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Sample records for cerevisiae diploid strain

  1. Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance

    NASA Technical Reports Server (NTRS)

    D'Elia, Riccardo; Allen, Patricia L.; Johanson, Kelly; Nickerson, Cheryl A.; Hammond, Timothy G.

    2005-01-01

    This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

  2. Improved stress resistance and ethanol production by segmental haploidization of the diploid genome in Saccharomyces cerevisiae.

    PubMed

    Kaboli, Saeed; Miyamoto, Tetsuya; Sunada, Keisuke; Sasano, Yu; Sugiyama, Minetaka; Harashima, Satoshi

    2016-06-01

    Saccharomyces cerevisiae strains from industrial and natural geographical environments are reported to show great variation in copy number of chromosomal regions. Such variation contributes to the mechanisms underlying adaptation to different environments. Here, we created and phenotypically analyzed segmentally haploidized strains, each harboring a deletion of one copy of approximately 100-300 kb of the left or right terminal region of 16 chromosomes in a diploid strain by using a PCR-mediated chromosomal deletion method. No haploidized strain of the 158-kb deleted right terminal region of chromosome III or the 172-kb deleted right terminal region of chromosome VI was produced; however, segmentally haploidized strains of the remaining 30 terminal regions were obtained. Among these 30 strains, two exhibited higher lactic acid resistance and two displayed higher thermo-tolerance at 41°C versus the host diploid strain. By contrast, four and two segmentally haploidized strains showed sensitivity to 6% lactic acid and low temperature at 13°C, respectively. The effect of the decreased copy number of the chromosomal terminal regions on ethanol production was analyzed. As compared with the host diploid strain, a 3.8% and 4.3% improvement in ethanol production in 10% glucose medium was observed for two strains in which one of two copies of the 197-kb left terminal region of chromosome V and one of two copies of the 195-kb left terminal region of chromosome X was deleted, respectively. These results indicate that artificial segmental haploidization might contribute to improvement of industrially important phenotypes and provide a new approach to breeding superior yeast strains. PMID:26690924

  3. Comparative Genome-Wide Screening Identifies a Conserved Doxorubicin Repair Network That Is Diploid Specific in Saccharomyces cerevisiae

    PubMed Central

    Westmoreland, Tammy J.; Wickramasekara, Sajith M.; Guo, Andrew Y.; Selim, Alice L.; Winsor, Tiffany S.; Greenleaf, Arno L.; Blackwell, Kimberly L.; Olson, John A.; Marks, Jeffrey R.; Bennett, Craig B.

    2009-01-01

    The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Δ, ctf4Δ, ctk1Δ, hfi1Δ,nup133Δ, tho2Δ) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Δ and hfi1Δ) or deficient (tho2Δ) for recombination. Using these and other “THO2-like” hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Δ, thp1Δ, thp2Δ) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases. PMID:19503795

  4. ``alternative Self-Diploidization'' or ``asd'' Homothallism in Saccharomyces Cerevisiae: Isolation of a Mutant, Nuclear-Cytoplasmic Interaction and Endomitotic Diploidization

    PubMed Central

    Ono, B. I.; Ishino-Arao, Y.; Takasugi, K.; Taniguchi, M.; Fukuda, M.; Fukui, M.; Miyakawa, I.; Sando, N.

    1990-01-01

    A mutant of Saccharomyces cerevisiae representing a novel life cycle, named ``alternative self-diploidization'' or ``ASD'' homothallism, was obtained fortuitously. In this life cycle, MATα (or MATa) haplophase and MATα/MATα (or MATa/MATa) diplophase alternate. Germinated cells are haploid and mating. They soon become nonmating and sporogenous as they vegetatively grow. They sooner or later diploidize presumably via endomitosis. The diploid cells haploidize via normal meiosis. A single recessive nuclear mutation, named asd1-1, is responsible for ``ASD'' homothallism. In the ρ(0) cytoplasm, asd1-1 cells mate even if at a low efficiency and fail to diploidize. Since pet mutations do not have such effects, we conclude that a certain mitochondrial function other than respiration is required for manifestation of ``ASD'' homothallism. That is, ``ASD'' homothallism is the result of some sort of nuclear-cytoplasmic interaction. PMID:2204579

  5. Sequential Logic of Polarity Determination during the Haploid-to-Diploid Transition in Saccharomyces cerevisiae

    PubMed Central

    Zapanta Rinonos, Serendipity; Rai, Urvashi; Vereb, Sydney; Wolf, Kyle; Yuen, Eric; Lin, Cindy

    2014-01-01

    In many organisms, the geometry of encounter of haploid germ cells is arbitrary. In Saccharomyces cerevisiae, the resulting zygotes have been seen to bud asymmetrically in several directions as they produce diploid progeny. What mechanisms account for the choice of direction, and do the mechanisms directing polarity change over time? Distinct subgroups of cortical “landmark” proteins guide budding by haploid versus diploid cells, both of which require the Bud1/Rsr1 GTPase to link landmarks to actin. We observed that as mating pairs of haploid cells form zygotes, bud site specification progresses through three phases. The first phase follows disassembly and limited scattering of proteins that concentrated at the zone of cell contact, followed by their reassembly to produce a large medial bud. Bud1 is not required for medial placement of the initial bud. The second phase produces a contiguous bud(s) and depends on axial landmarks. As the titer of the Axl1 landmark diminishes, the third phase ultimately redirects budding toward terminal sites and is promoted by bipolar landmarks. Thus, following the initial random encounter that specifies medial budding, sequential spatial choices are orchestrated by the titer of a single cortical determinant that determines whether successive buds will be contiguous to their predecessors. PMID:25172767

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

    PubMed

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

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

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

    PubMed Central

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

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

  8. Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production

    PubMed Central

    Argueso, Juan Lucas; Carazzolle, Marcelo F.; Mieczkowski, Piotr A.; Duarte, Fabiana M.; Netto, Osmar V.C.; Missawa, Silvia K.; Galzerani, Felipe; Costa, Gustavo G.L.; Vidal, Ramon O.; Noronha, Melline F.; Dominska, Margaret; Andrietta, Maria G.S.; Andrietta, Sílvio R.; Cunha, Anderson F.; Gomes, Luiz H.; Tavares, Flavio C.A.; Alcarde, André R.; Dietrich, Fred S.; McCusker, John H.; Petes, Thomas D.; Pereira, Gonçalo A.G.

    2009-01-01

    Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (∼2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies. PMID:19812109

  9. Understanding the Mechanism of Thermotolerance Distinct From Heat Shock Response Through Proteomic Analysis of Industrial Strains of Saccharomyces cerevisiae*

    PubMed Central

    Shui, Wenqing; Xiong, Yun; Xiao, Weidi; Qi, Xianni; Zhang, Yong; Lin, Yuping; Guo, Yufeng; Zhang, Zhidan; Wang, Qinhong; Ma, Yanhe

    2015-01-01

    Saccharomyces cerevisiae has been intensively studied in responses to different environmental stresses such as heat shock through global omic analysis. However, the S. cerevisiae industrial strains with superior thermotolerance have not been explored in any proteomic studies for elucidating the tolerance mechanism. Recently a new diploid strain was obtained through evolutionary engineering of a parental industrial strain, and it exhibited even higher resistance to prolonged thermal stress. Herein, we performed iTRAQ-based quantitative proteomic analysis on both the parental and evolved industrial strains to further understand the mechanism of thermotolerant adaptation. Out of ∼2600 quantifiable proteins from biological quadruplicates, 193 and 204 proteins were differentially regulated in the parental and evolved strains respectively during heat-stressed growth. The proteomic response of the industrial strains cultivated under prolonged thermal stress turned out to be substantially different from that of the laboratory strain exposed to sudden heat shock. Further analysis of transcription factors underlying the proteomic perturbation also indicated the distinct regulatory mechanism of thermotolerance. Finally, a cochaperone Mdj1 and a metabolic enzyme Adh1 were selected to investigate their roles in mediating heat-stressed growth and ethanol production of yeasts. Our proteomic characterization of the industrial strain led to comprehensive understanding of the molecular basis of thermotolerance, which would facilitate future improvement in the industrially important trait of S. cerevisiae by rational engineering. PMID:25926660

  10. The Transient Inactivation of the Master Cell Cycle Phosphatase Cdc14 Causes Genomic Instability in Diploid Cells of Saccharomyces cerevisiae.

    PubMed

    Quevedo, Oliver; Ramos-Pérez, Cristina; Petes, Thomas D; Machín, Félix

    2015-07-01

    Genomic instability is a common feature found in cancer cells . Accordingly, many tumor suppressor genes identified in familiar cancer syndromes are involved in the maintenance of the stability of the genome during every cell division and are commonly referred to as caretakers. Inactivating mutations and epigenetic silencing of caretakers are thought to be the most important mechanisms that explain cancer-related genome instability. However, little is known of whether transient inactivation of caretaker proteins could trigger genome instability and, if so, what types of instability would occur. In this work, we show that a brief and reversible inactivation, during just one cell cycle, of the key phosphatase Cdc14 in the model organism Saccharomyces cerevisiae is enough to result in diploid cells with multiple gross chromosomal rearrangements and changes in ploidy. Interestingly, we observed that such transient loss yields a characteristic fingerprint whereby trisomies are often found in small-sized chromosomes, and gross chromosome rearrangements, often associated with concomitant loss of heterozygosity, are detected mainly on the ribosomal DNA-bearing chromosome XII. Taking into account the key role of Cdc14 in preventing anaphase bridges, resetting replication origins, and controlling spindle dynamics in a well-defined window within anaphase, we speculate that the transient loss of Cdc14 activity causes cells to go through a single mitotic catastrophe with irreversible consequences for the genome stability of the progeny. PMID:25971663

  11. The Transient Inactivation of the Master Cell Cycle Phosphatase Cdc14 Causes Genomic Instability in Diploid Cells of Saccharomyces cerevisiae

    PubMed Central

    Quevedo, Oliver; Ramos-Pérez, Cristina; Petes, Thomas D.; Machín, Félix

    2015-01-01

    Genomic instability is a common feature found in cancer cells . Accordingly, many tumor suppressor genes identified in familiar cancer syndromes are involved in the maintenance of the stability of the genome during every cell division and are commonly referred to as caretakers. Inactivating mutations and epigenetic silencing of caretakers are thought to be the most important mechanisms that explain cancer-related genome instability. However, little is known of whether transient inactivation of caretaker proteins could trigger genome instability and, if so, what types of instability would occur. In this work, we show that a brief and reversible inactivation, during just one cell cycle, of the key phosphatase Cdc14 in the model organism Saccharomyces cerevisiae is enough to result in diploid cells with multiple gross chromosomal rearrangements and changes in ploidy. Interestingly, we observed that such transient loss yields a characteristic fingerprint whereby trisomies are often found in small-sized chromosomes, and gross chromosome rearrangements, often associated with concomitant loss of heterozygosity, are detected mainly on the ribosomal DNA-bearing chromosome XII. Taking into account the key role of Cdc14 in preventing anaphase bridges, resetting replication origins, and controlling spindle dynamics in a well-defined window within anaphase, we speculate that the transient loss of Cdc14 activity causes cells to go through a single mitotic catastrophe with irreversible consequences for the genome stability of the progeny. PMID:25971663

  12. Mead production: selection and characterization assays of Saccharomyces cerevisiae strains.

    PubMed

    Pereira, Ana Paula; Dias, Teresa; Andrade, João; Ramalhosa, Elsa; Estevinho, Letícia M

    2009-08-01

    Mead is a traditional drink, which results from the alcoholic fermentation of diluted honey carried out by yeasts. However, when it is produced in a homemade way, mead producers find several problems, namely, the lack of uniformity in the final product, delayed and arrested fermentations, and the production of "off-flavours" by the yeasts. These problems are usually associated with the inability of yeast strains to respond and adapt to unfavourable and stressful growth conditions. The main objectives of this work were to evaluate the capacity of Saccharomyces cerevisiae strains, isolated from honey of the Trás-os-Montes (Northeast Portugal), to produce mead. Five strains from honey, as well as one laboratory strain and one commercial wine strain, were evaluated in terms of their fermentation performance under ethanol, sulphur dioxide and osmotic stress. All the strains showed similar behaviour in these conditions. Two yeasts strains isolated from honey and the commercial wine strain were further tested for mead production, using two different honey (a dark and a light honey), enriched with two supplements (one commercial and one developed by the research team), as fermentation media. The results obtained in this work show that S. cerevisiae strains isolated from honey, are appropriate for mead production. However it is of extreme importance to take into account the characteristics of the honey, and supplements used in the fermentation medium formulation, in order to achieve the best results in mead production. PMID:19481129

  13. Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1.

    PubMed

    Babrzadeh, Farbod; Jalili, Roxana; Wang, Chunlin; Shokralla, Shadi; Pierce, Sarah; Robinson-Mosher, Avi; Nyren, Pål; Shafer, Robert W; Basso, Luiz C; de Amorim, Henrique V; de Oliveira, Antonio J; Davis, Ronald W; Ronaghi, Mostafa; Gharizadeh, Baback; Stambuk, Boris U

    2012-06-01

    The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains. PMID:22562254

  14. Diversity of Saccharomyces cerevisiae strains isolated from Borassus akeassii palm wines from Burkina Faso in comparison to other African beverages.

    PubMed

    Tapsoba, François; Legras, Jean-Luc; Savadogo, Aly; Dequin, Sylvie; Traore, Alfred Sababenedyo

    2015-10-15

    In South-West of Burkina Faso, palm wine is produced by spontaneous fermentation of the sap from a specific palm tree Borassus akeassii and plays an important role in people's lives. Saccharomyces cerevisiae is the main agent of this alcoholic fermentation but little is known about the diversity of the isolates from palm. In this work, 39 Saccharomyces cerevisiae strains were isolated from palm wine samples collected from 14 sites in Burkina Faso, as well as 7 isolates obtained from sorghum beer (Dolo) from 3 distant sites. Their diversity was analyzed at 12 microsatellite loci, and compared to the genotypes obtained for other African yeast populations isolated from Cocoa hulks from Ghana, sorghum beer from Ivory Coast, palm wine from Djibouti Republic, and to our database of strains from miscellaneous origins (bread, beer, wine, sake, oaks…). The ploidy of these strains has been assessed as well by flow cytometry. Our results show that B. akeassii palm wine contains a specific yeast population of diploid strains, different from Dolo produced in the same area and from other palm wine strains from Ivory Coast, Nigeria, or Djibouti Republic. In contrast, Dolo strains appeared as a group of related and mainly tetraploid strains despite being isolated from different countries. PMID:26202324

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

    PubMed Central

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

    2015-01-01

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

  16. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

    PubMed

    Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development. PMID:27470141

  17. Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Mortimer, R.K.; Contopoulou, R.; Schild, D.

    1981-09-01

    Cells of Saccharomyces cerevisiae with mutations in the RAD52 gene have previously been shown to be defective in meiotic and mitotic recombination, in sporulation, and in repair of radiation-induced damage to DNA. In this study we show that diploid cells homozygous for rad52 lose chromosomes at high frequencies and that these frequencies of loss can be increased dramatically by exposure of these cells to x-rays. Genetic analyses of survivors of x-ray treatment demonstrate that chromosome loss events result in the conversion of diploid cells to cells with near haploid chromosome numbers.

  18. Development of flocculent Saccharomyces cerevisiae strain GYK-10 for the selective fermentation of glucose/fructose in sugar mills.

    PubMed

    Kato, Taku; Ohara, Satoshi; Fukushima, Yasuhiro; Sugimoto, Akira; Masuda, Takayuki; Yasuhara, Takaomi; Yamagishi, Hiromi

    2016-07-01

    Advances in glucose/fructose-selective ethanol production have successfully enhanced raw sugar extraction from sugarcane juice by converting inhibitory substances (i.e., glucose/fructose) into ethanol, which is removed by subsequent operations in cane sugar mills. However, the commercial implementation of this breakthrough process in existing cane sugar mills requires a yeast strain that (i) can be used in food production processes, (ii) exhibits stable saccharometabolic selectivity, and (iii) can be easily separated from the saccharide solution. In this study, we developed a suitable saccharometabolism-selective and flocculent strain, Saccharomyces cerevisiae GYK-10. We obtained a suitable yeast strain for selective fermentation in cane sugar mills using a yeast mating system. First, we crossed a haploid strain defective in sucrose utilization with a flocculent haploid strain. Next, we performed tetrad dissection of the resultant hybrid diploid strain and selected GYK-10 from various segregants by investigating the sucrose assimilation and flocculation capacity phenotypes. Ten consecutive fermentation tests of the GYK-10 strain using a bench-scale fermentor confirmed its suitability for the implementation of practical selective fermentation in a commercial sugar mill. The strain exhibited complete saccharometabolic selectivity and sustained flocculation, where it maintained a high ethanol yield and conversion rate throughout the test. PMID:26811219

  19. A Genomic Screen Revealing the Importance of Vesicular Trafficking Pathways in Genome Maintenance and Protection against Genotoxic Stress in Diploid Saccharomyces cerevisiae Cells

    PubMed Central

    Krol, Kamil; Brozda, Izabela; Skoneczny, Marek; Bretne, Maria; Skoneczna, Adrianna

    2015-01-01

    The ability to survive stressful conditions is important for every living cell. Certain stresses not only affect the current well-being of cells but may also have far-reaching consequences. Uncurbed oxidative stress can cause DNA damage and decrease cell survival and/or increase mutation rates, and certain substances that generate oxidative damage in the cell mainly act on DNA. Radiomimetic zeocin causes oxidative damage in DNA, predominantly by inducing single- or double-strand breaks. Such lesions can lead to chromosomal rearrangements, especially in diploid cells, in which the two sets of chromosomes facilitate excessive and deleterious recombination. In a global screen for zeocin-oversensitive mutants, we selected 133 genes whose deletion reduces the survival of zeocin-treated diploid Saccharomyces cerevisiae cells. The screen revealed numerous genes associated with stress responses, DNA repair genes, cell cycle progression genes, and chromatin remodeling genes. Notably, the screen also demonstrated the involvement of the vesicular trafficking system in cellular protection against DNA damage. The analyses indicated the importance of vesicular system integrity in various pathways of cellular protection from zeocin-dependent damage, including detoxification and a direct or transitional role in genome maintenance processes that remains unclear. The data showed that deleting genes involved in vesicular trafficking may lead to Rad52 focus accumulation and changes in total DNA content or even cell ploidy alterations, and such deletions may preclude proper DNA repair after zeocin treatment. We postulate that functional vesicular transport is crucial for sustaining an integral genome. We believe that the identification of numerous new genes implicated in genome restoration after genotoxic oxidative stress combined with the detected link between vesicular trafficking and genome integrity will reveal novel molecular processes involved in genome stability in diploid cells

  20. Draft Genome Sequence of the Beer Spoilage Bacterium Megasphaera cerevisiae Strain PAT 1T

    PubMed Central

    Kutumbaka, Kirthi K.; Pasmowitz, Joshua; Mategko, James; Reyes, Dindo; Friedrich, Alex; Han, Sukkyun; Martens-Habbena, Willm; Neal-McKinney, Jason; Janagama, Harish K.; Nadala, Cesar

    2015-01-01

    The genus Megasphaera harbors important spoilage organisms that cause beer spoilage by producing off flavors, undesirable aroma, and turbidity. Megasphaera cerevisiae is mainly found in nonpasteurized low-alcohol beer. In this study, we report the draft genome of the type strain of the genus, M. cerevisiae strain PAT 1T. PMID:26358606

  1. Draft Genome Sequence of the Beer Spoilage Bacterium Megasphaera cerevisiae Strain PAT 1T.

    PubMed

    Kutumbaka, Kirthi K; Pasmowitz, Joshua; Mategko, James; Reyes, Dindo; Friedrich, Alex; Han, Sukkyun; Martens-Habbena, Willm; Neal-McKinney, Jason; Janagama, Harish K; Nadala, Cesar; Samadpour, Mansour

    2015-01-01

    The genus Megasphaera harbors important spoilage organisms that cause beer spoilage by producing off flavors, undesirable aroma, and turbidity. Megasphaera cerevisiae is mainly found in nonpasteurized low-alcohol beer. In this study, we report the draft genome of the type strain of the genus, M. cerevisiae strain PAT 1(T). PMID:26358606

  2. Induction of ploidy level increments in an asporogenous industrial strain of the yeast Saccharomyces cerevisiae by UV irradiation.

    PubMed Central

    Sasaki, T

    1992-01-01

    Cells of an asporogenous industrial strain of the yeast Saccharomyces cerevisiae were irradiated with UV light by using a method that was developed previously (T. Sasaki and Y. Ohshima, Appl. Environ. Microbiol. 53:1504-1511, 1987). This treatment gave rise to large-cell clones among the surviving cells, from which colonies consisting of cells with a normal morphology and a prototrophic property were obtained. The large-cell trait of these was stably inheritable, with the cell volumes being about twice that of the parent for 7 years on a slant agar medium at 4 degrees C with repeated transfers. The cellular DNA content of these clones, in comparison to those of two authentic haploid strains, was determined by chemical analysis. The ratio of the DNA contents showed that the parent and its large-cell derivatives were a diploid and tetraploids, respectively. No abnormality was found in the chromosomal DNA patterns of the large-cell clones, at least as determined by agarose gel electrophoresis with a CHEF-DR II pulsed-field electrophoresis system. These findings led to the conclusion that our UV light method is applicable for inducing ploidy level increments in the widely used yeast species S. cerevisiae. Images PMID:1575498

  3. Transcription analysis of recombinant industrial and laboratory Saccharomyces cerevisiae strains reveals the molecular basis for fermentation of glucose and xylose

    PubMed Central

    2014-01-01

    Background There has been much research on the bioconversion of xylose found in lignocellulosic biomass to ethanol by genetically engineered Saccharomyces cerevisiae. However, the rate of ethanol production from xylose in these xylose-utilizing yeast strains is quite low compared to their glucose fermentation. In this study, two diploid xylose-utilizing S. cerevisiae strains, the industrial strain MA-R4 and the laboratory strain MA-B4, were employed to investigate the differences between anaerobic fermentation of xylose and glucose, and general differences between recombinant yeast strains, through genome-wide transcription analysis. Results In MA-R4, many genes related to ergosterol biosynthesis were expressed more highly with glucose than with xylose. Additionally, these ergosterol-related genes had higher transcript levels in MA-R4 than in MA-B4 during glucose fermentation. During xylose fermentation, several genes related to central metabolic pathways that typically increase during growth on non-fermentable carbon sources were expressed at higher levels in both strains. Xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways, even under anaerobic conditions. In addition, several genes involved in spore wall metabolism and the uptake of ammonium, which are closely related to the starvation response, and many stress-responsive genes mediated by Msn2/4p, as well as trehalose synthase genes, increased in expression when fermenting with xylose, irrespective of the yeast strain. We further observed that transcript levels of genes involved in xylose metabolism, membrane transport functions, and ATP synthesis were higher in MA-R4 than in MA-B4 when strains were fermented with glucose or xylose. Conclusions Our transcriptomic approach revealed the molecular events underlying the response to xylose or glucose and differences between MA-R4 and MA-B4. Xylose-utilizing S. cerevisiae strains may recognize xylose as a non

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

    PubMed Central

    Zheng, Yan-Lin; Wang, Shi-An

    2015-01-01

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

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

    PubMed

    Zheng, Yan-Lin; Wang, Shi-An

    2015-01-01

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

  6. Adaptation of a Saccharomyces cerevisiae strain to high copper concentrations.

    PubMed

    Sarais, I; Manzano, M; De Bertoldi, M; Romandini, P; Beltramini, M; Salvato, B; Rocco, G P

    1994-07-01

    A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mM). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent. PMID:8043987

  7. A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae.

    PubMed

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

    2016-07-01

    The conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae. PMID:27070284

  8. Proteomic characterization of a wild-type wine strain of Saccharomyces cerevisiae.

    PubMed

    Trabalzini, Lorenza; Paffetti, Alessandro; Ferro, Elisa; Scaloni, Andrea; Talamo, Fabio; Millucci, Lia; Martelli, Paola; Santucci, Annalisa

    2003-12-01

    Saccharomyces cerevisiae is the optimal eukaryotic model system to study mammalian biological responses. At the same time Saccharomyces cerevisiae is also widely utilized as a biotechnological tool in the food industry. Enological Saccharomyces cerevisiae strains have been so far routinely analyzed for their microbiological aspects. Nevertheless, wine yeasts are gaining an increasing interest in the last years since they strongly affect both the vinification process and the organoleptic properties of the final product wine. The protein repertoire is responsible of such features and, consequently, 2D-PAGE can be an useful tool to evaluate and select optimal wine yeast strains. We present here the first proteomic map of a wild-type wine Saccharomyces cerevisiae strain selected for the guided fermentation of very high quality wines. PMID:15141481

  9. Diversity of Saccharomyces cerevisiae Strains Isolated from Two Italian Wine-Producing Regions.

    PubMed

    Capece, Angela; Granchi, Lisa; Guerrini, Simona; Mangani, Silvia; Romaniello, Rossana; Vincenzini, Massimo; Romano, Patrizia

    2016-01-01

    Numerous studies, based on different molecular techniques analyzing DNA polymorphism, have provided evidence that indigenous Saccharomyces cerevisiae populations display biogeographic patterns. Since the differentiated populations of S. cerevisiae seem to be responsible for the regional identity of wine, the aim of this work was to assess a possible relationship between the diversity and the geographical origin of indigenous S. cerevisiae isolates from two different Italian wine-producing regions (Tuscany and Basilicata). For this purpose, sixty-three isolates from Aglianico del Vulture grape must (main cultivar in the Basilicata region) and from Sangiovese grape must (main cultivar in the Tuscany region) were characterized genotypically, by mitochondrial DNA restriction analysis and MSP-PCR by using (GTG)5 primers, and phenotypically, by determining technological properties and metabolic compounds of oenological interest after alcoholic fermentation. All the S. cerevisiae isolates from each region were inoculated both in must obtained from Aglianico grape and in must obtained from Sangiovese grape to carry out fermentations at laboratory-scale. Numerical analysis of DNA patterns resulting from both molecular methods and principal component analysis of phenotypic data demonstrated a high diversity among the S. cerevisiae strains. Moreover, a correlation between genotypic and phenotypic groups and geographical origin of the strains was found, supporting the concept that there can be a microbial aspect to terroir. Therefore, exploring the diversity of indigenous S. cerevisiae strains can allow developing tailored strategies to select wine yeast strains better adapted to each viticultural area. PMID:27446054

  10. Diversity of Saccharomyces cerevisiae Strains Isolated from Two Italian Wine-Producing Regions

    PubMed Central

    Capece, Angela; Granchi, Lisa; Guerrini, Simona; Mangani, Silvia; Romaniello, Rossana; Vincenzini, Massimo; Romano, Patrizia

    2016-01-01

    Numerous studies, based on different molecular techniques analyzing DNA polymorphism, have provided evidence that indigenous Saccharomyces cerevisiae populations display biogeographic patterns. Since the differentiated populations of S. cerevisiae seem to be responsible for the regional identity of wine, the aim of this work was to assess a possible relationship between the diversity and the geographical origin of indigenous S. cerevisiae isolates from two different Italian wine-producing regions (Tuscany and Basilicata). For this purpose, sixty-three isolates from Aglianico del Vulture grape must (main cultivar in the Basilicata region) and from Sangiovese grape must (main cultivar in the Tuscany region) were characterized genotypically, by mitochondrial DNA restriction analysis and MSP-PCR by using (GTG)5 primers, and phenotypically, by determining technological properties and metabolic compounds of oenological interest after alcoholic fermentation. All the S. cerevisiae isolates from each region were inoculated both in must obtained from Aglianico grape and in must obtained from Sangiovese grape to carry out fermentations at laboratory-scale. Numerical analysis of DNA patterns resulting from both molecular methods and principal component analysis of phenotypic data demonstrated a high diversity among the S. cerevisiae strains. Moreover, a correlation between genotypic and phenotypic groups and geographical origin of the strains was found, supporting the concept that there can be a microbial aspect to terroir. Therefore, exploring the diversity of indigenous S. cerevisiae strains can allow developing tailored strategies to select wine yeast strains better adapted to each viticultural area. PMID:27446054

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  13. New amylolytic yeast strains for starch and dextrin fermentation. [Schwanniomyces alluvius, Saccharomyces cerevisiae var. diastaticus

    SciTech Connect

    Laluce, C.; Bertolini, M.C.; Ernandes, J.R. ); Martini, A.V.; Martini, A. )

    1988-10-01

    Yeast strains capable of fermenting starch and dextrin to ethanol were isolated from samples collected from Brazilian factories in which cassava flour is produced. Considerable alcohol production was observed for all the strains selected. One strain (DI-10) fermented starch rapidly and secreted 5 times as much amylolytic enzyme than that observed for Schwanniomyces alluvius UCD 54-83. This strain and three other similar isolates were classified as Saccharomyces cerevisiae var. diastaticus by morphological and physiological characteristics and molecular taxonomy.

  14. Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains.

    PubMed Central

    Lorenz, M C; Heitman, J

    1998-01-01

    Nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. Recognition of nitrogen starvation is mediated, at least in part, by the ammonium permease Mep2p and the Galpha subunit Gpa2p. Genetic activation of the pheromone-responsive MAP kinase cascade, which is also required for filamentous growth, only weakly suppresses the filamentation defect of Deltamep2/Deltamep2 and Deltagpa2/Deltagpa2 strain. Surprisingly, deletion of Mep1p, an ammonium permease not previously thought to regulate differentiation, significantly enhances the potency of MAP kinase activation, such that the STE11-4 allele induces filamentation to near wild-type levels in Deltamep1/Deltamep1 Deltamep2/Deltamep2 and Deltamep1/Deltamep1 Deltagpa2/Deltagpa2 strains. To identify additional regulatory components, we isolated high-copy suppressors of the filamentation defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant. Multicopy expression of TEC1, PHD1, PHD2 (MSS10/MSN1/FUP4), MSN5, CDC6, MSS11, MGA1, SKN7, DOT6, HMS1, HMS2, or MEP2 each restored filamentation in a Deltamep1/Deltamep1 Deltamep2/Deltamep2 strain. Overexpression of SRK1 (SSD1), URE2, DAL80, MEP1, or MEP3 suppressed only the growth defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant strain. Characterization of these genes through deletion analysis and epistasis underscores the complexity of this developmental pathway and suggests that stress conditions other than nitrogen deprivation may also promote filamentous growth. PMID:9832522

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

    PubMed

    Pérez-Torrado, Roberto; Querol, Amparo

    2015-01-01

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

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

    PubMed Central

    Pérez-Torrado, Roberto; Querol, Amparo

    2016-01-01

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

  17. Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p.

    PubMed

    Barua, Subit; Li, Li; Lipke, Peter N; Dranginis, Anne M

    2016-01-01

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

  18. Molecular Basis for Strain Variation in the Saccharomyces cerevisiae Adhesin Flo11p

    PubMed Central

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

    2016-01-01

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

  19. A Novel Saccharomyces cerevisiae Killer Strain Secreting the X Factor Related to Killer Activity and Inhibition of S. cerevisiae K1, K2 and K28 Killer Toxins.

    PubMed

    Melvydas, Vytautas; Bružauskaitė, Ieva; Gedminienė, Genovaitė; Šiekštelė, Rimantas

    2016-09-01

    It was determined that Kx strains secrete an X factor which can inhibit all known Saccharomyces cerevisiae killer toxins (K1, K2, K28) and some toxins of other yeast species-the phenomenon not yet described in the scientific literature. It was shown that Kx type yeast strains posess a killer phenotype producing small but clear lysis zones not only on the sensitive strain α'1 but also on the lawn of S. cerevisiae K1, K2 and K28 type killer strains at temperatures between 20 and 30 °C. The pH at which killer/antikiller effect of Kx strain reaches its maximum is about 5.0-5.2. The Kx yeast were identified as to belong to S. cerevisiae species. Another newly identified S. cerevisiae killer strain N1 has killer activity but shows no antikilller properties against standard K1, K2 and K28 killer toxins. The genetic basis for Kx killer/antikiller phenotype was associated with the presence of M-dsRNA which is bigger than M-dsRNA of standard S. cerevisiae K1, K2, K28 type killer strains. Killer and antikiller features should be encoded by dsRNA. The phenomenon of antikiller (inhibition) properties was observed against some killer toxins of other yeast species. The molecular weight of newly identified killer toxins which produces Kx type strains might be about 45 kDa. PMID:27407298

  20. Investigation of the dominance behavior of Saccharomyces cerevisiae strains during wine fermentation.

    PubMed

    Perrone, Benedetta; Giacosa, Simone; Rolle, Luca; Cocolin, Luca; Rantsiou, Kalliopi

    2013-07-15

    During wine fermentation, different strains of Saccharomyces cerevisiae compete in the same fermenting must and dominance takes place when one strain overcomes all the others. The purpose of this study was to investigate this phenomenon by identifying S. cerevisiae strains endowed with this feature and to test them in laboratory fermentations. First, autochthonous S. cerevisiae from Nebbiolo fermentations were isolated, molecularly identified and characterized. Genetically diverse S. cerevisiae strains were subsequently subjected to physiological characterization and to micro-scale fermentation, the weight loss kinetics was measured and HPLC analysis was performed at the end of the fermentation. Then, the strains that presented good fermentation characteristics were chosen for further analysis and to determine the dominance feature. For this purpose, couples of strains were co-inoculated in Nebbiolo must and the fermentations were monitored by microbiological and chemical analysis. Two different inoculation approaches were used: co-fermentations in flasks with mixed cells and reactor co-fermentations, in which the cells from the two different strains were kept separate by means of a 0.45 μm filter membrane, which allowed the fermenting must to move freely between the two compartments. During the flask co-fermentations, a minisatellite PCR protocol was applied, in order to differentiate the two strains and determine which one was able to dominate. The protocol included a culture-dependent approach and an independent one. In the first case, DNA extraction was performed on all the colonies scraped off the plates after sampling. In the second case, DNA extraction was performed directly on the fermenting must. The strains that were able to dominate were tested against several S. cerevisiae in order to confirm this dominance behavior. Dominance was observed in the early stages of fermentation, as early as 3days. Combinations of dominant and not-dominant strains were

  1. Characterization of an epithelial, nearly diploid liver cell strain, from Chinese hamster, able to activate promutagens.

    PubMed

    Turchi, G; Carluccio, M A; Oesch, F; Gemperlein, I; Glatt, H R

    1987-03-01

    Epithelial liver cells of the Chinese hamster (CHEL cells) were propagated in culture for 35 passages. At favourable cell densities, the population doubling time in normal medium, was 20 h. L-Tyrosine amino transferase activity was retained at a measurable level, but its enhancement by dexamethasone was detected solely in cells of early passages. Pyruvate kinase was strongly activated by fructose-1,6-biphosphate at low substrate concentrations. These enzymatic properties suggest that the CHEL cells are derived from a sub-population of parenchymal hepatocytes or from cells closely related to parenchymal hepatocytes. With a lag period of a few hours, CHEL cultures metabolized benzo[a]pyrene. In cell homogenates the various monooxygenase activities investigated were below the detection limits. However, other xenobiotic-metabolizing activities, such as cytochrome P-450 reductase, glutathione transferase and UDP-glucuronosyl-transferase were high, with levels comparable to those observed in freshly isolated rat parenchymal cells. Epoxide hydrolase activity was also detected, but was lower than in the liver. The CHEL cells were able to activate benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and aflatoxin B1 to mutagens, as shown in a co-culture assay with V79 cells, in which acquisition of resistance to 6-thioguanine was studied. At early passages, the CHEL cells had a near diploid set of chromosomes. Then, gradually the frequency of cells with slight changes in the number of chromosomes and the frequency of tetraploids were increased. During the observation period (up to passage 20) the modal number of chromosomes shifted from 22 to 23. No gross morphological changes in the cultures were noticed during the 20 passages.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2901026

  2. Label-Free Proteomic Analysis of Flavohemoglobin Deleted Strain of Saccharomyces cerevisiae

    PubMed Central

    Panja, Chiranjit; Setty, Rakesh K. S.; Vaidyanathan, Gopal; Ghosh, Sanjay

    2016-01-01

    Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in the nitrosative stress responses in Saccharomyces cerevisiae. It is still unclear how S. cerevisiae can withstand this NO level in the absence of flavohemoglobin. To better understand the physiological function of flavohemoglobin in yeast, in the present study a label-free differential proteomics study has been carried out in wild-type and YHB1 deleted strains of S. cerevisiae grown under fermentative conditions. From the analysis, 417 proteins in Y190 and 392 proteins in ΔYHB1 were identified with high confidence. Interestingly, among the differentially expressed identified proteins, 40 proteins were found to be downregulated whereas 41 were found to be upregulated in ΔYHB1 strain of S. cerevisiae (p value < 0.05). The differentially expressed proteins were also classified according to gene ontology (GO) terms. The most enriched and significant GO terms included nitrogen compound biosynthesis, amino acid biosynthesis, translational regulation, and protein folding. Interactions of differentially expressed proteins were generated using Search Tool for the Retrieval of Interacting Genes (STRING) database. This is the first report which offers a more complete view of the proteome changes in S. cerevisiae in the absence of flavohemoglobin. PMID:26881076

  3. The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific.

    PubMed

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

    2016-01-01

    The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae. PMID:27148191

  4. The Interaction between Saccharomyces cerevisiae and Non-Saccharomyces Yeast during Alcoholic Fermentation Is Species and Strain Specific

    PubMed Central

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

    2016-01-01

    The present study analyzes the lack of culturability of different non-Saccharomyces strains due to interaction with Saccharomyces cerevisiae during alcoholic fermentation. Interaction was followed in mixed fermentations with 1:1 inoculation of S. cerevisiae and ten non-Saccharomyces strains. Starmerella bacillaris, and Torulaspora delbrueckii indicated longer coexistence in mixed fermentations compared with Hanseniaspora uvarum and Metschnikowia pulcherrima. Strain differences in culturability and nutrient consumption (glucose, alanine, ammonium, arginine, or glutamine) were found within each species in mixed fermentation with S. cerevisiae. The interaction was further analyzed using cell-free supernatant from S. cerevisiae and synthetic media mimicking both single fermentations with S. cerevisiae and using mixed fermentations with the corresponding non-Saccharomyces species. Cell-free S. cerevisiae supernatants induced faster culturability loss than synthetic media corresponding to the same fermentation stage. This demonstrated that some metabolites produced by S. cerevisiae played the main role in the decreased culturability of the other non-Saccharomyces yeasts. However, changes in the concentrations of main metabolites had also an effect. Culturability differences were observed among species and strains in culture assays and thus showed distinct tolerance to S. cerevisiae metabolites and fermentation environment. Viability kit and recovery analyses on non-culturable cells verified the existence of viable but not-culturable status. These findings are discussed in the context of interaction between non-Saccharomyces and S. cerevisiae. PMID:27148191

  5. Social wasp intestines host the local phenotypic variability of Saccharomyces cerevisiae strains.

    PubMed

    Dapporto, Leonardo; Stefanini, Irene; Rivero, Damariz; Polsinelli, Mario; Capretti, Paolo; De Marchi, Paolo; Viola, Roberto; Turillazzi, Stefano; Cavalieri, Duccio

    2016-07-01

    Nowadays, the presence of Saccharomyces cerevisiae has been assessed in both wild and human-related environments. Social wasps have been shown to maintain and vector S. cerevisiae among different environments. The availability of strains isolated from wasp intestines represents a striking opportunity to assess whether the strains found in wasp intestines are characterized by peculiar traits. We analysed strains isolated from the intestines of social wasps and compared them with strains isolated from other sources, all collected in a restricted geographic area. We evaluated the production of volatile metabolites during grape must fermentation, the resistance to different stresses and the ability to exploit various carbon sources. Wasp strains, in addition to representing a wide range of S. cerevisiae genotypes, also represent large part of the phenotypes characterizing the sympatric set of yeast strains; their higher production of acetic acid and ethyl acetate could reflect improved ability to attract insects. Our findings suggest that the relationship between yeasts and wasps should be preserved, to safeguard not only the natural variance of this microorganism but also the interests of wine-makers, who could take advantage from the exploitation of their phenotypic variability. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27168222

  6. Historical Evolution of Laboratory Strains of Saccharomyces cerevisiae.

    PubMed

    Louis, Edward J

    2016-01-01

    Budding yeast strains used in the laboratory have had a checkered past. Historically, the choice of strain for any particular experiment depended on the suitability of the strain for the topic of study (e.g., cell cycle vs. meiosis). Many laboratory strains had poor fermentation properties and were not representative of the robust strains used for domestic purposes. Most strains were related to each other, but investigators usually had only vague notions about the extent of their relationships. Isogenicity was difficult to confirm before the advent of molecular genetic techniques. However, their ease of growth and manipulation in laboratory conditions made them "the model" model organism, and they still provided a great deal of fundamental knowledge. Indeed, more than one Nobel Prize has been won using them. Most of these strains continue to be powerful tools, and isogenic derivatives of many of them-including entire collections of deletions, overexpression constructs, and tagged gene products-are now available. Furthermore, many of these strains are now sequenced, providing intimate knowledge of their relationships. Recent collections, new isolates, and the creation of genetically tractable derivatives have expanded the available strains for experiments. But even still, these laboratory strains represent a small fraction of the diversity of yeast. The continued development of new laboratory strains will broaden the potential questions that can be posed. We are now poised to take advantage of this diversity, rather than viewing it as a detriment to controlled experiments. PMID:27371602

  7. An economical regimen of human diploid cell strain anti-rabies vaccine for post-exposure prophylaxis.

    PubMed

    Warrell, M J; Warrell, D A; Suntharasamai, P; Viravan, C; Sinhaseni, A; Udomsakdi, D; Phanfung, R; Xueref, C; Vincent-Falquet, J C; Nicholson, K G; Bunnag, D; Harinasuta, T

    1983-08-01

    Vaccine regimens using 0.1 ml human diploid cell strain vaccine (HDCSV) given intradermally (id) in single and multiple sites, or with aluminum hydroxide adjuvant given subcutaneously (sc), were compared with the regimens of HDCSV and Semple vaccine currently suggested by WHO. Some groups were also given human rabies-immune globulin (HRIG). Neutralising antibody titres were monitored for 3 months. Antibody was detected earliest in subjects given 0.1 ml HDCSV id at each of eight sites. The highest antibody titres from day 14 onwards were found after intramuscular (im) administration of HDCSV, but the multiple-site id regimen, which requires only one quarter of the volume of vaccine required for the im regimen, gave similar results, provided that a booster was given on day 91. This finding suggests that a treatment schedule based on this regimen would be suitable for post-exposure prophylaxis. Adjuvanted vaccine gave similar results to the same amount of antigen given id. Semple vaccine produced the lowest titres. HRIG, given at the high dose of 40 IU per kg, suppressed the antibody response to some of the regimens. PMID:6135830

  8. Improving 2-phenylethanol production via Ehrlich pathway using genetic engineered Saccharomyces cerevisiae strains.

    PubMed

    Yin, Sheng; Zhou, Hui; Xiao, Xiao; Lang, Tiandan; Liang, Jingru; Wang, Chengtao

    2015-05-01

    2-phenylethanol (2-PE) is an important aromatic compound with a rose-like fragrance widely used in food industry and cosmetic manufacture. In order to obtain "natural" 2-PE, the genetically modified budding yeasts were developed and applied for the 2-PE production. The gene ARO8 encoding transaminase and the gene ARO10 encoding decarboxylase in the Ehrlich pathway were expressed in Saccharomyces cerevisiae S288c. The activities of transaminase and decarboxylase were both enhanced in the corresponding recombinant strains. Consequently, the 2-PE yield in the recombinant strains with ARO8 and ARO10 were increased by 9.3 and 16.3 %, respectively, than that in the wild strain. A co-expression vector harboring ARO8 and ARO10 was then introduced into S. cerevisiae S288c, generating the recombinant strain SPO810. The fed-batch fermentation results indicated that the 2-PE yield in SPO810 reached 2.61 g L(-1) after 60 h of cultivation, which was 36.8 % higher than that in the wild strain. These results demonstrated that the 2-PE production was significantly improved by enhanced expression of the two key enzymes encoded by ARO8 and ARO10 in the Ehrlich pathway, providing new perspectives for enhancing "natural" 2-PE production in S. cerevisiae. PMID:25681107

  9. Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

    PubMed Central

    Laureau, Raphaëlle; Loeillet, Sophie; Salinas, Francisco; Bergström, Anders; Legoix-Né, Patricia; Liti, Gianni; Nicolas, Alain

    2016-01-01

    In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction. PMID:26828862

  10. Production of Volatile and Sulfur Compounds by 10 Saccharomyces cerevisiae Strains Inoculated in Trebbiano Must.

    PubMed

    Patrignani, Francesca; Chinnici, Fabio; Serrazanetti, Diana I; Vernocchi, Pamela; Ndagijimana, Maurice; Riponi, Claudio; Lanciotti, Rosalba

    2016-01-01

    In wines, the presence of sulfur compounds is the resulting of several contributions among which yeast metabolism. The characterization of the starter Saccharomyces cerevisiae needs to be performed also taking into account this ability even if evaluated together with the overall metabolic profile. In this perspective, principal aim of this experimental research was the evaluation of the volatile profiles, throughout GC/MS technique coupled with solid phase micro extraction, of wines obtained throughout the fermentation of 10 strains of S. cerevisiae. In addition, the production of sulfur compounds was further evaluated by using a gas-chromatograph coupled with a Flame Photometric Detector. Specifically, the 10 strains were inoculated in Trebbiano musts and the fermentations were monitored for 19 days. In the produced wines, volatile and sulfur compounds as well as amino acid concentrations were investigated. Also the physico-chemical characteristics of the wines and their electronic nose profiles were evaluated. PMID:26973621

  11. Production of Volatile and Sulfur Compounds by 10 Saccharomyces cerevisiae Strains Inoculated in Trebbiano Must

    PubMed Central

    Patrignani, Francesca; Chinnici, Fabio; Serrazanetti, Diana I.; Vernocchi, Pamela; Ndagijimana, Maurice; Riponi, Claudio; Lanciotti, Rosalba

    2016-01-01

    In wines, the presence of sulfur compounds is the resulting of several contributions among which yeast metabolism. The characterization of the starter Saccharomyces cerevisiae needs to be performed also taking into account this ability even if evaluated together with the overall metabolic profile. In this perspective, principal aim of this experimental research was the evaluation of the volatile profiles, throughout GC/MS technique coupled with solid phase micro extraction, of wines obtained throughout the fermentation of 10 strains of S. cerevisiae. In addition, the production of sulfur compounds was further evaluated by using a gas-chromatograph coupled with a Flame Photometric Detector. Specifically, the 10 strains were inoculated in Trebbiano musts and the fermentations were monitored for 19 days. In the produced wines, volatile and sulfur compounds as well as amino acid concentrations were investigated. Also the physico-chemical characteristics of the wines and their electronic nose profiles were evaluated. PMID:26973621

  12. [Construction of high sulphite-producing industrial strain of Saccharomyces cerevisiae].

    PubMed

    Qu, Na; He, Xiu-ping; Guo, Xue-na; Liu, Nan; Zhang, Bo-run

    2006-02-01

    In the process of beer storage and transportation, off-flavor can be produced for oxidation of beer. Sulphite is important for stabilizing the beer flavor because of its antioxidant activity. However, the low level of sulphite synthesized by the brewing yeast is not enough to stabilize beer flavor. Three enzymes involve sulphite biosynthesis in yeast. One of them, APS kinase (encoded by MET14) plays important role in the process of sulphite formation. In order to construct high sulphite-producing brewing yeast strain for beer production, MET14 gene was cloned and overexpressed in industrial strain of Saccharomyces cerevisiae. Primer 1 (5'-TGTGAATTCCTGTACACCAATGGCTACT-3', EcoR I) and primer 2 (5'-TATAAGCTTGATGA GGTGGATGAAGACG-3', HindIII) were designed according to the MET14 sequence in GenBank. A 1.1kb DNA fragment containing the open reading frame and terminator of MET14 gene was amplified from Saccharomyces cerevisiae YSF-5 by PCR, and inserted into YEp352 to generate recombinant plasmid pMET14. To express MET14 gene properly in S. cerevisiae, the recombinant expression plasmids pPM with URA3 gene as the selection marker and pCPM with URA3 gene and copper resistance gene as the selection marker for yeast transformation were constructed. In plasmid pPM, the PGK1 promoter from plasmid pVC727 was fused with the MET14 gene from pMET14, and the expression cassette was inserted into the plasmid YEp352. The dominant selection marker, copper-resistance gene expression cassette CUP1-MTI was inserted in plasmid pPM to result in pCPM. Restriction enzyme analysis showed that plasmids pPM and pCPM were constructed correctly. The laboratory strain of S. cerevisiae YS58 with ura3, trp1, leu2, his4 auxotroph was transformed with plasmid pPM. Yeast transformants were screened on synthetic minimal medium (SD) containing leucine, histidine and tryptophan. The sulphite production of the transformants carrying pPM was 2 fold of that in the control strain YS58, which showed that the

  13. Genome Sequencing and Comparative Analysis of Saccharomyces cerevisiae Strains of the Peterhof Genetic Collection

    PubMed Central

    Drozdova, Polina B.; Tarasov, Oleg V.; Matveenko, Andrew G.; Radchenko, Elina A.; Sopova, Julia V.; Polev, Dmitrii E.; Inge-Vechtomov, Sergey G.; Dobrynin, Pavel V.

