Enzymatic catalysts, such as lipases, have advantages over chemical catalysts for transesterification of triglycerides to produce biodiesel. A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomycescerevisiae. Western b...
We demonstrated that formaldehyde can be efficiently coutilized by an engineered Saccharomycescerevisiae strain that expresses Hansenula polymorpha genes encoding formaldehyde dehydrogenase (FLD1) and formate dehydrogenase (FMD), in contrast to wild-type strains. Initial chemostat experiments showe...
Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomycescerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high gl...
The RAD6 gene of the yeast Saccharomycescerevisiae is required for DNA repair, for DNA damage-induced mutagenesis, and for sporulation, and it encodes a ubiquitin-conjugating enzyme. We have cloned the RAD6 homolog from Drosophila melanogaster and find that its encoded protein displays a very high ...
We demonstrate here that SAP155 encodes a negative modulator of K+ efflux in the yeast Saccharomycescerevisiae. Overexpression of SAP155 decreases efflux, whereas deletion increases efflux. In contrast, a homolog of SAP155, called SAP185, encodes a positive modulator of K+ efflux: overexpression of...
The ADR6 gene of Saccharomycescerevisiae has an open reading frame which could encode a polypeptide of 1314 amino acids. The predicted mRNA encodes a protein with homopolymeric stretches of asparagine and threonine, particularly near its amino terminus and contains additional sequences consisting o...
The RAD6 gene of the yeast Saccharomycescerevisiae is required for DNA repair, for DNA damage-induced mutagenesis, and for sporulation, and it encodes a ubiquitin-conjugating enzyme. We have cloned the RAD6 homolog from Drosophila melanogaster and find that its encoded protein displays a very high ...
A single structural gene, SUC2, encodes both secreted and cytoplasmic invertase in Saccharomycescerevisiae. It is known that the unprocessed polypeptides which differ by a secretion signal sequence are encoded by separate mRNAs. This unusual transcriptional organization raises the question as to th...
An essential gene, designated HKE1/RAT1, has been isolated from the yeast Saccharomycescerevisiae and characterized. The gene encodes a protein of 116 kDa (p116) and has significant homology to another yeast gene (XRN1/KEM1) encoding a related protein (p175) with 5'-->3' exonuclease activity as wel...
In the yeast Saccharomycescerevisiae, the synthesis of endogenous trehalose is catalyzed by a trehalose synthase complex, TPS, and its hydrolysis relies on a cytosolic/neutral trehalase encoded by NTH1. In this work, we showed that NTH2, a paralog of NTH1, encodes a functional trehalase that is imp...
Detectable splicing by the Saccharomycescerevisiae mitochondrial bI3 group I intron RNA in vitro is shown to require both an intron-encoded protein, the bI3 maturase, and the nuclear-encoded protein, Mrs1. Both proteins bind independently to the bI3 RNA. The bI3 maturase binds as a monomer, wh...
Saccharomyces boulardii is a yeast, which is a type of fungus. Saccharomyces boulardii was previously identified as a unique species of ... be a strain of Saccharomycescerevisiae (baker's yeast). Saccharomyces boulardii is used as medicine. Saccharomyces boulardii is used ...
Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomycescerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.
The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomycescerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.
Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomycescerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3—a GTPase prot...
The SUP45 and SUP35 genes of Saccharomycescerevisiaeencode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+]...
In yeast Saccharomycescerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI+]. It is known that combination of [PSI+] with some mutant alle...
The susceptibility of Saccharomycescerevisiae JG436 multidrug transporter deletion mutant, ?pdr5, to several antifungal agents was compared to that of JG436-derived JGCDR1 and JGCaMDR1 transformants, harboring the CDR1 and CaMDR1 genes, encoding the main drug-extruding membrane proteins of Candida ...
The Saccharomycescerevisiae Rlp7 protein has extensive identity and similarity to the large ribosomal subunit L7 proteins and shares an RNA-binding domain with them. Rlp7p is not a ribosomal protein; however, it is encoded by an essential gene and therefore must perform a function essential for...
The PKC1 gene in the yeast Saccharomycescerevisiaeencodes for protein kinase C which is known to control a MAP kinase cascade consisting of different kinases: Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of pkc1? mutants suggests additional targets ...
The NDT80 gene of Saccharomycescerevisiae, which encodes a global activator of transcription of middle sporulation-specific genes, is first expressed after the activation of early meiotic genes but prior to activation of middle sporulation-specific genes. Both upstream repression sequence 1 (URS1) ...
The CDC9 gene of Saccharomycescerevisiaeencodes a DNA ligase, and we have determined the nucleotide sequence of a 3.85 kb fragment of DNA which encompasses the convergently transcribed CDC9 and CDC36 genes. S1 nuclease mapping has revealed a major 5' end for the CDC9 mRNA, and one major and one mi...
Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomycescerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism....
The Saccharomycescerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unc...
The gene encoding M2, the ion channel-forming protein of influenza virus A, was expressed under the control of an inducible promoter in Saccharomycescerevisiae. By using single and multicopy plasmids containing GAL promoter-M2 fusions, a correlation was observed between plasmid copy number and grow...
We have isolated a gene from the yeast Saccharomycescerevisiae that encodes a 2.0-kilobase heat-inducible mRNA. This gene, which we have designated STI1, for stress inducible, was also induced by the amino acid analog canavanine and showed a slight increase in expression as cells moved into station...
We have devised a general screen for isolating conditional lethal mutants defective in synthesis of mature tRNA in Saccharomycescerevisiae. Using this screen, we have identified several new genes in yeast that are required for production of mature tRNA. These genes most likely encode essential func...
The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomycescerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 maj...
The genome of the yeast Saccharomycescerevisiae contains a family of genes related to the HSP70 genes (encoding the 70-kDa heat shock protein) of other eukaryotes. Mutations in two of these yeast genes (SSC1 and SSD1), whose expression is increased a few fold after temperature upshift, were constru...
Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomycescerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been i...
An IMP dehydrogenase gene was isolated from Candida albicans on a approximately 2.9-kb XbaI genomic DNA fragment. The putative Candida IMP dehydrogenase gene (IMH3) encodes a protein of 521 amino acids with extensive sequence similarity to the IMP dehydrogenases of Saccharomycescerevisiae and vario...
The Saccharomycescerevisiae CHS7 gene encodes an integral membrane protein located in the ER which is directly involved in chitin synthesis through the regulation of chitin synthase III (CSIII) activity. In the absence of CHS7 product, Chs3p, but not other secreted proteins, is retained in the ...
A regulatory element has been identified in the promoter region of the gene encoding the 11 kDa subunit VIII of the ubiquinol-cytochrome c oxidoreductase in Saccharomycescerevisiae. The element, which is approximately 40 bp long and situated 185 bp upstream of the initiator ATG, is essential for in...
We have used DNA polymerase alpha affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomycescerevisiae. Two proteins that bound to the catalytic subunit of DNA polymerase alpha (Pol1 protein) are encoded by the essential genes CDC68/SPT16 and POB3....
The CDC68 gene (also called SPT16) encodes a transcription factor for the expression of a diverse set of genes in the budding yeast Saccharomycescerevisiae. To identify other proteins that are functionally related to the Cdc68 protein, we searched for genetic suppressors of a cdc68 mutation. Four s...
To identify genes encoding adhesins that mediate the binding of Candida albicans to endothelial cells, a genomic library from this organism was constructed and used to transform Saccharomycescerevisiae. These transformed organisms were screened for adherence to endothelial cells, and a highly adher...
Saccharomycescerevisiae strains bearing temperature-sensitive alleles of the cell division cycle gene CDC31 are specifically defective in duplication of the spindle pole body, the microtubule-organizing center of yeast. To define the function encoded by CDC31 more fully, we have isolated genomic cl...
The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomycescerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repa...
A gene encoding a fusion protein consisting of Escherichia coli iron superoxide dismutase (FeSOD) with the mitochondrial targeting presequence of yeast manganese superoxide dismutase (MnSOD) was cloned and expressed in E. coli and in Saccharomycescerevisiae DL1Mn- yeast cells deficient in MnSOD. In...
Abstract The Saccharomycescerevisiae gene PIF1 encodes a conserved eukaryotic DNA helicase required for both mitochondrial and nuclear DNA integrity. Our previous work revealed that a pif1D strain is tolerant to zinc overload. We demonstrate here that this effect is independent of the Pif1 ...
We identified a putative Saccharomycescerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segre...
We report the molecular cloning, sequencing and genetic characterization of the first gene encoding an organellar polypeptide chain release factor, the MRF1 gene of the yeast Saccharomycescerevisiae. The MRF1 gene was cloned by genetic complementation of a respiratory deficient mutant disturbed in ...
IME1 encodes a transcriptional activator required for the transcription of meiosis-specific genes and initiation of meiosis in Saccharomycescerevisiae. The transcription of IME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetat...
The SRS2 gene encodes a helicase that affects recombination, gene conversion and DNA damage repair in the yeast Saccharomycescerevisiae. Loss-of-function mutations in srs2 suppress the extreme sensitivity towards UV radiation of rad6 and rad18 mutants, both of which are impaired in post-replicat...
The APA1 gene in Saccharomycescerevisiaeencodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter. Enhanced expression of APA1 via the plasmids resulted ...
The SPT4, SPT5, and SPT6 gene products define a class of transcriptional repressors in Saccharomycescerevisiae that are thought to function through their effects on chromatin assembly or stability. Mutations in these genes confer a similar range of phenotypes to mutations in HIR genes, which encode...
The RAD6 gene of Saccharomycescerevisiaeencodes a ubiquitin-conjugating enzyme (E2) that is required for DNA repair, damage-induced mutagenesis, and sporulation. We have cloned the two human RAD6 homologs, designated HHR6A and HHR6B. The two 152-amino acid human proteins share 95% sequence identit...
The RAD6 gene of Saccharomycescerevisiaeencodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis. In addition, Rad6 functions in the N end rule pathway of protein degradation. Rad6 mediates its DNA repair role via its associa...
In Saccharomycescerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites. Oxidative DNA-damaging agents, such as H2O2, produce DNA strand breaks which contain 3?-phosphate or 3?-phosphoglycolate termini. Such 3? termini are inhib...
Antiserum was prepared against a synthetic peptide corresponding to the N-terminal 20 amino acids of the protein encoded by cauliflower mosaic virus (CaMV) open reading frame VII (ORF VII). This antiserum was used to detect the expression of CaMV ORF VII either in Saccharomycescerevisiae transforme...
Several secretion-defective variants of invertase from Saccharomycescerevisiae were generated by replacement of the wild-type signal sequence codons with DNA fragments with random sequences. Strains encoding these proteins failed to grow on medium containing sucrose as the sole source of carbon. Th...
Trehalose-6-P inhibits hexokinases in Saccharomycescerevisiae (M. A. Blázquez, R. Lagunas, C. Gancedo, and J. M. Gancedo, FEBS Lett. 329:51-54, 1993), and disruption of the TPS1 gene (formerly named CIF1 or FDP1) encoding trehalose-6-P synthase prevents growth in glucose. We have found that the hex...
The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomycescerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondria...
The Saccharomycescerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consens...
The MID2 gene of Saccharomycescerevisiaeencodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor. MID2 was isolated as a multicopy activator of the Skn7p transcription factor. Deletion of MID2 causes resistance to calcofluor white, diminished productio...
GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomycescerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader. uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions...
Zinc ion homeostasis in Saccharomycescerevisiae is controlled primarily through the transcriptional regulation of zinc uptake systems in response to intracellular zinc levels. A high-affinity uptake system is encoded by the ZRT1 gene, and its expression is induced more than 30-fold in zinc-limited ...
The GCR1 gene of Saccharomycescerevisiaeencodes a transcriptional activator that complexes with Rap1p and, through UAS(RPG) elements (Rap1p DNA binding sites), stimulates efficient expression of glycolytic and translational component genes. To map the functionally important domains in Gcr1p, we co...
Expression of the BAP3 gene of Saccharomycescerevisiae, encoding a branched chain amino acid permease, is induced in response to the availability of several naturally occurring amino acids in the medium. This induction is mediated via an upstream activating sequence (called UASaa) in the BAP3 promo...
We have cloned the Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomycescerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3?half of its coding region due to the overlap of ...
The TPS1 gene from Candida albicans, which encodes trehalose-6-phosphate synthase, has been cloned by functional complementation of a tps1 mutant from Saccharomycescerevisiae. In contrast with the wild-type strain, the double tps1/tps1 disruptant did not accumulate trehalose at stationary phase or ...
Deletion of trehalose-6-phosphate phosphatase, encoded by TPS2, in Saccharomycescerevisiae results in accumulation of trehalose-6-phosphate (Tre6P) instead of trehalose under stress conditions. Since trehalose is an important stress protectant and Tre6P accumulation is toxic, we have investigated w...
Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomycescerevisiae expressing 1a and 2a is capable of repl...
All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomycescerevisiae. BMV encodes RNA replication factors 1a, with doma...
In Saccharomycescerevisiae, the COX5a and COX5b genes encode two forms of cytochrome c oxidase subunit V, Va and Vb. We report here that heme increases COX5a expression and decreases COX5b expression and that the HAP2 and REO1 genes are involved in positive regulation of COX5a and negative regulati...
The core of the cytochrome c oxidase complex is composed of its three largest subunits, Cox1p, Cox2p, and Cox3p, which are encoded in mitochondrial DNA of Saccharomycescerevisiae and inserted into the inner membrane from the inside. Mitochondrial translation of the COX1, COX2, and COX3 mRNAs is act...
In Saccharomycescerevisiae, COX5a and COX5b encode two distinct forms of cytochrome c oxidase subunit V, Va and Vb, respectively. To determine the relative contribution of COX5a and COX5b to cytochrome c oxidase function, we have disrupted each gene. Cytochrome c oxidase activity levels and respira...
In Saccharomycescerevisiae UV radiation and a variety of chemical DNA-damaging agents induce the transcription of specific genes, including several involved in DNA repair. One of the best characterized of these genes is PHR1, which encodes the apoenzyme for DNA photolyase. Basal-level and damage-in...
The Saccharomycescerevisiae SWI4 gene encodes an essential transcription factor which controls gene expression at the G1/S transition of the cell cycle. SWI4 transcription itself is cell cycle regulated, and this periodicity is crucial for the normal cell cycle regulation of HO and at least two of ...
The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomycescerevisiae. Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation. We have recently shown that Pkc1p, a yeast homolog of mammalia...
In previous studies, we have shown that the PCF1-1 mutation of Saccharomycescerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second larg...
Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomycescerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase alpha subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol alp...
Cells of Saccharomycescerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential i...
The Ssa subfamily of Hsp70 molecular chaperones in the budding yeast Saccharomycescerevisiae has four members, encoded by SSA1, SSA2, SSA3, and SSA4. Deletion of the two constitutively expressed genes, SSA1 and SSA2, results in cells which are slow growing and temperature sensitive. In this study, ...
Why eukaryotes encode multiple Hsp70 isoforms is unclear. Saccharomycescerevisiae Ssa1p and Ssa2p are constitutive 98% identical Hsp70's. Stress-inducible Ssa3p and Ssa4p are 80% identical to Ssa1/2p. We show Ssa1p-4p have distinct functions affecting [PSI+] and [URE3] prions. When expressed as the...
We reported previously that heat or ethanol shock in Saccharomycescerevisiae leads to nuclear retention of most poly(A)+ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for...
Transcription factor UAF (upstream activation factor) is required for a high level of transcription, but not for basal transcription, of rDNA by RNA polymerase I (Pol I) in the yeast Saccharomycescerevisiae. RRN9 encodes one of the UAF subunits. We have found that rrn9 deletion mutants grow extreme...
The multifunctional DNA-binding proteins ABF1 and CPF1 bind in a mutually exclusive manner to the promoter region of the QCR8 gene, which encodes 11-kDa subunit VIII of the Saccharomycescerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR). We investigated the roles that the two fact...
This project deals with the characterization of DNA repair genes and their encoded proteins involved in the incision step of excision repair and in postreplication repair and mutagenesis following exposure to UV light in eukaryotes: the yeast Saccharomycescerevisiae and humans. Summarized in this report is progress in achieving the goals of this project.
The IPL1 gene is required for high-fidelity chromosome segregation in the budding yeast Saccharomycescerevisiae. Conditional ipl1ts mutants missegregate chromosomes severely at 37 degrees C. Here, we report that IPL1 encodes an essential putative protein kinase whose function is required during the...
The nucleotide sequence of the URA3 gene encoding orotidine-5?-phosphate decarboxylase (OMPDCase) in sourdough yeast Candida milleri CBS 8195 was determined by degenerate PCR and genome walking. Sequence analysis revealed the presence of an openreading frame of 810 bp, encoding 269 amino acid residue protein with the highest identity to the OMPDCase of the yeast Saccharomycescerevisiae. Phylogenetic analysis of deduced amino acid sequence revealed that it shares a high degree of identity with other yeast OMPDCases. The cloned URA3 gene successfully complemented the ura3 mutation in S. cerevisiae, indicating that it encodes a functional OMPDCase in C. millieri CBS 8195.
The RAD2 gene is one of at least seven indispensable for excision repair in the yeast Saccharomycescerevisiae, and its encoded protein shares remarkable homology with the xeroderma pigment to sum (XP) group-G gene product. The authors overproduced the RAD2- encoded protein in S. cerevisiae, purified it to near homogeneity, and showed that RAD2 protein in the presence of magnesium degrades circular single-stranded DNA. Given the absolute requirement for RAD2 in the incision step of excision repair, their findings implicate RAD2 protein and its human homologue XPG protein as a catalytic component that incises the damaged DNA strand during excision repair. (Author).
Robust industrial yeast strains are needed for profitable production of fuel ethanol from mixed biomass waste. USDA, ARS, NCAUR, RPT has been evaluating ethanol-producing yeasts, including Saccharomycescerevisiae, engineered GMAX Saccharomycescerevisiae, irradiated Kluyveromyces marxianus, and Pi...
Sequência nucleotídica, e respectiva tradução, do ortólogo de Pichia farinosa do gene GUP1 de Saccharomycescerevisiae. , Nucleotidic sequence, and its translation, of the Pichia farinosa ortholgue of GUP1 gene from Saccharomycescerevisiae.
Sequência nucleotídica, e respectiva tradução, do ortólogo de Kluyveromyces marxianus do gene FPS1 de Saccharomycescerevisiae. , Nucleotidic sequence, and its translation, of the Kluyveromyces marxianus ortholgue of FPS1 gene from Saccharomycescerevisiae.
Sequência nucleotídica, e respectiva tradução, do ortólogo de Kluyveromyces thermotolerans dos genes GPD de Saccharomycescerevisiae. , Nucleotidic sequence, and its translation, of the Kluyveromyces thermotolerans ortholgue of GPD genes from Saccharomycescerevisiae.
Sequência nucleotídica, e respectiva tradução, do ortólogo de Pichia farinosa do gene GUT1 de Saccharomycescerevisiae. , Nucleotidic sequence, and its translation, of the Pichia farinosa ortholgue of GUT1 gene from Saccharomycescerevisiae.
Sequência nucleotídica, e respectiva tradução, do ortólogo de Kluyveromyces lactis do gene FPS1 de Saccharomycescerevisiae. , Nucleotidic sequence, and its translation, of the Kluyveromyces lactis ortholgue of FPS1 gene from Saccharomycescerevisiae.
Sequência nucleotídica, e respectiva tradução, do ortólogo de Zygosaccharomyces rouxii do gene FPS1 de Saccharomycescerevisiae. , Nucleotidic sequence, and its translation, of the Zygosaccharomyces rouxii ortholgue of FPS1 gene from Saccharomycescerevisiae.
Wine strains of Saccharomycescerevisiae 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...
We show that difficulties in regulating cellular behavior with synthetic biological circuits may be circumvented using in silico feedback control. By tracking a circuit's output in Saccharomycescerevisiae in real time, we precisely control its behavior using an in silico feedback algorithm to compute regulatory inputs implemented through a genetically encoded light-responsive module. Moving control functions outside the cell should enable more sophisticated manipulation of cellular processes whenever real-time measurements of cellular variables are possible.
We show that difficulties in regulating cellular behavior with synthetic biological circuits may be circumvented using in silico feedback control. By tracking a circuit's output in Saccharomycescerevisiae in real time, we precisely control its behavior using an in silico feedback algorithm to compute regulatory inputs implemented through a genetically encoded light-responsive module. Moving control functions outside the cell should enable more sophisticated manipulation of cellular processes whenever real-time measurements of cellular variables are possible. PMID:22057053
Both terminators and promoters regulate gene expression. In Saccharomycescerevisiae, 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.
The budding yeast Saccharomycescerevisiae ?1278b contains the MPR1 gene encoding N-acetyltransferase, which detoxifies the L-proline analog L-azetidine-2-carboxylate (AZC). Of 131 yeasts tested, AZC acetyltransferase activity was detected in 17 strains of 41 strains that showed AZC resistance. Degenerate-PCR analysis revealed that two strains, i.e., Candida saitoana AKU4533 and Wickerhamia fluorescens AKU4722, contained a DNA fragment highly homologous to MPR1. This indicates that AZC acetyltransferases are widely distributed in yeasts.
Disruption of an SCS2 ortholog impaired the growth of the alkane-assimilating yeast Yarrowia lipolytica on n-alkanes, particularly on n-decane, although the mRNA level of the ALK1 gene encoding a highly inducible cytochrome P450ALK was not much affected. The same disruption did not cause inositol auxotrophy, implying that Y. lipolytica SCS2 has a different function from its Saccharomycescerevisiae counterpart. PMID:18685198
Disruption of an SCS2 ortholog impaired the growth of the alkane-assimilating yeast Yarrowia lipolytica on n-alkanes, particularly on n-decane, although the mRNA level of the ALK1 gene encoding a highly inducible cytochrome P450ALK was not much affected. The same disruption did not cause inositol auxotropy, implying that Y. lipolytica SCS2 has a different function from its Saccharomycescerevisiae counterpart.
Disruption of an SCS2 ortholog impaired the growth of the alkane-assimilating yeast Yarrowia lipolytica on n-alkanes, particularly on n-decane, although the mRNA level of the ALK1 gene encoding a highly inducible cytochrome P450ALK was not much affected. The same disruption did not cause inositol auxotrophy, implying that Y. lipolytica SCS2 has a different function from its Saccharomycescerevisiae counterpart.
The RHO1 gene encodes a homologue of mammalian RhoA small G-protein in the yeast Saccharomycescerevisiae. Rho1p is required for bud formation and is localized at a bud tip or a cytokinesis site. We have recently shown that Bni1p is a potential target of Rho1p. Bni1p shares the FH1 and FH2 domains w...
A DNA ligase-encoding gene (Ca CDC9) was cloned from Candida albicans by complementation of an ime-1 mutation in Saccharomycescerevisiae. In this system, IME1 function was assayed using a S. cerevisiae strain with a ime2-promoter-lacZ gene fusion such that following transformation with a C. albicans genomic library, the presence of positive clones was indicated upon the addition of X-gal to sporulation media. Transforming fragments were subcloned in pGEM7 and sequenced. Sequence homology with several ATP-dependent DNA ligases from viruses, fission yeast, human, baker yeast and bacteria was observed. PMID:8840507
Fungal species from extreme environments represent an underexploited source of stress-resistance genes. These genes have the potential to improve stress tolerance of economically important microorganisms and crops. An efficient high-throughput method for the identification of biotechnologically interesting genes of extremotolerant fungi was developed by constructing a cDNA expression library in Saccharomycescerevisiae and screening for gain-of-function transformants under stress conditions. The advantages and possible modifications of this method are discussed, and its efficiency is demonstrated using the stress-tolerant basidiomycetous yeast Rhodotorula mucilaginosa. Twelve R. mucilaginosa genes are described that increase halotolerance in S. cerevisiae. These include genes encoding a ph...
The recombinant xylose-utilizing Saccharomycescerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h(-1) for S. cerevisiae TMB 3399 to 0.14 h(-1) for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h(-1). All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose(-1) and 0.001, 0.10, and 0.16 g ethanol g biomass(-1) h(-1) for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein(-1), was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant. PMID:12689639
Fumarate reductase is a protein involved in the maintenance of redox balance during oxygen deficiency. This enzyme irreversibly catalyzes the reduction of fumarate to succinate and requires flavin cofactors as electron donors. Two examples are the soluble mitochondrial and the cytosolic fumarate reductases of Saccharomycescerevisiaeencoded by the OSM1 and FRDS1 genes, respectively. This work reports the identification and characterization of the gene encoding cytosolic fumarate reductase enzyme in the arbuscular mycorrhizal fungus, Glomus intraradices and the establishment of its physiological role. Using a yeast expression system, we demonstrate that G. intraradices GiFRD encodes a protein that has fumarate reductase activity which can functionally substitute for the S. cerevisiae fumar...
Inactivation of the key genes, which are responsible for the enzymes of polyphosphates degradation, exopolyphosphatases ppx1 and ppn1, caused both an increase of polyphosphates content in Saccharomycescerevisiae cells and an increase in chain length of acid-soluble and alkali-soluble fractions. It had no effect on the frequency of volutine granules metachromasy that was based on the interaction of dye molecules with ionic groups of polyphosphates. At the same time, a mutant strain reaction to nystatin differed from the reaction of the parental and wild-type strains when the metachromasy was absent. Obtained data may indicate a pleiotropic effect of ppx1 and ppn1 genes, which encode the major exopolyphosphatase of Saccharomycescerevisiae and affect the reaction of cells to external factors through changes in the metabolism of polyphosphates. PMID:23120983
It has been proposed that a bottom-fermenting yeast strain of Saccharomyces pastorianus is a natural hybrid between S. cerevisiae and S. bayanus and possesses at least two types of genome. In the process of conducting expressed sequence tag (EST) analysis, we isolated bottom-fermenting yeast-specific (BFY) genes that have no significant homology with sequences in the S288C database. One of the BFY genes, AMI1, encodes a protein with homology to an amidase conserved among plants, Bacillus subtilis, Neurospora crassa, Schizosaccharomyces pombe and Saccharomyces species, with the exception of S. cerevisiae S288C. In the bottom-fermenting yeast, three alleles of AMI1 (one AMI1-A and two AMI1-B alleles) were found on different chromosomes. AMI1-A on chromosome XIII is most homologous to the S. ...
The egI gene, encoding a major endoglucanase (EGI) of Scopulariopsis brevicaulis TOF-1212, was cloned and sequenced. The egI gene consisted of 868 bp with one intron and encoded a protein of 229 amino acids with a calculated molecular mass of 22,392 daltons. The EGI was assigned to a family 45 of glycosyl hydrolases and showed high similarity with other fungal endoglucanases, especially with those of Humicola grisea and Fusarium oxysporum, on the basis of hydrophobic cluster analysis. The egI gene was expressed under the promoter of the phosphoglycerate kinase gene (PGK) in Saccharomycescerevisiae. The transformed cells were able to secrete the enzyme efficiently in an active form.
Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomycescerevisiae strain, MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production fr...
A novel Kluyveromyces marxianus gene that encodes an acid phosphatase, Pho610, was cloned in Saccharomycescerevisiae. The deduced amino acid sequence was distinct from S. cerevisiae phosphatases but similar to some fungal enzymes. A peculiar feature of the sequence is that it has hydrophobic stretches both at the N- and C-termini, which is a characteristic of the precursors of glycosylphosphatidylinositol(GPI)-anchored proteins. When the gene was expressed in S. cerevisiae, the active enzyme was recovered in the periplasmic fraction by glucanase digestion. The Pho610 polypeptide was highly glycosylated and a significant portion was covalently linked to the cell-wall glucan. The enzyme was secreted when the C-terminal region was truncated to remove the GPI signal. Therefore, Pho610 is a novel cell-wall protein having an enzyme activity.
