Showing papers on "Yeast published in 2010"

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe

Abstract: We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast--the only other eukaryote for which a comprehensive knockout library exists--revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

Controlled mixed culture fermentation: a new perspective on the use of non-Saccharomyces yeasts in winemaking.

Abstract: Mixed fermentations using controlled inoculation of Saccharomyces cerevisiae starter cultures and non-Saccharomyces yeasts represent a feasible way towards improving the complexity and enhancing the particular and specific characteristics of wines. The profusion of selected starter cultures has allowed the more widespread use of inoculated fermentations, with consequent improvements to the control of the fermentation process, and the use of new biotechnological processes in winemaking. Over the last few years, as a consequence of the re-evaluation of the role of non-Saccharomyces yeasts in winemaking, there have been several studies that have evaluated the use of controlled mixed fermentations using Saccharomyces and different non-Saccharomyces yeast species from the wine environment. The combined use of different species often results in unpredictable compounds and/or different levels of fermentation products being produced, which can affect both the chemical and the aromatic composition of wines. Moreover, possible synergistic interactions between different yeasts might provide a tool for the implementation of new fermentation technologies. Thus, knowledge of the Saccharomyces and non-Saccharomyces wine yeast interactions during wine fermentation needs to be improved. To reach this goal, further investigations into the genetic and physiological background of such non-Saccharomyces wine yeasts are needed, so as to apply '-omics' approaches to mixed culture fermentations.

Apoptosis in yeast: triggers, pathways, subroutines

Abstract: A cell's decision to die is controlled by a sophisticated network whose deregulation contributes to the pathogenesis of multiple diseases including neoplastic and neurodegenerative disorders. The finding, more than a decade ago, that baker's yeast (Saccharomyces cerevisiae) can undergo apoptosis uncovered the possibility to investigate this mode of programmed cell death (PCD) in a model organism that combines both technical advantages and a eukaryotic 'cell room.' Since then, numerous exogenous and endogenous triggers have been found to induce yeast apoptosis and multiple yeast orthologs of crucial metazoan apoptotic regulators have been identified and characterized at the molecular level. Such apoptosis-relevant orthologs include proteases such as the yeast caspase as well as several mitochondrial and nuclear proteins that contribute to the execution of apoptosis in a caspase-independent manner. Additionally, physiological scenarios such as aging and failed mating have been discovered to trigger apoptosis in yeast, providing a teleological interpretation of PCD affecting a unicellular organism. Due to its methodological and logistic simplicity, yeast constitutes an ideal model organism that is efficiently helping to decipher the cell death regulatory network of higher organisms, including the switches between apoptotic, autophagic, and necrotic pathways of cellular catabolism. Here, we provide an overview of the current knowledge about the apoptotic subroutine of yeast PCD and its regulation.

An improved yeast transformation method for the generation of very large human antibody libraries

Abstract: Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (approximately 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl(2), MgCl(2), sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/microg vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.

Cellodextrin Transport in Yeast for Improved Biofuel Production

Abstract: Fungal degradation of plant biomass may provide insights for improving cellulosic biofuel production. We show that the model cellulolytic fungus Neurospora crassa relies on a high-affinity cellodextrin transport system for rapid growth on cellulose. Reconstitution of the N. crassa cellodextrin transport system in Saccharomyces cerevisiae promotes efficient growth of this yeast on cellodextrins. In simultaneous saccharification and fermentation experiments, the engineered yeast strains more rapidly convert cellulose to ethanol when compared with yeast lacking this system.

Engineering of protein secretion in yeast: Strategies and impact on protein production

Abstract: Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways. Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages for systematic engineering of yeast strains for effective protein secretion.

Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid

Abstract: Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5). Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to this weak acid. These are novel candidate genes for genetic engineering to obtain more robust yeast strains against acetic acid toxicity. Among these genes there are number of transcription factors that are documented regulators of a large percentage of the genes found to exert protection against acetic acid thus being considered interesting targets for subsequent genetic engineering. The increase of potassium concentration in the growth medium was found to improve the expression of maximal tolerance to acetic acid, consistent with the idea that the adequate manipulation of nutrient concentration of industrial growth medium can be an interesting strategy to surpass the deleterious effects of this weak acid in yeast cells.

Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair

Abstract: We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNACUAPyl pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNACUAPyl in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNACUAPyl pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNACUAPyl pairs to be site-specifically incorporated into proteins in yeast.