    2016-01-01

    The Peterhof genetic collection of Saccharomyces cerevisiae strains (PGC) is a large laboratory stock that has accumulated several thousands of strains for over than half a century. It originated independently of other common laboratory stocks from a distillery lineage (race XII). Several PGC strains have been extensively used in certain fields of yeast research but their genomes have not been thoroughly explored yet. Here we employed whole genome sequencing to characterize five selected PGC strains including one of the closest to the progenitor, 15V-P4, and several strains that have been used to study translation termination and prions in yeast (25-25-2V-P3982, 1B-D1606, 74-D694, and 6P-33G-D373). The genetic distance between the PGC progenitor and S288C is comparable to that between two geographically isolated populations. The PGC seems to be closer to two bakery strains than to S288C-related laboratory stocks or European wine strains. In genomes of the PGC strains, we found several loci which are absent from the S288C genome; 15V-P4 harbors a rare combination of the gene cluster characteristic for wine strains and the RTM1 cluster. We closely examined known and previously uncharacterized gene variants of particular strains and were able to establish the molecular basis for known phenotypes including phenylalanine auxotrophy, clumping behavior and galactose utilization. Finally, we made sequencing data and results of the analysis available for the yeast community. Our data widen the knowledge about genetic variation between Saccharomyces cerevisiae strains and can form the basis for planning future work in PGC-related strains and with PGC-derived alleles. PMID:27152522

  14. Comparison of Saccharomyces cerevisiae strains of clinical and nonclinical origin by molecular typing and determination of putative virulence traits

    PubMed Central

    Klingberg, Trine Danø; Lesnik, Urska; Arneborg, Nils; Raspor, Peter; Jespersen, Lene

    2008-01-01

    Saccharomyces cerevisiae strains of clinical and nonclinical origin were compared by pulse field gel electrophoresis. Complete separation between strains of clinical origin and food strains by their chromosome length polymorphism was not obtained even though there was a tendency for the clinical and food strains to cluster separately. All the investigated strains, except for one food strain, were able to grow at temperatures ≥37 °C but not at 42 °C. Great strain variations were observed in pseudohyphal growth and invasiveness, but the characters were not linked to strains of clinical origin. The adhesion capacities of the yeast strains to a human intestinal epithelial cell line (Caco-2) in response to different nutritional availabilities were determined, as were the effects of the strains on the transepithelial electrical resistance (TER) across polarized monolayers of Caco-2 cells. The yeast strains displayed very low adhesion capacities to Caco-2 cells (0.6–6.2%), and no significant difference was observed between the strains of clinical and nonclinical origin. Both S. cerevisiae strains of clinical and non-clinical origin increased the TER of polarized monolayers of Caco-2 cells. Based on the results obtained in this study, no specific virulence factor was found that clearly separated the strains of clinical origin from the strains of nonclinical origin. On the contrary, all investigated strains of S. cerevisiae were found to strengthen the epithelial barrier function. PMID:18355272

  15. [Breeding of robust industrial ethanol-tolerant Saccharomyces cerevisiae strain by artificial zinc finger protein library].

    PubMed

    Ma, Cui; Zhao, Xinqing; Li, Qian; Zhang, Mingming; Kim, Jin Soo; Bai, Fengwu

    2013-05-01

    Breeding of robust industrial Saccharomyces cerevisiae strains with high ethanol tolerance is of great significance for efficient fuel ethanol production. Zinc finger proteins play important roles in gene transcription and translation, and exerting control on the regulation of multiple genes. The sequence and localization of the zinc finger motif can be designed and engineered, and the artificial zinc finger protein can be used to regulate celluar metabolism. Stress tolerance of microbial strains is related to multiple genes. Therefore, it is possible to use artificially-designed zinc finger proteins to breed stress tolerant strains. In this study, a library containing artificial zinc finger protein encoding genes was transformed into the model yeast strain S288c. A recombinant strain named M01 with improved ethanol tolerance was obtained. The plasmid in M01 was isolated, and then transformed into the industrial yeast strain Sc4126. Ethanol tolerance of the recombinant strain of Sc4126 were significantly improved. When high gravity ethanol fermentation using 250 g/L glucose was performed, comparing with the wild-type strain, fermentation time of the recombinant strain was decreased by 24 h and the final ethanol concentration was enhanced by 6.3%. The results of this study demonstrate that artificial zinc finger proteins are able to exert control on stress tolerance of yeast strains, and these results provide basis to construct robust industrial yeast strains for efficient ethanol fermentation. PMID:24010359

  16. Response to different environmental stress conditions of industrial and laboratory Saccharomyces cerevisiae strains.

    PubMed

    Garay-Arroyo, A; Covarrubias, A A; Clark, I; Niño, I; Gosset, G; Martinez, A

    2004-02-01

    Two sets of Saccharomyces cerevisiae strains were compared for their physiological responses to different stress conditions. One group is composed of three strains adapted to controlled laboratory conditions (CEN.PK, LR88 and RS58), whereas the other consisted of five industrial strains (IND1101, SuperStart, LO24, LO41 and Azteca). Most industrial strains showed higher tolerance to heat shock and to an oxidative environment than laboratory strains. Excluding CEN.PK, a similar behavior was observed regarding ethanol production in high sugar concentrations (180 g/l glucose). Addition of acetate (10 g/l) or furfural (2 g/l), in concentrations similar to those found in sugar cane bagasse hydrolysates, decreased cell mass formation and growth rate in almost all strains. CEN.PK and SuperStart showed the highest sensitivity when grown in furfural-containing medium. Acetic acid treatment severely affected cell mass formation and reduced growth rate in all strains; CEN.PK and LO24 were the most resistant. The specific ethanol production rate was not affected by furfural addition. However, specific ethanol production rates decreased in response to acetic acid in four industrial strains, and increased in all laboratory strains and in LO24. No significant correlation was found between the stress tolerance of the strains tested and the transcript accumulation of genes selected by their involvement in the response to each of the stressful environments applied. PMID:12910327

  17. Functional expression of xylose isomerase in flocculating industrial Saccharomyces cerevisiae strain for bioethanol production.

    PubMed

    Li, Yun-Cheng; Li, Guo-Ying; Gou, Min; Xia, Zi-Yuan; Tang, Yue-Qin; Kida, Kenji

    2016-06-01

    Saccharomyces cerevisiae strains with xylose isomerase (XI) pathway were constructed using a flocculating industrial strain (YC-8) as the host. Both strains expressing wild-type xylA (coding XI) from the fungus Orpinomyces sp. and the bacterium Prevotella ruminicola, respectively, showed better growth ability and fermentation capacity when using xylose as the sole sugar than most of the reported strains expressing XI. Codon optimization of both XIs did not improve the xylose fermentation ability of the strains. Adaption significantly increased XI activity resulting in improved growth and fermentation. The strains expressing codon-optimized XI showed a higher increase in xylose consumption and ethanol production compared to strains expressing wild XI. Among all strains, the adapted strain YCPA2E expressing XI from P. ruminicola showed the best performance in the fermentation of xylose to ethanol. After 48 h of fermentation, YCPA2E assimilated 16.95 g/L xylose and produced 6.98 g/L ethanol. These results indicate that YC-8 is a suitable host strain for XI expression, especially for the codon-optimized XI originating from P. ruminicola. PMID:26645659

  18. Discrimination of Saccharomyces cerevisiae wine strains using microsatellite multiplex PCR and band pattern analysis.

    PubMed

    Vaudano, Enrico; Garcia-Moruno, Emilia

    2008-02-01

    We propose a rapid method for Saccharomyces cerevisiae strain identification based on multiplex PCR analysis of polymorphic microsatellite loci. Simple DNA extraction without the use of phenol, followed by a rapid PCR procedure optimised for multiplex amplification of loci SC8132X, YOR267C and SCPTSY7 and band pattern analysis of the fragments generated by agarose and polyacrylamide gel electrophoresis, has allowed us to distinguish among a panel of 30 tested commercial wine strains. This method was successfully performed in an ecological study where dominance between two strains was checked at two fermentation temperatures: 15 and 20 degrees C. The method should be useful for routine and low-budget discrimination of yeast strains, both in the wine and yeast production industries. PMID:17993377

  19. Responses of Saccharomyces cerevisiae Strains from Different Origins to Elevated Iron Concentrations

    PubMed Central

    Martínez-Garay, Carlos Andrés; de Llanos, Rosa; Romero, Antonia María; Martínez-Pastor, María Teresa

    2016-01-01

    Iron is an essential micronutrient for all eukaryotic organisms. However, the low solubility of ferric iron has tremendously increased the prevalence of iron deficiency anemia, especially in women and children, with dramatic consequences. Baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, a fermentative microorganism, and a feed supplement. In this report, we explore the genetic diversity of 123 wild and domestic strains of S. cerevisiae isolated from different geographical origins and sources to characterize how yeast cells respond to elevated iron concentrations in the environment. By using two different forms of iron, we selected and characterized both iron-sensitive and iron-resistant yeast strains. We observed that when the iron concentration in the medium increases, iron-sensitive strains accumulate iron more rapidly than iron-resistant isolates. We observed that, consistent with excess iron leading to oxidative stress, the redox state of iron-sensitive strains was more oxidized than that of iron-resistant strains. Growth assays in the presence of different oxidative reagents ruled out that this phenotype was due to alterations in the general oxidative stress protection machinery. It was noteworthy that iron-resistant strains were more sensitive to iron deficiency conditions than iron-sensitive strains, which suggests that adaptation to either high or low iron is detrimental for the opposite condition. An initial gene expression analysis suggested that alterations in iron homeostasis genes could contribute to the different responses of distant iron-sensitive and iron-resistant yeast strains to elevated environmental iron levels. PMID:26773083

  20. Responses of Saccharomyces cerevisiae Strains from Different Origins to Elevated Iron Concentrations.

    PubMed

    Martínez-Garay, Carlos Andrés; de Llanos, Rosa; Romero, Antonia María; Martínez-Pastor, María Teresa; Puig, Sergi

    2016-03-01

    Iron is an essential micronutrient for all eukaryotic organisms. However, the low solubility of ferric iron has tremendously increased the prevalence of iron deficiency anemia, especially in women and children, with dramatic consequences. Baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, a fermentative microorganism, and a feed supplement. In this report, we explore the genetic diversity of 123 wild and domestic strains of S. cerevisiae isolated from different geographical origins and sources to characterize how yeast cells respond to elevated iron concentrations in the environment. By using two different forms of iron, we selected and characterized both iron-sensitive and iron-resistant yeast strains. We observed that when the iron concentration in the medium increases, iron-sensitive strains accumulate iron more rapidly than iron-resistant isolates. We observed that, consistent with excess iron leading to oxidative stress, the redox state of iron-sensitive strains was more oxidized than that of iron-resistant strains. Growth assays in the presence of different oxidative reagents ruled out that this phenotype was due to alterations in the general oxidative stress protection machinery. It was noteworthy that iron-resistant strains were more sensitive to iron deficiency conditions than iron-sensitive strains, which suggests that adaptation to either high or low iron is detrimental for the opposite condition. An initial gene expression analysis suggested that alterations in iron homeostasis genes could contribute to the different responses of distant iron-sensitive and iron-resistant yeast strains to elevated environmental iron levels. PMID:26773083

  1. Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases.

    PubMed

    Favaro, Lorenzo; Viktor, Marko J; Rose, Shaunita H; Viljoen-Bloom, Marinda; van Zyl, Willem H; Basaglia, Marina; Cagnin, Lorenzo; Casella, Sergio

    2015-09-01

    The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch-based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon-optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α-amylase (SFA1) genes were δ-integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1-SFA1] and MEL2[TLG1-SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch-to-ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre-treatment or commercial enzyme addition) using industrial yeast strains co-secreting both a glucoamylase and α-amylase. PMID:25786804

  2. Engineering and Analysis of a Saccharomyces cerevisiae Strain That Uses Formaldehyde as an Auxiliary Substrate▿

    PubMed Central

    Baerends, Richard J. S.; de Hulster, Erik; Geertman, Jan-Maarten A.; Daran, Jean-Marc; van Maris, Antonius J. A.; Veenhuis, Marten; van der Klei, Ida J.; Pronk, Jack T.

    2008-01-01

    We demonstrated that formaldehyde can be efficiently coutilized by an engineered Saccharomyces cerevisiae strain that expresses Hansenula polymorpha genes encoding formaldehyde dehydrogenase (FLD1) and formate dehydrogenase (FMD), in contrast to wild-type strains. Initial chemostat experiments showed that the engineered strain coutilized formaldehyde with glucose, but these mixed-substrate cultures failed to reach steady-state conditions and did not exhibit an increased biomass yield on glucose. Subsequent transcriptome analyses of chemostat cultures of the engineered strain, grown on glucose-formaldehyde mixtures, indicated that the presence of formaldehyde in the feed caused biotin limitations. Further transcriptome analysis demonstrated that this biotin inactivation was prevented by using separate formaldehyde and vitamin feeds. Using this approach, steady-state glucose-limited chemostat cultures were obtained that coutilized glucose and formaldehyde. Coutilization of formaldehyde under these conditions resulted in an enhanced biomass yield of the glucose-limited cultures. The biomass yield was quantitatively consistent with the use of formaldehyde as an auxiliary substrate that generates NADH and subsequently, via oxidative phosphorylation, ATP. On an electron pair basis, the biomass yield increase observed with formaldehyde was larger than that observed previously for formate, which is tentatively explained by different modes of formate and formaldehyde transport in S. cerevisiae. PMID:18378663

  3. Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing.

    PubMed

    Hu, Nan; Yuan, Bo; Sun, Juan; Wang, Shi-An; Li, Fu-Li

    2012-09-01

    Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40 °C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200 g L(-1)) at 40 °C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2 g L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7 %, respectively. In the range of 30 to 40 °C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP. PMID:22760784

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

    PubMed Central

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

    2015-01-01

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

  5. GMAX-L Saccharomyces Cerevisiae Strains for Profitable Sustainable Cellulosic Ethanol and Biodiesel Production Concurrently using Engineered Workcell

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A stable GMAX-L strain of Saccharomyces cerevisiae is being constructed using pSUMO expression cassettes that are extremely high expression level plasmids designed for use on automated workcell. This strain expresses xylose isomerase, xylulokinase, XIB1, and XIG1 for anaerobic cellulosic ethanol pr...

  6. Saccharomyces cerevisiae mass transformed with FLEXGenes results in strain capable of anaerobic fermentation of pentose and hexose sugars

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Advanced automated high-throughput mass transformation of yeast full-genome libraries into Saccharomyces cerevisiae and screening for growth on xylose produced a yeast strain that is capable of fully utilizing pentose as well as hexose sugars anaerobically. This is the first yeast strain capable of...

  7. Impact of Commercial Strain Use on Saccharomyces cerevisiae Population Structure and Dynamics in Pinot Noir Vineyards and Spontaneous Fermentations of a Canadian Winery.

    PubMed

    Martiniuk, Jonathan T; Pacheco, Braydon; Russell, Gordon; Tong, Stephanie; Backstrom, Ian; Measday, Vivien

    2016-01-01

    Wine is produced by one of two methods: inoculated fermentation, where a commercially-produced, single Saccharomyces cerevisiae (S. cerevisiae) yeast strain is used; or the traditional spontaneous fermentation, where yeast present on grape and winery surfaces carry out the fermentative process. Spontaneous fermentations are characterized by a diverse succession of yeast, ending with one or multiple strains of S. cerevisiae dominating the fermentation. In wineries using both fermentation methods, commercial strains may dominate spontaneous fermentations. We elucidate the impact of the winery environment and commercial strain use on S. cerevisiae population structure in spontaneous fermentations over two vintages by comparing S. cerevisiae populations in aseptically fermented grapes from a Canadian Pinot Noir vineyard to S. cerevisiae populations in winery-conducted fermentations of grapes from the same vineyard. We also characterize the vineyard-associated S. cerevisiae populations in two other geographically separate Pinot Noir vineyards farmed by the same winery. Winery fermentations were not dominated by commercial strains, but by a diverse number of strains with genotypes similar to commercial strains, suggesting that a population of S. cerevisiae derived from commercial strains is resident in the winery. Commercial and commercial-related yeast were also identified in the three vineyards examined, although at a lower frequency. There is low genetic differentiation and S. cerevisiae population structure between vineyards and between the vineyard and winery that persisted over both vintages, indicating commercial yeast are a driver of S. cerevisiae population structure. We also have evidence of distinct and persistent populations of winery and vineyard-associated S. cerevisiae populations unrelated to commercial strains. This study is the first to characterize S. cerevisiae populations in Canadian vineyards. PMID:27551920

  8. Impact of Commercial Strain Use on Saccharomyces cerevisiae Population Structure and Dynamics in Pinot Noir Vineyards and Spontaneous Fermentations of a Canadian Winery

    PubMed Central

    Martiniuk, Jonathan T.; Pacheco, Braydon; Russell, Gordon; Tong, Stephanie; Backstrom, Ian; Measday, Vivien

    2016-01-01

    Wine is produced by one of two methods: inoculated fermentation, where a commercially-produced, single Saccharomyces cerevisiae (S. cerevisiae) yeast strain is used; or the traditional spontaneous fermentation, where yeast present on grape and winery surfaces carry out the fermentative process. Spontaneous fermentations are characterized by a diverse succession of yeast, ending with one or multiple strains of S. cerevisiae dominating the fermentation. In wineries using both fermentation methods, commercial strains may dominate spontaneous fermentations. We elucidate the impact of the winery environment and commercial strain use on S. cerevisiae population structure in spontaneous fermentations over two vintages by comparing S. cerevisiae populations in aseptically fermented grapes from a Canadian Pinot Noir vineyard to S. cerevisiae populations in winery-conducted fermentations of grapes from the same vineyard. We also characterize the vineyard-associated S. cerevisiae populations in two other geographically separate Pinot Noir vineyards farmed by the same winery. Winery fermentations were not dominated by commercial strains, but by a diverse number of strains with genotypes similar to commercial strains, suggesting that a population of S. cerevisiae derived from commercial strains is resident in the winery. Commercial and commercial-related yeast were also identified in the three vineyards examined, although at a lower frequency. There is low genetic differentiation and S. cerevisiae population structure between vineyards and between the vineyard and winery that persisted over both vintages, indicating commercial yeast are a driver of S. cerevisiae population structure. We also have evidence of distinct and persistent populations of winery and vineyard-associated S. cerevisiae populations unrelated to commercial strains. This study is the first to characterize S. cerevisiae populations in Canadian vineyards. PMID:27551920

  9. New integrative computational approaches unveil the Saccharomyces cerevisiae pheno-metabolomic fermentative profile and allow strain selection for winemaking.

    PubMed

    Franco-Duarte, Ricardo; Umek, Lan; Mendes, Inês; Castro, Cristiana C; Fonseca, Nuno; Martins, Rosa; Silva-Ferreira, António C; Sampaio, Paula; Pais, Célia; Schuller, Dorit

    2016-11-15

    During must fermentation by Saccharomyces cerevisiae strains thousands of volatile aroma compounds are formed. The objective of the present work was to adapt computational approaches to analyze pheno-metabolomic diversity of a S. cerevisiae strain collection with different origins. Phenotypic and genetic characterization together with individual must fermentations were performed, and metabolites relevant to aromatic profiles were determined. Experimental results were projected onto a common coordinates system, revealing 17 statistical-relevant multi-dimensional modules, combining sets of most-correlated features of noteworthy biological importance. The present method allowed, as a breakthrough, to combine genetic, phenotypic and metabolomic data, which has not been possible so far due to difficulties in comparing different types of data. Therefore, the proposed computational approach revealed as successful to shed light into the holistic characterization of S. cerevisiae pheno-metabolome in must fermentative conditions. This will allow the identification of combined relevant features with application in selection of good winemaking strains. PMID:27283661

  10. [Saccharomyces cerevisiae: porphobilinogenase activity in a wild-type strain and its heme-deficient mutant].

    PubMed

    Araujo, L S; Lombardo, M E; Rossetti, M V; Batlle, A M

    1987-01-01

    Properties of Porphobilinogenase (PBGase), the enzyme complex converting porphobilinogen (PBG) into uroporphyrinogens, were comparatively studied in a wild strain D273-10B and its mutant B231 of Saccharomyces cerevisiae, Figure 1 shows the growth curves for both strains. The basic pattern of growth was observed but, although S. cerevisiae is a facultative aerobe and was grown on dextrose, a diauxic growth curve was not observed. The beginning of the exponential phase was slightly delayed for the mutant, so, its generation time (G = 3.20 h) was greater than that for the wild strain (G = 1.26 h). Optimum conditions for extracting the enzyme from both strains were found to be sonication at 10 mu for 3 min (Table 1). Table 2 shows the effect of centrifugation at 24,000 xg for 30 min on activity. For both strains the amount of porphyrins formed was the same either in the absence or presence of air. It was found (Figure 2) that urogen formation was linear with protein over a wide range of concentrations and with incubation time up to 2h in agreement with previous results for the enzyme of different sources. Figure 3 shows the effect of pH on PBGase activity. An optimum pH of 7.4 was found for both strains employing sodium phosphate buffer pH 8.0. The shape of the pH curve as well as optimum pH were the same in both Tris-HCl and phosphate buffer, however PBGase was 15% less active in the former. When plots of velocity against PBG concentration were analyzed for PBGase, it was found that measuring the rate of the reaction on the basis of total urogen formation, saturation curves for wild and mutant strains harvested at the exponential phase, followed classical Michaelis-Menten kinetics. Saturation was reached at PBG concentration of about 70-90 microM. Therefore, double reciprocal plots (Figure 4) were linear and from these plots apparent Km's values of 20 and 14 microM were obtained for the wild and mutant strain respectively. It is known that in some organisms, the

  11. Impact of different spray-drying conditions on the viability of wine Saccharomyces cerevisiae strains.

    PubMed

    Aponte, Maria; Troianiello, Gabriele Danilo; Di Capua, Marika; Romano, Raffaele; Blaiotta, Giuseppe

    2016-01-01

    Spray-drying (SD) is widely considered a suitable method to preserve microorganisms, but data regarding yeasts are still scanty. In this study, the effect of growing media, process variables and carriers over viability of a wild wine Saccharomyces (S.) cerevisiae LM52 was evaluated. For biomass production, the strain was grown (batch and fed-batch fermentation) in a synthetic, as well as in a beet sugar molasses based-medium. Drying of cells resuspended in several combinations of soluble starch and maltose was performed at different inlet and outlet temperatures. Under the best conditions-suspension in soluble starch plus maltose couplet to inlet and outlet temperatures of 110 and 55 °C, respectively-the loss of viability of S. cerevisiae LM52 was 0.8 ± 0.1 and 0.5 ± 0.2 Log c.f.u. g(-1) for synthetic and molasses-based medium, respectively. Similar results were obtained when S. cerevisiae strains Zymoflore F15 and EC1118, isolated from commercial active dry yeast (ADY), were tested. Moreover, powders retained a high vitality and showed good fermentation performances up to 6 month of storage, at both 4 and -20 °C. Finally, fermentation performances of different kinds of dried formulates (SD and ADY) compared with fresh cultures did not show significant differences. The procedure proposed allowed a small-scale production of yeast in continuous operation with relatively simple equipment, and may thus represent a rapid response-on-demand for the production of autochthonous yeasts for local wine-making. PMID:26712628

  12. EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains.

    PubMed

    Stovicek, Vratislav; Borja, Gheorghe M; Forster, Jochen; Borodina, Irina

    2015-11-01

    Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway. PMID:26376869

  13. Enhanced xylose fermentation and ethanol production by engineered Saccharomyces cerevisiae strain.

    PubMed

    Vilela, Leonardo de Figueiredo; de Araujo, Verônica Parente Gomes; Paredes, Raquel de Sousa; Bon, Elba Pinto da Silva; Torres, Fernando Araripe Gonçalves; Neves, Bianca Cruz; Eleutherio, Elis Cristina Araújo

    2015-01-01

    We have recently demonstrated that heterologous expression of a bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia enabled a laboratorial Saccharomyces cerevisiae strain to ferment xylose anaerobically, without xylitol accumulation. However, the recombinant yeast fermented xylose slowly. In this study, an evolutionary engineering strategy was applied to improve xylose fermentation by the xylA-expressing yeast strain, which involved sequential batch cultivation on xylose. The resulting yeast strain co-fermented glucose and xylose rapidly and almost simultaneously, exhibiting improved ethanol production and productivity. It was also observed that when cells were grown in a medium containing higher glucose concentrations before being transferred to fermentation medium, higher rates of xylose consumption and ethanol production were obtained, demonstrating that xylose utilization was not regulated by catabolic repression. Results obtained by qPCR demonstrate that the efficiency in xylose fermentation showed by the evolved strain is associated, to the increase in the expression of genes HXT2 and TAL1, which code for a low-affinity hexose transporter and transaldolase, respectively. The ethanol productivity obtained after the introduction of only one genetic modification and the submission to a one-stage process of evolutionary engineering was equivalent to those of strains submitted to extensive metabolic and evolutionary engineering, providing solid basis for future applications of this strategy in industrial strains. PMID:25852993

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

    PubMed Central

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

    2012-01-01

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

  15. Near-freezing effects on the proteome of industrial yeast strains of Saccharomyces cerevisiae.

    PubMed

    Ballester-Tomás, Lidia; Pérez-Torrado, Roberto; Rodríguez-Vargas, Sonia; Prieto, Jose A; Randez-Gil, Francisca

    2016-03-10

    At near-freezing temperatures (0-4°C), the growth of the yeast Saccharomyces cerevisiae stops or is severely limited, and viability decreases. Under these conditions, yeast cells trigger a biochemical response, in which trehalose and glycerol accumulate and protect them against severe cold and freeze injury. However, the mechanisms that allow yeast cells to sustain this response have been not clarified. The effects of severe cold on the proteome of S. cerevisiae have been not investigated and its importance in providing cell survival at near-freezing temperatures and upon freezing remains unknown. Here, we have compared the protein profile of two industrial baker's yeast strains at 30°C and 4°C. Overall, a total of 16 proteins involved in energy-metabolism, translation and redox homeostasis were identified as showing increased abundance at 4°C. The predominant presence of glycolytic proteins among those upregulated at 4°C, likely represents a mechanism to maintain a constant supply of ATP for the synthesis of glycerol and other protective molecules. Accumulation of these molecules is by far the most important component in enhancing viability of baker's yeast strains upon freezing. Overexpression of genes encoding certain proteins associated with translation or redox homeostasis provided specifically protection against extreme cold damage, underlying the importance of these functions in the near-freezing response. PMID:26812658

  16. Construction of Novel Saccharomyces cerevisiae Strains for Bioethanol Active Dry Yeast (ADY) Production

    PubMed Central

    Gao, Kehui; Liu, Zewei; Zhang, Xing; Li, Ou; Sun, Jianguo; Zhang, Xiaoyang; Du, Fengguang; Sun, Peiyong; Qu, Aimin; Wu, Xuechang

    2013-01-01

    The application of active dry yeast (ADY) in bioethanol production simplifies operation processes and reduces the risk of bacterial contamination. In the present study, we constructed a novel ADY strain with improved stress tolerance and ethanol fermentation performances under stressful conditions. The industrial Saccharomyces cerevisiae strain ZTW1 showed excellent properties and thus subjected to a modified whole-genome shuffling (WGS) process to improve its ethanol titer, proliferation capability, and multiple stress tolerance for ADY production. The best-performing mutant, Z3-86, was obtained after three rounds of WGS, producing 4.4% more ethanol and retaining 2.15-fold higher viability than ZTW1 after drying. Proteomics and physiological analyses indicated that the altered expression patterns of genes involved in protein metabolism, plasma membrane composition, trehalose metabolism, and oxidative responses contribute to the trait improvement of Z3-86. This work not only successfully developed a novel S. cerevisiae mutant for application in commercial bioethanol production, but also enriched the current understanding of how WGS improves the complex traits of microbes. PMID:24376860

  17. Genetic diversity and geographical distribution of wild Saccharomyces cerevisiae strains from the wine-producing area of Charentes, France.

    PubMed Central

    Versavaud, A; Courcoux, P; Roulland, C; Dulau, L; Hallet, J N

    1995-01-01

    Electrophoretic karyotyping, mitochondrial DNA restriction fragment length polymorphism analysis, and PCR amplification of interspersed repeats were used to study the variability, phylogenetic affinities, and biogeographic distribution of wild Saccharomyces cerevisiae enological yeasts. The survey concentrated on 42 individual wine cellars in the Charentes area (Cognac region, France). A limited number (35) of predominant S. cerevisiae strains responsible for the fermentation process have been identified by the above molecular methods of differentiation. One strain (ACI) was found to be distributed over the entire area surveyed. There seemed to be little correlation between geographic location and genetic affinity. PMID:7486988

  18. Endomitotic effect of a cell cycle mutation of Saccharomyces cerevisiae

    SciTech Connect

    Schild, D.; Ananthaswamy, H.N.; Mortimer, R.K.

    1981-03-01

    A recessive temperature-sensitive mutation of Saccharomyces cerevisiae has been isolated and shown to cause an increase in ploidy in both haploids and diploids. Genetic analysis revealed that the strain carrying the mutation was an aa diploid, although MNNG mutagenesis had been done on an a haploid strain. When the mutant strain was crossed with an ..cap alpha cap alpha.. diploid and the resultant tetraploid sporulated, some of the meiotic progeny of this tetraploid were themselves tetraploid, as shown by both genetic analysis and DNA measurements, instead of diploid as expected of tetraploid meiosis. The ability of these tetraploids to continue to produce tetraploid meiotic progeny was followed for four generations. It was found that tetraploidization was independent of sporulation temperature, but was dependent on the temperature of germination and the growth of the spores. Increase in ploidy occurred when the spores were germinated and grown at 30/sup 0/, but did not occur at 23/sup 0/. Two cycles of sporulation and growth at 23/sup 0/ resulted in haploids, which were shown to diploidize within 24 hr when grown at 30/sup 0/.

  19. Genetic Diversity and Population Structure of Saccharomyces cerevisiae Strains Isolated from Different Grape Varieties and Winemaking Regions

    PubMed Central

    Schuller, Dorit; Cardoso, Filipa; Sousa, Susana; Gomes, Paula; Gomes, Ana C.; Santos, Manuel A. S.; Casal, Margarida

    2012-01-01

    We herein evaluate intraspecific genetic diversity of fermentative vineyard-associated S. cerevisiae strains and evaluate relationships between grape varieties and geographical location on populational structures. From the musts obtained from 288 grape samples, collected from two wine regions (16 vineyards, nine grape varieties), 94 spontaneous fermentations were concluded and 2820 yeast isolates were obtained that belonged mainly (92%) to the species S. cerevisiae. Isolates were classified in 321 strains by the use of ten microsatellite markers. A high strain diversity (8–43 strains per fermentation) was associated with high percentage (60–100%) of fermenting samples per vineyard, whereas a lower percentage of spontaneous fermentations (0–40%) corresponded to a rather low strain diversity (1–10 strains per fermentation). For the majority of the populations, observed heterozygosity (Ho) was about two to five times lower than the expected heterozygosity (He). The inferred ancestry showed a very high degree of admixture and divergence was observed between both grape variety and geographical region. Analysis of molecular variance showed that 81–93% of the total genetic variation existed within populations, while significant differentiation within the groups could be detected. Results from AMOVA analysis and clustering of allelic frequencies agree in the distinction of genetically more dispersed populations from the larger wine region compared to the less extended region. Our data show that grape variety is a driver of populational structures, because vineyards with distinct varieties harbor genetically more differentiated S. cerevisiae populations. Conversely, S. cerevisiae strains from vineyards in close proximity (5–10 km) that contain the same grape variety tend to be less divergent. Populational similarities did not correlate with the distance between vineyards of the two wine regions. Globally, our results show that populations of S. cerevisiae in

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

    PubMed

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

    2015-01-01

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

  1. Different strains of Saccharomyces cerevisiae differ in their effects on ruminal bacterial numbers in vitro and in sheep.

    PubMed

    Newbold, C J; Wallace, R J; Chen, X B; McIntosh, F M

    1995-06-01

    A ruminal simulation device (Rusitec) was used to compare the effects of Saccharomyces cerevisiae strains NCYC 240, NCYC 694, NCYC 1026, NCYC 1088, and Yea-Sacc (a commercial product containing S. cerevisiae) on ruminal fermentation. S. cerevisiae NCYC 240, NCYC 1088, NCYC 1026, and NCYC 694 were grown on malt extract at 30 degrees C in aerated fed-batch culture and harvested along with spent growth medium by freeze-drying. Each vessel received daily 20 g of a basal diet consisting of hay, barley, molasses, fishmeal, and a minerals/vitamins mixture at 500, 299.5, 100, 91, and 9.5 g/kg of DM, respectively. Yeast preparations (500 mg/d) were added along with the feed. S. cerevisiae NCYC 240, NCYC 1026, and Yea-Sacc stimulated total and cellulolytic bacterial numbers, whereas S. cerevisiae NCYC 694 and NCYC 1088 had no effect on the numbers of bacteria. The effects of S. cerevisiae NCYC 240, NCYC 1026, and Yea-Sacc on ruminal fermentation were further investigated in vivo using ruminally cannulated sheep fed 1.5 kg/d of the diet used in Rusitec, supplemented with 2 g/d of yeast culture. All treatments tended to stimulate total and cellulolytic bacterial numbers. However, the stimulation was only statistically significant for S. cerevisiae NCYC 1026 with total bacterial numbers and S. cerevisiae NCYC 240 with cellulolytic bacteria (P < .05). Increased bacterial numbers were associated with an increase in the rate of straw degradation in the rumen and a nonsignificant (P > .05) increase in the excretion of purine derivatives in the urine, measured as an index of microbial nitrogen leaving the rumen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7673076

  2. High-throughput profiling of amino acids in strains of the Saccharomyces cerevisiae deletion collection.

    PubMed

    Cooper, Sara J; Finney, Gregory L; Brown, Shauna L; Nelson, Sven K; Hesselberth, Jay; MacCoss, Michael J; Fields, Stanley

    2010-09-01

    The measurement of small molecule metabolites on a large scale offers the opportunity for a more complete understanding of cellular metabolism. We developed a high-throughput method to quantify primary amine-containing metabolites in the yeast Saccharomyces cerevisiae by the use of capillary electrophoresis in combination with fluorescent derivatization of cell extracts. We measured amino acid levels in the yeast deletion collection, a set of approximately 5000 strains each lacking a single gene, and developed a computational pipeline for data analysis. Amino acid peak assignments were validated by mass spectrometry, and the overall approach was validated by the result that expected pathway intermediates accumulate in mutants of the arginine biosynthetic pathway. Global analysis of the deletion collection was carried out using clustering methods. We grouped strains based on their metabolite profiles, revealing clusters of mutants enriched for genes encoding mitochondrial proteins, urea cycle enzymes, and vacuolar ATPase functions. One of the most striking profiles, common among several strains lacking ribosomal protein genes, accumulated lysine and a lysine-related metabolite. Mutations in the homologous ribosomal protein genes in the human result in Diamond-Blackfan anemia, demonstrating that metabolite data may have potential value in understanding disease pathology. This approach establishes metabolite profiling as capable of characterizing genes in a large collection of genetic variants. PMID:20610602

  3. Biodiversity of autolytic ability in flocculent Saccharomyces cerevisiae strains suitable for traditional sparkling wine fermentation.

    PubMed

    Perpetuini, Giorgia; Di Gianvito, Paola; Arfelli, Giuseppe; Schirone, Maria; Corsetti, Aldo; Tofalo, Rosanna; Suzzi, Giovanna

    2016-07-01

    Yeasts involved in secondary fermentation of traditional sparkling wines should show specific characteristics, such as flocculation capacity and autolysis. Recently it has been postulated that autophagy may contribute to the outcome of autolysis. In this study, 28 flocculent wine Saccahromyces cerevisiae strains characterized by different flocculation degrees were studied for their autolytic and autophagic activities. Autolysis was monitored in synthetic medium through the determination of amino acid nitrogen and total proteins released. At the same time, novel primer sets were developed to determine the expression of the genes ATG1, ATG17 and ATG29. Twelve strains were selected on the basis of their autolytic rate and ATG gene expressions in synthetic medium and were inoculated in a base wine. After 30, 60 and 180 days the autolytic process and ATG gene expressions were evaluated. The obtained data showed that autolysis and ATG gene expressions differed among strains and were independent of the degree of flocculation. This biodiversity could be exploited to select new starter stains to improve sparkling wine production. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26804203

  4. Genetic architecture of ethanol-responsive transcriptome variation in Saccharomyces cerevisiae strains.

    PubMed

    Lewis, Jeffrey A; Broman, Aimee T; Will, Jessica; Gasch, Audrey P

    2014-09-01

    Natural variation in gene expression is pervasive within and between species, and it likely explains a significant fraction of phenotypic variation between individuals. Phenotypic variation in acute systemic responses can also be leveraged to reveal physiological differences in how individuals perceive and respond to environmental perturbations. We previously found extensive variation in the transcriptomic response to acute ethanol exposure in two wild isolates and a common laboratory strain of Saccharomyces cerevisiae. Many expression differences persisted across several modules of coregulated genes, implicating trans-acting systemic differences in ethanol sensing and/or response. Here, we conducted expression QTL mapping of the ethanol response in two strain crosses to identify the genetic basis for these differences. To understand systemic differences, we focused on "hotspot" loci that affect many transcripts in trans. Candidate causal regulators contained within hotspots implicate upstream regulators as well as downstream effectors of the ethanol response. Overlap in hotspot targets revealed additive genetic effects of trans-acting loci as well as "epi-hotspots," in which epistatic interactions between two loci affected the same suites of downstream targets. One epi-hotspot implicated interactions between Mkt1p and proteins linked to translational regulation, prompting us to show that Mkt1p localizes to P bodies upon ethanol stress in a strain-specific manner. Our results provide a glimpse into the genetic architecture underlying natural variation in a stress response and present new details on how yeast respond to ethanol stress. PMID:24970865

  5. Physiological Effects of GLT1 Modulation in Saccharomyces cerevisiae Strains Growing on Different Nitrogen Sources.

    PubMed

    Brambilla, Marco; Adamo, Giusy Manuela; Frascotti, Gianni; Porro, Danilo; Branduardi, Paola

    2016-02-28

    Saccharomyces cerevisiae is one of the most employed cell factories for the production of bioproducts. Although monomeric hexose sugars constitute the preferential carbon source, this yeast can grow on a wide variety of nitrogen sources that are catabolized through central nitrogen metabolism (CNM). To evaluate the effects of internal perturbations on nitrogen utilization, we characterized strains deleted or overexpressed in GLT1, encoding for one of the key enzymes of the CNM node, the glutamate synthase. These strains, together with the parental strain as control, have been cultivated in minimal medium formulated with ammonium sulfate, glutamate, or glutamine as nitrogen source. Growth kinetics, together with the determination of protein content, viability, and reactive oxygen species (ROS) accumulation at the single cell level, revealed that GLT1 modulations do not significantly influence the cellular physiology, whereas the nitrogen source does. As important exceptions, GLT1 deletion negatively affected the scavenging activity of glutamate against ROS accumulation, when cells were treated with H2O2, whereas Glt1p overproduction led to lower viability in glutamine medium. Overall, this confirms the robustness of the CNM node against internal perturbations, but, at the same time, highlights its plasticity in respect to the environment. Considering that side-stream protein-rich waste materials are emerging as substrates to be used in an integrated biorefinery, these results underline the importance of preliminarily evaluating the best nitrogen source not only for media formulation, but also for the overall economics of the process. PMID:26528537

  6. Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae

    SciTech Connect

    Yang, Shihui; Land, Miriam L; Klingeman, Dawn Marie; Pelletier, Dale A; Lu, Tse-Yuan; Martin, S L.; Guo, Hao-Bo; Smith, Jeremy C; Brown, Steven D

    2010-01-01

    The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequencing limitations. Transcriptomics, genetic data and growth studies indicate that over-expression of the sodium-proton antiporter gene nhaA confers the elevated AcR sodium acetate tolerance phenotype. nhaA over-expression mostly confers enhanced sodium (Na{sup +}) tolerance and not acetate (Ac{sup -}) tolerance, unless both ions are present in sufficient quantities. NaAc is more inhibitory than potassium and ammonium acetate for Z. mobilis and the combination of elevated Na{sup +} and Ac{sup -} ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodium-proton antiporter is constructed to provide mechanistic insights. We demonstrate that Saccharomyces cerevisiae sodium-proton antiporter genes also contribute to sodium acetate, potassium acetate, and ammonium acetate tolerances. The present combination of classical and systems biology tools is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies.

  7. Characterization of a novel tyrosine permease of lager brewing yeast shared by Saccharomyces cerevisiae strain RM11-1a.

    PubMed

    Omura, Fumihiko; Hatanaka, Haruyo; Nakao, Yoshihiro

    2007-12-01

    In Saccharomyces cerevisiae yeast, the uptake of aromatic amino acids is mediated by the relatively specific permeases Tat1p, Tat2p, Bap2p, and Bap3p, as well as by two other permeases with broader specificities: Gap1p and Agp1p. Here, a novel permease gene TAT3 (Tyrosine Amino acid Transporter) identified in the S. cerevisiae-type subset genome of the lager brewing yeast strain Weihenstephan Nr.34 (34/70) is reported. The TAT3 sequence was also found in the genome of S. cerevisiae strain RM11-1a, but not in S. cerevisiae strain S288C. Tat3p showed a significant similarity to Penicillium chrysogenum ArlP permease, which has transport activity for aromatic amino acids and leucine. When overexpressed in ssy1Delta gap1Delta mutant cells, Tat3p exhibited a tyrosine transport activity with an apparent K(m) of 160 microM. TAT3 transcription in lager brewing yeast was subjected to nitrogen catabolite repression in a manner similar to that of GAP1. Furthermore, the subcellular localization of Tat3p-green fluorescent protein (GFP) fusion protein was dependent on the quality of the nitrogen source, indicating a post-translational control of Tat3p function. PMID:17825063

  8. Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37.

    PubMed

    Li, Yun-Cheng; Mitsumasu, Kanako; Gou, Zi-Xi; Gou, Min; Tang, Yue-Qin; Li, Guo-Ying; Wu, Xiao-Lei; Akamatsu, Takashi; Taguchi, Hisataka; Kida, Kenji

    2016-02-01

    Industrial yeast strains with good xylose fermentation ability and inhibitor tolerance are important for economical lignocellulosic bioethanol production. The flocculating industrial Saccharomyces cerevisiae strain NAPX37, harboring the xylose reductase-xylitol dehydrogenase (XR-XDH)-based xylose metabolic pathway, displayed efficient xylose fermentation during batch and continuous fermentation. During batch fermentation, the xylose consumption rates at the first 36 h were similar (1.37 g/L/h) when the initial xylose concentrations were 50 and 75 g/L, indicating that xylose fermentation was not inhibited even when the xylose concentration was as high as 75 g/L. The presence of glucose, at concentrations of up to 25 g/L, did not affect xylose consumption rate at the first 36 h. Strain NAPX37 showed stable xylose fermentation capacity during continuous ethanol fermentation using xylose as the sole sugar, for almost 1 year. Fermentation remained stable at a dilution rate of 0.05/h, even though the xylose concentration in the feed was as high as 100 g/L. Aeration rate, xylose concentration, and MgSO4 concentration were found to affect xylose consumption and ethanol yield. When the xylose concentration in the feed was 75 g/L, a high xylose consumption rate of 6.62 g/L/h and an ethanol yield of 0.394 were achieved under an aeration rate of 0.1 vvm, dilution rate of 0.1/h, and 5 mM MgSO4. In addition, strain NAPX37 exhibited good tolerance to inhibitors such as weak acids, furans, and phenolics during xylose fermentation. These findings indicate that strain NAPX37 is a promising candidate for application in the industrial production of lignocellulosic bioethanol. PMID:26603762

  9. Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

    PubMed Central

    Rossouw, D.; Heyns, E. H.; Setati, M. E.; Bosch, S.

    2013-01-01

    The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ and tdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines. PMID:23793638

  10. High ethanol fermentation performance of the dry dilute acid pretreated corn stover by an evolutionarily adapted Saccharomyces cerevisiae strain.

    PubMed

    Qureshi, Abdul Sattar; Zhang, Jian; Bao, Jie

    2015-01-01

    Ethanol fermentation was investigated at the high solids content of the dry dilute sulfuric acid pretreated corn stover feedstock using an evolutionary adapted Saccharomyces cerevisiae DQ1 strain. The evolutionary adaptation was conducted by successively transferring the S. cerevisiae DQ1 cells into the inhibitors containing corn stover hydrolysate every 12h and finally a stable yeast strain was obtained after 65 days' continuous adaptation. The ethanol fermentation performance using the adapted strain was significantly improved with the high ethanol titer of 71.40 g/L and the high yield of 80.34% in the simultaneous saccharification and fermentation (SSF) at 30% solids content. No wastewater was generated from pretreatment to fermentation steps. The results were compared with the published cellulosic ethanol fermentation cases, and the obvious advantages of the present work were demonstrated not only at the high ethanol titer and yield, but also the significant reduction of wastewater generation and potential cost reduction. PMID:25930238

  11. Ethanol production using a newly isolated Saccharomyces cerevisiae strain directly assimilating intact inulin with a high degree of polymerization.

    PubMed

    Yang, Fan; Liu, Zhicheng; Dong, Weifeng; Zhu, Linghuan; Chen, Xiaoyi; Li, Xianzhen

    2014-01-01

    An inulin-degrading strain L610, which was competent to directly convert inulin into ethanol, was isolated and identified as a strain of Saccharomyces cerevisiae according to physiological and phylogenetic analysis. Ion chromatography results showed that isolate L610 could assimilate the intact inulin completely without acidic or enzymatic pretreatment in contrast to the previously reported strains of S. cerevisiae, which could only ferment the fructo-oligosaccharides with a degree of polymerization less than 15. Strain L610 yielded 37.2 g/L ethanol within 48 H at a shake flask level under the evaluated culture conditions (11% inulin, 0.4% yeast extract, and 0.05% MgSO4 at 30 °C and pH 6.0). The conversion efficiency of inulin-type sugar to ethanol was 60% of the theoretical ethanol yield. Strain L610 produced 40.0 g/L of ethanol when directly fermented in Jerusalem artichoke (Helianthus tuberosus L.) powder suspension within 24 H, which was higher than the reported data, 28.9 g/L, produced by S. cerevisiae KCCM 50549. PMID:24237352

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

    PubMed Central

    2012-01-01

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

  13. A repressor activator protein1 homologue from an oleaginous strain of Candida tropicalis increases storage lipid production in Saccharomyces cerevisiae.