Differences between the recombinant xylose-utilizing Saccharomycescerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h?1, with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter?1, 10 g of glucose plus 10 g of xylose liter?1 or, for S. cerevisiae TMB 3400, 20 g of xylose liter?1. S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate?1 on glucose and 0.43 g of biomass g of consumed carbohydrate?1 on xylose. A Ks value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S. cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose. PMID:15269428
Differences between the recombinant xylose-utilizing Saccharomycescerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h(-1), with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter(-1), 10 g of glucose plus 10 g of xylose liter(-1) or, for S. cerevisiae TMB 3400, 20 g of xylose liter(-1). S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate(-1) on glucose and 0.43 g of biomass g of consumed carbohydrate(-1) on xylose. A K(s) value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S. cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose. PMID:12570990
Trehalose-6-P inhibits hexokinases in Saccharomycescerevisiae (M. A. Blázquez, R. Lagunas, C. Gancedo, and J. M. Gancedo, FEBS Lett. 329:51-54, 1993), and disruption of the TPS1 gene (formerly named CIF1 or FDP1) encoding trehalose-6-P synthase prevents growth in glucose. We have found that the hexokinase from Schizosaccharomyces pombe is not inhibited by trehalose-6-P even at a concentration of 3 mM. The highest internal concentration of trehalose-6-P that we measured in S. pombe was 0.75 mM after heat shock. We have isolated from S. pombe the tps1+ gene, which is homologous to the Saccharomycescerevisiae TPS1 gene. The DNA sequence from tps1+ predicts a protein of 479 amino acids with 65% identity with the protein of S. cerevisiae. The tps1+ gene expressed from its own promoter could complement the lack of trehalose-6-P synthase in S. cerevisiae tps1 mutants. The TPS1 gene from S. cerevisiae could also restore trehalose synthesis in S. pombe tps1 mutants. A chromosomal disruption of the tps1+ gene in S. pombe did not have a noticeable effect on growth in glucose, in contrast with the disruption of TPS1 in S. cerevisiae. However, the disruption prevented germination of spores carrying it. The level of an RNA hybridizing with an internal probe of the tps1+ gene reached a maximum after 20 min of heat shock treatment. The results presented support the idea that trehalose-6-P plays a role in the control of glycolysis in S. cerevisiae but not in S. pombe and show that the trehalose pathway has different roles in the two yeast species. PMID:8021171
The construction of Saccharomycescerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (?-galactosidase) genes of Kluyveromyces lacti...
The construction of Saccharomycescerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (?-galactosidase) genes of Kluyveromyces lacti...
Saccharomycescerevisiae is able to ferment xylose,when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However,xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed ...
Little information on the effects of cyclohexane at the cellular or subcellular level is available. In Saccharomycescerevisiae, cyclohexane inhibited respiration and diverse energy-dependent processes. In mitochondria isolated from S. cerevisiae, oxygen uptake and ATP synthesis were inhibited, alth...
Abstract A Cryptococcus flavus gene (AMY1) encoding an extracellular a-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) 144DVVVNH149, (II) 235GLRIDSLQQ243, (III) 263GEVFN267, (IV) 327FLENQD332, placed the enzyme in the GH13 a-amylase family. Furthermore, sequence comparison suggests that the C. flavusa-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomycescerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL-1) at ...
Pyruvate-decarboxylase (Pdc)-negative Saccharomycescerevisiae has been proven as a suitable metabolic engineering platform to produce organic acids. S. cerevisiae BY5419 Pdc^- strain cannot grow in batch cultures on synthetic medium with glucose as the sole carbon source, yet grows well on synthetic medium with ethanol or acetate. In this study, by combining adaptive evolution and cofactor engineering, we obtained a series of engineered yeasts that can produce pyruvate using glucose as sole carbon source. Differential expression of noxE, encoding a water-forming NADH oxidase from Lactococcus lactis, and udhA, encoding a soluble pyridine nucleotide transhydrogenase from Escherichia coli, was investigated. Of all the constructed recombinant strains, G2U1-A0 was able to produce 75.1gl^-^1 py...
The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomycescerevisiae Kyokai No. 7 (sake yeast). The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons. The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I. The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar. A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid. A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S. cerevisiae and brewery lager yeast have one ATF2 gene, while S. bayanus has no similar gene.
Comparative transcriptomics of Saccharomycescerevisiae and Candida glabrata revealed a remarkable conservation of response to drug-induced stress, despite underlying differences in the regulatory networks.
Abstract in portuguese O presente trabalho visou a aplicação da ?-1,3 glucanase lítica, obtida do microrganismo Cellulosimicrobium cellulans 191, na produção de protoplastos e na lise da parede celular de leveduras. A preparação bruta da enzima foi capaz de lisar as leveduras Kluyveromyces lodderi, Saccharomycescerevisiae (Fleischmann e Itaiquara), S. cerevisiae KL-88, S. diastaticus NCYC 713, S. cerevisiae NCYC 1001, Candida glabrata NCYC 388, Kluyveromyces marxianus NCYC 587 e Ha (more) nsenula mrakii NCYC 500. A ?-1,3 glucanase purificada foi capaz de lisar as leveduras Saccharomycescerevisiae KL-88, Saccharomyces capensis, Debaromyces vanriji, Pachysolen tannophillus, Kluyveromyces drosophilarum, Candida glabrata, Hansenula mrakii e Pichia membranaefaciens e formar protoplastos de Saccharomycescerevisiae KL-88. Abstract in english The aim of this work was the application of lytic ?-1,3 glucanase obtained from Cellulosimicrobium cellulans strain 191 in the production of protoplasts and lysis of yeast cell walls. The crude extract demonstrated lysis activity against the yeasts Kluyveromyces lodderi, Saccharomycescerevisiae (Fleischmann and Itaiquara), S. cerevisiae KL-88, S. diastaticus NCYC 713, S. cerevisiae NCYC 1001, Candida glabrata NCYC 388, Kluyveromyces marxianus NCYC 587, and Hansenula (more) mrakii NCYC 500. The purified ?-1,3 glucanase demonstrated lysis activity against the yeasts Saccharomycescerevisiae KL-88, Saccharomyces capensis, Debaromyces vanriji, Pachysolen tannophillus, Kluyveromyces drosophilarum, Candida glabrata, Hansenula mrakii, and Pichia membranaefaciens, and it was able to produce Saccharomycescerevisiae KL-88 protoplasts.
Abstract in portuguese A linhagem de Saccharomycescerevisiae Y500-4L com alta capacidade fermentativa e atividade "killer", previamente selecionada de mosto de fermentação de usina de álcool, foi caracterizada quanto ao espectro de atividade e quanto à perda do caracter "killer". A linhagem "killer" de S. cerevisiae Y500-4L, mostrou alta atividade "killer" contra as leveduras comerciais Fleischmann e Itaiquara, e também contra as linhagens "killer" padrões K2 (S. diastaticus NCYC 713), K (more) 4 (Candida glabrata NCYC 388) e K11 (Torulopsis glabrata ATCC 15126) e mostrou ser sensível às toxinas produzidas pelas leveduras padrões "killer" K8 (Hansenula anomala NCYC 435), K9 (Hansenula mrakii NCYC 500), K10 (Kluyveromyces drosophilarum NCYC 575) e K11 (Torulopsis glabrata ATCC 15126). A linhagem de S. cerevisiae Y500-4L não apresentou plasmídio M-dsRNA e, provavelmente, o caracter genético responsável pelo fenótipo "killer" é codificado por genes cromossomais. Em ensaios para a perda do fenótipo, a linhagem S. cerevisiae Y500-4L apresentou maior resistência ao tratamento com cicloheximida e a temperatura elevada (40oC) do que a levedura S. cerevisiae padrão "killer" K1. Abstract in english The strain Saccharomycescerevisiae Y500-4L, previously selected from the must of alcohol producing plants and showing high fermentative and killer capacities, was characterized according to the interactions between the yeasts and examined for curing and detection of dsRNA plasmids, which code for the killer character. The killer yeast S. cerevisiae Y500-4L showed considerable killer activity against the Fleischmann and Itaiquara commercial brands of yeast and also agains (more) t the standard killer yeasts K2 (S. diastaticus NCYC 713), K4 (Candida glabrata NCYC 388) and K11 (Torulopsis glabrata ATCC 15126). However S. cerevisiae Y500-4L showed sensitivity to the killer toxin produced by the standard killer yeasts K8 (Hansenula anomala NCYC 435), K9 (Hansenula mrakii NCYC 500), K10 (Kluyveromyces drosophilarum NCYC 575) and K11 (Torulopsis glabrata ATCC 15126). No M-dsRNA plasmid was detected in the S. cerevisiae Y500-4L strain and these results suggest that the genetic basis for toxin production is encoded by chromosomal DNA. The strain S. cerevisiae Y500-4L was more resistant to the loss of the phenotype killer with cycloheximide and incubation at elevated temperatures (40oC) than the standard killer yeast S. cerevisiae K1.
It is widely accepted that phosphatidylethanolamine (PE) is enriched in the cytosolic leaflet of the eukaryotic plasma membranes. To identify genes involved in the establishment and regulation of the asymmetric distribution of PE on the plasma membrane, we screened the deletion strain collection of the yeast Saccharomycescerevisiae for hypersensitive mutants to the lantibiotic peptide Ro09-0198 (Ro) that specifically binds to PE on the cell surface and inhibits cellular growth. Deletion mutants of VPS51, VPS52, VPS53, and VPS54 encoding the components of Golgi-associated retrograde protein (GARP) complex, YPT6 encoding a Rab family small GTPase that functions with GARP complex, RIC1 and RGP1 encoding its guanine nucleotide exchange factor (GEF), and TLG2 encoding t-SNARE exhibited hyperse...
The DNA sequences of the argG genes of Methanosarcina barkeri MS and Methanococcus vannielii were determined. The polypeptide products of these methanogen genes have amino acid sequences which are 50% identical to each other and 38% identical to the amino acid sequence encoded by the exons of the human argininosuccinate synthetase gene. Introns in the human chromosomal gene separate regions which encode amino acids conserved in both the archaebacterial and human gene products. An open reading frame immediately upstream of argG in Methanosarcina barkeri MS codes for an amino acid sequence which is 45 and 31% identical to the sequences of the large subunits of carbamyl phosphate synthetase in Escherichia coli and Saccharomycescerevisiae, respectively. If this gene encodes carbamyl phosphate synthetase in Methanosarcina barkeri, this is the first example, in an archaebacterium, of physical linkage of genes that encode enzymes which catalyze reactions in the same amino acid biosynthetic pathway.
Expression vectors were created in which the 5' end of the Saccharomycescerevisiae CDC9 gene, which encodes a mitochondrial targeting peptide, was cloned in-frame with the coding regions of the EcoR I, Hind III, and Pst I endonuclease genes. Expression of the EcoR I and Hind III fusion proteins inhibited growth of yeast on glycerol-containing media and resulted in the nearly quantitative restriction digestion of their mitochondrial DNA. In contrast, expression of Pst I, which does not recognize any sites within yeast mitochondrial DNA, had no effect on growth in glycerol-containing media, and did not affect the integrity of the mitochondrial genome. PMID:16120308
A virus that has properties consistent with inclusion in the virus family Narnaviridae was described in Phytophthora infestans, the oomycete that caused the Irish potato famine. The genome of phytophthora infestans RNA virus 4 (PiRV-4) is 2,984 nt with short complementary terminal sequences and a single open reading frame predicted to encode an RNA-dependent RNA polymerase (RdRp) most closely related to saccharomycescerevisiae narnavirus 20S (ScNV-20S) and ScNV-23S, the members of the genus Narnavirus, family Narnaviridae. This report constitutes the first description of a member of the family Narnaviridae from a host taxon outside of the kingdom Fungi.
The CCAAT-binding complex in Aspergillus species, known as the Hap complex, consists of at least three subunits, HapB, HapC, and HapE. Each Hap subunit contains an evolutionarily conserved core domain. In this study, a series of the truncated gene, which encodes the HapE subunit of Aspergillus oryzae, was constructed to survey the regions essential for the transcriptional enhancement of fungal genes. It was revealed that the non-conserved regions and the conserved region similar to the Hap4p recruiting domain of Saccharomycescerevisiae were not necessary for Hap complex-mediated transcriptional enhancement.
An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomycescerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.
The yeast Yarrowia lipolytica is one of the most intensively studied ?non-conventional? yeast species. Its ability to secrete various organic acids, like pyruvic (PA), citric, isocitric, and alpha-ketoglutaric (KGA) acid, in large amounts is of interest for biotechnological applications. We have studied the effect of the alpha-ketoglutarate dehydrogenase (KGDH) complex on the production process of KGA. Being well studied in Saccharomycescerevisiae this enzyme complex consists of three subunits: alpha-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and lipoamide dehydrogenase. Here we report the effect of overexpression of these subunits encoding genes and resulting increase of specific KGDH activity on organic acid production under several conditions of growth limitation and ...
The microbial additive MycoCell is a preparation of viable cells of Saccharomycescerevisiae. It is intended to be applied daily in dairy cows as a top dressing, or incorporated into a total mixed ration to provide 1*1010 CFU/head/day. The active agent, Saccharomycescerevisiae, is considered by EFS...
Saccharomycescerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions. Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 ?g/ml of L-tryptophan, a concentration that met the requirements of tryptophan-auxotrophic CHO1/PSS strains. The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations. This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2. These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S. cerevisiae at low tryptophan concentrations.
There is no high-resolution structure for the membrane domain of the human erythrocyte anion exchanger, AE1 (Band 3). In this report, we have developed an expression and purification strategy for AE1 to be used in crystallization trials. Saccharomycescerevisiae strain BJ5457 was transformed with an expression vector encoding the AE1 membrane domain (AE1MD, amino acids 388-911), fused C-terminally to an epitope tag, corresponding to the nine C-terminal amino acids of rhodopsin. The fusion protein, AE1MD-Rho, was expressed at a concentration of 0.3mg/l of culture. Confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation revealed that AE1MD-Rho did not process to the plasma membrane of S. cerevisiae, but was retained in an intracellular membrane fraction. Treatment wit...
The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomycescerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane ...
In the budding yeast Saccharomycescerevisiae, heterochromatic gene silencing has been found within HMR and HML silent mating type loci, the telomeres, and the rRNA-encoding DNA. There may be boundary elements that regulate the spread of silencing to protect genes adjacent to silenced domains from this epigenetic repressive effect. Many assays show that specific DNA regulatory elements separate a euchromatic locus from a neighboring heterochromatic domain and thereby function as a boundary. Alternatively, DNA-independent mechanisms such as competition between acetylated and deacetylated histones are also reported to contribute to gene insulation. However, the mechanism by which boundaries are formed is not clear. Here, the characteristics and functions of boundaries at silenced domains in S. cerevisiae are discussed.
Ethanol-sensitive mutants (es1 to es10) were isolated from sake yeast, Saccharomycescerevisiae SY-32. These mutants were unable to grow at 7% ethanol at which the wild type strain SY-32 does grow. The mutants had a variety of fermentation rates and viabilities in the presence of ethanol. The gene ERG6, com- plementing the ethanol-sensitive mutation of es5, was cloned from an SY-32 gene library. ERG6 encodes S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) in the ergosterol synthetic pathway. Mutant es5 had a reduced ability to synthesize ergosterol. An erg6 disruptant was also ethanol-sensitive. These results suggested that ERG6 plays an important role in the ethanol tolerance of S. cerevisiae.
Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote d-xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomycescerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing d-xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates d-xylose, its ethanol productivity is quite low during fermentation with d-xylose alone. In order to ascertain the key enzyme in ethanol production from d-xylose, we checked ...
The yeast Saccharomycescerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and ...
Fungal species from extreme environments represent an underexploited source of stress-resistance genes. These genes have the potential to improve stress tolerance of economically important microorganisms and crops. An efficient high-throughput method for the identification of biotechnologically interesting genes of extremotolerant fungi was developed by constructing a cDNA expression library in Saccharomycescerevisiae and screening for gain-of-function transformants under stress conditions. The advantages and possible modifications of this method are discussed, and its efficiency is demonstrated using the stress-tolerant basidiomycetous yeast Rhodotorula mucilaginosa. Twelve R. mucilaginosa genes are described that increase halotolerance in S. cerevisiae. These include genes encoding a phosphoglucomutase and a phosphomannomutase. All 12 investigated genes might be useful for the improvement of halotolerance in genetically modified crops or industrial microorganisms. PMID:22386631
In the yeast Saccharomycescerevisiae, the unfolded protein response (UPR) involves the unconventional splicing of HAC1 mRNA, which is mediated by the activated Ire1p transmembrane kinase/endonuclease. In this study, we isolated and characterized a Yarrowia lipolytica HAC1 (YlHAC1) encoding a basic-leucine zipper transcription factor. The null mutant strain of YlHAC1 (DYlhac1) displayed a significantly increased sensitivity to dithiothreitol (DTT) and tunicamycin (TM), along with a defect in hyphal growth, suggesting the essential function of YlHAC1 in UPR. The unconventional splicing of YlHAC1 mRNA occurred under the UPR conditions induced by DTT or TM treatment. Unlike S. cerevisiae HAC1 mRNA with an intron of 252 nt, YlHAC1 mRNA was shown to harbour a short intron of length 29 nt. The Y...
To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated. Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4. Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA). Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S. cerevisiae produced sufficient amounts of Ura+ transformants from the ura4 mutant of S. exiguus. This fact indicates that the S. exiguus URA4 gene encodes orotidine-5?-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S. cerevisiae are also usable in S. exiguus.
Three different transport systems exist to accumulate a sufficient amount of potassium cations in yeasts. The most common of these are Trk-type transporters, which are used by all yeast species. Though most yeast species employ two different types of transporters, we only identified one gene encoding a potassium uptake system (Trk-type) in the genome of the highly osmotolerant yeast Zygosaccharomyces rouxii, and our results showed that ZrTrk1 is its major (and probably only) specific potassium uptake system. When expressed in Saccharomycescerevisiae, the product of the ZrTRK1 gene is localized to the plasma membrane and its presence efficiently complements the phenotypes of S. cerevisiae trk1? trk2? cells. Deletion of the ZrTRK1 gene resulted in Z. rouxii cells being almost incapable of g...
Mitochondria are essential organelles required for a number of key cellular processes. As most mitochondrial proteins are nuclear encoded, their efficient translocation into the organelle is critical. Transport of proteins across the inner membrane is driven by a multicomponent, matrix-localized "import motor," which is based on the activity of the molecular chaperone Hsp70 and a J-protein cochaperone. In Saccharomycescerevisiae, two paralogous J-proteins, Pam18 and Mdj2, can form the import motor. Both contain transmembrane and matrix domains, with Pam18 having an additional intermembrane space (IMS) domain. Evolutionary analyses revealed that the origin of the IMS domain of S. cerevisiae Pam18 coincides with a gene duplication event that generated the PAM18/MDJ2 gene pair. The duplicati...
Abstract The yeast Saccharomycescerevisiae is able to adapt its metabolism to grow on different carbon sources and to shift to non fermentative growth on C2 or C3 carbon sources (ethanol, acetate, or glycerol) through the activation of gluconeogenesis. Here, we studied the response to the deletion of the glycolytic and gluconeogenic gene GPM1, encoding for phosphoglycerate mutase. It was previously shown that a S. cerevisiae strain with non functional copies of GPM1 can only grow when glycerol and ethanol are both present as carbon sources, whilst addition of glucose was shown to strongly inhibit growth. It was suggested that glycerol is needed to feed gluconeogenesis whilst ethanol is required for respiration. Here, we studied the physiological response of the GPM1 knock out mutant throu...
Abstract in english Saccharomycescerevisiae HAU-1, a time tested industrial yeast possesses most of the desirable fermentation characteristics like fast growth and fermentation rate, osmotolerance, high ethanol tolerance, ability to ferment molasses, and to ferment at elevated temperatures etc. However, this yeast was found to be sensitive against the killer strains of Saccharomycescerevisiae. In the present study, killer trait was introduced into Saccharomycescerevisiae HAU-1 by protopla (more) st fusion with Saccharomycescerevisiae MTCC 475, a killer strain. The resultant fusants were characterized for desirable fermentation characteristics. All the technologically important characteristics of distillery yeast Saccharomycescerevisiae HAU-1 were retained in the fusants, and in addition the killer trait was also introduced into them. Further, the killer activity was found to be stably maintained during hostile conditions of ethanol fermentations in dextrose or molasses, and even during biomass recycling.
We describe the identification and expression cloning of two novel enzymes, a beta-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-beta-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomycescerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0-6.0, and a temperature activity optimum around 40 degrees C. Both enzymes show only low sequence identity to other known enzymes. PMID:10091328
We describe the identification and expression cloning of two novel enzymes, a P-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-beta-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomycescerevisiae W3124. The bgl cDNA encodes a 424-residue precursor protein with a putative signal peptide. The prl cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0-6.0, and a temperature activity optimum around 40 degrees C. Both enzymes show only low sequence identity to other known enzymes.
The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRB1 gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomycescerevisiae PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRB1 genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4? and the pep4? prb1? strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.
Foam production is an essential characteristic of beer, generated mainly from the proteins present in the malt and, to a minor extent, from the mannoproteins in brewer's yeast cell walls. Here, we describe the isolation and characterization of the novel fermentation gene CFG1 (Carlsbergensis foaming gene) from Saccharomyces pastorianus . CFG1 encodes the cell wall protein Cfg1p, a 105 kDa protein highly homologous to Saccharomycescerevisiae cell wall mannoproteins, particularly those involved in foam formation, such as Awa1p and Fpg1p. Further characterization of Cfg1p revealed that this novel protein is responsible for beer foam stabilization. This report represents the first time that a brewing yeast foaming gene has been cloned and its action fully characterized. PMID:23039128
Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomycescerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system.
In Aspergillus nidulans a combination of null mutations in halA, encoding a protein kinase, and sltA, encoding a zinc-finger transcription factor having no yeast homologues, results in an elevated calcium requirement ('calcium auxotrophy') without impairing net calcium uptake. sltA(-) (+/-halA(-)) mutations result in hypertrophy of the vacuolar system. In halA(-)sltA(-) (and sltA(-)) strains, transcript levels for pmcA and pmcB, encoding vacuolar Ca(2+)-ATPase homologues, are highly elevated, suggesting a regulatory relationship between vacuolar membrane area and certain vacuolar membrane ATPase levels. Deletion of both pmcA and pmcB strongly suppresses the 'calcium auxotrophy'. Therefore the 'calcium auxotrophy' possibly results from excessive vacuolar calcium sequestration, causing cytosolic calcium deprivation. Null mutations in nhaA, homologous to Saccharomycescerevisiae NHA1, encoding a plasma membrane Na(+)/H(+) antiporter effluxing Na(+) and K(+), and a non-null mutation in trkB, homologous to S. cerevisiae TRK1, encoding a plasma membrane high affinity K(+) transporter, also suppress the calcium auxotrophy. PMID:20438880
Wine colour, phenolics and volatile fermentation-derived composition are the quintessential elements of a red wine. Many viticultural and winemaking factors contribute to wine aroma and colour with choice of yeast strain being a crucial factor. Besides the traditional Saccharomyces species S. cerevisiae, S. bayanus and several Saccharomyces interspecific hybrids are able to ferment grape juice to completion. This study examined the diversity in chemical composition, including phenolics and fermentation-derived volatile compounds, of an Australian Cabernet Sauvignon due to the use of different Saccharomyces strains. Eleven commercially available Saccharomyces strains were used in this study; S. cerevisiae (7), S. bayanus (2) and interspecific Saccharomyces hybrids (2). The eleven Cabernet S...
Saccharomycescerevisiae is widely used in microbial production of chemicals, metabolites and proteins, mainly because genetic manipulation of S. cerevisiae is relatively easy and experiences from its wide application in the existing industrial fermentations directly benefit new S. cerevisiae-based processes. This study has focused on metabolic engineering of the amino acid metabolism in S. cerevisiae for production of two types of chemicals of industrial interest. The first chemical is ?-(L-?-aminoadipyl)–L-cysteinyl–D-valine (LLD-ACV). ACV belongs to non-ribosomal peptides (NRPs), which are synthesized by specific peptide synthetases and have a broad range of biological and pharmacological properties. Due to the scarcity of the production of NRPs in nature and the difficulties in their chemical synthesis, it was initiated here to develop S. cerevisiae as a platform for microbial production of one type of NRPs – ACV. Production of ACV was achieved by introducing the Penicillium chrysogenum gene pcbAB, which encodes ACV synthetase (ACVS), and the Aspergillus nidulans gene npgA, which encodes phosphopantetheinyl transferase (PPTase) required for activation of ACVS, into S. cerevisiae on a high-copy plasmid. Several possible factors that could improve ACV production were investigated. Lowering the cultivation temperature from 30 to 20 oC led to a 30-fold enhancement. The ACVS and PPTase encoding genes were also integrated into the yeast genome. The second chemical is isobutanol, which is regarded as an important next generation biofuel. As a substitute for liquid fossil fuels, isobutanol is better than ethanol due to its higher energy density, lower hygroscopicity and lower vapor pressure. Isobutanol is also better than its isomer n-butanol due to a higer octane number. In this study, by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in S. cerevisiae, the isobutanol yield was improved from 0.16 to 0.97 mg per g glucose in anaerobic fermentation in mineral medium. Isobutanol yield was further improved by two times by the additional overexpression of BAT2. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by three times. The stoichiometric genome-scale model of S. cerevisiae was applied to find genetic manipulation targets. The BioOpt software was used for in silico cell metabolism simulation. A reasonable agreement was obtained between the experimental data and the in silico simulation results. Through the application of single gene overexpression and deletion options of BioOpt in the overexpression strain ILV2356_XCY605, several target genes are suggested for further overexpression or deletion.
A DNA ligase (CaLIG4) (formerly CaCDC9) of the human pathogen, Candida albicans, has been characterized. The encoded protein displayed a significant similarity to ligase IV from both Saccharomycescerevisiae and humans. In addition, whereas CaLIG4 did not complement a S. cerevisiae cdc9 mutant, it re-established non-homologous end-joining of DNA double-strand breaks in a S. cerevisiae lig4 deletant. CaLIG4 was assigned to chromosome 2. Several cis-acting effector sequences were identified in the promoter region of the CaLIG4, including the DNA sequence element ACGNG, which is required for periodic transcription of several DNA-replicating genes in S. cerevisiae. The level of transcription of CaLIG4 in C. albicans varies during the yeast cell cycle. Newly formed cells contained basal levels of transcript which increased to a maximum level when cells were in late G(1). Thereafter, levels of transcript dropped as DNA replication was initiated. Our results suggest that CaLIG4 may perform an important role during the mitotic cycle of C. albicans. PMID:10487922
Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomycescerevisiae strain, MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production from xylose, we constructed a set of recombinant isogenic strains harboring different combinations of genetic modifications present in MA-R4, and investigated the effect of constitutive expression of xylulokinase and of different levels of xylulokinase and xylose reductase activity on xylose fermentation. This strain comparison showed that constitutive expression of xylulokinase increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of xylose reductase resulted in an increased rate of xylose consumption and an increased glycerol yield. Moreover, strain MA-R6, which has moderate xylulokinase activity, grew slightly better but accumulated more xylitol than strain MA-R4. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol. PMID:22113018
The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high K(m) (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomycescerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus. PMID:18689525
Fumarate reductase is an enzyme involved in maintaining redox balance through regeneration of reduced cofactors during oxygen deficiency conditions. This work reports the identification and characterization of the gene and its promoter and terminator elements that encodes cytosolic fumarate reductase enzyme in the nonconventional yeast, Arxula adeninivorans. The gene harbours an ORF of 1446 bp, encoding a 482-amino acid protein. The deduced amino acid sequence is similar to those of fumarate reductases from other yeast and fungi, such as the two fumarate reductases of Saccharomycescerevisiae, Frd1p (44%) and Osm1p (41%). This enzyme is located in the cytosol and has a pH optimum of ca. 7.5 and a Michaelis constant (K(M) ) of 2.9 mM with fumarate as the substrate. Expression of AFRD1 is regulated by the cultivation conditions. A shift from NaCl-free to NaCl-supplemented media and aerobic to hypoxic growth conditions leads to reduced AFRD1 transcription levels, but not to alteration in the concentration of Afrd1p. The functional analyses of Afrd1p were performed in A. adeninivorans and S. cerevisiae disruption mutants. The A. adeninivorans fumarate reductase is capable of functional complementation of the missing S. cerevisiae genes during anoxia; however, it is not involved in yeast growth under osmotic stress. PMID:22900669
A series of 18 small overlapping restriction fragments has been cloned, covering the complete mitochondrial genome of Schizosaccharomyces pombe. By hybridizing mitochondrial gene probes from Saccharomycescerevisiae and Neurospora crassa with restriction fragments of Schizosaccharomyces pombe mitochondrial DNA, the following homologous genes were localized on the mitochondrial genome of S. pombe: cob, cox1, cox2 and cox3, ATPase subunit 6 and 9 genes, the large rRNA gene and both types of open reading frames occurring in mitochondrial introns of various ascomycetes. The region of the genome, hybridizing with cob exon probes is separated by an intervening sequence of about 2500 bp, which is homologous with the first two introns of the cox1 gene in Saccharomycescerevisiae (class II introns according to Michel et al. 1982). Similarly, in the cox1 homologous region, which covers about 4000 bp, two regions were detected hybridizing with class I intron probes, suggesting the existence of two cox1 introns in Schizosaccharomyces pombe. Hybridization with several specific exon probes with a determined order has revealed that cob, cox1, cox3 and the large rRNA gene are all transcribed from the same DNA strand. The low intensities of hybridization signals suggest a large evolutionary distance between Schizosaccharomyces pombe and Saccharomycescerevisiae or Neurospora crassa mitochondrial genes. Considering the length of the mitochondrial DNA of Schizosaccharomyces pombe (about 19.4 kbp) and the expected length of the localized genes and intron sequences there is enough space left for encoding the expected set of tRNAs and the small rRNA gene. The existence of leader-, trailer-, ori- and spacer sequences or further unassigned reading frames is then restricted to a total length of about 3000 bp only. PMID:6094974
The yeast 2 microns plasmid is found in the nucleus of almost all Saccharomycescerevisiae strains. Its replication is very similar to that of chromosomal DNA. Although the plasmid does not encode essential genes it is stably maintained in the yeast population and exhibits only a small, though detectable, loss rate. This stability is achieved by a plasmid-encoded copy-number control system which ensures constant plasmid levels. For the investigation of 2 microns replication, a yeast strain that is absolutely dependent on this plasmid was constructed. This was achieved by disruption of the chromosomal CDC9 gene, coding for DNA ligase and providing this essential gene on a 2 microns-derived plasmid. This plasmid is absolutely stable under all growth conditions tested. Using the temperature-sensitive mutant allele cdc9-1 we have developed an artificial control system which allows one to change the copy number of 2 microns-derived plasmids solely by changing the incubation temperature. PMID:1787802
To obtain cDNAs encoding oxidosqualene cyclase (OSC), we cloned two cDNAs, KcCAS and RsCAS, from roots of Kandelia candel (L.) Druce and leaves of Rhizophora stylosa Griff. by homology based PCR method respectively. The deduced amino acid sequences of both OSCs showed 82% homology to cycloartenol synthases from Lotus japonicus (OSC5) and Ricinus cummunis (RcCAS), suggesting that these are cycloartenol synthases of K. candel and R. stylosa. The genes obtained were expressed in a lanosterol synthase deficient Saccharomycescerevisiae (ERG7) strain, GIL77. GC–MS analysis identified the accumulated reaction product in the yeast transformant to be cycloartenol, indicating that both KcCAS and RsCAS encode cycloartenol synthase.
Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (?15 phosphate residues) were found in the mitochondria of Saccharomycescerevisiae. They comprised 7?10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca2+/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting Pi from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is...
Gene expression actualizes the organismal phenotypes encoded within the genome in an environment-dependent manner. Among all encoded phenotypes, cell population growth rate (cell population fitness) is perhaps the most important, since it determines how well-adapted a genotype is in various environments. Currently it remains unclear how a cell population's growth rate and its subpopulation fractions in specific environments emerge from the stochastic molecular-level kinetics of gene networks and the division rates of single cells. To address this question we developed and quantitatively characterized synthetic a gene circuit controlling the expression of a bifunctional antibiotic resistance gene in Saccharomycescerevisiae. We found that knowing the cell division rates and nongenetic (cellular) memory of gene expression states were necessary for predicting the overall fitness of cell populations in specific antibiotic- and inducer-containing environments. We validated these predictions experimentally and identified environmental conditions that determined a ``sweet spot'' of drug resistance.
In an effort to elucidate the molecular mechanisms of fatty acid desaturation in yeast, a complete gene encoding ?12 fatty acid desaturase of Saccharomyces kluyveri was cloned. The open reading frame of this gene (named Sk-FAD2) consists of 1,251 bp, encoding 416 amino acids. The deduced Sk-FAD2 protein had 37–55% identity with those from other filamentous fungi. Unlike the genes of these other fungi, S. cerevisiae expressing Sk-FAD2 was found to be capable of synthesizing the dienoic fatty acid hexadecadienoic acid as well as linoleic acid. Moreover, the Sk-FAD2-disrupted strain of S. kluyveri was unable to produce polyunsaturated fatty acids. These results suggested that Sk-FAD2 protein is a unique ?12 fatty acid desaturase in S. kluyveri. Analysis of transcriptional expression revealed that Sk-FAD2 was not repressed by exogenous unsaturated fatty acids but responded to low-temperature stress.
We report the isolation and sequencing of CHS3, a gene that encodes one of several chitin synthases in Paracoccidioides brasiliensis, a medically important fungus restricted geographically to Latin America. The gene contains a single open reading frame of 3817 bp with two introns (71 and 86 bp) and encodes a 1220 amino acid polypeptide with high similarity to other fungal chitin synthases. Northern analysis reveals a high expression of CHS3 in the pathogenic yeast-like phase of the fungus and at the end of the mycelium-yeast transition. Expression of P. brasiliensis CHS3 in a Saccharomycescerevisiae chs3 null mutant enhanced calcofluor white staining in parallel to an increase in total chitin synthase activity and chitin content in its cell wall. The sequence of P. brasiliensis CHS3 is av...
Isochrysis galbana, produces long chain polyunsaturated fatty acids including docosahexaenoic acid (DHA, 22:6n-3). A novel gene (IgFAD4-2), encoding a C22-?4 polyunsaturated fatty acid specific desaturase, has been isolated and characterized from I. galbana. A full-length cDNA of 1,302?bp was cloned by LA-PCR technique. The IgFAD4-2 encoded a protein of 433 amino acids that shares 78?% identity with a previously reported ?4-desaturase (IgFAD4-1) from I. galbana. The function of IgFAD4-2 was deduced by its heterologous expression in Saccharomycescerevisiae, which then desaturated docosapentaenoic acid (DPA, 22:5n-3) to DHA. The conversion ratio of DPA to DHA was 34?%, which is higher than other ?4-desaturases cloned from algae. However, IgFAD4-2 did not catalyze the desaturation or elongat...
The Zymomonas mobilis gene encoding acid phosphatase, phoC, has been cloned and sequenced. The gene spans 792 base pairs and encodes an M{sub r} 28,988 polypeptide. This protein was identified as the principal acid phosphatase activity in Z. mobilis by using zymograms and was more active and magnesium ions than with zinc ions. Its promoter region was similar to the -35 pho box region of the Escherichia coli pho genes as well as the regulatory sequences for Saccharomycescerevisiae acid phosphatase (PH05). A comparison of the gene structure of phoC with that of highly expressed Z. mobilis genes revealed that promoters for all genes were similar in degree of conservation of spacing and identity with the proposed Z. mobilis consensus sequence in the -10 region. The phoC gene contained a 5{prime} transcribed terminus which was AT rich, a weak ribosome-binding site, and less biased codon usage than the highly expressed Z. mobilis genes.
Catharanthus roseus is an important medicinal plant and the sole commercial source of monoterpenoid indole alkaloids (MIA), anticancer compounds. Recently, triterpenoids like ursolic acid and oleanolic acid have also been found in considerable amounts in C. roseus leaf cuticular wax layer. These simple pentacyclic triterpenoids exhibit various pharmacological activities such as anti-inflammatory, anti-tumor and anti-microbial properties. Using the EST collection from C. roseus leaf epidermome ( External Reference Not Shown ), we have successfully isolated a cDNA (CrAS) encoding 2,3-oxidosqualene cyclase (OSC) and a cDNA (CrAO) encoding amyrin C-28 oxidase from the leaves of C. roseus. The functions of CrAS and CrAO were analyzed in yeast (Saccharomycescerevisiae) systems. CrAS was charact...
The open reading frame YGL087c in the budding yeast Saccharomycescerevisiae genome encodes a polypeptide highly similar to the human UEV (ubiquitin-conjugating E2 enzyme variant) proteins, which have been proposed to belong to a family of putative dominant negative ubiquitin regulators. Deletion of the YGL087c open reading frame yields viable cells which are sensitive to UV irradiation or methyl methanesulfonate, but not to hydroxyurea. This phenotype is reminiscent of that of rad mutants and suggests that the YGL087c-encoded protein functions in a process related to tolerance to DNA damage. We also show that the mutant phenotype is fully complemented by expression of the human UEV-1A cDNA and we propose that UEV-1 proteins could also have a role in protecting higher eukaryotic cells from DNA damaging agents. PMID:9580084
The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomycescerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in ?-oxidation and peroxisome proliferation by oleate was distinctly diminished in the ?por1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica. PMID:20977884
Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomycescerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3D, which is disrupted for the gene ...
A cDNA sequence putatively encoding a Delta(6)-fatty acid desaturase was isolated from Rhizopus arrhizus using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1377 bp encoding 458 amino acids of 52 kDa. The deduced amino acid sequence showed high similarity to those of fungal Delta(6)-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich motifs and hydropathy profile. A cytochrome b(5)-like domain was observed at the N-terminus. To elucidate the function of this novel putative desaturase, the coding sequence was expressed heterologously in Saccharomycescerevisiae strain INVScl. The result demonstrated that the coding product of the sequence exhibited Delta(6)-fatty acid desaturase activity by the accumulation of gamma-linolenic acid. PMID:14706830
Mutation in the MAF1 gene was identified in a screen for decreased efficiency of tRNA suppressor SUP11 in the yeast Saccharomycescerevisiae (Sc). maf1-1 mutation exerts a dual phenotypic effect: antisuppression and temperature sensitive (ts) respiratory growth. MAF1, cloned by complementation of the ts phenotype of maf1-1, also alleviates the antisuppressor effect. The coding sequence of MAF1 is interrupted by an intron of 80 bp. The putative gene product, Maf1p, is a hydrophilic protein of 395 amino acids (aa) not showing significant similarity to known proteins which indicates that MAF1 encodes a novel protein. Maf1p may play a role in the tRNA biosynthetic pathway since a fragment of the RPO31/RPC160 gene encoding the largest subunit of RNA polymerase III was cloned as a multicopy suppressor of mafl-1. PMID:9055829
Many of the genes involved in aging have been identified in organisms ranging from yeast to human. Our previous study showed that deletion of the UGA3 gene-which encodes a zinc-finger transcription factor necessary for g-aminobutyric acid (GABA)-dependent induction of the UGA1 (GABA aminotransferase), UGA2 (succinate semialdehyde dehydrogenase), and UGA4 (GABA permease) genes-extends replicative lifespan in the budding yeast Saccharomycescerevisiae. Here, we found that deletion of UGA1 lengthened the lifespan, as did deletion of UGA3; in contrast, strains with UGA2 or UGA4 deletions exhibited no lifespan extension. The Duga1 strain cannot deaminate GABA to succinate semialdehyde. Deletion of GAD1, which encodes the glutamate decarboxylase that converts glutamate into GABA, also increased ...
To improve wine taste and flavor stability, a novel indigenous strain of Saccharomycescerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the ?-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes ?-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19?% in glycerol and GSH production. No heterologous DNA was harbored in t...
Abstract in portuguese Duas linhagens de Saccharomycescerevisiae foram testadas como iniciadoras em uma destilaria de cachaça. Foram utilizadas as linhagens de S. cerevisiae UFMG-A829, isolada de fermentação de cachaça, e S. cerevisiae K1-V1116, de origem vinícola. A permanência de cada linhagem durante a fermentação foi determinada por RAPD (Random Amplified Polymorphic DNA)-PCR, utilizando o iniciador M13. As duas linhagens predominaram nas dornas de fermentação por aproximadamente (more) 30 dias. Leveduras não-Saccharomyces e S. cerevisiae indígenas foram isoladas em menor proporção durante o experimento. As linhagens de S. cerevisiae indígenas apresentaram perfis moleculares distintos em relação às linhagens iniciadoras. As duas linhagens foram promissoras para serem utilizadas como iniciadoras do processo fermentativo para a produção da cachaça. Abstract in english Two Saccharomycescerevisiae strains were tested as the starter yeasts in a traditional cachaça distillery. The strains used were S. cerevisiae UFMG-A829, isolated from a cachaça fermentation process, and S. cerevisiae K1-V1116, obtained from the wine industry. The permanence of each strain in the fermentation must was determined by RAPD (Random Amplified Polymorphic DNA)-PCR, with primer M13. Both yeast strains were prevalent in the vats for approximately 30 days. Indi (more) genous non-Saccharomyces and indigenous S. cerevisiae strains were isolated in lower counts during the fermentation period. Indigenous S. cerevisiae strains were molecularly distinct when compared to the starter yeasts. The two yeasts appeared promising starter yeasts in the fermentation process to produce traditional cachaça.
3-ketoacyl-CoA synthase catalyses the initial condensation reaction during fatty acid elongation using malonyl-CoA and long-chain acyl-CoA as substrates. Previously, it was reported that several genes encoding putative cotton 3-ketoacyl-CoA synthases were significantly up-regulated during early cotton fibre development. In this study, GhCER6 cDNA that contains an open reading frame of 1479 bp, encoding a protein of 492 amino acid residues homologous to the Arabidopsis condensing enzyme CER6, was isolated and cloned. In situ hybridization results demonstrated that GhCER6 mRNA was detected only in the elongating wild-type cotton fibre cells. When GhCER6 was transformed to the Saccharomycescerevisiae elo3 deletion mutation strain that was deficient in the production of 26-carbon fatty acids and displayed a very slow-growth phenotype, the mutant cells were found to divide similarly compared with those of the wild-type cells. Further, heterologous expression of GhCER6 restored the viability of the S. cerevisiae haploid elo2 and elo3 double-deletion strain. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed that GhCER6 was enzymatically active since the yeast elo2 and elo3 double-deletion mutant expressing the cotton gene produced very-long-chain fatty acids that are essential for cell growth. The results suggest that GhCER6 encodes a functional 3-ketoacyl-CoA synthase. PMID:17122407
A cDNA encoding the DNA ligase I homologue has been isolated from Arabidopsis thaliana using a degenerate PCR approach. The ORF of this cDNA encodes an amino acid sequence of 790 residues, representing a protein with a theoretical molecular mass of 87.8 kDa and an isoelectric point (pi) of 8.20. Alignment of the A. thaliana DNA ligase protein sequence with the sequence of DNA ligases from human (Homo sapiens), murine (Mus musculus), clawed toad (Xenopus laevis) and the yeasts Schizosaccharomyces pombe and Saccharomycescerevisiae showed good sequence homology (42-45% identity, 61-66% similarity), particularly around the active site. Sequence data indicate that the Arabidopsis DNA ligase is the homologue of the animal DNA ligase I species. Functional analysis of the cDNA clone demonstrated its ability to complement the conditional lethal phenotype of an S. cerevisiae cdc9 mutant defective in DNA ligase activity, confirming that the cloned sequence encodes an active DNA ligase. The level of the DNA ligase transcript was not increased in A. thaliana seedlings in response to DNA damage induced by a period of enhanced UV-B irradiation. However, the cellular level of the DNA ligase mRNA transcript does correlate with the replicative state of plant cells. PMID:9681027
Non-ribosomal peptides (NRPs) are a diverse family of secondary metabolites with a broad range of biological activities. We started to develop an eukaryotic microbial platform based on the yeast Saccharomycescerevisiae for heterologous production of NRPs using ?-(l-?-aminoadipyl)–l-cysteinyl–d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 5? end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 °C. When ACVS and PPTase encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicatingthat gene copy number was one of the rate-limiting factors for ACV production in yeast.
Fumarate reductase is a protein involved in the maintenance of redox balance during oxygen deficiency. This enzyme irreversibly catalyzes the reduction of fumarate to succinate and requires flavin cofactors as electron donors. Two examples are the soluble mitochondrial and the cytosolic fumarate reductases of Saccharomycescerevisiaeencoded by the OSM1 and FRDS1 genes, respectively. This work reports the identification and characterization of the gene encoding cytosolic fumarate reductase enzyme in the arbuscular mycorrhizal fungus, Glomus intraradices and the establishment of its physiological role. Using a yeast expression system, we demonstrate that G. intraradices GiFRD encodes a protein that has fumarate reductase activity which can functionally substitute for the S. cerevisiae fumarate reductases. Additionally, we showed that GiFRD transformants are not affected by presence of salt in medium, indicating that the presence of this gene has no effect on yeast behavior under osmotic stress. The fact that GiFRD expression and enzymatic activity was present only in asymbiotic stage confirmed existence of at least one anaerobic metabolic pathway in this phase of fungus life cycle. This suggests that the AMF behave as facultative anaerobes in the asymbiotic stage. PMID:22343635
The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying different substrate specificities, patterns of gene expression and even non-mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans-membrane domain proteins. The number in other sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomycescerevisiae and comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of specific symmetry-related amino acid triplets. We assessed the predictive power of these triplets by comparing predictions with experimentally determined data for Arabidopsis MCs, and applied these predictions to the not yet functionally characterized mitochondrial carriers of the grass, Brachypodium distachyon, and the alga, Ostreococcus lucimarinus. We additionally studied evolutionary aspects of the plant MCF by comparing sequence data of the Arabidopsis MCF with those of Saccharomycescerevisiae and Homo sapiens, then with those of Brachypodium distachyon and Ostreococcus lucimarinus, employing intra- and inter-genome comparisons. Finally, we discussed the importance of the approaches of global gene expression analysis and in vivo characterizations in order to address the relevance of these vital carrier proteins. PMID:21443630
Gene-targeting efficiency in the land plant Physcomitrella patens (Bryophyta) can only be compared with that observed in Saccharomycescerevisiae. Sequencing programs and microbiological molecular genetic approaches are now being developed to unravel the precise function of plant genes...
Differences between the recombinant xylose-utilizing Saccharomycescerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and bioch...
Differences between the recombinant xylose-utilizing Saccharomycescerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and bioch...
We have identified five complementation groups of peroxisome biogenesis (peb) mutants in Saccharomycescerevisiae by a positive selection procedure. Three of these contained morphologically recognizable peroxisomes, and two appeared to lack the organelle altogether. The packaging of peroxisomal prot...
A rapid decrease of intracellular glutathione (GSH) was observed when exponentially growing cells of Saccharomycescerevisiae were treated with sublethal concentrations of either dimethyldithiocarbamic acid or thiram [bis(dimethylthiocarbamoyl) disulfide]. The underlying mechanism of this effect pos...
The effect of different pretreatment methods, temperature, and enzyme concentration on ethanol production from 8 lignocellulosic agrowaste by simultaneous saccharification and fermentation (SSF) using recombinant cellulase and Saccharomycescerevisiae were studied. Recombinant cellulase was isolated...
By combining cellulase production, cellulose hydrolysis, and sugar fermentation into a single step, consolidated bioprocessing (CBP) represents a promising technology for biofuel production. Here we report engineering of Saccharomycescerevisiae strains displaying a series of uni-, bi-, and trifunct...
We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomycescerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epoth...
The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomycescerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, 18O-labeling-aided shotgun comparative proteome analysis was applied to ...
Effects of furfural on the aerobic metabolism of the yeast Saccharomycescerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was ...
Effects of furfural on the aerobic metabolism of the yeast Saccharomycescerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was ...
Ari1p from Saccharomycescerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose...
Polyclonal antibodies elicited by injection into rabbits of a nystatin-bovine serum albumin conjugate were reactive with both nystatin and amphotericin B. Upon labeling of polyene-treated Saccharomycescerevisiae sterol auxotrophs grown on various sterols, nystatin reacted specifically with ergoster...
Genes complementing the glucose-negative fructose-negative Saccharomycescerevisiae triple mutant strain (hxkl hxk2 glk1), which lacks hexokinase PI, hexokinase PII, and glucokinase, were obtained from a pool of yeast DNA in the multicopy plasmid YEp13.
When confronted with a marked increase in external osmolarity, budding yeast (Saccharomycescerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high-osmolarity glycerol or HOG pathway) to elicit cellular responses necessary to permit continued growth....
Apr 6, 2011 ... Using modern advances in biotechnology and the weightlessness ... Left: The single-celled fungus Saccharomycescerevisiae, also known as brewer's yeast. ... of the proteins that constitute the molecular machinery of the cell.
A linear discrete dynamic system model is constructed to represent the temporal interactions among significantly expressed genes in response to bioethanol conversion inhibitor 5-hydroxymethylfurfural for ethanologenic yeast Saccharomycescerevisiae. This study identifies the most significant linear...
The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomycescerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intrig...
Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomycescerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome t...
Engineering the industrial ethanologen Saccharomycescerevisiae to utilize pentose sugars from lignocellulosic biomass is critical for commercializing cellulosic fuel ethanol production. Approaches to engineer pentose-fermenting yeasts have required expression of additional genes. We implemented a...
Acetate utilization and macromolecule synthesis during sporulation (meiosis) of Saccharomycescerevisiae were studied. When diploid cells are transferred from glucose nutrient medium to acetate sporulation medium at early stationary phase, respiration of the exogenously supplied acetate proceeds wit...
Saccharomycescerevisiae and S. ellipsoideus were grown on the juices diffused from apple and grape pomace. The amino acid composition of the yeasts was very similar to the amino acid composition of yeast grown on molasses.
Fifty-one human glycosyltransferases were expressed in Saccharomycescerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell...
Natural Saccharomycescerevisiae yeast strains exhibit very large genotypic and phenotypic diversity. However, the link between phenotype variation and genetic determinism is still difficult to identify, especially in wild populations. Using genome hybridization on DNA microarrays, it is now possibl...
Saccharomycescerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties...
The screening of twenty yeast strains for ethanol productivity at high osmotic pressure at temperatures ranging from 32/sup 0/C to 45/sup 0/C is described. Shake flask fermentations of 30/sup 0/, 40/sup 0/, and 50/sup 0/ Bx cane molasses were performed. The effect of temperature on productivity at a non-inhibitory ethanol level is weakly pronounced. Most strains fermented poorly at 50/sup 0/ Bx molasses but two Schizosaccharomyces pombe and one commercial baker's yeast, Saccharomycescerevisiae performed well at all concentrations of molasses. In an extended study with Schizosaccharomyces pombe (CBS 352) and Saccharomycescerevisiae (SJAB, fresh yeast), simulating a continuous run it was shown that Schizusaccharomyces pombe was less sensitive to high DS than Saccharomycescerevisiae. At 25% DS the productivity of Schizusaccharomyces pombe is almost twice that of Saccharomycescerevisiae.
The screening of 20 yeast strains for ethanol (EtOH) productivity at high osmotic pressure and temperatures of 32-45 degrees is described. Shake flask fermentations of 30 degrees, 40 degrees, and 50 degrees Brix cane molasses were performed. The effect of temperature on productivity at a noninhibitory EtOH level is weakly pronounced. Most strains fermented poorly on 50 degrees Brix molasses, but 2 Schizosaccharomyces pombe and 1 commercial bakers' yeast, Saccharomycescerevisiae, performed well at all concentrations of molasses. In an extended study with Schizosaccharomyces pombe (CBS 352) and Saccharomycescerevisiae (SJAB, fresh yeast) simulating a continuous run, Schizosaccharomyces pombe was less sensitive to high dissolved solids than Saccharomycescerevisiae. At 25% dissolved solids the productivity of Schizosaccharomyces pombe is almost twice that of Saccharomycescerevisiae.
Saccharomycescerevisiae 424A (LNH-ST) strain was used for fermentation of glucose and xylose. Growth kinetics and ethanol productivity were calculated for batch fermentation on media containing different combinations of glucose and xylose to give a final sugar concentra...
Hefen sind in der Natur mit vielen Arten weit verbreitet. Seit Urzeiten werden Hefen zur Herstellung von Brot, Bier, Essig, Wein und anderer Lebensmittel eingesetzt. Der Gärorganismus Saccharomycescerevisiae spielt besonders bei der Herstellung alkoholischer Getränke, insbesondere der Obstbrennerei...
...months.â (h) Selenium yeast is a dried, non-viable yeast (Saccharomycescerevisiae ) cultivated in a fed-batch fermentation which provides incremental...salts on the growth rate of the yeast and allows for optimal...
Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in Saccharomycescerevisiae from intermediates of the ?-oxidation cycle by expressing the polyhydroxyalkanoate synthases from Aeromonas caviae and Ralstonia eutropha in the peroxisomes. The quantity of polymer produced was...
Pyrrolnitrin has been reported to inhibit Bacillus megaterium primarily by forming complexes with phospholipids and to block electron transfer of Saccharomycescerevisiae between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. We found that pyrrolnitrin inhibited respir...
Three different transport systems exist to accumulate a sufficient amount of potassium cations in yeasts. The most common of these are Trk-type transporters, which are used by all yeast species. Though most yeast species employ two different types of transporters, we only identified one gene encoding a potassium uptake system (Trk-type) in the genome of the highly osmotolerant yeast Zygosaccharomyces rouxii, and our results showed that ZrTrk1 is its major (and probably only) specific potassium uptake system. When expressed in Saccharomycescerevisiae, the product of the ZrTRK1 gene is localized to the plasma membrane and its presence efficiently complements the phenotypes of S. cerevisiae trk1? trk2? cells. Deletion of the ZrTRK1 gene resulted in Z. rouxii cells being almost incapable of growth at low K(+) concentrations and it changed some cell physiological parameters in a way that differs from S. cerevisiae. In contrast to S. cerevisiae, Z. rouxii cells without the TRK1 gene contained less potassium than the control cells and their plasma membrane was significantly hyperpolarized compared with those of the parental strain when grown in the presence of 100 mM KCl. On the other hand, subsequent potassium starvation led to a substantial depolarization which is again different from S. cerevisiae. Plasma-membrane hyperpolarization did not prevent the efflux of potassium from Z. rouxii trk1? cells during potassium starvation, and the activity of ZrPma1 is less affected by the absence of ZrTRK1 than in S. cerevisiae. The use of a newly constructed Z. rouxii-specific plasmid for the expression of pHluorin showed that the intracellular pH of the Z. rouxii wild type and the trk1? mutant is not significantly different. Together with the fact that Z. rouxii cells contain a significantly lower amount of intracellular potassium than identically grown S. cerevisiae cells, our results suggest that this highly osmotolerant yeast species maintain its intracellular pH and potassium homeostasis in way(s) partially distinct from S. cerevisiae. PMID:22948499
Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomycescerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV. PMID:9092653
The response of Saccharomycescerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2? mutant of S. cerevisiae. Glutathione levels in the ure2? mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2-2.5-fold upon addition of either arsenate (As(V)) or arsenite (As(III)). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3'-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation. PMID:20740275
A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomycescerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved. PMID:10430583
The yeast Saccharomycescerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and ?-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases ?-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae. PMID:22265825
We have isolated a 3.7 kb EcoR1 fragment from a genomic library of Candida albicans which displayed a 65% level of identity with the PRS gene family (PRS) of Saccharomycescerevisiae. The PRS gene encodes a phosphoribosylpyrophosphate (PRPP) synthetase of S. cerevisiae, which catalyses the synthesis of purines, pyrimidines, and amino acids such as histidine and tryptophan. By Northern analyses, we observed that the entire 3.7 kb EcoR1 fragment as well as 1.1 kb KpnI-SacI internal fragment of the 3.7 kb EcoR1 fragment hybridized to the same 1.4 kb transcript. An internal 2.6 kb KpnI fragment was subcloned and sequenced. A deduced sequence of 321 amino acids representing a polypeptide of 35.2 kDa was determined. A FASTA search indicated that the C. albicans PRS (Ca PRS1) had an overall homology at the amino acid level of 91% with the S. cerevisiae PRS3. Putative transcriptional start and termination sequences as well as a cation-binding, PRPP synthetase signature sequence were identified. Ca PRS1 was localized to chromosome 2 of the C. albicans genome. Low stringency hybridizations indicates that the organism may possess multiple PRS genes. The function of these genes in nitrogen signaling is discussed. PMID:8553701
BackgroundIncreasing energy costs and environmental concerns have motivated engineering microbes for the production of ?second generation? biofuels that have better properties than ethanol.Results& ConclusionsSaccharomycescerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.
During growth of Saccharomycescerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.
The capability of immobilized Fusarium oxysporum lini, Mucor, and Saccharomycescerevisiae in fermenting pentose to EtOH was compared. Saccharomycescerevisiae had best fermentation rate on D-xylulose of 0.3 g/l-h. By using a separate isomerase column for converting D-xylose to D-xylulose and a yeast column for converting D-xylulose to EtOH, an EtOH concentration of 32 g/l was obtained from 10% D-xylose. The EtOH yield was calculated to be 64% of theoretical.
