low. These results demonstrate that glucose efflux can occur as a result of uncoordinated expression of the initial steps of maltose metabolism and the subsequent reactions in glucose dissimilation. This is a relevant phenomenon for selection of maltose-constitutive strains for baking and brewing.

Metabolic changes induced during adaptation of Saccharomyces cerevisiae to a water stress.

PubMed

Singh, K K; Norton, R S

1991-01-01

When exponentially growing Saccharomyces cerevisiae was transferred from a normal high water activity growth medium (aw 0.997) to a medium containing 8% NaCl low water activity growth medium (aw 0.955), glycerol accumulation during the first eight hours of the adaptation was both retarded and greatly diminished in magnitude. Investigation of the underlying reasons for the slow onset of glycerol accumulation revealed that not only was overall glycerol production reduced by salt transfer, but also the rates of ethanol production and glucose consumption were reduced. Measurement of glycolytic intermediates revealed an accumulation of glucose-6-phosphate, fructose-6-phosphate, fructose 1,6 bisphosphate and phosphoenolpyruvate in S. cerevisiae 3 to 4 h after transfer to salt, suggesting that one or more glycolytic enzymes were inhibited. Potassium ions accumulated in S. cerevisiae after salt transfer and reached a maximum about 6 h after transfer, whereas the sodium ion content increased progressively during the adaptation period. The trehalose content also increased in adapting cells. It is suggested that inhibition of glycerol production during the initial period of adaptation could be due to either the inhibition of glycerol-3-phosphate dehydrogenase by increased cation content or the inhibition of glycolysis, glycerol being produced glycolytically in S. cerevisiae. The increased accumulation of glycerol towards the end of the 8-h period suggests that the osmoregulatory response of S. cerevisiae involves complex sets of adjustments in which inhibition of glycerol-3-phosphate dehydrogenase must be relieved before glycerol functions as a major osmoregulator.

Transcriptome-Based Characterization of Interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in Lactose-Grown Chemostat Cocultures

PubMed Central

Mendes, Filipa; Sieuwerts, Sander; de Hulster, Erik; Almering, Marinka J. H.; Luttik, Marijke A. H.; Pronk, Jack T.; Smid, Eddy J.; Bron, Peter A.

2013-01-01

Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the cocultures, five mechanisms of interaction were identified. (i) Lb. delbrueckii subsp. bulgaricus hydrolyzes lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. delbrueckii subsp. bulgaricus, is excreted and provides a carbon source for yeast. (ii) In pure cultures, Lb. delbrueckii subsp. bulgaricus grows only in the presence of increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. (iii) Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacterium. (iv) A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. delbrueckii subsp. bulgaricus. (v) Transcriptome analysis of Lb. delbrueckii subsp. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipid metabolism, suggesting either a competition of the two microorganisms for fatty acids or a response to the ethanol produced by S. cerevisiae. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigate microbial interactions in mixed populations. PMID:23872557

Transcriptome-based characterization of interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in lactose-grown chemostat cocultures.

PubMed

Mendes, Filipa; Sieuwerts, Sander; de Hulster, Erik; Almering, Marinka J H; Luttik, Marijke A H; Pronk, Jack T; Smid, Eddy J; Bron, Peter A; Daran-Lapujade, Pascale

2013-10-01

Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the cocultures, five mechanisms of interaction were identified. (i) Lb. delbrueckii subsp. bulgaricus hydrolyzes lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. delbrueckii subsp. bulgaricus, is excreted and provides a carbon source for yeast. (ii) In pure cultures, Lb. delbrueckii subsp. bulgaricus grows only in the presence of increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. (iii) Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacterium. (iv) A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. delbrueckii subsp. bulgaricus. (v) Transcriptome analysis of Lb. delbrueckii subsp. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipid metabolism, suggesting either a competition of the two microorganisms for fatty acids or a response to the ethanol produced by S. cerevisiae. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigate microbial interactions in mixed populations.

Engineering of a Novel Saccharomyces cerevisiae Wine Strain with a Respiratory Phenotype at High External Glucose Concentrations

PubMed Central

Henricsson, C.; de Jesus Ferreira, M. C.; Hedfalk, K.; Elbing, K.; Larsson, C.; Bill, R. M.; Norbeck, J.; Hohmann, S.; Gustafsson, L.

2005-01-01

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Δ strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a non-ethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Δ strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain. PMID:16204537

Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations.

PubMed

Henricsson, C; de Jesus Ferreira, M C; Hedfalk, K; Elbing, K; Larsson, C; Bill, R M; Norbeck, J; Hohmann, S; Gustafsson, L

2005-10-01

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Delta strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a non-ethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Delta strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain.

Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae

PubMed Central

Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki

2015-01-01

Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved. PMID:26588105

Switching the mode of sucrose utilization by Saccharomyces cerevisiae

PubMed Central

Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-01-01

Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose

Switching the mode of sucrose utilization by Saccharomyces cerevisiae.

PubMed

Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-02-27

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells

Biodegradation of zearalenone by Saccharomyces cerevisiae: Possible involvement of ZEN responsive proteins of the yeast.

PubMed

Zhang, Hongyin; Dong, Manjia; Yang, Qiya; Apaliya, Maurice Tibiru; Li, Jun; Zhang, Xiaoyun

2016-06-30

The mycotoxin zearalenone, also known as F-2 mycotoxin or RAL is a potent estrogenic metabolite produced by some Gibberella and Fusarium species. It is a common contaminant of cereal crops, livestock and poultry products. However, detoxification of zearalenone (ZEN) remains a challenge. Recently, biological approach for ZEN detoxification is being explored. In this study, we investigated the biodegradation of ZEN by using Saccharomyces cerevisiae and the possible mechanisms involved. The findings revealed that, after 48h of incubation of S. cerevisiae in combination with ZEN, the ZEN was completely degraded by S. cerevisiae. On the contrary, heat-killed cells and cell-free culture filtrates of S. cerevisiae could not degrade ZEN. Furthermore, addition of cycloheximide to S. cerevisiae combined with ZEN at time 0h prevented ZEN degradation, while addition of cycloheximide at 12h significantly slowed down degradation. The results also indicated cellular proteomics of S. cerevisiae. Several differential proteins were identified, most of which were related to basic metabolism. The findings revealed that, after 48h of incubating ZEN together with S. cerevisiae, ZEN was completely degraded by S. cerevisiae. The mechanisms involved in the degradation of ZEN by S. cerevisiae may be the production of associated intracellular and extracellular enzymes, which have the ability to degrade ZEN. In addition, there were some functional proteins produced by S. cerevisiae, indicating that the basic metabolism of S. cerevisiae was improved when ZEN was added. This novel discovery by the authors, will greatly contribute to the field of biodegradation of mycotoxin by antagonists. The authors also believed this innovation will open the grounds for further research and improvement of S. cerevisiae in the field of biodegradation. Copyright © 2016 Elsevier B.V. All rights reserved.

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. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

The ASP3 locus in Saccharomyces cerevisiae originated by horizontal gene transfer from Wickerhamomyces.

PubMed

League, Garrett P; Slot, Jason C; Rokas, Antonis

2012-11-01

The asparagine degradation pathway in the S288c laboratory strain of Saccharomyces cerevisiae is comprised of genes located at two separate loci. ASP1 is located on chromosome IV and encodes for cytosolic l-asparaginase I, whereas ASP3 contains a gene cluster located on chromosome XII comprised of four identical genes, ASP3-1, ASP3-2, ASP3-3, and ASP3-4, which encode for cell wall-associated l-asparaginase II. Interestingly, the ASP3 locus appears to be only present, in variable copy number, in S. cerevisiae strains isolated from laboratory or industrial environments and is completely absent from the genomes of 128 diverse fungal species. Investigation of the evolutionary history of ASP3 across these 128 genomes as well as across the genomes of 43 S. cerevisiae strains shows that ASP3 likely arose in a S. cerevisiae strain via horizontal gene transfer (HGT) from, or a close relative of, the wine yeast Wickerhamomyces anomalus, which co-occurs with S. cerevisiae in several biotechnological processes. Thus, because the ASP3 present in the S288c laboratory strain of S. cerevisiae is induced in response to nitrogen starvation, its acquisition may have aided yeast adaptation to artificial environments. Our finding that the ASP3 locus in S. cerevisiae originated via HGT further highlights the importance of gene sharing between yeasts in the evolution of their remarkable metabolic diversity. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Synergic treatment for monosodium glutamate wastewater by Saccharomyces cerevisiae and Coriolus versicolor.

PubMed

Jia, Cuiying; Kang, Ruijuan; Zhang, Yuhui; Cong, Wei; Cai, Zhaoling

2007-03-01

Biodegradation and decolorization of monosodium glutamate wastewater were carried out by using an acidophilus yeast strain of Saccharomyces cerevisiae and Coriolus versicolor. For the yeast treatment, the highest COD removal and reducing sugar removal efficiency were 76.6% and 80.2%, respectively. The color removal was only 2%. For C. versicolor treatment, the highest COD removal, color removal and reducing sugar removal efficiencies were 78.7%, 56.5% and 90.9%, respectively. The synergic treatment process, in which the yeast and C. versicolor were successively applied,exhibited great advantage over the individual process.

Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

PubMed

Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

2015-11-01

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

Physico-chemical pretreatment and enzymatic hydrolysis of cotton stalk for ethanol production by Saccharomyces cerevisiae.