Transcriptional Regulation in Yeast during Diauxic Shift and Stationary Phase

Abstract: The preferred source of carbon and energy for yeast cells is glucose. When yeast cells are grown in liquid cultures, they metabolize glucose predominantly by glycolysis, releasing ethanol in the medium. When glucose becomes limiting, the cells enter diauxic shift characterized by decreased growth rate and by switching metabolism from glycolysis to aerobic utilization of ethanol. When ethanol is depleted from the medium, cells enter quiescent or stationary phase G0. Cells in diauxic shift and stationary phase are stressed by the lack of nutrients and by accumulation of toxic metabolites, primarily from the oxidative metabolism, and are differentiated in ways that allow them to maintain viability for extended periods of time. The transition of yeast cells from exponential phase to quiescence is regulated by protein kinase A, TOR, Snf1p, and Rim15p pathways that signal changes in availability of nutrients, converge on transcriptional factors Msn2p, Msn4p, and Gis1p, and elicit extensive reprogrammin...

Bulk segregant analysis by high-throughput sequencing reveals a novel xylose utilization gene from Saccharomyces cerevisiae.

Abstract: Fermentation of xylose is a fundamental requirement for the efficient production of ethanol from lignocellulosic biomass sources. Although they aggressively ferment hexoses, it has long been thought that native Saccharomyces cerevisiae strains cannot grow fermentatively or non-fermentatively on xylose. Population surveys have uncovered a few naturally occurring strains that are weakly xylose-positive, and some S. cerevisiae have been genetically engineered to ferment xylose, but no strain, either natural or engineered, has yet been reported to ferment xylose as efficiently as glucose. Here, we used a medium-throughput screen to identify Saccharomyces strains that can increase in optical density when xylose is presented as the sole carbon source. We identified 38 strains that have this xylose utilization phenotype, including strains of S. cerevisiae, other sensu stricto members, and hybrids between them. All the S. cerevisiae xylose-utilizing strains we identified are wine yeasts, and for those that could produce meiotic progeny, the xylose phenotype segregates as a single gene trait. We mapped this gene by Bulk Segregant Analysis (BSA) using tiling microarrays and high-throughput sequencing. The gene is a putative xylitol dehydrogenase, which we name XDH1, and is located in the subtelomeric region of the right end of chromosome XV in a region not present in the S288c reference genome. We further characterized the xylose phenotype by performing gene expression microarrays and by genetically dissecting the endogenous Saccharomyces xylose pathway. We have demonstrated that natural S. cerevisiae yeasts are capable of utilizing xylose as the sole carbon source, characterized the genetic basis for this trait as well as the endogenous xylose utilization pathway, and demonstrated the feasibility of BSA using high-throughput sequencing.

Cryptococcus: from human pathogen to model yeast.

Abstract: Cryptococcusfrom human pathogen to model yeast , Cryptococcusfrom human pathogen to model yeast , کتابخانه مرکزی دانشگاه علوم پزشکی تهران

Surface display of a functional minicellulosome by intracellular complementation using a synthetic yeast consortium and its application to cellulose hydrolysis and ethanol production.

Abstract: In this paper, we report the surface assembly of a functional minicellulosome by using a synthetic yeast consortium. The basic design of the consortium consisted of four different engineered yeast strains capable of either displaying a trifunctional scaffoldin, Scaf-ctf (SC), carrying three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f), or secreting one of the three corresponding dockerin-tagged cellulases (endoglucanase [AT], exoglucanase [EC/CB], or β-glucosidase [BF]). The secreted cellulases were docked onto the displayed Scaf-ctf in a highly organized manner based on the specific interaction of the three cohesin-dockerin pairs employed, resulting in the assembly of a functional minicellulosome on the yeast surface. By exploiting the modular nature of each population to provide a unique building block for the minicellulosome structure, the overall cellulosome assembly, cellulose hydrolysis, and ethanol production were easily fine-tuned by adjusting the ratio of different populations in the consortium. The optimized consortium consisted of a SC:AT:CB:BF ratio of 7:2:4:2 and produced almost twice the level of ethanol (1.87 g/liter) as a consortium with an equal ratio of the different populations. The final ethanol yield of 0.475 g of ethanol/g of cellulose consumed also corresponded to 93% of the theoretical value. This result confirms the use of a synthetic biology approach for the synergistic saccharification and fermentation of cellulose to ethanol by using a yeast consortium displaying a functional minicellulosome.