Zymomonas mobilis strains were compared with each other and with a Saccharomycescerevisiae strain for the production of EtOH from sugar cane molasses in batch fermentations. The effect of pH and temperature on EtOH production by Zymomonas was studied. The ability of Zymomonas mobilis to produce EtOH from molasses varied from 1 strain to another. At low sugar concentrations Zymomonas compared favorably with Saccharomycescerevisiae. However, at higher sugar concentrations the yeast produced considerably more EtOH than Zymomonas.
Abstract in english Cell suspension cultures of Tabernaemontana catharinensis were treated with autoclaved homogenates of Candida albicans, Fusarium oxysporum, Penicillium avelanium and Saccharomycescerevisiae. The effects caused by the concentration, exposure time and the type of elicitor on the accumulation of pentacyclic triterpenes were monitored. When exposed to biotic elicitors for longer periods, some cell lines redoubled the production of those triterpenes. Saccharomycescerevisiae homogenate was the best elicitor of triterpenes in all cell lines investigated.
The genome-wide transcriptional response of a Saccharomycescerevisiae strain deleted in GDH1 that encodes a NADP(+)-dependent glutamate dehydrogenase was compared to a wild-type strain under anaerobic steady-state conditions. The GDH1-deleted strain has a significantly reduced NADPH requirement, and therefore, an altered redox metabolism. Identification of genes with significantly changed expression using a t-test and a Bonferroni correction yielded only 16 transcripts when accepting two false-positives, and 7 of these were Open Reading Frames (ORFs) with unknown function. Among the 16 transcripts the only one with a direct link to redox metabolism was GND1, encoding phosphogluconate dehydrogenase. To extract additional information we analyzed the transcription data for a gene subset consisting of all known genes encoding metabolic enzymes that use NAD(+) or NADP(+). The subset was analyzed for genes with significantly changed expression again with a t-test and correction for multiple testing. This approach was found to enrich the analysis since GND1, ZWF1 and ALD6, encoding the most important enzymes for regeneration of NADPH under anaerobic conditions, were down-regulated together with eight other genes encoding NADP(H)-dependent enzymes. This indicates a possible common redox-dependent regulation of these genes. Furthermore, we showed that it might be necessary to analyze the expression of a subset of genes to extract all available information from global transcription analysis.
We have investigated the ability of transcripts of the Drosophila melanogaster alcohol dehydrogenase gene to be spliced in Saccharomycescerevisiae. The alcohol dehydrogenase gene was cloned in S. cerevisiae on a 2 micron DNA-based vector and a hybrid yeast actin-Drosophila alcohol dehydrogenase gen...
The Saccharomycescerevisiae Pif1p DNA helicase is the prototype member of a helicase subfamily conserved from yeast to humans. S. cerevisiae has two PIF1-like genes, PIF1 itself and RRM3, that have roles in maintenance of telomeric, ribosomal, and mitochondrial DNA. Here we describe the isolat...
A lot of studies have been carried out on Saccharomycescerevisiae, an yeast with a predominant fermentative metabolism under aerobic conditions, which allows exploring the complex response induced by oxidative stress. S. cerevisiae is considered a eukaryote model for these studies. We propose Kluyv...
Cyclase-associated protein (CAP), also called Srv2 in Saccharomycescerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CA...
We report here on the identification and characterization of novel 2-enoyl thioester reductases of fatty acid metabolism, Etr1p from Candida tropicalis and its homolog Ybr026p (Mrf1?p) from Saccharomycescerevisiae. Overexpression of these proteins in S. cerevisiae led to the development of signific...
A massive screening was performed to identify an acetate-tolerant strain of Saccharomycescerevisiae. We found that S. cerevisiae ATCC 38555 is acetate-tolerant, with a fermentation profile indicating that it has a high level of acetate adaptation. The global gene expression analysis indicated that AFT1- and HAA1-regulated genes are clearly up-regulated.
In the budding yeast Saccharomycescerevisiae the Srs2/RadH DNA helicase promotes survival after ultraviolet (UV) irradiation, and has been implicated in DNA repair, recombination and checkpoint signalling following DNA damage. A second helicase, Sgs1, is the S.cerevisiae homologue of the human B...
We constructed a set of recombinant Saccharomycescerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP+-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.
Specific Saccharomycescerevisiae strains were recently found capable of efficiently utilizing inulin, however, 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. PMID:23104410
We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomycescerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Thr...
A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomycescerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae...
ZEBRA has been shown to activate model reporter genes consisting of synthetic oligomerized ZEBRA response elements upstream of a minimal CYC1 promoter fused to beta-galactosidase in the yeast Saccharomycescerevisiae. Here it is shown that in S. cerevisiae ZEBRA activates transcription of natural Ep...
The effects of acetic acid and extracellular pH (pHex) on the intracellular pH (pHi) of nonfermenting, individual Saccharomycescerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system. S. cerevisiae cells grown in brewer’s wort to t...
Aerobic fermentation has evolved independently in two yeast lineages, the Saccharomycescerevisiae and the Schizosaccharomyces pombe lineages. In the S. cerevisiae lineage, the evolution of aerobic fermentation was shown to be associated with transcriptional reprogramming of the genes involved in re...
A nucleotide sequence encoding an exo-beta-(1,3)-glucanase was cloned from a library of genomic DNA of Candida albicans ATCC 10261. The sequenced gene encodes a protein of 438 amino acid residues. The amino terminal and an internal peptide sequence of the enzyme matched with deduced sequences within the cloned gene. Analysis of the sequence indicated that the nascent protein is processed during secretion by the signal peptidase and a Kex2-like proteinase, yielding a predicted mature enzyme of 400 residues. There is 58% identity and 85% similarity between the amino acid sequences of this exoglucanase and the homologous enzyme of Saccharomycescerevisiae. An antiserum to the purified exoglucanase cross-reacted with the S. cerevisiae exoglucanase and a similar protein secreted by other C. albicans strains and Candida species. There are no sites for N-linked glycosylation in the sequence and this is consistent with the carbohydrate content of the secreted enzyme. Putative upstream promoter elements are associated with the gene. Southern analysis of the gene indicated that it was present at one copy per genome and that the diploid genome of C. albicans ATCC 10261 is heterozygous at this locus for a BglII RFLP. A 2.5 kb mRNA transcript was detected by Northern analysis and gene expression, as monitored by Northern and Western blots, reflected the growth rates of the cultures. PMID:8436950
In a previous study, we reported that the Saccharomycescerevisiae gene YPC1 encodes an alkaline ceramidase with a dual activity, catalyzing both hydrolysis and synthesis of yeast ceramide (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876-6884). In this study, we have identified a YPC1 homologue in S. cerevisiae that also encodes an alkaline ceramidase. We show that these two ceramidases have different substrate specificity, such that YPC1p preferentially hydrolyzes phytoceramide, whereas the new ceramidase YDC1p hydrolyzes dihydroceramide preferentially and phytoceramide only slightly. Neither enzyme hydrolyzes unsaturated mammalian-type ceramide. In contrast to YPC1p, YDC1p had only minor in vitro reverse activity of catalyzing dihydroceramide formation from a free fatty acid and dihydrosphingosine and no activity with phytosphingosine. Overexpression of YDC1p had no reverse activity in non-stressed yeast cells, but like YPC1p suppressed the inhibition of growth by fumonisin B1 albeit more modestly. Deletion of YDC1 and YPC1 or both did not apparently affect growth, suggesting neither gene is essential. However, the Deltaydc1 deletion mutant but not the Deltaypc1 deletion mutant was sensitive to heat stress, indicating a role for dihydroceramide but not phytoceramide in heat stress responses, and suggesting that the two enzymes have distinct physiological functions. PMID:10900202
Alcoholic fermentations of starch hydrolysate by two different yeast strains, Saccharomycescerevisiae (var. Vinal) and Saccharomyces oviformis (IMAP 383), have been studied in batch runs. In order to evaluate the different inhibition phenomena due to both substrate and product, a new kinetic equation is suggested. 23 references.
Diploid hybrids of Saccharomycescerevisiae and its closest relative, Saccharomyces paradoxus, are viable, but the sexual gametes they produce are not. One of several possible causes of this gamete inviability is incompatibility between genes from different species—such incompatible genes are usuall...
As is the case for Saccharomyces boulardii, Saccharomycescerevisiae 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. cerevis...
The extracellular acid proteases of non-Saccharomyces wine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene from Metschnikowia pulcherrima IWBT Y1123, named MpAPr1, and the other gene from Candida apicola IWBT Y1384, named CaAPr1. In silico analysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression of MpAPr1 in Saccharomycescerevisiae YHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. The MpAPr1 gene was found to be present in 12 other M. pulcherrima strains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence. PMID:22820332
Transport-deficient strains of the yeast Saccharomycescerevisiae have recently proven useful for cloning, by functional complementation, of cDNAs encoding heterologous membrane transporters: specifically, H{sup +}-amino acid symporters and K{sup +} channels from the higher plant Arabidopsis thaliana. The present study uses whole-cell patch-clamp experiments to show that yeast strains which grow poorly on submillimolar K{sup +} due to the deletion of two K{sup +}-transporter genes (TRK1 and TRK2) are in fact missing a prominent K{sup +} inward current present in wild-type cells. Rescue of such strains for growth on low K{sup +} by transformation with a gene (KAT1) encoding an inward-rectifying K{sup +} channel from Arabidopsis is accompanied by the appearance of an inward current whose characteristics are in qualitative agreement with previous studies in the Xenopus oocyte system, but differ in quantitative details. The ability to make such measurements directly on Saccharomyces should facilitate structure-function studies of any electrogenic or electrophoretic ion transporters which can be expressed in the plasma membrane (or tonoplast) of that organism. 45 refs., 5 figs., 1 tab.
Staurosporine is a potent inhibitor of protein kinase C. To identify the genes that functionally interact with the Pkc1 pathway of the yeast Saccharomycescerevisiae, we screened for the genes that cause induced staurosporine sensitivity when overexpressed from a galactose-inducible promoter. The novel gene ISR1 encodes a predicted protein kinase with the highest sequence similarity to mammalian Raf in the kinase domain. Drug sensitivity induced by ISR1 overexpression is specific to staurosporine. Although ISR1 disruption causes no obvious phenotype, it does exacerbate the phenotypes of a temperature-sensitive allele (stt1-1) of PKC1, but not of the mpk1 and bck1 mutants of the Mpk1 MAP kinase pathway. These results suggest that Isr1 functions in an event important for growth in a manner redundant with a Mpk1-independent branch of the Pkc1 signalling pathways.
Metatranscriptomics applied to environmental transcripts provides unique opportunities to reveal microbial activity in the environment and to discover novel enzymes of potential use in biotechnological applications. Here, by functional complementation of a pho5^- mutation (affecting a repressible acid phosphatase) and a his3^- mutation in Saccharomycescerevisiae, we identified fungal genes encoding an acid phosphatase and an imidazoleglycerol-phosphate dehydratase in a metatranscriptomic library, which was obtained by reverse-transcribed polyA fraction of total RNA extracted from the organic layer of a sugar maple forest soil, constructed in the modified yeast secretion vector pTEF-MF-SfiI A/B. Yeast transformants exhibiting phosphatase activity were identified in a colony-staining assay ...
In Saccharomycescerevisiae, the genes encoding at least 10 enzymes involved in DNA replication are periodically expressed in the late G1 and S phases of the cell cycle. All of these genes have one copy or more of the sequence ACGCGT, which conforms to the recognition site for the Mlu I restriction endonuclease. For the CDC21, CDC9, and POL1 genes, the Mlu I site has been shown to be absolutely required for periodic transcription. Using nuclear extracts fractionated by conventional and oligonucleotide affinity chromatography, we have purified a 17-kDa protein that recognizes the Mlu I motif. Synthetic oligonucleotides containing mutated Mlu I sites do not bind the protein. In contrast, synthetic oligonucleotides derived from the CDC2, CDC6, and CDC21 genes, which are expressed with the same timing as POL1, bind purified protein efficiently. PMID:1871128
A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomycescerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.
We cloned a genomic fragment of a photolyase gene from Neurospora crassa by polymerase chain reaction using synthesized oligonucleotide primers designed from the most conserved amino acid sequences among photolyases of various organisms. Using the cloned fragment as a hybridization probe we isolated a genomic fragment and cDNA clones encoding the complete photolyase gene of this organism. The amino acid sequence of the photolyase deduced from the determined nucleotide sequence indicates a protein consisting of 615 amino acid residues (Mr 69,971), which is most similar to that of Saccharomycescerevisiae. Like yeast photolyase it contains a protruding amino terminus which is missing in photolyases of bacterial origin. Comparison of amino acids sequences among six photolyases suggests that the Neurospora crassa photolyase is more similar to photolyases of pterin type than those of deazaflavin type. PMID:1833725
The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5{prime} end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonas mobilis and 39.5% identity with ADH4 from Saccharomycescerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.
Dihydroflavonol 4-reductase (DFR), which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition. This study isolated a full length cDNA encoding DFR, designated as SmDFR (GenBank Accession No. EF600682), by screening a cDNA library from a red callus line of Saussurea medusa, which is an endangered, traditional Chinese medicinal plant with high pharmacological value. SmDFR was functionally expressed in yeast (Saccharomycescerevisiae) to confirm that SmDFR can readily reduce dihydroquercetin (DHQ) and dihydrokampferol (DHK), but it could not reduce dihydromyricetin (DHM). The deduced SmDFR structure shared extensive sequence similar...
An a-l-rhamnosidase gene (rhaL1) containing an open reading frame of 2046-bp encoding a 681-amino acid protein (RhaL1) was cloned from Alternaria sp. L1 for naringin hydrolysis on the cell surface of Saccharomycescerevisiae EBY-100. RhaL1 anchored to the yeast cell surface showed maximum enzyme activity at pH 6.0-6.5 and 70degreeC and was stable at pH 2.5-12.0 below 60degreeC. When the yeast cells were employed to hydrolyze naringin in grapefruit juice, about 85% naringin was hydrolyzed at 60degreeC in 10min. The yeast cells were harvested and recycled for the next batch. The hydrolysis rate of the naringin was maintained at over 80% for 10 batches. These results demonstrate the stability of the RhaL1-expressing yeast cells and effective in hydrolysis of naringin in juice. Thus, the syste...
An ?-l-rhamnosidase gene (rhaL1) containing an open reading frame of 2046-bp encoding a 681-amino acid protein (RhaL1) was cloned from Alternaria sp. L1 for naringin hydrolysis on the cell surface of Saccharomycescerevisiae EBY-100. RhaL1 anchored to the yeast cell surface showed maximum enzyme activity at pH 6.0-6.5 and 70°C and was stable at pH 2.5-12.0 below 60°C. When the yeast cells were employed to hydrolyze naringin in grapefruit juice, about 85% naringin was hydrolyzed at 60°C in 10min. The yeast cells were harvested and recycled for the next batch. The hydrolysis rate of the naringin was maintained at over 80% for 10 batches. These results demonstrate the stability of the RhaL1-expressing yeast cells and effective in hydrolysis of naringin in juice. Thus, the system could have promise for industrial bitterness reduction. PMID:22940311
A full-length cDNA (SsFAD3) for an ?-3 fatty acid desaturase (?-3 FAD) was cloned from Sapium sebiferum (L.) Roxb. using rapid amplification of cDNA ends and reverse transcription polymerase chain reaction methods. SsFAD3 contained a 1119-bp open reading frame encoding a 372-amino acid polypeptide. The genomic sequence region of the SsFAD3 ORF was composed of 8 exons and 7 introns, similar to other ?-3 FADs found in most plants. The amino acid sequence showed a higher identity with microsomal ?-3 FADs than plastidial ?-3 FADs. Southern blot analysis of SsFAD3 suggested the existence of a small gene family composed of several copies or closely linked genes. SsFAD3 transcripts were detected in shoots, roots, leaves, stems, and seeds, but were most abundant in shoots. The function of SsFAD3 was confirmed by the accumulation of ?-linolenic acid (?-18:3) in Saccharomycescerevisiae transformants.
The fission yeast Schizosaccharomyces pombe cannot use galactose as a carbon or energy source, and little is known about galactose metabolism in this species. Here we report isolation of a galactose-assimilating mutant that grows on a medium containing galactose as a sole carbon source through use of a proofreading-deficient DNA polymerase d variant encoded by cdc6-1. Based on comparative analysis of gene expression profiles in the wild-type and the mutant (FG2-8), we found that SPBPB2B2.10c (gal7^+), SPBPB2B2.12c (gal10^+) and SPBPB2B2.13 (gal1^+), homologous to Saccharomycescerevisiae GAL7, GAL10 and GAL1, respectively, and SPBPB2B2.08, SPBPB2B2.09c, and SPBPB2B2.11 that localize close to the gal genes, were highly expressed and dramatically induced by addition of galactose. The gal7D s...
Human ITPase, encoded by the ITPA gene, and its orthologs (RdgB in Escherichia coli and HAM1 in Saccharomycescerevisiae) exclude noncanonical nucleoside triphosphates (NTPs) from NTP pools. Deoxyinosine triphosphate (dITP) and 2'-deoxy-N-6-hydroxylaminopurine triphosphate are both hydrolyzed by ITPase to yield the corresponding deoxynucleoside monophosphate and pyrophosphate. In addition, metabolites of thiopurine drugs such as azathioprine have been shown to be substrates for ITPase. The ITPA 94C>A [P32T] variant is one of two polymorphisms associated with decreased ITPase activity. Furthermore, the ITPA 94C>A [P32T] variant is associated with an increased risk of adverse drug reactions for patients treated with azathioprine. The nature of the observed phenotypes for ITPA 94C>A [P32T] va...
Four genes encoding alcohol dehydrogenase (Adh) isozymes in the thermotolerant yeast Kluyveromyces marxianus, a potent candidate for ethanol production at high temperatures, were investigated. Of these, KmADH3 and KmADH4 were cloned and sequenced, and their deduced amino acid sequences were compared with those of KmAdh1 and KmAdh2 and other Adhs of Kluyveromyces lactis and Saccharomycescerevisiae. The four KmAdhs had high sequence similarity, though KmAdh3 and KmAdh4 possessed an amino-terminal extension as a mitochondrial targeting sequence, and appear to belong to the zinc-containing Adh family. These results and the results of Southern blot experiments suggest that there are at least four Adh isozymes in K. marxianus, two cytoplasmic enzymes and two mitochondrial enzymes. The expression profile revealed that KmADH genes are differently expressed depending on growth phase and carbon source, suggesting that these highly homologous Adhs play distinctive roles in cells.
The nuclear genome of Saccharomycescerevisiaeencodes 35 members of a family of membrane proteins. Known members transport substrates and products across the inner membranes of mitochondria. We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria. They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism. Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported [14C]oxoglutarate uptake. The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism. PMID:11013234
Explaining how the small molecule auxin triggers diverse yet specific responses is a long-standing challenge in plant biology. An essential step in auxin response is the degradation of Auxin/Indole-3-Acetic Acid (Aux/IAA, referred to hereafter as IAA) repressor proteins through interaction with auxin receptors. To systematically characterize diversity in degradation behaviors among IAA|receptor pairs, we engineered auxin-induced degradation of plant IAA proteins in yeast (Saccharomycescerevisiae). We found that IAA degradation dynamics vary widely, depending on which receptor is present, and are not encoded solely by the degron-containing domain II. To facilitate this and future studies, we identified a mathematical model able to quantitatively describe IAA degradation behavior in a single parameter. Together, our results demonstrate the remarkable tunability conferred by specific configurations of the auxin response pathway. PMID:22843664
Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomycescerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1?5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-ac...
Gentamicin is a highly efficacious antibiotic against Gram-negative bacteria. However, its usefulness in treating infections is compromised by its poorly understood renal toxicity. Toxic effects are also seen in a variety of other organisms. While the yeast Saccharomycescerevisiae is relatively insensitive to gentamicin, mutations in any one of ?20 genes cause a dramatic decrease in resistance. Many of these genes encode proteins important for translation termination or specific protein-trafficking complexes. Subsequent inspection of the physical and genetic interactions of the remaining gentamicin-sensitive mutants revealed a network centered on chitin synthase and the Arf GTPases. Further analysis has demonstrated that some conditional arf1 and gea1 alleles make cells hypersensitive to gentamicin under permissive conditions. These results suggest that one consequence of gentamicin exposure is disruption of Arf-dependent protein trafficking. PMID:20956596
Protoplast regeneration of a wild-type and two mutant strains of Candida glabrata defective in CHS3 homologues encoding class IV chitin synthase in Saccharomycescerevisiae was examined by scanning and negative-staining electron microscopy. In the wild-type strain, small particles and short filaments appeared on the protoplast surface at 10 min, filamentous materials covered the entire surface of the protoplast at 1 h, granular materials started filling interspaces of filamentous materials at 2 h and regeneration was completed at 6 h. The filamentous materials consisted of microfibrils of various widths ranging from chs3A and Dchs3B completed regeneration essentially by the same process as wild-type strain, although it took more time. These results suggest that CHS3A and CHS3B genes may ha...
In this study, in silico analysis of the Caenorhabditis elegans genome revealed six genes (cah-1, cah-2, cah-3, cah-4, cah-5, and cah-6) possibly encoding ? class CAs (carbonic anhydrase). Real-time RT-PCR analysis revealed the temporal expression pattern of each gene, as well as changes in expression levels under different atmospheric conditions (stress). Cah-3 and cah-4 showed the highest levels of transcript accumulation, while most genes responded to the stress conditions. Yeast complementation showed that cah-3 was able to complement the function of Saccharomycescerevisiae CA (NCE103) in vivo. Recombinant CAH-3, CAH-4a and CAH-5 enzymes, expressed in Escherichia coli were used for in vitro measurement of CA activity. However, in vitro activity was only detectable for CAH-4a. RNAi by ...
During the biosynthesis of N-glycans in multicellular eukaryotes, glycans with the compositions Man5GlcNAc2-3 are key intermediates. However, to reach this ?decision point?, these N-glycans are first processed from Glc3Man9GlcNAc2 through to Man5GlcNAc2 by a number of glycosidases, whereby up to four ?1-2-linked mannose residues are removed by class I mannosidases (glycohydrolase family 47). Whereas in the yeast Saccharomycescerevisiae there are maximally three members of this protein family, in higher organisms there are multiple class I mannosidases residing in the endoplasmic reticulum and Golgi apparatus. The genome of the model nematode Caenorhabditis elegans encodes seven members of this protein family, whereby four are predicted to be classical processing mannosidases and three are...
In the pathogen Candida albicans protein kinase A (PKA) catalytic subunit is encoded by two genes TPK1 and TPK2 and the regulatory subunit by one gene, BCY1. PKA mediates several cellular processes such as cell cycle regulation and the yeast to hyphae transition, a key factor for C. albicans virulence. The catalytic isoforms Tpk1p and Tpk2p share redundant functions in vegetative growth and hyphal development, though they differentially regulate glycogen metabolism, the stress response pathway and pseudohyphal formation. In Saccharomycescerevisiae it was earlier reported that BCY1 overexpression not only increased the amount of TPK3 mRNA but also its catalytic activity. In C. albicans a significant decrease in Bcy1p expression levels was already observed in tpk2D null strains. In this wor...
The gene SPI1, of Saccharomycescerevisiae, encodes a cell wall protein that is induced in several stress conditions, particularly in the postdiauxic and stationary phases of growth. It has a paralogue, SED1, which shows some common features in expression regulation and in the null mutant phenotype. In this work we have identified homologues in other species of yeasts and filamentous fungi, and we have also elucidated some aspects of the origin of SPI1, by duplication and diversification of SED1. In terms of regulation, we have found that the expression in the post-diauxic phase is regulated by genes related to the PKA pathway and stress response (MSN2/4, YAK1, POP2, SOK2, PHD1, and PHO84) and by genes involved in the PKC pathway (WSC2, PKC1, and MPK1).
IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of guanine, namely the oxidation of IMP to XMP with a concomitant reduction of NAD+. In Saccharomycescerevisiae, a family of four closely-related genes, IMD1, IMD2 (also known as PUR5), IMD3, and IMD4, encodes the putative IMPDH. Although IMPDH synthesizes guanine in the cytoplasm, it has also been found in the nucleus, where it associates with nucleic acids in human cells. Here, we further show that IMPDH is recruited to actively transcribed region of genes. A synthetic lethal screen using a deletion strain of Ctk1 kinase, a yeast homolog of mammalian Cdk9/P-TEFb that phosphorylates serine 2 within the RNA polymerase II (RNApII) C-terminal domain (CTD), identified that Imd2 genetically interacts with Ctk1...
A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to ?-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl ?-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomycescerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.
In Saccharomycescerevisiae, an endopolygalacturonase encoded by the PGL1 gene catalyzes the random hydrolysis of the ?-1,4 glycosidic linkages in polygalacturonic acid. To study the regulation of the PGL1 gene, we constructed a reporter vector containing the lacZ gene under the control of PGL1 promoter. Surprisingly, when Escherichia coli DH5? was transformed by this vector, cells harboring the constructed plasmid produced blue colonies. Sequence analysis of this promoter revealed that E. coli consensus sequences required to express an in-frame lacZ alpha product were present. We next decided to investigate how the PGL1 promoter is regulated in E. coli compared to yeast. In this study, we examined the modulation of the PGL1 promoter in E. coli, and the results indicated that its activity ...
FLR1 gene, encoding a multidrug resistance (MDR) transporter of the major facilitator superfamily (MFS) was found to confer resistance to the fungicide mancozeb in Saccharomycescerevisiae. This agrochemical has been linked to the development of Parkinson disease and cancer. Yeast response to mancozeb was proved to involve the strong activation of FLR1 transcription (20-fold) during the fungicide-induced growth latency. This activation of FLR1 transcription is fully dependent on Yap1p and is reduced (by 50%) in the absence of Rpn4p, Yrr1p or Pdr3p. A model for the coordinate action over FLR1 transcription activation, in response to mancozeb, of these transcription factors that mediate oxidative stress response (Yap1p), proteasome gene expression (Rpn4p), and pleiotropic drug resistance (Pdr3p and Yrr1p), is proposed. PMID:18086556
Abstract Two C-4 methyl sterol oxidase genes (Pcerg25A and Pcerg25B) that are involved in ergosterol biosynthesis have been cloned from the penicillin-producing fungus Penicillium chrysogenum. cDNAs of both Pcerg25A and Pcerg25B have an ORF 885 bp in length, encoding a peptide of 295 residues. The deduced amino acid sequences of PcErg25A and PcErg25B show 86% identity, and have high identities to the characterized C-4 methyl sterol oxidases from Candida albicans and Saccharomycescerevisiae. The function of Pcerg25A and Pcerg25B was identified by complementation of a yeast erg25-deficient strain. Pcerg25A is located in the DNA region containing the penicillin gene cluster, and thus its copy number is dependent on the patterns of the cluster region. Up to eight copies of Pcerg25A were found...
Abstract in english SnRKs (Sucrose non-fermenting-1 related kinases) is a family of protein kinases found in many crops, such as Arabidopsis, rice, sugarcane, tomato and several other plant species. This family of proteins is also present in other organisms like Saccharomycescerevisiae (sucrose non-fermenting-1 - Snf1) and in mammals (AMP-activated protein kinases - AMPKs). There is evidence that SnRKs play an important role in plant responses to nutritional and environmental stresses and t (more) hat SnRKs also play a major role in controlling key enzymes in the biosynthetic pathways of plants. In this work, we identified 18 contigs and two singletons encoding putative SnRKs in the CitEST database. All of them present highly conserved N-terminal catalytic domain, which is found in the SnRKs families of several plant species. Through comparison with known SnRKs, we were able to classify them into three subfamilies.
ABSTRACT. Small GTPase Rab (products of ras genes from rat brain) is a widely conserved molecular switch among eukaryotes and regulates membrane trafficking pathways. It is generally considered that the number of Rab encoded in the genome correlates with multicellularity; however, we found that unicellular ciliates Tetrahymena thermophila (Tt) and Paramecium tetraurelia (Pt) possess many more Rab genes in their genome than the 64 HsRab genes in the human genome. We succeeded in isolating 86 cDNA clones of 88 TtRab genes in the Tetrahymena genome. By comparing the amino acid sequence of Rab in humans and the budding yeast Saccharomycescerevisiae, 42 TtRab belonged to subfamilies functionally characterized and designated as conventional Rab, while the remaining 44 TtRab were considered to b...