PubMed

Singh, Anita; Bajar, Somvir; Bishnoi, Narsi R

2017-11-01

The aim of this work was to study the physico-chemical pretreatment and enzymatic hydrolysis of cotton stalk for ethanol production by Saccharomyces cerevisiae. Firstly, factors affecting pretreatment were screened out by Plackett-Burman design (PBD) and most significant factors were further optimized by Box-Behnken design (BBD). As shown by experimental study, most significant factors were FeCl 3 concentration (FC), irradiation time (IT) and substrate concentration (SC) affecting pretreatment of cotton stalk among all studied factors. Under optimum conditions of pretreatment FC 0.15mol/l, IT 20min and SC 55g/l, the release of reducing sugar was 6.6g/l. Hydrolysis of pretreated cotton stalk was done by crude on-site produced enzymes and hydrolysate was concentrated. Ethanol production by Saccharomyces cerevisiae using concentrated cotton stalk hydrolysate was 9.8g p /l, with ethanol yield 0.37g p /g s on consumed sugars. The data indicated that microwave FeCl 3 pretreated cotton stalk hydrolyses by crude unprocessed enzyme cocktail was good, and ethanol can be produced by fermentation of hydrolysate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effective inactivation of Saccharomyces cerevisiae in minimally processed Makgeolli using low-pressure homogenization-based pasteurization.

PubMed

Bak, Jin Seop

2015-01-01

In order to address the limitations associated with the inefficient pasteurization platform used to make Makgeolli, such as the presence of turbid colloidal dispersions in suspension, commercially available Makgeolli was minimally processed using a low-pressure homogenization-based pasteurization (LHBP) process. This continuous process demonstrates that promptly reducing the exposure time to excessive heat using either large molecules or insoluble particles can dramatically improve internal quality and decrease irreversible damage. Specifically, optimal homogenization increased concomitantly with physical parameters such as colloidal stability (65.0% of maximum and below 25-μm particles) following two repetitions at 25.0 MPa. However, biochemical parameters such as microbial population, acidity, and the presence of fermentable sugars rarely affected Makgeolli quality. Remarkably, there was a 4.5-log reduction in the number of Saccharomyces cerevisiae target cells at 53.5°C for 70 sec in optimally homogenized Makgeolli. This value was higher than the 37.7% measured from traditionally pasteurized Makgeolli. In contrast to the analytical similarity among homogenized Makgeollis, our objective quality evaluation demonstrated significant differences between pasteurized (or unpasteurized) Makgeolli and LHBP-treated Makgeolli. Low-pressure homogenization-based pasteurization, Makgeolli, minimal processing-preservation, Saccharomyces cerevisiae, suspension stability.

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

Adaptation and major chromosomal changes in populations of Saccharomyces cerevisiae.

PubMed

Adams, J; Puskas-Rozsa, S; Simlar, J; Wilke, C M

1992-07-01

Thirteen independent populations of Saccharomyces cerevisiae (nine haploid and four diploid) were maintained in continuous culture for up to approximately 1000 generations, with growth limited by the concentration of organic phosphates in medium buffered at pH 6. Analysis of clones isolated from these populations showed that a number (17) of large-scale chromosomal-length variants and rearrangements were present in the populations at their termination. Nine of the 16 yeast chromosomes were involved in such changes. Few of the changes could be explained by copy-number increases in the structural loci for acid phosphatase. Several considerations concerning the nature and frequency of the chromosome-length variants observed lead us to conclude that they are selectively advantageous.

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.

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates

USDA-ARS?s Scientific Manuscript database

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes needed for xylose-fermentation integrated into its genome was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than their parental strain (p < 0.05) and abl...

Effects of supplementing Saccharomyces cerevisiae fermentation product in sow diets on performance of sows and nursing piglets

USDA-ARS?s Scientific Manuscript database

Forty-two sows were used to determine the effects of adding Saccharomyces cerevisiae fermentation product to gestation and lactation diets on performance of sows and their progeny. On 5 d before breeding, sows were allotted to 2 dietary treatments representing: (1) sows fed a diet with 12.0 g fermen...

Identification of a Saccharomyces cerevisiae glucosidase that hydrolyzes flavonoid glucosides.

PubMed

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

2011-03-01

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

Impact of glutathione metabolism on zinc homeostasis in Saccharomyces cerevisiae.

PubMed

Steiger, Matthias G; Patzschke, Anett; Holz, Caterina; Lang, Christine; Causon, Tim; Hann, Stephan; Mattanovich, Diethard; Sauer, Michael

2017-06-01

Zinc is a crucial mineral for all organisms as it is an essential cofactor for the proper function of a plethora of proteins and depletion of zinc causes oxidative stress. Glutathione is the major redox buffering agent in the cell and therefore important for mitigation of the adverse effects of oxidative stress. In mammalian cells, zinc deficiency is accompanied by a glutathione depletion. In the yeast Saccharomyces cerevisiae, the opposite effect is observed: under low zinc conditions, an elevated glutathione concentration is found. The main regulator to overcome zinc deficiency is Zap1p. However, we show that Zap1p is not involved in this glutathione accumulation phenotype. Furthermore, we found that in glutathione-accumulating strains also the metal ion-binding phytochelatin-2, which is an oligomer of glutathione, is accumulated. This increased phytochelatin concentration correlates with a lower free zinc level in the vacuole. These results suggest that phytochelatin is important for zinc buffering in S. cerevisiae and thus explains how zinc homeostasis is connected with glutathione metabolism. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Ty1 LTR-retrotransposon of budding yeast, Saccharomyces cerevisiae

PubMed Central

Curcio, M. Joan; Lutz, Sheila; Lesage, Pascale

2015-01-01

Summary Long-terminal repeat (LTR)-retrotransposons generate a copy of their DNA (cDNA) by reverse transcription of their RNA genome in cytoplasmic nucleocapsids. They are widespread in the eukaryotic kingdom and are the evolutionary progenitors of retroviruses [1]. The Ty1 element of the budding yeast Saccharomyces cerevisiae was the first LTR-retrotransposon demonstrated to mobilize through an RNA intermediate, and not surprisingly, is the best studied. The depth of our knowledge of Ty1 biology stems not only from the predominance of active Ty1 elements in the S. cerevisiae genome but also the ease and breadth of genomic, biochemical and cell biology approaches available to study cellular processes in yeast. This review describes the basic structure of Ty1 and its gene products, the replication cycle, the rapidly expanding compendium of host co-factors known to influence retrotransposition and the nature of Ty1's elaborate symbiosis with its host. Our goal is to illuminate the value of Ty1 as a paradigm to explore the biology of LTR-retrotransposons in multicellular organisms, where the low frequency of retrotransposition events presents a formidable barrier to investigations of retrotransposon biology. PMID:25893143

Flor yeasts of Saccharomyces cerevisiae--their ecology, genetics and metabolism.

PubMed

Alexandre, Hervé

2013-10-15

The aging of certain white wines is dependent on the presence of yeast strains that develop a biofilm on the wine surface after the alcoholic fermentation. These strains belong to the genus Saccharomyces and are called flor yeasts. These strains possess distinctive characteristics compared with Saccharomyces cerevisiae fermenting strain. The most important one is their capacity to form a biofilm on the air-liquid interface of the wine. The major gene involved in this phenotype is FLO11, however other genes are also involved in velum formation by these yeast and will be detailed. Other striking features presented in this review are their aneuploidy, and their mitochondrial DNA polymorphism which seems to reflect adaptive evolution of the yeast to a stressful environment where acetaldehyde and ethanol are present at elevated concentration. The biofilm assures access to oxygen and therefore permits continued growth on non-fermentable ethanol. This specific metabolism explains the peculiar organoleptic profile of these wines, especially their content in acetaldehyde and sotolon. This review deals with these different specificities of flor yeasts and will also underline the existing gaps regarding these astonishing yeasts. © 2013.

Macromolecular Synthesis During the Germination of Saccharomyces cerevisiae Spores

PubMed Central

Rousseau, Paul; Halvorson, Harlyn O.

1973-01-01

After the dormancy of Saccharomyces cerevisiae ascospores had been broken, the synthesis of proteins was observed first, followed rapidly by synthesis of ribonucleic acid (RNA) and much later by deoxyribonucleic acid (DNA) synthesis. Phosphoglucomutase activity increased in a periodic (step) fashion, whereas the activity of five other enzymes increased linearly during germination and outgrowth. The rate of synthesis of these enzymes was highest at about the period of DNA replication. The amino acid pools of dormant spores contained high levels of proline, glutamic acid, and histidine. At 2 h after onset of germination, the pools of phenylalanine and methionine had disappeared and the other components had decreased significantly. By 3.5 h, with the exception of proline and cystine, most amino acid pool components had significantly increased. PMID:4570780

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

Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase

PubMed Central

Kozak, Barbara U.; van Rossum, Harmen M.; Niemeijer, Matthijs S.; van Dijk, Marlous; Benjamin, Kirsten; Wu, Liang; Daran, Jean-Marc G.; Pronk, Jack T.

2016-01-01

In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald− strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald− A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity—Vmax/Km for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields. PMID:26818854

The Impact of Saccharomyces cerevisiae on a Wine Yeast Consortium in Natural and Inoculated Fermentations

PubMed Central

Bagheri, Bahareh; Bauer, Florian F.; Setati, Mathabatha E.