Cdc14: a highly conserved family of phosphatases with non-conserved functions?

Abstract: CDC14 was originally identified by L. Hartwell in his famous screen for genes that regulate the budding yeast cell cycle. Subsequent work showed that Cdc14 belongs to a family of highly conserved dual-specificity phosphatases that are present in a wide range of organisms from yeast to human. Human CDC14B is even able to fulfill the essential functions of budding yeast Cdc14. In budding yeast, Cdc14 counteracts the activity of cyclin dependent kinase (Cdk1) at the end of mitosis and thus has important roles in the regulation of anaphase, mitotic exit and cytokinesis. On the basis of the functional conservation of other cell-cycle genes it seemed obvious to assume that Cdc14 phosphatases also have roles in late mitosis in mammalian cells and regulate similar targets to those found in yeast. However, analysis of the human Cdc14 proteins (CDC14A, CDC14B and CDC14C) by overexpression or by depletion using small interfering RNA (siRNA) has suggested functions that are quite different from those of ScCdc14. Recent studies in avian and human somatic cell lines in which the gene encoding either Cdc14A or Cdc14B had been deleted, have shown – surprisingly – that neither of the two phosphatases on its own is essential for viability, cell-cycle progression and checkpoint control. In this Commentary, we critically review the available data on the functions of yeast and vertebrate Cdc14 phosphatases, and discuss whether they indeed share common functions as generally assumed.

Comparative transcriptome profiling analyses during the lag phase uncover YAP1 , PDR1, PDR3, RPN4 , and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae

Abstract: The yeast Saccharomyces cerevisiae is able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and HMF. The length of lag phase for cell growth in response to the inhibitor challenge has been used to measure tolerance of strain performance. Mechanisms of yeast tolerance at the genome level remain unknown. Using systems biology approach, this study investigated comparative transcriptome profiling, metabolic profiling, cell growth response, and gene regulatory interactions of yeast strains and selective gene deletion mutations in response to HMF challenges during the lag phase of growth. We identified 365 candidate genes and found at least 3 significant components involving some of these genes that enable yeast adaptation and tolerance to HMF in yeast. First, functional enzyme coding genes such as ARI1, ADH6, ADH7, and OYE3, as well as gene interactions involved in the biotransformation and inhibitor detoxification were the direct driving force to reduce HMF damages in cells. Expressions of these genes were regulated by YAP1 and its closely related regulons. Second, a large number of PDR genes, mainly regulated by PDR1 and PDR3, were induced during the lag phase and the PDR gene family-centered functions, including specific and multiple functions involving cellular transport such as TPO1, TPO4, RSB1, PDR5, PDR15, YOR1, and SNQ2, promoted cellular adaptation and survival in order to cope with the inhibitor stress. Third, expressed genes involving degradation of damaged proteins and protein modifications such as SHP1 and SSA4, regulated by RPN4, HSF1, and other co-regulators, were necessary for yeast cells to survive and adapt the HMF stress. A deletion mutation strain Δrpn4 was unable to recover the growth in the presence of HMF. Complex gene interactions and regulatory networks as well as co-regulations exist in yeast adaptation and tolerance to the lignocellulose derived inhibitor HMF. Both induced and repressed genes involving diversified functional categories are accountable for adaptation and energy rebalancing in yeast to survive and adapt the HMF stress during the lag phase of growth. Transcription factor genes YAP1, PDR1, PDR3, RPN4, and HSF1 appeared to play key regulatory rules for global adaptation in the yeast S. cerevisiae.

Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences

Abstract: The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776–782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The β-aggregation predictor TANGO found highly β-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were β-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High β-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

Abstract: The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration. In cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression. To our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.

Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica

Abstract: Biotechnological production of weak organic acids such as succinic acid is most economically advantageous when carried out at low pH. Among naturally occurring microorganisms, several bacterial strains are known to produce considerable amounts of succinic acid under anaerobic conditions but they are inefficient in performing the low-pH fermentation due to their physiological properties. We have proposed therefore a new strategy for construction of an aerobic eukaryotic producer on the basis of the yeast Yarrowia lipolytica with a deletion in the gene coding one of succinate dehydrogenase subunits. Firstly, an original in vitro mutagenesis-based approach was proposed to construct strains with Ts mutations in the Y. lipolytica SDH1 gene. These mutants were used to optimize the composition of the media for selection of transformants with the deletion in the Y. lipolytica SDH2 gene. Surprisingly, the defects of each succinate dehydrogenase subunit prevented the growth on glucose but the mutant strains grew on glycerol and produced succinate in the presence of the buffering agent CaCO3. Subsequent selection of the strain with deleted SDH2 gene for increased viability allowed us to obtain a strain capable of accumulating succinate at the level of more than 45 g L−1 in shaking flasks with buffering and more than 17 g L−1 without buffering. The possible effect of the mutations on the utilization of different substrates and perspectives of constructing an industrial producer is discussed. Biotechnol. Bioeng. 2010;107:673–682. © 2010 Wiley Periodicals, Inc.