Myrmecia incisa Reisigl H4301 is a green coccoid freshwater microalga that is rich in arachidonic acid (20:4n-6, ArA) especially grown under a nitrogen starvation stress. A fatty acid elongase gene, MiFAE, was cloned based on a selected expressed sequence tag (EST) from a M. incisa cDNA library. To examine the function, the MiFAE gene was heterologously expressed in Saccharomycescerevisiae. The fatty acid profile of the transgenic yeast was analyzed by gas chromatography-mass spectrometry (GC-MS), and the results illustrated that the enzyme encoded by MiFAE was able to elongate g-linolenic acid (18:3n-6, GLA) and stearidonic acid (18:4n-3, SDA) to di-homo-g-linolenic acid (20:3n-6, DGLA) and eicosatetraenoic acid (20:4n-3, ETA), respectively, suggesting that the cloned MiFAE gene seemed t...
Sodium/hydrogen transporters transfer ions across membranes and thus play an important role in pH and electrolyte homeostasis. To further understand the mechanism and function of the plant vacuolar Na+/H+, a new Na+/H+ antiporter, named DmNHX1, was isolated from chrysanthemum (Dendranthema morifolium) and characterized. The total length of DmNHX1 is 1,897?bp, with an open reading frame of 1,653?bp. Functional complementation studies in Saccharomycescerevisiae strains with mutated endogenous NHX genes showed that the encoded protein, DmNHX1, has a broad cation specificity (Na+, K+, Li+) and confered hygromycin B tolerance. Quantitative RT-PCR revealed that the transcription level of DmNHX1 was induced by salt stress in all studied tissues, especially expressed highest in the leaves. Agroba...
The RAD3 gene of the yeast Saccharomycescerevisiae is required for excision repair of damaged DNA and for cell viability. A protein of approx. = 89 kDa was purified to near homogeneity from yeast strains harboring multicopy plasmids that overproduce RAD3 protein; this protein corresponds closely to the expected size of the RAD3 protein and crossreacts with the antiserum raised against a truncated RAD3 protein produced in Escherichia coli. The purified RAD3 protein shows a single-stranded DNA-dependent ATPase activity that catalyzes hydrolysis of ATP to ADP and P/sub i/. The ATPase activity was coincident with the RAD3 protein during purification and is inhibited by anti-RAD3 antibodies, indicating that the RAD3 gene encodes this activity
Abstract The biochemical characterization of sugar uptake in yeasts started five decades ago and led to the early production of abundant kinetic and mechanistic data. However, the first accurate overview of the underlying sugar transporter genes was obtained relatively late, due mainly to the genetic complexity of hexose uptake in the model yeast Saccharomycescerevisiae. The genomic era generated in turn a massive amount of information, allowing the identification of a multitude of putative sugar transporter and sensor-encoding genes in yeast genomes, many of which are phylogenetically related. This review aims to briefly summarize our current knowledge on the biochemical and molecular features of the transporters of hexoses and pentoses in yeasts, when possible establishing links between...
To improve the ability of recombinant Saccharomycescerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due ...
Many genes encoding exo- and endo-inulinases from bacteria, yeasts and filamentous fungi have been cloned and characterized. All the inulinases have several conserved motifs, such as WMND(E)PNGL, RDP, EC(V)P, SVEVF, Q and FS(T), which play an important role in inulinase catalysis and substrate binding. However, the exo-inulinases produced by yeasts has no conserved motif SVEVF and the yeasts do not produce any endo-inulinase. Exo- and endo-inulinases found in different microorganisms cluster separately at distant positions from each other. Most of the cloned inulinase genes have been expressed in Yarrowia lipolytica, Saccharomycescerevisiae, Pichia pastoris, Klyuveromyces lactis and Escherichia coli, respectively. The recombinant inulinases produced and the engineered hosts using the cloned inulinase genes have many potential applications. Expression of most of the inulinase genes is repressed by glucose and fructose and induced by inulin and sucrose. However, the detailed mechanisms of the repression and induction are still unknown. PMID:22734928
Eukaryotic DNA polymerases ? and ?, both of which are required for chromosomal DNA replication, contain proofreading 3’?5’exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase ? variant encoded by pol3-01 in the yeast Saccharomycescerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38°C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40°C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40°C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA ? polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.
In order to take full advantage of the industrially important yeast Candida utilis, we developed a practical recombinant DNA tool for multiple gene disruption in C. utilis based on the Cre-loxP system, which makes possible the reuse of selection markers. For this purpose, two plasmids were constructed: one harbored a heterologous loxP-flanked selection marker cassette carrying the gene responsible for hygromycin B-resistance, and the other had an autonomous replication sequence (ARS) and a Cre-recombinase expression module. Multiple disruption of C. utilis NBRC0988 URA3 genes (CuURA3), encoding orotidine-5?-phosphate decarboxylase, validated the efficiency of the system. The fourth round of deletion yielded a null mutant, i.e., a uracil auxotroph, giving some support to the possibility that C. utilis NBRC0988 is a tetraploid. This agreed very well with the outcomes of FACS analysis, which showed that various strains of this yeast contained 3–5 times more DNA than a Saccharomycescerevisiae haploid.
In order to take full advantage of the industrially important yeast Candida utilis, we developed a practical recombinant DNA tool for multiple gene disruption in C. utilis based on the Cre-loxP system, which makes possible the reuse of selection markers. For this purpose, two plasmids were constructed: one harbored a heterologous loxP-flanked selection marker cassette carrying the gene responsible for hygromycin B-resistance, and the other had an autonomous replication sequence (ARS) and a Cre-recombinase expression module. Multiple disruption of C. utilis NBRC0988 URA3 genes (CuURA3), encoding orotidine-5?-phosphatate decarboxylase, validated the efficiency of the system. The fourth round of deletion yielded a null mutant, i.e., a uracil auxotroph, giving some support to the possibility that C. utilis NBRC0988 is a tetraploid. This agreed very well with the outcomes of FACS analysis, which showed that various strains of this yeast contained 3–5 times more DNA than a Saccharomycescerevisiae haploid.
Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomycescerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from se...
In higher plants, long-chain fatty acid hydroperoxides are intermediates in the synthesis of a diverse group of bioactive compounds. We used the reverse trascriptase-polymerase chain reaction to isolate a gene responsible for the oxidization of fatty acids from Petunia hybrida. A P450 cDNA not isolated earlier, CYP92B1, contained an open reading frame predicted to encode a polypeptide consisting of 510 amino acid residues. The transcript of the cyp92B1 gene was expressed at a high level in the early stage of flower development. CYP92B1 cDNA was expressed in a yeast, Saccharomycescerevisiae, and recombinant yeast microsomes containing CYP92B1, a hemoprotein, metabolized lauric acid, linoleic acid, and linolenic acid.
The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomycescerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid. Whereas Dga1p requires acyl-CoA as a substrate for acylation of diacylglycerol, Lro1p is an acyl-CoA independent triacylglycerol synthase using phospholipids as acyl-donor. Growth of Yarrowia lipolytica strains deleted of DGA1 and/or LRO1 on glucose containing medium significantly decreases triacylg...
The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomycescerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in b-oxidation and peroxisome proliferation by oleate was ...
Summary The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying different substrate specificities, patterns of gene expression and even non-mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans-membrane domain proteins. The number in other sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomycescerevisiae and comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of specific symmetry-related amino acid triplets. We assessed the predictive power of these triplets by comparing predictions with experimentally de...
Saccharomycescerevisiae strain TIY20, which produces a mammalian high-mannose type N-glycan, exhibits a severe growth defect due to disruption of yeast-specific outer chain mannosyltransferases. We have isolated a more fit strain, YAB103, from TIY20 by the use of a novel mutagenesis technique based on the disparity theory of evolution. To determine why YAB103 lacked the growth defect and the hygromycin B sensitivity of its parent, TIY20, gene expression profiles of YAB103 and TIY20 were analyzed using DNA microarrays. Expression of genes that encode enzymes in the gluconeogenesis pathway and glyoxylate cycle, which produce glucose 6-phophate and its derivatives, was up-regulated in YAB103. Up-regulation of these genes suppressed the drug sensitivity of the TIY20 strain. Furthermore, we found that YAB103 had a thicker cell-wall due to an increase in glucan content. To our knowledge, this is first report linking N-glycosylation to gluconeogenesis and the glyoxylate cycle.
Pseudomonas putida benF, benK, benE1, and benE2 genes encode proteins belonging to benzoate transporter super family, but those functions have not yet been elucidated. In this study we analyzed the functions of these gene products using the yeast Saccharomycescerevisiae. P. putida gene products expressed in yeast cells were localized to the yeast plasma membrane and were involved in taking up benzoate into the cells. According to the sensitivity of yeast cell-growth to benzoate, it is proposed that benK, benE1, and benE2 gene products function as transporters, that take up benzoate into the cells, whereas the benF gene product functions as an efflux pump of benzoate.
The sister chromatid cohesion complex of Saccharomycescerevisiae is composed of proteins termed cohesins. The complex forms a ring structure that entraps sister DNAs, probably following replication. The mechanism of cohesion is universal and the proteins participating in this process are evolutionarily highly conserved. We investigated the Irr1p/Scc3p cohesin subunit, an under-studied protein. We show that the presence of a mutated copy of IRR1 gene, encoding the F658G substitution in Irr1p, changes the sensitivity of the heterozygous irr1-1/IRR1 diploid to cell wall-affecting compounds. Microscopic images indicate that chitin distribution in the mutant cell wall is affected, although the biochemical composition of the cell wall is not drastically changed. This observation suggests that irr1-1 mutation in heterozygous state may influence the cell wall integrity and indicates a possible link between mechanisms regulating the cell wall biosynthesis, nuclear migration and chromosome segregation.
The budding yeast Saccharomycescerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations. Using this novel approach, we show that depletion of a single deoxyribonucleoside causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase, Rad53. We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse-chase labelling.
The Arabidopsis HCC1 gene is a homologue of the copper chaperone SCO1 from the yeast Saccharomycescerevisiae. SCO1 (synthesis of cytochrome c oxidase 1) encodes a mitochondrial protein that is essential for the correct assembly of complex IV in the respiratory chain. GUS analyses showed HCC1 promoter activity in vascular tissue, guard cells, hydathodes, trichome support cells, and embryos. HCC1 function was studied in two hcc1 T-DNA insertion lines, hcc1-1 and hcc1-2. Gametophyte development was not affected by the disruption of HCC1, but homozygous hcc1-1 and hcc1-2 embryos became arrested at various developmental stages, mostly at the heart stage. Both the wild-type HCC1 gene and the modified gene coding for the C-terminally SNAP-tagged HCC1 were able to complement the embryo-lethal phe...
HAP4 encodes a transcriptional activator of respiration-related genes and so, redirection from fermentation to respiration flux should give rise to an increase in biomass production in Saccharomycescerevisiae transformants that overexpress HAP4. With this aim, three bakers' yeasts, that is, V1 used for lean doughs, its 2-deoxy-D-glucose resistant derivative DOG21, and V3 employed for sweet doughs, were transformed with integrative cassettes that carried HAP4 gene under the control of constitutive promoter pTEF2; in addition VTH, DTH and 3TH transformants were selected and characterized. Transformants showed increased expression of HAP4 and respiration-related genes such as QCR7 and QCR8 with regard to parental, and similar expression of SUC2 and MAL12; these genes are relevant in bakers' ...
Abstract Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis, in contrast to Saccharomycescerevisiae, where two homologous genes are present. Mutant cells had lost the ability to grow with the amino acids glutamate, proline or aspartate and other non-fermentable carbon sources, such as acetic acid and ethanol, as sole carbon sources. Biomass formation of the mutant cells in medium containing glucose or xylose as carbon source was lower comp...
Oxidative damage, mitochondrial dysfunction, genomic instability, and telomere shortening represent all molecular processes proposed as causal factors in aging. Lifespan can be increased by metabolism through an influence on such processes. Glucose reduction extends chronological lifespan (CLS) of Saccharomycescerevisiae through metabolic adaptation to respiration. To answer the question if the reduced CLS could be ascribed to glucose per se or to glucose repression of respiratory enzymes, we used the Kluyveromyces lactis yeast, where glucose repression does not affect the respiratory function. We identified the unique hexokinase, encoded by RAG5 gene, as an important player in influencing yeast lifespan by modulating mitochondrial functionality and the level of the mitochondrial chaperonin Hsp60. In this context, this hexokinase might have a regulatory role in the influence of CLS, shedding new light on the complex regulation played by hexokinases. PMID:22675632
Upon glucose depletion, a massive reprogramming of gene expression occurs in the yeast Saccharomycescerevisiae for the use of alternate carbon sources such as the nonfermentable compounds ethanol and glycerol. This process is mediated by the master kinase Snf1 that controls the activity of various targets including the transcriptional regulators Cat8, Sip4 and Adr1. We have recently identified Rds2 as an additional player in this pathway. Here, we have performed genome-wide location analysis of Rds2 in cells grown in the presence of glycerol. We show that Rds2 binds to promoters of genes involved in gluconeogenesis, the glyoxylate shunt, and the TCA cycle as well as some genes encoding mitochondrial components or some involved in the stress response. Interestingly, we also detected Rds2 a...
Abstract. Coenzyme Q is a lipid molecule required for respiration and antioxidant protection. Q biosynthesis in Saccharomycescerevisiae requires nine proteins (Coq1p?Coq9p). We demonstrate in this study that Q levels are modulated during growth by its conversion from demethoxy-Q (DMQ), a late intermediate. Similar conversion was produced when cells were subjected to oxidative stress conditions. Changes in Q6/DMQ6 ratio were accompanied by changes in COQ7 gene mRNA levels encoding the protein responsible for the DMQ hydroxylation, the penultimate step in Q biosynthesis pathway. Yeast coq null mutant failed to accumulate any Q late biosynthetic intermediate. However, in coq7 mutants the addition of exogenous Q produces the DMQ synthesis. Similar effect was produced by over-expressing ABC1/C...
Codon usage data for 56 Bacillus subtilis genes show that synonymous codon usage in B. subtilis is less biased than in Escherichia coli, or in Saccharomycescerevisiae. Nevertheless, certain genes with a high codon bias can be identified by correspondence analysis, and also by various indices of codon bias. These genes are very highly expressed, and a general trend (a decrease) in codon bias across genes seems to correspond to decreasing expression level. This, then, may be a general phenomenon in unicellular organisms. The unusually small effect of translational selection on the pattern of codon usage in lowly expressed genes in B. subtilis yields similar dinucleotide frequencies among different codon positions, and on complementary strands. These patterns could arise through selection on DNA structure, but more probably are largely determined by mutation. This prevalence of mutational bias could lead to difficulties in assessing whether open reading frames encode proteins. PMID:3118331
Triglycerides (TG) are major storage lipids for eukaryotic cells. In this study, we characterized three genes of fission yeast Schizosaccharomyces pombe, SPCC1450.16c, SPAC1786.01c, and SPAC1A6.05c, that show high homology to Saccharomycescerevisiae TG lipase genes, TGL3, TGL4, and TGL5. Deletion of each gene increased TG content by approximately 1.7-fold compared to the parental wild-type strain, and their triple deletion mutant further increased TG content to 2.7-fold of the wild-type strain, suggesting that all three genes encode TG lipase and are functioning in S. pombe. The triple deletion mutant showed no growth defect in rich and synthetic medium, but its growth was sensitive to cerulenin, an inhibitor of fatty acid synthesis. This growth defect by cerulenin was restored by adding ...
In previous studies, the authors have described a human genomic DNA fragment of about 21 kbp which was found immediately upstream of the fes/fps proto-oncogene and contained genetic sequences of a new gene that they called fur. Now they present the complete nucleotide sequence of the fur coding sequences and the deduced amino acid sequence of the fur product furin. From computer analysis it appeared that the deduced furin sequence exhibited a striking homology to the subtilisin-type serine protease encoded by the KEX2 gene of yeast Saccharomycescerevisiae. In the subtilisin-like domain structural similarity is also found with the catalytic domains of the subtilisins thermitase and subtilisin BPN. These observations suggests that furin is a prime candidate for a mammalian precursor protein processing enzyme.
Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomycescerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomycescerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild-type strains, among them FY1679, CEN.PK2-1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism. PMID:10341423
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 Saccharomycescerevisiae 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 simultan...
Persimmon (Diospyros kaki) is a seasonal fruit with important health benefits. In this study, persimmon use in wine and condiment production was investigated using molecular methods to identify the yeast and acetic acid bacteria (AAB) isolated from the alcoholic fermentation and acetification of the fruit. Alcoholic fermentation was allowed to occur either spontaneously, or by inoculation with a commercial Saccharomycescerevisiae wine strain, while acetification was always spontaneous; all these processes were performed in triplicates. Non-Saccharomyces yeast species were particularly abundant during the initial and mid-alcoholic fermentation stages, but S. cerevisiae became dominant toward the end of these processes. During spontaneous fermentation, S. cerevisiae Sc1 was the predominant ...
The phytopathogen Glomerella cingulata (anamorph: Colletotrichum gloeosporioides) infects host tissue by means of a specialised infection structure, the appressorium. The Saccharomycescerevisiae alpha-mating factor pheromone, the Saccharomyces kluyveri alpha-mating factor pheromone and a hendecapeptide produced by G. cingulata inhibit appressorium development. The amino acid sequence of the G. cingulata peptide, GYFSYPHGNLF, is different from that of the mature pheromone peptides of other filamentous fungi. The peptide has sequence similarity with the Saccharomyces alpha-mating factor pheromones, but is unable to elicit growth arrest in S. cerevisiae. PMID:12007806
It is widely accepted that phosphatidylethanolamine (PE) is enriched in the cytosolic leaflet of the eukaryotic plasma membranes. To identify genes involved in the establishment and regulation of the asymmetric distribution of PE on the plasma membrane, we screened the deletion strain collection of the yeast Saccharomycescerevisiae for hypersensitive mutants to the lantibiotic peptide Ro09-0198 (Ro) that specifically binds to PE on the cell surface and inhibits cellular growth. Deletion mutants of VPS51, VPS52, VPS53, and VPS54 encoding the components of Golgi-associated retrograde protein (GARP) complex, YPT6 encoding a Rab family small GTPase that functions with GARP complex, RIC1 and RGP1 encoding its guanine nucleotide exchange factor (GEF), and TLG2 encoding t-SNARE exhibited hypersensitivity to Ro. The mutants deleted for VPS51, VPS52, VPS53, and VPS54 were impaired in the uptake of fluorescently labeled PE. In addition, aberrant intracellular localization of the EGFP-tagged Dnf2p, the putative inward-directed phospholipid translocase (flippase) of the plasma membrane, was observed in the mutant defective in the GARP complex, Ypt6p, its GEF proteins, or Tlg2p. Our results suggest that the GARP complex is involved in the recycling of Dnf flippases. PMID:22177957
An extracellular endo-1,4-?-xylanase was purified from the culture supernatant of Aureobasidium pullulans var. melanigenum (ATCC 20524) grown on oat-spelt xylan. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with an apparent Mr of 24 kDa and had an isoelectric point of 6.7. Xylanase activity was optimal at pH 2.0 and 50°C. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. Southern blot analysis indicated that the xylanase gene (xynI) was present as a single copy in the genome. An open reading frame, consisting of 663 bp, encoded a presumed prepropeptide of 34 amino acids and a mature protein of 187 amino acid. The DNA region encoding the prepeptide was interrupted by a 59-bp intron. A single transcription start point was observed at position ?46 (A) from the start codon. The 5?-noncoding region had a putative TATA box at ?91 (TATATAA) and two possible CCAAT boxes at ?247 (CAAT) and ?283 (CCAAT). A cloned xynI cDNA was expressed in Saccharomycescerevisiae. The deduced amino acid sequence showed 94% identity with that of a previously reported equivalent gene (xynA) encoding a xylanase with an optimal pH of 4.8 from a color variant strain, NRRL Y-2311-1, of A. pullulans. A neighbor-joining tree showed that the Aureobasidium enzymes are closely related to several other family-11 xylanases from black aspergilli and Penicillium purpurogenum.
Creating Saccharomyces yeasts capable of efficient fermentation of pentoses such as xylose remains a key challenge in the production of ethanol from lignocellulosic biomass. Metabolic engineering of industrial Saccharomycescerevisiae strains has yielded xylose-fermenting strains, but these strains have not yet achieved industrial viability due largely to xylose fermentation being prohibitively slower than that of glucose. Recently, it has been shown that naturally occurring xylose-utilizing Saccharomyces species exist. Uncovering the genetic architecture of such strains will shed further light on xylose metabolism, suggesting additional engineering approaches or possibly even enabling the development of xylose-fermenting yeasts that are not genetically modified. We previously identified a hybrid yeast strain, the genome of which is largely Saccharomyces uvarum, which has the ability to grow on xylose as the sole carbon source. To circumvent the sterility of this hybrid strain, we developed a novel method to genetically characterize its xylose-utilization phenotype, using a tetraploid intermediate, followed by bulk segregant analysis in conjunction with high-throughput sequencing. We found that this strain’s growth in xylose is governed by at least two genetic loci, within which we identified the responsible genes: one locus contains a known xylose-pathway gene, a novel homolog of the aldo-keto reductase gene GRE3, while a second locus contains a homolog of APJ1, which encodes a putative chaperone not previously connected to xylose metabolism. Our work demonstrates that the power of sequencing combined with bulk segregant analysis can also be applied to a nongenetically tractable hybrid strain that contains a complex, polygenic trait, and identifies new avenues for metabolic engineering as well as for construction of nongenetically modified xylose-fermenting strains. PMID:15063853
Creating Saccharomyces yeasts capable of efficient fermentation of pentoses such as xylose remains a key challenge in the production of ethanol from lignocellulosic biomass. Metabolic engineering of industrial Saccharomycescerevisiae strains has yielded xylose-fermenting strains, but these strains have not yet achieved industrial viability due largely to xylose fermentation being prohibitively slower than that of glucose. Recently, it has been shown that naturally occurring xylose-utilizing Saccharomyces species exist. Uncovering the genetic architecture of such strains will shed further light on xylose metabolism, suggesting additional engineering approaches or possibly even enabling the development of xylose-fermenting yeasts that are not genetically modified. We previously identified a hybrid yeast strain, the genome of which is largely Saccharomyces uvarum, which has the ability to grow on xylose as the sole carbon source. To circumvent the sterility of this hybrid strain, we developed a novel method to genetically characterize its xylose-utilization phenotype, using a tetraploid intermediate, followed by bulk segregant analysis in conjunction with high-throughput sequencing. We found that this strain's growth in xylose is governed by at least two genetic loci, within which we identified the responsible genes: one locus contains a known xylose-pathway gene, a novel homolog of the aldo-keto reductase gene GRE3, while a second locus contains a homolog of APJ1, which encodes a putative chaperone not previously connected to xylose metabolism. Our work demonstrates that the power of sequencing combined with bulk segregant analysis can also be applied to a nongenetically tractable hybrid strain that contains a complex, polygenic trait, and identifies new avenues for metabolic engineering as well as for construction of nongenetically modified xylose-fermenting strains. PMID:22426884
The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from ''Primitivo'' grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic materi...
We have developed a genetic screen of the yeast Saccharomycescerevisiae to identify genes that act to coordinate DNA replication so that each part of the genome is copied exactly once per cell cycle. A mutant was recovered in this screen that accumulates aberrantly high DNA contents but does not complete a second round of synthesis. The mutation principally responsible for this phenotype is in the DOA4 gene, which encodes a ubiquitin hydrolase, one of several yeast genes that encode enzymes that can remove the signalling polypeptide ubiquitin hydrolase, one of several yeast genes that encode enzymes that can remove the signaling polypeptide ubiquitin from its covalently linked conjugated forms. DOA4 is nonessential, and deleting this gene causes uncoordinated replication. Overreplication does not occur in cells with limiting amounts of Cdc7 protein kinase, suggesting that entry into S phase is required for this phenotype. The DNA formed in doa4 mutants is not highly unusual in the sense that mitotic recombination rates are normal, implying that a high level of repair is not induced. The temperature sensitivity of doa4 mutations is partially suppressed by extra copies of the polyubiquitin gene UB14, but overreplication still occurs in the presence of this suppressor. Mutations in DOA4 cause loss of the free ubiquitin pool in cells under heat stress conditions, and extra copies of UB14 restore this pool without restoring coordination of replication. We conclude that a ubiquitin-mediated signaling event directly involving the ubiquitin hydrolase encoded by DOA4 is needed in S. cerevisiae to prevent uncoordinated DNA replication. PMID:8657109
Saccharomycescerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain. PMID:21642010
In metabolically engineered Saccharomycescerevisiae expressing Pichia stipitis XYL1 and XYL2 genes, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, xylitol is excreted as the major product during anaerobic xylose fermentation and only low yields of ethanol are produced. This has been interpreted as a result of the dual cofactor dependence of XR and the exclusive use of NAD{sup +} by XDH. The excretion of xylitol was completely stopped and the formation of glycerol and acetic acid were reduced in xylose utilising S. cerevisiae strains cultivated in oxygen-limited conditions by expressing lower levels of XR than of XDH. The expression level of XYL1 and XYL2 were controlled by changing the promoters and transcription directions of the genes. A new functional metabolic pathway was established when Thermus thermophilus xylA gene was expressed in S. cerevisiae. The recombinant strain was able to ferment xylose to ethanol when cultivated on a minimal medium containing xylose as only carbon source. In order to create a channeled metabolic transfer in the two first steps of the xylose metabolism, XYL1 and XYL2 were fused in-frame and expressed in S. cerevisiae. When the fusion protein, containing a linker of three amino acids, was co expressed together with native XR and XDH monomers, enzyme complexes consisting of chimeric and native subunits were formed. The total activity of these complexes exhibited 10 and 9 times higher XR and XDH activity, respectively, than the original conjugates, consisting of only chimeric subunits. This strain produced less xylitol and the xylitol yield was lower than with strains only expressing native XR and XDH monomers. In addition, more ethanol and less acetic acid were formed. A new gene encoding the cytoplasmic transhydrogenase from Azotobacter vinelandii was cloned. The enzyme showed high similarity to the family of pyridine nucleotide-disulphide oxidoreductase. To analyse the physiological effect of transhydrogenation between the two coenzyme systems NADP(H) and NAD(H) during anaerobic growth, S. cerevisiae was transformed with a plasma membrane bound AB-transhydrogenase from E. coli and with a cytoplasmic BB-transhydrogenase from A. vinelandii. Expression of both types changed the intracellular nucleotide levels. The NADPH/NADP{sup +} ratio was reduced while the NADH/NAD{sup +} ratio was almost constant. An increased formation of 2-oxoglutarate, glycerol and acetate was observed during anaerobic glucose fermentation 206 refs, 8 figs, 3 tabs
Due to declining drug discovery rates from organic synthetic libraries, pharmaceutical companies are turning their attention towards secondary metabolites. Isoprenoids, also known as terpenoids, constitute the largest known group of secondary metabolites isolated to date, encompassing more than 55,000 different compounds including several blockbuster drugs such as paclitaxel and artemisinin. All molecules within this group are biosynthesized from the same precursor called isopentenyl pyrophosphate (IPP), which is repeatedly polymerized and diversified giving rise to enormous chemical and structural diversity. The most common way of producing these compounds is by organic synthesis. Organic synthesis does however have several disadvantages for production of secondary metabolites such as low yields due to the complex structures, which makes this way of production economically unfeasible. Microbial production can easily be scaled to meet current demands and it is also an environmental benign production method compared to organic synthesis. Thus it would be attractive to engineer a microorganism to produce high amounts of IPP and other immediate prenyl precursors such as geranyl pyrophosphate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate for large-scale microbial production of terpenoids. Saccharomycescerevisiae was chosen as production platform due to its widespread use in industrial production and the waste number of molecular biology tools which is available for its manipulation. The effort for discovering new genetic perturbations, which would results in and increased production of isoprenoids by S. cerevisiae has been very limited. This project is focus on creating diversity within a lycopene producing S. cerevisiae strain by construction of gDNA-, cDNA-, and transposon-libraries. The diversified population of S. cerevisiae clones will afterwards be screened using the isoprenoid molecule lycopene as a model compound, hereby enabling the isolation of phenotypes producing higher amounts of isoprenoid. The property making lycopene ideal for screening is its system of 11 conjugated double bonds, which absorbs light within the visible range resulting in the red color of lycopene. This feature is the cause for the orange/red phenotype of S. cerevisiae strains transformed with the genes encoding lycopene and enables visual screening of yeast colonies, by searching for colonies with more intense red colony coloration which is the result of higher amount of lycopene is being produced and hence high amount of isoprenoid precursor being available. This will elucidate novel genetic targets for increasing isoprenoid production in S. cerevisiae
Autophagy is conserved throughout the eukaryotes and for many years, work in Saccharomycescerevisiae has been at the forefront of autophagy research. However as our knowledge of the autophagic machinery has increased, differences between S. cerevisiae and mammalian cells have become apparent. Recent work in other organisms, such as the amoeba Dictyostelium discoideum, indicate an autophagic pathway much more similar to mammalian cells than S. cerevisiae, despite its earlier evolutionary divergence. S. cerevisiae therefore appear to have significantly specialized, and the autophagic pathway in mammals is much more ancient than previously appreciated, which has implications for how we interpret data from organisms throughout the eukaryotic tree. PMID:22722653
There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomycescerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleot...
