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Acorrect classification of fungi, including yeasts, is of prime importance to understand fungal biodiversity and to communicate about this diversity. Fungal genera are mainly defined based on phenotypic characteristics and the results of single or multigene-based phylogenetic analyses. However, because yeasts often have less phenotypic characters, their classification experienced a strong move towards DNA-based data, from short riboso)mal sequences to multigene phylogenies and more recently to phylogenomics. Here, we explore the usefulness of various genomics-based parameters to circumscribe fungal genera more correctly taking the yeast domain as an example. Therefore, we compared the results of a phylogenomic analysis, average amino acid identity (AAI) values, the presence of conserved signature indels (CSIs), the percentage of conserved proteins (POCP) and the presence-absence patterns of orthologs (PAPO). These genome-based metrics were used to investigate their usefulness in demarcating 13 hitherto relatively well accepted genera in Saccharomycetaceae, namely Eremothecium, Grigorovia, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Saccharomyces, Tetrapisispora, Torulaspora, Vanderwaltozyma, Zygosaccharomyces and Zygotorulaspora. As a result, most of these genera are supported by the genomics-based metrics, but the genera Kazachstania, Nakaseomyces and Tetrapisispora were shown to be genetically highly diverse based on the above listed analyses. Considering the results obtained for the presently recognized genera, a range of 80–92 % POCP values and a range of 60–70 % AAI values might be valuable thresholds to discriminate genera in Saccharomycetaceae. Furthermore, the genus-specific genes identified in the PAPO analysis and the CSIs were found to be useful as synapomorphies to characterize and define genera in Saccharomycetaceae. Our results indicate that the combined monophyly-based phylogenomic analysis together with genomic relatedness indices and synapomorphies provide promising approaches to delineating yeast genera and likely those of filamentous fungi as well. The genera Kazachstania, Nakaseomyces and Tetrapisispora are revised and we propose eight new genera and 41 new combinations. [ABSTRACT FROM AUTHOR]

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of enzymes. Production cost of enzymes may be brought down by multifaceted approaches which include use of cheap lignocellulosic substrates for fermentation and production of enzymes, and use of cost efficient fermentation strategies like solid state fermentation (SSF). The focus of this study, firstly different agro-waste was washed with tap water for three times. Next wheat bran was screened from different agro-wastes. Then enzymatic saccharification of pre-treated wheat bran for high yield of reducing sugar using a purified enzymatic treatment from fungal isolate Aspergillus flavus PUF5. Central Composite Design was employed to plan experiments and optimize the enzymatic saccharification of wheat bran. Experimental results show that substrate amount, fermentation time and pH of the medium were main factors governing the enzymatic saccharification of wheat bran. The Model F value of 52.73 implied the Model was significant. Model P value in this study was < 0.01, which also indicated that the model was significant. The "Pred R--Squared" of 0.8982 was in reasonable agreement with the "Adj R-squared" of 0.9668, which indicated a good fitness of the model. On the basis of medium optimization, the quadratic model predicted that the maximum amount of reducing sugar was produced (36.4 mg/g) when the substrate amount, fermentation time and pH were 5.7 g, 16 h, and pH 6.5, respectively. The fermentation of the concentrated hydrolyzate (20 gL-1) by Saccharomyces cerevesiae resulted in ethanol concentration of 7.9 gL-1. [ABSTRACT FROM AUTHOR]

Yeasts have been used for centuries for the leavening of bread. The main emphasis on the selection of yeast strains has been in relation to wheat products. This study is the first evaluation of different yeasts coming from the baking and brewing industry in a gluten-free system. Five different yeast strains (US-05, WB-06, T-58, S-23 and baker's yeast) of the species Saccharomyces cerevisiae were evaluated for their suitability to leaven gluten-free dough. A wide range of dough quality characteristics such as the time and temperature-dependent rising behaviour, the chemical composition of the dough and the pH were determined. In addition to this, the bread quality attributes like, volume, texture, structure, aroma and flavour were evaluated. Obtained results indicated different activity levels between the selected yeast strains. Doughs prepared with US-05 showed a slower dough rise during proofing and a decreased height, in comparison to the baker's yeast control. The application of WB-06 and T-58, however, resulted in a faster dough rise and increased dough height with greater gas cells (p < 0.05). These observations were also found in the baked breads, where these two yeasts reached a higher specific volume and a softer breadcrumb than the baker's yeast bread (p < 0.05). Statistical analysis revealed strong correlations (p < 0.05) between activity level, dough properties and bread properties. Results obtained showed that the selected yeast strains reached different level of activity due to diverse preferences in temperature, time and sugars. Yeast strains which originated from the brewing industry were found to be suitable for gluten-free bread making. [ABSTRACT FROM AUTHOR]

Controlled fermentation with a pure Saccharomyces cerevisiae culture is a frequent practice in modern wineries. Use of other fermentation strategies could positively influence the aroma of the wine product. However, the background and application of these approaches are often very difficult and complex. The use of axenic S. cerevisiae culture originating from the particular terroir remains the best compromise for modern winemaking. In our research, 48 S. cerevisiae strains from 7 viticultural regions of Czech Republic and Slovakia were isolated, identified and studied. For the first time, newly-bred vine varieties Devín, Pálava, Moravian Muscat and Dunaj were used for isolation of new yeast strains. The aim of this work was to find a convenient method for identification and selection of autochthonous S. cerevisiae yeasts. Isolated strains were identified using conventional microbiological tests, sequencing of the internal transcribed spacer region and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). From the results of this research, the selection of 17 yeast strains for potential use as a starter culture in the production of typical wines was performed. Six of them, namely, PAC34, PAE58, PAG63, DUH135, PAH48 and DEH53, were isolated from newly-bred varieties. [ABSTRACT FROM AUTHOR]