Microbial communities and chemical changes during fermentation of sugary Brazilian kefir

Abstract: The microorganisms associated with sugary Brazilian kefir beverage were investigated using a combination of culture-dependent and -independent methods. A total of 289 bacteria and 129 yeasts were identified via phenotypic and genotypic methods. Lb. paracasei (23.8%) was the major bacterial isolate identified, followed by Acetobacter lovaniensis (16.31%), Lactobacillus parabuchneri (11.71%), Lactobacillus kefir (10.03%) and Lactococcus lactis (10.03%). Saccharomyces cerevisiae (54.26%) and Kluyveromyces lactis (20.15%) were the most common yeast species isolated. Scanning electron microscopy showed that the microbiota was dominated by lemon-shaped yeast cells growing in close association with Lactobacillus (long and curved). Some lactic acid bacteria detected by sequence analysis of DGGE (denaturing gradient gel electrophoresis) bands were not recovered at any time through fermentation by plating. Conversely, DGGE fingerprints did not reveal bands corresponding to some of the species isolated by culturing methods. The bacteria Acetobacter lovaniensis and the yeast Kazachstania aerobia are described for the first time in sugary kefir. During the 24 h of fermentation, the concentration of lactic acid ranged from 0.2 to 1.80 mg/ml, and that of acetic acid increased from 0.08 to 1.12 mg/ml. The production of ethanol was limited, reaching a final mean value of 1.24 mg/ml.

Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes

Abstract: To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification-fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 degrees C and 37 degrees C, while the activity of cellulolytic enzymes is highest at around 50 degrees C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus beta-glucosidase on the cell surface, which successfully converts a cellulosic beta-glucan to ethanol directly at 48 degrees C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of beta-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.

Capric Acid Secreted by S. boulardii Inhibits C. albicans Filamentous Growth, Adhesion and Biofilm Formation

Abstract: Candidiasis are life-threatening systemic fungal diseases, especially of gastro intestinal track, skin and mucous membranes lining various body cavities like the nostrils, the mouth, the lips, the eyelids, the ears or the genital area. Due to increasing resistance of candidiasis to existing drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. One promising strategy is the use of the probiotic microorganisms, which when administered in adequate amounts confer a health benefit. Such a probiotic microorganism is yeast Saccharomyces boulardii, a close relative of baker yeast. Saccharomyces boulardii cells and their extract affect the virulence factors of the important human fungal pathogen C. albicans, its hyphae formation, adhesion and biofilm development. Extract prepared from S. boulardii culture filtrate was fractionated and GC-MS analysis showed that the active fraction contained, apart from 2-phenylethanol, caproic, caprylic and capric acid whose presence was confirmed by ESI-MS analysis. Biological activity was tested on C. albicans using extract and pure identified compounds. Our study demonstrated that this probiotic yeast secretes into the medium active compounds reducing candidal virulence factors. The chief compound inhibiting filamentous C. albicans growth comparably to S. boulardii extract was capric acid, which is thus responsible for inhibition of hyphae formation. It also reduced candidal adhesion and biofilm formation, though three times less than the extract, which thus contains other factors suppressing C. albicans adherence. The expression profile of selected genes associated with C. albicans virulence by real-time PCR showed a reduced expression of HWP1, INO1 and CSH1 genes in C. albicans cells treated with capric acid and S. boulardii extract. Hence capric acid secreted by S. boulardii is responsible for inhibition of C. albicans filamentation and partially also adhesion and biofilm formation.