Proline protects yeast cells from damage caused by various stresses. A yeast Saccharomycescerevisiae mutant with high levels of intracellular proline grown in a minimal medium accumulated proline in its vacuole, but when grown in a nutrient medium, accumulated proline mainly in the cytosol. To understand the role of the proline pool in the vacuole, we examined the stress-protective effect of proline in proline-accumulating yeast cells deficient in vacuolar functions. The disruption of PEP3 encoding a vacuolar membrane protein required for vacuolar biogenesis caused hypersensitivity to heat shock and ethanol stresses, probably due to disappearance of normal vacuoles. The vph1-disrupted cells lacking vacuolar-ATPase activity showed resistance to heat shock without any change in proline localization, but showed severe growth defects in an ethanol-containing medium. These results indicate that vacuolar functions are involved in the stress-protective effect of proline in S. cerevisiae. Also, it appears that excess proline is transported to the vacuole in an ATP-independent manner.
Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [alpha-32P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomycescerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis. PMID:3907711
The Saccharomycescerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4). The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage. PMID:11266561
Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomycescerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1-1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae. PMID:11851922
In Kluyveromyces lactis, the expression of the major glucose permease gene RAG1 is controlled by extracellular glucose through a signaling cascade similar to the Saccharomycescerevisiae Snf3/Rgt2/Rgt1 pathway. We have identified a key component of the K. lactis glucose signaling pathway by characterizing a new mutation, rag20-1, which impairs the regulation of RAG1 and hexokinase RAG5 genes by glucose. Functional complementation of the rag20-1 mutation identified the KlSNF2 gene, which encodes a protein 59% identical to S. cerevisiae Snf2, the major subunit of the SWI/SNF chromatin remodeling complex. Reverse transcription-quantitative PCR and chromatin immunoprecipitation analyses confirmed that the KlSnf2 protein binds to RAG1 and RAG5 promoters and promotes the recruitment of the basic helix-loop-helix Sck1 activator. Besides this transcriptional effect, KlSnf2 is also implicated in the glucose signaling pathway by controlling Sms1 and KlRgt1 posttranscriptional modifications. When KlSnf2 is absent, Sms1 is not degraded in the presence of glucose, leading to constitutive RAG1 gene repression by KlRgt1. Our work points out the crucial role played by KlSnf2 in the regulation of glucose transport and metabolism in K. lactis, notably, by suggesting a link between chromatin remodeling and the glucose signaling pathway. PMID:23002104
Reducing xylitol formation is necessary in engineering xylose utilization in recombinant Saccharomycescerevisiae for ethanol production through xylose reductase/xylitol dehydrogenase pathway. To balance the expression of XYL1 and mutant XYL2 encoding xylose reductase (XR) and NADP+-dependent xylitol dehydrogenase (XDH), respectively, we utilized a strategy combining chassis selection and direct fine-tuning of XYL1 and XYL2 expression in this study. A XYL1 gene under the control of various promoters of ADH1, truncated ADH1 and PGK1, and a mutated XYL2 with different copy numbers were constructed into different xylose-utilizing modules, which were then expressed in two yeast chassises W303a and L2612. The strategy enabled an improved L2612-derived recombinant strain with XYL1 controlled by promoter PGK1 and with two copies of XYL2. The strain exhibited a 21.3% lower xylitol yield and a 40.0% higher ethanol yield. The results demonstrate the feasibility of the combinatorial strategy for construction of an efficient xylose-fermenting S. cerevisiae. PMID:18690426
A leuB strain of Thermus thermophilus TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomycescerevisiaeencoded by the LEU2 gene. The original strain could not grow at 50 degrees C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50 degrees, 60 degrees, 62 degrees, 65 degrees, 67 degrees, and 70 degrees C, step by step. All the mutant strains except for one isolated at 50 degrees C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Alal 12Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits. PMID:11302498
The fission yeast rad31-1 mutant is sensitive to both UV and ionising radiation and exhibits a growth defect at 35 degrees C. In addition, the mutant displays defects in cell morphology and nuclear division at 26 degrees C which are exaggerated at 35 degrees C. We have cloned the rad31 gene and have shown that it is not essential for viability, although cells containing a disrupted rad31 gene grow slowly. The null allele has similar cell and nuclear morphologies to the original allele and displays an extremely high frequency of loss of minichromosomes. rad31 is not required for either the S/M or G2/M checkpoint, however double mutant analysis indicates that rad31 acts in a process which is defective in the checkpoint rad mutants and which involves hus5 . Sequence analysis indicates that rad31 encodes a protein which is related to ubiquitin activating proteins and more particularly to an ORF in Saccharomycescerevisiae and to the Arabidopsis thaliana AXR1 and human APP-BP1 genes. We have isolated the S.cerevisiae sequence, which we have named RHC31 ( ad31homologue in S. erevisiae), since we show that it can complement the slow growth phenotype and radiation sensitivity of S.pombe rad31. PMID:9092625
The partial substitution of fossil fuels with bioethanol has become an important strategy for the use of renewable energy. Ethanol production is generally achieved through fermentation of starch or sugar-based feedstock by Saccharomycescerevisiae. In order to meet the growing demand for ethanol, there is a need for new yeast strains that can produce ethanol more efficiently and cost effectively. This paper presented a new genome engineering approach that was developed to improve ethanol production by S. cerevisiae. In this study, the aneuploid strain constructed on the base of tetraploid cells was shown to have favourable metabolic traits in very high gravity (VHG) fermentation with 300 g/L glucose as the carbon source. The tetraploid strain was constructed using the plasmid YCplac33-GHK, which comprised the HO gene encoding the site-specific HO endonucleases. The aneuploid strain, WT4-M, was chosen and screened once the tetraploid cells were treated with methyl benzimidazole-2-yl-carbamate to induce loss of mitotic chromosomes. The aneuploid strain WT4-M increased ethanol production as well as osmotic and thermal tolerance. The sugar to ethanol conversion rate also improved. It was concluded that this new approach is valuable for creating yeast strains with better fermentation characteristics. 25 refs., 3 figs.
All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomycescerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.
A Na+/H+ antiporter gene (CvNHA1) was cloned from the salt-tolerant yeast Candida versatilis. CvNHA1 encodes an antiporter with a typical yeast plasma membrane Na+/H+ antiporter structure. Transcription of CvNHA1 in C. versatilis cells was dependent on the salinity of the culture. When CvNHA1 was expressed in salt-sensitive Saccharomycescerevisiae cells, increased salt-tolerance was observed, indicating that Cvnha1p possesses an Na+/H+ antiporter function, because the increased salt-tolerance was dependent on the extracellular pH. It appears that Cvnha1p mediates only the transport of Na+. In an S. cerevisiae transformant harboring a CvNHA1-EGFP fusion plasmid in which the greater part of the C-terminal hydrophilic region of Cvnha1p was deleted by fusion with enhanced green fluorescent protein (EGFP), the Cvnha1-EGFP fusion protein was localized mainly in the plasma membrane, and the NaCl-tolerance of this transformant was greater than that of a strain harboring the entire CvNHA1 gene.
Brome mosaic virus (BMV) RNA replication has been examined in a number of systems, including Saccharomycescerevisiae. We developed an efficient T-DNA-based gene delivery system using Agrobacterium tumefaciens to transiently express BMV RNAs in Nicotiana benthamiana. The expressed RNAs can systemically infect plants and provide material to extract BMV replicase that can perform template-dependent RNA-dependent RNA synthesis in vitro. We also expressed the four BMV-encoded proteins from nonreplicating RNAs and analyzed their effects on BMV RNA accumulation. The capsid protein that coinfiltrated with constructs expressing RNA1 and RNA2 suppressed minus-strand levels but increased plus-strand RNA accumulation. The replication proteins 1a and 2a could function in trans to replicate and transcribe the BMV RNAs. None of the BMV proteins or RNA could efficiently suppress posttranscriptional silencing. However, 1a expressed in trans will suppress the production of a recombinant green fluorescent protein expressed from the nontranslated portions of BMV RNA1 and RNA2, suggesting that 1a may regulate translation from BMV RNAs. BMV replicase proteins 1a did not affect the accumulation of the BMV RNAs in the absence of RNA replication, unlike the situation reported for S. cerevisiae. This work demonstrates that the Agrobacterium-mediated gene delivery system can be used to study the cis- and trans-acting requirements for BMV RNA replication in plants and that significant differences can exist for BMV RNA replication in different hosts. PMID:16254357
In the yeast Saccharomycescerevisiae, the unfolded protein response (UPR) involves the unconventional splicing of HAC1 mRNA, which is mediated by the activated Ire1p transmembrane kinase/endonuclease. In this study, we isolated and characterized a Yarrowia lipolytica HAC1 (YlHAC1) encoding a basic-leucine zipper transcription factor. The null mutant strain of YlHAC1 (DeltaYlhac1) displayed a significantly increased sensitivity to dithiothreitol (DTT) and tunicamycin (TM), along with a defect in hyphal growth, suggesting the essential function of YlHAC1 in UPR. The unconventional splicing of YlHAC1 mRNA occurred under the UPR conditions induced by DTT or TM treatment. Unlike S. cerevisiae HAC1 mRNA with an intron of 252 nt, YlHAC1 mRNA was shown to harbour a short intron of length 29 nt. The YlHAC1 mRNA harboured the nucleotides CAG, conserved at the intron borders in the filamentous fungi hac1/hacA and mammalian XBP1, as well as a conserved bipartite element within the 3' untranslated region. The expression of the spliced form of YlHAC1 mRNA in the wild-type andDeltaYlhac1 strains resulted in an increased resistance to DTT, thereby indicating that the spliced form is translated into a functional YlHac1p. PMID:20162530
Laboratory evolution is a powerful approach in applied and fundamental yeast research, but complete elucidation of the molecular basis of evolved phenotypes remains a challenge. In this study, DNA microarray-based transcriptome analysis and whole-genome resequencing were used to investigate evolution of novel lactate transporters in Saccharomycescerevisiae that can replace Jen1p, the only documented S. cerevisiae lactate transporter. To this end, a jen1? mutant was evolved for growth on lactate in serial batch cultures. Two independent evolution experiments yielded growth on lactate as sole carbon source (0.14 and 0.18 h(-1) , respectively). Transcriptome analysis did not provide leads, but whole-genome resequencing showed different single-nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Introduction of these ADY2 alleles in a jen1? ady2? strain enabled growth on lactate (0.14 h(-1) for Ady2p(Leu219Val) and 0.12 h(-1) for Ady2p(Ala252Gly) ), demonstrating that these alleles of ADY2 encode efficient lactate transporters. Depth of coverage of DNA sequencing, combined with karyotyping, gene deletions and diagnostic PCR, showed that an isochromosome III (c. 475 kb) with two additional copies of ADY2(C755G) had been formed via crossover between retrotransposons YCLW?15 and YCRC?6. The isochromosome formation shows how even short periods of selective pressure can cause substantial karyotype changes. PMID:22257278
The past decades witnessed extensive efforts to study the relationship among proteins. Particularly, sequence-based protein-protein interactions (PPIs) prediction is fundamentally important in speeding up the process of mapping interactomes of organisms. High-throughput experimental methodologies make many model organism's PPIs known, which allows us to apply machine learning methods to learn understandable rules from the available PPIs. Under the machine learning framework, the composition vectors are usually applied to encode proteins as real-value vectors. However, the composition vector value might be highly correlated to the distribution of amino acids, i.e., amino acids which are frequently observed in nature tend to have a large value of composition vectors. Thus formulation to estimate the noise induced by the background distribution of amino acids may be needed during representations. Here, we introduce two kinds of denoising composition vectors, which were successfully used in construction of phylogenetic trees, to eliminate the noise. When validating these two denoising composition vectors on Escherichia coli (E. coli), Saccharomycescerevisiae (S. cerevisiae) and human PPIs datasets, surprisingly, the predictive performance is not improved, and even worse than non-denoised prediction. These results suggest that the noise in phylogenetic tree construction may be valuable information in PPIs prediction. PMID:22999977
A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomycescerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta. PMID:8262953
We describe a new Escherichia coli vector (pON5) that allows positive selection for recombinant clones. In this plasmid, the bla gene from pBR322 is permanently active, whereas the neo gene from transposon Tn5 is repressed by the cI-encoded lambda repressor. When DNA is inserted into the Bc/I or HindIII restriction sites situated within the cI gene, the neo gene becomes transcribed from the lambda pR promoter. We have also made a Schizosaccharomyces pombe derivative of pON5 (= pON163) by introducing the fission yeast ars1 and ura4+ sequences. We show that this plasmid is capable of transforming Sc. pombe ura4 strains, as well as ura 3 strains of the distantly related budding yeast Saccharomycescerevisiae. We have used pON163 for the construction of two fission yeast genomic libraries. From these gene banks clones were isolated that were able to complement fission yeast his2 mutants. Such plasmids could also rescue his4C mutants of Sa. cerevisiae, defective in the histidinol dehydrogenase activity of the multifunctional HIS4 gene product. Finally, we describe the plasmid pDW232 which is useful for functional analysis of fission yeast genes. It is a pGEM3 derivative adapted to fission yeast, carrying multiple cloning sites between the T7 and SP6 promoters, together with ars1 and ura4+ from Sc. pombe.
Mitochondria are essential organelles required for a number of key cellular processes. As most mitochondrial proteins are nuclear encoded, their efficient translocation into the organelle is critical. Transport of proteins across the inner membrane is driven by a multicomponent, matrix-localized "import motor," which is based on the activity of the molecular chaperone Hsp70 and a J-protein cochaperone. In Saccharomycescerevisiae, two paralogous J-proteins, Pam18 and Mdj2, can form the import motor. Both contain transmembrane and matrix domains, with Pam18 having an additional intermembrane space (IMS) domain. Evolutionary analyses revealed that the origin of the IMS domain of S. cerevisiae Pam18 coincides with a gene duplication event that generated the PAM18/MDJ2 gene pair. The duplication event and origin of the Pam18 IMS domain occurred at the relatively ancient divergence of the fungal subphylum Saccharomycotina. The timing of the duplication event also corresponds with a number of additional functional changes related to mitochondrial function and respiration. Physiological and genetic studies revealed that the IMS domain of Pam18 is required for efficient growth under anaerobic conditions, even though it is dispensable when oxygen is present. Thus, the gene duplication was beneficial for growth capacity under particular environmental conditions as well as diversification of the import motor components. PMID:21245414
Candida albicans, Saccharomycescerevisiae and their cell wall components, zymosan and glucan, have been shown to stimulate interleukin-8 (IL-8/CXCL-8) production by intestinal epithelial cell-like Caco-2 cells pre-cultured with 10 mM butyric acid. We examined in this study whether these yeasts also altered the production of other cytokines and cyclooxygenases (COXs) by Caco-2 cells. Culturing Caco-2 cells with 10 mM butyric acid and 15% FBS for 4 days enhanced the basal levels of mRNA encoding IL-6, IL-8, IL-18, monocyte chemoattractant protein (MCP)-1, stem cell factor, transforming growth factor (TGF)-?1, TGF-?3, tumor necrosis factor (TNF)-?, COX-1, and COX-2, but not of granulocyte-macrophage colony-stimulating factor (GM-CSF) and TGF-?2. The inclusion of live S. cerevisiae or C. albicans further enhanced the production of IL-8, but not of the other cytokines and COXs. The non-pathogenic yeasts, C. kefyr, C. utilis, C. versatilis, Kluyveromyces lactis, K. marxianus, Schizosaccharomyces pombe and Zygosaccharomyces rouxii, used for the production of fermented foods and probiotics, and the opportunistic pathogens, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis, isolated from human tissue samples also enhanced IL-8 secretion by Caco-2 cells.
Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies was asked to provide a scientific opinion on a health claim pursuant to Article 13 of Regulation (EC) No 1924/2006 in the framework of further assessment related to Saccharomycescerevisiae var. boulardii CNCM I-1079 and defence against pathogenic gastro-intestinal microorganisms. The food constituent that is the subject of the health claim, Saccharomycescerevisiae var. boulardii CNCM I-1079, is sufficiently characterised. The claimed effect, defence against pathogenic gastro-intestinal microorganisms, is a beneficial physiological effect. The proposed target population is the general population. The Panel notes that the evidence provided is not sufficient to establish that the strains Saccharomycescerevisiae var. boulardii CNCM I-1079 and Saccharomycescerevisiae var. boulardii Hansen CBS 5926, which were used in the studies provided, are identical, and considers that owing to the possibility that the effects are strain-specific, results obtained with one strain cannot be extrapolated to another strain. On the basis of the data presented, the Panel concludes that a cause and effect relationship has not been established between the consumption of Saccharomycescerevisiae var. boulardii CNCM I-1079 and defence against pathogenic gastro-intestinal microorganisms.
DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I-IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomycescerevisiae. Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian DNA ligase IV. Unlike CDC9, LIG4 is not essential for DNA replication, RAD52-dependent homologous recombination nor the repair of UV light-induced DNA damage. Instead, it encodes a crucial component of the non-homologous end-joining (NHEJ) apparatus, which repairs DNA double-strand breaks that are generated by ionizing radiation or restriction enzyme digestion: a function which cannot be complemented by CDC9. Lig4p acts in the same DNA repair pathway as the DNA end-binding protein Ku. However, unlike Ku, it does not function in telomere length homeostasis. These findings indicate diversification of function between different eukaryotic DNA ligases. Furthermore, they provide insights into mechanisms of DNA repair and suggest that the NHEJ pathway is highly conserved throughout the eukaryotic kingdom. PMID:9303323
Nutrient-limited Saccharomycescerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G(1). PMID:12582131
A pyrithiamine (PT) resistance gene (ptrA) was cloned from a genomic DNA library prepared from a PT resistant mutant of Aspergillus oryzae. It conferred high resistance to PT on an A. oryzae industrial strain as well as A. nidulans. Nucleotide sequence analysis showed that the ptrA gene contained one intron (58-bp) and encodes 327 amino acid (aa) residues. Additionally, the deduced aa sequence has 72% and 63% identity to Fusarium solani sti35 encoding a stress-inducible protein and Saccharomycescerevisiae THI4 encoding an enzyme involved in thiamine biosynthesis, respectively, indicating that ptrA is a mutated allele of a gene belonging to the THI4 family. The mutation point was identified in the conserved motif in 5?-flanking region of these three THI4 homologous genes (ptrA, sti35, and THI4). The introduction of the ptrA gene allowed an A. oryzae industrial strain to grow on the minimum medium containing PT (0.1 mg/l) on which an untransformed strain did not grow. This result indicates that the ptrA is applicable as a dominant selectable marker for transformation of A. oryzae.
The Ustilago maydis viral (UmV) genome consists of three distinct size groups of double-stranded RNA (dsRNA) segments: H (heavy), M (medium), and L (light). The H segments have been suggested to encode all essential viral proteins, but without any molecular evidences. As a preliminary step to understand viral genomic organization and the molecular mechanism governing gene expression in UmV, we determined the complete nucleotide sequence of the H1 dsRNA genome in P1 viral killer subtype. The H1 dsRNA genome (designated UmV-H1) contained a single open reading frame that encodes a polyprotein of 1820 residues, which is predicted to be autocatalytically processed by a viral papain-like protease to generate viral proteins. The amino-terminal region is the capsid polypeptide with a predicted molecular mass of 79.9 kDa. The carboxy-terminal region is the RNA-dependent RNA polymerase (RDRP) that has a high sequence homology to those of the totiviruses. The H2 dsRNA also encodes a distinct RDRP, suggesting that UmV is a complex virus system like the Saccharomycescerevisiae viruses ScV-L1 and -La. PMID:11410317
We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomycescerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (YP/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the YP/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the YP/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H+ to NADPH+H+. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ?aceE ?pqo ?ilvE ?ldhA ?mdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h?1, and showed an overall YP/S of about 0.48 mol per mol of glucose in the production phase.
Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomycescerevisiae, PAP is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1 dpp1 lpp1 triple mutant indicated another gene(s) encoding the enzyme. We purified PAP from the pah1 dpp1 lpp1 triple mutant by salt-extraction of mitochondria, followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, we found that APP1, a gene whose molecular function has been unknown, confers ~30 % PAP activity of wild type cells. The overexpression of APP1 in the pah1 dpp1 lpp1 mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1 mutation into the pah1 dpp1 lpp1 triple mutant resulted in a complete loss of PAP activity, indicating that distinct PAP enzymes in S. cerevisiae are encoded by APP1, PAH1, DPP1, and LPP1. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity. PMID:23071111
Wine colour, phenolics and volatile fermentation-derived composition are the quintessential elements of a red wine. Many viticultural and winemaking factors contribute to wine aroma and colour with choice of yeast strain being a crucial factor. Besides the traditional Saccharomyces species S. cerevisiae, S. bayanus and several Saccharomyces interspecific hybrids are able to ferment grape juice to completion. This study examined the diversity in chemical composition, including phenolics and fermentation-derived volatile compounds, of an Australian Cabernet Sauvignon due to the use of different Saccharomyces strains. Eleven commercially available Saccharomyces strains were used in this study; S. cerevisiae (7), S. bayanus (2) and interspecific Saccharomyces hybrids (2). The eleven Cabernet Sauvignon wines varied greatly in their chemical composition. Nine yeast strains completed alcoholic fermentation in 19 days; S. bayanus AWRI 1375 in 26 days, and S. cerevisiae AWRI 1554 required 32 days. Ethanol concentrations varied in the final wines (12.7-14.2 %). The two S. bayanus strains produced the most distinct wines, with the ability to metabolise malic acid, generate high glycerol concentrations and distinctive phenolic composition. Saccharomyces hybrid AWRI 1501 and S. cerevisiae AWRI 1554 and AWRI 1493 also generated distinctive wines. This work demonstrates that the style of a Cabernet Sauvignon can be clearly modulated by choice of commercially available wine yeast. PMID:22878903
In the yeast Saccharomycescerevisiae inactivation of trehalose-6-phosphate (Tre6P) synthase (Tps1) encoded by the TPS1 gene causes a specific growth defect in the presence of glucose in the medium. The growth inhibition is associated with deregulation of the initial part of glycolysis. Sugar phosphates, especially fructose-1,6-bisphosphate (Fru1,6bisP), hyperaccumulate while the levels of ATP, Pi and downstream metabolites are rapidly depleted. This was suggested to be due to the absence of Tre6P inhibition on hexokinase. Here we show that overexpression of Tre6P (as well as glucose-6-phosphate (Glu6P))-insensitive hexokinase from Schizosaccharomyces pombe in a wild-type strain does not affect growth on glucose but still transiently enhances initial sugar phosphate accumulation. We have in addition replaced the three endogenous glucose kinases of S. cerevisiae by the Tre6P-insensitive hexokinase from S. pombe. High hexokinase activity was measured in cell extracts and growth on glucose was somewhat reduced compared to an S. cerevisiae wild-type strain but expression of the Tre6P-insensitive S. pombe hexokinase never caused the typical tps1Delta phenotype. Moreover, deletion of TPS1 in this strain expressing only the Tre6P-insensitive S. pombe hexokinase still resulted in a severe drop in growth capacity on glucose as well as sensitivity to millimolar glucose levels in the presence of excess galactose. In this case, poor growth on glucose was associated with reduced rather than enhanced glucose influx into glycolysis. Initial glucose transport was not affected. Apparently, deletion of TPS1 causes reduced activity of the S. pombe hexokinase in vivo. Our results show that Tre6P inhibition of hexokinase is not the major mechanism by which Tps1 controls the influx of glucose into glycolysis or the capacity to grow on glucose. In addition, they show that a Tre6P-insensitive hexokinase can still be controlled by Tps1 in vivo. PMID:14507429
Bioethanol produced by microbial fermentations of plant biomass hydrolysates consisting of hexose and pentose mixtures is an excellent alternative to fossil transportation fuels. However, the yeast Saccharomycescerevisiae, commonly used in bioethanol production, can utilize pentose sugars like l-arabinose or d-xylose only after heterologous expression of corresponding metabolic pathways from other organisms. Here we report the improvement of a bacterial l-arabinose utilization pathway consisting of l-arabinose isomerase from Bacillus subtilis and l-ribulokinase and l-ribulose-5-P 4-epimerase from Escherichia coli after expression of the corresponding genes in S. cerevisiae. l-Arabinose isomerase from B. subtilis turned out to be the limiting step for growth on l-arabinose as the sole carbon source. The corresponding enzyme could be effectively replaced by the enzyme from Bacillus licheniformis, leading to a considerably decreased lag phase. Subsequently, the codon usage of all the genes involved in the l-arabinose pathway was adapted to that of the highly expressed genes encoding glycolytic enzymes in S. cerevisiae. Yeast transformants expressing the codon-optimized genes showed strongly improved l-arabinose conversion rates. With this rational approach, the ethanol production rate from l-arabinose could be increased more than 2.5-fold from 0.014 g ethanol h(-1) (g dry weight)(-1) to 0.036 g ethanol h(-1) (g dry weight)(-1) and the ethanol yield could be increased from 0.24 g ethanol (g consumed l-arabinose)(-1) to 0.39 g ethanol (g consumed l-arabinose)(-1). These improvements make up a new starting point for the construction of more-efficient industrial l-arabinose-fermenting yeast strains by evolutionary engineering. PMID:18263741
The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14?-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomycescerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance. PMID:21478305
The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14?-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomycescerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance. PMID:15809418
The ChrA membrane protein belongs to the CHR superfamily of chromate ion transporters, which includes homologues from bacteria, archaea and eukaryotes. Bacterial ChrA homologues confer chromate resistance by exporting chromate ions from the cell's cytoplasm. The Neurospora crassa strain 74-A chr-1 gene encodes a putative CHR-1 protein of 507 amino acid residues, which belongs to the CHR superfamily. RT-PCR assays showed that expression of the chr-1 gene was up-regulated by chromate exposure of N. crassa cultures. Introduction in N. crassa of sense and antisense fragments of the chr-1 gene, as part of a silencing module within the pSilent-1 vector, produced transformants with a phenotype of resistance to chromate and diminished accumulation of chromium, as compared with the control strain containing only the vector. A chromate-resistance phenotype was also observed in N crassa strains deleted in the genomic chr-1 gene, thus confirming that the absence of CHR-1 protein confers chromate resistance to the fungus. The cDNA from N. crassa chr-1 gene (Ncchr-1) was cloned into the pYES2 vector under the control of a GAL promoter and the resulting recombinant plasmid was transferred to the yeast Saccharomycescerevisiae. Galactose-induced S. cerevisiae transformants expressing Ncchr-1 were more sensitive to chromate and accumulated 2.5 times more chromium than the induced strain containing only the vector. Excess sulfate, a chromate analog, was unable to protect S. cerevisiae chr-1 transformants from chromate toxicity. These data indicate that the N. crassa CHR-1 protein functions as a transporter that takes up chromate; it also appears that this transport occurs in a sulfate-independent fashion. This is the first report assigning a role as a chromate transporter to a nonbacterial CHR protein. PMID:23085746
The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/ADP(+) and has a significant influence on product formation in microorganisms. The enzyme pyridine nucleotide transhydrogenase, which can catalyse transfer of reducing equivalents between the two nucleotide systems, occurs in several organisms, but not in yeasts, The purpose of this work was to analyse how metabolism during anaerobic growth of Saccharomycescerevisiae might be altered when transfer of reducing equivalents between the two systems is made possible by expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii. We therefore cloned sth, encoding this enzyme and expressed it under the control of a S, cerevisiae promoter in a strain derived from the industrial model strain S, cerevisiae CBS8066, Anaerobic batch cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein was measured in the extracts from the strain expressing the sth gene from A. vinelandii, while no transhydrogenase activity could be detected in control strains without the gene. Production of the transhydrogenase caused a significant increase in formation of glycerol and 2-oxoglutarate. Since NADPH is used to convert 2-oxoglutarate to glutamate while glycerol formation increases when excess NADH is formed, this suggested that transhydrogenase converted NADH and NADP(+) to NAD+ and NADPH. This was further supported by measurements of the intracellular nucleotide concentrations. Thus, the (NADPH/ NADP(+)):(NADH/NAD(+)) ratio was reduced from 35 to 17 by the transhydrogenase. The increased formation of 2-oxoglutarate was accompanied by a two-fold decrease in the maximal specific growth rate. Also the biomass and ethanol yields were significantly lowered by the transhydrogenase. Copyright (C) 2000 John Whey & Sons, Ltd.