    PubMed

    Chattopadhyay, Atrayee; Dey, Prabuddha; Barik, Amita; Bahadur, Ranjit P; Maiti, Mrinal K

    2015-06-01

    The repressor activator protein1 (Rap1) has been studied over the years as a multifunctional regulator in Saccharomyces cerevisiae. However, its role in storage lipid accumulation has not been investigated. This report documents the identification and isolation of a putative transcription factor CtRap1 gene from an oleaginous strain of Candida tropicalis, and establishes the direct effect of its expression on the storage lipid accumulation in S. cerevisiae, usually a non-oleaginous yeast. In silico analysis revealed that the CtRap1 polypeptide binds relatively more strongly to the promoter of fatty acid synthase1 (FAS1) gene of S. cerevisiae than ScRap1. The expression level of CtRap1 transcript in vivo was found to correlate directly with the amount of lipid produced in oleaginous native host C. tropicalis. Heterologous expression of the CtRap1 gene resulted in ∼ 4-fold enhancement of storage lipid content (57.3%) in S. cerevisiae. We also showed that the functionally active CtRap1 upregulates the endogenous ScFAS1 and ScDGAT genes of S. cerevisiae, and this, in turn, might be responsible for the increased lipid production in the transformed yeast. Our findings pave the way for the possible utility of the CtRap1 gene in suitable microorganisms to increase their storage lipid content through transcription factor engineering. PMID:25805842

  14. Saccharomyces cerevisiae BY4741 and W303-1A laboratory strains differ in salt tolerance.

    PubMed

    Petrezselyova, Silvia; Zahradka, Jaromir; Sychrova, Hana

    2010-01-01

    Saccharomyces cerevisiae yeast cells serve as a model to elucidate the bases of salt tolerance and potassium homeostasis regulation in eukaryotic cells. In this study, we show that two widely used laboratory strains, BY4741 and W303-1A, differ not only in cell size and volume but also in their relative plasma-membrane potential (estimated with a potentiometric fluorescent dye diS-C3(3) and as Hygromycin B sensitivity) and tolerance to alkali-metal cations. W303-1A cells and their mutant derivatives lacking either uptake (trk1 trk2) or efflux (nha1) systems for alkali-metal cations are more tolerant to toxic sodium and lithium cations but also more sensitive to higher external concentrations of potassium than BY4741 cells and their mutants. Moreover, our results suggest that though the two strains do not differ in the total potassium content, the regulation of intracellular potassium homeostasis is probably not the same in BY4741 and W303-1A cells. PMID:20960970

  15. Development of minimal fermentation media supplementation for ethanol production using two Saccharomyces cerevisiae strains.

    PubMed

    Tropea, Alessia; Wilson, David; Cicero, Nicola; Potortì, Angela G; La Torre, Giovanna L; Dugo, Giacomo; Richardson, David; Waldron, Keith W

    2016-01-01

    Ethanol production by fermentation is strongly dependent on media composition. Specific nutrients, such as trace elements, vitamins and nitrogen will affect the physiological state and, consequently, the fermentation performance of the micro-organism employed. The purpose of this study has been to assess the highest ethanol production by a minimal medium, instead of the more complex nutrients supplementation used during alcoholic fermentation. All fermentation tests were carried out using a microwell plate reader to monitor the processes. Two Saccharomyces cerevisiae strains (NCYC 2826 and NCYC 3445) were tested using three nitrogen sources, supplied with different vitamin and salts. The results show that solutions made of urea phosphate, KCl, MgSO4·7H2O, Ca-panthothenate, biotin allowed an ethanol yield of 22.9 and 23.4 g/L for strain NCYC 2826 and NCYC 3445, respectively, representing 90 and 92% of the theoretical yield. All tests were carried out using glucose as common reference carbon source. PMID:26469871

  16. Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae

    SciTech Connect

    Avery, S.V.; Tobin, J.M. )

    1992-12-01

    Concern over transfer of toxic metals from microorgansims to higher organisms and interest in the biotechnological potential of microorganisms for metal removal and/or recovery has increased interest in the processes involved in heavy metal uptake. Strontium is a trace element with no know essential biological role, but a long half-live and discharge as a constituent of radioactive wastewaters from nuclear reactors and in fall-out make its fate in the environment a concern. In this study, strontium uptake in biomass obtained from laboratory and industrial sources was examined. The mechanisms of Sr[sup 2+] uptake were examined and uptake capacities for Sr[sup 2+] were compared in both live and denatured forms of laboratory and brewery-derived strains of Saccharomyces cerevisiae. Release of cellular Ca[sup 2+], Mg[sup 2+], and H[sup +] in response to metabolism-independent and -dependent Sr[sup 2+] uptake processes, was determined for all biomass types. The results indicate clear differences in the mechanisms of both Sr[sup 2+] adsorption and intracellular Sr[sup 2+] accumulation between the yeasts examined. They point out the strong influence that the differential ecophysiology of strains from a single genus may exert on metal uptake characteristics and on external binding and intracellular distribution of essential ions.

  17. Heterosis Is Prevalent Among Domesticated but not Wild Strains of Saccharomyces cerevisiae

    PubMed Central

    Plech, Marcin; de Visser, J. Arjan G. M.; Korona, Ryszard

    2013-01-01

    Crosses between inbred but unrelated individuals often result in an increased fitness of the progeny. This phenomenon is known as heterosis and has been reported for wild and domesticated populations of plants and animals. Analysis of heterosis is often hindered by the fact that the genetic relatedness between analyzed organisms is only approximately known. We studied a collection of Saccharomyces cerevisiae isolates from wild and human-created habitats whose genomes were sequenced and thus their relatedness was fully known. We reasoned that if these strains accumulated different deleterious mutations at an approximately constant rate, then heterosis should be most visible in F1 heterozygotes from the least related parents. We found that heterosis was substantial and positively correlated with sequence divergence, but only in domesticated strains. More than 80% of the heterozygous hybrids were more fit than expected from the mean of their homozygous parents, and approximately three-quarters of those exceeded even the fittest parent. Our results support the notion that domestication brings about relaxation of selection and accumulation of deleterious mutations. However, other factors may have contributed as well. In particular, the observed build-up of genetic load might be facilitated by a decrease, and not increase, in the rate of inbreeding. PMID:24347627

  18. Surfome analysis of a wild-type wine Saccharomyces cerevisiae strain.

    PubMed

    Braconi, Daniela; Amato, Loredana; Bernardini, Giulia; Arena, Simona; Orlandini, Maurizio; Scaloni, Andrea; Santucci, Annalisa

    2011-09-01

    The yeast Saccharomyces cerevisiae, besides being an eukaryotic cell model, plays a fundamental role in the production of fermented foods. In the winemaking industry, yeast cell walls may be involved in numerous processes and contribute substantially to the final chemical and sensorial profiles of wines. Nonetheless, apart from mannoproteins, little is known on the protein components of the yeast cell wall and their changes during the fermentation of must into wine. In this work, we performed a dynamic analysis of the cell surface proteome (surfome) of an autochthonous wine yeast strain (previously selected as a wine fermentation starter) by shaving intact cells with trypsin and identifying tryptic peptides by means of nLC-ESI-LIT-MS/MS. Out of the 42 identified proteins, 16 and 14 were found to be specifically expressed in wine yeast surfome at the beginning and at the end of fermentation, respectively. The molecular functions of these specifically expressed proteins might help in explaining their roles in the cell wall as a response to the alcoholic fermentation-related stresses. Additionally, we provided the identification of 20 new potential cell wall related proteins. Globally, our results might provide new useful data for the selection and characterization of yeast strains to be used in the winemaking industry. PMID:21645823

  19. Genome Sequence and Analysis of a Stress-Tolerant, Wild-Derived Strain of Saccharomyces cerevisiae Used in Biofuels Research

    PubMed Central

    McIlwain, Sean J.; Peris, David; Sardi, Maria; Moskvin, Oleg V.; Zhan, Fujie; Myers, Kevin S.; Riley, Nicholas M.; Buzzell, Alyssa; Parreiras, Lucas S.; Ong, Irene M.; Landick, Robert; Coon, Joshua J.; Gasch, Audrey P.; Sato, Trey K.; Hittinger, Chris Todd

    2016-01-01

    The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics. PMID:27172212

  20. Genome Sequence and Analysis of a Stress-Tolerant, Wild-Derived Strain of Saccharomyces cerevisiae Used in Biofuels Research.

    PubMed

    McIlwain, Sean J; Peris, David; Sardi, Maria; Moskvin, Oleg V; Zhan, Fujie; Myers, Kevin S; Riley, Nicholas M; Buzzell, Alyssa; Parreiras, Lucas S; Ong, Irene M; Landick, Robert; Coon, Joshua J; Gasch, Audrey P; Sato, Trey K; Hittinger, Chris Todd

    2016-01-01

    The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics. PMID:27172212

  1. Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

    DOE PAGESBeta

    McIlwain, Sean J.; Peris, Davis; Sardi, Maria; Moskvin, Oleg V.; Zhan, Fujie; Myers, Kevin S.; Riley, Nicholas M.; Buzzell, Alyssa; Parreiras, Lucas S.; Ong, Irene M.; et al

    2016-04-20

    The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assemblymore » approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. Lastly, the Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.« less

  2. Biotechnological process for obtaining new fermented products from cashew apple fruit by Saccharomyces cerevisiae strains.

    PubMed

    Araújo, Suzane Macêdo; Silva, Cristina Ferraz; Moreira, Jane Jesus Silveira; Narain, Narendra; Souza, Roberto Rodrigues

    2011-09-01

    In Brazil, the use of cashew apple (Anacardium occidentale L.) to obtain new products by biotechnological process represents an important alternative to avoid wastage of a large quantity of this fruit, which reaches about 85% of the annual production of 1 million tons. This work focuses on the development of an alcoholic product obtained by the fermentation of cashew apple juice. The inoculation with two different strains of yeast Saccharomyces cerevisiae viz. SCP and SCT, were standardized to a concentration of 10(7 )cells ml(-1). Each inoculum was added to 1,500 ml of cashew must. Fermentation was performed at 28 ± 3°C and aliquots were withdrawn every 24 h to monitor soluble sugar concentrations, pH, and dry matter contents. The volatile compounds in fermented products were analyzed using the gas chromatography/mass spectrometry (GC/MS) system. After 6 days, the fermentation process was completed, cells removed by filtration and centrifugation, and the products were stabilized under refrigeration for a period of 20 days. The stabilized products were stored in glass bottles and pasteurized at 60 ± 5°C/30 min. Both fermented products contained ethanol concentration above 6% (v v(-1)) while methanol was not detected and total acidity was below 90 mEq l(-1), representing a pH of 3.8-3.9. The volatile compounds were characterized by the presence of aldehyde (butyl aldehyde diethyl acetal, 2,4-dimethyl-hepta-2,4-dienal, and 2-methyl-2-pentenal) and ester (ethyl α-methylbutyrate) representing fruity aroma. The strain SCT was found to be better and efficient and this produced 10% more alcohol over that of strain SCP. PMID:21069555

  3. Assessment of Inactivating Stop Codon Mutations in Forty Saccharomyces cerevisiae Strains: Implications for [PSI+] Prion- Mediated Phenotypes

    PubMed Central

    Fitzpatrick, David A.; O'Brien, Jennifer; Moran, Ciara; Hasin, Naushaba; Kenny, Elaine; Cormican, Paul; Gates, Amy; Morris, Derek W.; Jones, Gary W.

    2011-01-01

    The yeast prion [PSI+] has been implicated in the generation of novel phenotypes by a mechanism involving a reduction in translation fidelity causing readthrough of naturally occurring stop codons. Some [PSI+] associated phenotypes may also be generated due to readthrough of inactivating stop codon mutations (ISCMs). Using next generation sequencing we have sequenced the genomes of two Saccharomyces cerevisiae strains that are commonly used for the study of the yeast [PSI+] prion. We have identified approximately 26,000 and 6,500 single nucleotide polymorphisms (SNPs) in strains 74-D694 and G600 respectively, compared to reference strain S288C. In addition to SNPs that produce non-synonymous amino acid changes we have also identified a number of SNPs that cause potential ISCMs in these strains, one of which we show is associated with a [PSI+]-dependent stress resistance phenotype in strain G600. We identified twenty-two potential ISCMs in strain 74-D694, present in genes involved in a variety of cellular processes including nitrogen metabolism, signal transduction and oxidative stress response. The presence of ISCMs in a subset of these genes provides possible explanations for previously identified [PSI+]-associated phenotypes in this strain. A comparison of ISCMs in strains G600 and 74-D694 with S. cerevisiae strains sequenced as part of the Saccharomyces Genome Resequencing Project (SGRP) shows much variation in the generation of strain-specific ISCMs and suggests this process is possible under complex genetic control. Additionally we have identified a major difference in the abilities of strains G600 and 74-D694 to grow at elevated temperatures. However, this difference appears unrelated to novel SNPs identified in strain 74-D694 present in proteins involved in the heat shock response, but may be attributed to other SNP differences in genes previously identified as playing a role in high temperature growth. PMID:22194885

  4. Selection of autochthonous Saccharomyces cerevisiae strains as wine starters using a polyphasic approach and ochratoxin A removal.

    PubMed

    Petruzzi, Leonardo; Bevilacqua, Antonio; Corbo, Maria Rosaria; Garofalo, Carmela; Baiano, Antonietta; Sinigaglia, Milena

    2014-07-01

    Over the last few years, the selection of autochthonous strains of Saccharomyces cerevisiae as wine starters has been studied; however, researchers have not focused on the ability to remove ochratoxin A (OTA) as a possible trait to use in oenological characterization. In this article, a polyphasic approach, including yeast genotyping, evaluation of phenotypic traits, and fermentative performance in a model system (temperature, 25 and 30°C; sugar level, 200 and 250 g liter(-1)), was proposed as a suitable approach to select wine starters of S. cerevisiae from 30 autochthonous isolates from Uva di Troia cv., a red wine grape variety grown in the Apulian region (Southern Italy). The ability to remove OTA, a desirable trait to improve the safety of wine, was also assessed using enzyme-linked immunosorbent assay. The isolates, identified by PCR-restriction fragment length polymorphism analysis of the internal transcribed spacer region and DNA sequencing, were differentiated at strain level through the amplification of the interdelta region; 11 biotypes (I to XI) were identified and further studied. Four biotypes (II, III, V, VIII) were able to reduce OTA, with the rate of toxin removal from the medium (0.6 to 42.8%, wt/vol) dependent upon the strain and the temperature, and biotypes II and VIII were promising in terms of ethanol, glycerol, and volatile acidity production, as well as for their enzymatic and stress resistance characteristics. For the first time, the ability of S. cerevisiae to remove OTA during alcoholic fermentation was used as an additional trait in the yeast-selection program; the results could have application for evaluating the potential of autochthonous S. cerevisiae strains as starter cultures for the production of typical wines with improved quality and safety. PMID:24988024

  5. Physicochemical characterization of pomegranate wines fermented with three different Saccharomyces cerevisiae yeast strains.

    PubMed

    Berenguer, María; Vegara, Salud; Barrajón, Enrique; Saura, Domingo; Valero, Manuel; Martí, Nuria

    2016-01-01

    Three commercial Saccharomyces cerevisiae yeast strains: Viniferm Revelación, Viniferm SV and Viniferm PDM were evaluated for the production of pomegranate wine from a juice coupage of the two well-known varieties Mollar and Wonderfull. Further malolactic fermentation was carried out spontaneously. The same fermentation patterns were observed for pH, titratable acidity, density, sugar consumption, and ethanol and glycerol production. Glucose was exhausted while fructose residues remained at the end of alcoholic fermentation. A high ethanol concentration (10.91 ± 0.27% v/v) in combination with 1.49 g/L glycerol was achieved. Citric acid concentration increased rapidly a 31.7%, malic acid disappeared as result of malolactic fermentation and the lactic acid levels reached values between 0.40 and 0.96 g/L. The analysis of CIEa parameter and total anthocyanin content highlights a lower degradation of monomeric anthocyanins during winemaking with Viniferm PDM yeast. The resulting wine retains a 34.5% of total anthocyanin content of pomegranate juice blend. PMID:26213048

  6. Regulation of hydrogen sulfide liberation in wine-producing Saccharomyces cerevisiae strains by assimilable nitrogen.

    PubMed Central

    Jiranek, V; Langridge, P; Henschke, P A

    1995-01-01

    Saccharomyces cerevisiae wine-producing yeast cultures grown under model winemaking conditions could be induced to liberate hydrogen sulfide (H2S) by starvation for assimilable nitrogen. The amount of H2S produced was dependent on the yeast strain, the sulfur precursor compound, the culture growth rate, and the activity of the sulfite reductase enzyme (EC 1.8.1.2) immediately before nitrogen depletion. Increased H2S formation relative to its utilization by metabolism was not a consequence of a de novo synthesis of sulfite reductase. The greatest amount of H2S was produced when nitrogen became depleted during the exponential phase of growth or during growth on amino acids capable of supporting short doubling times. Both sulfate and sulfite were able to act as substrates for the generation of H2S in the absence of assimilable nitrogen; however, sulfate reduction was tightly regulated, leading to limited H2S liberation, whereas sulfite reduction appeared to be uncontrolled. In addition to ammonium, most amino acids were able to suppress the liberation of excess H2S when added as sole sources of nitrogen, particularly for one of the strains studied. Cysteine was the most notable exception, inducing the liberation of H2S at levels exceeding that of the nitrogen-depleted control. Threonine and proline also proved to be poor substitutes for ammonium. These data suggest that any compound that can efficiently generate sulfide-binding nitrogenous precursors of organic sulfur compounds will prevent the liberation of excess H2S. PMID:7574581

  7. Regulation of hydrogen sulfide liberation in wine-producing Saccharomyces cerevisiae strains by assimilable nitrogen.

    PubMed

    Jiranek, V; Langridge, P; Henschke, P A

    1995-02-01

    Saccharomyces cerevisiae wine-producing yeast cultures grown under model winemaking conditions could be induced to liberate hydrogen sulfide (H2S) by starvation for assimilable nitrogen. The amount of H2S produced was dependent on the yeast strain, the sulfur precursor compound, the culture growth rate, and the activity of the sulfite reductase enzyme (EC 1.8.1.2) immediately before nitrogen depletion. Increased H2S formation relative to its utilization by metabolism was not a consequence of a de novo synthesis of sulfite reductase. The greatest amount of H2S was produced when nitrogen became depleted during the exponential phase of growth or during growth on amino acids capable of supporting short doubling times. Both sulfate and sulfite were able to act as substrates for the generation of H2S in the absence of assimilable nitrogen; however, sulfate reduction was tightly regulated, leading to limited H2S liberation, whereas sulfite reduction appeared to be uncontrolled. In addition to ammonium, most amino acids were able to suppress the liberation of excess H2S when added as sole sources of nitrogen, particularly for one of the strains studied. Cysteine was the most notable exception, inducing the liberation of H2S at levels exceeding that of the nitrogen-depleted control. Threonine and proline also proved to be poor substitutes for ammonium. These data suggest that any compound that can efficiently generate sulfide-binding nitrogenous precursors of organic sulfur compounds will prevent the liberation of excess H2S. PMID:7574581

  8. Whole Genome Comparison Reveals High Levels of Inbreeding and Strain Redundancy Across the Spectrum of Commercial Wine Strains of Saccharomyces cerevisiae

    PubMed Central

    Borneman, Anthony R.; Forgan, Angus H.; Kolouchova, Radka; Fraser, James A.; Schmidt, Simon A.

    2016-01-01

    Humans have been consuming wines for more than 7000 yr . For most of this time, fermentations were presumably performed by strains of Saccharomyces cerevisiae that naturally found their way into the fermenting must . In contrast, most commercial wines are now produced by inoculation with pure yeast monocultures, ensuring consistent, reliable and reproducible fermentations, and there are now hundreds of these yeast starter cultures commercially available. In order to thoroughly investigate the genetic diversity that has been captured by over 50 yr of commercial wine yeast development and domestication, whole genome sequencing has been performed on 212 strains of S. cerevisiae, including 119 commercial wine and brewing starter strains, and wine isolates from across seven decades. Comparative genomic analysis indicates that, despite their large numbers, commercial strains, and wine strains in general, are extremely similar genetically, possessing all of the hallmarks of a population bottle-neck, and high levels of inbreeding. In addition, many commercial strains from multiple suppliers are nearly genetically identical, suggesting that the limits of effective genetic variation within this genetically narrow group may be approaching saturation. PMID:26869621

  9. Whole Genome Comparison Reveals High Levels of Inbreeding and Strain Redundancy Across the Spectrum of Commercial Wine Strains of Saccharomyces cerevisiae.

    PubMed

    Borneman, Anthony R; Forgan, Angus H; Kolouchova, Radka; Fraser, James A; Schmidt, Simon A

    2016-01-01

    Humans have been consuming wines for more than 7000 yr . For most of this time, fermentations were presumably performed by strains of Saccharomyces cerevisiae that naturally found their way into the fermenting must . In contrast, most commercial wines are now produced by inoculation with pure yeast monocultures, ensuring consistent, reliable and reproducible fermentations, and there are now hundreds of these yeast starter cultures commercially available. In order to thoroughly investigate the genetic diversity that has been captured by over 50 yr of commercial wine yeast development and domestication, whole genome sequencing has been performed on 212 strains of S. cerevisiae, including 119 commercial wine and brewing starter strains, and wine isolates from across seven decades. Comparative genomic analysis indicates that, despite their large numbers, commercial strains, and wine strains in general, are extremely similar genetically, possessing all of the hallmarks of a population bottle-neck, and high levels of inbreeding. In addition, many commercial strains from multiple suppliers are nearly genetically identical, suggesting that the limits of effective genetic variation within this genetically narrow group may be approaching saturation. PMID:26869621

  10. In situ near infrared spectroscopy monitoring of cyprosin production by recombinant Saccharomyces cerevisiae strains.

    PubMed

    Sampaio, Pedro N; Sales, Kevin C; Rosa, Filipa O; Lopes, Marta B; Calado, Cecília R

    2014-10-20

    Near infrared (NIR) spectroscopy was used to in situ monitoring the cultivation of two recombinant Saccharomyces cerevisiae strains producing heterologous cyprosin B. NIR spectroscopy is a fast and non-destructive technique, that by being based on overtones and combinations of molecular vibrations requires chemometrics tools, such as partial least squares (PLS) regression models, to extract quantitative information concerning the variables of interest from the spectral data. In the present work, good PLS calibration models based on specific regions of the NIR spectral data were built for estimating the critical variables of the cyprosin production process: biomass concentration, cyprosin activity, cyprosin specific activity, the carbon sources glucose and galactose concentration and the by-products acetic acid and ethanol concentration. The PLS models developed are valid for both recombinant S. cerevisiae strains, presenting distinct cyprosin production capacities, and therefore can be used, not only for the real-time control of both processes, but also in optimization protocols. The PLS model for biomass yielded a R(2)=0.98 and a RMSEP=0.46 g dcw l(-1), representing an error of 4% for a calibration range between 0.44 and 13.75 g dcw l(-1). A R(2)=0.94 and a RMSEP=167 Um l(-1) were obtained for the cyprosin activity, corresponding to an error of 6.7% of the experimental data range (0-2509 Um l(-1)), whereas a R(2)=0.93 and RMSEP=672 U mg(-1) were obtained for the cyprosin specific activity, corresponding to an error of 7% of the experimental data range (0-11,690 Um g(-1)). For the carbon sources glucose and galactose, a R(2)=0.96 and a RMSECV of 1.26 and 0.55 g l(-1), respectively, were obtained, showing high predictive capabilities within the range of 0-20 g l(-1). For the metabolites resulting from the cell growth, the PLS model for acetate was characterized by a R(2)=0.92 and a RMSEP=0.06 g l (-1), which corresponds to a 6.1% error within the range of 0

  11. Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

    PubMed

    Parreiras, Lucas S; Breuer, Rebecca J; Avanasi Narasimhan, Ragothaman; Higbee, Alan J; La Reau, Alex; Tremaine, Mary; Qin, Li; Willis, Laura B; Bice, Benjamin D; Bonfert, Brandi L; Pinhancos, Rebeca C; Balloon, Allison J; Uppugundla, Nirmal; Liu, Tongjun; Li, Chenlin; Tanjore, Deepti; Ong, Irene M; Li, Haibo; Pohlmann, Edward L; Serate, Jose; Withers, Sydnor T; Simmons, Blake A; Hodge, David B; Westphall, Michael S; Coon, Joshua J; Dale, Bruce E; Balan, Venkatesh; Keating, David H; Zhang, Yaoping; Landick, Robert; Gasch, Audrey P; Sato, Trey K

    2014-01-01

    The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH. PMID:25222864

  12. Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant Saccharomyces cerevisiae Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

    PubMed Central

    Parreiras, Lucas S.; Breuer, Rebecca J.; Avanasi Narasimhan, Ragothaman; Higbee, Alan J.; La Reau, Alex; Tremaine, Mary; Qin, Li; Willis, Laura B.; Bice, Benjamin D.; Bonfert, Brandi L.; Pinhancos, Rebeca C.; Balloon, Allison J.; Uppugundla, Nirmal; Liu, Tongjun; Li, Chenlin; Tanjore, Deepti; Ong, Irene M.; Li, Haibo; Pohlmann, Edward L.; Serate, Jose; Withers, Sydnor T.; Simmons, Blake A.; Hodge, David B.; Westphall, Michael S.; Coon, Joshua J.; Dale, Bruce E.; Balan, Venkatesh; Keating, David H.; Zhang, Yaoping; Landick, Robert; Gasch, Audrey P.; Sato, Trey K.

    2014-01-01

    The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH. PMID:25222864

  13. Standard YPD, even supplemented with extra nutrients, does not always compensate growth defects of Saccharomyces cerevisiae auxotrophic strains.

    PubMed

    Corbacho, Isaac; Teixidó, Francisco; Velázquez, Rocío; Hernández, Luis M; Olivero, Isabel

    2011-03-01

    Conventional complex media are routinely used to grow auxotrophic strains under the assumption that they can compensate the latter's nutritional deficiencies. We here demonstrate that this is not always true. This study compares the growth parameters of Saccharomyces cerevisiae (S288C) and its derived auxotrophic strains FY1679-14C and BY4741 in synthetic minimal medium (SD), standard YPD medium from two of the most commonly used suppliers, or modified YPD medium. Maximum specific growth rates of auxotrophic strains were slightly lower than the prototrophic case in all growth conditions tested. Also, the biomass production of auxotrophic strains in synthetic medium was slightly less than the prototrophic case. However in both of the two standard YPD media used, the biomass production of both auxotrophic strains was markedly lower than that of the prototrophic one. The extent of the differences depended on the medium used. Indeed in one of the two YPD media, the lower biomass production of auxotrophic strains was evident even at the diauxic shift. Uracil seems to be the main limiting growth factor for both auxotrophic strains growing in the two standard YPD medium tested. No YPD media or specific supplement was able to compensate for the effect of the auxotrophic mutations in the multiple auxotrophic marker strain BY4741. The fact that auxotrophic strains grew poorly on YPD when compared to their prototrophic counterpart indicates that standard YPD medium is not sufficient to overcome the effect of auxotrophic mutations. PMID:21120607

  14. Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

    PubMed Central

    Bettiga, Maurizio; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2008-01-01

    Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells)-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)-1 h-1 compared with 0.01 g (g cells)-1 h-1 for the xylose reductase

  15. Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Brettanomyces bruxellensis

    PubMed Central

    Thornton, Mark A.; Thornton, Roy J.

    2013-01-01

    The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%. PMID:23913433

  16. Expression of a Heterologous Xylose Transporter in a Saccharomyces cerevisiae Strain Engineered to Utilize Xylose Improves Aerobic Xylose Co-consumption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strains of Saccharomyces cerevisiae have been engineered to utilize xylose by expression of the genes for xylose reductase and xylitol dehydrogenase, or xylose isomerase. These strains are still limited in their ability to efficiently use xylose. Unlike native xylose assimilating yeasts such as Pi...

  17. Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose increases xylose uptake and improves xylose/glucose co-consumption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strains of Saccharomyces cerevisiae have been engineered to utilize xylose by expressing either the genes for xylose reductase and xylitol dehydrogenase, or for xylose isomerase. These strains still use xylose at sub-optimal rates for industrial fermentation. Unlike natural xylose fermenting yeast...

  18. Genome Sequences of Industrially Relevant Saccharomyces cerevisiae Strain M3707, Isolated from a Sample of Distillers Yeast and Four Haploid Derivatives

    SciTech Connect

    Brown, Steven D.; Klingeman, Dawn M.; Johnson, Courtney M.; Clum, Alicia; Aerts, Andrea; Salamov, Asaf; Sharma, Aditi; Zane, Matthew; Barry, Kerrie; Grigoriev, Igor V.; Davison, Brian H.; Lynd, Lee R.; Gilna, Paul; Hau, Heidi; Hogsett, David A.; Froehlich, Allan C.

    2013-04-19

    Saccharomyces cerevisiae strain M3707 was isolated from a sample of commercial distillers yeast, and its genome sequence together with the genome sequences for the four derived haploid strains M3836, M3837, M3838, and M3839 has been determined. Yeasts have potential for consolidated bioprocessing (CBP) for biofuel production, and access to these genome sequences will facilitate their development.

  19. Mechanism of imidazolium ionic liquids toxicity in Saccharomyces cerevisiae and rational engineering of a tolerant, xylose-fermenting strain

    DOE PAGESBeta

    Dickinson, Quinn; Bottoms, Scott; Hinchman, Li; McIlwain, Sean; Li, Sheena; Myers, Chad L.; Boone, Charles; Coon, Joshua J.; Hebert, Alexander; Sato, Trey K.; et al

    2016-01-20

    In this study, imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae. As a result, we found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effectsmore » of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2Δ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance. In conclusion, this work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.« less

  20. Diploid versus Haploid Organisms

    NASA Astrophysics Data System (ADS)

    Ticona, Armando; de Oliveira, Paulo Murilo C.

    Using a bit string model, we show that asexual reproduction for diploids is more efficient than for haploids: it improves genetic material producing new individuals with less deleterious mutations. We also see that in a system where competition is present, diploids dominate, even though we consider some dominant loci.

  1. Metabolomic and 13C-Metabolic Flux Analysis of a Xylose-Consuming Saccharomyces cerevisiae Strain Expressing Xylose Isomerase

    PubMed Central

    Wasylenko, Thomas M.; Stephanopoulos, Gregory

    2016-01-01

    Over the past two decades significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative PPP is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis. PMID:25311863

  2. Analysis of the Saccharomyces cerevisiae pan-genome reveals a pool of copy number variants distributed in diverse yeast strains from differing industrial environments

    PubMed Central

    Dunn, Barbara; Richter, Chandra; Kvitek, Daniel J.; Pugh, Tom; Sherlock, Gavin

    2012-01-01

    Although the budding yeast Saccharomyces cerevisiae is arguably one of the most well-studied organisms on earth, the genome-wide variation within this species—i.e., its “pan-genome”—has been less explored. We created a multispecies microarray platform containing probes covering the genomes of several Saccharomyces species: S. cerevisiae, including regions not found in the standard laboratory S288c strain, as well as the mitochondrial and 2-μm circle genomes–plus S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum, S. kluyveri, and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) on 83 different S. cerevisiae strains collected across a wide range of habitats; of these, 69 were commercial wine strains, while the remaining 14 were from a diverse set of other industrial and natural environments. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole-genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae. PMID:22369888

  3. Oxidative stress response and nitrogen utilization are strongly variable in Saccharomyces cerevisiae wine strains with different fermentation performances.

    PubMed

    Treu, Laura; Campanaro, Stefano; Nadai, Chiara; Toniolo, Chiara; Nardi, Tiziana; Giacomini, Alessio; Valle, Giorgio; Blondin, Bruno; Corich, Viviana

    2014-05-01

    We used RNA-sequencing (RNA-seq) to analyze the expression profile of four vineyard strains of Saccharomyces cerevisiae having different fermentation performances. The expression profiles obtained in two steps of the fermentation process were compared with those obtained for the industrial wine strain EC1118 and for the laboratory strain S288c. The two strains with low fermentation efficiency, namely, S288c and the vineyard strain R103, exhibited markedly different expression profiles when compared to the other four strains. We also found that the vineyard strains P283 and P301 are characterized by a high expression of the transcription factor Met32p in the first step of the fermentation. Met32p, in coordination with the Hap4p transcription factor, determined the over-expression of the genes involved in the respiration processes, in the response to oxidative stress and in the sulfur amino acids biosynthesis. These combined actions are likely to increase the level of antioxidants whose protective effect could contribute to improve the fermentation process. Gene expression and phenotypic data revealed that the vineyard strain P301 has low nitrogen utilization in comparison to the other wine strains, combined with high fermentation efficiency. Analysis of the genes involved in fermentation stress response revealed a lower expression in strains characterized by low fermentation efficiency, particularly in the first fermentation phase. These findings evidenced the high variability of transcriptional profiles among different wine yeast strains and clarify their connection with complex phenotypic traits, such as the fermentation efficiency and the nitrogen sources utilization. PMID:24695828

  4. NUCLEOLI OF DIPLOID CELL STRAINS

    PubMed Central

    Phillips, Stephanie G.; Phillips, David M.

    1971-01-01

    Nucleoli of cultured Chinese hamster or mouse cells in early passages had a loosely reticular substructure. Within the reticulum small, irregularly shaped, light fibrillar zones occurred which were contiguous with denser fibrillar zones. These denser zones appeared to be connected in some places to the particulate material which composed the mass of the nucleolus. Generally, electron-transparent spaces separated the particulate zones from the fibrillar areas. Treatment with toyocamycin, an agent which is reported to cause a blockage in the processing of ribosomal RNA, greatly inhibited the accumulation of newly synthesized RNA in the cytoplasm, as monitored by radioautography. Toyocamycin treatment caused the gradual disappearance of the granules from the particulate region of the nucleoli, and resulted ultimately in the nucleoli appearing homogeneously fibrillar. Actinomycin D treatment, which inhibited virtually all RNA synthesis, caused a segregation, and finally a disaggregation, of nucleolar components. PMID:5104724

  5. Diploid yeast cells yield homozygous spontaneous mutations

    NASA Technical Reports Server (NTRS)

    Esposito, M. S.; Bruschi, C. V.; Brushi, C. V. (Principal Investigator)

    1993-01-01

    A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

  6. Comparative study of Saccharomyces cerevisiae wine strains to identify potential marker genes correlated to desiccation stress tolerance.

    PubMed

    Capece, Angela; Votta, Sonia; Guaragnella, Nicoletta; Zambuto, Marianna; Romaniello, Rossana; Romano, Patrizia

    2016-05-01

    The most diffused formulation of starter for winemaking is active dry yeast (ADY). ADYs production process is essentially characterized by air-drying stress, a combination of several stresses, including thermal, hyperosmotic and oxidative and cell capacity to counteract such multiple stresses will determine its survival. The molecular mechanisms underlying cell stress response to desiccation have been mostly studied in laboratory and commercial yeast strains, but a growing interest is currently developing for indigenous yeast strains which represent a valuable and alternative source of genetic and molecular biodiversity to be exploited. In this work, a comparative study of different Saccharomyces cerevisiae indigenous wine strains, previously selected for their technological traits, has been carried out to identify potentially relevant genes involved in desiccation stress tolerance. Cell viability was evaluated along desiccation treatment and gene expression was analyzed by real-time PCR before and during the stress. Our data show that the observed differences in individual strain sensitivity to desiccation stress could be associated to specific gene expression over time. In particular, either the basal or the stress-induced mRNA levels of certain genes, such as HSP12, SSA3, TPS1, TPS2, CTT1 and SOD1, result tightly correlated to the strain survival advantage. This study provides a reliable and sensitive method to predict desiccation stress tolerance of indigenous wine yeast strains which could be preliminary to biotechnological applications. PMID:26882930

  7. High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces cerevisiae distillery strain

    PubMed Central

    Bravim, Fernanda; Lippman, Soyeon I.; da Silva, Lucas F.; Souza, Diego T.; Fernandes, A. Alberto R.; Masuda, Claudio A.; Broach, James R.

    2016-01-01

    High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries. PMID:22915193

  8. High Level Ethanol from Sugar Cane Molasses by a New Thermotolerant Saccharomyces cerevisiae Strain in Industrial Scale

    PubMed Central

    Fadel, M.; Keera, Abeer A.; Mouafi, Foukia E.; Kahil, Tarek

    2013-01-01

    A new local strain of S. cerevisiae F-514, for ethanol production during hot summer season, using Egyptian sugar cane molasses was applied in Egyptian distillery factory. The inouluum was propagated through 300 L, 3 m3, and 12 m3 fermenters charged with diluted sugar cane molasses containing 4%-5% sugars. The yeast was applied in fermentation vessels 65 m3 working volume to study the varying concentrations of urea, DAP, orthophosphoric acid (OPA), and its combinations as well as magnesium sulfate and inoculum size. The fermenter was allowed to stay for a period of 20 hours to give time for maximum conversion of sugars into ethanol. S. cerevisiae F-514 at molasses sugar level of 18% (w/v), inoculum size of 20% (v/v) cell concentration of 3.0 × 108/mL, and combinations of urea, diammonium phosphate (DAP), orthophosphoric acid (OPA), and magnesium sulfate at amounts of 20, 10, 5, and 10 kg/65 m3 working volume fermenters, respectively, supported maximum ethanol production (9.8%, v/v), fermentation efficiency (FE) 88.1%, and remaining sugars (RS) 1.22%. The fermentation resulted 13.4 g dry yeast/L contained 34.6% crude protein and 8.2% ash. By selecting higher ethanol yielding yeast strain and optimizing, the fermentation parameters both yield and economics of the fermentation process can be improved. PMID:24363937

  9. Efficient Bioethanol Production by a Recombinant Flocculent Saccharomyces cerevisiae Strain with a Genome-Integrated NADP+-Dependent Xylitol Dehydrogenase Gene▿

    PubMed Central

    Matsushika, Akinori; Inoue, Hiroyuki; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke; Sawayama, Shigeki

    2009-01-01

    The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP+-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips. PMID:19329659

  10. Nuclear and mitochondrial genome instability induced by senna (Cassia angustifolia Vahl.) aqueous extract in Saccharomyces cerevisiae strains.

    PubMed

    Silva, C R; Caldeira-de-Araújo, A; Leitão, A C; Pádula, M

    2014-01-01

    Cassia angustifolia Vahl. (senna) is commonly used in self-medication and is frequently used to treat intestine constipation. A previous study involving bacteria and plasmid DNA suggested the possible toxicity of the aqueous extract of senna (SAE). The aim of this study was to extend the knowledge concerning SAE genotoxicity mechanisms because of its widespread use and its risks to human health. We investigated the impact of SAE on nuclear DNA and on the stability of mitochondrial DNA in Saccharomyces cerevisiae (wt, ogg1, msh6, and ogg1msh6) strains, monitoring the formation of petite mutants. Our results demonstrated that SAE specifically increased Can(R) mutagenesis only in the msh6 mutant, supporting the view that SAE can induce misincorporation errors in DNA. We observed a significant increase in the frequency of petite colonies in all studied strains. Our data indicate that SAE has genotoxic activity towards both mitochondrial and nuclear DNA. PMID:25501195

  11. Zearalenone and Its Derivatives α-Zearalenol and β-Zearalenol Decontamination by Saccharomyces cerevisiae Strains Isolated from Bovine Forage

    PubMed Central

    Keller, Luiz; Abrunhosa, Luís; Keller, Kelly; Rosa, Carlos Alberto; Cavaglieri, Lilia; Venâncio, Armando

    2015-01-01

    Zearalenone (ZEA) and its derivatives are mycotoxins with estrogenic effects on mammals. The biotransformation for ZEA in animals involves the formation of two major metabolites, α- and β-zearalenol (α-ZOL and β-ZOL), which are subsequently conjugated with glucuronic acid. The capability of Saccharomyces cerevisiae strains isolated from silage to eliminate ZEA and its derivatives α-ZOL and β-ZOL was investigated as, also, the mechanisms involved. Strains were grown on Yeast Extract-Peptone-Dextrose medium supplemented with the mycotoxins and their elimination from medium was quantified over time by HPLC-FL. A significant effect on the concentration of ZEA was observed, as all the tested strains were able to eliminate more than 90% of the mycotoxin from the culture medium in two days. The observed elimination was mainly due to ZEA biotransformation into β-ZOL (53%) and α-ZOL (8%) rather than to its adsorption to yeast cells walls. Further, the biotransformation of α-ZOL was not observed but a small amount of β-ZOL (6%) disappeared from culture medium. ZEA biotransformation by yeasts may not be regarded as a full detoxification process because both main end-products are still estrogenic. Nonetheless, it was observed that the biotransformation favors the formation of β-ZOL which is less estrogenic than ZEA and α-ZOL. This metabolic effect is only possible if active strains are used as feed additives and may play a role in the detoxification performance of products with viable S. cerevisiae cells. PMID:26308051

  12. Zearalenone and Its Derivatives α-Zearalenol and β-Zearalenol Decontamination by Saccharomyces cerevisiae Strains Isolated from Bovine Forage.

    PubMed

    Keller, Luiz; Abrunhosa, Luís; Keller, Kelly; Rosa, Carlos Alberto; Cavaglieri, Lilia; Venâncio, Armando

    2015-08-01

    Zearalenone (ZEA) and its derivatives are mycotoxins with estrogenic effects on mammals. The biotransformation for ZEA in animals involves the formation of two major metabolites, α- and β-zearalenol (α-ZOL and β-ZOL), which are subsequently conjugated with glucuronic acid. The capability of Saccharomyces cerevisiae strains isolated from silage to eliminate ZEA and its derivatives α-ZOL and β-ZOL was investigated as, also, the mechanisms involved. Strains were grown on Yeast Extract-Peptone-Dextrose medium supplemented with the mycotoxins and their elimination from medium was quantified over time by HPLC-FL. A significant effect on the concentration of ZEA was observed, as all the tested strains were able to eliminate more than 90% of the mycotoxin from the culture medium in two days. The observed elimination was mainly due to ZEA biotransformation into β-ZOL (53%) and α-ZOL (8%) rather than to its adsorption to yeast cells walls. Further, the biotransformation of α-ZOL was not observed but a small amount of β-ZOL (6%) disappeared from culture medium. ZEA biotransformation by yeasts may not be regarded as a full detoxification process because both main end-products are still estrogenic. Nonetheless, it was observed that the biotransformation favors the formation of β-ZOL which is less estrogenic than ZEA and α-ZOL. This metabolic effect is only possible if active strains are used as feed additives and may play a role in the detoxification performance of products with viable S. cerevisiae cells. PMID:26308051

  13. Investigating host dependence of xylose utilization in recombinant Saccharomyces cerevisiae strains using RNA-seq analysis

    PubMed Central

    2013-01-01

    Background Xylose-based ethanol production by recombinant S. cerevisiae is of great interest to basic and applied bioenergy research. By expressing three different fungal pathways in two S. cerevisiae hosts respectively, we found that the xylose utilization efficiency by recombinant S. cerevisiae depends not only on the choice of xylose pathway but also on the choice of host, exhibiting an obvious host or context dependence. To investigate molecular mechanisms of this context dependence, we applied RNA-seq analysis in this study for a systematic characterization of the xylose utilization via different pathways in different S. cerevisiae hosts. Results Based on the RNA-seq analysis, the transcripts that were regulated during xylose utilization have been identified. Three transcription factors involved in regulation of amino acid metabolism, responses to oxidative stresses, and degradation of aggregated proteins, respectively, were found to participate in xylose metabolism regulation regardless of which pathway was expressed and which host the xylose pathway was expressed in. Nine transcription factors, involved in homeostasis, regulation of amino acid metabolism, and stress responses, were identified as the key modules responsible for the host-specific responses to the same xylose pathway. In addition, the transcriptional regulations of xylose utilization in different yeast hosts were compared to two reference regulation patterns, which indicated that diverse regulation strategies were adopted by different hosts for improved xylose utilization. Conclusions This study provides the first transcriptomic study of the host dependence of xylose utilization in S. cerevisiae. Both the conserved regulatory modules for xylose metabolism and the key modules responsible for host dependence were identified. As indicated by the functions of the conserved transcription factors involved in xylose metabolism regulation, the xylose utilization in recombinant S. cerevisiae may be

  14. Direct observation of oxidative stress on the cell wall of Saccharomyces cerevisiae strains with atomic force microscopy.

    PubMed

    de Souza Pereira, R; Geibel, J

    1999-11-01

    We imaged pores on the surface of the cell wall of three different industrial strains of Saccharomyces cerevisiae using atomic force microscopy. The pores could be enlarged using 10 mM diamide, an SH residue oxidant that attacks surface proteins. We found that two strains showed signs of oxidative damage via changes in density and diameter of the surface pores. We found that the German strain was resistant to diamide induced oxidative damage, even when the concentration of the oxidant was increased to 50 mM. The normal pore size found on the cell walls of American strains had diameters of about 200 nm. Under conditions of oxidative stress the diameters changed to 400 nm. This method may prove to be a useful rapid screening process (45-60 min) to determine which strains are oxidative resistant, as well as being able to screen for groups of yeast that are sensitive to oxidative stress. This rapid screening tool may have direct applications in molecular biology (transference of the genes to inside of living cells) and biotechnology (biotransformations reactions to produce chiral synthons in organic chemistry. PMID:10630618

  15. The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains.

    PubMed

    Jeppsson, Marie; Johansson, Björn; Jensen, Peter Ruhdal; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2003-11-01

    Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD(+) into NADP(+) and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H(2)O(2) than the control strain TMB3001. PMID:14618564

  16. Hardness does not affect the physiological responses of wild and domestic strains of diploid and triploid rainbow trout Oncorhynchus mykiss to short-term exposure to pH 9.5.

    PubMed

    Thompson, W A; Rodela, T M; Richards, J G

    2016-08-01

    This study examined the effects of water hardness on the physiological responses associated with high pH exposure in multiple strains of diploid and triploid rainbow trout Oncorhynchus mykiss. To accomplish this, three wild strains and one domesticated strain of diploid and triploid O. mykiss were abruptly transferred from control soft water (City of Vancouver dechlorinated tap water; pH 6·7; [CaCO3 ] < 17·9 mg l(-1) ) to control soft water (handling control), high pH soft water (pH 9·5; [CaCO3 ] < 17·9 mg l(-1) ), or high pH hard water (pH 9·5; [CaCO3 ] = 320 mg l(-1) ) followed by sampling at 24 h for physiological measurements. There was a significant effect of ploidy on loss of equilibrium (LOE) over the 24 h exposure, with only triploid O. mykiss losing equilibrium at high pH in both soft and hard water. Furthermore, exposure to pH 9·5 resulted in significant decreases in plasma sodium and chloride, and increases in plasma and brain ammonia with no differences between soft and hard water. There was no significant effect of strain on LOE, but there were significant differences between strains in brain ammonia and plasma cortisol. Overall, there were no clear protective effects of hardness on high pH exposure in these strains of O. mykiss. PMID:27325291

  17. Isolation of mannan-protein complexes from viable cells of Saccharomyces cerevisiae X2180-1A wild type and Saccharomyces cerevisiae X2180-1 A-5 mutant strains by the action of Zymolyase-60,000.

    PubMed Central

    Shibata, N; Mizugami, K; Takano, K; Suzuki, S

    1983-01-01

    The viable whole cells of Saccharomyces cerevisiae X2180-1A wild type and its mannan mutant strain S. cerevisiae X2180-1A-5, were treated with an Arthrobacter sp. beta-1,3-glucanase in the presence of a serine protease inhibitor, phenyl-methylsulfonyl fluoride. Fractionation of the solubilized materials of each strain with Cetavlon (cetyltrimethylammonium bromide) yielded one mannan-protein complex. Molecular weights of these complexes were almost the same as that of the mannoprotein of the mutant strain prepared by Nakajima and Ballou, which had a molecular weight of 133,000 and were approximately three times larger than those of the mannans isolated from the same cells by hot-water extraction. Each mannan-protein complex contained up to 2% glucose residue, which was not removed by specific precipitation with anti-mannan sera or by affinity chromatography on a column of concanavalin A-Sepharose. Treatment of these complexes with alkaline NaBH4 produced peptide-free mannan containing small amounts of glucose nearly identical to those of the parent complexes. The above findings provide evidence that the glucose residues exist in a covalently linked form to the mannan moiety. Fractionation of the mannan-protein complex of the S. cerevisiae wild-type strain by DEAE-Sephadex chromatography yielded five subfractions of different phosphate content, indicating that these highly intact mannan-protein complexes were of heterogeneous material consisting of many molecular species of different phosphate content. PMID:6355061

  18. Transcriptomes of a xylose-utilizing industrial flocculating Saccharomyces cerevisiae strain cultured in media containing different sugar sources.