Genetically identical cells exhibit extensive phenotypic variation even under constant and benign conditions. This so-called nongenetic heterogeneity has important clinical implications: within tumors and microbial infections, cells show nongenetic heterogeneity in growth rate and in susceptibility to drugs or stress. The budding yeast, Saccharomyces cerevisiae, shows a similar form of nongenetic heterogeneity in which growth rate correlates positively with susceptibility to acute heat stress at the single-cell level. Using genetic and chemical perturbations, combined with high-throughput single-cell assays of yeast growth and gene expression, we show here that heterogeneity in intracellular cyclic AMP (cAMP) levels acting through the conserved Ras/cAMP/protein kinase A (PKA) pathway and its target transcription factors, Msn2 and Msn4, underlies this nongenetic heterogeneity. Lower levels of cAMP correspond to slower growth, as shown by direct comparison of cAMP concentration in subpopulations enriched for slower vs. faster growing cells. Concordantly, an endogenous reporter of this pathway’s activity correlates with growth in individual cells. The paralogs Msn2 and Msn4 differ in their roles in nongenetic heterogeneity in a way that demonstrates slow growth and stress tolerance are not inevitably linked. Heterogeneity in growth rate requires each, whereas only Msn2 is required for heterogeneity in expression of Tsl1, a subunit of trehalose synthase that contributes to acute-stress tolerance. Perturbing nongenetic heterogeneity by mutating genes in this pathway, or by culturing wild-type cells with the cell-permeable cAMP analog 8-bromo-cAMP or the PKA inhibitor H89, significantly impacts survival of acute heat stress. Perturbations that increase intracellular cAMP levels reduce the slower-growing subpopulation and increase susceptibility to acute heat stress, whereas PKA inhibition slows growth and decreases susceptibility to acute heat stress. Loss of Msn2 reduces, but does not completely eliminate, the correlation in individual cells between growth rate and acute-stress survival, suggesting a major role for the Msn2 pathway in nongenetic heterogeneity but also a residual benefit of slow growth. Our results shed light on the genetic control of nongenetic heterogeneity and suggest a possible means of defeating bet-hedging pathogens or tumor cells by making them more uniformly susceptible to treatment. [ABSTRACT FROM AUTHOR]

The study aims to isolate the yeast strains that could be used effectively as baker’s yeast and compare them with the commercial baker’s yeast available in the market of Nepal. A total of 10 samples including locally available sources like fruits, Murcha, and a local tree “Dar” were collected from different localities of Bhaktapur, Kavre, and Syangja districts of Nepal, respectively. Following enrichment and fermentation of the samples, 26 yeast strains were isolated using selective medium Wallerstein Laboratory Nutrient Agar. From the differential tests which included morphological and microscopic observation and physiological and biochemical characterization such as nitrate reduction and lactose utilization tests, 8 strains were selected as possible Saccharomyces strain. The selected strains were further assessed for their efficient leavening ability by tests such as ethanol tolerance, osmotolerance, invertase test, and stress exclusion test. The three most potent strains ENG, MUR3B, and SUG1 isolated from grape, Murcha, and sugarcane, respectively, were used in the fermentation and baking of dough. These strains also carried a possibility of being used as industrial baker’s yeast. [ABSTRACT FROM AUTHOR]

Exploring the evolutionary patterns of mitochondrial genomes is important for our understanding of the Saccharomyces sensu stricto (SSS) group, which is a model system for genomic evolution and ecological analysis. In this study, we first obtained the complete mitochondrial sequences of two important species, Saccharomyces mikatae and Saccharomyces kudriavzevii. We then compared the mitochondrial genomes in the SSS group with those of close relatives, and found that the non-coding regions evolved rapidly, including dramatic expansion of intergenic regions, fast evolution of introns and almost 20-fold higher rearrangement rates than those of the nuclear genomes. However, the coding regions, and especially the protein-coding genes, are more conserved than those in the nuclear genomes of the SSS group. The different evolutionary patterns of coding and non-coding regions in the mitochondrial and nuclear genomes may be related to the origin of the aerobic fermentation lifestyle in this group. Our analysis thus provides novel insights into the evolution of mitochondrial genomes. [ABSTRACT FROM AUTHOR]