2017-01-01

Natural, also referred to as spontaneous wine fermentations, are carried out by the native microbiota of the grape juice, without inoculation of selected, industrially produced yeast or bacterial strains. Such fermentations are commonly initiated by non-Saccharomyces yeast species that numerically dominate the must. Community composition and numerical dominance of species vary significantly between individual musts, but Saccharomyces cerevisiae will in most cases dominate the late stages of the fermentation and complete the process. Nevertheless, non-Saccharomyces species contribute significantly, positively or negatively, to the character and quality of the final product. The contribution is species and strain dependent and will depend on each species or strain’s absolute and relative contribution to total metabolically active biomass, and will therefore, be a function of its relative fitness within the microbial ecosystem. However, the population dynamics of multispecies fermentations are not well understood. Consequently, the oenological potential of the microbiome in any given grape must, can currently not be evaluated or predicted. To better characterize the rules that govern the complex wine microbial ecosystem, a model yeast consortium comprising eight species commonly encountered in South African grape musts and an ARISA based method to monitor their dynamics were developed and validated. The dynamics of these species were evaluated in synthetic must in the presence or absence of S. cerevisiae using direct viable counts and ARISA. The data show that S. cerevisiae specifically suppresses certain species while appearing to favor the persistence of other species. Growth dynamics in Chenin blanc grape must fermentation was monitored only through viable counts. The interactions observed in the synthetic must, were upheld in the natural must fermentations, suggesting the broad applicability of the observed ecosystem dynamics. Importantly, the presence of

The Impact of Saccharomyces cerevisiae on a Wine Yeast Consortium in Natural and Inoculated Fermentations.

PubMed

Bagheri, Bahareh; Bauer, Florian F; Setati, Mathabatha E

2017-01-01

Natural, also referred to as spontaneous wine fermentations, are carried out by the native microbiota of the grape juice, without inoculation of selected, industrially produced yeast or bacterial strains. Such fermentations are commonly initiated by non- Saccharomyces yeast species that numerically dominate the must. Community composition and numerical dominance of species vary significantly between individual musts, but Saccharomyces cerevisiae will in most cases dominate the late stages of the fermentation and complete the process. Nevertheless, non- Saccharomyces species contribute significantly, positively or negatively, to the character and quality of the final product. The contribution is species and strain dependent and will depend on each species or strain's absolute and relative contribution to total metabolically active biomass, and will therefore, be a function of its relative fitness within the microbial ecosystem. However, the population dynamics of multispecies fermentations are not well understood. Consequently, the oenological potential of the microbiome in any given grape must, can currently not be evaluated or predicted. To better characterize the rules that govern the complex wine microbial ecosystem, a model yeast consortium comprising eight species commonly encountered in South African grape musts and an ARISA based method to monitor their dynamics were developed and validated. The dynamics of these species were evaluated in synthetic must in the presence or absence of S. cerevisiae using direct viable counts and ARISA. The data show that S. cerevisiae specifically suppresses certain species while appearing to favor the persistence of other species. Growth dynamics in Chenin blanc grape must fermentation was monitored only through viable counts. The interactions observed in the synthetic must, were upheld in the natural must fermentations, suggesting the broad applicability of the observed ecosystem dynamics. Importantly, the presence of

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

Phenotypic selection of a wild Saccharomyces cerevisiae strain for simultaneous saccharification and co-fermentation of AFEX pretreated corn stover

Treesearch

Mingie Jin; Cory Sarks; Christa Gunawan; Benjamin D. Bice; Shane P. Simonett; Ragothaman Avanasi Narasimhan; Laura B. Willis; Bruce E. Dale; Venkatesh Balan; Trey K. Sato

2013-01-01

Simultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover,...

Dynamic study of yeast species and Saccharomyces cerevisiae strains during the spontaneous fermentations of Muscat blanc in Jingyang, China.

PubMed

Wang, Chunxiao; Liu, Yanlin

2013-04-01

The evolution of yeast species and Saccharomyces cerevisiae genotypes during spontaneous fermentations of Muscat blanc planted in 1957 in Jingyang region of China was followed in this study. Using a combination of colony morphology on Wallerstein Nutrient (WLN) medium, sequence analysis of the 26S rDNA D1/D2 domain and 5.8S-ITS-RFLP analysis, a total of 686 isolates were identified at the species level. The six species identified were S. cerevisiae, Hanseniaspora uvarum, Hanseniaspora opuntiae, Issatchenkia terricola, Pichia kudriavzevii (Issatchenkia orientalis) and Trichosporon coremiiforme. This is the first report of T. coremiiforme as an inhabitant of grape must. Three new colony morphologies on WLN medium and one new 5.8S-ITS-RFLP profile are described. Species of non-Saccharomyces, predominantly H. opuntiae, were found in early stages of fermentation. Subsequently, S. cerevisiae prevailed followed by large numbers of P. kudriavzevii that dominated at the end of fermentations. Six native genotypes of S. cerevisiae were determined by interdelta sequence analysis. Genotypes III and IV were predominant. As a first step in exploring untapped yeast resources of the region, this study is important for monitoring the yeast ecology in native fermentations and screening indigenous yeasts that will produce wines with regional characteristics. Copyright © 2012 Elsevier Ltd. All rights reserved.

Widespread Use of Non-productive Alternative Splice Sites in Saccharomyces cerevisiae

PubMed Central

Kawashima, Tadashi; Douglass, Stephen; Gabunilas, Jason; Pellegrini, Matteo; Chanfreau, Guillaume F.

2014-01-01

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3′ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3′-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity. PMID:24722551

An energy-saving glutathione production method from low-temperature cooked rice using amylase-expressing Saccharomyces cerevisiae.

PubMed

Hara, Kiyotaka Y; Kim, Songhee; Kiriyama, Kentaro; Yoshida, Hideyo; Arai, Shogo; Ishii, Jun; Ogino, Chiaki; Fukuda, Hideki; Kondo, Akihiko

2012-05-01

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is industrially produced by fermentation using Saccharomyces cerevisiae. Before the glutathione fermentation process with S. cerevisiae, a glucose extraction process from starchy materials is required. This glucose extraction is usually carried out by converting starchy materials to starch using high-temperature cooking and subsequent hydrolysis by amylases to convert starch to glucose. In this study, to develop an energy-saving glutathione production process by reducing energy consumption during the cooking step, we efficiently produced glutathione from low-temperature cooked rice using amylase-expressing S. cerevisiae. The combination of the amylase-expressing yeast with low-temperature cooking is potentially applicable to a variety of energy-saving bio-production methods of chemicals from starchy bio-resources. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of Saccharomyces cerevisiae To Reduce the Bioaccessibility of Mercury from Food.

PubMed

Jadán-Piedra, Carlos; Baquedano, Marta; Puig, Sergi; Vélez, Dinoraz; Devesa, Vicenta

2017-04-05

Food is the main pathway of exposure to inorganic mercury [Hg(II)] and methylmercury (CH 3 Hg). Intestinal absorption of these mercury species is influenced by their chemical form, the luminal pH, and the composition of the diet. In this regard, strategies have been proposed for reducing mercury absorption using dietary components. This study evaluates the capacity of Saccharomyces cerevisiae to reduce the amount of mercury solubilized after gastrointestinal digestion that is available for intestinal absorption (bioaccessibility). The results show that S. cerevisiae strains reduce mercury bioaccessibility from aqueous solutions of Hg(II) (89 ± 6%) and CH 3 Hg (83 ± 4%), and from mushrooms (19-77%), but not from seafood. The formation of mercury-cysteine or mercury-polypeptide complexes in the bioaccessible fraction may contribute to the reduced effect of yeasts on mercury bioaccessibility from seafood. Our study indicates that budding yeasts could be useful for reducing the extent of intestinal absorption of mercury present in water and some food matrices.

European derived Saccharomyces cerevisiae colonisation of New Zealand vineyards aided by humans

PubMed Central

Gayevskiy, Velimir; Lee, Soon

2016-01-01

Humans have acted as vectors for species and expanded their ranges since at least the dawn of agriculture. While relatively well characterised for macrofauna and macroflora, the extent and dynamics of human-aided microbial dispersal is poorly described. We studied the role which humans have played in manipulating the distribution of Saccharomyces cerevisiae, one of the world's most important microbes, using whole genome sequencing. We include 52 strains representative of the diversity in New Zealand to the global set of genomes for this species. Phylogenomic approaches show an exclusively European origin of the New Zealand population, with a minimum of 10 founder events mostly taking place over the last 1000 years. Our results show that humans have expanded the range of S. cerevisiae and transported it to New Zealand where it was not previously present, where it has now become established in vineyards, but radiation to native forests appears limited. PMID:27744274

Enhanced production of para-hydroxybenzoic acid by genetically engineered Saccharomyces cerevisiae.

PubMed

Averesch, Nils J H; Prima, Alex; Krömer, Jens O

2017-08-01

Saccharomyces cerevisiae is a popular organism for metabolic engineering; however, studies aiming at over-production of bio-replacement precursors for the chemical industry often fail to overcome proof-of-concept stage. When intending to show real industrial attractiveness, the challenge is twofold: formation of the target compound must be increased, while minimizing the formation of side and by-products to maximize titer, rate and yield. To tackle these, the metabolism of the organism, as well as the parameters of the process, need to be optimized. Addressing both we show that S. cerevisiae is well-suited for over-production of aromatic compounds, which are valuable in chemical industry and are particularly useful in space technology. Specifically, a strain engineered to accumulate chorismate was optimized for formation of para-hydroxybenzoic acid. Then a fed-batch bioreactor process was developed, which delivered a final titer of 2.9 g/L, a maximum rate of 18.625 mg pHBA /(g CDW  × h) and carbon-yields of up to 3.1 mg pHBA /g glucose .

Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture

NASA Technical Reports Server (NTRS)

Johanson, Kelly; Allen, Patricia L.; Lewis, Fawn; Cubano, Luis A.; Hyman, Linda E.; Hammond, Timothy G.

2002-01-01

This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.

Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae

PubMed Central

Henderson, Clark M.; Zeno, Wade F.; Lerno, Larry A.; Longo, Marjorie L.

2013-01-01

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner. PMID:23811519

The ecology and evolution of non-domesticated Saccharomyces species.