    PubMed

    Zeng, Wei-Yi; Tang, Yue-Qin; Gou, Min; Xia, Zi-Yuan; Kida, Kenji

    2016-12-01

    Lignocellulosic hydrolysates used for bioethanol production contain a mixture of sugars, with xylose being the second most abundant after glucose. Since xylose is not a natural substrate for Saccharomyces cerevisiae, recombinant S. cerevisiae strongly prefers glucose over xylose, and the fermentation rate and ethanol yield with xylose are both lower than those with glucose. To determine the molecular basis for glucose and xylose fermentation, we used microarrays to investigate the transcriptional difference of a xylose-utilizing industrial strain cultured in both single sugar media and a mixed sugar medium of glucose and xylose. The transcriptomes were nearly identical between glucose metabolizing cells in the glucose alone medium and those in the glucose fermentation phase in the mixed-sugar medium. Whereas the transcriptomes highly differed between the xylose metabolizing cells in the xylose alone medium and those in the xylose fermentation phase in the mixed sugar medium, and the differences mainly involved sulfur metabolism. When the transcriptional profiles were compared between glucose fermentation state and xylose fermentation state, we found the expression patterns of hexose transporters and glucose signaling pathway differed in response to different sugar sources, and the expression levels of the genes involved in gluconeogenesis, the glyoxylate and tricarboxylic acid cycles and respiration increased with xylose, indicating that the xylose-metabolizing cells had high requirements for maintenance energy and lacked the carbon catabolite repression capability. The effect of carbon catabolite repression by glucose lasted after glucose depletion for specific genes to different extents. PMID:27485516

  19. Multiple Ty-mediated chromosomal translocations lead to karyotype changes in a wine strain of Saccharomyces cerevisiae.

    PubMed

    Rachidi, N; Barre, P; Blondin, B

    1999-06-01

    Enological strains of Saccharomyces cerevisiae display a high level of chromosome length polymorphism, but the molecular basis of this phenomenon has not yet been clearly defined. In order to gain further insight into the molecular mechanisms responsible for the karyotypic variability, we examined the chromosomal constitution of a strain known to possess aberrant chromosomes. Our data revealed that the strain carries four rearranged chromosomes resulting from two reciprocal translocations between chromosomes III and I, and chromosomes III and VII. The sizes of the chromosomal fragments exchanged through translocation range from 40 to 150 kb. Characterization of the breakpoints indicated that the translocations involved the RAHS of chromosome III, a transposition hot-spot on the right arm of chromosome I and a region on the left arm of chromosome VII. An analysis of the junctions showed that in all cases Ty elements were present and suggested that the translocations result from recombination between transposable Ty elements. The evidence for multiple translocations mediated by Ty elements in a single strain suggests that spontaneous Ty-driven rearrangement could be quite common and may play a major role in the alteration of karyotypes in natural and industrial yeasts. PMID:10394922

  20. WASP suppresses the growth defect of Saccharomyces cerevisiae las17Delta strain in the presence of WIP.

    PubMed

    Rajmohan, Rajamuthiah; Meng, Lei; Yu, Shangjuan; Thanabalu, Thirumaran

    2006-04-01

    Wiskott-Aldrich syndrome is caused by alterations in the Wiskott-Aldrich syndrome protein (WASP) and several of these mutations affect WASP's interaction with WIP (WASP-interacting protein), suggesting that loss of interaction between WASP and WIP is causal to the disease. Las17p is the yeast homologue of WASP and las17Delta strain is unable to grow at 37 degrees C. We show that Human WASP suppresses the growth defect of Saccharomyces cerevisiae las17Delta strain, only in the presence of WIP. WIP mediates cortical localisation of WASP as well as stabilise WASP in yeast cells. Mutations which affected WASP-WIP interaction abolished WASP's ability to suppress the growth defect of las17Delta strain. We have demonstrated that WASP-WIP is an active complex and WASP's ability to suppress the growth defect of las17Delta strain is dependent on the presence of a functional Arp2/3 activating domain of WASP and also the Verprolin domain (V) of WIP. PMID:16488394

  1. Effect of fermentation with Saccharomyces cerevisiae strain PJ69-4 on the phytic acid, raffinose, and stachyose contents of soybean meal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three experiments were conducted to determine the impact of submerged fermentation procedures using Saccharomyces cerevisiae baker’s yeast strain PJ69-4a on degradation of phytic acid and the raffinosaccharides, raffinose, and stachyose, in soybean meal. The goal of the research was to identify a n...

  2. Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations

    PubMed Central

    Guadalupe-Medina, Víctor; Metz, Benjamin; Oud, Bart; van Der Graaf, Charlotte M; Mans, Robert; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd– strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd– mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h−1. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l−1). However, these glycerol concentrations were below 10% of those observed with a Gpd+ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth. PMID:24004455

  3. Molecular-genetic biodiversity in a natural population of the yeast Saccharomyces cerevisiae from "Evolution Canyon": microsatellite polymorphism, ploidy and controversial sexual status.

    PubMed

    Ezov, T Katz; Boger-Nadjar, E; Frenkel, Z; Katsperovski, I; Kemeny, S; Nevo, E; Korol, A; Kashi, Y

    2006-11-01

    The yeast S. cerevisiae is a central model organism in eukaryotic cell studies and a major component in many food and biotechnological industrial processes. However, the wide knowledge regarding genetics and molecular biology of S. cerevisiae is based on an extremely narrow range of strains. Studies of natural populations of S. cerevisiae, not associated with human activities or industrial fermentation environments, are very few. We isolated a panel of S. cerevisiae strains from a natural microsite, "Evolution Canyon" at Mount Carmel, Israel, and studied their genomic biodiversity. Analysis of 19 microsatellite loci revealed high allelic diversity and variation in ploidy level across the panel, from diploids to tetraploids, confirmed by flow cytometry. No significant differences were found in the level of microsatellite variation between strains derived from the major localities or microniches, whereas strains of different ploidy showed low similarity in allele content. Maximum genetic diversity was observed among diploids and minimum among triploids. Phylogenetic analysis revealed clonal, rather than sexual, structure of the triploid and tetraploid subpopulations. Viability tests in tetrad analysis also suggest that clonal reproduction may predominate in the polyploid subpopulations. PMID:16980391

  4. Xylose and xylose/glucose co-fermentation by recombinant Saccharomyces cerevisiae strains expressing individual hexose transporters.

    PubMed

    Gonçalves, Davi L; Matsushika, Akinori; de Sales, Belisa B; Goshima, Tetsuya; Bon, Elba P S; Stambuk, Boris U

    2014-09-01

    Since the uptake of xylose is believed to be one of the rate-limiting steps for xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) with an integrative plasmid to overexpress the genes for xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using xylose, glucose, or xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose/glucose co-fermentations, but was incapable to allow xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient xylose fermentation, but during xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing xylose/glucose blends, whereas the HXT7 permease would be a better choice for xylose-enriched sugar streams. PMID:25039054

  5. High Level Ethanol from Sugar Cane Molasses by a New Thermotolerant Saccharomyces cerevisiae Strain in Industrial Scale.

    PubMed

    Fadel, M; Keera, Abeer A; Mouafi, Foukia E; Kahil, Tarek

    2013-01-01

    A new local strain of S. cerevisiae F-514, for ethanol production during hot summer season, using Egyptian sugar cane molasses was applied in Egyptian distillery factory. The inouluum was propagated through 300 L, 3 m(3), and 12 m(3) fermenters charged with diluted sugar cane molasses containing 4%-5% sugars. The yeast was applied in fermentation vessels 65 m(3) working volume to study the varying concentrations of urea, DAP, orthophosphoric acid (OPA), and its combinations as well as magnesium sulfate and inoculum size. The fermenter was allowed to stay for a period of 20 hours to give time for maximum conversion of sugars into ethanol. S. cerevisiae F-514 at molasses sugar level of 18% (w/v), inoculum size of 20% (v/v) cell concentration of 3.0 × 10(8)/mL, and combinations of urea, diammonium phosphate (DAP), orthophosphoric acid (OPA), and magnesium sulfate at amounts of 20, 10, 5, and 10 kg/65 m(3) working volume fermenters, respectively, supported maximum ethanol production (9.8%, v/v), fermentation efficiency (FE) 88.1%, and remaining sugars (RS) 1.22%. The fermentation resulted 13.4 g dry yeast/L contained 34.6% crude protein and 8.2% ash. By selecting higher ethanol yielding yeast strain and optimizing, the fermentation parameters both yield and economics of the fermentation process can be improved. PMID:24363937

  6. Improved sugar co-utilisation by encapsulation of a recombinant Saccharomyces cerevisiae strain in alginate-chitosan capsules

    PubMed Central

    2014-01-01

    Background Two major hurdles for successful production of second-generation bioethanol are the presence of inhibitory compounds in lignocellulosic media, and the fact that Saccharomyces cerevisiae cannot naturally utilise pentoses. There are recombinant yeast strains that address both of these issues, but co-utilisation of glucose and xylose is still an issue that needs to be resolved. A non-recombinant way to increase yeast tolerance to hydrolysates is by encapsulation of the yeast. This can be explained by concentration gradients occuring in the cell pellet inside the capsule. In the current study, we hypothesised that encapsulation might also lead to improved simultaneous utilisation of hexoses and pentoses because of such sugar concentration gradients. Results In silico simulations of encapsulated yeast showed that the presence of concentration gradients of inhibitors can explain the improved inhibitor tolerance of encapsulated yeast. Simulations also showed pronounced concentration gradients of sugars, which resulted in simultaneous xylose and glucose consumption and a steady state xylose consumption rate up to 220-fold higher than that found in suspension culture. To validate the results experimentally, a xylose-utilising S. cerevisiae strain, CEN.PK XXX, was constructed and encapsulated in semi-permeable alginate-chitosan liquid core gel capsules. In defined media, encapsulation not only increased the tolerance of the yeast to inhibitors, but also promoted simultaneous utilisation of glucose and xylose. Encapsulation of the yeast resulted in consumption of at least 50% more xylose compared with suspended cells over 96-hour fermentations in medium containing both sugars. The higher consumption of xylose led to final ethanol titres that were approximately 15% higher. In an inhibitory dilute acid spruce hydrolysate, freely suspended yeast cells consumed the sugars in a sequential manner after a long lag phase, whereas no lag phase was observed for the

  7. Independent production of two molecular forms of a recombinant Rhizopus oryzae lipase by KEX2-engineered strains of Saccharomyces cerevisiae.

    PubMed

    Takahashi, S; Ueda, M; Tanaka, A

    1999-10-01

    A mixture of rProROL having the full-length prosequence (97 amino acids) for a recombinant lipase of Rhizopus oryzae (rROL) and r28ROL having 28 amino acids of the same prosequence has been produced as active forms by Saccharomyces cerevisiae [Takahashi et al. (1998) J Ferment Bioeng 86: 164-168]. However, the separation of rProROL and r28ROL has not been successful due to their identical behavior on column chromatographs, presumably because of the similarity of their surface properties. The independent production of two different molecular forms of rROL was carried out using KEX2-engineered strains of S. cerevisiae, since r28ROL was predicted to be a product from rProROL by a Kex2-like protease. rProROL was successfully obtained by expression of the ROL gene in the S. cerevisiae kex2 strain in which the KEX2 gene encoding Kex2p was disrupted, while r28ROL was obtained by co-expression of the gene (KEX2 delta 613) encoding the soluble form of the C-terminal truncated Kex2 protease (sKex2p). The specific lipase activities of rProROL and r28ROL were 92.9 U/mg and 140 U/mg, respectively. rProROL was stable at pH 2.2-8.0, and showed the optimal reaction temperature to be 30-35 degrees C with a T50 of 55 degrees C (T50 is the temperature resulting in 50% loss of activity). The values for r28ROL were pH 3.0-10.0, 25-30 degrees C, and 40 degrees C, respectively. rProROL was an N-linked glycosylated form, but r28ROL was not. The enhanced thermostability of rProROL did not seem to be due to the N-linked glycosylation, as judged by the results of the Endo H treatment. rProROL had the highest esterase activity toward p-nitrophenyl laurate (C12), whereas r28ROL had the highest esterase activity toward p-nitrophenyl caprylate (C8) and stearate (C18). These results suggest that the distinct properties of these two forms of lipase are caused by the different length of the ROL prosequence. PMID:10570801

  8. A Simple and Reliable Method for Hybridization of Homothallic Wine Strains of Saccharomyces cerevisiae

    PubMed Central

    Ramírez, Manuel; Peréz, Francisco; Regodón, José A.

    1998-01-01

    A procedure was developed for the hybridization and improvement of homothallic industrial wine yeasts. Killer cycloheximide-sensitive strains were crossed with killer-sensitive cycloheximide-resistant strains to get killer cycloheximide-resistant hybrids, thereby enabling hybrid selection and identification. This procedure also allows backcrossing of spore colonies from the hybrids with parental strains. PMID:9835605

  9. Robust cellulosic ethanol production from SPORL-pretreated lodgepole pine using an adapted strain Saccharomyces cerevisiae without detoxification.

    PubMed

    Tian, S; Luo, X L; Yang, X S; Zhu, J Y

    2010-11-01

    This study reports an ethanol yield of 270L/ton wood from lodgepole pine pretreated with sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) using an adapted strain, Saccharomyces cerevisiae Y5, without detoxification. The enzymatic hydrolysate produced from pretreated cellulosic solids substrate was combined with pretreatment hydrolysate before fermentation. Detoxification of the pretreatment hydrolysate using overliming or XAD-4 resin before being combined with enzymatic hydrolysate improved ethanol productivity in the first 4h of fermentation and overall fermentation efficiency. However, detoxification did not improve final ethanol yield because of sugar losses. The Y5 strain showed excellent ethanol productivities of 2.0 and 0.8g/L/h averaged over a period of 4 and 24h, respectively, in the undetoxified run. The furan metabolization rates of the Y5 strain were significantly higher for the undetoxified run than those for the detoxidfied runs, suggesting it can tolerate even higher furan concentrations than those studied. Preliminary mass and energy balances were conducted. SPORL produced an excellent monomeric sugar recovery value of about 85% theoretical and a net energy output of 4.05GJ/ton wood with an ethanol energy production efficiency of 178% before distillation. PMID:20620049

  10. A computational pipeline to discover highly phylogenetically informative genes in sequenced genomes: application to Saccharomyces cerevisiae natural strains.

    PubMed

    Ramazzotti, Matteo; Berná, Luisa; Stefanini, Irene; Cavalieri, Duccio

    2012-05-01

    The quest for genes representing genetic relationships of strains or individuals within populations and their evolutionary history is acquiring a novel dimension of complexity with the advancement of next-generation sequencing (NGS) technologies. In fact, sequencing an entire genome uncovers genetic variation in coding and non-coding regions and offers the possibility of studying Saccharomyces cerevisiae populations at the strain level. Nevertheless, the disadvantageous cost-benefit ratio (the amount of details disclosed by NGS against the time-expensive and expertise-demanding data assembly process) still precludes the application of these techniques to the routinely assignment of yeast strains, making the selection of the most reliable molecular markers greatly desirable. In this work we propose an original computational approach to discover genes that can be used as a descriptor of the population structure. We found 13 genes whose variability can be used to recapitulate the phylogeny obtained from genome-wide sequences. The same approach that we prove to be successful in yeasts can be generalized to any other population of individuals given the availability of high-quality genomic sequences and of a clear population structure to be targeted. PMID:22266652

  11. Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers

    PubMed Central

    Otero, Jose M.; Koetter, Peter; Nielsen, Jens; Ebizuka, Yutaka; Kushiro, Tetsuo; Panagiotou, Gianni

    2011-01-01

    Background Metabolic engineering is an attractive approach in order to improve the microbial production of drugs. Triterpenes is a chemically diverse class of compounds and many among them are of interest from a human health perspective. A systematic experimental or computational survey of all feasible gene modifications to determine the genotype yielding the optimal triterpene production phenotype is a laborious and time-consuming process. Methodology/Principal Findings Based on the recent genome-wide sequencing of Saccharomyces cerevisiae CEN.PK 113-7D and its phenotypic differences with the S288C strain, we implemented a strategy for the construction of a β-amyrin production platform. The genes Erg8, Erg9 and HFA1 contained non-silent SNPs that were computationally analyzed to evaluate the changes that cause in the respective protein structures. Subsequently, Erg8, Erg9 and HFA1 were correlated with the increased levels of ergosterol and fatty acids in CEN.PK 113-7D and single, double, and triple gene over-expression strains were constructed. Conclusions The six out of seven gene over-expression constructs had a considerable impact on both ergosterol and β-amyrin production. In the case of β-amyrin formation the triple over-expression construct exhibited a nearly 500% increase over the control strain making our metabolic engineering strategy the most successful design of triterpene microbial producers. PMID:21445244

  12. Xylulokinase Overexpression in Two Strains of Saccharomyces cerevisiae Also Expressing Xylose Reductase and Xylitol Dehydrogenase and Its Effect on Fermentation of Xylose and Lignocellulosic Hydrolysate

    PubMed Central

    Johansson, Björn; Christensson, Camilla; Hobley, Timothy; Hahn-Hägerdal, Bärbel

    2001-01-01

    Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae. PMID:11526030

  13. Novel starters for old processes: use of Saccharomyces cerevisiae strains isolated from artisanal sourdough for craft beer production at a brewery scale.

    PubMed

    Marongiu, Antonella; Zara, Giacomo; Legras, Jean-Luc; Del Caro, Alessandra; Mascia, Ilaria; Fadda, Costantino; Budroni, Marilena

    2015-01-01

    The deliberate inoculation of yeast strains isolated from food matrices such as wine or bread, could allow the transfer of novel properties to beer. In this work, the feasibility of the use of baker's yeast strains as starters for craft beer production has been evaluated at laboratory and brewery scale. Nine out of 12 Saccharomyces cerevisiae strains isolated from artisanal sourdoughs metabolized 2 % maltose, glucose and trehalose and showed growth rates and cell populations higher than those of the brewer's strain Safbrew-S33. Analysis of allelic variation at 12 microsatellite loci clustered seven baker's strains and Safbrew-S33 in the main group of bread isolates. Chemical analyses of beers produced at a brewery scale showed significant differences among the beers produced with the baker's strain S38 or Safbrew-S33, while no significant differences were observed when S38 or the brewer's strain Safbrew-F2 was used for re-fermentation. The sensory profile of beers obtained with S38 or the brewer's yeasts did not show significant differences, thus suggesting that baker's strains of S. cerevisiae could represent a reservoir of biodiversity for the selection of starter strains for craft beer production. PMID:25387611

  14. Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production

    PubMed Central

    2013-01-01

    Background Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance. Results Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse. Conclusions This study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and

  15. Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation

    PubMed Central

    Zhu, Yuan O.; Sherlock, Gavin; Petrov, Dmitri A.

    2016-01-01

    Budding yeast has undergone several independent transitions from commercial to clinical lifestyles. The frequency of such transitions suggests that clinical yeast strains are derived from environmentally available yeast populations, including commercial sources. However, despite their important role in adaptive evolution, the prevalence of polyploidy and aneuploidy has not been extensively analyzed in clinical strains. In this study, we have looked for patterns governing the transition to clinical invasion in the largest screen of clinical yeast isolates to date. In particular, we have focused on the hypothesis that ploidy changes have influenced adaptive processes. We sequenced 144 yeast strains, 132 of which are clinical isolates. We found pervasive large-scale genomic variation in both overall ploidy (34% of strains identified as 3n/4n) and individual chromosomal copy numbers (36% of strains identified as aneuploid). We also found evidence for the highly dynamic nature of yeast genomes, with 35 strains showing partial chromosomal copy number changes and eight strains showing multiple independent chromosomal events. Intriguingly, a lineage identified to be baker’s/commercial derived with a unique damaging mutation in NDC80 was particularly prone to polyploidy, with 83% of its members being triploid or tetraploid. Polyploidy was in turn associated with a >2× increase in aneuploidy rates as compared to other lineages. This dataset provides a rich source of information on the genomics of clinical yeast strains and highlights the potential importance of large-scale genomic copy variation in yeast adaptation. PMID:27317778

  16. Whole Genome Analysis of 132 Clinical Saccharomyces cerevisiae Strains Reveals Extensive Ploidy Variation.

    PubMed

    Zhu, Yuan O; Sherlock, Gavin; Petrov, Dmitri A

    2016-01-01

    Budding yeast has undergone several independent transitions from commercial to clinical lifestyles. The frequency of such transitions suggests that clinical yeast strains are derived from environmentally available yeast populations, including commercial sources. However, despite their important role in adaptive evolution, the prevalence of polyploidy and aneuploidy has not been extensively analyzed in clinical strains. In this study, we have looked for patterns governing the transition to clinical invasion in the largest screen of clinical yeast isolates to date. In particular, we have focused on the hypothesis that ploidy changes have influenced adaptive processes. We sequenced 144 yeast strains, 132 of which are clinical isolates. We found pervasive large-scale genomic variation in both overall ploidy (34% of strains identified as 3n/4n) and individual chromosomal copy numbers (36% of strains identified as aneuploid). We also found evidence for the highly dynamic nature of yeast genomes, with 35 strains showing partial chromosomal copy number changes and eight strains showing multiple independent chromosomal events. Intriguingly, a lineage identified to be baker's/commercial derived with a unique damaging mutation in NDC80 was particularly prone to polyploidy, with 83% of its members being triploid or tetraploid. Polyploidy was in turn associated with a >2× increase in aneuploidy rates as compared to other lineages. This dataset provides a rich source of information on the genomics of clinical yeast strains and highlights the potential importance of large-scale genomic copy variation in yeast adaptation. PMID:27317778

  17. Engineering industrial Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass conversion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces physiology and fermentation related properties vary broadly among industrial strains. In this study, six industrial strains of varied genetic background were engineered to ferment xylose. Aerobic growth rates on xylose were 0.040 h**-1 to 0.167 h**-1. Fermentation of xylose, glucose/xy...

  18. Rumen fermentation and acetogen population changes in response to an exogenous acetogen TWA4 strain and Saccharomyces cerevisiae fermentation product*

    PubMed Central

    Yang, Chun-lei; Guan, Le-luo; Liu, Jian-xin; Wang, Jia-kun

    2015-01-01

    The presence of yeast cells could stimulate hydrogen utilization of acetogens and enhance acetogenesis. To understand the roles of acetogens in rumen fermentation, an in vitro rumen fermentation experiment was conducted with addition of acetogen strain (TWA4) and/or Saccharomyces cerevisiae fermentation product (XP). A 2×2 factorial design with two levels of TWA4 (0 or 2×107 cells/ml) and XP (0 or 2 g/L) was performed. Volatile fatty acids (VFAs) were increased (P<0.05) in XP and TWA4XP, while methane was increased only in TWA4XP (P<0.05). The increase rate of microorganisms with formyltetrahydrofolate synthetase, especially acetogens, was higher than that of methanogens under all treatments. Lachnospiraceae was predominant in all acetogen communities, but without close acetyl-CoA synthase (ACS) amino acid sequences from cultured isolates. Low-Acetitomaculum ruminis-like ACS was predominant in all acetogen communities, while four unique phylotypes in XP treatment were all amino acid identified low-Eubacterium limosum-like acetogens. It differs to XP treatment that more low-A. ruminis-like and less low-E. limosum-like sequences were identified in TWA4 and TWA4XP treatments. Enhancing acetogenesis by supplementation with an acetogen strain and/or yeast cells may be an approach to mitigate methane, by targeting proper acetogens such as uncultured low-E. limosum-like acetogens. PMID:26238546

  19. Using mixed inocula of Saccharomyces cerevisiae killer strains to improve the quality of traditional sparkling-wine.

    PubMed

    Velázquez, Rocío; Zamora, Emiliano; Álvarez, Manuel; Álvarez, María L; Ramírez, Manuel

    2016-10-01

    The quality of traditional sparkling-wine depends on the aging process in the presence of dead yeast cells. These cells undergo a slow autolysis process thereby releasing some compounds, mostly colloidal polymers such as polysaccharides and mannoproteins, which influence the wine's foam properties and mouthfeel. Saccharomyces cerevisiae killer yeasts were tested to increase cell death and autolysis during mixed-yeast-inoculated second fermentation and aging. These yeasts killed sensitive strains in killer plate assays done under conditions of low pH and temperature similar to those used in sparkling-wine making, although some strains showed a different killer behaviour during the second fermentation. The fast killer effect improved the foam quality and mouthfeel of the mixed-inoculated wines, while the slow killer effect gave small improvements over single-inoculated wines. The effect was faster under high-pressure than under low-pressure conditions. Wine quality improvement did not correlate with the polysaccharide, protein, mannan, or aromatic compound concentrations, suggesting that the mouthfeel and foaming quality of sparkling wine are very complex properties influenced by other wine compounds and their interactions, as well as probably by the specific chemical composition of a given wine. PMID:27375256

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

    PubMed

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

    2011-06-30

    The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol. PMID:21531033

  1. Comparative Transcriptomic Analysis Reveals Similarities and Dissimilarities in Saccharomyces cerevisiae Wine Strains Response to Nitrogen Availability

    PubMed Central

    Barbosa, Catarina; García-Martínez, José; Pérez-Ortín, José E.; Mendes-Ferreira, Ana

    2015-01-01

    Nitrogen levels in grape-juices are of major importance in winemaking ensuring adequate yeast growth and fermentation performance. Here we used a comparative transcriptome analysis to uncover wine yeasts responses to nitrogen availability during fermentation. Gene expression was assessed in three genetically and phenotypically divergent commercial wine strains (CEG, VL1 and QA23), under low (67 mg/L) and high nitrogen (670 mg/L) regimes, at three time points during fermentation (12h, 24h and 96h). Two-way ANOVA analysis of each fermentation condition led to the identification of genes whose expression was dependent on strain, fermentation stage and on the interaction of both factors. The high fermenter yeast strain QA23 was more clearly distinct from the other two strains, by differential expression of genes involved in flocculation, mitochondrial functions, energy generation and protein folding and stabilization. For all strains, higher transcriptional variability due to fermentation stage was seen in the high nitrogen fermentations. A positive correlation between maximum fermentation rate and the expression of genes involved in stress response was observed. The finding of common genes correlated with both fermentation activity and nitrogen up-take underlies the role of nitrogen on yeast fermentative fitness. The comparative analysis of genes differentially expressed between both fermentation conditions at 12h, where the main difference was the level of nitrogen available, showed the highest variability amongst strains revealing strain-specific responses. Nevertheless, we were able to identify a small set of genes whose expression profiles can quantitatively assess the common response of the yeast strains to varying nitrogen conditions. The use of three contrasting yeast strains in gene expression analysis prompts the identification of more reliable, accurate and reproducible biomarkers that will facilitate the diagnosis of deficiency of this nutrient in the grape

  2. Two chromosomal genes required for killing expression in killer strains of Saccharomyces cerevisiae.

    PubMed

    Wickner, R B; Leibowitz, M J

    1976-03-25

    The killer character of yeast is determined by a 1.4 X 10(6) molecular weight double-stranded RNA plasmid and at least 12 chromosomal genes. Wild-type strains of yeast that carry this plasmid (killers) secret a toxin which is lethal only to strains not carrying this plasmid (sensitives).--We have isolated 28 independent recessive chromosomal mutants of a killer strain that have lost the ability to secrete an active toxin but remain resistant to the effects of the toxin and continue to carry the complete cytoplasmic killer genome. These mutants define two complementation groups, kex1 and kex2. Kex1 is located on chromosome VII between ade5 and lys5. Kex2 is located on chromosome XIV, but it does not show meiotic linkage to any gene previously located on this chromosome.--When the killer plasmid of kex1 or kex2 strains is eliminated by curing with heat or cycloheximide, the strains become sensitive to killing. The mutant phenotype reappears among the meiotic segregants in a cross with a normal killer. Thus, the kex phenotype does not require an alteration of the killer plasmid.--Kex1 and kex2 strains each contain near-normal levels of the 1.4 x 10(6) molecular weight double-stranded RNA, whose presence is correlated with the presence of the killer genome. PMID:773743

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

    PubMed

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

    2016-02-01

    Wine fermentation and innovation have focused mostly on Saccharomyces cerevisiae strains. However, recent studies have shown that other Saccharomyces species can also be involved in wine fermentation or are useful for wine bouquet, such as Saccharomyces uvarum and Saccharomyces paradoxus. Many interspecies hybrids have also been isolated from wine fermentation, such as S. cerevisiae × Saccharomyces kudriavzevii hybrids. In this study, we explored the genetic diversity and fermentation performance of Spanish S. kudriavzevii strains, which we compared to other S. kudriavzevii strains. Fermentations of red and white grape musts were performed, and the phenotypic differences between Spanish S. kudriavzevii strains under different temperature conditions were examined. An ANOVA analysis suggested striking similarity between strains for glycerol and ethanol production, although a high diversity of aromatic profiles among fermentations was found. The sources of these phenotypic differences are not well understood and require further investigation. Although the Spanish S. kudriavzevii strains showed desirable properties, particularly must fermentations, the quality of their wines was no better than those produced with a commercial S. cerevisiae. We suggest hybridization or directed evolution as methods to improve and innovate wine. PMID:26678127

  4. Synergistic effects of TAL1 over-expression and PHO13 deletion on the weak acid inhibition of xylose fermentation by industrial Saccharomyces cerevisiae strain.

    PubMed

    Li, Yun-Cheng; Gou, Zi-Xi; Liu, Ze-Shen; Tang, Yue-Qin; Akamatsu, Takashi; Kida, Kenji

    2014-10-01

    In the industrial production of bioethanol from lignocellulosic biomass, a strain of Saccharomyces cerevisiae that can ferment xylose in the presence of inhibitors is of utmost importance. The recombinant, industrial-flocculating S. cerevisiae strain NAPX37, which can ferment xylose, was used as the parent to delete the gene encoding p-nitrophenylphosphatase (PHO13) and overexpress the gene encoding transaldolase (TAL1) to evaluate the synergistic effects of these two genes on xylose fermentation in the presence of weak acid inhibitors, including formic, acetic, or levulinic acids. TAL1 over-expression or PHO13 deletion improved xylose fermentation as well as the tolerance of NAPX37 to all three weak acids. The simultaneous deletion of PHO13 and the over-expression of TAL1 had synergistic effects and improved ethanol production and reduction of xylitol accumulation in the absence and presence of weak acid inhibitors. PMID:24966040

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

    PubMed Central

    Engel, Stacia R.; Cherry, J. Michael

    2013-01-01

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

  6. Draft Genome Sequence of the Probiotic Yeast Saccharomyces cerevisiae var. boulardii Strain ATCC MYA-796

    PubMed Central

    Marques, E. T. A.; Franco, G. R.

    2014-01-01

    Saccharomyces boulardii is the only yeast approved as a probiotic for human consumption. Here, we report the draft genome sequence of the strain ATCC MYA-796, derived from the French Ultra Levure probiotic drug. The genome has a size of 11.6 Mb with 5,305 putative open reading frames predicted. PMID:25523784

  7. Comparisons of five Saccharomyces cerevisiae strains for ethanol production from SPORL pretreated lodgepole pine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The performances of 5 yeast strains under three levels of toxicity were evaluated using hydrolysates from lodgepole pine pretreated by Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL). The highest level of toxicity was represented by the whole pretreated biomass slurry, ...

  8. Population Structure and Comparative Genome Hybridization of European Flor Yeast Reveal a Unique Group of Saccharomyces cerevisiae Strains with Few Gene Duplications in Their Genome

    PubMed Central

    Legras, Jean-Luc; Erny, Claude; Charpentier, Claudine

    2014-01-01

    Wine biological aging is a wine making process used to produce specific beverages in several countries in Europe, including Spain, Italy, France, and Hungary. This process involves the formation of a velum at the surface of the wine. Here, we present the first large scale comparison of all European flor strains involved in this process. We inferred the population structure of these European flor strains from their microsatellite genotype diversity and analyzed their ploidy. We show that almost all of these flor strains belong to the same cluster and are diploid, except for a few Spanish strains. Comparison of the array hybridization profile of six flor strains originating from these four countries, with that of three wine strains did not reveal any large segmental amplification. Nonetheless, some genes, including YKL221W/MCH2 and YKL222C, were amplified in the genome of four out of six flor strains. Finally, we correlated ICR1 ncRNA and FLO11 polymorphisms with flor yeast population structure, and associate the presence of wild type ICR1 and a long Flo11p with thin velum formation in a cluster of Jura strains. These results provide new insight into the diversity of flor yeast and show that combinations of different adaptive changes can lead to an increase of hydrophobicity and affect velum formation. PMID:25272156

  9. Short-term response of different Saccharomyces cerevisiae strains to hyperosmotic stress caused by inoculation in grape must: RT-qPCR study and metabolite analysis.

    PubMed

    Noti, Olta; Vaudano, Enrico; Pessione, Enrica; Garcia-Moruno, Emilia

    2015-12-01

    During the winemaking process, glycerol synthesis represents the first adaption response of Saccharomyces cerevisiae to osmotic stress after inoculation in grape must. We have implemented an RT-qPCR (Reverse Transcription-quantitative PCR) methodology with a preventive evaluation of candidate reference genes, to study six target genes related to glycerol synthesis (GPD1, GPD2, GPP2 and GPP1) and flux (STL1 and FPS1), and three ALD genes coding for aldehyde dehydrogenase involved in redox equilibrium via acetate production. The mRNA level in three strains, characterized by different metabolite production, was monitored in the first 120 min from inoculation into natural grape must. Expression analysis shows a transient response of genes GPD1, GPD2, GPP2, GPP1 and STL1 with differences among strains in term of mRNA abundance, while FPS1 was expressed constitutively. The transient response and different expression intensity among strains, in relation to the intracellular glycerol accumulation pattern, prove the negative feedback control via the HOG (High Osmolarity Glycerol) signalling pathway in S. cerevisiae wine strains under winery conditions. Among the ALD genes, only ALD6 was moderately induced in the hyperosmotic environment but not in all strains tested, while ALD3 and ALD4 were drastically glucose repressed. The intensity of transcription of ALD6 and ALD3 seems to be related to different acetate production found among the strains. PMID:26338116

  10. [Ethanol fermentation from Jerusalem artichoke tubers by a genetically-modified Saccharomyces cerevisiae strain capable of secreting inulinase].

    PubMed

    Li, Nannan; Yuan, Wenjie; Wang, Na; Xin, Chengxun; Ge, Xumeng; Bai, Fengwu

    2011-07-01

    Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value. PMID:22016987

  11. Engineering of a Nepetalactol-Producing Platform Strain of Saccharomyces cerevisiae for the Production of Plant Seco-Iridoids.

    PubMed

    Campbell, Alex; Bauchart, Philippe; Gold, Nicholas D; Zhu, Yun; De Luca, Vincenzo; Martin, Vincent J J

    2016-05-20

    The monoterpene indole alkaloids (MIAs) are a valuable family of chemicals that include the anticancer drugs vinblastine and vincristine. These compounds are of global significance-appearing on the World Health Organization's list of model essential medicines-but remain exorbitantly priced due to low in planta levels. Chemical synthesis and genetic manipulation of MIA producing plants such as Catharanthus roseus have so far failed to find a solution to this problem. Synthetic biology holds a potential answer, by building the pathway into more tractable organisms such as Saccharomyces cerevisiae. Recent work has taken the first steps in this direction by producing small amounts of the intermediate strictosidine in yeast. In order to help improve on these titers, we aimed to optimize the early biosynthetic steps of the MIA pathway to the metabolite nepetalactol. We combined a number of strategies to create a base strain producing 11.4 mg/L of the precursor geraniol. We also show production of the critical intermediate 10-hydroxygeraniol and demonstrate nepetalactol production in vitro. Lastly we demonstrate that activity of the iridoid synthase toward the intermediates geraniol and 10-hydroxygeraniol results in the synthesis of the nonproductive intermediates citronellol and 10-hydroxycitronellol. This discovery has serious implications for the reconstruction of the MIA in heterologous organisms. PMID:26981892

  12. Combining inhibitor tolerance and D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production

    PubMed Central

    2013-01-01

    Background In addition to efficient pentose utilization, high inhibitor tolerance is a key trait required in any organism used for economically viable industrial bioethanol production with lignocellulose biomass. Although recent work has succeeded in establishing efficient xylose fermentation in robust industrial Saccharomyces cerevisiae strains, the resulting strains still lacked sufficient inhibitor tolerance for efficient sugar fermentation in lignocellulose hydrolysates. The aim of the present work was to combine high xylose fermentation activity and high inhibitor tolerance in a single industrial yeast strain. Results We have screened 580 yeast strains for high inhibitor tolerance using undetoxified acid-pretreated spruce hydrolysate and identified a triploid industrial baker’s yeast strain as having the highest inhibitor tolerance. From this strain, a mating competent diploid segregant with even higher inhibitor tolerance was obtained. It was crossed with the recently developed D-xylose fermenting diploid industrial strain GS1.11-26, with the Ethanol Red genetic background. Screening of 819 diploid segregants from the tetraploid hybrid resulted in two strains, GSF335 and GSF767, combining high inhibitor tolerance and efficient xylose fermentation. In a parallel approach, meiotic recombination of GS1.11-26 with a haploid segregant of Ethanol Red and screening of 104 segregants resulted in a similar inhibitor tolerant diploid strain, GSE16. The three superior strains exhibited significantly improved tolerance to inhibitors in spruce hydrolysate, higher glucose consumption rates, higher aerobic growth rates and higher maximal ethanol accumulation capacity in very-high gravity fermentation, compared to GS1.11-26. In complex medium, the D-xylose utilization rate by the three superior strains ranged from 0.36 to 0.67 g/g DW/h, which was lower than that of GS1.11-26 (1.10 g/g DW/h). On the other hand, in batch fermentation of undetoxified acid-pretreated spruce

  13. Saccharomyces Cerevisiae Rad52 Alleles Temperature-Sensitive for the Repair of DNA Double-Strand Breaks

    PubMed Central

    Kaytor, M. D.; Livingston, D. M.

    1994-01-01

    We have screened for mutations of the Saccharomyces cerevisiae RAD52 gene which confer a temperature-sensitive (ts) phenotype with respect to either the repair of DNA lesions caused by methyl methanesulfonate (MMS) or the recombination of an intrachromosomal recombination reporter. We were readily able to isolate alleles ts for the repair of lesions caused by MMS but were unable to find alleles with a severe ts deficiency in intrachromosomal recombination. We extensively characterized four strains conferring ts growth on MMS agar. These strains also exhibit ts survival when exposed to γ-radiation or when the HO endonuclease is constitutively expressed. Although none of the four alleles confers a severe ts defect in intrachromosomal recombination, two confer significant defects in tests of mitotic, interchromosomal recombination carried out in diploid strains. The mutant diploids sporulate, but the two strains with defects in interchromosomal recombination have reduced spore viability. Meiotic recombination is not depressed in the two diploids with reduced spore viability. Thus, in the two strains with reduced spore viability, defects in mitotic and meiotic recombination do not correlate. Sequence analysis revealed that in three of the four ts alleles the causative mutations are in the first one-third of the open reading frame while the fourth is in the C-terminal third. PMID:7982574

  14. Enhanced 3-Sulfanylhexan-1-ol Production in Sequential Mixed Fermentation with Torulaspora delbrueckii/Saccharomyces cerevisiae Reveals a Situation of Synergistic Interaction between Two Industrial Strains

    PubMed Central

    Renault, Philippe; Coulon, Joana; Moine, Virginie; Thibon, Cécile; Bely, Marina

    2016-01-01

    The aim of this work was to study the volatile thiol productions of two industrial strains of Torulaspora delbrueckii and Saccharomyces cerevisiae during alcoholic fermentation (AF) of Sauvignon Blanc must. In order to evaluate the influence of the inoculation procedure, sequential and simultaneous mixed cultures were carried out and compared to pure cultures of T. delbrueckii and S. cerevisiae. The results confirmed the inability of T. delbrueckii to release 4-methyl-4-sulfanylpentan-2-one (4MSP) and its low capacity to produce 3-sulfanylhexyl acetate (3SHA), as already reported in previous studies. A synergistic interaction was observed between the two species, resulting in higher levels of 3SH (3-sulfanylhexan-1-ol) and its acetate when S. cerevisiae was inoculated 24 h after T. delbrueckii, compared to the pure cultures. To elucidate the nature of the interactions between these two species, the yeast population kinetics were examined and monitored, as well as the production of 3SH, its acetate and their related non-odorous precursors: Glut-3SH (glutathionylated conjugate precursor) and Cys-3SH (cysteinylated conjugate precursor). For the first time, it was suggested that, unlike S. cerevisiae, which is able to metabolize the two precursor forms, T. delbrueckii was only able to metabolize the glutathionylated precursor. Consequently, the presence of T. delbrueckii during mixed fermentation led to an increase in Glut-3SH degradation and Cys-3SH production. This overproduction was dependent on the T. delbrueckii biomass. In sequential culture, thus favoring T. delbrueckii development, the higher availability of Cys-3SH throughout AF resulted in more abundant 3SH and 3SHA production by S. cerevisiae. PMID:27014216

  15. Enhanced 3-Sulfanylhexan-1-ol Production in Sequential Mixed Fermentation with Torulaspora delbrueckii/Saccharomyces cerevisiae Reveals a Situation of Synergistic Interaction between Two Industrial Strains.

    PubMed

    Renault, Philippe; Coulon, Joana; Moine, Virginie; Thibon, Cécile; Bely, Marina

    2016-01-01

    The aim of this work was to study the volatile thiol productions of two industrial strains of Torulaspora delbrueckii and Saccharomyces cerevisiae during alcoholic fermentation (AF) of Sauvignon Blanc must. In order to evaluate the influence of the inoculation procedure, sequential and simultaneous mixed cultures were carried out and compared to pure cultures of T. delbrueckii and S. cerevisiae. The results confirmed the inability of T. delbrueckii to release 4-methyl-4-sulfanylpentan-2-one (4MSP) and its low capacity to produce 3-sulfanylhexyl acetate (3SHA), as already reported in previous studies. A synergistic interaction was observed between the two species, resulting in higher levels of 3SH (3-sulfanylhexan-1-ol) and its acetate when S. cerevisiae was inoculated 24 h after T. delbrueckii, compared to the pure cultures. To elucidate the nature of the interactions between these two species, the yeast population kinetics were examined and monitored, as well as the production of 3SH, its acetate and their related non-odorous precursors: Glut-3SH (glutathionylated conjugate precursor) and Cys-3SH (cysteinylated conjugate precursor). For the first time, it was suggested that, unlike S. cerevisiae, which is able to metabolize the two precursor forms, T. delbrueckii was only able to metabolize the glutathionylated precursor. Consequently, the presence of T. delbrueckii during mixed fermentation led to an increase in Glut-3SH degradation and Cys-3SH production. This overproduction was dependent on the T. delbrueckii biomass. In sequential culture, thus favoring T. delbrueckii development, the higher availability of Cys-3SH throughout AF resulted in more abundant 3SH and 3SHA production by S. cerevisiae. PMID:27014216

  16. An event-specific method for the detection and quantification of ML01, a genetically modified Saccharomyces cerevisiae wine strain, using quantitative PCR.

    PubMed

    Vaudano, Enrico; Costantini, Antonella; Garcia-Moruno, Emilia

    2016-10-01

    The availability of genetically modified (GM) yeasts for winemaking and, in particular, transgenic strains based on the integration of genetic constructs deriving from other organisms into the genome of Saccharomyces cerevisiae, has been a reality for several years. Despite this, their use is only authorized in a few countries and limited to two strains: ML01, able to convert malic acid into lactic acid during alcoholic fermentation, and ECMo01 suitable for reducing the risk of carbamate production. In this work we propose a quali-quantitative culture-independent method for the detection of GM yeast ML01 in commercial preparations of ADY (Active Dry Yeast) consisting of efficient extraction of DNA and qPCR (quantitative PCR) analysis based on event-specific assay targeting MLC (malolactic cassette), and a taxon-specific S. cerevisiae assay detecting the MRP2 gene. The ADY DNA extraction methodology has been shown to provide good purity DNA suitable for subsequent qPCR. The MLC and MRP2 qPCR assay showed characteristics of specificity, dynamic range, limit of quantification (LOQ) limit of detection (LOD), precision and trueness, which were fully compliant with international reference guidelines. The method has been shown to reliably detect 0.005% (mass/mass) of GM ML01 S. cerevisiae in commercial preparations of ADY. PMID:27367966

  17. Alcoholic chestnut fermentation in mixed culture. Compatibility criteria between Aspergillus oryzae and Saccharomyces cerevisiae strains.

    PubMed

    Murado, Miguel Anxo; Pastrana, Lorenzo; Vázquez, José Antonio; Mirón, Jesús; González, María Pilar

    2008-10-01

    The main objective of the present work consisted in the transfer to the case of the chestnut of a rice fermentative process that carried out to the Japanese traditional way to lead to an alcoholic bagasse, the moromi, capable of obtaining distilled. This way, selection assays of amylolitic Aspergillus oryzae strains and studies of compatibility between microfungi and yeast were carried out. These mixed cultivations were performed operating in batch submerged culture. Later on, using solid state system (chestnut, microfungi, yeast), a fermentative fed-batch process (koji, moto, moromi) was defined. By means of this approach a yield of 70% was reached in the conversion of total carbohydrates in ethanol. Also, the time required by the traditional operation was reduced in half. PMID:18289846

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii

    PubMed Central

    Sasaki, Takashi; Ohshima, Yoshinobu

    1987-01-01

    The yeast Torulaspora delbrueckii, which propagates as a haploid, was made into a diploid by treatment with dimethyl sulfoxide (DMSO) on the regeneration of protoplasts. The diploid state was stably inherited; the cell volume was three times that of the parent strain and the cellular DNA content was two times that of the parental strain. No essential difference was found between diploids induced by DMSO and those formed through intraspecific protoplast fusion. The diploid strains sporulated fairly well, with their cells converting directly into asci. Random spore analysis revealed that diploids induced through protoplast fusion gave rise to auxotrophic segregants (haploids) with the parental genetic marker or to segregants formed by recombination, while diploids induced by DMSO from a doubly auxotrophic parent gave rise to no recombinant, indicating that it was chromosomally homoallelic in nature. The magnesium level in the protoplast regeneration medium was found to be an important factor for inducing diploid formation. At 0.2 mM magnesium diploids appeared even in the absence of DMSO, while at 2 mM magnesium diploids never appeared unless DMSO was added to the regeneration medium. Evidence is provided that the diploids induced by DMSO or a low magnesium level are due to direct diploidization but not protoplast fusion. UV light irradiation of intact cells (without protoplasts), 10% of which survived, also produced diploids among this surviving population. From these results we conclude that the perturbation of protoplast regeneration or of cell division by the treatments mentioned above somehow induced direct diploidization of T. delbrueckii. Images PMID:16347380

  20. Characterization of the Saccharomyces cerevisiae sec6-4 mutation and tools to create S. cerevisiae strains containing the sec6-4 allele.

    PubMed

    Lamping, Erwin; Tanabe, Koichi; Niimi, Masakazu; Uehara, Yoshimasa; Monk, Brian C; Cannon, Richard D

    2005-11-21

    The highly conserved exocyst complex of eukaryotic cells allows the polarized transport and fusion of late secretory vesicles with the plasma membrane. In Saccharomyces cerevisiae the Sec6p component of the exocyst complex is essential for cell growth. The sec6-4 temperature-sensitive mutation of the S. cerevisiae SEC6 gene leads to the accumulation of large amounts of mature late post-Golgi secretory vesicles in the cytosol of mutant cells at the restrictive temperature of 37 degrees C. These readily isolated, inside-out and tightly sealed vesicles contain mature post-translationally modified plasma membrane and secretory proteins and provide a valuable tool for the study of plasma membrane protein function. This study shows that the single point mutation L633P in the SEC6 coding region defines the sec6-4 phenotype. We followed the localization of the wild type Sec6p and the mutant Sec6-4p proteins (C-terminally tagged with the green fluorescent protein yEGfp3p) in the presence or absence of heterologously over-expressed Candida albicans plasma membrane ATP-binding cassette (ABC) transporter CaCdr1p (C-terminally tagged with the red fluorescent protein mRfp1p). The Sec6-4p protein localized to buds and septa, like wild type Sec6p, at the permissive temperature of 23 degrees C and the sec6-4 mutant cells grew at the same rate as the wild type control cells. Sec6-4p was mislocalized at the restrictive temperature of 37 degrees C and heterogenous vesicles accumulated in cells but sec6-4 cells also accumulated homogenous secretory vesicles at the permissive temperature. PMID:16185821

  1. Saccharomyces cerevisiae strain improvement using selection, mutation, and adaptation for the resistance to lignocellulose-derived fermentation inhibitor for ethanol production.