The characterization of mutational spectra is usually carried out in one of three ways–by direct observation through mutation accumulation (MA) experiments, through parent-offspring sequencing, or by indirect inference from sequence data. Direct observations of spontaneous mutations with MA experiments are limited, given (i) the rarity of spontaneous mutations, (ii) applicability only to laboratory model species with short generation times, and (iii) the possibility that mutational spectra under lab conditions might be different from those observed in nature. Trio sequencing is an elegant solution, but it is not applicable in all organisms. Indirect inference, usually from divergence data, faces no such technical limitations, but rely upon critical assumptions regarding the strength of natural selection that are likely to be violated. Ideally, new mutational events would be directly observed before the biased filter of selection, and without the technical limitations common to lab experiments. One approach is to identify very young mutations from population sequencing data. Here we do so by leveraging two characteristics common to all new mutations—new mutations are necessarily rare in the population, and absent in the genomes of immediate relatives. From 132 clinical yeast strains, we were able to identify 1,425 putatively new mutations and show that they exhibit extremely low signatures of selection, as well as display a mutational spectrum that is similar to that identified by a large scale MA experiment. We verify that population sequencing data are a potential wealth of information for inferring mutational spectra, and should be considered for analysis where MA experiments are infeasible or especially tedious. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the effects of dietary symbiotic supplementation on growth performance, digestive tract development, ilealhistomorphology, caecaltotalColiform, E coli andEnterobactericaeacounts in different chick weights. A blend of mannanoligosaccharide and Saccharomyces cerevisiae mixture was used as symbiotic. Two initial body weight groups (L= Light and H= Heavy chicks) and two feeds (B= basal diet and S= 0,2% symbiotic supplemented diet) were tested in 2x2 factorial experimental design. One hundred and twenty newly hatched chicks obtained from a healthy broiler parent stock aged 40 wk old. Each treatment group had 3 replicates consist of 10 chicks. Trial lasted for 21 days. Daily body weight gain (DBWG), daily feed intake (DFI), feed conversion ratio (FCR) (g feed/g gain) was recorded weekly. The DBWG, DFI, FCR and digestive tract development (heart, liver, gizzard, proventriculus, pancreas, bursa fabricius weight and gut length) were not affected by treatments. Symbiotic supplementation increased villi length. Symbiotic supplementation decreased pathogenic microorganisms (Escherichia Coli, Coliform and Enterobactericaea) in caeca irrespective to the DBWG of chicks. To conclude, symbiotic supplementation can be used to improve villi morphology and to suppress pathogenic microorganisms in caeca. [ABSTRACT FROM AUTHOR]

Simple and efficient genotyping methods are widely used to assess the diversity of a large number of microbial strains, e.g. wine yeasts isolated from a specific geographical area or a vintage. Such methods are often also the first to be applied, to decrease the number of strains deemed interesting for a more time-consuming physiological characterization. Here, we aimed to use a physiologically characterized strain collection of 69 Saccharomyces cerevisiae strains from Hungarian wine regions to determine whether geographical origin or physiological similarity can be recovered by clustering the strains with one or two simultaneously used variations of interdelta genotyping. Our results indicate that although a detailed clustering with high resolution can be achieved with this method, the clustering of strains is largely contrasting when different primer sets are used and it does not recover geographical or physiological groups. Significance and Impact of the Study Genotyping is routinely used for assessing the diversity of a large number of isolates/strains of a single species, e.g. a collection of wine yeasts. We tested the efficiency of interdelta genotyping on a collection of Saccharomyces wine yeasts from four wine regions of Hungary that was previously characterized physiologically. Interdelta fingerprinting recovered neither physiological nor geographical similarities, and in addition, the two different primer pairs widely used for this method showed conflicting and barely comparable results. Thus, this method does not necessarily represent the true diversity of a strain collection, but detailed clustering may be achieved by the combined use of primer sets. [ABSTRACT FROM AUTHOR]

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

The aim of the work was to evaluate the application potential of a glycosidase extract of one indigenous non- Saccharomyces strain in wine aroma enhancement. The isolate was selected from a local winemaking region in China for its high β-glucosidase level and was identified as Rhodotorula mucilaginosa. The tolerance of the glycosidase extract to the typical winemaking conditions was assessed using the activity of its β-glucosidase. After that, the hydrolysis capacity of R. mucilaginosa glycosidase for liberation of grape aroma glycosides was characterized in comparison to commercial enzyme preparations. Results of this work revealed that glycosidase extract from R. mucilaginosa proved to be active in the presence of 0-20% (w/v) glucose, 0-20% (v/v) ethanol and at pH 3·0-5·0. In the hydrolysis of aroma precursors, enzymes obtained from different origins possessed various levels of specificity and activity, showing high origin dependence ( α = 0·05). Compared to commercial enzymes, the indigenous R. mucilaginosa glycosidase extract presented better catalytic preference for the 'fruity and floral' glycosides of benzenic compounds and C13-norisoprenoids, but less sensitivity to the glycosides of C6 compounds and volatile phenols. Significance and Impact of the Study This work presents a novel extracellular glycosidase preparation from an indigenous Rhodotorula mucilaginosa strain selected from a local winemaking region in China. This enzyme extract exhibits strong tolerance towards winemaking conditions. It shows hydrolysis specificity for glycosides of benzenic compounds and C13-norisoprenoids, proving a potential candidate for improving floral and fruity aroma characteristics of wine. [ABSTRACT FROM AUTHOR]

Motivation: Genome-scale metabolic models (GEMs) are tools that allow predicting a phenotype from a genotype under certain environmental conditions. GEMs have been developed in the last ten years for a broad range of organisms, and are used for multiple purposes such as discovering new properties of metabolic networks, predicting new targets for metabolic engineering, as well as optimizing the cultivation conditions for biochemicals or recombinant protein production. Pichia pastoris is one of the most widely used organisms for heterologous protein expression. There are different GEMs for this methylotrophic yeast of which the most relevant and complete in the published literature are iPP668, PpaMBEL1254 and iLC915. However, these three models differ regarding certain pathways, terminology for metabolites and reactions and annotations. Moreover, GEMs for some species are typically built based on the reconstructed models of related model organisms. In these cases, some organism-specific pathways could be missing or misrepresented. Results: In order to provide an updated and more comprehensive GEM for P. pastoris, we have reconstructed and validated a consensus model integrating and merging all three existing models. In this step a comprehensive review and integration of the metabolic pathways included in each one of these three versions was performed. In addition, the resulting iMT1026 model includes a new description of some metabolic processes. Particularly new information described in recently published literature is included, mainly related to fatty acid and sphingolipid metabolism, glycosylation and cell energetics. Finally the reconstructed model was tested and validated, by comparing the results of the simulations with available empirical physiological datasets results obtained from a wide range of experimental conditions, such as different carbon sources, distinct oxygen availability conditions, as well as producing of two different recombinant proteins. In these simulations, the iMT1026 model has shown a better performance than the previous existing models. [ABSTRACT FROM AUTHOR]

DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved “steric gate” residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of Saccharomyces cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo . Unlike the related pol ι enzyme, wild-type pol η does not readily incorporate NMPs in vitro . In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol η F35A ( rad30 -F35A) that is also deficient for nucleotide excision repair ( rad1Δ ) and the TLS polymerase, pol ζ ( rev3Δ ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201 + counterpart. Interestingly the rad1Δ rev3Δ rad30 -F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1 bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell’s replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells. [ABSTRACT FROM AUTHOR]

The experimental evidence from the last decade made telomerase a prominent member of a family of moonlighting proteins performing different functions at various cellular loci. However, the study of extratelomeric functions of the catalytic subunit of mammalian telomerase (TERT) is often complicated by the fact that it is sometimes difficult to distinguish them from its role(s) at the chromosomal ends. Here, we present an experimental model for studying the extranuclear function(s) of mammalian telomerase in the yeast Saccharomyces cerevisiae. We demonstrate that the catalytic subunit of mammalian telomerase protects the yeast cells against oxidative stress and affects the stability of the mitochondrial genome. The advantage of using S. cerevisiae to study of mammalian telomerase is that (1) mammalian TERT does not interfere with its yeast counterpart in the maintenance of telomeres, (2) yeast telomerase is not localized in mitochondria and (3) it does not seem to be involved in the protection of cells against oxidative stress and stabilization of mtDNA. Thus, yeast cells can be used as a 'test tube' for reconstitution of mammalian TERT extranuclear function(s). [ABSTRACT FROM AUTHOR]

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

The cell wall polysaccharides of brewers spent yeast Saccharomyces pastorianus (BSY) and the inoculum yeast (IY) were studied in order to understand the changes induced by the brewing process. The hot water and alkali extractions performed solubilized mainly mannoproteins, more branched for BSY than those of IY. Also, 31 P solid state NMR showed that the BSY mannoproteins were 3 times more phosphorylated. By electron microscopy it was observed that the final residues of alkali sequential extraction until 4 M KOH preserved the yeast three-dimensional structure. The final residues, composed mainly by glucans (92%), showed that the BSY, when compared with IY, contained higher amount of (1 → 4)-linked Glc (43% for BSY and 16% for IY) and lower (1 → 3)-linked Glc (17% for BSY and 42% for IY). The enzymatic treatment of final residue showed that both BSY and IY had (α1 → 4)-linked Glc and (β1 → 4)-linked Glc, in a 2:1 ratio, showing that S. pastorianus increases their cellulose-like linkages with the brewing process. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae, an ascosporogenous yeast commonly used in the production of food, is an emerging infection in immunocompromised patients. We report the case of a 60-year-old man whose orthotopic liver transplant was complicated by S. cerevisiae fungemia and peritoneal abscess, successfully treated with caspofungin and drainage. We also review the literature of invasive saccharomycoses in recipients of hematologic and solid organ transplants. [ABSTRACT FROM AUTHOR]

Background & Aims: Probiotics, including Saccharomyces boulardii, have been advocated for the prevention of Clostridium difficile infection. The aim of this project was to evaluate the effects of the removal of S. boulardii from an automatic antibiotic order set and hospital formulary on hospital onset C. difficile infection rates. Method. Design: A retrospective chart review was performed on all patients with hospital onset C. difficile infection during the 13 months prior (control group) and the 13 months after (study group) removal of an automatic order set linking S. boulardii capsules to certain broad spectrum antibiotics. Setting: A large 800+ bed tertiary hospital Results: Among all hospitalized patients, the rate of hospital onset C. difficile infection was 0.99 per 1000 patient days while the S. boulardii protocol was active compared with 1.04 per 1000 patient days (p=0.10) after S. boulardii was removed from the formulary. No difference in the rate of hospital onset C. difficile infection was detected in patients receiving the linked broad spectrum antibiotics during and after the removal of the protocol (1.25% vs. 1.51%, respectively; p=0.70). Conclusions: Removal of S. boulardii administration to patients receiving broad spectrum antibiotics and the hospital formulary did not impact the rate of hospital onset C. difficile infection in either the hospital population or patients receiving broad spectrum antibiotics. [ABSTRACT FROM AUTHOR]