PubMed

Boynton, Primrose J; Greig, Duncan

2014-12-01

Yeast researchers need model systems for ecology and evolution, but the model yeast Saccharomyces cerevisiae is not ideal because its evolution has been affected by domestication. Instead, ecologists and evolutionary biologists are focusing on close relatives of S. cerevisiae, the seven species in the genus Saccharomyces. The best-studied Saccharomyces yeast, after S. cerevisiae, is S. paradoxus, an oak tree resident throughout the northern hemisphere. In addition, several more members of the genus Saccharomyces have recently been discovered. Some Saccharomyces species are only found in nature, while others include both wild and domesticated strains. Comparisons between domesticated and wild yeasts have pinpointed hybridization, introgression and high phenotypic diversity as signatures of domestication. But studies of wild Saccharomyces natural history, biogeography and ecology are only beginning. Much remains to be understood about wild yeasts' ecological interactions and life cycles in nature. We encourage researchers to continue to investigate Saccharomyces yeasts in nature, both to place S. cerevisiae biology into its ecological context and to develop the genus Saccharomyces as a model clade for ecology and evolution. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd.

The ecology and evolution of non-domesticated Saccharomyces species

PubMed Central

Boynton, Primrose J; Greig, Duncan

2014-01-01

Yeast researchers need model systems for ecology and evolution, but the model yeast Saccharomyces cerevisiae is not ideal because its evolution has been affected by domestication. Instead, ecologists and evolutionary biologists are focusing on close relatives of S. cerevisiae, the seven species in the genus Saccharomyces. The best-studied Saccharomyces yeast, after S. cerevisiae, is S. paradoxus, an oak tree resident throughout the northern hemisphere. In addition, several more members of the genus Saccharomyces have recently been discovered. Some Saccharomyces species are only found in nature, while others include both wild and domesticated strains. Comparisons between domesticated and wild yeasts have pinpointed hybridization, introgression and high phenotypic diversity as signatures of domestication. But studies of wild Saccharomyces natural history, biogeography and ecology are only beginning. Much remains to be understood about wild yeasts' ecological interactions and life cycles in nature. We encourage researchers to continue to investigate Saccharomyces yeasts in nature, both to place S. cerevisiae biology into its ecological context and to develop the genus Saccharomyces as a model clade for ecology and evolution. © 2014 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:25242436

Split luciferase complementation assay for the analysis of G protein-coupled receptor ligand response in Saccharomyces cerevisiae.

PubMed

Fukutani, Yosuke; Ishii, Jun; Kondo, Akihiko; Ozawa, Takeaki; Matsunami, Hiroaki; Yohda, Masafumi

2017-06-01

The budding yeast Saccharomyces cerevisiae is equipped with G protein-coupled receptors (GPCR). Because the yeast GPCR signaling mechanism is partly similar to that of the mammalian system, S. cerevisiae can be used for a host of mammalian GPCR expression and ligand-mediated activation assays. However, currently available yeast systems require several hours to observe the responses because they depend on the expression of reporter genes. In this study, we attempted to develop a simple GPCR assay system using split luciferase and β-arrestin, which are independent of the endogenous S. cerevisiae GPCR signaling pathways. We applied the split luciferase complementation assay method to S. cerevisiae and found that it can be used to analyze the ligand response of the human somatostatin receptor in S. cerevisiae. On the contrary, the response of the pheromone receptor Ste2 was not observed by the assay. Thus, the split luciferase complementation should be free from the effect of the endogenous GPCR signaling. Biotechnol. Bioeng. 2017;114: 1354-1361. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae.

PubMed

Ito, Yoichiro; Kitagawa, Takao; Yamanishi, Mamoru; Katahira, Satoshi; Izawa, Shingo; Irie, Kenji; Furutani-Seiki, Makoto; Matsuyama, Takashi

2016-11-15

Post-transcriptional upregulation is an effective way to increase the expression of transgenes and thus maximize the yields of target chemicals from metabolically engineered organisms. Refractory elements in the 3' untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae, several terminator regions have shown activity in increasing the production of proteins by upstream coding genes; among these terminators the DIT1 terminator has the highest activity. Here, we found in Saccharomyces cerevisiae that two resident trans-acting RNA-binding proteins (Nab6p and Pap1p) enhance the activity of the DIT1 terminator through the cis element GUUCG/U within the 3'-UTR. These two RNA-binding proteins could upregulate a battery of cell-wall-related genes. Mutagenesis of the DIT1 terminator improved its activity by a maximum of 500% of that of the standard PGK1 terminator. Further understanding and improvement of this system will facilitate inexpensive and stable production of complicated organism-derived drugs worldwide.

Oxygen availability and strain combination modulate yeast growth dynamics in mixed culture fermentations of grape must with Starmerella bacillaris and Saccharomyces cerevisiae.

PubMed

Englezos, Vasileios; Cravero, Francesco; Torchio, Fabrizio; Rantsiou, Kalliopi; Ortiz-Julien, Anne; Lambri, Milena; Gerbi, Vincenzo; Rolle, Luca; Cocolin, Luca

2018-02-01

Starmerella bacillaris (synonym Candida zemplinina) is a non-Saccharomyces yeast that has been proposed as a co-inoculant of selected Saccharomyces cerevisiae strains in mixed culture fermentations to enhance the analytical composition of the wines. In order to acquire further knowledge on the metabolic interactions between these two species, in this study we investigated the impact of oxygen addition and combination of Starm. bacillaris with S. cerevisiae strains on the microbial growth and metabolite production. Fermentations were carried out under two different conditions of oxygen availability. Oxygen availability and strain combination clearly influenced the population dynamics throughout the fermentation. Oxygen concentration increased the survival time of Starm. bacillaris and decreased the growth rate of S. cerevisiae strains in mixed culture fermentations, whereas it did not affect the growth of the latter in pure culture fermentations. This study reveals new knowledge about the influence of oxygen availability on the successional evolution of yeast species during wine fermentation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Replication profile of Saccharomyces cerevisiae chromosome VI.

PubMed

Friedman, K L; Brewer, B J; Fangman, W L

1997-11-01

An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae. Chromosome VI shows a wide variation in the percentage of cell cycles in which different replication origins are utilized. Most of the origins are activated in only a fraction of cells, suggesting that the pattern of origin usage on chromosome VI varies greatly within the cell population. The replication times of fragments containing chromosome VI origins show a temporal pattern that has been recognized on other chromosomes--the telomeres replicate late in S phase, while the central region of the chromosome replicates early. As demonstrated here for chromosome VI, analysis of the direction of replication fork movement along a chromosome and determination of replication time by measuring a period of hemimethylation may provide an efficient means of surveying origin activity over large regions of the genome.

Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae.

PubMed

Elbing, Karin; Larsson, Christer; Bill, Roslyn M; Albers, Eva; Snoep, Jacky L; Boles, Eckhard; Hohmann, Stefan; Gustafsson, Lena

2004-09-01

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.

Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae

PubMed Central

Elbing, Karin; Larsson, Christer; Bill, Roslyn M.; Albers, Eva; Snoep, Jacky L.; Boles, Eckhard; Hohmann, Stefan; Gustafsson, Lena

2004-01-01

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations. PMID:15345416

Air-liquid biofilm formation is dependent on ammonium depletion in a Saccharomyces cerevisiae flor strain.

PubMed

Zara, Giacomo; Budroni, Marilena; Mannazzu, Ilaria; Zara, Severino

2011-12-01

Air-liquid biofilm formation appears to be an adaptive mechanism that promotes foraging of Saccharomyces cerevisiae flor strains in response to nutrient starvation. The FLO11 gene plays a central role in this phenotype as its expression allows yeast cells to rise to the liquid surface. Here, we investigated the role of ammonium depletion in air-liquid biofilm formation and FLO11 expression in a S. cerevisiae flor strain. The data obtained show that increasing ammonium concentrations from 0 to 450 m m reduce air-liquid biofilm in terms of biomass and velum formation and correlate with a reduction of FLO11 expression. Rapamycin inhibition of the TOR pathway and deletion of RAS2 gene significantly reduced biofilm formation and FLO11 expression. Taken together, these data suggest that ammonium depletion is a key factor in the induction of air-liquid biofilm formation and FLO11 expression in S. cerevisiae flor strains. Copyright © 2011 John Wiley & Sons, Ltd.

Controlled mixed fermentation at winery scale using Zygotorulaspora florentina and Saccharomyces cerevisiae.

PubMed

Lencioni, Livio; Romani, Cristina; Gobbi, Mirko; Comitini, Francesca; Ciani, Maurizio; Domizio, Paola

2016-10-03

Over the last few years the use of multi-starter inocula has become an attractive biotechnological practice in the search for wine with high flavour complexity or distinctive characters. This has been possible through exploiting the particular oenological features of some non-Saccharomyces yeast strains, and the effects that derive from their specific interactions with Saccharomyces. In the present study, we evaluated the selected strain Zygotorulaspora florentina (formerly Zygosaccharomyces florentinus) in mixed culture fermentations with Saccharomyces cerevisiae, from the laboratory scale to the winery scale. The scale-up fermentation and substrate composition (i.e., white or red musts) influenced the analytical composition of the mixed fermentation. At the laboratory scale, mixed fermentation with Z. florentina exhibited an enhancement of polysaccharides and 2-phenylethanol content and a reduction of volatile acidity. At the winery scale, different fermentation characteristics of Z. florentina were observed. Using Sangiovese red grape juice, sequential fermentation trials showed a significantly higher concentration of glycerol and esters while the sensorial analysis of the resulting wines showed higher floral notes and lower perception of astringency. To our knowledge, this is the first time that this yeasts association has been evaluated at the winery scale indicating the potential use of this mixed culture in red grape varieties. Copyright © 2016. Published by Elsevier B.V.

Genomic structural variation contributes to phenotypic change of industrial bioethanol yeast Saccharomyces cerevisiae.