S-adenosyl-L-methionine (SAM) is an essential metabolite in all living cells, and which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. The current study was carried out to obtain an industrial strain with overproduction of SAM. The wild-type strain, Saccharomycescerevisiae CGMCC 1226, was subjected to successive mutagenic with ultraviolet irradiation (UV) coupled with ethionine-resistant screening procedure to achieve a rapid improvement of S-adenosyl-L-methionine production in Saccharomycescerevisiae. A high SAM yield strain, designated as Saccharomycescerevisiae CGMCC 2842, was successfully selected and exhibited higher SAM synthetase activity which was increased by 2.7-fold in comparison with the wild-type strain. Meanwhile, t...
One hundred and seventy yeast strains belonging to 14 genera and 29 species were isolated from 112 water samples of the river Danube in the area of Bratislava. The samples were collected through the year from April to March. Saccharomycescerevisiae, Candida maltosa, Aureobasidium pullulans, Cystofilobasidium capitatum, Rhodotorula glutinis, Geotrichum candidum, and Candida krusei were the most frequent. The basidiomycetous yeasts and yeast-like organisms with oxidative metabolism were present in approximately equal numbers to those with fermentative metabolism. Saccharomycescerevisiae was the dominant yeast and was isolated from 50% of all samples examined and represented approximately one quarter of the yeast community. Yeast densities ranged from 100 to 21,100 CFU per litre. The highest population density was observed in October. Cryptococcus albidus, Saccharomycescerevisiae, Rhodotorula glutinis, and Aureobasidium pullulans formed the main part of the yeast population in this month. PMID:9298185
Through genome-wide transcript analysis of a reference strain and two recombinant Saccharomycescerevisiae strains with different rates of galactose uptake, we obtained information about the global transcriptional response to metabolic engineering of the GAL gene regulatory network. One of the recombinant strains overexpressed the gene encoding the transcriptional activator Gal4, and in the other strain the genes encoding Gal80, Gal6, and Mig1, which are negative regulators of the GAL system, were deleted. Even though the galactose uptake rates were significantly different in the three strains, we surprisingly did not find any significant changes in the expression of the genes encoding the enzymes catalyzing the first steps of the pathway (i.e., the genes encoding Gal2, Gal1, Gal7, and Gal10). We did, however, find that PGM2, encoding the major isoenzyme of phosphoglucomutase, was slightly up-regulated in the two recombinant strains with higher galactose uptake rates. This indicated that PGM2 is a target for overexpression in terms of increasing the flux through the Leloir pathway, and through overexpression of PGM2 the galactose uptake rate could be increased by 70% compared to that of the reference strain. Based on our findings, we concluded that phosphoglucomutase plays a key role in controlling the flux through the Leloir pathway, probably due to increased conversion of glucose-l-phosphate to glucose-6-phosphate. This conclusion was supported by measurements of sugar phosphates, which showed that there were increased concentrations of glucose-6-phosphate, galactose-6-phosphate, and fructose-6-phosphate in the strain construct overexpressing PGM2.
Yeasts Candida kefyr NCYC143, Candida utilis CUM, Kluyveromyces lactis KL71, Saccharomyces bayanus SB1, Saccharomycescerevisiae EC1118, Saccharomyces chevalieri CCICC1028, Candida famata (previously Torulopsis candida) CCICC1041 and Williopsis saturnus var. saturnus CBS254 were screened for their ability to produce flavour-active methionol (3-methylthio-1-propanol) in coconut cream supplemented with l-methionine. The yeasts varied with their ability to produce methionol from methionine with Saccharomycescerevisiae EC1118 producing the most, followed by Kluyveromyces lactis KL71. Little methionol was produced by the other yeasts. Methionol production by Kluyveromyces lactis KL71 was subjected to further studies under different conditions of initial pH (4.0-6.3), temperature (20-33^oC), l-...
Abstract The effect of fungal suspensions on the disinfectant solution (Actisept with sodium dichloroisocyanurate as active substance, which releases 140 ppm active chlorine) and the corrosion behavior of type AISI 304 stainless steel (SS) were evaluated through linear polarization. The work presents a study of biocide Actisept solution as reference solution and the mixed solutions: reference biocide Actisept solution with three fungal suspensions (10 vol%), Aspergillus niger, Candida mycoderma, and Saccharomycescerevisiae. The fungal suspension has an important influence on the synergistic effect of the SS corrosion in the following order: Saccharomycescerevisiae > Candida mycoderma > Aspergillus niger. The Ecorr values from the mixed solutions decreased with immersion time of the sampl...
Abstract in portuguese RACIONAL: Anticorpos anti-Saccharomycescerevisiae antibodies, considerados marcadores sorológicos para a doença de Crohn, foram descritos em pacientes com doença celíaca, desaparecendo após dieta isenta de glúten. OBJETIVOS: Avaliação da positividade de anti-Saccharomycescerevisiae antibodies em pacientes com doença de Crohn e doença celíaca, em relação a indivíduos sadios da mesma área geográfica. MÉTODOS: Foram estudados 145 pacientes, 36 com doença (more) de Crohn e 52 com doença celíaca que preencheram os critérios diagnósticos para ambas as afecções, e 57 indivíduos sadios para controle. Os pacientes celíacos foram divididos como segue: ao diagnóstico (grupo doença celíaca I: n = 34), obedientes à dieta isenta de glúten (grupo doença celíaca II: n = 13) e não-aderentes à dieta isenta de glúten (grupo doença celíaca III: n = 5). Anti-Saccharomycescerevisiae antibodies IgA e IgG foram determinados por ELISA. RESULTADOS: Anti-Saccharomycescerevisiae antibodies IgA foi positivo na doença de Crohn, nos celíacos ao diagnóstico e nos transgressores à dieta, com significado estatístico. Anti-Saccharomycescerevisiae antibodies IgG foi positivo na doença de Crohn e em todos os grupos de celíacos, com significado estatístico. CONCLUSÕES: A detecção de anti-Saccharomycescerevisiae antibodies em pacientes com doença celíaca permite sugerir que o mesmo não seja marcador específico para a doença de Crohn, mas que esteja associado à inflamação do intestino delgado. A positividade de anti-Saccharomycescerevisiae antibodies pode ser decorrente de fatores genéticos e aumento da permeabilidade intestinal. Abstract in english CONTEXT: Anti-Saccharomycescerevisiae antibodies (ASCA), considered serologic markers for Crohn's disease, were described in patients with celiac disease, disappearing after a gluten-free diet. OBJECTIVES: Evaluation of ASCA positivity in patients with Crohn's disease and celiac disease in relation to healthy individuals. METHODS: A total of 145 individuals were studied: 36 with Crohn's disease and 52 with celiac disease, that fulfilled the diagnostic criteria for both a (more) ffections, and 57 healthy individuals for control. The celiac patients were divided as follow: group CeD I at diagnosis (n = 34), group CeD II with gluten-free diet compliance (n = 13) and group CeD III with transgressions to the diet (n = 5). ASCA IgA and IgG were determined by ELISA. RESULTS: With statistical significance, ASCA IgA were positive in Crohn's disease, celiac disease at diagnosis and celiac disease with diet transgressions; ASCA IgG in Crohn's disease and in all groups with celiac disease. CONCLUSIONS: The detection of ASCA in patients with celiac disease allows to suggest that ASCA is not a specific marker for Crohn's disease, but was associated with the inflammation of the small intestine. The increased levels of positive ASCA may be due to genetic factors and increased intestinal permeability.
The transcriptional activator protein Gcn4p increases the transcription of many genes that code for amino acid synthesis genes during amino acid starvation in Saccharomycescerevisiae. Here we showed that after the disruption of C-GCN4, a homolog in Candida maltosa of GCN4 in S. cerevisiae, formed pseudohyphae in minimal medium. This is the first report that a GCN4 homolog is involved in the control of morphological transition.
For direct and efficient ethanol production from cellulosic materials, we screened optimal cellulases from symbiotic protists of termites through heterologous expression with Saccharomycescerevisiae. 11 cellulases, belonging to glycoside hydrolase families 5, 7, and 45 endoglucanases (EGs), were confirmed to produce with S. cerevisiae for the first time. A recombinant yeast expressing SM2042B24 EG I was more efficient at degrading carboxylmethyl cellulose than was Trichoderma reesei EG I, a major EG with high cellulolytic activity.
Examines the potential of sugar beet as a raw material for ethanol fermentation. The ethanol production from sugarbeet by 23 strains of Saccharomyces belonging to cerevisiae and uvarum species and four strains of Zymomonas mobilis was studied in batch fermentation. After screening, S. cerevisiae CDRI NTG was compared. The economics of ethanol production from sugar beet was calculated and the cost was found to be Rs. 2.50 per liter (excluding excise duty). (Refs. 187).
Large differences in colony forming capacity, ethanol production and inhibitor conversion were noted between nine different strains of Saccharomycescerevisiae in anaerobic batch and fed-batch cultures on dilute acid wood hydrolysate. S. cerevisiae ATCC 96581 was able to metabolize all added glucose and mannose in fed-batch experiments. The choice of production strain will have a significant effect on the performance of a hydrolysate-based ethanol production plant.
Mouse pancreatic ..cap alpha..-amylase complementary DNA was inserted into a yeast shuttle vector after the Saccharomycescerevisiae MF..cap alpha..1 promoter and secretion signals coding sequences. When transformed with the recombinant plasmid, S. cerevisiae cells were able to synthesize and secrete functional ..cap alpha..-amylase, efficiently hydrolyzing starch present in the culture medium. Stable amylolytic cells were obtained from different yeast strains. This work represents a significant step towards producing yeast that can convert starchy materials directly to ethanol.
This study has had the purpose of enhancing the knowledge on the dominant strains of ethanol fermentation process biomass to use as bio fuel. The attributes studied were: numerical taxonomy (Griffits, 1981) and fermentative performance. The results have showed that all strains have presented good fermentative characteristics. All strains have been classified as Saccharomyces. In this genus were found the follow species: chevalieri, coreanus cerevisiae. If the classification according Barnet (1992) is considered, all of them are representative of S. cerevisiae. (author)
The addition of 0.1% caffeine to the plating medium markedly reduced the ozone-survival of the wild-type and the rad1 and rad6 mutants of Saccharomycescerevisiae, whereas no effect was observed in the rad52 mutant. Since, in S. cerevisiae, caffeine has been reported to interfere with the recombinational repair pathway under the control of the RAD52 gene, these results support previous observations suggesting that this pathway is involved in the repair of ozone-induced DNA damage.
Mixed-species biofilm was remarkably formed in a static co-culture of Lactobacillus plantarum ML11-11 and Saccharomycescerevisiae Y11-43 isolated from brewing samples of Fukuyama pot vinegar. Mixed-species biofilm is probably formed by direct cell-cell contact between ML11-11 and S. cerevisiae including Y11-43 and laboratory yeast strains. Scanning electron microscopic observation suggested that the mixed-species biofilm had a thick, bi-layer structure.
The crt gene clusters responsible for the biosynthesis of carotenoids such as lycopene, beta-carotene and astaxanthin have been isolated from carotenogenic bacteria such as Erwinia species and the marine bacterium Agrobacterium aurantiacum. The functions of the individual genes have been identified. The first substrate of the enzymes encoded by the Erwinia crt clusters is farnesyl pyrophosphate which is not only the precursor for carotenoid biosynthesis but also sterols, dolichols and other numerous isoprenoid compounds. Escherichia coli does not naturally synthesize carotenoids, but by using the carotenogenic genes recombinant strains accumulating lycopene, beta-carotene and astaxanthin have been produced. Other non-carotenogenic bacteria such as Zymomonas mobilis have also been engineered to produce beta-carotene by the introduction of the corresponding crt genes. A gene capable of enhancing carotenoid levels in E. coli has also been isolated from cDNA libraries of the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. This gene has been found to encode an isopentenyl pyrophophate isomerase. It has further been shown that the edible yeasts Candida utilis as well as Saccharomycescerevisiae, which possess no carotenoid biosynthetic pathway, acquire the ability to produce carotenoids, when the carotenogenic genes are expressed under the control of yeast-derived promoters and terminators. It has been observed in the yeasts S. cerevisiae and C. utilis carrying the lycopene biosynthesis genes that ergosterol content is decreased by 10 and 35%, respectively. It is therefore likely that the carbon flux for the ergosterol biosynthesis has been partially directed from farnesyl pyrophosphate to a new pathway for the lycopene biosynthesis. Further, the expression of a truncated gene which codes for the catalytic domain of the endogenous 3-hydroxy-3-methylglutaryl coenzyme. A reductase, has been found to be effective for enhancing carotenoid levels in the yeast C. utilis. PMID:9519479
When Saccharomyces oviformis and Saccharomycescerevisiae were cultivated aerobically and anaerobically in a synthetic liquid medium containing low concentrations (7.0-12.6 g/L) of glucose, more EtOH was produced under anaerobic conditions. In aerobiosis, most EtOH was utilized by yeast for the biosynthesis of AcCoA and citrate (incomplete glycolysis). In anaerobiosis, 24-30% of glucose was assimilated to AcCoA and citrate, and 70-76% was fermented to EtOH.
The effect of cadmium chloride on growth and metabolism was tested in Saccharomycescerevisiae and Rhodotorula rubra. The uptake of cadmium in both yeast strains is the same, but Saccharomyces cells are much more sensitive to cadmium than Rhodotorula cells. In both yeast strains the effect of cadmium on protein synthesis and on transport of glucose or adenine through membranes is very low, the effect on RNA - and ribosome synthesis very high. 28 references, 7 figures.
Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomycescerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis. PMID:9271115
In Saccharomycescerevisiae, the PUT1-encoded proline oxidase and the PUT2-encoded ?1-pyrroline-5-carboxylate dehydrogenase are required to convert proline to glutamate. We recently showed that a put1 disruptant accumulated higher levels of proline intracellularly and conferred higher resistance to freezing stress. Here, we determined the effect of put2 disruption on yeast cell viability under freezing stress. When grown on arginine as the sole nitrogen source, the put2 disruptant showed a significant decrease in cell viability after freezing despite the high proline and arginine contents. This result suggests that ?1-pyrroline-5-carboxylate or glutamate-?-semialdehyde, a proline catabolism intermediate, is toxic to yeast cells under freezing stress. In contrast, the survival rate of the wild-type and the put1-disruptant strains was found to increase after freezing in proportion to their arginine contents. This indicates that arginine has a cryoprotective function in yeast. Furthermore, the yeast cells accumulated proline as well as arginine in the vacuole, suggesting that there is a system for the transport of excess proline to the vacuole and that this vacuolar accumulation may be important in the freezing resistance of yeast cells.
Membrane trafficking via targeted exocytosis to the Saccharomycescerevisiae bud neck provides new membrane and membrane-associated factors that are critical for cytokinesis. It remains unknown whether yeast plasma membrane abscission, the final step of cytokinesis, occurs spontaneously following extensive vesicle fusion, as in plant cells, or requires dedicated membrane fission machinery, as in cultured mammalian cells. Components of the endosomal sorting complexes required for transport (ESCRT) pathway, or close relatives thereof, appear to participate in cytokinetic abscission in various cell types, but roles in cell division had not been documented in budding yeast, where ESCRTs were first characterized. By contrast, the septin family of filament-forming cytoskeletal proteins were first identified by their requirement for yeast cell division. We show here that mutations in ESCRT-encoding genes exacerbate the cytokinesis defects of cla4? or elm1? mutants, in which septin assembly is perturbed at an early stage in cell division, and alleviate phenotypes of cells carrying temperature-sensitive alleles of a septin-encoding gene, CDC10. Elevated chitin synthase II (Chs2) levels coupled with aberrant morphogenesis and chitin deposition in elm1? cells carrying ESCRT mutations suggest that ESCRTs normally enhance the efficiency of cell division by promoting timely endocytic turnover of key cytokinetic enzymes. PMID:21824003
In an attempt to clarify the involvement of fatty acid desaturases (FADs) in the freezing tolerance of Chlorella vulgaris IAM C-27, developed by hardening, we have isolated cDNA clones for two types of FADs from the Chlorella strain, based on the sequence information of genes for ?12 and ?-3 FADs, respectively desaturating oleic acid (18:1) to linoleic acid (18:2) and linoleic acid (18:2) to linolenic acid (18:3). The deduced amino acid sequence of the first clone, designated CvFad2, showed about 66% similarity to the microsomal ?12 FADs from several higher plants and this gene had ?12 FAD activity when expressed in Saccharomycescerevisiae. The predicted protein encoded by a second gene, designated CvFad3, showed about 60% similarity to the microsomal and plastidial ?-3 FADs from several higher plants. The features of the amino acid sequences of the C- and N-terminal regions of CvFAD3 and fatty acid analysis of polar lipids in transgenic tobacco plant expressing the CvFad3 gene suggested that this gene encodes the microsomal ?-3 FAD. Southern blot analysis showed that both genes were single-copy genes in the genome of the Chlorella strain. Different transcriptional patterns were observed with the two genes during hardening in Northern blot analysis.
Strong evidence indicates that transcription elongation by RNA polymerase II (pol II) is a highly regulated process. Here we present genetic results that indicate a role for the Saccharomycescerevisiae Rtf1 protein in transcription elongation. A screen for synthetic lethal mutations was carried out with an rtf1 deletion mutation to identify factors that interact with Rtf1 or regulate the same process as Rtf1. The screen uncovered mutations in SRB5, CTK1, FCP1, and POB3. These genes encode an Srb/mediator component, a CTD kinase, a CTD phosphatase, and a protein involved in the regulation of transcription by chromatin structure, respectively. All of these gene products have been directly or indirectly implicated in transcription elongation, indicating that Rtf1 may also regulate this process. In support of this view, we show that RTF1 functionally interacts with genes that encode known elongation factors, including SPT4, SPT5, SPT16, and PPR2. We also show that a deletion of RTF1 causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated with a transcription elongation defect. Collectively, our results suggest that Rtf1 may function as a novel transcription elongation factor in yeast. PMID:11014804
Bloom syndrome (BS) is rare autosomal recessive disorder associated with chromosomal instability. The gene responsible for BS, BLM, encodes a protein belonging to the RecQ helicase family. Disruptions of the SGS1 gene of Saccharomycescerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both genes independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function independently to repair DNA or induce tolerance to the lesions induced by CPT.
Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomycescerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.
The RAD6 and RAD18 genes of Saccharomycescerevisiae are required for postreplication repair of discontinuities occurring in newly synthesized DNA following exposure to uv light. In addition, rad6 mutants are highly defective in mutagenesis induced by uv and other DNA damaging agents and in sporulation. RAD6 encodes a protein of 172 amino acids with a highly acidic carboxyl terminus. Deletion of the carboxyl terminal 23 residues, 20 of which are acidic, has little or no effect on uv sensitivity or uv mutagenesis, but sporulation is greatly reduced. Addition of the first four residues of the polyacidic tail restores sporulation to 50% the level observed in RAD+/RAD+ diploids. RAD6 protein has been previously shown to be a ubiquitin-conjugating (E2) enzyme that attaches ubiquitin to histones H2A and H2B in vitro. Our experiments show that deletion of varying lengths of the polyacidic tail of RAD6 protein greatly reduces its ubiquitin-conjugating activity. The RAD18 encoded protein contains features which suggest that it binds DNA and nucleotides. Ten of the 12 cysteine residues occur in regions that could form zinc finger domains for nucleic acid binding. The other interesting feature in RAD18 protein is the presence of a putative nucleotide binding sequence. The possible in vivo functions of the RAD6 and RAD18 proteins are discussed. PMID:2698834
In higher plants, very-long-chain fatty acids (VLCFAs) are the main constituents of hydrophobic polymers that prevent dessication at the leaf surface and provide stability to pollen grains. Of the 21 genes encoding VLCFA elongases (VLCFAEs) from Arabidopsis thaliana, 17 were expressed heterologously in Saccharomycescerevisiae. Six VLCFAEs, including three known elongases (FAE1, KCS1, and KCS2) and three previously uncharacterized gene products (encoded by At5g43760, At1g04220, and At1g25450) were found to be enzymatically active with endogenous yeast fatty acid substrates and to some extent with externally supplied unsaturated substrates. The spectrum of VLCFAs accumulated in expressing yeast strains was determined by gas chromatography/mass spectrometry. Marked specificity was found among elongases tested with respect to their elongation products, which encompassed saturated and monounsaturated fatty acids 20-30 carbon atoms in length. The active VLCFAEs revealed highly distinct patterns of differential sensitivity to oxyacetamides, chloroacetanilides, and other compounds tested, whereas yeast endogenous VLCFA production, which involves its unrelated elongase (ELO) in sphingolipid synthesis, was unaffected. Several compounds inhibited more than one VLCFAE, and some inhibited all six active enzymes. These findings pinpoint VLCFAEs as the target of the widely used K(3) class herbicides, which have been in commercial use for 50 years, provide important clues as to why spontaneous resistance to this class is rare, and point to complex patterns of substrate specificity and product spectrum among members of the Arabidopsis VLCFAE family. PMID:15277688
The nucleotide sequencing of 8887 bp of the left arm of chromosome XIV is described. The sequence includes the centromeric region. Both strands were sequenced with an average redundancy of 5.09 per base pair. The overall G+C content is 37.3% (39.2% for putative coding regions versus 32.5% for non-coding regions). Six open reading frames (ORFs) greater than 100 amino acids were detected, all of which are completely confined to the 8.9 kbp region. Codon frequencies of the six ORFs agree with codon usage in Saccharomycescerevisiae and all show the characteristics of low-level expressed genes. Comparison of the translated sequences with protein sequences in data bases suggests the presence of two ORFs (N2014 and N2007) encoding ribosomal proteins, the latter of which is the previously sequenced MRP7 gene. Another ORF (N2012) could encode a membrane-associated protein since it contains secretory signal sequence and two presumed transmembrane helices. This protein might be involved in mitochondrial energy transfer. ORF N2016 is immediately adjacent to the centromere, suggesting that it corresponds to the SPO1 gene, which is very tightly linked to the centromere at the left arm side of chromosome XIV (Mortimer et al., 1989). PMID:7985421
Isochrysis galbana, produces long chain polyunsaturated fatty acids including docosahexaenoic acid (DHA, 22:6n-3). A novel gene (IgFAD4-2), encoding a C22-?4 polyunsaturated fatty acid specific desaturase, has been isolated and characterized from I. galbana. A full-length cDNA of 1,302 bp was cloned by LA-PCR technique. The IgFAD4-2 encoded a protein of 433 amino acids that shares 78 % identity with a previously reported ?4-desaturase (IgFAD4-1) from I. galbana. The function of IgFAD4-2 was deduced by its heterologous expression in Saccharomycescerevisiae, which then desaturated docosapentaenoic acid (DPA, 22:5n-3) to DHA. The conversion ratio of DPA to DHA was 34 %, which is higher than other ?4-desaturases cloned from algae. However, IgFAD4-2 did not catalyze the desaturation or elongation reactions with other fatty acids. These results confirm that IgFAD4-2 has C22-?4-PUFAs-specific desaturase activity. PMID:22941368
Walleye dermal sarcoma (WDS) and walleye epidermal hyperplasia (WEH) are skin diseases of walleye fish that appear and regress on a seasonal basis. We report here that the complex retroviruses etiologically associated with WDS (WDS virus [WDSV]) and WEH (WEH viruses 1 and 2 [WEHV1 and WEHV2, respectively]) encode D-type cyclin homologs. The retroviral cyclins (rv-cyclins) are distantly related to one another and to known cyclins and are not closely related to any walleye cellular gene based on low-stringency Southern blotting. Since aberrant expression of D-type cyclins occurs in many human tumors, we suggest that expression of the rv-cyclins may contribute to the development of WDS or WEH. In support of this hypothesis, we show that rv-cyclin transcripts are made in developing WDS and WEH and that the rv-cyclin of WDSV induces cell cycle progression in yeast (Saccharomycescerevisiae). WEHV1, WEHV2, and WDSV are the first examples of retroviruses that encode cyclin homologs. WEH and WDS and their associated retroviruses represent a novel paradigm of retroviral tumor induction and, importantly, tumor regression. PMID:9765420
Based on the sequence information of Delta12-fatty acid desaturase genes (from Mucor circinelloides, Mortierella alpina, Mucor rouxii and Aspergillus nidulans), which were involved in the conversion from C18:1 to C18:2, a cDNA sequence putatively encoding a Delta12-fatty acid desaturase was isolated from Rhizopus arrhizus using the combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Sequence analysis indicated that it had an open reading frame (ORF) of 1170 bp, coding for 389 amino acid residues of 45 kDa, pI of the deduced protein was 7.01. The deduced amino acid sequence of this cloned cDNA showed high identity to those filamentous fungal Delta12-desaturases mentioned above, including three conserved histidine-rich motifs and two hydrophobic domains. Functional identification was done heterologously in Saccharomycescerevisiae strain INVScl. The result demonstrated that the deduced amino acid sequence exhibited Delta12-fatty acid desaturase activity, suggesting that this gene encoded for a membrane-bound desaturase, Delta12-fatty acid desaturase. PMID:15327973
Stearoyl-acyl-carrier-protein (ACP) desaturase was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomycescerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the {Delta}{sup 9} desaturase is developmentally regulated.
We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomycescerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain. PMID:8065914
Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomycescerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation. PMID:19940927
We have increased the contents of several amino acids in the seeds of Arabidopsis thaliana by introduction of aspartate aminotransferase (AAT), an enzyme of the aspartate biosynthetic pathway. mRNA was prepared from one-week-old seedlings of Glycine max cv. enrei and the cDNA encoding AAT5 was isolated and linked to the CaMV35S promoter in the plant vector pBI121. The AAT5 gene encodes a protein of 462 amino acid residues that shows 51% amino acid sequence similarity to A. thaliana chloroplast Asp3. The soybean AAT5 also contains a chloroplast transit peptide and is able to functionally complement a Saccharomycescerevisiae mutant lacking the Asp5 gene. A. thaliana was transformed with the AAT5 gene from Agrobacterium tumefaciens by the vacuum infiltration method. The AAT5 gene was detected in the transcript and genomic DNA from the transgenic T2 plants. The T3 progeny showed a 3:1 segregation ratio indicating the presence of a single integration. Expression of G. max AAT5 in A. thaliana transformants caused 3-, 4-, 23-, and 50-fold increases in the contents of free glycine, alanine, asparagine, and glutamine, respectively, in the T3 seeds. A decrease in the contents of valine, tyrosine, isoleucine, leucine, and phenylalanine by several folds was also observed. Thus, it is of interest that a key gene expression resulted in marked changes of metabolites in plant seeds.
Recombinant strains altered in the ammonium assimilation pathways were constructed with the purpose of increasing NADPH availability. The NADPH-dependent glutamate dehydrogenase encoded by GDH1, which accounts for a major fraction of the NADPH consumption during growth on ammonium, was deleted, and alternative pathways for ammonium assimilation were overexpressed: GDH2 (NADH-consuming) or GLN1 and GLT1 (the GS-GOGAT system). The flux through the pentose phosphate pathway during aerobic growth on glucose decreased to about half that of the reference strain Saccharomycescerevisiae CEN.KK113-7D, indicating a major redox alteration in the strains. The basic growth characteristics of the recombinant strains were not affected to a great extent, but the dilution rate at which the onset of aerobic fermentation occurred decreased, suggesting a relation between the onset of the Crabtree effect and the flux through the Embden-Meyerhof-Parnas pathway downstream of glucose 6-phosphate. No redox effect was observed in a strain containing a deletion of GLR1, encoding glutathione reductase, an enzyme that is NADPH-consuming.