The Saccharomyces sensu stricto group encompasses species ranging from the industrially ubiquitous yeast Saccharomyces cerevisiae to those that are confined to geographically limited environmental niches. The wealth of genomic data that are now available for the Saccharomyces genus is providing unprecedented insights into the genomic processes that can drive speciation and evolution, both in the natural environment and in response to human-driven selective forces during the historical "domestication" of these yeasts for baking, brewing, and winemaking. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae naturally produces small amounts of isobutanol and 3-methyl-1-butanol via Ehrlich pathway from the catabolism of valine and leucine, respectively. In this study, we engineered CEN.PK2-1C, a leucine auxotrophic strain having a LEU2 gene mutation, for the production of isobutanol and 3-methyl-1-butanol. First, ALD6 encoding aldehyde dehydrogenase and BAT1 involved in valine synthesis were deleted to eliminate competing pathways. We also increased transcription of endogenous genes in the valine and leucine biosynthetic pathways by expressing Leu3Δ601, a constitutively active form of Leu3 transcriptional activator. For the production of isobutanol, genes involved in isobutanol production ( ILV2, ILV3, ILV5, ARO10, and ADH2) were additionally overexpressed in ald6Δbat1Δ strain expressing LEU3Δ601, resulting in 376.9 mg/L isobutanol production from 100 g/L glucose. To increase 3-methyl-1-butanol production, leucine biosynthetic genes were additionally overexpressed in the final isobutanol-production strain. The resulting strain overexpressing LEU2 and LEU4, a feedback inhibition-insensitive mutant of LEU4, showed a 34-fold increase in 3-methyl-1-butanol synthesis compared with CEN.PK2-1C control strain, producing 765.7 mg/L 3-methyl-1-butanol. [ABSTRACT FROM AUTHOR]

TOR kinase complex I (TORC1) is a key regulator of cell growth and metabolism in all eukaryotes. Previous studies in yeast have shown that three GTPases-Gtr1, Gtr2, and Rho1-bind to TORC1 in nitrogen and amino acid starvation conditions to block phosphorylation of the S6 kinase Sch9 and activate protein phosphatase 2A (PP2A). This leads to downregulation of 450 Sch9- dependent protein and ribosome synthesis genes and upregulation of 100 PP2A-dependent nitrogen assimilation and amino acid synthesis genes. Here, using bandshift assays and microarray measurements, we show that the TORC1 pathway also populates three other stress/starvation states. First, in glucose starvation conditions, the AMP-activated protein kinase (AMPK/Snf1) and at least one other factor push the TORC1 pathway into an off state, in which Sch9-branch signaling and PP2A-branch signaling are both inhibited. Remarkably, the TORC1 pathway remains in the glucose starvation (PP2A inhibited) state even when cells are simultaneously starved for nitrogen and glucose. Second, in osmotic stress, the MAPK Hog1/p38 drives the TORC1 pathway into a different state, in which Sch9 signaling and PP2A-branch signaling are inhibited, but PP2A-branch signaling can still be activated by nitrogen starvation. Third, in oxidative stress and heat stress, TORC1-Sch9 signaling is blocked while weak PP2A-branch signaling occurs. Together, our data show that the TORC1 pathway acts as an information-processing hub, activating different genes in different conditions to ensure that available energy is allocated to drive growth, amino acid synthesis, or a stress response, depending on the needs of the cell. [ABSTRACT FROM AUTHOR]

Aerobic glycolysis is a metabolic pathway utilized by human cancer cells and also by yeast cells when they ferment glucose to ethanol. Both cancer cells and yeast cells are inhibited by the presence of low concentrations of 2-deoxyglucose (2DG). Genetic screens in yeast used resistance to 2-deoxyglucose to identify a small set of genes that function in regulating glucose metabolism. A recent high throughput screen for 2-deoxyglucose resistance identified a much larger set of seemingly unrelated genes. Here, we demonstrate that these newly identified genes do not in fact confer significant resistance to 2-deoxyglucose. Further, we show that the relative toxicity of 2-deoxyglucose is carbon source dependent, as is the resistance conferred by gene deletions. Snf1 kinase, the AMPactivated protein kinase of yeast, is required for 2-deoxyglucose resistance in cells growing on glucose. Mutations in the SNF1 gene that reduce kinase activity render cells hypersensitive to 2-deoxyglucose, while an activating mutation in SNF1 confers 2-deoxyglucose resistance. Snf1 kinase activated by 2-deoxyglucose does not phosphorylate the Mig1 protein, a known Snf1 substrate during glucose limitation. Thus, different stimuli elicit distinct responses from the Snf1 kinase. [ABSTRACT FROM AUTHOR]

The nuclear RNA and DNA helicase Sen1 is essential in the yeast Saccharomyces cerevisiae and is required for efficient termination of RNA polymerase II transcription of many short noncoding RNA genes. However, the mechanism of Sen1 function is not understood. We created a plasmid-based genetic system to study yeast Sen1 in vivo. Using this system, we show that (1) the minimal essential region of Sen1 corresponds to the helicase domain and one of two flanking nuclear localization sequences; (2) a previously isolated terminator readthrough mutation in the Sen1 helicase domain, E1597K, is rescued by a second mutation designed to restore a salt bridge within the first RecA domain; and (3) the human ortholog of yeast Sen1, Senataxin, cannot functionally replace Sen1 in yeast. Guided by sequence homology between the conserved helicase domains of Sen1 and Senataxin, we tested the effects of 13 missense mutations that cosegregate with the inherited disorder ataxia with oculomotor apraxia type 2 on Sen1 function. Ten of the disease mutations resulted in transcription readthrough of at least one of three Sen1-dependent termination elements tested. Our genetic system will facilitate the further investigation of structure--function relationships in yeast Sen1 and its orthologs. [ABSTRACT FROM AUTHOR]