    PubMed

    Jang, Youri; Lim, Younghoon; Kim, Keun

    2014-05-01

    Twenty-five Saccharomyces cerevisiae strains were screened for the highest sugar tolerance, ethanol-tolerance, ethanol production, and inhibitor resistance, and S. cerevisiae KL5 was selected as the best strain. Inhibitor cocktail (100%) was composed of 75 mM formic acid, 75 mM acetic acid, 30 mM furfural, 30 mM hydroxymethyl furfural (HMF), and 2.7 mM vanillin. The cells of strain KL5 were treated with γ-irradiation, and among the survivals, KL5- G2 with improved inhibitor resistance and the highest ethanol yield in the presence of inhibitor cocktail was selected. The KL5-G2 strain was adapted to inhibitor cocktail by sequential transfer of cultures to a minimal YNB medium containing increasing concentrations of inhibitor cocktail. After 10 times of adaptation, most of the isolated colonies could grow in YNB with 80% inhibitor cocktail, whereas the parental KL5 strain could not grow at all. Among the various adapted strains, the best strain (KL5-G2-A9) producing the highest ethanol yield in the presence of inhibitor cocktail was selected. In a complex YP medium containing 60% inhibitor cocktail and 5% glucose, the theoretical yield and productivity (at 48 h) of KL5- G2-A9 were 81.3% and 0.304 g/l/h, respectively, whereas those of KL5 were 20.8% and 0.072 g/l/h, respectively. KL5-G2-A9 reduced the concentrations of HMF, furfural, and vanillin in the medium in much faster rates than KL5. PMID:24608567

  2. Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three generations of recombinant Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    Background Lignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2. Results With all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50

  3. Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity

    SciTech Connect

    Flikweert, M.T.; Kuyper, M.; Maris, A.J.A. van; Koetter, P.; Dijken, J.P. van; Pronk, J.T.

    1999-07-01

    Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN.PK113-7D. This caused a 3--4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h{sup {minus}1}. Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2{Delta} strain, formation of ethanol and acetate was reduced by 60--70%. In contrast to the wild type, the pdc2{Delta} strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2{Delta} cultures was about 40% lower than in wild-type cultures.

  4. Effects of pH and temperature on growth and glycerol production kinetics of two indigenous wine strains of Saccharomyces cerevisiae from Turkey

    PubMed Central

    Yalcin, Seda Karasu; Yesim Ozbas, Z.

    2008-01-01

    The study was performed in a batch system in order to determine the effects of pH and temperature on growth and glycerol production kinetics of two indigenous wine yeast strains Saccharomyces cerevisiae Kalecik 1 and Narince 3. The highest values of dry mass and specific growth rate were obtained at pH 4.00 for both of the strains. Maximum specific glycerol production rates were obtained at pH 5.92 and 6.27 for the strains Kalecik 1 and Narince 3, respectively. Kalecik 1 strain produced maximum 8.8 gL−1 of glycerol at pH 6.46. Maximum glycerol concentration obtained by the strain Narince 3 was 9.1 gL−1 at pH 6.48. Both yeasts reached maximum specific growth rate at 30°C. Optimum temperature range for glycerol production was determined as 25-30°C for the strain Kalecik 1. The strain Narince 3 reached maximum specific glycerol production rate at 30°C. Maximum glycerol concentrations at 30°C were obtained as 8.5 and 7.6 gL−1 for Kalecik 1 and Narince 3, respectively. PMID:24031225

  5. Anaerobic and sequential aerobic production of high-titer ethanol and single cell protein from NaOH-pretreated corn stover by a genome shuffling-modified Saccharomyces cerevisiae strain.

    PubMed

    Ren, Xueliang; Wang, Juncong; Yu, Hui; Peng, Chunlan; Hu, Jinlong; Ruan, Zhiyong; Zhao, Shumiao; Liang, Yunxiang; Peng, Nan

    2016-10-01

    In this study, a Saccharomyces cerevisiae recombinant strain 14 was constructed through genome shuffling method by transferring the whole genomic DNA of Candida intermedia strain 23 into a thermo-tolerant S. cerevisiae strain. The recombinant strain 14 combined the good natures of both parent strains that efficiently produced ethanol from glucose and single cell protein from xylose with 54.6% crude protein and all essential amino acids except cysteine at 35°C. Importantly, the recombinant strain 14 produced 64.07g/L ethanol from 25%(w/v) NaOH-pretreated and washed corn stover with the ethanol yield of 0.26g/g total stover by fed-batch simultaneous saccharification and fermentation and produced 66.50g/L dry cell mass subsequently from the residual hydrolysate and ethanol. Therefore, this study represents a feasible method to comprehensively utilize hexose and pentose in lignocellulosic materials. PMID:27416512

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Evaluation of engineered xylose-fermenting industrial strains of Saccharomyces cerevisiae for improved ethanol production from lignocellulosic feedstocks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. Hemicellulose can make up to 20-30% of biomass and is primarily composed of xylose. Enzymes from native xylose-assimi...

  8. Biochemical and Molecular Characterization of Saccharomyces cerevisiae Strains Obtained from Sugar-Cane Juice Fermentations and Their Impact in Cachaça Production▿

    PubMed Central

    Oliveira, Valdinéia Aparecida; Vicente, Maristela Araújo; Fietto, Luciano Gomes; de Miranda Castro, Ieso; Coutrim, Maurício Xavier; Schüller, Dorit; Alves, Henrique; Casal, Margarida; de Oliveira Santos, Juliana; Araújo, Leandro Dias; da Silva, Paulo Henrique Alves; Brandão, Rogelio Lopes

    2008-01-01

    Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H2S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5′,5"-trifluor-d,l-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard. PMID:18065624

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

    PubMed Central

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

    2001-01-01

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

  10. Identification of yeasts isolated from raffia wine (Raphia hookeri) produced in Côte d'Ivoire and genotyping of Saccharomyces cerevisiae strains by PCR inter-delta.

    PubMed

    Tra Bi, Charles Y; N'guessan, Florent K; Kouakou, Clémentine A; Jacques, Noemie; Casaregola, Serge; Djè, Marcellin K

    2016-08-01

    Raffia wine is a traditional alcoholic beverage produced in several African countries where it plays a significant role in traditional customs and population diet. Alcoholic fermentation of this beverage is ensured by a complex natural yeast flora which plays a decisive role in the quality of the final product. This present study aims to evaluate the distribution and the diversity of the yeast strains isolated in raffia wine from four sampling areas (Abengourou, Alépé, Grand-Lahou and Adzopé) in Côte d'Ivoire. Based on the D1/D2 domain of the LSU rDNA sequence analysis, nine species belonging to six genera were distinguished. With a percentage of 69.5 % out of 171 yeast isolates, Saccharomyces cerevisiae was the predominant species in the raffia wine, followed by Kodamaea ohmeri (20.4 %). The other species isolated were Candida haemulonii (4.1 %), Candida phangngensis (1.8 %), Pichia kudriavzevii (1.2 %), Hanseniaspora jakobsenii (1.2 %), Candida silvae (0.6 %), Hanseniaspora guilliermondii (0.6 %) and Meyerozyma caribbica (0.6 %). The molecular characterization of S. cerevisiae isolates at the strain level using the PCR-interdelta method revealed the presence of 21 profiles (named I to XXI) within 115 isolates. Only four profiles (I, III, V and XI) were shared by the four areas under study. Phenotypic characterization of K. ohmeri strains showed two subgroups for sugar fermentation and no diversity for the nitrogen compound assimilations and the growth at different temperatures. PMID:27339306

  11. Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

    PubMed

    Pratter, S M; Eixelsberger, T; Nidetzky, B

    2015-12-01

    A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. PMID:26452180

  12. Conversion of starch to ethanol in a recombinant saccharomyces cerevisiae strain expressing rice [alpha]-amylase from a novel Pichia pastoris alcohol oxidase promoter

    SciTech Connect

    Kumagai, M.H.; Sverlow, G.G.; della-Cioppa, G.; Grill, L.K. )

    1993-05-01

    A recombinant Saccharomyces cerevisiae, expressing and secreting rice [alpha]-amylase, converts starch to ethanol. The rice [alpha]-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N[sub 3]-GCTTCCAA-N[sub 5]-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida biodinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch. 45 refs., 8 figs.

  13. Comparative analysis of the effects of locally used herbicides and their active ingredients on a wild-type wine Saccharomyces cerevisiae strain.

    PubMed

    Braconi, Daniela; Sotgiu, Michele; Millucci, Lia; Paffetti, Alessandro; Tasso, Flavia; Alisi, Chiara; Martini, Silvia; Rappuoli, Roberto; Lusini, Paola; Sprocati, Anna Rosa; Rossi, Claudio; Santucci, Annalisa

    2006-04-19

    Herbicides are released to the environment with potential ecotoxicological risks for mammals. Yeast is a good model to elucidate toxicity mechanisms. We investigated how three commercial herbicides (Proper Energy, Pointer, and Silglif) and their active ingredients (respectively, fenoxaprop-P-ethyl, tribenuron methyl, and glyphosate) can affect biological activities of an oenological Saccharomyces cerevisiae strain, which may be resident on grape vineyards of the same geographical areas where herbicides are used. The use of commercial grade herbicides employed in Italy allowed us to reproduce the same conditions applied in crops; at the same time, assaying pure single active compounds made it possible to compare the effects obtained with commercial formulations. Interestingly, we found that while pure active compounds affect cell growth and metabolism at a lower extent, commercial preparations have a significant major negative influence on yeast biology. PMID:16608247

  14. Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development.

    PubMed Central

    Gimeno, C J; Fink, G R

    1994-01-01

    When starved for nitrogen, MATa/MAT alpha cells of the budding yeast Saccharomyces cerevisiae undergo a dimorphic transition to pseudohyphal growth. A visual genetic screen, called PHD (pseudohyphal determinant), for S. cerevisiae pseudohyphal growth mutants was developed. The PHD screen was used to identify seven S. cerevisiae genes that when overexpressed in MATa/MAT alpha cells growing on nitrogen starvation medium cause precocious and unusually vigorous pseudohyphal growth. PHD1, a gene whose overexpression induced invasive pseudohyphal growth on a nutritionally rich medium, was characterized. PHD1 maps to chromosome XI and is predicted to encode a 366-amino-acid protein. PHD1 has a SWI4- and MBP1-like DNA binding motif that is 73% identical over 100 amino acids to a region of Aspergillus nidulans StuA. StuA regulates two pseudohyphal growth-like cell divisions during conidiophore morphogenesis. Epitope-tagged PHD1 was localized to the nucleus by indirect immunofluorescence. These facts suggest that PHD1 may function as a transcriptional regulatory protein. Overexpression of PHD1 in wild-type haploid strains does not induce pseudohyphal growth. Interestingly, PHD1 overexpression enhances pseudohyphal growth in a haploid strain that has the diploid polar budding pattern because of a mutation in the BUD4 gene. In addition, wild-type diploid strains lacking PHD1 undergo pseudohyphal growth when starved for nitrogen. The possible functions of PHD1 in pseudohyphal growth and the uses of the PHD screen to identify morphogenetic regulatory genes from heterologous organisms are discussed. Images PMID:8114741

  15. Yeast ecology of vineyards within Marsala wine area (western Sicily) in two consecutive vintages and selection of autochthonous Saccharomyces cerevisiae strains.

    PubMed

    Settanni, Luca; Sannino, Ciro; Francesca, Nicola; Guarcello, Rosa; Moschetti, Giancarlo

    2012-12-01

    In this work, the yeast ecology associated with the spontaneous fermentation of Grillo cultivar grapes from 10 vineyards was analyzed from grape harvest till complete consumption of must sugars. The microbiological investigation started with the plate count onto two culture media to distinguish total yeasts (TY) and presumptive Saccharomyces (PS). Yeasts were randomly isolated and identified by a combined genotypic approach consisting of restriction fragment length polymorphism (RFLP) of 5.8S rRNA gene and 26S rRNA and sequencing of D1/D2 domain of the 26S rRNA gene, which resulted in the recognition of 14 species belonging to 10 genera. The distribution of the yeasts within the vineyards showed some differences in species composition and concentration levels among 2008 and 2009 vintages. Due to the enological relevance, all Saccharomyces cerevisiae isolates were differentiated applying two genotypic tools (interdelta analysis and microsatellite multiplex PCR of polymorphic microsatellite loci) that recognized 51 strains. Based on the low production of H(2)S, acetic acid and foam, ethanol resistance, growth in presence of high concentrations of potassium metabisulphite (KMBS) and CuSO(4) and at low temperatures, 14 strains were selected and used as starter to ferment grape must at 13 °C and 17 °C in presence of 100 mg/L of KMBS. Three strains (CS160, CS165 and CS182) showed optimal technological aptitudes. PMID:22877686

  16. The Improvement of SAM Accumulation by Integrating the Endogenous Methionine Adenosyltransferase Gene SAM2 in Genome of the Industrial Saccharomyces cerevisiae Strain.

    PubMed

    Zhao, Weijun; Shi, Feng; Hang, Baojian; Huang, Lei; Cai, Jin; Xu, Zhinan

    2016-03-01

    S-Adenosyl-L-methionine (SAM) plays important roles in trans-methylation, trans-sulfuration, and polyamine synthesis in all living cells, and it is also an effective cure for liver disease, depressive syndromes, and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, a multiple-copy integrative plasmid pYMIKP-SAM2 was introduced into the chromosome of wild-type Saccharomyces cerevisiae strain ZJU001 to construct the recombined strain R1-ZJU001. Further studies showed that the recombinant yeast exhibited higher enzymatic activity of methionine adenosyltransferase and improved its SAM biosynthesis. With a three-phase fed-batch strategy in 15-liter bench-top fermentor, 8.81 g/L SAM was achieved after 52 h cultivation of R1-ZJU001, about 27.1 % increase over its parent strain ZJU001, whereas the SAM content was also improved from 64.6 mg/g DCW to 91.0 mg/g DCW. Our results shall provide insights into the metabolic engineering of SAM pathway in yeast for improved productivity of SAM and subsequent industrial applications. PMID:26728652

  17. Triploid-diploid mosaic chicken embryo.

    PubMed

    Bloom, S E; Buss, E G

    1966-08-12

    Cytological analysis of an underdeveloped chicken embryo at 6 days of incubation revealed a triploid-diploid mosaic condition. Of the 30 metaphases observed, 19 were triploid and 11 diploid. The triploid cells were 3A-ZZZ and diploid cells 2A-ZZ, as determined for the six largest pairs of chromnosomes. PMID:5328678

  18. Identification of MET10-932 and characterization as an allele reducing hydrogen sulfide formation in wine strains of Saccharomyces cerevisiae.

    PubMed

    Linderholm, Angela; Dietzel, Kevin; Hirst, Marissa; Bisson, Linda F

    2010-12-01

    A vineyard isolate of the yeast Saccharomyces cerevisiae, UCD932, was identified as a strain producing little or no detectable hydrogen sulfide during wine fermentation. Genetic analysis revealed that this trait segregated as a single genetic determinant. The gene also conferred a white colony phenotype on BiGGY agar (bismuth-glucose-glycine-yeast agar), which is thought to indicate low basal levels of sulfite reductase activity. However, this isolate does not display a requirement for S-containing amino acids, indicating that the sulfate reduction pathway is fully operational. Genetic crosses against known mutations conferring white colony color on BiGGY agar identified the gene leading to reduced H(2)S formation as an allele of MET10 (MET10-932), which encodes a catalytic subunit of sulfite reductase. Sequence analysis of MET10-932 revealed several corresponding amino acid differences in relation to laboratory strain S288C. Allele differences for other genes of the sulfate reduction pathway were also detected in UCD932. The MET10 allele of UCD932 was found to be unique in comparison to the sequences of several other vineyard isolates with differing levels of production of H(2)S. Replacing the MET10 allele of high-H(2)S-producing strains with MET10-932 prevented H(2)S formation by those strains. A single mutative change, corresponding to T662K, in MET10-932 resulted in a loss of H(2)S production. The role of site 662 in sulfide reduction was further analyzed by changing the encoded amino acid at this position. A change back to threonine or to the conservative serine fully restored the H(2)S formation conferred by this allele. In addition to T662K, arginine, tryptophan, and glutamic acid substitutions similarly reduced sulfide formation. PMID:20889780

  19. A Novel Wild-Type Saccharomyces cerevisiae Strain TSH1 in Scaling-Up of Solid-State Fermentation of Ethanol from Sweet Sorghum Stalks

    PubMed Central

    Feng, Quanzhou; Li, Peipei; Zhang, Lei; Chang, Sandra; Li, Shizhong

    2014-01-01

    The rising demand for bioethanol, the most common alternative to petroleum-derived fuel used worldwide, has encouraged a feedstock shift to non-food crops to reduce the competition for resources between food and energy production. Sweet sorghum has become one of the most promising non-food energy crops because of its high output and strong adaptive ability. However, the means by which sweet sorghum stalks can be cost-effectively utilized for ethanol fermentation in large-scale industrial production and commercialization remains unclear. In this study, we identified a novel Saccharomyces cerevisiae strain, TSH1, from the soil in which sweet sorghum stalks were stored. This strain exhibited excellent ethanol fermentative capacity and ability to withstand stressful solid-state fermentation conditions. Furthermore, we gradually scaled up from a 500-mL flask to a 127-m3 rotary-drum fermenter and eventually constructed a 550-m3 rotary-drum fermentation system to establish an efficient industrial fermentation platform based on TSH1. The batch fermentations were completed in less than 20 hours, with up to 96 tons of crushed sweet sorghum stalks in the 550-m3 fermenter reaching 88% of relative theoretical ethanol yield (RTEY). These results collectively demonstrate that ethanol solid-state fermentation technology can be a highly efficient and low-cost solution for utilizing sweet sorghum, providing a feasible and economical means of developing non-food bioethanol. PMID:24736641

  20. Contribution of PRS3, RPB4 and ZWF1 to the resistance of industrial Saccharomyces cerevisiae CCUG53310 and PE-2 strains to lignocellulosic hydrolysate-derived inhibitors.

    PubMed

    Cunha, Joana T; Aguiar, Tatiana Q; Romaní, Aloia; Oliveira, Carla; Domingues, Lucília

    2015-09-01

    PRS3, RPB4 and ZWF1 were previously identified as key genes for yeast tolerance to lignocellulose-derived inhibitors. To better understand their contribution to yeast resistance to the multiple stresses occurring during lignocellulosic hydrolysate fermentations, we overexpressed these genes in two industrial Saccharomyces cerevisiae strains, CCUG53310 and PE-2, and evaluated their impact on the fermentation of Eucalyptus globulus wood and corn cob hydrolysates. PRS3 overexpression improved the fermentation rate (up to 32%) and productivity (up to 48%) in different hydrolysates. ZWF1 and RPB4 overexpression did not improve the fermentation performance, but their increased expression in the presence of acetic acid, furfural and hydroxymethylfurfural was found to contribute to yeast adaptation to these inhibitors. This study expands our understanding about the molecular mechanisms involved in industrial yeast tolerance to the stresses occurring during lignocellulosic bioethanol production and highlights the importance of selecting appropriate strain backgrounds/hydrolysates combinations when addressing further improvement of these processes. PMID:25974617

  1. A strain of Saccharomyces cerevisiae evolved for fermentation of lignocellulosic biomass displays improved growth and fermentative ability in high solids concentrations and in the presence of inhibitory compounds

    PubMed Central

    2011-01-01

    Background Softwoods are the dominant source of lignocellulosic biomass in the northern hemisphere, and have been investigated worldwide as a renewable substrate for cellulosic ethanol production. One challenge to using softwoods, which is particularly acute with pine, is that the pretreatment process produces inhibitory compounds detrimental to the growth and metabolic activity of fermenting organisms. To overcome the challenge of bioconversion in the presence of inhibitory compounds, especially at high solids loading, a strain of Saccharomyces cerevisiae was subjected to evolutionary engineering and adaptation for fermentation of pretreated pine wood (Pinus taeda). Results An industrial strain of Saccharomyces, XR122N, was evolved using pretreated pine; the resulting daughter strain, AJP50, produced ethanol much more rapidly than its parent in fermentations of pretreated pine. Adaptation, by preculturing of the industrial yeast XR122N and the evolved strains in 7% dry weight per volume (w/v) pretreated pine solids prior to inoculation into higher solids concentrations, improved fermentation performance of all strains compared with direct inoculation into high solids. Growth comparisons between XR122N and AJP50 in model hydrolysate media containing inhibitory compounds found in pretreated biomass showed that AJP50 exited lag phase faster under all conditions tested. This was due, in part, to the ability of AJP50 to rapidly convert furfural and hydroxymethylfurfural to their less toxic alcohol derivatives, and to recover from reactive oxygen species damage more quickly than XR122N. Under industrially relevant conditions of 17.5% w/v pretreated pine solids loading, additional evolutionary engineering was required to decrease the pronounced lag phase. Using a combination of adaptation by inoculation first into a solids loading of 7% w/v for 24 hours, followed by a 10% v/v inoculum (approximately equivalent to 1 g/L dry cell weight) into 17.5% w/v solids, the final

  2. Genetic profiling of yeast industrial strains using in situ comparative genomic hybridization (CGH).

    PubMed

    Wnuk, Maciej; Panek, Anita; Golec, Ewelina; Magda, Michal; Deregowska, Anna; Adamczyk, Jagoda; Lewinska, Anna

    2015-09-20

    The genetic differences and changes in genomic stability may affect fermentation processes involving baker's, brewer's and wine yeast strains. Thus, it seems worthwhile to monitor the changes in genomic DNA copy number of industrial strains. In the present study, we developed an in situ comparative genomic hybridization (CGH) to investigate the ploidy and genetic differences between selected industrial yeast strains. The CGH-based system was validated using the laboratory Saccharomyces cerevisiae yeast strains (haploid BY4741 and diploid BY4743). DNA isolated from BY4743 cells was considered a reference DNA. The ploidy and DNA gains and losses of baker's, brewer's and wine strains were revealed. Taken together, the in situ CGH was shown a helpful molecular tool to identify genomic differences between yeast industrial strains. Moreover, the in situ CGH-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale. PMID:26116136

  3. Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118

    PubMed Central

    Novo, Maite; Bigey, Frédéric; Beyne, Emmanuelle; Galeote, Virginie; Gavory, Frédérick; Mallet, Sandrine; Cambon, Brigitte; Legras, Jean-Luc; Wincker, Patrick; Casaregola, Serge; Dequin, Sylvie

    2009-01-01

    Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations. PMID:19805302

  4. Harnessing Genetic Diversity in Saccharomyces cerevisiae for Fermentation of Xylose in Hydrolysates of Alkaline Hydrogen Peroxide-Pretreated Biomass

    PubMed Central

    Liu, Tongjun; Parreiras, Lucas S.; Williams, Daniel L.; Wohlbach, Dana J.; Bice, Benjamin D.; Ong, Irene M.; Breuer, Rebecca J.; Qin, Li; Busalacchi, Donald; Deshpande, Shweta; Daum, Chris; Gasch, Audrey P.

    2014-01-01

    The fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverse S. cerevisiae strains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na+, acetate, and p-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts of pCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production. PMID:24212571

  5. Effect of Cymbopogon citratus L. essential oil on growth and morphogenesis of Saccharomyces cerevisiae ML2-strain.

    PubMed

    Helal, G A; Sarhan, M M; Abu Shahla, A N K; Abou El-Khair, E K

    2006-01-01

    The growth of Saccharomyces cerevisiae was completely inhibited using 2.0 microl/ml or 4.0 microl/ml of Cymbopogon citratus essential oil applied by fumigation or contact method in Sabouraud's broth medium, respectively. This oil was found also to be fungicidal at the same concentrations. The sublethal doses 1.0 and 3.0 microl/ml inhibited about 98% of yeast growth after 24 hr of incubation as compared with the control. Microscopic observations using Light Microscope (LM), Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) showed morphogenic and ultrastructure changes in the fumigated cells with 1.0 microl/ml of the oil. These changes including decrease in cell size, depressions on the surface of the cells, alteration in cell wall thickness and disruption of plasma membrane. Moreover, Ca(+2), K(+) and Mg(+2) leakages increased from the fumigated cells and its total lipid content decreased. Also, the fatty acid composition was altered with decrease in the amount of saturated fatty acids and increase in the amount of unsaturated fatty acids. PMID:17009293

  6. Extraction and purification of hepatitis B virus-like M particles from a recombinant Saccharomyces cerevisiae strain using alumina powder.

    PubMed

    Hadiji-Abbes, Nadia; Martin, Marta; Benzina, Wafa; Karray-Hakim, Hella; Gergely, Csilla; Gargouri, Ali; Mokdad-Gargouri, Raja

    2013-01-01

    A recombinant hepatitis B surface antigen (HBsAg) has been produced in the yeast Saccharomyces cerevisiae and used as a vaccine against hepatitis B virus (HBV) infection. The present study aimed to optimize the extraction of recombinant virus-like particles (rVLPs) to develop a simple purification procedure based on gel filtration and high performance size-exclusion chromatography. The findings showed that disruption of yeast cells with alumina powder increased the yield of the total proteins (290mg/l) and rVLPs (1mg/l) compared to the values for glass beads (171mg/l and 0.5mg/l), as estimated by quantitative ELISA. The purification of rVLPs was performed by two consecutive gel filtration chromatographic steps, namely Sephacryl S-200 followed by SEC-250 HPLC. The purified M protein was analyzed by atomic force microscopy and shown to assemble in particles that were able to recognize HBV antibodies in the sera of patients with chronic hepatitis B, providing evidence for their immunoreactivity. PMID:23059550

  7. Industrial Saccharomyces cerevisiae Yeast Strain Engineered to Convert Glucose, Mannose, Arabinose, and Xylose (GMAX) to Ethanol Anaerobically

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technology for engineering an industrial yeast strain for production of ethanol from glucose, mannose, arabinose, and xylose (GMAX-yeast) using both corn starch and cellulosic feedstocks with simultaneous production of valuable coproducts, including biodiesel, will be discussed. A stable industrial...

  8. Diploid males, diploid sperm production, and triploid females in the ant Tapinoma erraticum

    NASA Astrophysics Data System (ADS)

    Cournault, Laurent; Aron, Serge

    2009-12-01

    Under complementary sex determination (CSD), females of Hymenoptera arise from diploid, fertilized eggs and males from haploid, unfertilized eggs. Incidentally, fertilized eggs that inherit two identical alleles at the CSD locus will develop into diploid males. Diploid males are usually unviable or sterile. In a few species, however, they produce diploid sperm and father a triploid female progeny. Diploid males have been reported in a number of social Hymenoptera, but the occurrence of triploid females has hardly ever been documented. Here, we report the presence of triploid females, diploid males, and diploid sperm (produced by diploid males and stored in queen spermathecae) in the ant Tapinoma erraticum. Moreover, we show variations in the frequency of triploids among female castes: Triploid females are more frequent among workers than virgin queens; they are absent among mated, reproductive queens. The frequency of triploid workers also varies between populations and between nests within populations.

  9. Variation in Indole-3-Acetic Acid Production by Wild Saccharomyces cerevisiae and S. paradoxus Strains from Diverse Ecological Sources and Its Effect on Growth

    PubMed Central

    Liu, Yen-Yu; Chen, Hung-Wei; Chou, Jui-Yu

    2016-01-01

    Phytohormone indole-3-acetic acid (IAA) is the most common naturally occurring and most thoroughly studied plant growth regulator. Microbial synthesis of IAA has long been known. Microbial IAA biosynthesis has been proposed as possibly occurring through multiple pathways, as has been proven in plants. However, the biosynthetic pathways of IAA and the ecological roles of IAA in yeast have not been widely studied. In this study, we investigated the variation in IAA production and its effect on the growth of Saccharomyces cerevisiae and its closest relative Saccharomyces paradoxus yeasts from diverse ecological sources. We found that almost all Saccharomyces yeasts produced IAA when cultured in medium supplemented with the primary precursor of IAA, L-tryptophan (L-Trp). However, when cultured in medium without L-Trp, IAA production was only detected in three strains. Furthermore, exogenous added IAA exerted stimulatory and inhibitory effects on yeast growth. Interestingly, a negative correlation was observed between the amount of IAA production in the yeast cultures and the IAA inhibition ratio of their growth. PMID:27483373

  10. Origin and Genetic Diversity of Diploid Parthenogenetic Artemia in Eurasia

    PubMed Central

    Maccari, Marta; Amat, Francisco; Gómez, Africa

    2013-01-01

    There is wide interest in understanding how genetic diversity is generated and maintained in parthenogenetic lineages, as it will help clarify the debate of the evolution and maintenance of sexual reproduction. There are three mechanisms that can be responsible for the generation of genetic diversity of parthenogenetic lineages: contagious parthenogenesis, repeated hybridization and microorganism infections (e.g. Wolbachia). Brine shrimps of the genus Artemia (Crustacea, Branchiopoda, Anostraca) are a good model system to investigate evolutionary transitions between reproductive systems as they include sexual species and lineages of obligate parthenogenetic populations of different ploidy level, which often co-occur. Diploid parthenogenetic lineages produce occasional fully functional rare males, interspecific hybridization is known to occur, but the mechanisms of origin of asexual lineages are not completely understood. Here we sequenced and analysed fragments of one mitochondrial and two nuclear genes from an extensive set of populations of diploid parthenogenetic Artemia and sexual species from Central and East Asia to investigate the evolutionary origin of diploid parthenogenetic Artemia, and geographic origin of the parental taxa. Our results indicate that there are at least two, possibly three independent and recent maternal origins of parthenogenetic lineages, related to A. urmiana and Artemia sp. from Kazakhstan, but that the nuclear genes are very closely related in all the sexual species and parthenogegetic lineages except for A. sinica, who presumable took no part on the origin of diploid parthenogenetic strains. Our data cannot rule out either hybridization between any of the very closely related Asiatic sexual species or rare events of contagious parthenogenesis via rare males as the contributing mechanisms to the generation of genetic diversity in diploid parthenogenetic Artemia lineages. PMID:24376692

  11. Computer simulation of arising of diploid genomes

    NASA Astrophysics Data System (ADS)

    Tretyakov, Victor S.; Tretyakov, Nikolay P.

    2008-07-01

    The haploid-diploid cycle where, under unfavorable conditions the population becomes diploid, is modeled by a Monte-Carlo method in the framework of the Jan-Stauffer-Moseley hypothesis. Diploidy and sex may have first arisen as a way to escape death, when a simple unicellular individual is threatened by too many deleterious mutations. Using a bit string model, we find that in a system where competition is present (through the Verhulst factor), diploids dominate. In this case the transition from haploid to essentially diploid population takes place in a short time interval reminiscent of phase transitions in physical systems.

  12. Application of the Saccharomyces cerevisiae FLP/FRT recombination system in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes.

    PubMed

    Kopke, Katarina; Hoff, Birgit; Kück, Ulrich

    2010-07-01

    To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT recombination system from the yeast Saccharomyces cerevisiae for marker recycling. We tested this system in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in P. chrysogenum. As a proof of application of this system, we constructed a DeltaPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT system, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT recombination system in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT recombination system as a molecular tool for the genetic manipulation of filamentous fungi. PMID:20472720

  13. Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance. Results In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid. Conclusions Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering. PMID:21219616

  14. Proteomics and redox-proteomics of the effects of herbicides on a wild-type wine Saccharomyces cerevisiae strain.

    PubMed

    Braconi, Daniela; Bernardini, Giulia; Possenti, Silvia; Laschi, Marcella; Arena, Simona; Scaloni, Andrea; Geminiani, Michela; Sotgiu, Michele; Santucci, Annalisa

    2009-01-01

    Several toxicological and environmental problems are associated with the extensive use of agricultural pesticides, such as herbicides. Nevertheless, little is known about the toxic effects of formulated herbicides, since many studies have been carried out using pure active molecules alone. In this work, we used as an eukaryotic model system an autochthonous wine yeast strain to investigate the effects of three commercial herbicides, currently used in the same geographical area from where this strain had been isolated. We carried out a comparative proteomic analysis to study the effects at the protein level of the herbicide-related stress, and found that the herbicides tested can alter the yeast proteome producing responses that share homologies with those observed treating yeast cells with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) or with well-known oxidizing agents. We evaluated, through redox-proteomic techniques, protein carbonylation as a biomarker of oxidative stress. This analysis showed that herbicide-induced carbonylation is a dynamic phenomenon with degrees of selectivity. PMID:19032026

  15. Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

    PubMed Central

    2009-01-01

    Background Saccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain. Results Our results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of S. cerevisiae BY4741 [PIR4-IL1β] in fed-batch reactor. Even in the presence of a proper ACA supplementation, S. cerevisiae BY4741 [PIR4-IL1β] did not achieve a high cell density. The Δyca1 deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the kd value (0.0045 h-1), negligible if compared to that of the parental strain (0.028 h-1). However, independently of their robustness, both the parental and the Δyca1 mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for

  16. Switching of a Mating-Type a Mutant Allele in Budding Yeast SACCHAROMYCES CEREVISIAE

    PubMed Central

    Klar, Amar J. S.; Fogel, Seymour; Radin, David N.

    1979-01-01

    Aimed at investigating the recovery of a specific mutant allele of the mating type locus (MAT) by switching a defective MAT allele, these experiments provide information bearing on several models proposed for MAT interconversion in bakers yeast, Saccharomyces cerevisiae. Hybrids between heterothallic (ho) cells carrying a mutant MATa allele, designated mata-2, and MATα ho strains show a high capacity for mating with MATa strains. The MATα/mata-2 diploids do not sporulate. However, zygotic clones obtained by mating MATα homothallic (HO) cells with mata-2 ho cells are unable to mate and can sporulate. Tetrad analysis of such clones revealed two diploid (MATα/MATa):two haploid segregants. Therefore, MAT switches occur in MATα/mata-2 HO/ho cells to produce MATα/MATa cells capable of sporulation. In heterothallic strains, the mata-2 allele can be switched to a functional MATα and subsequently to a functional MATa. Among 32 MATα to MATa switches tested, where the MATα was previously derived from the mata-2 mutant, only one mata-2 like isolate was observed. However, the recovered allele, unlike the parental allele, conplements the matα ste1–5 mutant, suggesting that these alleles are not identical and that the recovered allele presumably arose as a mutation of the MATα locus. No mata-2 was recovered by HO-mediated switching of MATα (previously obtained from mata-2 by HO) in 217 switches analyzed. We conclude that in homothallic and heterothallic strains, the mata-2 allele can be readily switched to a functional MATα and subsequently to a functional MATa locus. Overall, the results are in accord with the cassette model (Hicks, Strathern and Herskowitz 1977b) proposed to explain MAT interconversions. PMID:395020

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

    PubMed

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

    2015-07-01

    Improved supply of farnesyl diphosphate (FPP) is often considered as a typical strategy for engineering Saccharomyces cerevisiae towards efficient terpenoid production. However, in the engineered strains with enhanced precursor supply, the production of the target metabolite is often impeded by insufficient capacity of the heterologous terpenoid pathways, which limits further conversion of FPP. Here, we tried to assemble an unimpeded biosynthesis pathway by combining directed evolution and metabolic engineering in S. cerevisiae for lycopene-overproduction. First, the catalytic ability of phytoene syntheses from different sources was investigated based on lycopene accumulation. Particularly, the lycopene cyclase function of the bifunctional enzyme CrtYB from Xanthophyllomyces dendrorhous was inactivated by deletion of functional domain and directed evolution to obtain mutants with solely phytoene synthase function. Coexpression of the resulting CrtYB11M mutant along with the CrtE and CrtI genes from X. dendrorhous, and the tHMG1 gene from S. cerevisiae led to production of 4.47 mg/g DCW (Dry cell weight) of lycopene and 25.66 mg/g DCW of the by-product squalene. To further increase the FPP competitiveness of the lycopene synthesis pathway, we tried to enhance the catalytic performance of CrtE by directed evolution and created a series of pathway variants by varying the copy number of Crt genes. Finally, fed-batch fermentation was conducted for the diploid strain YXWPD-14 resulting in accumulation of 1.61 g/L (24.41 mg/g DCW) of lycopene, meanwhile, the by-production of squalene was reduced to below 1 mg/g DCW. PMID:25959020

  18. Characterization of Chromosome Stability in Diploid, Polyploid and Hybrid Yeast Cells

    PubMed Central

    Kumaran, Rajaraman; Yang, Shi-Yow; Leu, Jun-Yi

    2013-01-01

    Chromosome instability is a key component of cancer progression and many heritable diseases. Understanding why some chromosomes are more unstable than others could provide insight into understanding genome integrity. Here we systematically investigate the spontaneous chromosome loss for all sixteen chromosomes in Saccharomyces cerevisiae in order to elucidate the mechanisms underlying chromosome instability. We observed that the stability of different chromosomes varied more than 100-fold. Consistent with previous studies on artificial chromosomes, chromosome loss frequency was negatively correlated to chromosome length in S. cerevisiae diploids, triploids and S. cerevisiae-S. bayanus hybrids. Chromosome III, an equivalent of sex chromosomes in budding yeast, was found to be the most unstable chromosome among all cases examined. Moreover, similar instability was observed in chromosome III of S. bayanus, a species that diverged from S. cerevisiae about 20 million years ago, suggesting that the instability is caused by a conserved mechanism. Chromosome III was found to have a highly relaxed spindle checkpoint response in the genome. Using a plasmid stability assay, we found that differences in the centromeric sequence may explain certain aspects of chromosome instability. Our results reveal that even under normal conditions, individual chromosomes in a genome are subject to different levels of pressure in chromosome loss (or gain). PMID:23874507

  19. mRNA transcription in nuclei isolated from Saccharomyces cerevisiae.

    PubMed Central

    Jerome, J F; Jaehning, J A

    1986-01-01

    We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II. Images PMID:3537708

  20. Sporulation in the Budding Yeast Saccharomyces cerevisiae

    PubMed Central

    Neiman, Aaron M.

    2011-01-01

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

  1. Population studies in microorganisms. I. Evolution of diploidy in Saccharomyces cerevisiae.

    PubMed

    Adams, J; Hansche, P E

    1974-02-01

    The relative adaptation of isogenic haploid and diploid strains of yeast was investigated in different sets of physiological conditions. When all nutrients were present in excess, no difference in the reproductive rates of isogenic haploid and diploid strains of yeast was detected in both optimal and non-optimal growth conditions. Competition between haploid and diploid strains of yeast was observed when growth was limited by the concentration of a single nutrilite. Under certain conditions when fitness (reproductive rate) is determined by transport of an essential nutrilite that exists in very low concentrations, diploid cells were selected against. These environmental conditions are similar to those found in offshore marine environments where nutrients are often present in extremely low concentrations. The fitness of diploid cells was estimated to be.93 +/-.02 (haploid fitness = 1). The reduced fitness of diploid cells in this environment can be explained by the reduced surface area/volume ratio possessed by diploid cells in comparison to haploid cells. The fitnesses of haploid and diploid cells in these environments are closely correlated with geometric variations in these strains. These results are consistent with the hypothesis that diploid cells are simply double haploids, and diploidy per se does not confer any direct adaptive advantage. The mechanism of the evolution of diploidy as a dominant phase in the life cycle of higher plants and animals remains obscure. PMID:4595645

  2. Population Studies in Microorganisms I. Evolution of Diploidy in SACCHAROMYCES CEREVISIAE

    PubMed Central

    Adams, Julian; Hansche, Paul E.

    1974-01-01

    The relative adaptation of isogenic haploid and diploid strains of yeast was investigated in different sets of physiological conditions. When all nutrients were present in excess, no difference in the reproductive rates of isogenic haploid and diploid strains of yeast was detected in both optimal and non-optimal growth conditions. Competition between haploid and diploid strains of yeast was observed when growth was limited by the concentration of a single nutrilite. Under certain conditions when fitness (reproductive rate) is determined by transport of an essential nutrilite that exists in very low concentrations, diploid cells were selected against. These environmental conditions are similar to those found in offshore marine environments where nutrients are often present in extremely low concentrations. The fitness of diploid cells was estimated to be.93 ±.02 (haploid fitness = 1). The reduced fitness of diploid cells in this environment can be explained by the reduced surface area/volume ratio possessed by diploid cells in comparison to haploid cells. The fitnesses of haploid and diploid cells in these environments are closely correlated with geometric variations in these strains. These results are consistent with the hypothesis that diploid cells are simply double haploids, and diploidy per se does not confer any direct adaptive advantage. The mechanism of the evolution of diploidy as a dominant phase in the life cycle of higher plants and animals remains obscure. PMID:4595645

  3. Analysis and Dynamics of the Chromosomal Complements of Wild Sparkling-Wine Yeast Strains

    PubMed Central

    Nadal, Dolors; Carro, David; Fernández-Larrea, Juan; Piña, Benjamin

    1999-01-01

    We isolated Saccharomyces cerevisiae yeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed in S. cerevisiae wild populations. PMID:10103269

  4. Fed-batch SSCF using steam-exploded wheat straw at high dry matter consistencies and a xylose-fermenting Saccharomyces cerevisiae strain: effect of laccase supplementation

    PubMed Central

    2013-01-01

    Background Lignocellulosic bioethanol is expected to play an important role in fossil fuel replacement in the short term. Process integration, improvements in water economy, and increased ethanol titers are key considerations for cost-effective large-scale production. The use of whole steam-pretreated slurries under high dry matter (DM) conditions and conversion of all fermentable sugars offer promising alternatives to achieve these goals. Results Wheat straw slurry obtained from steam explosion showed high concentrations of degradation compounds, hindering the fermentation performance of the evolved xylose-recombinant Saccharomyces cerevisiae KE6-12 strain. Fermentability tests using the liquid fraction showed a higher number of colony-forming units (CFU) and higher xylose consumption rates when treating the medium with laccase. During batch simultaneous saccharification and co-fermentation (SSCF) processes, cell growth was totally inhibited at 12% DM (w/v) in untreated slurries. However, under these conditions laccase treatment prior to addition of yeast reduced the total phenolic content of the slurry and enabled the fermentation. During this process, an ethanol concentration of 19 g/L was obtained, corresponding to an ethanol yield of 39% of the theoretical yield. By changing the operation from batch mode to fed-batch mode, the concentration of inhibitors at the start of the process was reduced and 8 g/L of ethanol were obtained in untreated slurries with a final consistency of 16% DM (w/v). When fed-batch SSCF medium was supplemented with laccase 33 hours after yeast inoculation, no effect on ethanol yield or cell viability was found compared to untreated fermentations. However, if the laccase supplementation (21 hours after yeast inoculation) took place before the first addition of substrate (at 25 hours), improved cell viability and an increased ethanol titer of up to 32 g/L (51% of the theoretical) were found. Conclusions Laccase treatment in SSCF processes

  5. The Parasexual Cycle in Candida albicans Provides an Alternative Pathway to Meiosis for the Formation of Recombinant Strains

    PubMed Central

    Forche, Anja; Alby, Kevin; Schaefer, Dana; Johnson, Alexander D; Berman, Judith; Bennett, Richard J

    2008-01-01

    Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and α strains. The product of mating is a tetraploid a/α cell that must undergo a reductional division to return to the diploid state. Despite the presence of several “meiosis-specific” genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle. PMID:18462019

  6. Photorepair of ultraviolet-induced petite mutational damage in Saccharomyces cerevisiae requires the product of the PHR1 gene

    SciTech Connect

    Green, G.; MacQuillan, A.M.

    1980-11-01

    A wild-type (phr/sup +/) diploid yeast strain showed photorepair of petite mutational damage, whereas a photoreactivation-deficient (phr1/phr1) diploid strain did not, indicating that the PHR1 gene product was required for mitochondrial photorepair.

  7. Genetic Polymorphism and Evolution in Parthenogenetic Animals. II. Diploid and Polyploid SOLENOBIA TRIQUETRELLA (Lepidoptera: Psychidae)

    PubMed Central

    Lokki, Juhani; Suomalainen, Esko; Saura, Anssi; Lankinen, Pekka

    1975-01-01

    Genic polymorphism at sixteen enzyme loci of four different chromosomal races of Solenobia triquetrella (bisexual, two diploid parthenogenetic races and tetraploid parthenogenetic) has been studied by starch gel electrophoresis. Isolated small diploid bisexual populations have rather uniform allele frequencies at all loci which we have studied. Diploid and tetraploid parthenogenetic individuals of this species are in general as heterozygous as bisexual ones. All parthenogenetic local populations are different from each other in the Alps. These parthenogenetic genotypes cannot be derived from a common ancestor through single mutations but rather bear evidence for a polyphyletic origin of parthenogenesis in Solenobia triquetrella. In the marginal distribution areas of the species in northern Europe single genotypes are spread over far larger areas than in the mountain regions of central Europe. This may be due to the old origin of parthenogenesis and polyploidy in northern Europe. No new parthenogenetic and polyploid strains have lately arisen in the regions outside of the Alps. PMID:1126629

  8. On the Genealogy of Asexual Diploids

    NASA Astrophysics Data System (ADS)

    Lam, Fumei; Langley, Charles H.; Song, Yun S.

    Given molecular genetic data from diploid individuals that, at present, reproduce mostly or exclusively asexually without recombination, an important problem in evolutionary biology is detecting evidence of past sexual reproduction (i.e., meiosis and mating) and recombination (both meiotic and mitotic). However, currently there is a lack of computational tools for carrying out such a study. In this paper, we formulate a new problem of reconstructing diploid genealogies under the assumption of no sexual reproduction or recombination, with the ultimate goal being to devise genealogy-based tools for testing deviation from these assumptions. We first consider the infinite-sites model of mutation and develop linear-time algorithms to test the existence of an asexual diploid genealogy compatible with the infinite-sites model of mutation, and to construct one if it exists. Then, we relax the infinite-sites assumption and develop an integer linear programming formulation to reconstruct asexual diploid genealogies with the minimum number of homoplasy (back or recurrent mutation) events. We apply our algorithms on simulated data sets with sizes of biological interest.

  9. Recombination-aware alignment of diploid individuals

    PubMed Central

    2014-01-01

    Background Traditionally biological similarity search has been studied under the abstraction of a single string to represent each genome. The more realistic representation of diploid genomes, with two strings defining the genome, has so far been largely omitted in this context. With the development of sequencing techniques and better phasing routines through haplotype assembly algorithms, we are not far from the situation when individual diploid genomes could be represented in their full complexity with a pair-wise alignment defining the genome. Results We propose a generalization of global alignment that is designed to measure similarity between phased predictions of individual diploid genomes. This generalization takes into account that individual diploid genomes evolve through a mutation and recombination process, and that predictions may be erroneous in both dimensions. Even though our model is generic, we focus on the case where one wants to measure only the similarity of genome content allowing free recombination. This results into efficient algorithms for direct application in (i) evaluation of variation calling predictions and (ii) progressive multiple alignments based on labeled directed acyclic graphs (DAGs) to represent profiles. The latter may be of more general interest, in connection to covering alignment of DAGs. Extensions of our model and algorithms can be foreseen to have applications in evaluating phasing algorithms, as well as more fundamental role in phasing child genome based on parent genomes. PMID:25572943

  10. Breeding potato at the diploid level

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In most regions of the world, potato cultivars are tetraploid. However, complexities due to tetraploid genetics have slowed breeding progress and limited the implementation of breeding strategies commonly used in other major crops. We are developing diploid genetics resources, including partially in...

  11. Automated Yeast Transformation Protocol to Engineer S. cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames that Express Proteins Binding to Xylose Isomerase Identified using Robotic Two-hybrid Screen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. Since S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI...

  12. Heterozygous screen in Saccharomyces cerevisiae identifies dosage-sensitive genes that affect chromosome stability.

    PubMed

    Strome, Erin D; Wu, Xiaowei; Kimmel, Marek; Plon, Sharon E

    2008-03-01

    Current techniques for identifying mutations that convey a small increased cancer risk or those that modify cancer risk in carriers of highly penetrant mutations are limited by the statistical power of epidemiologic studies, which require screening of large populations and candidate genes. To identify dosage-sensitive genes that mediate genomic stability, we performed a genomewide screen in Saccharomyces cerevisiae for heterozygous mutations that increase chromosome instability in a checkpoint-deficient diploid strain. We used two genome stability assays sensitive enough to detect the impact of heterozygous mutations and identified 172 heterozygous gene disruptions that affected chromosome fragment (CF) loss, 45% of which also conferred modest but statistically significant instability of endogenous chromosomes. Analysis of heterozygous deletion of 65 of these genes demonstrated that the majority increased genomic instability in both checkpoint-deficient and wild-type backgrounds. Strains heterozygous for COMA kinetochore complex genes were particularly unstable. Over 50% of the genes identified in this screen have putative human homologs, including CHEK2, ERCC4, and TOPBP1, which are already associated with inherited cancer susceptibility. These findings encourage the incorporation of this orthologous gene list into cancer epidemiology studies and suggest further analysis of heterozygous phenotypes in yeast as models of human disease resulting from haplo-insufficiency. PMID:18245329

  13. Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae.