The influence of yeast strains on the volatile flavour compounds of Chinese rice wine

Abstract: In order to assess the influence of yeast strains on volatile flavour profiles of Chinese rice wine, small-scale Chinese rice wine brewing was carried out with eight yeast strains from three different typical Chinese rice wine brewing regions. The volatile flavour compounds were extracted by headspace solid phase microextraction (HS-SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). The volatile flavour profiles of the different Chinese rice wines showed statistically significant differences depending on the yeast strains used. Yeast strains from the Shaoxing region showed a higher capacity to produce the esters 2-phenylethanol and 3-methylthiopropanol, while yeast strains from the Shanghai region stood out for their production of branched-chain higher alcohols. Chinese rice wine fermented with a yeast strain from the Jiangsu region had the highest levels of organic acids. Using principal component analysis of the Chinese rice wine volatile flavour compounds, the eight yeast strains could be classified into three groups according to their origins. This is the first report about the volatile flavour characteristics of Chinese rice wine yeast. The data obtained in this work shows that the yeast strains contributed significantly to the flavour differences of the Chinese rice wines from the different regions.

Microbiological approaches to lowering ethanol concentration in wine

Abstract: There is increasing demand by the wine industry for technologies to reduce the alcohol content of wine without compromising product quality. This review describes biological-based approaches that influence ethanol yield during fermentation, most of which have attempted to partially redirect yeast metabolism away from ethanol production. Genetic engineering approaches have been used to develop yeast strains that produce less ethanol, and this has provided insight into how yeast respond to metabolic rerouting. However, public attitudes towards the use of GMOs (Genetically Modified Organisms) in food products suggests that novel yeast strains will have to be generated using non-GM approaches, such as evolutionary engineering.

The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae

Abstract: Magnaporthe oryzae is the most damaging fungal pathogen of rice (Oryza sativa). In this study, we characterized the TIG1 transducin β-like gene required for infectious growth and its interacting genes that are required for plant infection in this model phytopathogenic fungus. Tig1 homologs in yeast and mammalian cells are part of a conserved histone deacetylase (HDAC) transcriptional corepressor complex. The tig1 deletion mutant was nonpathogenic and defective in conidiogenesis. It had an increased sensitivity to oxidative stress and failed to develop invasive hyphae in plant cells. Using affinity purification and coimmunoprecipitation assays, we identified several Tig1-associated proteins, including two HDACs that are homologous to components of the yeast Set3 complex. Functional analyses revealed that TIG1, SET3, SNT1, and HOS2 were core components of the Tig1 complex in M. oryzae. The set3, snt1, and hos2 deletion mutants displayed similar defects as those observed in the tig1 mutant, but deletion of HST1 or HOS4 had no detectable phenotypes. Deletion of any of these core components of the Tig1 complex resulted in a significant reduction in HDAC activities. Our results showed that TIG1, like its putative yeast and mammalian orthologs, is one component of a conserved HDAC complex that is required for infectious growth and conidiogenesis in M. oryzae and highlighted that chromatin modification is an essential regulatory mechanism during plant infection.

Streptococcus mutans inhibits Candida albicans hyphal formation by the fatty acid signaling molecule trans-2-decenoic acid (SDSF).

Abstract: In the human mouth, fungi and several hundred species of bacteria coexist. Here we report a case of interkingdom signaling in the oral cavity: A compound excreted by the caries bacterium Streptococcus mutans inhibits the morphological transition from yeast to hyphae, an important virulence trait, in the opportunistic fungus Candida albicans. The compound excreted by S. mutans was originally studied because it inhibited signaling by the universal bacterial signal autoinducer-2 (AI-2), determined by the luminescence of a Vibrio harveyi sensor strain. The inhibitor was purified from cell-free culture supernatants of S. mutans guided by its activity. Its chemical structure was elucidated by using NMR spectroscopy and GC-MS and proved to be trans-2-decenoic acid. We show that trans-2-decenoic acid does not inhibit AI-2-specific signaling, but rather the luciferase reaction used for its detection. A potential biological role of trans-2-decenoic acid was then discovered. It is able to suppress the transition from yeast to hyphal morphology in the opportunistic human pathogen Candida albicans at concentrations that do not affect growth. The expression of HWP1, a hyphal-specific signature gene of C. albicans, is abolished by trans-2-decenoic acid. trans-2-Decenoic acid is structurally similar to the diffusible signal factor (DSF) family of interkingdom-signaling molecules and is the first member of this family from a Gram-positive organism (Streptococcus DSF, SDSF). SDSF activity was also found in S. mitis, S. oralis, and S. sanguinis, but not in other oral bacteria. SDSF could be relevant in shaping multispecies Candida bacteria biofilms in the human body.