In the yeast Saccharomyces cerevisiae, fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. This adaptation results in massive reprogramming of gene expression. Increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced. The zinc cluster proteins Cat8, Sip4, and Rds2, as well as Adr1, have been shown to mediate this reprogramming of gene expression. In this study, we have characterized the gene YBR239C encoding a putative zinc cluster protein and it was named ERT1 (ethanol regulated transcription factor 1). ChIP-chip analysis showed that Ert1 binds to a limited number of targets in the presence of glucose. The strongest enrichment was observed at the promoter of PCK1 encoding an important gluconeogenic enzyme. With ethanol as the carbon source, enrichment was observed with many additional genes involved in gluconeogenesis and mitochondrial function. Use of lacZ reporters and quantitative RT-PCR analyses demonstrated that Ert1 regulates expression of its target genes in a manner that is highly redundant with other regulators of gluconeogenesis. Interestingly, in the presence of ethanol, Ert1 is a repressor of PDC1 encoding an important enzyme for fermentation. We also show that Ert1 binds directly to the PCK1 and PDC1 promoters. In summary, Ert1 is a novel factor involved in the regulation of gluconeogenesis as well as a key fermentation gene. [ABSTRACT FROM AUTHOR]

Retrotransposons are mobile DNA elements present throughout eukaryotic genomes that can cause mutations and genome rearrangements when they replicate through reverse transcription. Increased expression and/or mobility of retrotransposons has been correlated with aging in yeast, Caenorhabditis elegans, Drosophila melanogaster, and mammals. The many copies of retrotransposons in humans and various model organisms complicate further pursuit of this relationship. The Saccharomyces cerevisiae Ty1 retrotransposon was introduced into a strain of S. paradoxus that completely lacks retrotransposons to compare chronological lifespans (CLSs) of yeast strains with zero, low, or high Ty1 copy number. Yeast chronological lifespan reflects the progressive loss of cell viability in a nondividing state. Chronological lifespans for the strains were not different in rich medium, but were extended in high Ty1 copy-number strains in synthetic medium and in rich medium containing a low dose of hydroxyurea (HU), an agent that depletes deoxynucleoside triphosphates. Lifespan extension was not strongly correlated with Ty1 mobility or mutation rates for a representative gene. Buffering deoxynucleoside triphosphate levels with threonine supplementation did not substantially affect this lifespan extension, and no substantial differences in cell cycle arrest in the nondividing cells were observed. Lifespan extension was correlated with reduced reactive oxygen species during early stationary phase in high Ty1 copy strains, and antioxidant treatment allowed the zero Ty1 copy strain to live as long as high Ty1 copy-number strains in rich medium with hydroxyurea. This exceptional yeast system has identified an unexpected longevity-promoting role for retrotransposons that may yield novel insights into mechanisms regulating lifespan. [ABSTRACT FROM AUTHOR]

Carbonic anhydrases ( CAs) are metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, β, γ, δ, ζ) that are unrelated in sequence and structure. The fungal β-class has only recently attracted attention. In the present study, we investigated the structure and function of the plant-like β- CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae β- CA Nce103 and exhibit an in vitro CO2 hydration activity ( kcat/ Km of CAS1: 1.30 × 106 m−1·s−1; CAS2: 1.21 × 106 m−1·s−1). To further investigate the structural properties of CAS1 and CAS2, we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With the exception of the active site composition, no further major differences have been found. In both enzymes, the Zn2+ -ion is tetrahedrally coordinated; in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions, making them good candidates for industrial applications. Structured digital abstract and by (), Database Structural data have been deposited in the Protein Data Bank database under accession numbers for CAS1 and for CAS2. [ABSTRACT FROM AUTHOR]

The 33 genes in the Saccharomyces cerevisia emitotic CLB2 transcription cluster have been known to be downregulated by the DNA damage checkpoint for many years. Here, we show that this is mediated by the checkpoint kinase Rad53 and the dedicated transcriptional activator of the cluster, Ndd1. Ndd1 is phosphorylated in response to DNA damage, which blocks recruitment to promoters and leads to the transcriptional downregulation of the CLB2 cluster. Finally, we show that downregulation of Ndd1 is an essential function of Rad53, as a hypomorphic ndd1 allele rescues RAD53 deletion. [ABSTRACT FROM AUTHOR]

Phenotypic traits may be gained and lost together because of pleiotropy, the involvement of common genes and networks, or because of simultaneous selection for multiple traits across environments (multiple-trait coevolution). However, the extent to which network pleiotropy versus environmental coevolution shapes shared responses has not been addressed. To test these alternatives, we took advantage of the fact that the genus Saccharomyces has variation in habitat usage and diversity in the carbon sources that a given strain can metabolize. We examined patterns of gain and loss in carbon utilization traits across 488 strains of Saccharomyces to investigate whether the structure of metabolic pathways or selection pressure from common environments may have caused carbon utilization traits to be gained and lost together. While most carbon sources were gained and lost independently of each other, we found four clusters that exhibit non-random patterns of gain and loss across strains. Contrary to the network pleiotropy hypothesis, we did not find that these patterns are explained by the structure of metabolic pathways or shared enzymes. Consistent with the hypothesis that common environments shape suites of phenotypes, we found that the environment a strain was isolated from partially predicts the carbon sources it can assimilate. [ABSTRACT FROM AUTHOR]