    PubMed

    Freeman, Kathryn M; Hoffmann, George R

    2007-03-01

    Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, beta-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv(+) revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state. PMID:17156798

  14. EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9.

    PubMed

    Jessop-Fabre, Mathew M; Jakočiūnas, Tadas; Stovicek, Vratislav; Dai, Zongjie; Jensen, Michael K; Keasling, Jay D; Borodina, Irina

    2016-08-01

    Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available. PMID:27166612

  15. Growth-inhibitory activity of the D-mannan of Saccharomyces cerevisiae X2180-1A-5 mutant strain against mouse-implanted sarcoma 180 and Ehrlich-carcinoma solid tumor.

    PubMed

    Matsumoto, T; Takanohashi, M; Okubo, Y; Suzuki, M; Suzuki, S

    1980-08-15

    The D-mannan of Saccharomyces cerevisiae X2180-1A-5 mutant strain, which possesses a main chain composed of alpha-(1 yields 6) linked D-mannopyranosyl residues and a small proportion of branches composed of alpha-(1 yields 2)- and alpha-(1 yields 3)-linked D-mannopyranosyl residues, showed strong growth-inhibitory activity against mouse-implanted Sarcoma 180 and Ehrlich-carcinoma solid tumor. The observation that the level of this activity was nearly identical with that of the D-mannan of a wild-type strain of bakers' yeast, which possesses a high proportion of branches composed of alpha-(1 yields 2)-and alpha-(1 yields 3)-linked D-mannopyranosyl residues, suggests that the branches are not essential for antitumor activity. The partial acid-degradation products of both D-mannans, the molecular weight of which was one-third of that of each parent D-mannan, had only one half of the antitumor activity of the parent D-mannans. This suggests that molecular size is the most important factor for the differences in acitvity of the polysaccharides of wild and mutant strains. PMID:6996813

  16. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.

    PubMed

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

    2008-12-01

    Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P < 0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (P < 0.05). The spaceflight mutant of S. cerevisiae 2.0016 showed 46.7%, 62.6%, and 146.0% increment in biomass, cell wall thickness and beta-glucan content, respectively, when compared to the ground strain. Moreover, growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production. PMID:18797865

  17. Physiological Characterization of Adaptive Clones in Evolving Populations of the Yeast, SACCHAROMYCES CEREVISIAE

    PubMed Central

    Adams, Julian; Paquin, Charlotte; Oeller, Paul W.; Lee, Lester W.

    1985-01-01

    Populations of a diploid strain of S. cerevisiae were grown in glucose-limited continuous culture for more than 260 generations. A series of seven sequential adaptive changes were identified by monitoring the frequency of cycloheximide resistance in these populations. Samples were taken from the continuous cultures following each adaptive shift and characterized physiologically to determine (1) the range of phenotypes that can be selected in a precisely defined constant environment and (2) the order and predictability of the occurrence of the adaptive mutations in evolving populations. The clones were characterized with respect to the growth parameters, maximum growth rate, saturation coefficient and yield, as well as for changes in cell size and geometry and rate of glucose uptake. The maximum growth rates of the seven adaptive clones were very similar, but in contrast the saturation coefficients differed substantially. Surprisingly, not all clones showed reductions in the saturation coefficients, in comparison to the immediately preceding clones, as would be predicted from classical continuous culture kinetics. In addition, yield estimates first increased and then decreased for later isolated adaptive clones. In general, the results suggest epistatic interactions between the adaptive clones, consistent with earlier published results. The rate of glucose uptake, as measured by 14Cxylose uptake, increased dramatically after the selection and fixation of seven adaptive clones. Progressive decreases in cell volume and changes in cell geometry, resulting in increased surface area to volume ratios, were also observed in the adaptive clones, but these changes were not always seen in other haploid and diploid yeast populations evolving under the same conditions. Such changes may be easily explainable in terms of the characteristics of the glucose-limited environment. The significance of the results to the evolution of microorganisms under nutrient-limiting conditions is

  18. Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase

    PubMed Central

    Remize, F.; Roustan, J. L.; Sablayrolles, J. M.; Barre, P.; Dequin, S.

    1999-01-01

    Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of the GPD1 gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressing GPD1 produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained by GPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation. PMID:9872772

  19. Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

    PubMed Central

    2013-01-01

    Background The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions An industrial yeast strain for

  20. Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p.

    PubMed Central

    Newman, S M; Zelenaya-Troitskaya, O; Perlman, P S; Butow, R A

    1996-01-01

    DNA-protein complexes (nucleoids) are believed to be the segregating unit of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. A mitochondrial HMG box protein, Abf2p, is needed for maintenance of mtDNA in cells grown on rich dextrose medium, but is dispensible in glycerol grown cells. As visualized by 4',6'-diamino-2-phenylindole staining, mtDNA nucleoids in mutant cells lacking Abf2p ( delta abf2) are diffuse compared with those in wild-type cells. We have isolated mtDNA nucleoids and characterized two mtDNA-protein complexes, termed NCLDp-2 and NCLDs-2, containing distinct but overlapping sets of polypeptides. This protocol yields similar nucleoid complexes from the delta abf2 mutant, although several proteins appear lacking from NCLDs-2. Segments of mtDNA detected with probes to COXII, VAR1 and ori5 sequences are equally sensitive to DNase I digestion in NCLDs-2 and NCLDp-2 from wild-type cells and from the delta abf2 mutant. However, COXII and VAR1 sequences are 4-to 5-fold more sensitive to DNase I digestion of mtDNA in toluene-permeabilized mitochondria from the delta abf2 mutant than from wild-type cells, but no difference in DNase I sensitivity was detected with the ori5 probe. These results provide a first indication that Abf2p influences differential organization of mtDNA sequences. PMID:8628667

  1. Optimization of ethanol, citric acid, and α-amylase production from date wastes by strains of Saccharomyces cerevisiae, Aspergillus niger, and Candida guilliermondii.

    PubMed

    Acourene, S; Ammouche, A

    2012-05-01

    The present study deals with submerged ethanol, citric acid, and α-amylase fermentation by Saccharomyces cerevisiae SDB, Aspergillus niger ANSS-B5, and Candida guilliermondii CGL-A10, using date wastes as the basal fermentation medium. The physical and chemical parameters influencing the production of these metabolites were optimized. As for the ethanol production, the optimum yield obtained was 136.00 ± 0.66 g/l under optimum conditions of an incubation period of 72 h, inoculum content of 4% (w/v), sugars concentration of 180.0 g/l, and ammonium phosphate concentration of 1.0 g/l. Concerning citric acid production, the cumulative effect of temperature (30°C), sugars concentration of 150.0 g/l, methanol concentration of 3.0%, initial pH of 3.5, ammonium nitrate concentration of 2.5 g/l, and potassium phosphate concentration of 2.5 g/l during the fermentation process of date wastes syrup did increase the citric acid production to 98.42 ± 1.41 g/l. For the production of α-amylase, the obtained result shows that the presence of starch strongly induces the production of α-amylase with a maximum at 5.0 g/l. Among the various nitrogen sources tested, urea at 5.0 g/l gave the maximum biomass and α-amylase estimated at 5.76 ± 0.56 g/l and 2,304.19 ± 31.08 μmol/l/min, respectively after 72 h incubation at 30°C, with an initial pH of 6.0 and potassium phosphate concentration of 6.0 g/l. PMID:22193823

  2. [Tolerance of Saccharomyces cerevisiae to monoterpenes--a review].

    PubMed

    Liu, Jidong; Zhou, Jingwen; Chen, Jian

    2013-06-01

    Tolerance of Saccharomyces cerevisiae to monoterpenes is important in both metabolic engineering of the yeast to produce these chemicals de novo and efficient use of biomass containing these chemicals. Understanding the mechanisms in the tolerance of S. cerevisiae to monoterpenes could facilitate the construction of yeast strains with enhanced monoterpenes resistance, and therefore improve related bioprocesses. Monoterpenes could disturb the redox balance in S. cerevisiae, therefore increase the accumulation of reactive oxygen species (ROS) and result in cell death. S. cerevisiae has to systematically improve its antioxidative ability to deal with the ROS induced damage. The current review summarized the recent developments in demonstration of the tolerance of S. cerevisiae to different typical monoterpenes mainly from the aspect of the antioxidative mechanisms. Based on the analysis of the previous works, further attempts to demonstrate the mechanisms were proposed. PMID:24028054

  3. A hypothesis on the origin of genetic heterozygosity in diploids and triploids in Japanese Cayratia japonica species complex (Vitaceae).

    PubMed

    Tsukaya, Hirokazu; Ishikawa, Naoko; Okada, Hiroshi

    2012-07-01

    We previously reported the occurrence of triploid strains in Japanese populations of Cayratia japonica (Thunb.) Gagnep. Interestingly, the triploid and most diploid strains had variably reduced pollen fertility. Two questions emerged from this earlier work: (1) How do triploids arise, and are they allotriploids or autotriploids? and (2) Why is there low pollen fertility in some diploid plants? We used a molecular genetic approach to determine the phylogenetic origins of triploids in C. japonica and the closely related species Cayratia tenuifolia (Wight & Arn.) Gagnep. In our analysis, we compared the sequences of the nuclear single-copy genes LEAFY and ASYMMETRIC LEAVES1. As a result, most triploids and diploids were heterozygous for the loci examined; the triploid genome shared an allele with the diploid genome, but other alleles differed between the ploidies. Therefore, Japanese populations of C. japonica and C. tenuifolia almost certainly arose from repeated hybridization events among genetically differentiated strains. Using our sequence data, we discuss possible scenarios accounting for the occurrence of triploids in the two species of Cayratia. PMID:22200910

  4. GMAX Yeast Background Strain Made from Industrial Tolerant Saccharomyces cerevisiae Engineered to Convert Sucrose, Starch and Cellulosic Sugars Universally to Ethanol Anaerobically with Concurrent Coproduct Expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tailored GMAX yeast background strain technology for universal ethanol production industrially. Production of the stable baseline glucose, mannose, arabinose, xylose-utilizing (GMAX) yeast will be evaluated by taking the genes identified in high-throughput screening for a plasmid-based yeast to uti...

  5. Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a co-product in an ethanologenix Saccharomyces cerevisiae strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinse...

  6. GMAX Yeast Background Strain Made from Industrial Tolerant Saccharomyces Cerevisiae Engineered to Convert Pretreated Lignocellulosic Starch and Cellulosic Sugars Universally to Ethanol Anaerobically

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tailored GMAX yeast background strain technology for universal ethanol production industrially: Production of the stable baseline glucose, mannose, arabinose, xylose-utilizing (GMAX) yeast will be evaluated by taking the genes identified in high-throughput screening for a plasmid-based yeast to util...

  7. Screening of High-Level 4-Hydroxy-2 (or 5)-Ethyl-5 (or 2)-Methyl-3(2H)-Furanone-Producing Strains from a Collection of Gene Deletion Mutants of Saccharomyces cerevisiae

    PubMed Central

    Watanabe, Jun; Akao, Takeshi; Watanabe, Daisuke; Mogi, Yoshinobu; Shimoi, Hitoshi

    2014-01-01

    4-Hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3(2H)-furanone (HEMF) is an important flavor compound that contributes to the sensory properties of many natural products, particularly soy sauce and soybean paste. The compound exhibits a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. HEMF is produced by yeast species in soy sauce manufacturing; however, the enzymes involved in HEMF production remain unknown, hindering efforts to breed yeasts with high-level HEMF production. In this study, we identified high-level HEMF-producing mutants among a Saccharomyces cerevisiae gene deletion mutant collection. Fourteen deletion mutants were screened as high-level HEMF-producing mutants, and the ADH1 gene deletion mutant (adh1Δ) exhibited the maximum HEMF production capacity. Further investigations of the adh1Δ mutant implied that acetaldehyde accumulation contributes to HEMF production, agreeing with previous findings. Therefore, acetaldehyde might be a precursor for HEMF. The ADH1 gene deletion mutant of Zygosaccharomyces rouxii, which is the dominant strain of yeast found during soy sauce fermentation, also produces HEMF effectively, suggesting that acetaldehyde accumulation might be a benchmark for breeding industrial yeasts with excellent HEMF production abilities. PMID:25362059

  8. Screening of high-level 4-hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3(2H)-furanone-producing strains from a collection of gene deletion mutants of Saccharomyces cerevisiae.

    PubMed

    Uehara, Kenji; Watanabe, Jun; Akao, Takeshi; Watanabe, Daisuke; Mogi, Yoshinobu; Shimoi, Hitoshi

    2015-01-01

    4-Hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3(2H)-furanone (HEMF) is an important flavor compound that contributes to the sensory properties of many natural products, particularly soy sauce and soybean paste. The compound exhibits a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. HEMF is produced by yeast species in soy sauce manufacturing; however, the enzymes involved in HEMF production remain unknown, hindering efforts to breed yeasts with high-level HEMF production. In this study, we identified high-level HEMF-producing mutants among a Saccharomyces cerevisiae gene deletion mutant collection. Fourteen deletion mutants were screened as high-level HEMF-producing mutants, and the ADH1 gene deletion mutant (adh1Δ) exhibited the maximum HEMF production capacity. Further investigations of the adh1Δ mutant implied that acetaldehyde accumulation contributes to HEMF production, agreeing with previous findings. Therefore, acetaldehyde might be a precursor for HEMF. The ADH1 gene deletion mutant of Zygosaccharomyces rouxii, which is the dominant strain of yeast found during soy sauce fermentation, also produces HEMF effectively, suggesting that acetaldehyde accumulation might be a benchmark for breeding industrial yeasts with excellent HEMF production abilities. PMID:25362059

  9. Comparison of aroma-active compounds and sensory characteristics of durian (Durio zibethinus L.) wines using strains of Saccharomyces cerevisiae with odor activity values and partial least-squares regression.

    PubMed

    Zhu, JianCai; Chen, Feng; Wang, LingYing; Niu, YunWei; Shu, Chang; Chen, HeXing; Xiao, ZuoBing

    2015-02-25

    The study evaluated the effects of five different strains (GRE, RC212, Lalvin D254, CGMCC2.4, and CGMCC2.23) of the yeast Saccharomyces cerevisiae on the aromatic characteristics of fermented durian musts. In this work, 38 and 43 compounds in durian juices and wines were analyzed by gas chromatography-mass spectrometry (GC-MS) and GC-pulsed flame photometric detection (GC-PFPD) with the aid of stir bar sorptive extraction (SBSE), respectively. According to the measured odor activity values (OAV), only 11 and 15 aroma compounds had OAVs >1 in durian juices or wines, among which 2,3-butanedione, 3-methylbutanol, dimethyl sulfide, dimethyl disulfide, methyl ethyl disulfide, ethyl 2-methylbutanoate, ethyl butanoate, and ethyl octanoate were major contributors to the aroma of juices and wines. Partial least-squares regression (PLSR) was used to detect positive correlations between sensory analysis and aroma compounds. The results showed that the attributes were closely related to aroma compounds. PMID:25620380

  10. Evolutionary advantage of diploidal over polyploidal sexual reproduction

    NASA Astrophysics Data System (ADS)

    Sousa, A. O.; Moss de Oliveira, S.; Sá Martins, J. S.

    2003-03-01

    We modify the Penna model for biological aging, which is based on the mutation-accumulation theory, in order to verify if there would be any evolutionary advantage of triploid over diploid organisms. We show that this is not the case, and that diploidal sex is always better than that involving three individuals.

  11. Ethanol fermentation from Jerusalem artichoke powder using Saccharomyces cerevisiae KCCM50549 without pretreatment for inulin hydrolysis.

    PubMed

    Lim, Seok-Hwan; Ryu, Ji-Myoung; Lee, Hongweon; Jeon, Jae Heung; Sok, Dai-Eun; Choi, Eui-Sung

    2011-01-01

    A strain of Saccharomyces cerevisiae, KCCM50549, was found to efficiently ferment the inulin-containing carbohydrates in Jerusalem artichoke without acidic or enzymatic pretreatment prior to fermentation. S. cerevisiae KCCM50549 could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke (up to degree of polymerization (DP) of 15), in contrast to the other S. cerevisiae strain such as NCYC625 that fermented the fructo-oligosaccharides with DP of up to around six. Inulin-fermenting S. cerevisiae KCCM50549 produced c.a. 1.6 times more ethanol from Jerusalem artichoke compared with S. cerevisiae NCYC625. Direct ethanol fermentation of Jerusalem artichoke flour at 180 g/L without any supplements or pretreatments by S. cerevisiae KCCM50549 in a 5 L jar fermentor yielded 36.2 g/L of ethanol within 36 h. The conversion efficiency of inulin-type sugars to ethanol was 70% of the theoretical ethanol yield. PMID:20833540

  12. Evolution of haploid selection in predominantly diploid organisms.

    PubMed

    Otto, Sarah P; Scott, Michael F; Immler, Simone

    2015-12-29

    Diploid organisms manipulate the extent to which their haploid gametes experience selection. Animals typically produce sperm with a diploid complement of most proteins and RNA, limiting selection on the haploid genotype. Plants, however, exhibit extensive expression in pollen, with actively transcribed haploid genomes. Here we analyze models that track the evolution of genes that modify the strength of haploid selection to predict when evolution intensifies and when it dampens the "selective arena" within which male gametes compete for fertilization. Considering deleterious mutations, evolution leads diploid mothers to strengthen selection among haploid sperm/pollen, because this reduces the mutation load inherited by their diploid offspring. If, however, selection acts in opposite directions in haploids and diploids ("ploidally antagonistic selection"), mothers evolve to reduce haploid selection to avoid selectively amplifying alleles harmful to their offspring. Consequently, with maternal control, selection in the haploid phase either is maximized or reaches an intermediate state, depending on the deleterious mutation rate relative to the extent of ploidally antagonistic selection. By contrast, evolution generally leads diploid fathers to mask mutations in their gametes to the maximum extent possible, whenever masking (e.g., through transcript sharing) increases the average fitness of a father's gametes. We discuss the implications of this maternal-paternal conflict over the extent of haploid selection and describe empirical studies needed to refine our understanding of haploid selection among seemingly diploid organisms. PMID:26669442

  13. A diploid wheat TILLING resource for wheat functional genomics

    PubMed Central

    2012-01-01

    Background Triticum monococcum L., an A genome diploid einkorn wheat, was the first domesticated crop. As a diploid, it is attractive genetic model for the study of gene structure and function of wheat-specific traits. Diploid wheat is currently not amenable to reverse genetics approaches such as insertion mutagenesis and post-transcriptional gene silencing strategies. However, TILLING offers a powerful functional genetics approach for wheat gene analysis. Results We developed a TILLING population of 1,532 M2 families using EMS as a mutagen. A total of 67 mutants were obtained for the four genes studied. Waxy gene mutation frequencies are known to be 1/17.6 - 34.4 kb DNA in polyploid wheat TILLING populations. The T. monococcum diploid wheat TILLING population had a mutation frequency of 1/90 kb for the same gene. Lignin biosynthesis pathway genes- COMT1, HCT2, and 4CL1 had mutation frequencies of 1/86 kb, 1/92 kb and 1/100 kb, respectively. The overall mutation frequency of the diploid wheat TILLING population was 1/92 kb. Conclusion The mutation frequency of a diploid wheat TILLING population was found to be higher than that reported for other diploid grasses. The rate, however, is lower than tetraploid and hexaploid wheat TILLING populations because of the higher tolerance of polyploids to mutations. Unlike polyploid wheat, most mutants in diploid wheat have a phenotype amenable to forward and reverse genetic analysis and establish diploid wheat as an attractive model to study gene function in wheat. We estimate that a TILLING population of 5, 520 will be needed to get a non-sense mutation for every wheat gene of interest with 95% probability. PMID:23134614

  14. The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].

    PubMed

    Ellul, P; Ríos, G; Atarés, A; Roig, L A; Serrano, R; Moreno, V

    2003-08-01

    An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species. PMID:12783167

  15. Carboxypeptidase S- and carboxypeptidase Y-deficient mutants of Saccharomyces cerevisiae.

    PubMed

    Wolf, D H; Ehmann, C

    1981-08-01

    A new carboxypeptidase (carboxypeptidase S) was found in a Saccharomyces cerevisiae strain lacking carboxypeptidase Y (D. H. Wolf and U. Weiser, Eur. J. Biochem. 73:553-556, 1977). Mutants devoid of carboxypeptidase S activity were isolated from a mutant strain that was also deficient in carboxypeptidase Y. Four mutants were analyzed in detail and fell into one complementation group. The defect segregated 2:2 in meiotic tetrads. Gene dosage experiments indicated that the mutation might reside in the structural gene of carboxypeptidase S. The absence of both enzymes, carboxypeptidases Y and S, did not affect mitotic growth. Ascopore formation was only slightly affected by the absence of both carboxypeptidases. Protein degradation under conditions of nutrient deprivation and under sporulation conditions showed no obvious alteration in the absence of carboxypeptidases Y and S. When a proteinase B mutation, which led to the absence of proteinase B activity and resulted in the partial reduction of sporulation, was introduced into a mutant lacking both carboxypeptidases, the ability of diploid cells to sporulate was nearly completely lost. Mutants lacking both carboxypeptidases were unable to grow on the dipeptide benzyloxycarbonylglycyl-l-leucine as a sole nitrogen source, which indicates an additional function for carboxypeptidases Y and S in supplying nutrients from exogenous peptides. Catabolite inactivation of fructose-1,6-bisphosphatase, cytoplasmic malate dehydrogenase, and phosphoenolpyruvate carboxykinase and inactivation of nicotin-amide adenine dinucleotide phosphate-dependent, glutamate dehydrogenase, events which have been proposed to involve proteolysis in vivo, were not dependent on the presence of carboxypeptidase Y and S. In a mutant lacking both carboxypeptidases, four new proteolytic enzymes with carboxypeptidase activity were detected. PMID:7021530

  16. Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids.

    PubMed

    De Jesus-Gonzalez, L; Weathers, P J

    2003-04-01

    Hairy root cultures of diploid Artemisia annua L. (clone YUT16) grow rapidly and produce the antimalarial sesquiterpene artemisinin. Little is known about how polyploidy affects the growth of transformed hairy roots and the production of secondary metabolites. Using colchicine, we produced four stable tetraploid clones of A. annua L. from the YUT16 hairy root clone. Analysis showed major differences in growth and artemisinin production compared to the diploid clone. Tetraploid clones produced up to six times more artemisinin than the diploid parent. This study provides an initial step in increasing our understanding of the role of polyploidy in secondary metabolite production, especially in hairy roots. PMID:12789527

  17. dipSPAdes: Assembler for Highly Polymorphic Diploid Genomes

    PubMed Central

    Bankevich, Anton; Pevzner, Pavel A.

    2015-01-01

    Abstract While the number of sequenced diploid genomes have been steadily increasing in the last few years, assembly of highly polymorphic (HP) diploid genomes remains challenging. As a result, there is a shortage of tools for assembling HP genomes from the next generation sequencing (NGS) data. The initial approaches to assembling HP genomes were proposed in the pre-NGS era and are not well suited for NGS projects. To address this limitation, we developed the first de Bruijn graph assembler, dipSPAdes, for HP genomes that significantly improves on the state-of-the-art assemblers for HP diploid genomes. PMID:25734602

  18. New phenotypes of functional expression of the mKir2.1 channel in potassium efflux-deficient Saccharomyces cerevisiae strains.

    PubMed

    Kolacna, Lucie; Zimmermannova, Olga; Hasenbrink, Guido; Schwarzer, Sarah; Ludwig, Jost; Lichtenberg-Fraté, Hella; Sychrova, Hana

    2005-12-01

    The functional expression of the mouse Kir2.1 potassium channel in yeast cells lacking transport systems for potassium and sodium efflux (ena1-4delta nha1delta) resulted in increased cell sensitivity to high external concentrations of potassium. The phenotype depended on the level of Kir2.1 expression and on the external pH. The activity of Kir2.1p in the yeast cells was almost negligible at pH 3.0 and the highest at pH 7.0. Kir2.1p was permeable for both potassium and rubidium cations, but neither sodium nor lithium were transported via the channel. Measurements of the cation contents in cells confirmed the higher concentration of potassium in cells with Kir2.1p. Specific inhibition of the mKir2.1 channel activity by Ba2+ cations was observed. The use of a mutant strain lacking both potassium efflux and uptake transporters (ena1-4delta nha1delta trk1delta trk2delta) enabled the monitoring of channel activity on two levels--the provision of the necessary amount of intracellular K+ in media with low potassium concentrations, and simultaneously, the channel's contribution to cell potassium sensitivity in the presence of high external K+. This combination of mutations proved to be a new, sensitive and practical tool for characterizing the properties of heterologously expressed transporters mediating both the efflux and influx of alkali-metal-cations. PMID:16358319

  19. Methionine catabolism in Saccharomyces cerevisiae.

    PubMed

    Perpète, Philippe; Duthoit, Olivier; De Maeyer, Simon; Imray, Louise; Lawton, Andrew I; Stavropoulos, Konstantinos E; Gitonga, Virginia W; Hewlins, Michael J E; Dickinson, J Richard

    2006-01-01

    The catabolism of methionine to methionol and methanethiol in Saccharomyces cerevisiae was studied using (13)C NMR spectroscopy, GC-MS, enzyme assays and a number of mutants. Methionine is first transaminated to alpha-keto-gamma-(methylthio)butyrate. Methionol is formed by a decarboxylation reaction, which yields methional, followed by reduction. The decarboxylation is effected specifically by Ydr380wp. Methanethiol is formed from both methionine and alpha-keto-gamma-(methylthio)butyrate by a demethiolase activity. In all except one strain examined, demethiolase was induced by the presence of methionine in the growth medium. This pathway results in the production of alpha-ketobutyrate, a carbon skeleton, which can be re-utilized. Hence, methionine catabolism is more complex and economical than the other amino acid catabolic pathways in yeast, which use the Ehrlich pathway and result solely in the formation of a fusel alcohol. PMID:16423070

  20. Creation of a synthetic xylose-inducible promoter for Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae is currently used to produce ethanol from glucose, but it cannot utilize five-carbon sugars contained in the hemicellulose component of biomass feedstocks. S. cerevisiae strains engineered for xylose fermentation have been made using constitutive promoters to express the req...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. A new model for disruption of the ornithine decarboxylase gene, SPE1, in Saccharomyces cerevisiae exhibits growth arrest and genetic instability at the MAT locus.

    PubMed Central

    Schwartz, B; Hittelman, A; Daneshvar, L; Basu, H S; Marton, L J; Feuerstein, B G

    1995-01-01

    Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination. Images Figure 3 Figure 10 PMID:7492339

  3. The canonical equation of adaptive dynamics for Mendelian diploids and haplo-diploids

    PubMed Central

    Metz, Johan A. J.; de Kovel, Carolien G. F.

    2013-01-01

    One of the powerful tools of adaptive dynamics is its so-called canonical equation (CE), a differential equation describing how the prevailing trait vector changes over evolutionary time. The derivation of the CE is based on two simplifying assumptions, separation of population dynamical and mutational time scales and small mutational steps. (It may appear that these two conditions rarely go together. However, for small step sizes the time-scale separation need not be very strict.) The CE was derived in 1996, with mathematical rigour being added in 2003. Both papers consider only well-mixed clonal populations with the simplest possible life histories. In 2008, the CE's reach was heuristically extended to locally well-mixed populations with general life histories. We, again heuristically, extend it further to Mendelian diploids and haplo-diploids. Away from strict time-scale separation the CE does an even better approximation job in the Mendelian than in the clonal case owing to gene substitutions occurring effectively in parallel, which obviates slowing down by clonal interference. PMID:24516713

  4. Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption.

    PubMed

    Scalcinati, Gionata; Otero, José Manuel; Van Vleet, Jennifer R H; Jeffries, Thomas W; Olsson, Lisbeth; Nielsen, Jens

    2012-08-01

    Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S. cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source. PMID:22487265

  5. Isolation and properties of genetically defined strains of the methylotrophic yeast Hansenula polymorpha CBS4732.

    PubMed

    Lahtchev, Kantcho L; Semenova, Vika D; Tolstorukov, Ilia I; van der Klei, Ida; Veenhuis, Marten

    2002-02-01

    Genetically defined strains of the yeast Hansenula polymorpha were constructed from a clone of H. polymorpha CBS4732 with very low mating and sporulation abilities. Mating, spore viability, and the percentage of four-spore-containing asci were increased to a level at which tetrad analysis was possible. Auxotrophic mutations in 30 genes were isolated and used to construct strains with multiple markers for mapping studies, transformation with plasmid DNA, and mutant screening. Various other types of mutants were isolated and characterized, among them mutants that displayed an altered morphology, methanol-utilization deficient mutants and strains impaired in the biosynthesis of alcohol oxidase and catalase. Also, the mutability of H. polymorpha CBS4732 vs H. polymorpha NCYC495 was compared. The data revealed clear differences in frequencies of appearance and mutational spectra of some mutants isolated. Many of the mutants isolated had good mating abilities, and diploids resulting from their crossing displayed high sporulation frequencies and high spore viability. Most of the markers used revealed normal Mendelian segregation during meiosis. The frequency of tetratype spore formation was lower than in Saccharomyces cerevisiae suggesting a lower frequency of recombination during the second meiotic division. The properties of genetically defined strains of H. polymorpha CBS4732 as well as their advantages for genetics and molecular studies are discussed. PMID:11807564

  6. Ancestries of a recombining diploid population.

    PubMed

    Sainudiin, R; Thatte, B; Véber, A

    2016-01-01

    We derive the exact one-step transition probabilities of the number of lineages that are ancestral to a random sample from the current generation of a bi-parental population that is evolving under the discrete Wright-Fisher model with n diploid individuals. Our model allows for a per-generation recombination probability of r . When r = 1, our model is equivalent to Chang's (Adv Appl Probab 31:1002-1038, 1999) model for the karyotic pedigree. When r = 0, our model is equivalent to Kingman's (Stoch Process Appl 13:235-248, 1982) discrete coalescent model for the cytoplasmic tree or sub-karyotic tree containing a DNA locus that is free of intra-locus recombination. When 0 < r < 1 our model can be thought to track a sub-karyotic ancestral graph containing a DNA sequence from an autosomal chromosome that has an intra-locus recombination probability r . Thus, our family of models indexed by r ∈ [0, 1] connects Kingman's discrete coalescent to Chang's pedigree in a continuous way as r goes from 0 to 1. For large populations, we also study three properties of the ancestral process corresponding to a given r ∈ (0, 1): the time Tn to a most recent common ancestor (MRCA) of the population, the time Un at which all individuals are either common ancestors of all present day individuals or ancestral to none of them, and the fraction of individuals that are common ancestors at time Un. These results generalize the three main results of Chang's (Adv Appl Probab 31:1002-1038, 1999). When we appropriately rescale time and recombination probability by the population size, our model leads to the continuous time Markov chain called the ancestral recombination graph of Hudson (Theor Popul Biol 23:183-201, 1983) and Griffiths (The two-locus ancestral graph, Institute of Mathematical Statistics 100-117, 1991). PMID:25925241

  7. Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and. cap alpha. factor pheromones

    SciTech Connect

    Chan, R.K.; Otte, C.A.

    1982-01-01

    Eight independently isolated mutants which are supersensitive (Sst/sup -/) to the G1 arrest induced by the tridecapeptide pheromone ..cap alpha.. factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by ..cap alpha.. factor. These mutants carries lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to ..cap alpha.. factor, but MAT..cap alpha.. sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT..cap alpha.. cells. Even in the absence of added ..cap alpha.. pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology (''shmoo'' shape) that normally develops only after MATa cells are exposed to ..cap alpha.. factor. This ''self-shmooing'' phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT..cap alpha.. diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT..cap alpha.. sst2-1/sst2-1) were still insensitive to ..cap alpha.. factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked nor centromere distal to MAT on the right arm of chromosome III.

  8. Sex-determination system in the diploid yeast Zygosaccharomyces sapae.

    PubMed

    Solieri, Lisa; Dakal, Tikam Chand; Giudici, Paolo; Cassanelli, Stefano

    2014-06-01

    Sexual reproduction and breeding systems are driving forces for genetic diversity. The mating-type (MAT) locus represents a mutation and chromosome rearrangement hotspot in yeasts. Zygosaccharomyces rouxii complex yeasts are naturally faced with hostile low water activity (aw) environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy. Here, we investigated sex-determination system in Zygosaccharomyces sapae diploid strain ABT301(T), a member of the Z. rouxii complex. We cloned three divergent mating type-like (MTL) α-idiomorph sequences and designated them as ZsMTLα copies 1, 2, and 3. They encode homologs of Z. rouxii CBS 732(T) MATα2 (amino acid sequence identities spanning from 67.0 to 99.5%) and MATα1 (identity range 81.5-99.5%). ABT301(T) possesses two divergent HO genes encoding distinct endonucleases 100% and 92.3% identical to Z. rouxii HO. Cloning of MATA: -idiomorph resulted in a single ZsMTLA: locus encoding two Z. rouxii-like proteins MATA: 1 and MATA: 2. To assign the cloned ZsMTLα and ZsMTLA: idiomorphs as MAT, HML, and HMR cassettes, we analyzed their flanking regions. Three ZsMTLα loci exhibited the DIC1-MAT-SLA2 gene order canonical for MAT expression loci. Furthermore, four putative HML cassettes were identified, two containing the ZsMTLα copy 1 and the remaining harboring ZsMTLα copies 2 and 3. Finally, the ZsMTLA: locus was 3'-flanked by SLA2, suggesting the status of MAT expression locus. In conclusion, Z. sapae ABT301(T) displays an aααα genotype missing of the HMR silent cassette. Our results demonstrated that mating-type switching is a hypermutagenic process in Z. rouxii complex that generates genetic diversity de novo. This error-prone mechanism could be suitable to generate progenies more rapidly adaptable to hostile environments. PMID:24939186

  9. Development of rapidly fermenting strains of Saccharomyces diastaticus for direct conversion of starch and dextrins to ethanol

    SciTech Connect

    Laluce, C.; Mattoon, J.R.

    1984-07-01

    Alcoholic fermentation, growth, and glucoamylase production by 12 strains of Saccharomyces diastaticus were compared by using starch and dextrins as substrates. Haploid progeny produced from a rapidly fermenting strain, SD2, were used for hybridization with other S. diastaticus and Saccharomyces cerevisiae haploids. Alcoholic fermentation and enzyme production by hybrid diploids and their haploid parents were evaluated. Although the dosage of the STA or DEX (starch or dextrin fermentation) genes may enhance ethanol production, epistatic effects in certain strain combinations caused decreases in starch-fermenting activity. Both the nature of the starch or dextrin used and the fermentation medium pH had substantial effects on alcohol production. Commercial dextrin was not as good a substrate as dextrins prepared by digesting starch with ..cap alpha..-amylase. Crude manioc starch digested by ..cap alpha..-amylase was fermented directly by selected hybrids with almost 100% conversion efficiency. The manioc preparation contained adequate minerals and growth factors. This procedure should be suitable for direct commercial application in manioc-producing regions in Brazil and elsewhere. A rapidly fermenting haploid strain, SD2-A8, descended from strain SD2, contains two unlinked genes controlling formation of extracellular amylase. A convenient method for detecting these genes (STA genes) in replica plates containing large numbers of meiotic progeny was developed.

  10. Genotoxicity of Achillea millefolium essential oil in diploid cells of Aspergillus nidulans.

    PubMed

    de Sant'anna, Juliane Rocha; Franco, Claudinéia Conationi da Silva; Miyamoto, Claudia Tiemi; Cunico, Miriam Machado; Miguel, Obdulio Gomes; Côcco, Lílian Cristina; Yamamoto, Carlos Itsuo; Junior, Cirino Corrêa; de Castro-Prado, Marialba Avezum Alves

    2009-02-01

    The essential oil of Achillea millefolium is commonly used in folk medicine for the treatment of several diseases and has been demonstrated previously to exert an in vitro antimicrobial activity against human pathogens. Current study investigates the genotoxic activity of A. millefolium oil. The oil's major constituents are: chamazulene (42.15%), sabinene (19.72%), terpin-4-ol (5.22%), beta-caryophyllene (4.44%) and eucalyptol (3.10%), comprising 74.63% of the total. The oil's genotoxic evaluation was performed at concentrations of 0.13 microL/mL, 0.19 microL/mL and 0.25 microL/mL with a heterozygous diploid strain of Aspergillus nidulans, named A757//UT448, with green conidia. A statistically significant increasing number of yellow and white mitotic recombinants, per colony, of the diploid strain was reported after oil treatment with 0.19 microL/mL and 0.25 microL/mL concentrations. The genotoxicity of the oil was associated with the induction of mitotic non-disjunction or crossing-over by oil. PMID:18803228

  11. Investigation of fatty acid accumulation in the engineered Saccharomyces cerevisiae under nitrogen limited culture condition.

    PubMed

    Tang, Xiaoling; Chen, Wei Ning

    2014-06-01

    In this study, the Saccharomyces cerevisiae wild type strain and engineered strain with an overexpressed heterologous ATP-citrate lyase (acl) were cultured in medium with different carbon and nitrogen concentrations, and their fatty acid production levels were investigated. The results showed that when the S. cerevisiae engineered strain was cultivated under nitrogen limited culture condition, the yield of mono-unsaturated fatty acids showed higher than that under non-nitrogen limited condition; with the carbon concentration increased, the accumulation become more apparent, whereas in the wild type strain, no such correlation was found. Besides, the citrate level in the S. cerevisiae under nitrogen limited condition was found to be much higher than that under non-nitrogen limited condition, which indicated a relationship between the diminution of nitrogen and accumulation of citrate in the S. cerevisiae. The accumulated citrate could be further cleaved by acl to provide substrate for fatty acid synthesis. PMID:24755317

  12. Effects of the rad52 gene on recombination in Saccharomyces cerevisiae

    SciTech Connect

    Prakash, S.; Prakash, L.; Burke, W.; Montelone, B.A.

    1980-01-01

    Effects of the rad 52 mutation in Saccharomyces cerevisiae on meiotic, ..gamma..-ray-induced, uv-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the his1-1/his1-315 and trp-5-2/trp5-48 heteroalleles. Gene-centromere recombination also was not observed in rad/52/rad52 diploids. No ..gamma..-ray- or uv-induced intragenic mitotic recombination is seen in rad52/rad52 diploids. The rate of spontaneous mitotic recombination is lowered five-fold at the his1-1/his1-315 and leu1-c/leu1-12 heteroalleles. Spontaneous reversion rates of both his1-1 and his1-315 were elevated 10 to 20 fold in rad52/rad52 diploids. The RAD52 gene function is required for spontaneous mitotic recombination, uv- and ..gamma..-ray-induced mitotic recombination and mitotic recombination.

  13. The Induction of Mitotic Gene Conversion by X-Irradiation of Haploid SACCHAROMYCES CEREVISIAE

    PubMed Central

    Campbell, D. A.

    1973-01-01

    Mitotic recombination in Saccharomyces cerevisiae was examined by means of experiments in which one of the haploid parents was X-irradiated prior to zygote formation. By this method radiation-induced lesions are restricted to only one of the two non-sister chromatids that may be expected to undergo mitotic exchange in the diploid. The principal results of this work are: (1) X-irradiated haploid cells that are incapable of further vegetative growth (colony formation) are efficiently rescued into viable diploids by mating with unirradiated haploid cells. (2) X-rays delivered to only one of the two haploid parents are recombinogenic in the resultant diploid. The frequency of detected recombinational events increases as a probable linear function of the X-ray dose. (3) A majority of the induced recombinational events are nonreciprocal in nature (mitotic gene conversion). These results complement those obtained from X-irradiation of the vegetative diploid itself, where the induced genetic exchanges are principally reciprocal. PMID:17248615

  14. Evaluation of yeast strains for production of fuel ethanol from biomass hydrolysates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Robust industrial yeast strains are needed for profitable production of fuel ethanol from mixed biomass waste. USDA, ARS, NCAUR, RPT has been evaluating ethanol-producing yeasts, including Saccharomyces cerevisiae, engineered GMAX Saccharomyces cerevisiae, irradiated Kluyveromyces marxianus, and Pi...

  15. Escape from Het-6 Incompatibility in Neurospora Crassa Partial Diploids Involves Preferential Deletion within the Ectopic Segment

    PubMed Central

    Smith, M. L.; Yang, C. J.; Metzenberg, R. L.; Glass, N. L.

    1996-01-01

    Self-incompatible het-6(OR)/het-6(PA) partial diploids of Neurospora crassa were selected from a cross involving the translocation strain, T(IIL -> IIIR)AR18, and a normal sequence strain. About 25% of the partial diploids exhibited a marked increase in growth rate after 2 weeks, indicating that ``escape'' from het-6 incompatibility had occurred. Near isogenic tester strains with different alleles (het-6(OR) and het-6(PA)) were constructed and used to determine that 80 of 96 escape strains tested were het-6(PA), retaining the het-6 allele found in the normal-sequence LGII position; 16 were het-6(OR), retaining the allele in the translocated position. Restriction fragment length polymorphisms in 45 escape strains were examined with probes made from cosmids that spanned the translocated region. Along with electrophoretic analysis of chromosomes from three escape strains, RFLPs showed that escape is associated with deletion of part of one or the other of the duplicated DNA segments. Deletions ranged in size from ~70 kbp up to putatively the entire 270-kbp translocated region but always included a 35-kbp region wherein we hypothesize het-6 is located. The deletion spectrum at het-6 thus resembles other cases where mitotic deletions occur such as of tumor suppressor genes and of the hprt gene (coding for hypoxanthine-guanine phosphoribosyl-transferase) in humans. PMID:8889517

  16. Cell death caused by excision of centromeric DNA from a chromosome in Saccharomyces cerevisiae.

    PubMed

    Miyamoto, Akihiro; Yanamoto, Toshiaki; Matsumoto, Takehiro; Hatano, Takushi; Matsuzaki, Hiroaki

    2013-01-01

    If genetically modified organisms (GMOs) are spread through the natural environment, it might affect the natural environment. To help prevent the spread of GMOs, we examined whether it is possible to introduce conditional lethality by excising centromeric DNA from a chromosome by site-specific recombination in Saccharomyces cerevisiae as model organism. First, we constructed haploid cells in which excision of the centromeric DNA from chromosome IV can occur due to recombinase induced by galactose. By this excision, cell death can occur. In diploid cells, cell death can also occur by excision from both homologous chromosomes IV. Furthermore, cell death can occur in the case of chromosome V. A small number of surviving cells appeared with excision of centromeric DNA, and the diploid showed greater viability than the haploid in both chromosomes IV and V. The surviving cells appeared mainly due to deletion of a recombination target site (RS) from the chromosome. PMID:24018677

  17. Apparent Epigenetic Meiotic Double-Strand-Break Disparity in Saccharomyces cerevisiae: A Meta-Analysis

    PubMed Central

    Stahl, Franklin W.; Rehan, Maryam Binti Mohamed; Foss, Henriette M.; Borts, Rhona H.

    2016-01-01

    Previously published, and some unpublished, tetrad data from budding yeast (Saccharomyces cerevisiae) are analyzed for disparity in gene conversion, in which one allele is more often favored than the other (conversion disparity). One such disparity, characteristic of a bias in the frequencies of meiotic double-strand DNA breaks at the hotspot near the His4 locus, is found in diploids that undergo meiosis soon after their formation, but not in diploids that have been cloned and frozen. Altered meiotic DNA breakability associated with altered metabolism-related chromatin states has been previously reported. However, the above observations imply that such differing parental chromatin states can persist through at least one chromosome replication, and probably more, in a common environment. This conclusion may have implications for interpreting changes in allele frequencies in populations. PMID:27356614

  18. Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae.

    PubMed

    Mormeneo, María; Pastor, Fi Javier; Zueco, Jesús

    2012-01-01

    The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western immunoblot using Pir-specific antibodies, while enzymatic activity on carboxymethyl cellulose was demonstrated on plate assays, zymographic analysis and colorimetric assays. Hyperglycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not affect activity, and values of 1.2 U/ml were obtained in growth medium supernatants in ordinary batch cultures after 24 h. These values compare quite favorably with those described for other recombinant endoglucanases expressed in S. cerevisiae. This is one of the few reports describing the expression of Bacillus cellulases in S. cerevisiae, since yeast expressed recombinant cellulases have been mostly of fungal origin. It is also the first report of the yeast expression of this particular endoglucanase. PMID:21701899

  19. Genome size variation in diploid and tetraploid wild wheats

    PubMed Central

    Özkan, Hakan; Tuna, Metin; Kilian, Benjamin; Mori, Naoki; Ohta, Shoji

    2010-01-01

    Background and aims Intra- and interspecific variations of C-values and the relationship between habitat factors and genome size were studied in natural populations of diploid and tetraploid wild wheats. Methodology The 1C nuclear DNA content of 376 individual plants representing 41 populations of diploid and tetraploid wild wheats was determined by flow cytometry (FCM) and correlated with geographical and bioclimate variables. Principal results Based on analysis of variance, significant differences between diploid and tetraploid Triticum species were found. Differences among populations of T. boeoticum and T. dicoccoides were also statistically significant and argue for isolation between populations, except for T. araraticum. However, the variation among individuals of the same population was not statistically significant. Maximum genome size differences among populations for T. boeoticum (0.143 pg; 2.32 %), T. dicoccoides (0.314 pg; 2.49 %) and T. araraticum (0.116 pg; 0.98 %) argue for genome constancy in these species. There was no significant correlation between intra-population variance and geographical and bioclimate variables for T. boeoticum and T. dicoccoides. In contrast to the limited genome size variation at the intraspecific level, the interspecific variation was large: ∼0.5 pg/1C (8 %) at the diploid level (T. boeoticum vs. T. urartu) and ∼1 pg/1C (9.7 %) at the tetraploid level (T. dicoccoides vs. T. araraticum). Conclusions Low intraspecific genome size variation occurs in diploid and tetraploid wild wheats, and this limited variation is not correlated with geographical and climate variables. However, interspecific variation is significant at the diploid and tetraploid level. It can be concluded that the genome size of wild self-fertilizing Triticum species is generally stable, despite the presence of many potentially active retroelements. In natural habitats, it is very difficult to distinguish wild wheats from each other. However, all four

  20. Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae

    PubMed Central

    Pablo-Hernando, M Evangelina; Arnaiz-Pita, Yolanda; Tachikawa, Hiroyuki; del Rey, Francisco; Neiman, Aaron M; Vázquez de Aldana, Carlos R

    2008-01-01

    Background In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. Results Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. Conclusion Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources. PMID:18826657

  1. Characterization of Alcohol-induced Filamentous Growth in Saccharomyces cerevisiae

    PubMed Central

    Lorenz, Michael C.; Cutler, N. Shane; Heitman, Joseph

    2000-01-01

    Diploid cells of the budding yeast Saccharomyces cerevisiae starved for nitrogen differentiate into a filamentous growth form. Poor carbon sources such as starches can also stimulate filamentation, whereas haploid cells undergo a similar invasive growth response in rich medium. Previous work has demonstrated a role for various alcohols, by-products of amino acid metabolism, in altering cellular morphology. We found that several alcohols, notably isoamyl alcohol and 1-butanol, stimulate filamentous growth in haploid cells in which this differentiation is normally repressed. Butanol also induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology, even in liquid medium. The filamentous colony morphology and cell elongation require elements of the pheromone-responsive MAPK cascade and TEC1, whereas components of the nutrient-sensing machinery, such as MEP2, GPA2, and GPR1, do not affect this phenomenon. A screen for 1-butanol–insensitive mutants identified additional proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG1), mitochondrial function (MSM1, MRP21, and HMI1), and a transcriptional regulator (CHD1). Furthermore, we have also found that ethanol stimulates hyperfilamentation in diploid cells, again in a MAPK-dependent manner. Together, these results suggest that yeast may sense a combination of nutrient limitation and metabolic by-products to regulate differentiation. PMID:10637301

  2. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Nevoigt, Elke

    2008-01-01

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

  3. Killer systems of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Nesterova, G.F.

    1989-01-01

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

  4. Differential gene expression and alternative splicing between diploid and tetraploid watermelon lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Synthetic tetraploid plants have been used for production of seedless triploid watermelon lines being pollinated with diploid plants. When compared to their diploid or triploid counterparts, the tetraploid exhibit wide phenotypic differences. Though many factors, including alternative splicing (AS),...