PubMed

Zhang, Ke; Zhang, Li-Jie; Fang, Ya-Hong; Jin, Xin-Na; Qi, Lei; Wu, Xue-Chang; Zheng, Dao-Qiong

2016-03-01

Genomic structural variation (GSV) is a ubiquitous phenomenon observed in the genomes of Saccharomyces cerevisiae strains with different genetic backgrounds; however, the physiological and phenotypic effects of GSV are not well understood. Here, we first revealed the genetic characteristics of a widely used industrial S. cerevisiae strain, ZTW1, by whole genome sequencing. ZTW1 was identified as an aneuploidy strain and a large-scale GSV was observed in the ZTW1 genome compared with the genome of a diploid strain YJS329. These GSV events led to copy number variations (CNVs) in many chromosomal segments as well as one whole chromosome in the ZTW1 genome. Changes in the DNA dosage of certain functional genes directly affected their expression levels and the resultant ZTW1 phenotypes. Moreover, CNVs of large chromosomal regions triggered an aneuploidy stress in ZTW1. This stress decreased the proliferation ability and tolerance of ZTW1 to various stresses, while aneuploidy response stress may also provide some benefits to the fermentation performance of the yeast, including increased fermentation rates and decreased byproduct generation. This work reveals genomic characters of the bioethanol S. cerevisiae strain ZTW1 and suggests that GSV is an important kind of mutation that changes the traits of industrial S. cerevisiae strains. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effect of supplementing a diet with monensin sodium and Saccharomyces Cerevisiae on reproductive performance of Ghezel ewes.

PubMed

Ahmadzadeh, Leila; Hosseinkhani, Ali; Daghigh Kia, Hossein

2018-01-01

Effect of supplementing a diet, in an attempt to enhance reproduction, with monensin sodium and Saccharomyces cerevisiae yeast on reproductive performance was investigated during the breeding season using 44 Ghezel ewes (body weight 56.97±7.47kg, age 2-5 years and body condition score (BCS) 2.5) which were allocated randomly in equal numbers to the four dietary treatments as follows: 1) Basal diet plus supplemental feed (450g/ewe/d) plus monensin sodium (30mg/ewe/d) (MS); 2) Basal diet plus supplemental feed (450 g/ewe/d) plus Saccharomyces cerevisiae yeast (4×10 9 CFU/ewe/d) (SC); 3) Basal diet plus supplemental feed (450g/ewe/d) (FG); 4) Basal diet (only grazing on pasture, Control; G). Estrous synchronization of all ewes was done using controlled internal drug release (CIDR) and all ewes were mated with purebred Ghezel rams after CIDR removal. The results indicated that MS and SC treatments with 15 lambs had greater number of lambs than ewes of the other two treatment groups. Ewes in MS group with 50% twining rate had the greatest value followed by the FG, SC and G treatment groups (P<0.05). The lambs from ewes in MS and SC groups were heavier in weight than those in FG and G treatments (P<0.01). Blood sample analysis provided evidence that ewes in MS and SC groups had greater concentrations of 17β-estradiol (E2), progesterone (P4), blood urea nitrogen (P<0.05), insulin, glucose, cholesterol and total protein (P<0.01) than ewes of the other groups. These results indicated that using a diet for enhancing reproduction, including monensin sodium and Saccharomyces cerevisiae yeast in the breeding season could have beneficial effects on reproductive performance of Ghezel ewes. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of supplementing a Saccharomyces cerevisiae fermentation product in sow diets on performance of sows and nursing piglets

USDA-ARS?s Scientific Manuscript database

Forty-two sows (Camborough-22, PIC) were used to determine the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; Diamond V Original XPC) in gestation and lactation diets on performance of sows and their progeny. On 5 d before breeding, sows were allotted to 2 dietary tr...

Hxt-Carrier-Mediated Glucose Efflux upon Exposure of Saccharomyces cerevisiae to Excess Maltose

PubMed Central

Jansen, Mickel L. A.; De Winde, Johannes H.; Pronk, Jack T.

2002-01-01

When wild-type Saccharomyces cerevisiae strains pregrown in maltose-limited chemostat cultures were exposed to excess maltose, release of glucose into the external medium was observed. Control experiments confirmed that glucose release was not caused by cell lysis or extracellular maltose hydrolysis. To test the hypothesis that glucose efflux involved plasma membrane glucose transporters, experiments were performed with an S. cerevisiae strain in which all members of the hexose transporter (HXT) gene family had been eliminated and with an isogenic reference strain. Glucose efflux was virtually eliminated in the hexose-transport-deficient strain. This constitutes experimental proof that Hxt transporters facilitate export of glucose from S. cerevisiae cells. After exposure of the hexose-transport-deficient strain to excess maltose, an increase in the intracellular glucose level was observed, while the concentrations of glucose 6-phosphate and ATP remained relatively low. These results demonstrate that glucose efflux can occur as a result of uncoordinated expression of the initial steps of maltose metabolism and the subsequent reactions in glucose dissimilation. This is a relevant phenomenon for selection of maltose-constitutive strains for baking and brewing. PMID:12200274

Increased ethanol production by deletion of HAP4 in recombinant xylose-assimilating Saccharomyces cerevisiae.

PubMed

Matsushika, Akinori; Hoshino, Tamotsu

2015-12-01

The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.

ATLAS: An advanced PCR-method for routine visualization of telomere length in Saccharomyces cerevisiae.

PubMed

Zubko, Elena I; Shackleton, Jennifer L; Zubko, Mikhajlo K

2016-12-01

Measuring telomere length is essential in telomere biology. Southern blot hybridization is the predominant method for measuring telomere length in the genetic model Saccharomyces cerevisiae. We have further developed and refined a telomere PCR approach, which was rarely used previously (mainly in specific telomeric projects), into a robust method allowing direct visualisation of telomere length differences in routine experiments with S. cerevisiae, and showing a strong correlation of results with data obtained by Southern blot hybridization. In this expanded method denoted as ATLAS (A-dvanced T-elomere L-ength A-nalysis in S. cerevisiae), we have introduced: 1) set of new primers annealing with high specificity to telomeric regions on five different chromosomes; 2) new approach for designing reverse telomere primers that is based on the ligation of an adaptor of a fixed size to telomeric ends. ATLAS can be used at the scale of individual assays and high-throughput approaches. This simple, time/cost-effective and reproducible methodology will complement Southern blot hybridization and facilitate further progress in telomere research. Copyright © 2016 Elsevier B.V. All rights reserved.

Fermentation of Saccharomyces cerevisiae - Combining kinetic modeling and optimization techniques points out avenues to effective process design.

PubMed

Scheiblauer, Johannes; Scheiner, Stefan; Joksch, Martin; Kavsek, Barbara

2018-09-14

A combined experimental/theoretical approach is presented, for improving the predictability of Saccharomyces cerevisiae fermentations. In particular, a mathematical model was developed explicitly taking into account the main mechanisms of the fermentation process, allowing for continuous computation of key process variables, including the biomass concentration and the respiratory quotient (RQ). For model calibration and experimental validation, batch and fed-batch fermentations were carried out. Comparison of the model-predicted biomass concentrations and RQ developments with the corresponding experimentally recorded values shows a remarkably good agreement for both batch and fed-batch processes, confirming the adequacy of the model. Furthermore, sensitivity studies were performed, in order to identify model parameters whose variations have significant effects on the model predictions: our model responds with significant sensitivity to the variations of only six parameters. These studies provide a valuable basis for model reduction, as also demonstrated in this paper. Finally, optimization-based parametric studies demonstrate how our model can be utilized for improving the efficiency of Saccharomyces cerevisiae fermentations. Copyright © 2018 Elsevier Ltd. All rights reserved.

Saccharomyces and non-Saccharomyces Competition during Microvinification under Different Sugar and Nitrogen Conditions

PubMed Central

Lleixà, Jessica; Manzano, Maria; Mas, Albert; Portillo, María del C.

2016-01-01

The inoculation of wines with autochthonous yeast allows obtaining complex wines with a peculiar microbial footprint characteristic from a wine region. Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. However, the interactions between these yeasts are not well understood, especially those regarding the availability of nutrients. The aim of the present study was to analyze the effect of nitrogen and sugar concentration on the evolution of mixed yeast populations on controlled laboratory-scale fermentations monitored by density, plate culturing, PCR-DGGE and sugar and nitrogen consumption. Furthermore, the effect of the time of inoculation of Saccharomyces cerevisiae respect the initial co-inoculation of three non-Saccharomyces yeasts was evaluated over the evolution of fermentation. Our results have shown that S. cerevisiae inoculation during the first 48 h conferred a stabilizing effect over the fermentations with non-Saccharomyces strains tested and, generally, reduced yeast diversity at the end of the fermentation. On the other hand, nitrogen limitation increased the time of fermentation and also the proportion of non-Saccharomyces yeasts at mid and final fermentation. High sugar concentration resulted in different proportions of the inoculated yeast depending on the time of S. cerevisiae inoculation. This work emphasizes the importance of the concentration of nutrients on the evolution of mixed fermentations and points to the optimal conditions for a stable fermentation in which the inoculated yeasts survived until the end. PMID:27994585

Phenotypic Landscape of Saccharomyces cerevisiae during Wine Fermentation: Evidence for Origin-Dependent Metabolic Traits

PubMed Central

Camarasa, Carole; Sanchez, Isabelle; Brial, Pascale; Bigey, Frédéric; Dequin, Sylvie

2011-01-01

The species Saccharomyces cerevisiae includes natural strains, clinical isolates, and a large number of strains used in human activities. The aim of this work was to investigate how the adaptation to a broad range of ecological niches may have selectively shaped the yeast metabolic network to generate specific phenotypes. Using 72 S. cerevisiae strains collected from various sources, we provide, for the first time, a population-scale picture of the fermentative metabolic traits found in the S. cerevisiae species under wine making conditions. Considerable phenotypic variation was found suggesting that this yeast employs diverse metabolic strategies to face environmental constraints. Several groups of strains can be distinguished from the entire population on the basis of specific traits. Strains accustomed to growing in the presence of high sugar concentrations, such as wine yeasts and strains obtained from fruits, were able to achieve fermentation, whereas natural yeasts isolated from “poor-sugar” environments, such as oak trees or plants, were not. Commercial wine yeasts clearly appeared as a subset of vineyard isolates, and were mainly differentiated by their fermentative performances as well as their low acetate production. Overall, the emergence of the origin-dependent properties of the strains provides evidence for a phenotypic evolution driven by environmental constraints and/or human selection within S. cerevisiae. PMID:21949874

Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries.