Gene transcription by RNA polymerase (Pol) II requires the coactivator complex Mediator. Mediator connects transcriptional regulators and Pol II, and is linked to human disease. Mediator from the yeast Saccharomyces cerevisiae has a molecular mass of 1.4?megadaltons and comprises 25?subunits that form the head, middle, tail and kinase modules. The head module constitutes one-half of the essential Mediator core, and comprises the conserved subunits Med6, Med8, Med11, Med17, Med18, Med20 and Med22. Recent X-ray analysis of the S. cerevisiae head module at 4.3?Å resolution led to a partial architectural model with three submodules called neck, fixed jaw and moveable jaw. Here we determine de novo the crystal structure of the head module from the fission yeast Schizosaccharomyces pombe at 3.4?Å resolution. Structure solution was enabled by new structures of Med6 and the fixed jaw, and previous structures of the moveable jaw and part of the neck, and required deletion of Med20. The S. pombe head module resembles the head of a crocodile with eight distinct elements, of which at least four are mobile. The fixed jaw comprises tooth and nose domains, whereas the neck submodule contains a helical spine and one limb, with shoulder, arm and finger elements. The arm and the essential shoulder contact other parts of Mediator. The jaws and a central joint are implicated in interactions with Pol?II and its carboxy-terminal domain, and the joint is required for transcription in vitro. The S. pombe head module structure leads to a revised model of the S. cerevisiae module, reveals a high conservation and flexibility, explains known mutations, and provides the basis for unravelling a central mechanism of gene regulation. [ABSTRACT FROM AUTHOR]

Background: Fecal antibodies against bacterial products may directly reflect the interaction between luminal bacteria and mucosal immunity, and assays for these antibodies may be clinically useful in the diagnosis and differential diagnosis of Crohn's disease-like (CDL) condition of the pouch. Aims: This study aims to evaluate stool and serum anti- Saccharomyces cerevisiae antibodies (ASCA) in normal and diseased pouches, to assess the correlation between ASCA levels and endoscopic disease activity, and to ascertain the diagnostic utility of ASCA for CDL of the pouch. Methods: One hundred eighty-nine patients with ileal pouches were prospectively enrolled and corresponding serum and pouch aspirate samples were collected. Fecal and serum ASCA levels were measured with enzyme-linked immunosorbent assay in a blinded fashion. Statistical analysis was then conducted using the signed rank test, Spearman correlation coefficients, and analysis of variance. Results: Forty-three patients (22.8 %) had irritable pouch syndrome or normal pouches, 74 (39.2 %) had pouchitis/cuffitis, 52 (27.5 %) had CDL, 9 (4.8 %) had familial adenomatous polyposis, and 11 (5.8 %) had surgical complications of the pouch. Receiver operating characteristic curves to distinguish CDL from other categories of pouch dysfunction had an area under the curve (AUC) of 0.608 for fecal ASCA and an AUC of 0.517 for serum ASCA. Neither fecal nor serum ASCA correlated with endoscopic disease activity scores. There was a significant difference in the mean values of fecal ASCA between inflammatory and fistulizing CDL (0.27 vs. 0.03 ELISA units/ml, P < 0.05). Conclusions: Fecal ASCA appears to be better than serum ASCA in differentiating CDL from other pouch disorders, although this distinction may be of limited clinical utility. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated the microbiota of sour rotten wine grapes and its impact on wine fermentations. Yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were enumerated and identified on sound and sour rot grapes during the ripening stage. The alteration of the ecological balance induced by sour rot was particularly evidenced by the unequivocal increase of yeast and AAB counts on rotten grapes, since the beginning of ripening. Yeast and AAB species diversity in rotten grape samples were much higher than those found in sound grapes. LAB populations were low detected from both healthy and sour rotten grapes. The yeast species Issatchenkia occidentalis, Zygoascus hellenicus and Zygosaccharomyces bailii and the AAB species Gluconacetobacter hansenii, Gluconacetobacter intermedius and Acetobacter malorum, were recovered from damaged grapes and resulting grape juices in the winery. Acetobacter orleaniensis and Acetobacter syzygii were only recovered from sour rotten grapes. Dekkera bruxellensis and Oenococcus oeni were only recovered after wine fermentation induced by starter inoculation, irrespective of grape health, probably originating from cellar environment. After malolactic fermentation, racking and sulphur dioxide addition the only remaining species were the yeast Trigonopsis cantarellii and Saccharomyces cerevisiae, independently of the grape health status. [Copyright &y& Elsevier]

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high-energy-pulse-electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L33 orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l−1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l−1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g−1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial-scale ethanol production. Significance and Impact of the Study: High-energy-pulse-electron radiation is a new efficient technology in breeding micro-organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress. [ABSTRACT FROM AUTHOR]