  5. Identification of the genetic locus for the structural gene and a new regulatory gene for the synthesis of repressible alkaline phosphatase in Saccharomyces cerevisiae

    SciTech Connect

    Kaneko, Y.; Toh-e, A.; Oshima, Y.

    1982-02-01

    Two lines of evidence showed that the PHO8 gene encodes the structure of repressible, nonspecific alkaline phosphatase in Saccharomyces cerevisiae: (I) the enzyme produced by a temperature-sensitive pho8 mutant at the permissive temperature (25/sup 0/C) was more thermolabile than that of the wild-type strain, and (II) the PHO8 gene showed a gene dosage effect on the enzyme activity. The pho8 locus has been mapped on chromosome IV, 8 centimorgans distal to rna3. A new mutant carrying the pho9 gene was isolated which lacks repressible alkaline phosphatase, but has the normal phenotype for the synthesis of repressible acid phosphatase. The pho9 gene segregated independently of all known pho-regulatory genes and did not show the gene dosage effect on repressible alkaline phosphatase activity. The pho9/pho9 diploid hardly sporulated and showed no commitment to intragenic recombination when it was inoculated on sporulation medium. Hence the pho9 mutant has a phenotype similar to the pep4 mutant, which was isolated as a pleiotropic mutant with reduced levels of proteinases A and B carboxypeptidase Y. An allelism test indicated that pho9 and pep4 are allelic.

  6. Fumaric Acid Production in Saccharomyces cerevisiae by In Silico Aided Metabolic Engineering

    PubMed Central

    Xu, Guoqiang; Zou, Wei; Chen, Xiulai; Xu, Nan; Liu, Liming; Chen, Jian

    2012-01-01

    Fumaric acid (FA) is a promising biomass-derived building-block chemical. Bio-based FA production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here we report on FA production by direct fermentation using metabolically engineered Saccharomyces cerevisiae with the aid of in silico analysis of a genome-scale metabolic model. First, FUM1 was selected as the target gene on the basis of extensive literature mining. Flux balance analysis (FBA) revealed that FUM1 deletion can lead to FA production and slightly lower growth of S. cerevisiae. The engineered S. cerevisiae strain obtained by deleting FUM1 can produce FA up to a concentration of 610±31 mg L–1 without any apparent change in growth in fed-batch culture. FT-IR and 1H and 13C NMR spectra confirmed that FA was synthesized by the engineered S. cerevisiae strain. FBA identified pyruvate carboxylase as one of the factors limiting higher FA production. When the RoPYC gene was introduced, S. cerevisiae produced 1134±48 mg L–1 FA. Furthermore, the final engineered S. cerevisiae strain was able to produce 1675±52 mg L–1 FA in batch culture when the SFC1 gene encoding a succinate–fumarate transporter was introduced. These results demonstrate that the model shows great predictive capability for metabolic engineering. Moreover, FA production in S. cerevisiae can be efficiently developed with the aid of in silico metabolic engineering. PMID:23300594

  7. Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae strains expressing xylose isomerase (XI) produce some of the highest reported ethanol yields from xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are not functional, require additional strain modification, and have low affinity for xylose...

  8. Inheritance of carotenoid content in tetraploid and diploid potato crosses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carotenoids have a wide range of human health benefits. Yellow-fleshed tetraploid potato cultivars have more than twice the concentration of carotenoids as white-fleshed cultivars. However, carotenoid concentrations in some diploid potatoes have been reported to be up to 13 times higher than in ‘Y...

  9. Genotyping by sequencing of a diploid potato F2 population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genotyping-by-sequencing (GBS) of multiplexed, restriction-site associated DNA (RAD) libraries is an attractive technology for generating genome-wide markers because of its technical simplicity and low costs per sample. To investigate its feasibility for potato, a diploid F2 population (S. tuberosum...

  10. Selection on Meiosis Genes in Diploid and Tetraploid Arabidopsis arenosa

    PubMed Central

    Wright, Kevin M.; Arnold, Brian; Xue, Katherine; Šurinová, Maria; O’Connell, Jeremy; Bomblies, Kirsten

    2015-01-01

    Meiotic chromosome segregation is critical for fertility across eukaryotes, and core meiotic processes are well conserved even between kingdoms. Nevertheless, recent work in animals has shown that at least some meiosis genes are highly diverse or strongly differentiated among populations. What drives this remains largely unknown. We previously showed that autotetraploid Arabidopsis arenosa evolved stable meiosis, likely through reduced crossover rates, and that associated with this there is strong evidence for selection in a subset of meiosis genes known to affect axis formation, synapsis, and crossover frequency. Here, we use genome-wide data to study the molecular evolution of 70 meiosis genes in a much wider sample of A. arenosa. We sample the polyploid lineage, a diploid lineage from the Carpathian Mountains, and a more distantly related diploid lineage from the adjacent, but biogeographically distinct Pannonian Basin. We find that not only did selection act on meiosis genes in the polyploid lineage but also independently on a smaller subset of meiosis genes in Pannonian diploids. Functionally related genes are targeted by selection in these distinct contexts, and in two cases, independent sweeps occurred in the same loci. The tetraploid lineage has sustained selection on more genes, has more amino acid changes in each, and these more often affect conserved or potentially functional sites. We hypothesize that Pannonian diploid and tetraploid A. arenosa experienced selection on structural proteins that mediate sister chromatid cohesion, the formation of meiotic chromosome axes, and synapsis, likely for different underlying reasons. PMID:25543117

  11. 'Don' a Diploid Falcata Alfalfa for Western US Rangelands

    Technology Transfer Automated Retrieval System (TEKTRAN)

    'Don' (Reg. No. CV-______, PI _______) a diploid falcata alfalfa (Medicago sativa subsp falcata L.) developed by the Forage and Range Research Laboratory in Logan, Utah, in cooperation with the Utah Agricultural Experiment Station, Utah State University. Recent interest in falcata alfalfa has been ...

  12. Does hybridization drive the transition to asexuality in diploid Boechera?

    PubMed

    Beck, James B; Alexander, Patrick J; Allphin, Loreen; Al-Shehbaz, Ihsan A; Rushworth, Catherine; Bailey, C Donovan; Windham, Michael D

    2012-04-01

    Gametophytic apomixis is a common form of asexual reproduction in plants. Virtually all gametophytic apomicts are polyploids, and some view polyploidy as a prerequisite for the transition to apomixis. However, any causal link between apomixis and polyploidy is complicated by the fact that most apomictic polyploids are allopolyploids, leading some to speculate that hybridization, rather than polyploidy, enables apomixis. Diploid apomixis presents a rare opportunity to isolate the role of hybridization, and a number of diploid apomicts have been documented in the genus Boechera (Brassicaceae). Here, we present the results of a microsatellite study of 1393 morphologically and geographically diverse diploid individuals, evaluating the hypothesis that diploid Boechera apomicts are hybrids. This genus-wide dataset was made possible by the applicability of a core set of microsatellite loci in 69 of the 70 diploid Boechera species and by our ability to successfully genotype herbarium specimens of widely varying ages. With few exceptions, diploid apomicts exhibited markedly high levels of heterozygosity resulting from the combination of disparate genomes. This strongly suggests that most apomictic diploid Boechera lineages are of hybrid origin, and that the genomic consequences of hybridization allow for the transition to gametophytic apomixis in this genus. PMID:22486684

  13. Hybridization barriers between diploid Solanum tuberosum and wild Solanum raphanifolium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wild potato germplasm represents a unique, diverse and accessible resource for disease and pest resistance, along with useful agronomic traits that may be introgressed into the cultivated potato (Solanum tuberosum L.). Hybridization of diploid wild Solanum species with haploids (2x) of cultivated po...

  14. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    DOEpatents

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2001-10-01

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

  17. No Need to Discriminate? Reproductive Diploid Males in a Parasitoid with Complementary Sex Determination

    PubMed Central

    Elias, Jan; Mazzi, Dominique; Dorn, Silvia

    2009-01-01

    Diploid males in hymenopterans are generally either inviable or sterile, thus imposing a severe genetic load on populations. In species with the widespread single locus complementary sex determination (sl-CSD), sex depends on the genotype at one single locus with multiple alleles. Haploid (hemizygous) individuals are always males. Diploid individuals develop into females when heterozygous and into males when homozygous at the sex determining locus. Our comparison of the mating and reproductive success of haploid and diploid males revealed that diploid males of the braconid parasitoid Cotesia glomerata sire viable and fertile diploid daughters. Females mated to diploid males, however, produced fewer daughters than females mated to haploid males. Nevertheless, females did not discriminate against diploid males as mating partners. Diploid males initiated courtship display sooner than haploid males and were larger in body size. Although in most species so far examined diploid males were recognized as genetic dead ends, we present a second example of a species with sl-CSD and commonly occurring functionally reproductive diploid males. Our study suggests that functionally reproductive diploid males might not be as rare as hitherto assumed. We argue that the frequent occurrence of inbreeding in combination with imperfect behavioural adaptations towards its avoidance promote the evolution of diploid male fertility. PMID:19551142

  18. Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

    PubMed Central

    Paulovich, A. G.; Thompson, J. R.; Larkin, J. C.; Li, Z.; Woolford-Jr., J. L.

    1993-01-01

    The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele. PMID:8293976

  19. Sex-Determination System in the Diploid Yeast Zygosaccharomyces sapae

    PubMed Central

    Solieri, Lisa; Dakal, Tikam Chand; Giudici, Paolo; Cassanelli, Stefano

    2014-01-01

    Sexual reproduction and breeding systems are driving forces for genetic diversity. The mating-type (MAT) locus represents a mutation and chromosome rearrangement hotspot in yeasts. Zygosaccharomyces rouxii complex yeasts are naturally faced with hostile low water activity (aw) environments and are characterized by gene copy number variation, genome instability, and aneuploidy/allodiploidy. Here, we investigated sex-determination system in Zygosaccharomyces sapae diploid strain ABT301T, a member of the Z. rouxii complex. We cloned three divergent mating type-like (MTL) α-idiomorph sequences and designated them as ZsMTLα copies 1, 2, and 3. They encode homologs of Z. rouxii CBS 732T MATα2 (amino acid sequence identities spanning from 67.0 to 99.5%) and MATα1 (identity range 81.5–99.5%). ABT301T possesses two divergent HO genes encoding distinct endonucleases 100% and 92.3% identical to Z. rouxii HO. Cloning of MATa-idiomorph resulted in a single ZsMTLa locus encoding two Z. rouxii-like proteins MATa1 and MATa2. To assign the cloned ZsMTLα and ZsMTLa idiomorphs as MAT, HML, and HMR cassettes, we analyzed their flanking regions. Three ZsMTLα loci exhibited the DIC1-MAT-SLA2 gene order canonical for MAT expression loci. Furthermore, four putative HML cassettes were identified, two containing the ZsMTLα copy 1 and the remaining harboring ZsMTLα copies 2 and 3. Finally, the ZsMTLa locus was 3′-flanked by SLA2, suggesting the status of MAT expression locus. In conclusion, Z. sapae ABT301T displays an aααα genotype missing of the HMR silent cassette. Our results demonstrated that mating-type switching is a hypermutagenic process in Z. rouxii complex that generates genetic diversity de novo. This error-prone mechanism could be suitable to generate progenies more rapidly adaptable to hostile environments. PMID:24939186

  20. The relationship between enzyme activity, cell geometry, and fitness in Saccharomyces cerevisiae.

    PubMed Central

    Weiss, R L; Kukora, J R; Adams, J

    1975-01-01

    The relationship between enzyme activity, cell geometry, and the ploidy levels has been investigated in Saccharomyces cerevisiae. Diploid cells have 1.57 times the volume of haploid cells under nonlimiting growth conditions (minimal medium). However, when diploid cells are grown under conditions of carbon limitation, they have the same volume as haploid cells. Thus, by altering the environmental conditions, cell size can be varied independently of the degree of ploidy. The results indicate that the basic biochemical parameters of the cell are primarily determined by cell geometry rather than ploidy level. RNA content, protein content, and ornithine transcarbamylase (carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3), tryptophan synthetase [L-serine hydro-lyase (adding indole), EC 4.2.1.20], and invertase (alpha-D-glucoside glucohydrolase, Ec 3.2.1.20) activity are related to cell volume, whereas acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) activity, a cell surface enzyme, is related to the surface area of the cells. Fitness is determined by the activity of certain cell surface enzymes, such as acid phosphatase, diploids would be expected to have a lower fitness than haploids because of the lower surface area/volume ratio. However, when fitness is determined by the activity of an internal enzyme, diploids would be expected to have the same fitness as haploids. Results from competition experiments between haploids and diploids are consistent with these predictions. The significance of these results to the evolution of diploidy as the predominant phase of the life cycle of higher plants and animals is discussed. PMID:1093169

  1. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  2. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  3. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... techniques, antibodies to S. cerevisiae (baker's or brewer's yeast) in human serum or plasma. Detection of S... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test...

  5. Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae.

    PubMed

    Shen, Qing-Ji; Kassim, Hakimi; Huang, Yong; Li, Hui; Zhang, Jing; Li, Guang; Wang, Peng-Ye; Yan, Jun; Ye, Fangfu; Liu, Ji-Long

    2016-06-20

    Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filament-forming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frame-GFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases (Ura7p and Ura8p) and two asparagine synthetases (Asn1p and Asn2p) form filaments both in the cytoplasm and in the nucleus. Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated. PMID:27312010

  6. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth. Images PMID:6217414

  7. [Production of rabies vaccine in animal diploid cells].

    PubMed

    Lucas, G; Reculard, P; Adamowicz, P; Vacher, B; Prunet, P

    1982-01-01

    Modalities for production of inactivated rabies vaccine derived from diploid hamster cell cultures are reported. The inactivated concentrated virus, purified by zonal centrifugation, is utilised for the preparation of vaccines destinated to carnivores, either in the form of monovalent vaccine or associated with distemper and canine contagious hepatitis vaccines. The inactivated concentrated virus is utilised for the preparation of bovine vaccine. The procedure is compatible with industrial production. The results concerning safety and potency tests of the experimental lots are presented. PMID:7128072

  8. Sexual conflict and the alternation of haploid and diploid generations

    PubMed Central

    Haig, David; Wilczek, Amity

    2006-01-01

    Land plants possess a multicellular diploid stage (sporophyte) that begins development while attached to a multicellular haploid progenitor (gametophyte). Although the closest algal relatives of land plants lack a multicellular sporophyte, they do produce a zygote that grows while attached to the maternal gametophyte. The diploid offspring shares one haploid set of genes with the haploid mother that supplies it with resources and a paternal haploid complement that is not shared with the mother. Sexual conflict can arise within the diploid offspring because the offspring's maternal genome will be transmitted in its entirety to all other sexual and asexual offspring that the mother may produce, but the offspring's paternally derived genes may be absent from these other offspring. Thus, the selective forces favouring the evolution of genomic imprinting may have been present from the origin of modern land plants. In bryophytes, where gametophytes are long-lived and capable of multiple bouts of asexual and sexual reproduction, we predict strong sexual conflict over allocation to sporophytes. Female gametophytes of pteridophytes produce a single sporophyte and often lack means of asexual reproduction. Therefore, sexual conflict is predicted to be attenuated. Finally, we explore similarities among models of mate choice, offspring choice and segregation distortion. PMID:16612891

  9. Candida zemplinina Can Reduce Acetic Acid Produced by Saccharomyces cerevisiae in Sweet Wine Fermentations

    PubMed Central

    Rantsiou, Kalliopi; Dolci, Paola; Giacosa, Simone; Torchio, Fabrizio; Tofalo, Rosanna; Torriani, Sandra; Suzzi, Giovanna; Rolle, Luca

    2012-01-01

    In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ∼0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response. PMID:22247148

  10. Characters that differ between diploid and haploid honey bee (Apis mellifera) drones.

    PubMed

    Herrmann, Matthias; Trenzcek, Tina; Fahrenhorst, Hartmut; Engels, Wolf

    2005-01-01

    Diploid males have long been considered a curiosity contradictory to the haplo-diploid mode of sex determination in the Hymenoptera. In Apis mellifera, 'false' diploid male larvae are eliminated by worker cannibalism immediately after hatching. A 'cannibalism substance' produced by diploid drone larvae to induce worker-assisted suicide has been hypothesized, but it has never been detected. Diploid drones are only removed some hours after hatching. Older larvae are evidently not regarded as 'false males' and instead are regularly nursed by the brood-attending worker bees. As the pheromonal cues presumably are located on the surface of newly hatched bee larvae, we extracted the cuticular secretions and analyzed their chemical composition by gas chromatograph-mass spectrometry (GC-MS) analyses. Larvae were sexed and then reared in vitro for up to three days. The GC-MS pattern that was obtained, with alkanes as the major compounds, was compared between diploid and haploid drone larvae. We also examined some physical parameters of adult drones. There was no difference between diploid and haploid males in their weight at the day of emergence. The diploid adult drones had fewer wing hooks and smaller testes. The sperm DNA content was 0.30 and 0.15 pg per nucleus, giving an exact 2:1 ratio for the gametocytes of diploid and haploid drones, respectively. Vitellogenin was found in the hemolymph of both types of imaginal drones at 5 to 6 days, with a significantly lower titer in the diploids. PMID:16475107

  11. Molecular genetic study of introgression between Saccharomyces bayanus and S. cerevisiae.

    PubMed

    Naumova, Elena S; Naumov, Gennadi I; Masneuf-Pomarède, Isabelle; Aigle, Michel; Dubourdieu, Denis

    2005-10-30

    The genomic constitution of different S. bayanus strains and natural interspecific Saccharomyces hybrids has been studied by genetic and molecular methods. Unlike S. bayanus var. uvarum, some S. bayanus var. bayanus strains (the type culture CBS 380, CBS 378, CBS 425, CBS 1548) harbour a number of S. cerevisiae subtelomeric sequences: Y', pEL50, SUC, RTM and MAL. The two varieties, having 86-100% nDNA-nDNA reassociation, are partly genetically isolated from one another but completely isolated from S. cerevisiae. Genetic and molecular data support the maintaining of var. bayanus and var. uvarum strains in the species S. bayanus. Using Southern hybridization with species-specific molecular markers, RFLP of the MET2 gene and flow cytometry analysis, we showed that the non-S. cerevisiae parents are different in lager brewing yeasts and in wine hybrid strains. Our results suggest that S. pastorianus is a hybrid between S. cerevisiae and S. bayanus var. bayanus, while S. bayanus var. uvarum contributed to the formation of the wine hybrids S6U and CID1. According to the partial sequence of ACT1 gene and flow cytometry analysis, strain CID1 is a triple hybrid between S. cerevisiae, S. kudriavzevii and S. bayanus var. uvarum. PMID:16240458

  12. ROG1 encodes a monoacylglycerol lipase in Saccharomyces cerevisiae.

    PubMed

    Vishnu Varthini, Lakshmanaperumal; Selvaraju, Kandasamy; Srinivasan, Malathi; Nachiappan, Vasanthi

    2015-01-01

    Lipid metabolism is extensively studied in Saccharomyces cerevisiae. Here, we report that revertant of glycogen synthase kinase mutation-1 (Rog1p) possesses monoacylglycerol (MAG) lipase activity in S. cerevisiae. The lipase activity of Rog1p was confirmed in two ways: through analysis of a strain with a double deletion of ROG1 and monoglyceride lipase YJU3 (yju3Δrog1Δ) and by site-directed mutagenesis of the ROG1 lipase motif (GXSXG). Rog1p is localized in both the cytosol and the nucleus. Overexpression of ROG1 in a ROG1-deficient strain resulted in an accumulation of reactive oxygen species. These results suggest that Rog1p is a MAG lipase that regulates lipid homeostasis. PMID:25433290

  13. Post-zygotic sterility and cytonuclear compatibility limits in S. cerevisiae xenomitochondrial cybrids

    PubMed Central

    Špírek, Mário; Poláková, Silvia; Jatzová, Katarína; Sulo, Pavol

    2015-01-01

    Nucleo-mitochondrial interactions, particularly those determining the primary divergence of biological species, can be studied by means of xenomitochondrial cybrids, which are cells where the original mitochondria are substituted by their counterparts from related species. Saccharomyces cerevisiae cybrids are prepared simply by the mating of the ρ0 strain with impaired karyogamy and germinating spores from other Saccharomyces species and fall into three categories. Cybrids with compatible mitochondrial DNA (mtDNA) from Saccharomyces paradoxus CBS 432 and Saccharomyces cariocanus CBS 7994 are metabolically and genetically similar to cybrids containing mtDNA from various S. cerevisiae. Cybrids with mtDNA from other S. paradoxus strains, S. cariocanus, Saccharomyces kudriavzevii, and Saccharomyces mikatae require a period of adaptation to establish efficient oxidative phosphorylation. They exhibit a temperature-sensitive phenotype, slower growth rate on a non-fermentable carbon source and a long lag phase after the shift from glucose. Their decreased respiration capacity and reduced cytochrome aa3 content is associated with the inefficient splicing of cox1I3β, the intron found in all Saccharomyces species but not in S. cerevisiae. The splicing defect is compensated in cybrids by nuclear gain-of-function and can be alternatively suppressed by overexpression of MRP13 gene for mitochondrial ribosomal protein or the MRS2, MRS3, and MRS4 genes involved in intron splicing. S. cerevisiae with Saccharomyces bayanus mtDNA is unable to respire and the growth on ethanol–glycerol can be restored only after mating to some mit− strains. The nucleo-mitochondrial compatibility limit of S. cerevisiae and other Saccharomyces was set between S. kudriavzevii and S. bayanus at the divergence from S. cerevisiae about 15 MYA. The MRS1-cox1 S. cerevisiae/S. paradoxus cytonuclear Dobzhansky–Muller pair has a neglible impact on the separation of species since its imperfection is

  14. Tetraploid citrus rootstocks are more tolerant to salt stress than diploid.

    PubMed

    Saleh, Basel; Allario, Thierry; Dambier, Dominique; Ollitrault, Patrick; Morillon, Raphaël

    2008-09-01

    Citrus trees are subject to several abiotic constraints such as salinity. Providing new rootstocks more tolerant is thus a requirement. In this article, we investigated salt stress tolerance of three tetraploid rootstock genotypes when compared to their respective diploid rootstocks (Poncirus trifoliata, Carrizo citrange, Cleopatra mandarin). Plant growth, leaf fall and ion contents were investigated. At the end of the experiment, leaf fall was observed only for diploid Poncirus trifoliata plants as well as chlorosis symptoms for Poncirus trifoliata and Carrizo citrange diploid plants. The diploid Cleopatra mandarin plants growth rate was not affected by salt stress and has even been increased for tetraploid Cleopatra mandarin. Ion contents investigation has shown lower accumulations of chloride ions in leaves of the tetraploid plants when compared to diploid plants. Our results suggest that citrus tetraploid rootstocks are more tolerant to salt stress than their corresponding diploid. PMID:18722990

  15. The importance of effective sampling for exploring the population dynamics of haploid-diploid seaweeds.

    PubMed

    Krueger-Hadfield, Stacy A; Hoban, Sean M

    2016-02-01

    The mating system partitions genetic diversity within and among populations and the links between life history traits and mating systems have been extensively studied in diploid organisms. As such most evolutionary theory is focused on species for which sexual reproduction occurs between diploid male and diploid female individuals. However, there are many multicellular organisms with biphasic life cycles in which the haploid stage is prolonged and undergoes substantial somatic development. In particular, biphasic life cycles are found across green, brown and red macroalgae. Yet, few studies have addressed the population structure and genetic diversity in both the haploid and diploid stages in these life cycles. We have developed some broad guidelines with which to develop population genetic studies of haploid-diploid macroalgae and to quantify the relationship between power and sampling strategy. We address three common goals for studying macroalgal population dynamics, including haploid-diploid ratios, genetic structure and paternity analyses. PMID:26987084

  16. Phenotypic Landscape of Saccharomyces cerevisiae during Wine Fermentation: Evidence for Origin-Dependent Metabolic Traits

    PubMed Central

    Camarasa, Carole; Sanchez, Isabelle; Brial, Pascale; Bigey, Frédéric; Dequin, Sylvie

    2011-01-01

    The species Saccharomyces cerevisiae includes natural strains, clinical isolates, and a large number of strains used in human activities. The aim of this work was to investigate how the adaptation to a broad range of ecological niches may have selectively shaped the yeast metabolic network to generate specific phenotypes. Using 72 S. cerevisiae strains collected from various sources, we provide, for the first time, a population-scale picture of the fermentative metabolic traits found in the S. cerevisiae species under wine making conditions. Considerable phenotypic variation was found suggesting that this yeast employs diverse metabolic strategies to face environmental constraints. Several groups of strains can be distinguished from the entire population on the basis of specific traits. Strains accustomed to growing in the presence of high sugar concentrations, such as wine yeasts and strains obtained from fruits, were able to achieve fermentation, whereas natural yeasts isolated from “poor-sugar” environments, such as oak trees or plants, were not. Commercial wine yeasts clearly appeared as a subset of vineyard isolates, and were mainly differentiated by their fermentative performances as well as their low acetate production. Overall, the emergence of the origin-dependent properties of the strains provides evidence for a phenotypic evolution driven by environmental constraints and/or human selection within S. cerevisiae. PMID:21949874

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

    PubMed Central

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

    2013-01-01

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

  18. [Production of β-carotene by metabolically engineered Saccharomyces cerevisiae].

    PubMed

    Wang, Beibei; Shi, Mingyu; Wang, Dong; Xu, Jiaoyang; Liu, Yi; Yang, Hongjiang; Dai, Zhubo; Zhang, Xueli

    2014-08-01

    β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production. PMID:25507473

  19. [Production of β-carotene by metabolically engineered Saccharomyces cerevisiae].

    PubMed

    Wang, Beibei; Shi, Mingyu; Wang, Dong; Xu, Jiaoyang; Liu, Yi; Yang, Hongjiang; Dai, Zhubo; Zhang, Xueli

    2014-08-01

    β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production. PMID:25423750

  20. Performance comparisons between diploid and triploid sunshine bass in fresh water ponds

    USGS Publications Warehouse

    Kerby, J.H.; Everson, J.M.; Harrell, R.M.; Geiger, J.G.; Starling, C.C.; Revels, H.

    2002-01-01

    Diploid and triploid sunshine bass (white bass ??? x striped bass ???) were produced in 1990 at Florida's Richloam Fish Hatchery. Triploidy was induced with hydrostatic pressure. Fry were cultured to phase I in earthen ponds in Webster and Gainesville, FL, and transported to Leetown, WV, where they were held in circular flow-through fiberglass tanks. Ploidy of treated fish was determined with a Coulter counter and triploids were segregated from diploids. In April 1991, control diploid and triploid populations were graded to remove the largest and smallest individuals, and four 0.2-ha hypalon-lined ponds were stocked with 600 fish each; two ponds contained triploids and two contained diploids. Triploids and diploids were not significantly different in average fork length (FL) or weight at stocking. Triploids averaged 231 mm and 181.2 g, compared to diploid averages of 233 mm and 188.9 g. Monthly samples indicated that diploids grew faster than triploids; mean weights and lengths were both significantly different after 3 months. When harvested in October, triploids averaged 358 mm and 867.9 g, whereas diploids averaged 381 mm and 1153.5 g. Survival of triploids and diploids was 97.0% and 95.9%, respectively. Mean standing crop was 2496.3 kg/ha for triploids and 3280.6 kg/ha for diploids. Male diploids and most female diploids were sexually mature at 2 years of age. Sterility of triploids was confirmed as gonads remained reduced and dysfunctional at 5 years of age. ?? 2002 Elsevier Science B.V. All rights reserved.

  1. Diploid biological evolution models with general smooth fitness landscapes and recombination.

    PubMed

    Saakian, David B; Kirakosyan, Zara; Hu, Chin-Kun

    2008-06-01

    Using a Hamilton-Jacobi equation approach, we obtain analytic equations for steady-state population distributions and mean fitness functions for Crow-Kimura and Eigen-type diploid biological evolution models with general smooth hypergeometric fitness landscapes. Our numerical solutions of diploid biological evolution models confirm the analytic equations obtained. We also study the parallel diploid model for the simple case of recombination and calculate the variance of distribution, which is consistent with numerical results. PMID:18643300

  2. Saccharomyces cerevisiae Genes Involved in Survival of Heat Shock

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Diezmann, Stephanie; Dietrich, Fred S.

    2009-01-01

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

  4. Lactose fermentation by engineered Saccharomyces cerevisiae capable of fermenting cellobiose.

    PubMed

    Liu, Jing-Jing; Zhang, Guo-Chang; Oh, Eun Joong; Pathanibul, Panchalee; Turner, Timothy L; Jin, Yong-Su

    2016-09-20

    Lactose is an inevitable byproduct of the dairy industry. In addition to cheese manufacturing, the growing Greek yogurt industry generates excess acid whey, which contains lactose. Therefore, rapid and efficient conversion of lactose to fuels and chemicals would be useful for recycling the otherwise harmful acid whey. Saccharomyces cerevisiae, a popular metabolic engineering host, cannot natively utilize lactose. However, we discovered that an engineered S. cerevisiae strain (EJ2) capable of fermenting cellobiose can also ferment lactose. This finding suggests that a cellobiose transporter (CDT-1) can transport lactose and a β-glucosidase (GH1-1) can hydrolyze lactose by acting as a β-galactosidase. While the lactose fermentation by the EJ2 strain was much slower than the cellobiose fermentation, a faster lactose-fermenting strain (EJ2e8) was obtained through serial subcultures on lactose. The EJ2e8 strain fermented lactose with a consumption rate of 2.16g/Lh. The improved lactose fermentation by the EJ2e8 strain was due to the increased copy number of cdt-1 and gh1-1 genes. Looking ahead, the EJ2e8 strain could be exploited for the production of other non-ethanol fuels and chemicals from lactose through further metabolic engineering. PMID:27457698

  5. Methionine restriction slows down senescence in human diploid fibroblasts

    PubMed Central

    Kozieł, Rafał; Ruckenstuhl, Christoph; Albertini, Eva; Neuhaus, Michael; Netzberger, Christine; Bust, Maria; Madeo, Frank; Wiesner, Rudolf J; Jansen-Dürr, Pidder

    2014-01-01

    Methionine restriction (MetR) extends lifespan in animal models including rodents. Using human diploid fibroblasts (HDF), we report here that MetR significantly extends their replicative lifespan, thereby postponing cellular senescence. MetR significantly decreased activity of mitochondrial complex IV and diminished the accumulation of reactive oxygen species. Lifespan extension was accompanied by a significant decrease in the levels of subunits of mitochondrial complex IV, but also complex I, which was due to a decreased translation rate of several mtDNA-encoded subunits. Together, these findings indicate that MetR slows down aging in human cells by modulating mitochondrial protein synthesis and respiratory chain assembly. PMID:25273919

  6. Genome Size Variations in Diploid African Coffea Species

    PubMed Central

    NOIROT, M.; PONCET, V.; BARRE, P.; HAMON, P.; HAMON, S.; DE KOCHKO, A.

    2003-01-01

    Flow cytometry was conducted to evaluate genome size diversity among African diploid species of the Coffea genus. The study included 15 species and six new taxa from Congolese and Cameroonian forest regions which have yet to be botanically characterized. Between‐population differences were also recorded in some cases. These evaluations using an internal standard were highly correlated with previous results obtained with an external standard, but differences of up to 18 % existed for some species, involving stoichiometric errors. Consequently, genome size variation between species and within species are discussed as true genome size differences or stoichiometric errors. Environmental and phenotypic correlations with genome size are also discussed. PMID:14573524

  7. Delimination of brewing yeast strains using different molecular techniques.

    PubMed

    Tornai-Lehoczki, J; Dlauchy, D

    2000-12-01

    In general, the genetic characteristics, the phenotype and the microbial purity of the production brewing yeast strains are among the most important factors in maintaining a consistently good quality of products. Analysis of restriction fragment length polymorphism (RFLP) patterns of 18S rRNA-coding DNA was investigated to group ale and lager strains. All production brewing yeast strains showed the same RFLP pattern as the type strain and synonym type strains of S. cerevisiae, and were quite different from the type and synonym type strains of S. pastorianus. Based on these data, all production brewing yeast strains investigated in this study appeared to belong to S. cerevisiae. Electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis appeared to be suitable methods for distinguishing not only the type and synonym type strain of S. cerevisiae and S. pastorianus, but also the ale and the lager strains. PMID:11139020

  8. Noncomplementing Diploids from Bacillus Subtilis Protoplast Fusion: Relationship between Maintenance of Chromosomal Inactivation and Segregation Capacity

    PubMed Central

    Grandjean, V.; Hauck, Y.; Le-Derout, J.; Hirschbein, L.

    1996-01-01

    Fusions of Bacillus subtilis protoplasts from two genetically marked strains produce noncomplementing heterodiploid bacteria. These noncomplementing diploids (Ncds) carry both parental chromosomes, but only one is expressed. Fusion products of strains polymorphic for NotI restriction sites provide new physical evidence to support the conclusion that Ncds are not an artifact of cross feeding or cell adhesion. We show that reversible chromosomal inactivation can only account for the biparental trait of unstable Ncds. Two types of cells were recovered from the late progeny of unstable Ncds: Ncds with irreversible chromosome silencing (stable Ncds) and secondary recombinants that displayed a genomic mosaic NotI profile. Segregants from an unstable Ncd population gave rise to two viable haploid cell types. By contrast, stable Ncds segregated into a population of viable and inviable haploid cells. We propose that the latter are derived from irreversible chromosome silencing. Our results indicate that clonal populations of stable Ncds are heterogenous and suggest that segregation and inactivation are independent parameters. PMID:8913734

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

    PubMed

    Borodina, Irina; Nielsen, Jens

    2014-05-01

    Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up the development of yeast cell factories. We also present an overview of metabolic engineering strategies for developing yeast strains for production of polymer monomers: lactic, succinic, and cis,cis-muconic acids. S. cerevisiae has already firmly established itself as a cell factory in industrial biotechnology and the advances in yeast strain engineering will stimulate development of novel yeast-based processes for chemicals production. PMID:24677744

  10. Metabolic Engineering of Saccharomyces cerevisiae

    PubMed Central

    Ostergaard, Simon; Olsson, Lisbeth; Nielsen, Jens

    2000-01-01

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

  11. Kinetics of staurosporine-arrest of human diploid fibroblasts

    SciTech Connect

    Stevenson, A.P.; Bustos, L.D.; Dickson, J.A.; Habbersett, R.C.; Crissman, H.A. )

    1993-01-01

    The authors have previously shown that the general protein kinase inhibitor, staurosporine (Stsp), has a differential effect on the progression of normal and transformed cells. Low levels of Stsp (1.0 ng/ml) reversibly arrested normal diploid cells in early G1 phase, whereas concentrations of Stsp as high as 50-75 ng/ml had no effect on G1 progression in transformed cells. High concentrations of Stsp arrested normal cells in G1 and G2 phases but blocked transformed cells only in G2 phase of the cell cycle. To follow the accumulation of cells in G1 and/or G2 phases, asynchronous cultures of human diploid fibroblasts were treated with 0, 1, 5, 10, 20, 30 or 50 ng/ml staurosporine for 18 hours in the presence of bromodeoxyuridine. The kinetics of labeling with BrdU were determined with a two-laser flow cytometric technique that monitored emission of fluorescence from DNA-species stains that differed in the degree of BrdU-induced quenching of their fluorescence signals. Bivariate plots of Hoechst/mithramycin fluorescence signal ratio vs. Hoechst fluorescence identified the arrested cells as cohorts of cells in different positions across cell cycle at the time of Stsp addition.

  12. Partial diploidization of meiosis in autotetraploid Arabidopsis thaliana.

    PubMed Central

    Santos, J L; Alfaro, D; Sanchez-Moran, E; Armstrong, S J; Franklin, F C H; Jones, G H

    2003-01-01

    Meiosis was analyzed cytogenetically in autotetraploids of Arabidopsis, including both established lines and newly generated autotetraploid plants. Fluorescent in situ hybridization with 5S and 45S rDNA probes was used to identify the different chromosomes at metaphase I of meiosis. Multivalents were observed frequently in all the lines analyzed, but there were significant differences in multivalent frequency not only between the newly generated tetraploids and the established lines but also among the different established lines. The new tetraploids showed high multivalent frequencies, exceeding the theoretical 66.66% predicted by the simple random-end pairing model, in some cases significantly, thus indicating that Arabidopsis autotetraploids have more than two autonomous pairing sites per chromosome, despite their small sizes. The established lines showed fewer multivalents than the new autotetraploids did, but the extent of this reduction was strongly line and chromosome dependent. One line in particular showed a large reduction in multivalents and a concomitant increase in bivalents, while the other lines showed lesser reductions in multivalents. The reduction in multivalents was not uniformly distributed across chromosomes. The smaller chromosomes, especially chromosomes 2 and 4, showed the most marked reductions while the largest chromosome (1) showed virtually no reduction compared to the new tetraploids. It is concluded that the established autotetraploid lines have undergone a partial diploidization of meiosis, but not necessarily genetical diploidization, since their creation. Possible mechanisms for the resulting change in meiotic chromosome behavior are discussed. PMID:14668400

  13. A monoecious and diploid Moran model of random mating.

    PubMed

    Hössjer, Ola; Tyvand, Peder A

    2016-04-01

    An exact Markov chain is developed for a Moran model of random mating for monoecious diploid individuals with a given probability of self-fertilization. The model captures the dynamics of genetic variation at a biallelic locus. We compare the model with the corresponding diploid Wright-Fisher (WF) model. We also develop a novel diffusion approximation of both models, where the genotype frequency distribution dynamics is described by two partial differential equations, on different time scales. The first equation captures the more slowly varying allele frequencies, and it is the same for the Moran and WF models. The other equation captures departures of the fraction of heterozygous genotypes from a large population equilibrium curve that equals Hardy-Weinberg proportions in the absence of selfing. It is the distribution of a continuous time Ornstein-Uhlenbeck process for the Moran model and a discrete time autoregressive process for the WF model. One application of our results is to capture dynamics of the degree of non-random mating of both models, in terms of the fixation index fIS. Although fIS has a stable fixed point that only depends on the degree of selfing, the normally distributed oscillations around this fixed point are stochastically larger for the Moran than for the WF model. PMID:26807805

  14. Microencapsulation of human diploid fibroblasts in cationic polyacrylates.

    PubMed

    Mallabone, C L; Crooks, C A; Sefton, M V

    1989-08-01

    Human diploid fibroblasts and Chinese hamster ovary cells were encapsulated in several copolymers of dimethylaminoethyl methacrylate with methacrylic acid and/or methyl methacrylate. Copolymers containing 16 to 25% dimethylaminoethyl methacrylate and less than or equal to 2.2% methacrylic acid (based on monomer mol%) supported human diploid fibroblast growth when the polymer was cast as a film on glass or polystyrene. The cells survived encapsulation and grew, but growth was only observed in those capsules which appeared to be flawed; the flaws were detected as an early loss of fluorescence, due to leakage of the FITC-dextran added as a marker to the encapsulated cell suspension. Presumably the capsule wall had too low a permeability to allow for unrestricted growth. Chinese hamster ovary cells behaved similarly in dimethylaminoethyl methacrylate/methyl methacrylate capsules. Increasing the water content, by addition of methacrylic acid, did not improve matters, since these materials were not as good a substrate for cell growth as the others. Preparing materials that are sufficiently permeable, with low toxicity and high processability and which support the growth of anchorage-dependent cells is difficult, yet it remains an appropriate goal for further study. PMID:2804227

  15. Deciphering the diploid ancestral genome of the Mesohexaploid Brassica rapa.

    PubMed

    Cheng, Feng; Mandáková, Terezie; Wu, Jian; Xie, Qi; Lysak, Martin A; Wang, Xiaowu

    2013-05-01

    The genus Brassica includes several important agricultural and horticultural crops. Their current genome structures were shaped by whole-genome triplication followed by extensive diploidization. The availability of several crucifer genome sequences, especially that of Chinese cabbage (Brassica rapa), enables study of the evolution of the mesohexaploid Brassica genomes from their diploid progenitors. We reconstructed three ancestral subgenomes of B. rapa (n = 10) by comparing its whole-genome sequence to ancestral and extant Brassicaceae genomes. All three B. rapa paleogenomes apparently consisted of seven chromosomes, similar to the ancestral translocation Proto-Calepineae Karyotype (tPCK; n = 7), which is the evolutionarily younger variant of the Proto-Calepineae Karyotype (n = 7). Based on comparative analysis of genome sequences or linkage maps of Brassica oleracea, Brassica nigra, radish (Raphanus sativus), and other closely related species, we propose a two-step merging of three tPCK-like genomes to form the hexaploid ancestor of the tribe Brassiceae with 42 chromosomes. Subsequent diversification of the Brassiceae was marked by extensive genome reshuffling and chromosome number reduction mediated by translocation events and followed by loss and/or inactivation of centromeres. Furthermore, via interspecies genome comparison, we refined intervals for seven of the genomic blocks of the Ancestral Crucifer Karyotype (n = 8), thus revising the key reference genome for evolutionary genomics of crucifers. PMID:23653472

  16. Asymmetrical division of Saccharomyces cerevisiae.

    PubMed Central

    Lord, P G; Wheals, A E

    1980-01-01

    The unequal division model proposed for budding yeast (L. H. Hartwell and M. W. Unger, J. Cell Biol. 75:422-435, 1977) was tested by bud scar analyses of steady-state exponential batch cultures of Saccharomyces cerevisiae growing at 30 degrees C at 19 different rates, which were obtained by altering the carbon source. The analyses involved counting the number of bud scars, determining the presence or absence of buds on at least 1,000 cells, and independently measuring the doubling times (gamma) by cell number increase. A number of assumptions in the model were tested and found to be in good agreement with the model. Maximum likelihood estimates of daughter cycle time (D), parent cycle time (P), and the budded phase (B) were obtained, and we concluded that asymmetrical division occurred at all growth rates tested (gamma, 75 to 250 min). D, P, and B are all linearly related to gamma, and D, P, and gamma converge to equality (symmetrical division) at gamma = 65 min. Expressions for the genealogical age distribution for asymmetrically dividing yeast cells were derived. The fraction of daughter cells in steady-state populations is e-alpha P, and the fraction of parent cells of age n (where n is the number of buds that a cell has produced) is (e-alpha P)n-1(1-e-alpha P)2, where alpha = IN2/gamma; thus, the distribution changes with growth rate. The frequency of cells with different numbers of bud scars (i.e., different genealogical ages) was determined for all growth rates, and the observed distribution changed with the growth rate in the manner predicted. In this haploid strain new buds formed adjacent to the previous buds in a regular pattern, but at slower growth rates the pattern was more irregular. The median volume of the cells and the volume at start in the cell cycle both increased at faster growth rates. The implications of these findings for the control of the cell cycle are discussed. PMID:6991494

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

    PubMed Central

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

    2015-01-01

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

  18. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    PubMed

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions. PMID:27221740

  19. S. cerevisiae × S. eubayanus interspecific hybrid, the best of both worlds and beyond.

    PubMed

    Hebly, Marit; Brickwedde, Anja; Bolat, Irina; Driessen, Maureen R M; de Hulster, Erik A F; van den Broek, Marcel; Pronk, Jack T; Geertman, Jan-Maarten; Daran, Jean-Marc; Daran-Lapujade, Pascale

    2015-05-01

    Saccharomyces pastorianus lager-brewing yeasts have descended from natural hybrids of S. cerevisiae and S. eubayanus. Their alloploidy has undoubtedly contributed to successful domestication and industrial exploitation. To understand the early events that have led to the predominance of S. pastorianus as lager-brewing yeast, an interspecific hybrid between S. cerevisiae and S. eubayanus was experimentally constructed. Alloploidy substantially improved the performance of the S. cerevisiae × S. eubayanus hybrid as compared to either parent regarding two cardinal features of brewing yeasts: tolerance to low temperature and oligosaccharide utilization. The hybrid's S. eubayanus subgenome conferred better growth rates and biomass yields at low temperature, both on glucose and on maltose. Conversely, the ability of the hybrid to consume maltotriose, which was absent in the S. eubayanus CBS12357 type strain, was inherited from its S. cerevisiae parent. The S. cerevisiae × S. eubayanus hybrid even outperformed its parents, a phenomenon known as transgression, suggesting that fast growth at low temperature and oligosaccharide utilization may have been key selective advantages of the natural hybrids in brewing environments. To enable sequence comparisons of the parental and hybrid strains, the genome of S. eubayanus CBS12357 type strain (Patagonian isolate) was resequenced, resulting in an improved publicly available sequence assembly. PMID:25743788

  20. Diploid males and their triploid offspring in the paper wasp Polistes dominulus

    PubMed Central

    Liebert, Aviva E; Sumana, Annagiri; Starks, Philip T

    2005-01-01

    Although the hymenopteran sex-determining mechanism generally results in haploid males and diploid females, diploid males can be produced via homozygosity at the sex-determining locus. Diploid males have low fitness because they are effectively sterile or produce presumably sterile triploid offspring. Previously, triploid females were observed in three species of North American Polistes paper wasps, and this was interpreted as indirect evidence of diploid males. Here we report what is, to our knowledge, the first direct evidence: four of five early male-producing Polistes dominulus nests from three populations contained diploid males. Because haploid males were also found, however, the adaptive value of early males cannot be ignored. Using genetic and morphological data from triploid females, we also present evidence that both diploid males and triploid females remain undetected throughout the colony cycle. Consequently, diploid male production may result in a delayed fitness cost for two generations. This phenomenon is particularly relevant for introduced populations with few alleles at the sex-determining locus, but cannot be ignored in native populations without supporting genetic data. Future research using paper wasp populations to test theories of social evolution should explicitly consider the potential impacts of diploid males. PMID:17148166

  1. [Surface display of phytase on Saccharomyces cerevisiae for efficient bioethanol production from corn starch].

    PubMed

    Xiao, Yan; Chen, Xianzhong; Shen, Wei; Yang, Haiquan; Fan, You

    2015-12-01

    Production of bioethanol using starch as raw material has become a very prominent technology. However, phytate in the raw material not only decreases ethanol production efficiency, but also increases phosphorus discharge. In this study, to decrease phytate content in an ethanol fermentationprocess, Saccharomyces cerevisiae was engineered forheterologous expression of phytase on the cell surface. The phy gene encoding phytase gene was fused with the C-terminal-half region of α-agglutinin and then inserted downstream of the secretion signal gene, to produce a yeast surface-display expression vector pMGK-AG-phy, which was then transformed into S. cerevisiae. The recombinant yeast strain, PHY, successfully displayed phytase on the surface of cells producing 6.4 U/g wet cells and its properties were further characterized. The growthrate and ethanol production of the PHY strain were faster than the parent S. cerevisiae strain in the fermentation medium by simultaneous saccharification and fermentation. Moreover, the phytate concentration decreased by 91% in dry vinasse compared to the control. In summary, we constructed recombinant S. cerevisiae strain displaying phytase on the cell surface, which could effectively reduce the content of phytate, improve the utilization value of vinasse and reduce the discharge of phosphorus. The strain reported here represents a useful novel engineering platform for developing an environment-friendly system for bioethanol production from a corn substrate. PMID:27093833

  2. Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response.