PubMed

Hong, Kuk-Ki; Nielsen, Jens

2012-08-01

Metabolic engineering is the enabling science of development of efficient cell factories for the production of fuels, chemicals, pharmaceuticals, and food ingredients through microbial fermentations. The yeast Saccharomyces cerevisiae is a key cell factory already used for the production of a wide range of industrial products, and here we review ongoing work, particularly in industry, on using this organism for the production of butanol, which can be used as biofuel, and isoprenoids, which can find a wide range of applications including as pharmaceuticals and as biodiesel. We also look into how engineering of yeast can lead to improved uptake of sugars that are present in biomass hydrolyzates, and hereby allow for utilization of biomass as feedstock in the production of fuels and chemicals employing S. cerevisiae. Finally, we discuss the perspectives of how technologies from systems biology and synthetic biology can be used to advance metabolic engineering of yeast.

Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.

PubMed

Duina, Andrea A; Miller, Mary E; Keeney, Jill B

2014-05-01

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.

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

Aspergillus oryzae–Saccharomyces cerevisiae Consortium Allows Bio-Hybrid Fuel Cell to Run on Complex Carbohydrates

PubMed Central

Jahnke, Justin P.; Hoyt, Thomas; LeFors, Hannah M.; Sumner, James J.; Mackie, David M.

2016-01-01

Consortia of Aspergillus oryzae and Saccharomyces cerevisiae are examined for their abilities to turn complex carbohydrates into ethanol. To understand the interactions between microorganisms in consortia, Fourier-transform infrared spectroscopy is used to follow the concentrations of various metabolites such as sugars (e.g., glucose, maltose), longer chain carbohydrates, and ethanol to optimize consortia conditions for the production of ethanol. It is shown that with proper design A. oryzae can digest food waste simulants into soluble sugars that S. cerevisiae can ferment into ethanol. Depending on the substrate and conditions used, concentrations of 13% ethanol were achieved in 10 days. It is further shown that a direct alcohol fuel cell (FC) can be coupled with these A. oryzae-enabled S. cerevisiae fermentations using a reverse osmosis membrane. This “bio-hybrid FC” continually extracted ethanol from an ongoing consortium, enhancing ethanol production and allowing the bio-hybrid FC to run for at least one week. Obtained bio-hybrid FC currents were comparable to those from pure ethanol—water mixtures, using the same FC. The A. oryzae–S. cerevisiae consortium, coupled to a bio-hybrid FC, converted food waste simulants into electricity without any pre- or post-processing. PMID:27681904

Aspergillus oryzae-Saccharomyces cerevisiae Consortium Allows Bio-Hybrid Fuel Cell to Run on Complex Carbohydrates.

PubMed

Jahnke, Justin P; Hoyt, Thomas; LeFors, Hannah M; Sumner, James J; Mackie, David M

2016-02-04

Consortia of Aspergillus oryzae and Saccharomyces cerevisiae are examined for their abilities to turn complex carbohydrates into ethanol. To understand the interactions between microorganisms in consortia, Fourier-transform infrared spectroscopy is used to follow the concentrations of various metabolites such as sugars (e.g., glucose, maltose), longer chain carbohydrates, and ethanol to optimize consortia conditions for the production of ethanol. It is shown that with proper design A. oryzae can digest food waste simulants into soluble sugars that S. cerevisiae can ferment into ethanol. Depending on the substrate and conditions used, concentrations of 13% ethanol were achieved in 10 days. It is further shown that a direct alcohol fuel cell (FC) can be coupled with these A. oryzae-enabled S. cerevisiae fermentations using a reverse osmosis membrane. This "bio-hybrid FC" continually extracted ethanol from an ongoing consortium, enhancing ethanol production and allowing the bio-hybrid FC to run for at least one week. Obtained bio-hybrid FC currents were comparable to those from pure ethanol-water mixtures, using the same FC. The A. oryzae-S. cerevisiae consortium, coupled to a bio-hybrid FC, converted food waste simulants into electricity without any pre- or post-processing.

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae.

PubMed

Chalissery, Jisha; Jalal, Deena; Al-Natour, Zeina; Hassan, Ahmed H

2017-03-01

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

Catheter-related Saccharomyces cerevisiae Fungemia Following Saccharomyces boulardii Probiotic Treatment: In a child in intensive care unit and review of the literature.

PubMed

Atıcı, Serkan; Soysal, Ahmet; Karadeniz Cerit, Kıvılcım; Yılmaz, Şerife; Aksu, Burak; Kıyan, Gürsu; Bakır, Mustafa

2017-03-01

Although Saccharomyces boulardii is usually a non-pathogenic fungus, in rare occasions it can cause invasive infection in children. We present the case of an 8-year-old patient in pediatric surgical intensive care unit who developed S. cerevisiae fungemia following probiotic treatment containing S. boulardii . Caspofungin was not effective in this case and he was treated with amphotericin B. We want to emphasize that physicians should be careful about probiotic usage in critically ill patients.

Polyphosphatase PPN1 of Saccharomyces cerevisiae: Switching of Exopolyphosphatase and Endopolyphosphatase Activities

PubMed Central

Andreeva, Nadezhda; Trilisenko, Ludmila; Eldarov, Mikhail; Kulakovskaya, Tatiana

2015-01-01

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co2+ and Mg2+, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co2+ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg2+ was activated by ADP and arginine but inhibited by ATP. PMID:25742176

Intracellular metabolite profiling of Saccharomyces cerevisiae evolved under furfural.

PubMed

Jung, Young Hoon; Kim, Sooah; Yang, Jungwoo; Seo, Jin-Ho; Kim, Kyoung Heon

2017-03-01

Furfural, one of the most common inhibitors in pre-treatment hydrolysates, reduces the cell growth and ethanol production of yeast. Evolutionary engineering has been used as a selection scheme to obtain yeast strains that exhibit furfural tolerance. However, the response of Saccharomyces cerevisiae to furfural at the metabolite level during evolution remains unknown. In this study, evolutionary engineering and metabolomic analyses were applied to determine the effects of furfural on yeasts and their metabolic response to continuous exposure to furfural. After 50 serial transfers of cultures in the presence of furfural, the evolved strains acquired the ability to stably manage its physiological status under the furfural stress. A total of 98 metabolites were identified, and their abundance profiles implied that yeast metabolism was globally regulated. Under the furfural stress, stress-protective molecules and cofactor-related mechanisms were mainly induced in the parental strain. However, during evolution under the furfural stress, S. cerevisiae underwent global metabolic allocations to quickly overcome the stress, particularly by maintaining higher levels of metabolites related to energy generation, cofactor regeneration and recovery from cellular damage. Mapping the mechanisms of furfural tolerance conferred by evolutionary engineering in the present study will be led to rational design of metabolically engineered yeasts. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Post-zygotic sterility and cytonuclear compatibility limits in S. cerevisiae xenomitochondrial cybrids

PubMed Central

Špírek, Mário; Poláková, Silvia; Jatzová, Katarína; Sulo, Pavol

2015-01-01

Nucleo-mitochondrial interactions, particularly those determining the primary divergence of biological species, can be studied by means of xenomitochondrial cybrids, which are cells where the original mitochondria are substituted by their counterparts from related species. Saccharomyces cerevisiae cybrids are prepared simply by the mating of the ρ0 strain with impaired karyogamy and germinating spores from other Saccharomyces species and fall into three categories. Cybrids with compatible mitochondrial DNA (mtDNA) from Saccharomyces paradoxus CBS 432 and Saccharomyces cariocanus CBS 7994 are metabolically and genetically similar to cybrids containing mtDNA from various S. cerevisiae. Cybrids with mtDNA from other S. paradoxus strains, S. cariocanus, Saccharomyces kudriavzevii, and Saccharomyces mikatae require a period of adaptation to establish efficient oxidative phosphorylation. They exhibit a temperature-sensitive phenotype, slower growth rate on a non-fermentable carbon source and a long lag phase after the shift from glucose. Their decreased respiration capacity and reduced cytochrome aa3 content is associated with the inefficient splicing of cox1I3β, the intron found in all Saccharomyces species but not in S. cerevisiae. The splicing defect is compensated in cybrids by nuclear gain-of-function and can be alternatively suppressed by overexpression of MRP13 gene for mitochondrial ribosomal protein or the MRS2, MRS3, and MRS4 genes involved in intron splicing. S. cerevisiae with Saccharomyces bayanus mtDNA is unable to respire and the growth on ethanol–glycerol can be restored only after mating to some mit− strains. The nucleo-mitochondrial compatibility limit of S. cerevisiae and other Saccharomyces was set between S. kudriavzevii and S. bayanus at the divergence from S. cerevisiae about 15 MYA. The MRS1-cox1 S. cerevisiae/S. paradoxus cytonuclear Dobzhansky–Muller pair has a neglible impact on the separation of species since its imperfection is

Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

PubMed Central

Salusjärvi, Laura; Kankainen, Matti; Soliymani, Rabah; Pitkänen, Juha-Pekka; Penttilä, Merja; Ruohonen, Laura

2008-01-01

Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains

Behavior of Lactobacillus plantarum and Saccharomyces cerevisiae in fresh and thermally processed orange juice.