Upon glucose depletion, a massive reprogramming of gene expression occurs in the yeast Saccharomycescerevisiae for the use of alternate carbon sources such as the nonfermentable compounds ethanol and glycerol. This process is mediated by the master kinase Snf1 that controls the activity of various targets including the transcriptional regulators Cat8, Sip4 and Adr1. We have recently identified Rds2 as an additional player in this pathway. Here, we have performed genome-wide location analysis of Rds2 in cells grown in the presence of glycerol. We show that Rds2 binds to promoters of genes involved in gluconeogenesis, the glyoxylate shunt, and the TCA cycle as well as some genes encoding mitochondrial components or some involved in the stress response. Interestingly, we also detected Rds2 at the promoters of SIP4, ADR1 and HAP4 which encodes the limiting subunit of the Hap2/3/4/5 complex, a regulator of respiration. Strikingly, we observed an important overlap between the targets of Rds2 and Adr1. Finally, we provide a model to account for the complex interplay among these transcriptional regulators. PMID:22687600
Background A well known example of oscillatory phenomena is the transient oscillations of glycolytic intermediates in Saccharomycescerevisiae, their regulation being predominantly investigated by mathematical modeling. To our knowledge there has not been a genetic approach to elucidate the regulatory role of the different enzymes of the glycolytic pathway. Results We report that the laboratory strain BY4743 could also be used to investigate this oscillatory phenomenon, which traditionally has been studied using S. cerevisiae X2180. This has enabled us to employ existing isogenic deletion mutants and dissect the roles of isoforms, or subunits of key glycolytic enzymes in glycolytic oscillations. We demonstrate that deletion of TDH3 but not TDH2 and TDH1 (encoding glyceraldehyde-3-phosphate dehydrogenase: GAPDH) abolishes NADH oscillations. While deletion of each of the hexokinase (HK) encoding genes (HXK1 and HXK2) leads to oscillations that are longer lasting with lower amplitude, the effect of HXK2 deletion on the duration of the oscillations is stronger than that of HXK1. Most importantly our results show that the presence of beta (Pfk2) but not that of alpha subunits (Pfk1) of the hetero-octameric enzyme phosphofructokinase (PFK) is necessary to achieve these oscillations. Furthermore, we report that the cAMP-mediated PKA pathway (via some of its components responsible for feedback down-regulation) modulates the activity of glycoytic enzymes thus affecting oscillations. Deletion of both PDE2 (encoding a high affinity cAMP-phosphodiesterase) and IRA2 (encoding a GTPase activating protein- Ras-GAP, responsible for inactivating Ras-GTP) abolished glycolytic oscillations. Conclusions The genetic approach to characterising the glycolytic oscillations in yeast has demonstrated differential roles of the two types of subunits of PFK, and the isoforms of GAPDH and HK. Furthermore, it has shown that PDE2 and IRA2, encoding components of the cAMP pathway responsible for negative feedback regulation of PKA, are required for glycolytic oscillations, suggesting an enticing link between these cAMP pathway components and the glycolysis pathway enzymes shown to have the greatest role in glycolytic oscillation. This study suggests that a systematic genetic approach combined with mathematical modelling can advance the study of oscillatory phenomena. PMID:11428892
ABSTRACT: BACKGROUND: A well known example of oscillatory phenomena is the transient oscillations of glycolytic intermediates in Saccharomycescerevisiae, their regulation being predominantly investigated by mathematical modeling. To our knowledge there has not been a genetic approach to elucidate the regulatory role of the different enzymes of the glycolytic pathway. RESULTS: We report that the laboratory strain BY4743 could also be used to investigate this oscillatory phenomenon, which traditionally has been studied using S. cerevisiae X2180. This has enabled us to employ existing isogenic deletion mutants and dissect the roles of isoforms, or subunits of key glycolytic enzymes in glycolytic oscillations. We demonstrate that deletion of TDH3 but not TDH2 and TDH1 (encoding glyceraldehyde-3-phosphate dehydrogenase: GAPDH) abolishes NADH oscillations. While deletion of each of the hexokinase (HK) encoding genes (HXK1 and HXK2) leads to oscillations that are longer lasting with lower amplitude, the effect of HXK2 deletion on the duration of the oscillations is stronger than that of HXK1. Most importantly our results show that the presence of beta (Pfk2) but not that of alpha subunits (Pfk1) of the hetero-octameric enzyme phosphofructokinase (PFK) is necessary to achieve these oscillations. Furthermore, we report that the cAMP-mediated PKA pathway (via some of its components responsible for feedback down-regulation) modulates the activity of glycoytic enzymes thus affecting oscillations. Deletion of both PDE2 (encoding a high affinity cAMP-phosphodiesterase) and IRA2 (encoding a GTPase activating protein- Ras-GAP, responsible for inactivating Ras-GTP) abolished glycolytic oscillations. CONCLUSIONS: The genetic approach to characterising the glycolytic oscillations in yeast has demonstrated differential roles of the two types of subunits of PFK, and the isoforms of GAPDH and HK. Furthermore, it has shown that PDE2 and IRA2, encoding components of the cAMP pathway responsible for negative feedback regulation of PKA, are required for glycolytic oscillations, suggesting an enticing link between these cAMP pathway components and the glycolysis pathway enzymes shown to have the greatest role in glycolytic oscillation. This study suggests that a systematic genetic approach combined with mathematical modelling can advance the study of oscillatory phenomena. PMID:22920924
ATP-binding cassette transporters (ABC) are important detoxification proteins and were proposed to play important roles in monoterpene resistance in Saccharomycescerevisiae. In this work, the transcriptional levels of typical ABC transporters of S. cerevisiae under 85?mg d-limonene/l were evaluated using real-time quantitative PCR. Only the transcriptional level of PDR5, YOR1 and PDR15 were upregulated but overexpression of these genes in S. cerevisiae failed to improve d-limonene tolerance suggesting that other mechanisms are involved in tolerance of yeast to monoterpenes.
The studying and monitoring of physiological and metabolic changes in Saccharomycescerevisiae (S. cerevisiae) has been a key research area for the brewing, baking, and biofuels industries, which rely on these economically important yeasts to produce their products. Specifically for breweries, physiological and metabolic parameters such as viability, vitality, glycogen, neutral lipid, and trehalose content can be measured to better understand the status of S. cerevisiae during fermentation. Traditionally, these physiological and metabolic changes can be qualitatively observed using fluorescence microscopy or flow cytometry for quantitative fluorescence analysis of fluorescently labeled cellular components associated with each parameter. However, both methods pose known challenges to the en...
Abstract Key distinguishing characteristics of yeast glucose metabolism are the relative proportions of fermentation and respiration. Crabtree-positive yeast species exhibit a respirofermentative metabolism, whereas aerobic species respire fully without secretion of fermentation byproducts. Physiological data suggest a gradual transition in different species between these two states. Here, we investigate whether this gradual transition also occurs at the intracellular level by quantifying the intracellular metabolism of Saccharomycescerevisiae, Saccharomyces bayanus, Saccharomyces exiguus, Kluyveromyces thermotolerans, Yarrowia lipolytica, Pichia angusta and Candida rugosa by 13C-flux analysis and metabolomics. Different from the extracellular physiology, the intracellular fluxes through ...
The probiotic potential of IS Saccharomycescerevisiae strains used for production of foods or bevel-ages or isolated from such, and eight strains of Saccharomycescerevisiae var. boulardii, was investigated. All strains included were able to withstand pH 2.5 and 0.3% Ox-all. Adhesion to the nontumorigenic porcine jejunal epithelial cell line (IPEC-J2) was investigated by incorporation of H-3-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-1? 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. © 2004 Elsevier B.V. All rights reserved.
Saccharomycescerevisiae 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 epithelialbarrier 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 epithelialbarrier integrity is previously compromised. --------------------------------------------------------------------------------
Yeasts, used for beer production, can be divided into two groups, top fermenters and bottom fermenters and Saccharomyces carlsbergensis has been chosen as the name for the bottom fermenting yeasts which are used in lager beer production. The key for the analysis of the chromosomes of Saccharomyces carlsbergensis was provided by the discovery that single chromosomes of meiotic segregants of these strains can be transferred to genetically marked Saccharomycescerevisiae strains and subsequently analyzed by tetrad analysis and molecular hybridization. It is proposed that Saccharomyces carlsbergensis is composed of two rather divergent genotypes. Breeding can be accomplished by cross breeding and mutagenesis and possibly by introducing in vitro modified cloned genes into meiotic segregants of Saccharomyces carlsbergensis.
Termination translation in Saccharomycescerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed an...
Translation termination in eukaryotes requires a stop codon-responsive (class-I) release factor, eRF1, and a guanine nucleotide-responsive (class-II) release factor, eRF3. Schizosaccharomyces pombe eRF3 has an N-terminal polypeptide similar in size to the prion-like domain of Saccharomycescerevisia...
The small GTPase Arf1 is a crucial regulator of vesicle formation at many steps of the secretory pathway in the yeast Saccharomycescerevisiae as well as in higher eukaryotes. Currently best understood is the role of Arf1 in the formation of COPI-coated vesicles at different levels of the Golgi appa...
Peroxiredoxins are a family of antioxidant enzymes conserved from bacteria to humans. In Saccharomycescerevisiae, there exist five peroxiredoxins, among which Tsa2p shares striking homology with the well described Tsa1p but has not been extensively studied. Here we report on the functional characte...
Expression of retroviral Gag protein in yeast has previously shown Gag targeting to the plasma membrane but little or no production of Gag virus-like particles (VLPs). Here we show that, after removal of the cell wall, the expression of HIV type 1 Gag protein in Saccharomycescerevisiae spheroplasts...
Choline transport of Saccharomycescerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N...
Most ribosomal proteins play important roles in ribosome biogenesis and function. Herein, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomycescerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impai...
Ribosomal protein L1 from Saccharomycescerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we ...
Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomycescerevisiae rpS5 but lacks a negativel...
Meiotic recombination requires the action of several gene products in both Saccharomycescerevisiae and Drosophila melanogaster. Genetic studies in D. melanogaster have shown that the mei-W68 gene is required for all meiotic gene conversion and crossing-over. We cloned mei-W68 using a new genetic ma...
The Saccharomycescerevisiae meiosis-specific transcription factor Ndt80 is responsible for the induction of a class of genes referred to as middle sporulation genes. Among the members of this family are the B-type cyclins and other genes whose products are required for meiotic chromosome division a...
A key transition in meiosis is the exit from prophase and entry into the nuclear divisions, which in the yeast Saccharomycescerevisiae depends upon induction of the middle sporulation genes. Ndt80 is the primary transcriptional activator of the middle sporulation genes and binds to a DNA sequence e...
Ndt80 is a transcriptional activator required for meiosis in the yeast Saccharomycescerevisiae. Here, we report the crystal structure at 2.3 ? resolution of the DNA-binding domain of Ndt80 experimentally phased by using the anomalous and isomorphous signal from a single ordered Se atom per molecule...
We describe the identification of a new meiosis-specific gene of Saccharomycescerevisiae, NDT80. The ndt80 null and point mutants arrest at the pachytene stage of meiosis, with homologs connected by full-length synaptonemal complexes and spindle pole bodies duplicated but unseparated. Meiotic recom...
The Ndt80 protein of the yeast Saccharomycescerevisiae is the founding member of a new sub-family of proteins in the Ig-fold superfamily of transcription factors. The crystal structure of Ndt80 bound to DNA shows that it makes contacts through several loops on one side of the protein that connect ?...
The NDT80/PhoG transcription factor family includes ScNdt80p, a key modulator of the progression of meiotic division in Saccharomycescerevisiae. In Candida albicans, a member of this family, CaNdt80p, modulates azole sensitivity by controlling the expression of ergosterol biosynthesis genes. We pre...
Using cultures synchronised by three independent procedures, we have shown that the CDC9 gene, coding for DNA ligase, is periodically expressed in the Saccharomycescerevisiae cell cycle. The level of CDC9 transcript increases many fold in late G1 reaching a peak at about the G1/S phase boundary and...
In contrast to ligase-deficient (cdc9) Saccharomycescerevisiae, which did not rejoin bleomycin-induced DNA breaks, ligase-proficient (CDC9) yeast cells eliminated approximately 90% of DNA breaks within 90 to 120 min after treatment. Experimental conditions restricted enzymatic removal of the unusua...
The functional compatibility of vaccinia virus DNA ligase with eukaryotic counterparts was demonstrated by its ability to complement Saccharomycescerevisiae cdc9. The vaccinia DNA ligase is a 63 kDa protein expressed early during infection that is non-essential for virus DNA replication and recombi...
The Saccharomycescerevisiae cdc9 gene has been cloned in the vector YRp12 by complementation of the temperature-sensitive lesion in vivo. The gene is contained within a 3300-base-pair fragment of DNA, which maps to the chromosomal locus of cdc9 and which is able to complement a DNA-ligase-deficient mutant of the fission yeast Schizosaccharomyces pombe. PMID:6347688
The temperature-sensitive Saccharomycescerevisiae cell cycle mutant cdc9 is defective in DNA ligase, and the DNA synthesized at the restrictive temperature contains many single-strand breaks. We find that holding a diploid homozygous for cdc9 at the restrictive temperature and then plating cells at...
We report that the NAD-dependent Escherichia coli DNA ligase can support the growth of Saccharomycescerevisiae strains deleted singly for CDC9 or doubly for CDC9 plus LIG4. Alanine-scanning mutagenesis of E.coli DNA ligase led to the identification of seven amino acids (Lys115, Asp117, Asp285, Lys3...
We have cloned CDC9, the structural gene for Saccharomycescerevisiae DNA ligase, and investigated its transcriptional regulation both as a function of cell cycle stage and after UV irradiation. The steady-state level of DNA ligase mRNA increases at least fourfold in late G1, after the completion of...
DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus. In the budding yeast Saccharomycescerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair. In this report, dual nuclear–mitochondrial localization is ...
Conditional ligase-deficient mutants of Saccharomycescerevisiae were more sensitive than their parental (CDC9) strain to dose-dependent killing by bleomycin, even when mutant cells were pregrown and exposed to the antibiotic at permissive temperatures. Pretreatment incubation at the restrictive tem...
Apresentação efectuada no 2nd Annual Workshop of the PhD program BioPlant, em Braga, 2011 , In this work we used the model organism Saccharomycescerevisiae to characterise the biological activity and the mechanism of action of phytochemicals. We have assessed DNA damage and repair using the comet assa...
In this work we used the model organism Saccharomycescerevisiae to characterise the biological activity and the mechanism of action of phytochemicals. We have assessed DNA damage and repair using the comet assay, which displayed a dose-response relationship with different DNA-damaging agents. Subse...
In this work we used the model organism Saccharomycescerevisiae to characterise the biological activity and the mechanism of action of phytochemicals. One of the goals is to use mutant strains affected in basic mechanisms of oxidative stress response and DNA repair in order to uncover the molecular...
Hybrid sterility is thought to be due to deleterious epistatic interactions between genes from different species. Here we demonstrate that dominant genic incompatibility does not contribute to sterility in hybrids between Saccharomycescerevisiae and five closely related species. Sterile diploids we...
Tetraploid strains of Saccharomycescerevisiae carrying different dosages of the CYR1+ gene have been constructed. Adenylate cyclase activity observed in these tetraploid strains was proportional to the dosage of the active CYR1+ gene. Of the 57 mutants requiring adenosine 3',5'-monophosphate for gr...
We analyzed the effects of the location and number of stress response elements (STREs) on gene expression in Saccharomycescerevisiae. Genes containing STRE between 51 and 300 bp upstream from translational start codon tended to be up-regulated and genes with multiple STREs exhibited higher up-regulation under stress conditions.
Zygote formation occurs through tightly coordinated cell and nuclear fusion events. Genetic evidence suggests that the FUS2 gene product promotes cell fusion during zygote formation in Saccharomycescerevisiae, functioning with the Fus1 plasma membrane protein at or before cell wall and plasma membr...
Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomycescerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in ...
Saccharomycescerevisiae harbors two cyclophilin 40-type enzymes, Cpr6 and Cpr7, which are components of the Hsp90 molecular chaperone machinery. Cpr7 is required for normal growth and is required for maximal activity of heterologous Hsp90-dependent substrates, including glucocorticoid receptor (GR)...
We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomycescerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous ...
In this study, we have evaluated the incorporation of two types of protein hydrolysates at 9 and 12% levels of inclusion, one from yeast (Saccharomycescerevisiae, YPH) and another one from pig blood (PBPH), in microdiets for gilthead sea bream (Sparus aurora) larvae, and compared these results to a...
Eukaryotic cells use multiple replication origins to replicate their large genomes. Some origins fire early during S phase whereas others fire late. In Saccharomycescerevisiae, initiator sequences (ARSs) are bound by the origin recognition complex (ORC). Cdc6p synthesized at the end of mitosis join...
We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomycescerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase ...
Saccharomycescerevisiae cells are exquisitely sensitive to altered dosage of the spindle pole body duplication gene, NDC1. We show that the NDC1 locus is haploinsufficient because diploid yeast cells cannot survive with a single chromosomal copy of the NDC1 gene. Diploid cells with a single copy of...
We have investigated the chromatin structure of wild-type and mutationally altered centromere sequences in the yeast Saccharomycescerevisiae by using an indirect end-labeling mapping strategy. Wild-type centromere DNA from chromosome III (CEN3) exhibits a nuclease-resistant chromatin structure 220-...
Genomic DNA of the yeast, Saccharomycescerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-1-cytochrome c were generated by transforming a cyc1 mutant with oligonucleoti...
Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomycescerevisiae, an evolutionarily conserved spindle checkpoin...
We have screened for proteins that interact with v-SNAREs of the late secretory pathway in the yeast Saccharomycescerevisiae. A novel protein, designated Vsm1, binds tightly to the Snc2 v-SNARE in the two-hybrid system and can be coimmunoprecipitated with Snc1 or Snc2 from solubilized yeast cell ex...
Ddi1/Vsm1 is an ubiquitin receptor involved in regulation of the cell cycle and late secretory pathway in Saccharomycescerevisiae. Ddi1 possesses three domains: an NH2-terminal ubiquitin-like domain (UBL), a COOH-terminal ubiquitin-associated domain (UBA), and a retroviral aspartyl-protease domain ...
The abilities of 22,23-epoxy-2-aza-2,3-dihydrosqualene and the corresponding N-oxide, 22,23-epoxy-2-aza-2,3-dihydrosqualene-N-oxide, to inhibit sterol biosynthesis were studied in microsomes and cells of Saccharomycescerevisiae and Candida albicans. 22,23-Epoxy-2-aza-2,3-dihydrosqualene, which diff...
Hsp90 interacts with Sti1 (p60) in lysates of yeast and vertebrate cells. Here we provide the first analysis of their interaction in vivo. Saccharomycescerevisiae mutations that eliminate Sti1 or reduce intracellular concentrations of Hsp90 individually have little or no effect on growth at normal ...
Apresentação efectuada nas "XXXI Jornadas Portuguesas de Genética", em Oeiras, Portugal, em 2004. , Wine production by the use of selected Saccharomycescerevisiae strains, commercially available as active dry yeast is widely accepted, being an enological practice extensively applied nowadays. The use ...
Apresentação efectuada nas "12as Jornadas de Biologia de Leveduras Professor Nicolau van Uden", em Aveiro, Portugal, em 2004. , Wine production by the use of selected Saccharomycescerevisiae strains, commercially available as active dry yeast is widely accepted, being an enological practice extensivel...
Newly isolated temperature-sensitive cdc35 mutants of Saccharomycescerevisiae have been characterized. They show the morphology, growth and conjugation characteristics typical of class-A or class-II start mutants. The cdc35 mutation induces a significant decrease of the intracellular cAMP level and...
Mutants of the yeast Saccharomycescerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic an...
Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomycescerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase ? that results in increased rates of mutagenesis, recombination, and chro...
An application of flow cytometric sorting is used for isolation of Saccharomycescerevisiae mutants that mislocalize vacuolar vital dyes. This screen is based on the ability of a lipophilic styryl compound, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (F...
A screen was designed to identify Saccharomycescerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independe...
As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomycescerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification. Vacuoles were labeled with 6-carboxyfluorescein with the membrane-perme...
In Saccharomycescerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants ...
Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomycescerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arreste...
A system is described for isolating temperature-sensitive mutants of Saccharomycescerevisiae with defects in early meiotic events. We used an otherwise haploid strain disomic (n+1) for chromosome III, and heteroallelic at the leucine-2 locus. Meiotic development was initiated by exposure of the st...
Many Saccharomycescerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 ?m circle plasmid (2 ?m). Here we show that accumulation of 2 ?m in the SUMO pathway mutants siz1? siz2?, slx5?, and slx8? is associated with formation of an aberrant high-molecular-weight ...
Spontaneous recessive orthovanadate-resistant mutants of Saccharomycescerevisiae were obtained in five complementation groups, and all show defects in protein glycosylation that mimic the previously isolated mnn mutants. Three of the groups are allelic to the known mnn8, mnn9, and mnn10 mutants, wh...
The actin gene in yeast Saccharomycescerevisiae is interrupted by a 309-base-pair intron within the protein-coding region. By using nuclease BAL-31, several intron deletion mutants were constructed to define sequences at the 5' splice junction that are required for RNA splicing. Extensive parts of ...
Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomycescerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is ...
In haploid Saccharomycescerevisiae, mating occurs by activation of the pheromone response pathway. A genetic selection for mutants that activate this pathway uncovered a class of mutants defective in cell wall integrity. Partial loss-of-function alleles of PGI1, PMI40, PSA1, DPM1, ALG1, MNN10, SPT1...
The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomycescerevisiae as a model system for better understanding t...
The mitochondrial matrix of the yeast Saccharomycescerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported int...
The product of the Saccharomycescerevisiae LEU3 gene, Leu3p, is a transcriptional activator which regulates leucine biosynthesis in response to intracellular levels of leucine through the biosynthetic intermediate alpha-isopropylmalate. We devised a novel assay to examine the DNA site occupancy of ...
A variety of leucine-containing di- and tripeptides and two lysine-containing dipeptides supported the growth of strain Z1-2D, a leucine, lysine auxotroph of Saccharomycescerevisiae. However, (Lys)2, (Lys)3, (Lys)4, and (Lys)5 as well as Gly-Leu-Gly, three tetra- and one pentapeptide containing leu...
A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomycescerevisiae. When a cell surface display system based on ?-agglutinin wa...
The capability of multi-stress-tolerant Saccharomycescerevisiae diploid strain TJ14 for the production of cellulosic bio-ethanol by semi-simultaneous saccharification and fermentation (SSSF) technology was evaluated under high-temperature conditions. At 39°C, the TJ14 produced 45 g/l ethanol by SSSF of 100 g (w/v)/l cellulose - a significantly higher concentration than reported in prevailing literature. PMID:22925900
In the budding yeast Saccharomycescerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G1 to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins ar...
A wild-type strain of the yeast Saccharomycescerevisiae grown at a medium [Cu] of less than or equal to 50 nM contained less Cu,Zn superoxide dismutase (SOD) mRNA (60%), protein (50%), and activity (50%) in comparison with control cultures grown in normal synthetic dextrose medium ([Cu] approximate...
Saccharomycescerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachac¸a, the Brazilian sugar cane spirit. The methodology established took into account the screening for bioche...
Comparative studies of the fermentation of cane molasses into ethanol by Saccharomycescerevisiae in the presence or absence of fungal invertase were performed. When cane molasses was fermented by the yeast at 30°C and pH 5.0, the presence of the enzyme had no effect on ethanol production. At pH 3.5...
Progression through the middle phase of sporulation in Saccharomycescerevisiae is promoted by the successful completion of recombination at the end of prophase I. Completion of meiotic recombination allows the activation of the sporulation-specific transcription factor Ndt80, which binds to a speci...
We have identified, cloned, and sequenced the gene for the small subunit of ribonucleotide diphosphate reductase of Saccharomycescerevisiae. The protein and its transcript are induced about 10-fold by the alkylating agent methyl methanesulfonate, a result which suggests that the gene is induced by ...
The small subunit of ribonucleotide reductase in Saccharomycescerevisiae (RNR2) was induced 3- to 20-fold by a variety of DNA-damaging agents. Induction of the RNR2 transcript by at least one of these agents, methyl methanesulfonate, did not require protein synthesis. To identify sequences involved...
For more than three decades micro-organisms have been employed as hosts for recombinant protein production, with the most popular organisms being Escherichia coli and Saccharomycescerevisiae (1). One of the crucial factors to obtain high product yields in recombinant protein bioprocesses is the bio...
The carboxy-terminal part of the Saccharomycescerevisiae SDC25 gene product (SDC25 C domain) can elicit activation of mammalian Ras proteins. Specifically, SDC25 C domain functions as an exchange factor for cellular Ras proteins in CHO cells. In this study, we used the dominant inhibitory Ha-Ras As...
The SDC25 C-domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomycescerevisiae which acts equally well on Ha-ras p21 and yeast RAS2. These properties make the SDC25 C-domain a suitable tool to study the basic mechanism of a GDS. The action of the SDC25 C-...
She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomycescerevisiae. We show herein that She4p/Dim1p is involved in endocytosis and actin polarization thr...
Comparison of the chitin synthase genes of Saccharomycescerevisiae CHS1 and CHS2 with the Candida albicans CHS1 gene (UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyltransferase, EC 2.4.1.16) revealed two small regions of complete amino acid sequence conservation that were used to desi...
The deposition of the polysaccharide chitin in the Saccharomycescerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p...
The CHS5 locus of Saccharomycescerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5...
In Saccharomycescerevisiae, chitin forms the primary division septum and the bud scar in the walls of vegetative cells. Three chitin synthetic activities have been detected. Two of them, chitin synthase I and chitin synthase II, are not required for synthesis of most of the chitin present in vivo. ...
In Saccharomycescerevisiae, the synthesis of chitin is temporally and spatially regulated through the transport of Chs3p (chitin synthase III) to the plasma membrane in the bud neck region. Traffic of Chs3p from the trans-Golgi network (TGN)/early endosome to the plasma membrane requires the functi...
Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomycescerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the...
Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomycescerevisiae by localization of the putativ...
Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomycescerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for t...
During cell stress, Saccharomycescerevisiae increases the synthesis of chitin and glucans to strengthen and repair the cell wall. In this study, we show that under conditions of cell stress, the steady-state localization of chitin synthase III (Chs3p) shifts from internal stores (chitosomes) to...
The morphology of three Saccharomycescerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were v...
Chitin in the cyst wall of Entamoeba histolytica is made by two chitin synthases (Chs), one of which is unique (EhCHS-1) and one of which resembles those of insects and nematodes (EhCHS-2). EhCHS-1 is deposited chitin in the lateral wall of transformed Saccharomycescerevisiae Chs mutants, independe...
In Saccharomycescerevisiae, the polysaccharide chitin forms the primary division septum between mother cell and bud. Two related enzymes, chitin synthase I and chitin synthase II (UDP-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase, EC 2.4.1.16), have been identified and...
Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomycescerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuc...
ABSTRACT: BACKGROUND: To explore chitin synthesis initiation, the effect of addition of exogenous oligosaccharides on in vitro chitin synthesis was studied. Oligosaccharides of various natures and lengths were added to a chitin synthase assay performed on a Saccharomycescerevisiae membrane fraction...
Abstract Saccharomycescerevisiae chitin synthase 2 (Chs2) synthesizes the primary septum after mitosis is completed. It is essential for proper cell separation and expected to be highly regulated. We have expressed Chs2 and a mutant lacking the N-terminal region in Pichia pastoris in an act...
In Saccharomycescerevisiae, the polysaccharide chitin is deposited at the mother bud junction by an integral membrane enzyme, chitin synthase 3 (Chs3p). The traffic of Chs3p to the cell surface from the trans-Golgi network (TGN) depends on two proteins, Chs5p and Chs6p, which sort selected cargo pr...
The growths of Saccharomycescerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3), defective in chitin synthase 1 (Chs1) but showing a new chitin synthase activity (Chs2), were affected by Calcofluor. To be effective, the interaction ...
Nystatin (5 to 10 ?g/ml) was fungicidal to Saccharomycescerevisiae NCYC 361. There was an initial rapid uptake of nystatin by the yeast cells at 30 C, the uptake being pH dependent, with a maximum at low pH values (3 to 4). Photomicrographs indicated that nystatin-treated cells became granular. The...
The cytoplasmic viral RNA sensors RIG-I and MDA5 are important for the production of type I interferon and other inflammatory cytokines. DDX60 is an uncharacterized DEXD/H box RNA helicase similar to Saccharomycescerevisiae Ski2, a cofactor of RNA exosome, which is a protein complex required for th...
The hexokinase A (HKA) and hexokinase B (HKB) genes of Saccharomycescerevisiae have been cloned from a library of yeast genomic DNA. Using an in vitro glucose phosphorylation assay, the HKB gene was located on a plasmid carrying a 13.6 kb fragment of yeast DNA. After subcloning the relevant restric...