Background: Salmonella causes acute systemic inflammation by using its virulence factors to invade the intestinal epithelium. But, prolonged inflammation may provoke severe body catabolism and immunological diseases. Salmonella has become more life-threatening due to emergence of multiple-antibiotic resistant strains. Mannose-rich oligosaccharides (MOS) from cells walls of Saccharomyces cerevisiae have shown to bind mannose-specific lectin of Gram-negative bacteria including Salmonella, and prevent their adherence to intestinal epithelial cells. However, whether MOS may potentially mitigate systemic inflammation is not investigated yet. Moreover, molecular events underlying innate immune responses and metabolic activities during late inflammation, in presence or absence of MOS, are unknown. Methods and Principal Findings: Using a Salmonella LPS-induced systemic inflammation chicken model and microarray analysis, we investigated the effects of MOS and virginiamycin (VIRG, a sub-therapeutic antibiotic) on innate immunity and glucose metabolism during late inflammation. Here, we demonstrate that MOS and VIRG modulated innate immunity and metabolic genes differently. Innate immune responses were principally mediated by intestinal IL-3, but not TNF-α, IL-1 or IL- 6, whereas glucose mobilization occurred through intestinal gluconeogenesis only. MOS inherently induced IL-3 expression in control hosts. Consequent to LPS challenge, IL-3 induction in VIRG hosts but not differentially expressed in MOS hosts revealed that MOS counteracted LPS's detrimental inflammatory effects. Metabolic pathways are built to elucidate the mechanisms by which VIRG host's higher energy requirements were met: including gene up-regulations for intestinal gluconeogenesis (PEPCK) and liver glycolysis (ENO2), and intriguingly liver fatty acid synthesis through ATP citrate synthase (CS) down-regulation and ATP citrate lyase (ACLY) and malic enzyme (ME) up-regulations. However, MOS host's lower energy demands were sufficiently met through TCA citrate-derived energy, as indicated by CS up-regulation. Conclusions: MOS terminated inflammation earlier than VIRG and reduced glucose mobilization, thus representing a novel biological strategy to alleviate Salmonella-induced systemic inflammation in human and animal hosts. [ABSTRACT FROM AUTHOR]

The de novo biosynthetic machinery for both sphingolipid and ergosterol production in yeast is localized in the endoplasmic reticulum (ER) and Golgi. The interconnections between the two pathways are still poorly understood, but they may be connected in specialized membrane domains, and specific knockouts strongly suggest that both routes have different layers of mutual control and are co-affected by drugs. With the goal of shedding light on the functional integration of the yeast sphingolipid-ergosterol (SL-E) pathway, we constructed a dynamic model of the ergosterol pathway using the guidelines of Biochemical Systems Theory (BST) (Savageau., J. theor. Biol., 25, 365-9, 1969). The resulting model was merged with a previous mathematical model of sphingolipid metabolism in yeast (Alvarez-Vasquez et al., J. theor. Biol., 226, 265-91, 2004; Alvarez-Vasquez et al., Nature 433, 425-30, 2005). The S-system format within BST was used for analyses of consistency, stability, and sensitivity of the SL-E model, while the GMA format was used for dynamic simulations and predictions. Model validation was accomplished by comparing predictions from the model with published results on sterol and sterol-ester dynamics in yeast. The validated model was used to predict the metabolomic dynamics of the SL-E pathway after drug treatment. Specifically, we simulated the action of drugs affecting sphingolipids in the endoplasmic reticulum and studied changes in ergosterol associated with microdomains of the plasma membrane (PM). [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae proteasome comprises a 19-subunit regulatory particle and a 28-subunit core particle. To be degraded, substrates must cross the core particle-regulatory particle interface, a site for complex conformational changes and regulatory events. This interface includes two aligned heteromeric rings, one formed by the six ATPase (Rpt) subunits of the regulatory particle and the other by the seven ? subunits of the core particle. The Rpt C termini bind to intersubunit cavities in the ?-ring, thus directing core particle gating and proteasome assembly. We mapped the Rpt C termini to the ? subunit pockets, using a cross-linking approach that revealed an unexpected asymmetry: one side of the ring shows 1:1 contacts of Rpt2-?4, Rpt6-?3 and Rpt3-?2, whereas on the opposite side, the Rpt1, Rpt4 and Rpt5 tails each cross-link to multiple ? pockets. Rpt-core particle cross-links are all sensitive to nucleotides, implying that ATP hydrolysis drives dynamic alterations at the core particle-regulatory particle interface. [ABSTRACT FROM AUTHOR]

Background: Fatty acids synthesized in chloroplast are transported to endoplasmic reticulum (ER) for triacylglycerols (TAGs) resembling. The development of chloroplast also requires lipids trafficking from ER to chloroplast. The membrane contact sites (MCSs) between ER and chloroplast has been demonstrated to be involved for the trafficking of lipids and proteins. Lipids trafficking between ER and chloroplast is often accompanied by lipids interconversion. However, it is rarely known how lipids interconversion happens during their trafficking. Methodology/Principal Findings: We cloned a lipase gene from Brassica napus L., designated as BnCLIP1. Green fluorescence protein (GFP)-tagged BnCLIP1 was shown to locate at the MCSs between ER and chloroplasts in tobacco leaves. Heterogeneous expression of BnCLIP1 in Saccharomyces cerevisiae (pep4) reduced the total amount of fatty acid. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the truncated BnCLIP1 had a substrate preference for C16:0 lipids in Saccharomyces cerevisiae (pep4). To probe the physiological function of BnCLIP1, two Brassica napus lines with different oil-content were introduced to investigate the transcript patterns of BnCLIP1 during seed development. Intriguingly, the transcript level of BnCLIP1 was found to be immediately up-regulated during the natural seed senescence of both lines; the transcription response of BnCLIP1 in the high oil-content seeds was faster than the lower ones, suggesting a potential role of BnCLIP1 in affecting seed oil synthesis via regulating chloroplast integrity. Further researches showed that chemical disruption of leaf chloroplast also activated the transcription of BnCLIP1. Conclusions/Significance: The findings of this study show that BnCLIP1 encodes a lipase, localizes at the MCSs and involves in chloroplast development. [ABSTRACT FROM AUTHOR]