PubMed

Alwazeer, Duried; Cachon, Remy; Divies, Charles

2002-10-01

Lactobacillus plantarum and Saccharomyces cerevisiae are acid-tolerant microorganisms that are able to spoil citrus juices before and after pasteurization. The growth of these microorganisms in orange juice with and without pasteurization was investigated. Two samples of orange juice were inoculated with ca. 10(5) CFU/ml of each microorganism. Others were inoculated with ca. 10(7) CFU/ml of each microorganism and then thermally treated. L. plantarum populations were reduced by 2.5 and <1 log10 CFU/ml at 60 degrees C for 40 s and at 55 degrees C for 40 s, respectively. For the same treatments, S. cerevisiae populations were reduced by >6 and 2 log10 CFU/ml, respectively. Samples of heated and nonheated juice were incubated at 15 degrees C for 20 days. Injured populations of L. plantarum decreased by ca. 2 log10 CFU/ml during the first 70 h of storage, but those of S. cerevisiae did not decrease. The length of the lag phase after pasteurization increased 6.2-fold for L. plantarum and 1.9-fold for S. cerevisiae, and generation times increased by 41 and 86%, respectively. The results of this study demonstrate the differences in the capabilities of intact and injured cells of spoilage microorganisms to spoil citrus juice and the different thermal resistance levels of cells. While L. plantarum was more resistant to heat treatment than S. cerevisiae was, growth recovery after pasteurization was faster for the latter microorganism.

Oxygen requirements for formation and activity of the squalene expoxidase in Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Jahnke, L.; Klein, H. P.

1983-01-01

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the 'mitochondrial' fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the 'microsomal' fraction. At 0.03 percent (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent K sub m for oxygen of 0.38 percent (vol/vol) was determined from a crude particulate preparation for the epoxidase.

Saccharomyces cerevisiae Boulardii Reduces the Deoxynivalenol-Induced Alteration of the Intestinal Transcriptome.

PubMed

Alassane-Kpembi, Imourana; Pinton, Philippe; Hupé, Jean-François; Neves, Manon; Lippi, Yannick; Combes, Sylvie; Castex, Mathieu; Oswald, Isabelle P

2018-05-15

Type B trichothecene mycotoxin deoxynivalenol (DON) is one of the most frequently occurring food contaminants. By inducing trans-activation of a number of pro-inflammatory cytokines and increasing the stability of their mRNA, trichothecene can impair intestinal health. Several yeast products, especially Saccharomyces cerevisiae , have the potential for improving the enteric health of piglets, but little is known about the mechanisms by which the administration of yeast counteracts the DON-induced intestinal alterations. Using a pig jejunum explant model, a whole-transcriptome analysis was performed to decipher the early response of the small intestine to the deleterious effects of DON after administration of S. cerevisiae boulardii strain CNCM I-1079. Compared to the control condition, no differentially expressed gene (DE) was observed after treatment by yeast only. By contrast, 3619 probes-corresponding to 2771 genes-were differentially expressed following exposure to DON, and 32 signaling pathways were identified from the IPA software functional analysis of the set of DE genes. When the intestinal explants were treated with S. cerevisiae boulardii prior to DON exposure, the number of DE genes decreased by half (1718 probes corresponding to 1384 genes). Prototypical inflammation signaling pathways triggered by DON, including NF-κB and p38 MAPK, were reversed, although the yeast demonstrated limited efficacy toward some other pathways. S. cerevisiae boulardii also restored the lipid metabolism signaling pathway, and reversed the down-regulation of the antioxidant action of vitamin C signaling pathway. The latter effect could reduce the burden of DON-induced oxidative stress. Altogether, the results show that S. cerevisiae boulardii reduces the DON-induced alteration of intestinal transcriptome, and point to new mechanisms for the healing of tissue injury by yeast.

Saccharomyces cerevisiae boulardii Reduces the Deoxynivalenol-Induced Alteration of the Intestinal Transcriptome

PubMed Central

Alassane-Kpembi, Imourana; Hupé, Jean-François; Neves, Manon; Lippi, Yannick; Combes, Sylvie; Castex, Mathieu

2018-01-01

Type B trichothecene mycotoxin deoxynivalenol (DON) is one of the most frequently occurring food contaminants. By inducing trans-activation of a number of pro-inflammatory cytokines and increasing the stability of their mRNA, trichothecene can impair intestinal health. Several yeast products, especially Saccharomyces cerevisiae, have the potential for improving the enteric health of piglets, but little is known about the mechanisms by which the administration of yeast counteracts the DON-induced intestinal alterations. Using a pig jejunum explant model, a whole-transcriptome analysis was performed to decipher the early response of the small intestine to the deleterious effects of DON after administration of S. cerevisiae boulardii strain CNCM I-1079. Compared to the control condition, no differentially expressed gene (DE) was observed after treatment by yeast only. By contrast, 3619 probes—corresponding to 2771 genes—were differentially expressed following exposure to DON, and 32 signaling pathways were identified from the IPA software functional analysis of the set of DE genes. When the intestinal explants were treated with S. cerevisiae boulardii prior to DON exposure, the number of DE genes decreased by half (1718 probes corresponding to 1384 genes). Prototypical inflammation signaling pathways triggered by DON, including NF-κB and p38 MAPK, were reversed, although the yeast demonstrated limited efficacy toward some other pathways. S. cerevisiae boulardii also restored the lipid metabolism signaling pathway, and reversed the down-regulation of the antioxidant action of vitamin C signaling pathway. The latter effect could reduce the burden of DON-induced oxidative stress. Altogether, the results show that S. cerevisiae boulardii reduces the DON-induced alteration of intestinal transcriptome, and point to new mechanisms for the healing of tissue injury by yeast. PMID:29762474

Genomic diversity of Saccharomyces cerevisiae yeasts associated with alcoholic fermentation of bacanora produced by artisanal methods.

PubMed

Álvarez-Ainza, M L; Zamora-Quiñonez, K A; Moreno-Ibarra, G M; Acedo-Félix, E

2015-03-01

Bacanora is a spirituous beverage elaborated with Agave angustifolia Haw in an artisanal process. Natural fermentation is mostly performed with native yeasts and bacteria. In this study, 228 strains of yeast like Saccharomyces were isolated from the natural alcoholic fermentation on the production of bacanora. Restriction analysis of the amplified region ITS1-5.8S-ITS2 of the ribosomal DNA genes (RFLPr) were used to confirm the genus, and 182 strains were identified as Saccharomyces cerevisiae. These strains displayed high genomic variability in their chromosomes profiles by karyotyping. Electrophoretic profiles of the strains evaluated showed a large number of chromosomes the size of which ranged between 225 and 2200 kpb approximately.

Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

PubMed Central

2012-01-01

Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene

An insight into the complex prion-prion interaction network in the budding yeast Saccharomyces cerevisiae.

PubMed

Du, Zhiqiang; Valtierra, Stephanie; Li, Liming

2014-01-01

The budding yeast Saccharomyces cerevisiae is a valuable model system for studying prion-prion interactions as it contains multiple prion proteins. A recent study from our laboratory showed that the existence of Swi1 prion ([SWI(+)]) and overproduction of Swi1 can have strong impacts on the formation of 2 other extensively studied yeast prions, [PSI(+)] and [PIN(+)] ([RNQ(+)]) (Genetics, Vol. 197, 685-700). We showed that a single yeast cell is capable of harboring at least 3 heterologous prion elements and these prions can influence each other's appearance positively and/or negatively. We also showed that during the de novo [PSI(+)] formation process upon Sup35 overproduction, the aggregation patterns of a preexisting inducer ([RNQ(+)] or [SWI(+)]) can undergo significant remodeling from stably transmitted dot-shaped aggregates to aggregates that co-localize with the newly formed Sup35 aggregates that are ring/ribbon/rod- shaped. Such co-localization disappears once the newly formed [PSI(+)] prion stabilizes. Our finding provides strong evidence supporting the "cross-seeding" model for prion-prion interactions and confirms earlier reports that the interactions among different prions and their prion proteins mostly occur at the initiation stages of prionogenesis. Our results also highlight a complex prion interaction network in yeast. We believe that elucidating the mechanism underlying the yeast prion-prion interaction network will not only provide insight into the process of prion de novo generation and propagation in yeast but also shed light on the mechanisms that govern protein misfolding, aggregation, and amyloidogenesis in higher eukaryotes.

TPS1 terminator increases mRNA and protein yield in a Saccharomyces cerevisiae expression system.

PubMed

Yamanishi, Mamoru; Katahira, Satoshi; Matsuyama, Takashi

2011-01-01

Both terminators and promoters regulate gene expression. In Saccharomyces cerevisiae, the TPS1 terminator (TPS1t), coupled to a gene encoding a fluorescent protein, produced more transgenic mRNA and protein than did similar constructs containing other terminators, such as CYC1t, TDH3t, and PGK1t. This suggests that TPS1t can be used as a general terminator in the development of metabolically engineered yeast in high-yield systems.

Functional expression of Schizosaccharomyces pombe Vba2p in the vacuolar membrane of Saccharomyces cerevisiae.

PubMed

Pongcharoen, Pongsanat; Kawano-Kawada, Miyuki; Iwaki, Tomoko; Sugimoto, Naoko; Sekito, Takayuki; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

2013-01-01

A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.

Crystallization and preliminary X-ray analysis of CTP:phosphoethanolamine cytidylyltransferase (ECT) from Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohtsuka, Jun; Nagata, Koji; Lee, Woo Cheol

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} = 2.2more » Å{sup 3} Da{sup −1}), with a solvent content of 43%.« less

Secretory production of cell wall components by Saccharomyces cerevisiae protoplasts in static liquid culture.

PubMed

Aoyagi, Hideki; Ishizaka, Mikiko; Tanaka, Hideo

2012-04-01

When protoplasts of Saccharomyces cerevisiae T7 and IFO 0309 are cultured in a static liquid culture at 2.5 × 10(6) protoplasts/ml, cell wall regeneration does not occur and cell wall components (CWC) are released into the culture broth. By using a specialized fluorometer, the concentrations of CWC could be measured on the basis of the fluorescence intensity of the CWC after staining with Fluostain I. The inoculum concentration, pH, and osmotic pressure of the medium were important factors for the production of CWC in culture. Under optimal culture conditions, S. cerevisiae T7 protoplasts produced 0.91 mg/ml CWC after 24 h. The CWC induced the tumor necrosis factor-α production about 1.3 times higher than that of the commercially available β-1,3/1,6-glucan from baker's yeast cells.

Ethanol production using immobilized Saccharomyces cerevisiae in lyophilized cellulose gel.

PubMed

Winkelhausen, Eleonora; Velickova, Elena; Amartey, Samuel A; Kuzmanova, Slobodanka

2010-12-01

A new lyophilization technique was used for immobilization of Saccharomyces cerevisiae cells in hydroxyethylcellulose (HEC) gels. The suitability of the lyophilized HEC gels to serve as immobilization matrices for the yeast cells was assessed by calculating the immobilization efficiency and the cell retention in three consecutive batches, each in duration of 72 h. Throughout the repeated batch fermentation, the immobilization efficiency was almost constant with an average value of 0.92 (12-216 h). The maximum value of cell retention was 0.24 g immobilized cells/g gel. Both parameters indicated that lyophilized gels are stable and capable of retaining the immobilized yeast cells. Showing the yeast cells propagation within the polymeric matrix, the scanning electron microscope images also confirmed that the lyophilization technique for immobilization of S. cerevisiae cells in the HEC gels was successful. The activity of the immobilized yeast cells was demonstrated by their capacity to convert glucose to ethanol. Ethanol yield of 0.40, 0.43 and 0.30 g ethanol/g glucose corresponding to 79%, 84% and 60% of the theoretical yield was attained in the first, second and third batches, respectively. The cell leakage was less than 10% of the average concentration of the immobilized cells.

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

USDA-ARS?s Scientific Manuscript database

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

Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

PubMed

Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

2017-07-14

The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

β-Glucan from Saccharomyces cerevisiae Induces IFN-γ Production In Vivo in BALB/c Mice.

PubMed

Javmen, Artur; Nemeikaitė-Čėnienė, Aušra; Bratchikov, Maksim; Grigiškis, Saulius; Grigas, Fortūnatas; Jonauskienė, Irena; Zabulytė, Danguolė; Mauricas, Mykolas

2015-01-01

β-Glucan is one of the most abundant polymers in nature and has been established as an immunomodulator. This compound has notable physiological effects on mammalian immune systems, including anti-tumor and anti-infective activities and can activate the immune response. It is considered that the immune-stimulating activities of β-glucan can depend on physicochemical parameters, such as molecular size. Saccharomyces cerevisiae, also known as baker's yeast, is a frequently used source of β-glucan. The aim of the experiments was to investigate how different Saccharomyces cerevisiae β-glucan preparations with different molecular size affect interferon-gamma (IFN-γ) production in BALB/c mice. In vivo and in vitro BALB/c mouse models were used for the investigations. Different β-glucan preparations were orally administrated in the in vivo experiments. IFN-γ production in BALB/c mice was analyzed by enzyme-linked immunosorbent assay and measuring interferon-γ RNA concentration. The results showed that orally-administered β-glucan from S. cerevisiae enhanced IFN-γ production in BALB/c mice in the in vivo model, but not by mouse leukocytes in vitro. Moreover, water-soluble β-glucan enhanced IFN-γ production more effectively than did particulate β-glucan. IFN-γ plays an important role in immunity against viral and bacterial infections. Our experiments have shown that β-glucan preparations enhance IFN-γ production in BALB/c mice and can be potentially used for immune system stimulation in mammals. Current results may be used to develop soluble β-glucan nutritional supplements. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Use of Torulaspora delbrueckii Co-fermentation With Two Saccharomyces cerevisiae Strains With Different Aromatic Characteristic to Improve the Diversity of Red Wine Aroma Profile

PubMed Central

Zhang, Bo-Qin; Luan, Yu; Duan, Chang-Qing; Yan, Guo-Liang

2018-01-01

The use of selected Saccharomyces and non-Saccharomyces strains as mixed starters has advantages over pure fermentation due to achieving wine products with distinctive and diversified aroma expected by consumers. To obtain a way to improve the aroma diversity and increase the differentiation of wine product, in this study, the aromatic effect of multi-culture of indigenous Torulaspora delbrueckii (TD12), simultaneous and sequential inoculation with two Saccharomyces strains (indigenous icewine yeast SC45 and commercial yeast BDX) with different enological characteristics were investigated in laboratory-scale 20 L fermenter, respectively. The results showed that T. delbrueckii co-fermented with different S. cerevisiae strain could generate diversified physicochemical and aromatic quality of wine as evidenced by PCA. Mixed fermentation of SC45/TD12 produced higher contents of higher alcohol (3-methyl-1-pentanol and phenylethyl alcohol), ethyl esters (ethyl decanoate and ethyl butanoate), terpenes and phenylacetaldehyde with less fatty acids (hexanoic acid, octanoic acid) and acetic acid, while BDX/TD12 generated more C6 alcohol (1-hexanol) and acetate esters (ethyl acetate and isoamyl acetate). Compared to simultaneous inoculation, sequential inoculation could achieve higher aroma diversity, and generate higher intensity of fruity, flowery and sweet attributes of wine as assessed by calculating the odor activity values. The different S. cerevisiae strain and inoculation method in alcoholic fermentation could further influence the formations of aromatic compounds in malolactic fermentation. Our results highlighted the importance of S. cerevisiae strain in shaping the aromatic quality of wine in mixed fermentation, and also suggested that using different S. cerevisiae strains with distinct aromatic characteristics co-fermentation with specific non-Saccharomyces strain is a potential way to increase the aromatic diversity and quality of wine product, which could provide

Evaluation of Saccharomyces cerevisiae as an antiaflatoxicogenic agent in broiler feedstuffs.

PubMed

Pizzolitto, R P; Armando, M R; Salvano, M A; Dalcero, A M; Rosa, C A

2013-06-01

Aflatoxins (AF) are the most important mycotoxins produced by toxigenic strains of various Aspergillus spp. Biological decontamination of mycotoxins using microorganisms is a well-known strategy for the management of mycotoxins in feeds. Saccharomyces cerevisiae strains have been reported to bind aflatoxin B1 (AFB1). The aim of this study was to evaluate the ability of S. cerevisiae CECT 1891 in counteracting the deleterious effects of AFB1 in broiler chicks. Experimental aflatoxicosis was induced in 6-d-old broilers by feeding them 1.2 mg of AFB1/kg of feed for 3 wk, and the yeast strain was administrated in feed (10(10) cells/kg), in the drinking water (5 × 10(9) cells/L), or a combination of both treatments. A total of 160 chicks were randomly divided into 8 treatments (4 repetitions per treatment). Growth performance was measured weekly from d 7 to 28, and serum biochemical parameters, weights, and histopathological examination of livers were determined at d 28. The AFB1 significantly decreased the BW gain, feed intake, and impaired feed conversion rate. Moreover, AFB1 treatment decreased serum protein concentration and increased liver damage. The addition of S. cerevisiae strain to drinking water, to diets contaminated with AFB1, showed a positive protection effect on the relative weight of the liver, histopathology, and biochemical parameters. Furthermore, dietary addition of the yeast strain to drinking water alleviated the negative effects of AFB1 on growth performance parameters. In conclusion, this study suggests that in feed contaminated with AFB1, the use of S. cerevisiae is an alternative method to reduce the adverse effects of aflatoxicosis. Thus, apart from its excellent nutritional value, yeast can also be used as a mycotoxin adsorbent.

Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

PubMed

Wang, Meng; Li, Sijin; Zhao, Huimin

2016-01-01

The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. © 2015 Wiley Periodicals, Inc.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of Salidroside from Glucose.

PubMed

Jiang, Jingjie; Yin, Hua; Wang, Shuai; Zhuang, Yibin; Liu, Shaowei; Liu, Tao; Ma, Yanhe

2018-05-02

Salidroside is an important plant-derived aromatic compound with diverse biological properties. Because of inadequate natural resources, the supply of salidroside is currently limited. In this work, we engineered the production of salidroside in yeast. First, the aromatic aldehyde synthase (AAS) from Petroselinum crispum was overexpressed in Saccharomyces cerevisiae when combined with endogenous Ehrlich pathway to produce tyrosol from tyrosine. Glucosyltransferases from different resources were tested for ideal production of salidroside in the yeast. Metabolic flux was enhanced toward tyrosine biosynthesis by overexpressing pathway genes and eliminating feedback inhibition. The pathway genes were integrated into yeast chromosome, leading to a recombinant strain that produced 239.5 mg/L salidroside and 965.4 mg/L tyrosol. The production of salidroside and tyrosol reached up to 732.5 and 1394.6 mg/L, respectively, by fed-batch fermentation. Our work provides an alternative way for industrial large-scale production of salidroside and tyrosol from S. cerevisiae.

Saccharomyces cerevisiae expressing Gp43 protects mice against Paracoccidioides brasiliensis infection.

PubMed