    PubMed

    Verwaal, René; Jiang, Yang; Wang, Jing; Daran, Jean-Marc; Sandmann, Gerhard; van den Berg, Johan A; van Ooyen, Albert J J

    2010-12-01

    To obtain insight into the genome-wide transcriptional response of heterologous carotenoid production in Saccharomyces cerevisiae, the transcriptome of two different S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous grown in carbon-limited chemostat cultures was analysed. The strains exhibited different absolute carotenoid levels as well as different intermediate profiles. These discrepancies were further sustained by the difference of the transcriptional response exhibited by the two strains. Transcriptome analysis of the strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ABC-type and major facilitator transporters which are reported to be involved in secretion of toxic compounds out of cells. β-Carotene was found to be secreted when sunflower oil was added to the medium of S. cerevisiae cells producing high levels of carotenoids, which was not observed when added to X. dendrorhous cells. Deletion of pdr10, one of the induced ABC transporters, decreased the transformation efficiency of a plasmid containing carotenogenic genes. The few transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to a pdr5 deletion and a reference strain transformed with the same genes. Our results suggest that production of high amounts of carotenoids in S. cerevisiae leads to membrane stress, in which Pdr10 might play an important role, and a cellular response to secrete carotenoids out of the cell. PMID:20632327

  3. Engineering the robustness of Saccharomyces cerevisiae by introducing bifunctional glutathione synthase gene.

    PubMed

    Qiu, Zhiqi; Deng, Zujun; Tan, Hongming; Zhou, Shining; Cao, Lixiang

    2015-04-01

    Robust, high-yielding Saccharomyces cerevisiae is highly desirable for cost-effective cellulosic ethanol production. In this study, the bifunctional glutathione (GSH) synthetase genes GCSGS at high copy number was integrated into ribosomal DNA of S. cerevisiae by Cre-LoxP system. Threefold higher GSH contents (54.9 μmol/g dry weight) accumulated in the engineered strain BY-G compared to the reference strain. Tolerance of BY-G to H2O2 (3 mM), temperature (40 °C), furfural (10 mM), hydroxymethylfurfural (HMF, 10 mM) and 0.5 mM Cd(2+) increased compared to reference strain. Twofold higher ethanol concentration was obtained by BY-G in simultaneous saccharification and fermentation of corn stover compared to the reference strain. The results showed that intracellular GSH content of S. cerevisiae has an influence on robustness. The strategy is used to engineer S. cerevisiae strains adaptive to a combination of tolerance to inhibitors and raised temperature that may occur in high solid simultaneous saccharification and fermentation of lignocellulosic feedstocks. PMID:25561319

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

    PubMed

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

    2014-03-01

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

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

    PubMed

    Kelly, Amy C; Wickner, Reed B

    2013-01-01

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

  6. Interorganelle signaling is a determinant of longevity in Saccharomyces cerevisiae.

    PubMed Central

    Kirchman, P A; Kim, S; Lai, C Y; Jazwinski, S M

    1999-01-01

    Replicative capacity, which is the number of times an individual cell divides, is the measure of longevity in the yeast Saccharomyces cerevisiae. In this study, a process that involves signaling from the mitochondrion to the nucleus, called retrograde regulation, is shown to determine yeast longevity, and its induction resulted in postponed senescence. Activation of retrograde regulation, by genetic and environmental means, correlated with increased replicative capacity in four different S. cerevisiae strains. Deletion of a gene required for the retrograde response, RTG2, eliminated the increased replicative capacity. RAS2, a gene previously shown to influence longevity in yeast, interacts with retrograde regulation in setting yeast longevity. The molecular mechanism of aging elucidated here parallels the results of genetic studies of aging in nematodes and fruit flies, as well as the caloric restriction paradigm in mammals, and it underscores the importance of metabolic regulation in aging, suggesting a general applicability. PMID:10224252

  7. The Reference Genome Sequence of Saccharomyces cerevisiae: Then and Now

    PubMed Central

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

    2014-01-01

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

  8. The Influence of Microgravity on Invasive Growth in Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Van Mulders, Sebastiaan E.; Stassen, Catherine; Daenen, Luk; Devreese, Bart; Siewers, Verena; van Eijsden, Rudy G. E.; Nielsen, Jens; Delvaux, Freddy R.; Willaert, Ronnie

    2011-01-01

    This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Σ1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Σ1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Σ1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium.

  9. Construction of a flocculent Saccharomyces cerevisiae fermenting lactose.

    PubMed

    Domingues, L; Teixeira, J A; Lima, N

    1999-05-01

    A flocculent Saccharomyces cerevisiae strain with the ability to express both the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus was constructed. This recombinant strain is not only able to grow on lactose, but it can also ferment this substrate. To our knowledge this is the first time that a recombinant S. cervisiae has been found to ferment lactose in a way comparable to that of the existing lactose-fermenting yeast strains. Moreover, the flocculating capacity of the strain used in this work gives the process several advantages. On the one hand, it allows for operation in a continuous mode at high cell concentration, thus increasing the system's overall productivity; on the other hand, the biomass concentration in the effluent is reduced, thus decreasing product separation/purification costs. PMID:10390820

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

    PubMed Central

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

    1995-01-01

    The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci [Bang et al. (1992) Nucleic Acids Res. 20, 3925-3931]. More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae [Waters et al. (1993) Mol. Gen. Genet. 239, 28-32]. We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation. Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA. In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced. The basal level of the RAD16 transcript is constant during the mitotic cell cycle. G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation. However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle. Images PMID:7784171

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

    PubMed Central

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

    2002-01-01

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

  12. Mating-Type Genes and MAT Switching in Saccharomyces cerevisiae

    PubMed Central

    Haber, James E.

    2012-01-01

    Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATα. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATα1, and MATα2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break. PMID:22555442

  13. Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance.

    PubMed

    Garcia Sanchez, Rosa; Solodovnikova, Natalia; Wendland, Jürgen

    2012-08-01

    Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. PMID:22887121

  14. Functional Analysis of Phosphorylation on Saccharomyces cerevisiae Syntaxin 1 Homologues Sso1p and Sso2p

    PubMed Central

    Yuan, Qiang; Jäntti, Jussi

    2010-01-01

    Background The Saccharomyces cerevisiae syntaxin1 homologues Sso1p and Sso2p perform an essential function in membrane fusion in exocytosis. While deletion of either SSO1 or SSO2 causes no obvious phenotype in vegetatively grown cells, deletion of both genes is lethal. In sporulating diploid S. cerevisiae cells only Sso1p, but not Sso2p, is needed for membrane fusion during prospore membrane formation. Mass spectrometry and in vivo labeling data suggest that serines 23, 24, and 79 in Sso1p and serines 31 and 34 in Sso2p can be phosphorylated in vivo. Here we set out to assess the contribution of phosphorylation on Sso protein in vivo function. Principal Findings Different mutant versions of SSO1 and SSO2 were generated to target the phosphorylation sites in Sso1p and Sso2p. Basal or overexpression of phospho-mimicking or putative non-phosphorylated Sso1p or Sso2p mutants resulted in no obvious growth phenotype. However, S79A and S79E mutations caused a mild defect in the ability of Sso1p to complement the temperature-sensitive growth phenotype of sso2-1 sso1Δ cells. Combination of all mutations did not additionally compromise Sso1p in vivo function. When compared to the wild type SSO1 and SSO2, the phosphoamino acid mutants displayed similar genetic interactions with late acting sec mutants. Furthermore, diploid cells expressing only the mutant versions of Sso1p had no detectable sporulation defects. In addition to sporulation, also pseudohyphal and invasive growth modes are regulated by the availability of nutrients. In contrast to sporulating diploid cells, deletion of SSO1 or SSO2, or expression of the phospho-mutant versions of SSO1 or SSO2 as the sole copies of SSO genes caused no defects in haploid or diploid pseudohyphal and invasive growth. Conclusions The identified phosphorylation sites do not significantly contribute to the in vivo functionality of Sso1p and Sso2p in S. cerevisiae. PMID:20948969

  15. Regulation of Phosphatidylcholine Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Waechter, Charles J.; Lester, Robert L.

    1971-01-01

    Evidence is presented which indicates that the biosynthesis of phosphatidylcholine by the methylation pathway in growing cultures of Saccharomyces cerevisiae is repressed by the presence of choline in the growth medium. This result, obtained previously for glucose-grown cells, was also observed for lactate-grown cells, of which half of the phosphatidylcholine is mitochondrial. A respiration-deficient mutant of the parent wild-type strain has been studied, and its inability to form functional mitochondria cannot be due to an impaired methylation pathway, as it has been shown to incorporate 14C-CH3-methionine into all of the methylated glycerophosphatides. The incorporation rate is depressed by the inclusion of 1 mm choline in the growth medium, suggesting a regulatory effect similar to that demonstrated for the wild-type strain. The effects of choline on the glycerophospholipid composition of lactate and glucose-grown cells is presented. The repressive effects of the two related bases, mono- and dimethylethanolamine, were examined, and reduced levels of 14C-CH3-methionine incorporation were found for cells grown in the presence of these bases. The effect of choline on the methylation rates is reversible and glucosegrown cells regain the nonrepressed level of methylation activity in 60 to 80 min after removal of choline from the growth medium. Images PMID:5547992

  16. Characterization of Insertion Mutations in the Saccharomyces Cerevisiae Msh1 and Msh2 Genes: Evidence for Separate Mitochondrial and Nuclear Functions

    PubMed Central

    Reenan, RAG.; Kolodner, R. D.

    1992-01-01

    The MSH1 and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSH1 gene caused a petite phenotype which was established rapidly. A functional MSH1 gene present on a single-copy centromere plasmid was incapable of rescuing the established msh1 petite phenotype. Analysis of msh1 strains demonstrated that mutagenesis and large-scale rearrangement of mitochondrial DNA had occurred. 4',6-Diamidino-2-phenylindole (DAPI) staining of msh1 yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5' end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (<0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his4b showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (<0.26%) PMS events were observed. These results indicate that MSH1 plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus. PMID:1334021

  17. Specialization of B-Type Cyclins for Mitosis or Meiosis in S. Cerevisiae

    PubMed Central

    Dahmann, C.; Futcher, B.

    1995-01-01

    The CLB1, CLB2, and CLB3 genes encode B-type cyclins important for mitosis in Saccharomyces cerevisiae, while a fourth B-type cyclin gene, CLB4, has no clear role. The effects of homozygous clb mutations on meiosis were examined. Mutants homozygous for clb1 clb3, or for clb1 clb4, gave high levels of sporulation, but produced mainly two-spored asci instead of four-spored asci. The cells had completed meiosis I but not meiosis II, producing viable diploid ascospores. CLB1 and CLB4 seem to be much more important for meiosis than for mitosis and may play some special role in meiosis II. In contrast, CLB2 is important for mitosis but not meiosis. The level of Cdc28-Clb activity may be important in determining whether meiosis II will occur. PMID:7672594

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

    PubMed Central

    2013-01-01

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

  19. Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.

    PubMed

    Guimarães, Pedro M R; François, Jean; Parrou, Jean Luc; Teixeira, José A; Domingues, Lucília

    2008-03-01

    The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (beta-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media. PMID:18245248

  20. Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris.

    PubMed Central

    Soares-Silva, Isabel; Schuller, Dorit; Andrade, Raquel P; Baltazar, Fátima; Cássio, Fernanda; Casal, Margarida

    2003-01-01

    In Saccharomyces cerevisiae the activity for the lactate-proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a V (max) of 2.1 nmol x s(-1) x mg of dry weight(-1). Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest V (max) (0.84 nmol x s(-1) x mg of dry weight(-1)) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels. PMID:12962538

  1. A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism

    PubMed Central

    Solis-Escalante, Daniel; Kuijpers, Niels G. A.; Barrajon-Simancas, Nuria; van den Broek, Marcel; Pronk, Jack T.; Daran, Jean-Marc

    2015-01-01

    As a result of ancestral whole-genome and small-scale duplication events, the genomes of Saccharomyces cerevisiae and many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of the Saccharomyces lineage, the duplication of glycolytic genes is purported to have played an important role leading to S. cerevisiae's current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modern S. cerevisiae strains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the “minimal glycolysis” [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic of S. cerevisiae and essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community. PMID:26071034

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

    PubMed Central

    Konovalovas, Aleksandras

    2016-01-01

    We cloned and sequenced the complete genome of the L-A-28 virus from the Saccharomyces cerevisiae K28 killer strain. This sequence completes the set of currently identified L-A helper viruses required for expression of double-stranded RNA-originated killer phenotypes in baking yeast. PMID:27313294

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

    PubMed

    Konovalovas, Aleksandras; Serviené, Elena; Serva, Saulius

    2016-01-01

    We cloned and sequenced the complete genome of the L-A-28 virus from the Saccharomyces cerevisiae K28 killer strain. This sequence completes the set of currently identified L-A helper viruses required for expression of double-stranded RNA-originated killer phenotypes in baking yeast. PMID:27313294

  4. Construction of ploidy series of Saccharomyces cerevisiae by the plasmid YCplac33-GHK.

    PubMed

    Hou, Lihua; Li, Xiaoyang; Wang, Cong; Cao, Xiaohong; Wang, Haiyong

    2013-04-01

    An effective approach, using the plasmid YCplac33-GHK, is developed to construct a ploidy series of Saccharomyces cerevisiae. YCplac33-GHK harbors the HO gene under the control of galactose-inducible promoter and KanMX4 as the selective marker. The simple method can solve the problem of industrial applications of strains with resistance genes. PMID:23430413

  5. Genetic variation of the repeated MAL loci in natural populations of Saccharomyces cerevisiae and Saccharomyces paradoxus.

    PubMed

    Naumov, G I; Naumova, E S; Michels, C A

    1994-03-01

    In Saccharomyces cerevisiae, the gene functions required to ferment the disaccharide maltose are encoded by the MAL loci. Any one of five highly sequence homologous MAL loci identified in various S. cerevisiae strains (called MAL1, 2, 3, 4 and 6) is sufficient to ferment maltose. Each is a complex of three genes encoding maltose permease, maltase and a transcription activator. This family of loci maps to telomere-linked positions on different chromosomes and most natural strains contain more than one MAL locus. A number of naturally occurring, mutant alleles of MAL1 and MAL3 have been characterized which lack one or more of the gene functions encoded by the fully functional MAL loci. Loss of these gene functions appears to have resulted from mutation and/or rearrangement within the locus. Studies to date concentrated on the standard maltose fermenting strains of S. cerevisiae available from the Berkeley Yeast Stock Center collection. In this report we extend our genetic analysis of the MAL loci to a number of maltose fermenting and nonfermenting natural strains of S. cerevisiae and Saccharomyces paradoxus. No new MAL loci were discovered but several new mutant alleles of MAL1 were identified. The evolution of this gene family is discussed. PMID:8005435

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

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

    PubMed

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

    2015-08-01

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

  9. Cytotype distribution at a diploid-tetraploid contact zone in Chamerion (Epilobium) angustifolium (Onagraceae).

    PubMed

    Husband, B C; Schemske, D W

    1998-12-01

    In North America, the geographic distributions of diploid and tetraploid Chamerion (formerly Epilobium) angustifolium overlap in a narrow zone along the southern border of the boreal forest and along the Rocky Mountains. We examined the frequency and distribution of diploid and tetraploid cytotypes in a narrow (5 km) zone of sympatry across an elevational gradient and in putatively uniform diploid and tetraploid reference populations on the Beartooth Pass, in the Rocky Mountains of southern Montana-northern Wyoming. All five reference populations sampled were dominated by a single cytotype, but only one was completely uniform. In the zone of sympatry, 27 transects were sampled every 2 m for a total of 238 plants. Reproductive status (vegetative, flower buds, open flowers) was recorded, and the ploidy of each plant was determined by flow cytometry. Diploid and tetraploid plants predominated (36 and 55%, respectively) but were heterogeneously distributed among the transects. Six of the 27 transects were fixed for a single cytotype (four transects, diploid; two transects, tetraploid), and in seven others either diploids or tetraploids predominated (frequency >75%). Triploids represented 9% of the total sample and occurred most frequently in transects containing both diploids and tetraploids (G = 3.4, df = 2, P = 0.07). Diploids were more often reproductive (in bud, flower, or fruit) than either triploids or tetraploids (G = 12.0, df = 2, P < 0.001) and were the only cytotype to have produced open flowers. These results suggest that the zone of sympatry is best characterized as a mosaic rather than a cline, with diploid and tetraploids in close proximity and that the distribution of polyploidy is regulated by ecological sorting in a heterogeneous physical environment. PMID:21719413

  10. Molecular epidemiology of clonal diploids: a quick overview and a short DIY (do it yourself) notice.

    PubMed

    De Meeûs, Thierry; Lehmann, Laurent; Balloux, François

    2006-03-01

    In this short review we report the basic notions needed for understanding the population genetics of clonal diploids. We focus on the consequences of clonality on the distribution of genetic diversity within individuals, between individuals and between populations. We then summarise how to detect clonality in mainly sexual populations, conversely, how to detect sexuality in mainly clonal populations and also how genetic differentiation between populations is affected by clonality in diploids. This information is then used for building recipes on how to analyse and interpret genetic polymorphism data in molecular epidemiology studies of clonal diploids. PMID:16290062

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

    PubMed

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

    2007-05-01

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

  12. Peptidase activities in Saccharomyces cerevisiae.

    PubMed Central

    Rose, B; Becker, J M; Naider, F

    1979-01-01

    At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives. Images PMID:378955

  13. Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts.

    PubMed

    Gallone, Brigida; Steensels, Jan; Prahl, Troels; Soriaga, Leah; Saels, Veerle; Herrera-Malaver, Beatriz; Merlevede, Adriaan; Roncoroni, Miguel; Voordeckers, Karin; Miraglia, Loren; Teiling, Clotilde; Steffy, Brian; Taylor, Maryann; Schwartz, Ariel; Richardson, Toby; White, Christopher; Baele, Guy; Maere, Steven; Verstrepen, Kevin J

    2016-09-01

    Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP. PMID:27610566

  14. PET genes of Saccharomyces cerevisiae.

    PubMed Central

    Tzagoloff, A; Dieckmann, C L

    1990-01-01

    We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae. PMID:2215420

  15. Transcriptional regulation by ergosterol in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Smith, S J; Crowley, J H; Parks, L W

    1996-01-01

    Sterol biosynthesis in the yeast Saccharomyces cerevisiae is an energy-expensive, aerobic process, requiring heme and molecular oxygen. Heme, also synthesized exclusively during aerobic growth, not only acts as an enzymatic cofactor but also is directly and indirectly responsible for the transcriptional control of several yeast genes. Because of their biosynthetic similarities, we hypothesized that ergosterol, like heme, may have a regulatory function. Sterols are known to play a structural role in membrane integrity, but regulatory roles have not been characterized. To test possible regulatory roles of sterol, the promoter for the ERG3 gene, encoding the sterol C-5 desaturase, was fused to the bacterial lacZ reporter gene. This construct was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as ninefold in erg mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifungal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate for the first time that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae. PMID:8816455

  16. Genetic determinants for enhanced glycerol growth of Saccharomyces cerevisiae.

    PubMed

    Swinnen, Steve; Ho, Ping-Wei; Klein, Mathias; Nevoigt, Elke

    2016-07-01

    The yeast Saccharomyces cerevisiae generally shows a low natural capability to utilize glycerol as the sole source of carbon, particularly when synthetic medium is used and complex supplements are omitted. Nevertheless, wild type isolates have been identified that show a moderate growth under these conditions. In the current study we made use of intraspecies diversity to identify targets suitable for reverse metabolic engineering of the non-growing laboratory strain CEN.PK113-1A. A genome-wide genetic mapping experiment using pooled-segregant whole-genome sequence analysis was conducted, and one major and several minor genetic loci were identified responsible for the superior glycerol growth phenotype of the previously selected S. cerevisiae strain CBS 6412-13A. Downscaling of the major locus by fine-mapping and reciprocal hemizygosity analysis allowed the parallel identification of two superior alleles (UBR2CBS 6412-13A and SSK1CBS 6412-13A). These alleles together with the previously identified GUT1CBS 6412-13A allele were used to replace the corresponding alleles in the strain CEN.PK113-1A. In this way, glycerol growth could be established reaching a maximum specific growth rate of 0.08h(-1). Further improvement to a maximum specific growth rate of 0.11h(-1) could be achieved by heterologous expression of the glycerol facilitator FPS1 from Cyberlindnera jadinii. PMID:26971668

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

    PubMed

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

    2016-04-01

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

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

    SciTech Connect

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

    1996-06-01

    Previous experiments in vitro have demonstrated the ability of acylphosphatase to increase the rate of glucose fermentation in yeast. To evaluate the possibility of increasing fermentation in vivo also, a chemically synthesized DNA sequence coding for human muscle acylphosphatase was expressed at high level in Saccharomyces cerevisiae. Ethanol production was measured in these engineered strains in comparison with a control. Acylphosphatase expression strongly increased the rate of ethanol production both in aerobic and anaerobic culture. This finding may be potentially important for the development of more efficient industrial fermentation processes. 20 refs., 5 figs.

  19. A G-protein alpha subunit from asexual Candida albicans functions in the mating signal transduction pathway of Saccharomyces cerevisiae and is regulated by the a1-alpha 2 repressor.

    PubMed Central

    Sadhu, C; Hoekstra, D; McEachern, M J; Reed, S I; Hicks, J B

    1992-01-01

    We have isolated a gene, designated CAG1, from Candida albicans by using the G-protein alpha-subunit clone SCG1 of Saccharomyces cerevisiae as a probe. Amino acid sequence comparison revealed that CAG1 is more homologous to SCG1 than to any other G protein reported so far. Homology between CAG1 and SCG1 not only includes the conserved guanine nucleotide binding domains but also spans the normally variable regions which are thought to be involved in interaction with the components of the specific signal transduction pathway. Furthermore, CAG1 contains a central domain, previously found only in SCG1. cag1 null mutants of C. albicans created by gene disruption produced no readily detectable phenotype. The C. albicans CAG1 gene complemented both the growth and mating defects of S. cerevisiae scg1 null mutants when carried on either a low- or high-copy-number plasmid. In diploid C. albicans, the CAG1 transcript was readily detectable in mycelial and yeast cells of both the white and opaque forms. However, the CAG1-specific transcript in S. cerevisiae transformants containing the C. albicans CAG1 gene was observed only in haploid cells. This transcription pattern matches that of SCG1 in S. cerevisiae and is caused by a1-alpha 2 mediated repression in diploid cells. That is, CAG1 behaves as a haploid-specific gene in S. cerevisiae, subject to control by the a1-alpha 2 mating-type regulation pathway. We infer from these results that C. albicans may have a signal transduction system analogous to that controlling mating type in S. cerevisiae or possibly even a sexual pathway that has so far remained undetected. Images PMID:1569935

  20. Haematological parameters in Umbrina cirrosa (Teleostei, Sciaenidae): a comparison between diploid and triploid specimens.

    PubMed

    Ballarin, Loriano; Dall'Oro, Manuela; Bertotto, Daniela; Libertini, Angelo; Francescon, Antonia; Barbaro, Alvise

    2004-05-01

    Haematological features were compared between diploid and triploid specimens of the ray-finned fish Umbrina cirrosa. No significant differences between diploids and triploids were reported in haematocrit and total haemoglobin concentration, but erythrocytes and thrombocytes were significantly greater in size in triploids. Glycaemia was significantly lower in diploids, whereas triploid erythrocytes were more resistant to osmotic stress. In triploids, a greater fraction of leukocytes was positive for alkaline phosphatase activity, when stimulated with Bacillus clausii spores, otherwise no significant increase of oxygen consumption was observed in triploid leukocytes after stimulation, based on assays for superoxide anions. Triploids were characterized by a lower concentration of circulating blood cells with a lower surface/volume ratio when compared with diploids. These features may lead to a general disadvantage of triploids in withstanding stress conditions: a situation that needs to be taken into account in aquaculture practice. PMID:15165570

  1. Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells

    SciTech Connect

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. )

    1993-07-01

    Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli [beta]-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. The authors showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

  2. Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae

    SciTech Connect

    Sarthy, A.V.; McConaughy, B.L.; Lobo, Z.; Sundstrom, J.A.; Furlong, C.E.; Hall, B.D.

    1987-09-01

    Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10/sup 3/-fold less active.

  3. Diploid and triploid African catfish (Clarias gariepinus) differ in biomarker responses to the pesticide chlorpyrifos.

    PubMed

    Karami, Ali; Goh, Yong-Meng; Jahromi, Mohammad Faseleh; Lazorchak, James M; Abdullah, Maha; Courtenay, Simon C

    2016-07-01

    The impacts of environmental stressors on polyploid organisms are largely unknown. This study investigated changes in morphometric, molecular, and biochemical parameters in full-sibling diploid and triploid African catfish (Clarias gariepinus) in response to chlorpyrifos (CPF) exposures. Juvenile fish were exposed to three concentrations of CPF (mean measured μg/L (SD): 9.71 (2.27), 15.7 (3.69), 31.21 (5.04)) under a static-renewal condition for 21days. Diploid control groups had higher hepatosomatic index (HSI), plasma testosterone (T), and brain GnRH and cyp19a2 expression levels than triploids. In CPF-exposed groups, changes in HSI, total weight and length were different between the diploid and triploid fish. In contrast, condition factor did not alter in any of the treatments, while visceral-somatic index (VSI) changed only in diploids. In diploid fish, exposure to CPF did not change brain 11β-hsd2, ftz-f1, foxl2, GnRH or cyp19a2 mRNA levels, while reduced tph2 transcript levels compared to the control group. In contrast, 11β-hsd2 and foxl2 expression levels were changed in triploids following CPF exposures. In diploids, plasma T levels showed a linear dose-response reduction across CPF treatments correlating with liver weight and plasma total cholesterol concentrations. In contrast, no changes in plasma cholesterol and T concentrations were observed in triploids. Plasma cortisol and 17-β estradiol (E2) showed no response to CPF exposure in either ploidy. Results of this first comparison of biomarker responses to pesticide exposure in diploid and polyploid animals showed substantial differences between diploid and triploid C. gariepinus. PMID:26994807

  4. The population genetics of clonal and partially clonal diploids.

    PubMed Central

    Balloux, François; Lehmann, Laurent; de Meeûs, Thierry

    2003-01-01

    The consequences of variable rates of clonal reproduction on the population genetics of neutral markers are explored in diploid organisms within a subdivided population (island model). We use both analytical and stochastic simulation approaches. High rates of clonal reproduction will positively affect heterozygosity. As a consequence, nearly twice as many alleles per locus can be maintained and population differentiation estimated as F(ST) value is strongly decreased in purely clonal populations as compared to purely sexual ones. With increasing clonal reproduction, effective population size first slowly increases and then points toward extreme values when the reproductive system tends toward strict clonality. This reflects the fact that polymorphism is protected within individuals due to fixed heterozygosity. Contrarily, genotypic diversity smoothly decreases with increasing rates of clonal reproduction. Asexual populations thus maintain higher genetic diversity at each single locus but a lower number of different genotypes. Mixed clonal/sexual reproduction is nearly indistinguishable from strict sexual reproduction as long as the proportion of clonal reproduction is not strongly predominant for all quantities investigated, except for genotypic diversities (both at individual loci and over multiple loci). PMID:12930767

  5. The diploid genome sequence of an Asian individual

    PubMed Central

    Wang, Jun; Wang, Wei; Li, Ruiqiang; Li, Yingrui; Tian, Geng; Goodman, Laurie; Fan, Wei; Zhang, Junqing; Li, Jun; Zhang, Juanbin; Guo, Yiran; Feng, Binxiao; Li, Heng; Lu, Yao; Fang, Xiaodong; Liang, Huiqing; Du, Zhenglin; Li, Dong; Zhao, Yiqing; Hu, Yujie; Yang, Zhenzhen; Zheng, Hancheng; Hellmann, Ines; Inouye, Michael; Pool, John; Yi, Xin; Zhao, Jing; Duan, Jinjie; Zhou, Yan; Qin, Junjie; Ma, Lijia; Li, Guoqing; Yang, Zhentao; Zhang, Guojie; Yang, Bin; Yu, Chang; Liang, Fang; Li, Wenjie; Li, Shaochuan; Li, Dawei; Ni, Peixiang; Ruan, Jue; Li, Qibin; Zhu, Hongmei; Liu, Dongyuan; Lu, Zhike; Li, Ning; Guo, Guangwu; Zhang, Jianguo; Ye, Jia; Fang, Lin; Hao, Qin; Chen, Quan; Liang, Yu; Su, Yeyang; san, A.; Ping, Cuo; Yang, Shuang; Chen, Fang; Li, Li; Zhou, Ke; Zheng, Hongkun; Ren, Yuanyuan; Yang, Ling; Gao, Yang; Yang, Guohua; Li, Zhuo; Feng, Xiaoli; Kristiansen, Karsten; Wong, Gane Ka-Shu; Nielsen, Rasmus; Durbin, Richard; Bolund, Lars; Zhang, Xiuqing; Li, Songgang; Yang, Huanming; Wang, Jian

    2009-01-01

    Here we present the first diploid genome sequence of an Asian individual. The genome was sequenced to 36-fold average coverage using massively parallel sequencing technology. We aligned the short reads onto the NCBI human reference genome to 99.97% coverage, and guided by the reference genome, we used uniquely mapped reads to assemble a high-quality consensus sequence for 92% of the Asian individual's genome. We identified approximately 3 million single-nucleotide polymorphisms (SNPs) inside this region, of which 13.6% were not in the dbSNP database. Genotyping analysis showed that SNP identification had high accuracy and consistency, indicating the high sequence quality of this assembly. We also carried out heterozygote phasing and haplotype prediction against HapMap CHB and JPT haplotypes (Chinese and Japanese, respectively), sequence comparison with the two available individual genomes (J. D. Watson and J. C. Venter), and structural variation identification. These variations were considered for their potential biological impact. Our sequence data and analyses demonstrate the potential usefulness of next-generation sequencing technologies for personal genomics. PMID:18987735

  6. Enhancer Runaway and the Evolution of Diploid Gene Expression

    PubMed Central

    Fyon, Frédéric; Cailleau, Aurélie; Lenormand, Thomas

    2015-01-01

    Evidence is mounting that the evolution of gene expression plays a major role in adaptation and speciation. Understanding the evolution of gene regulatory regions is indeed an essential step in linking genotypes and phenotypes and in understanding the molecular mechanisms underlying evolutionary change. The common view is that expression traits (protein folding, expression timing, tissue localization and concentration) are under natural selection at the individual level. Here, we use a theoretical approach to show that, in addition, in diploid organisms, enhancer strength (i.e., the ability of enhancers to activate transcription) may increase in a runaway process due to competition for expression between homologous enhancer alleles. These alleles may be viewed as self-promoting genetic elements, as they spread without conferring a benefit at the individual level. They gain a selective advantage by getting associated to better genetic backgrounds: deleterious mutations are more efficiently purged when linked to stronger enhancers. This process, which has been entirely overlooked so far, may help understand the observed overrepresentation of cis-acting regulatory changes in between-species phenotypic differences, and sheds a new light on investigating the contribution of gene expression evolution to adaptation. PMID:26561855

  7. The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

    PubMed Central

    Suzuki, Yo; Stam, Jason; Novotny, Mark; Yachie, Nozomu; Lasken, Roger S.; Roth, Frederick P.

    2012-01-01

    Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative. In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated. Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction. The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci—either Green Monster GMToolkit-a or GMToolkit-α at the

  8. The green monster process for the generation of yeast strains carrying multiple gene deletions.

    PubMed

    Suzuki, Yo; Stam, Jason; Novotny, Mark; Yachie, Nozomu; Lasken, Roger S; Roth, Frederick P

    2012-01-01

    Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition(1,2). There are examples where many genes need to be deleted to unmask hidden gene functions(3,4). Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative. In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker(5,6,7,8,9,10). However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated. Here we describe the Green Monster method for routinely engineering multiple deletions in yeast(11). In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction. The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci-either Green Monster GMToolkit-a or GMToolkit

  9. Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

    PubMed

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

    2016-03-01

    Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces. PMID:26671616

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

    PubMed

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

    2015-12-01

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

  11. Molecular population genetics and evolution of a prion-like protein in Saccharomyces cerevisiae.

    PubMed Central

    Jensen, M A; True, H L; Chernoff, Y O; Lindquist, S

    2001-01-01

    The prion-like behavior of Sup35p, the eRF3 homolog in the yeast Saccharomyces cerevisiae, mediates the activity of the cytoplasmic nonsense suppressor known as [PSI(+)]. Sup35p is divided into three regions of distinct function. The N-terminal and middle (M) regions are required for the induction and propagation of [PSI(+)] but are not necessary for translation termination or cell viability. The C-terminal region encompasses the termination function. The existence of the N-terminal region in SUP35 homologs of other fungi has led some to suggest that this region has an adaptive function separate from translation termination. To examine this hypothesis, we sequenced portions of SUP35 in 21 strains of S. cerevisiae, including 13 clinical isolates. We analyzed nucleotide polymorphism within this species and compared it to sequence divergence from a sister species, S. paradoxus. The N domain of Sup35p is highly conserved in amino acid sequence and is highly biased in codon usage toward preferred codons. Amino acid changes are under weak purifying selection based on a quantitative analysis of polymorphism and divergence. We also conclude that the clinical strains of S. cerevisiae are not recently derived and that outcrossing between strains in S. cerevisiae may be relatively rare in nature. PMID:11606530

  12. Effect of Domestication on the Spread of the [PIN+] Prion in Saccharomyces cerevisiae

    PubMed Central

    Kelly, Amy C.; Busby, Ben; Wickner, Reed B.

    2014-01-01

    Prions (infectious proteins) cause fatal neurodegenerative diseases in mammals. In the yeast Saccharomyces cerevisiae, many toxic and lethal variants of the [PSI+] and [URE3] prions have been identified in laboratory strains, although some commonly studied variants do not seem to impair cell growth. Phylogenetic analysis has revealed four major clades of S. cerevisiae that share histories of two prion proteins and largely correspond to different ecological niches of yeast. The [PIN+] prion was most prevalent in commercialized niches, infrequent among wine/vineyard strains, and not observed in ancestral isolates. As previously reported, the [PSI+] and [URE3] prions are not found in any of these strains. Patterns of heterozygosity revealed genetic mosaicism and indicated extensive outcrossing among divergent strains in commercialized environments. In contrast, ancestral isolates were all homozygous and wine/vineyard strains were closely related to each other and largely homozygous. Cellular growth patterns were highly variable within and among clades, although ancestral isolates were the most efficient sporulators and domesticated strains showed greater tendencies for flocculation. [PIN+]-infected strains had a significantly higher likelihood of polyploidy, showed a higher propensity for flocculation compared to uninfected strains, and had higher sporulation efficiencies compared to domesticated, uninfected strains. Extensive phenotypic variability among strains from different environments suggests that S. cerevisiae is a niche generalist and that most wild strains are able to switch from asexual to sexual and from unicellular to multicellular growth in response to environmental conditions. Our data suggest that outbreeding and multicellular growth patterns adapted for domesticated environments are ecological risk factors for the [PIN+] prion in wild yeast. PMID:24812307

  13. Trait responses of invasive aquatic macrophyte congeners: colonizing diploid outperforms polyploid

    PubMed Central

    Grewell, Brenda J.; Skaer Thomason, Meghan J.; Futrell, Caryn J.; Iannucci, Maria; Drenovsky, Rebecca E.

    2016-01-01

    Understanding traits underlying colonization and niche breadth of invasive plants is key to developing sustainable management solutions to curtail invasions at the establishment phase, when efforts are often most effective. The aim of this study was to evaluate how two invasive congeners differing in ploidy respond to high and lowresource availability following establishment from asexual fragments. Because polyploids are expected to have wider niche breadths than diploid ancestors, we predicted that a decaploid species would have superior ability to maximize resource uptake and use, and outperform a diploid congener when colonizing environments with contrasting light and nutrient availability. A mesocosm experiment was designed to test the main and interactive effects of ploidy (diploid and decaploid) and soil nutrient availability (low and high) nested within light environments (shade and sun) of two invasive aquatic plant congeners. Counter to our predictions, the diploid congener outperformed the decaploid in the early stage of growth. Although growth was similar and low in the cytotypes at low nutrient availability, the diploid species had much higher growth rate and biomass accumulation than the polyploid with nutrient enrichment, irrespective of light environment. Our results also revealed extreme differences in time to anthesis between the cytotypes. The rapid growth and earlier flowering of the diploid congener relative to the decaploid congener represent alternate strategies for establishment and success. PMID:26921139

  14. Ribosomal DNA locus variation and REMAP analysis of the diploid and triploid complexes of Lilium lancifolium.

    PubMed

    Nguyen, Truong Xuan; Lee, Sung-Il; Rai, Rameshwar; Kim, Nam-Soo; Kim, Jong Hwa

    2016-08-01

    Lilium lancifolium Thunb. (2n = 2x = 24) is a cytologically conspicuous species with both diploids and triploids in nature. Cytological and molecular genetic analyses were carried out in both diploids and triploids that were collected from 55 geographical locations in Korea, Japan, and China. While the 5S rRNA gene loci were located at duplicated loci on the long arm of chromosome 2, the 45S rRNA gene loci were present in chromosomes 1, 2, 4, 6, 7, and 11. While the loci on chromosomes 1 and 7 were constant, the loci on chromosomes 2, 4, 6, 7, and 11 were variable in some plants so that the L. lancifolium accessions were grouped into 7 cytotypes in diploids and 12 cytotypes in triploids. REMAP marker analysis revealed that the diploids were classified into seven clusters, and the triploids were classified into a large cluster. Geographic, cytological, and genetic differentiations were not related in both the diploid and triploid accessions of L. lancifolium. Thus, current genetic variations occurred prior to the geographic differentiation in both diploids and triploids, and the 45S rDNA cytotype variations occurred after geographic differentiation in the current habitats of L. lancifolium. PMID:27458741

  15. The evolutionary advantage of haploid versus diploid microbes in nutrient-poor environments.

    PubMed

    Bessho, Kazuhiro; Iwasa, Yoh; Day, Troy

    2015-10-21

    Sexual eukaryotic organisms are characterized by haploid and diploid nuclear phases. In many organisms, growth and development occur in both haploid and diploid phases, and the relative length of these phases exhibits considerable diversity. A number of hypotheses have been put forward to explain the maintenance of this diversity of life cycles and the advantage of being haploid versus that of being diploid. The nutrient-limitation hypothesis postulates that haploid cells, because they are small and thus have a higher surface area to volume ratio, are advantageous in nutrient-poor environments. In this paper, we examine this hypothesis theoretically and determine the conditions under which it holds. On the basis of our analysis, we make the following predictions. First, the relative advantages of different ploidy levels strongly depend on the ploidy-dependent energy conversion efficiency and the scaling of mortality with cell size. Specifically, haploids enjoy a higher intrinsic population growth rate than diploids do under nutrient-poor conditions, but under nutrient-rich conditions the intrinsic population growth rate of diploids is higher, provided that the energy conversion efficiency of diploids is higher than that of haploids and the scaling of mortality with cell size is weak. Second, differences in nutrient concentration in the inflowing medium have almost no effect on the relative advantage of ploidy levels at population equilibrium. Our study illustrates the importance of explicit modeling of microbial life history and population dynamics to understand the evolution of ploidy levels. PMID:26247141

  16. The presence of the enhanced K/Na discrimination trait in diploid Triticum species.

    PubMed

    Gorham, J; Bristol, A; Young, E M; Wyn Jones, R G

    1991-10-01

    A number of accessions of the three species of diploid wheat, Triticum boeoticum, T. monococcum, and T. urartu, were grown in 50 mol m(-3) NaCl+2.5 mol m(-3) CaCl2. Sodium accumulation in the leaves was low and potassium concentrations remained high. This was not the case in T. durum grown under the same conditions, and indicates the presence in diploid wheats of the enhanced K/Na discrimination character which has previously been found in Aegilops squarrosa and hexaploid wheat. None of the accessions of diploid wheat showed poor K/Na discrimination, which suggests that if the A genome of modern tetraploid wheats was derived from a diploid Triticum species, then the enhanced K/Na discrimination character became altered after the formation of the original allopolyploid. Another possibility is that a diploid wheat that did not have the enhanced K/Na discrimination character was involved in the hybridization event which produced tetraploid wheat, and that this diploid is now extinct or has not yet been discovered. PMID:24213448

  17. Somatic cell nuclear transfer: Infinite reproduction of a unique diploid genome

    SciTech Connect

    Kishigami, Satoshi Wakayama, Sayaka; Hosoi, Yoshihiko; Iritani, Akira; Wakayama, Teruhiko

    2008-06-10

    In mammals, a diploid genome of an individual following fertilization of an egg and a spermatozoon is unique and irreproducible. This implies that the generated unique diploid genome is doomed with the individual ending. Even as cultured cells from the individual, they cannot normally proliferate in perpetuity because of the 'Hayflick limit'. However, Dolly, the sheep cloned from an adult mammary gland cell, changes this scenario. Somatic cell nuclear transfer (SCNT) enables us to produce offspring without germ cells, that is, to 'passage' a unique diploid genome. Animal cloning has also proven to be a powerful research tool for reprogramming in many mammals, notably mouse and cow. The mechanism underlying reprogramming, however, remains largely unknown and, animal cloning has been inefficient as a result. More momentously, in addition to abortion and fetal mortality, some cloned animals display possible premature aging phenotypes including early death and short telomere lengths. Under these inauspicious conditions, is it really possible for SCNT to preserve a diploid genome? Delightfully, in mouse and recently in primate, using SCNT we can produce nuclear transfer ES cells (ntES) more efficiently, which can preserve the eternal lifespan for the 'passage' of a unique diploid genome. Further, new somatic cloning technique using histone-deacetylase inhibitors has been developed which can significantly increase the previous cloning rates two to six times. Here, we introduce SCNT and its value as a preservation tool for a diploid genome while reviewing aging of cloned animals on cellular and individual levels.

  18. Trait responses of invasive aquatic macrophyte congeners: colonizing diploid outperforms polyploid.

    PubMed

    Grewell, Brenda J; Skaer Thomason, Meghan J; Futrell, Caryn J; Iannucci, Maria; Drenovsky, Rebecca E

    2016-01-01

    Understanding traits underlying colonization and niche breadth of invasive plants is key to developing sustainable management solutions to curtail invasions at the establishment phase, when efforts are often most effective. The aim of this study was to evaluate how two invasive congeners differing in ploidy respond to high and lowresource availability following establishment from asexual fragments. Because polyploids are expected to have wider niche breadths than diploid ancestors, we predicted that a decaploid species would have superior ability to maximize resource uptake and use, and outperform a diploid congener when colonizing environments with contrasting light and nutrient availability. A mesocosm experiment was designed to test the main and interactive effects of ploidy (diploid and decaploid) and soil nutrient availability (low and high) nested within light environments (shade and sun) of two invasive aquatic plant congeners. Counter to our predictions, the diploid congener outperformed the decaploid in the early stage of growth. Although growth was similar and low in the cytotypes at low nutrient availability, the diploid species had much higher growth rate and biomass accumulation than the polyploid with nutrient enrichment, irrespective of light environment. Our results also revealed extreme differences in time to anthesis between the cytotypes. The rapid growth and earlier flowering of the diploid congener relative to the decaploid congener represent alternate strategies for establishment and success. PMID:26921139

  19. Insights into the Evolution of Cotton Diploids and Polyploids from Whole-Genome Re-sequencing

    PubMed Central

    Page, Justin T.; Huynh, Mark D.; Liechty, Zach S.; Grupp, Kara; Stelly, David; Hulse, Amanda M.; Ashrafi, Hamid; Van Deynze, Allen; Wendel, Jonathan F.; Udall, Joshua A.

    2013-01-01

    Understanding the composition, evolution, and function of the Gossypium hirsutum (cotton) genome is complicated by the joint presence of two genomes in its nucleus (AT and DT genomes). These two genomes were derived from progenitor A-genome and D-genome diploids involved in ancestral allopolyploidization. To better understand the allopolyploid genome, we re-sequenced the genomes of extant diploid relatives that contain the A1 (Gossypium herbaceum), A2 (Gossypium arboreum), or D5 (Gossypium raimondii) genomes. We conducted a comparative analysis using deep re-sequencing of multiple accessions of each diploid species and identified 24 million SNPs between the A-diploid and D-diploid genomes. These analyses facilitated the construction of a robust index of conserved SNPs between the A-genomes and D-genomes at all detected polymorphic loci. This index is widely applicable for read mapping efforts of other diploid and allopolyploid Gossypium accessions. Further analysis also revealed locations of putative duplications and deletions in the A-genome relative to the D-genome reference sequence. The approximately 25,400 deleted regions included more than 50% deletion of 978 genes, including many involved with starch synthesis. In the polyploid genome, we also detected 1,472 conversion events between homoeologous chromosomes, including events that overlapped 113 genes. Continued characterization of the Gossypium genomes will further enhance our ability to manipulate fiber and agronomic production of cotton. PMID:23979935

  20. Insights into the evolution of cotton diploids and polyploids from whole-genome re-sequencing.

    PubMed

    Page, Justin T; Huynh, Mark D; Liechty, Zach S; Grupp, Kara; Stelly, David; Hulse, Amanda M; Ashrafi, Hamid; Van Deynze, Allen; Wendel, Jonathan F; Udall, Joshua A

    2013-10-01

    Understanding the composition, evolution, and function of the Gossypium hirsutum (cotton) genome is complicated by the joint presence of two genomes in its nucleus (AT and DT genomes). These two genomes were derived from progenitor A-genome and D-genome diploids involved in ancestral allopolyploidization. To better understand the allopolyploid genome, we re-sequenced the genomes of extant diploid relatives that contain the A1 (Gossypium herbaceum), A2 (Gossypium arboreum), or D5 (Gossypium raimondii) genomes. We conducted a comparative analysis using deep re-sequencing of multiple accessions of each diploid species and identified 24 million SNPs between the A-diploid and D-diploid genomes. These analyses facilitated the construction of a robust index of conserved SNPs between the A-genomes and D-genomes at all detected polymorphic loci. This index is widely applicable for read mapping efforts of other diploid and allopolyploid Gossypium accessions. Further analysis also revealed locations of putative duplications and deletions in the A-genome relative to the D-genome reference sequence. The approximately 25,400 deleted regions included more than 50% deletion of 978 genes, including many involved with starch synthesis. In the polyploid genome, we also detected 1,472 conversion events between homoeologous chromosomes, including events that overlapped 113 genes. Continued characterization of the Gossypium genomes will further enhance our ability to manipulate fiber and agronomic production of cotton. PMID:23979935

  1. Fatal Saccharomyces Cerevisiae Aortic Graft Infection

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  2. Saccharomyces cerevisiae osteomyelitis in an immunocompetent baker.

    PubMed

    Seng, Piseth; Cerlier, Alexandre; Cassagne, Carole; Coulange, Mathieu; Legré, Regis; Stein, Andreas

    2016-01-01

    Invasive infection caused by Saccharomyces cerevisiae is rare. We report the first case of osteomyelitis caused by S. cerevisiae (baker's yeast) in a post-traumatic patient. The clinical outcome was favorable after surgical debridement, prolonged antifungal treatment and hyperbaric oxygen therapy. PMID:27347482

  3. Stationary phase in the yeast Saccharomyces cerevisiae.

    PubMed Central

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

    1993-01-01

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

  4. Expression of exoinulinase genes in Saccharomyces cerevisiae to improve ethanol production from inulin sources.

    PubMed

    Yuan, Bo; Wang, Shi-An; Li, Fu-Li

    2013-10-01

    To improve inulin utilization and ethanol fermentation, exoinulinase genes from the yeast Kluyveromyces marxianus and the recently identified yeast, Candida kutaonensis, were expressed in Saccharomyces cerevisiae. S. cerevisiae harboring the exoinulinase gene from C. kutaonensis gave higher ethanol yield and productivity from both inulin (0.38 vs. 0.34 g/g and 1.35 vs. 1.22 g l(-1) h(-1)) and Jerusalem artichoke tuber flour (0.47 vs. 0.46 g/g and 1.62 vs. 1.54 g l(-1) h(-1)) compared with the strain expressing the exoinulinase gene from K. marxianus. Thus, the exoinulinase gene from C. kutaonensis is advantageous for engineering S. cerevisiae to improve ethanol fermentation from inulin sources. PMID:23743955

  5. Genetics of Somatic Fusion in PHYSARUM POLYCEPHALUM: the Ppii Strain

    PubMed Central

    Collins, O'Neil Ray; Haskins, Edward F.

    1972-01-01

    Plasmodial (somatic) fusion in a strain of Physarum polycephalum, a true slime mold, is controlled by four loci, each of which displays simple dominance. Two diploid plasmodia fuse with each other only if they are phenotypically or genotypically identical for all four fusion loci. PMID:17248576

  6. Cadmium biosorption by Saccharomyces cerevisiae

    SciTech Connect

    Volesky, B.; May, H.; Holan, Z.R. )

    1993-04-01

    Cadmium uptake by nonliving and resting cells of Saccharomyces cerevisiae obtained from aerobic or anaerobic cultures from pure cadmium-bearing solutions was examined. The highest cadmium uptake exceeding 70 mg Cd/g was observed with aerobic baker's yeast biomass from the exponential growth phase. Nearly linear sorption isotherms featured by higher sorbing resting cells together with metal deposits localized exclusively in vacuoles indicate the possibility of a different metal-sequestering mechanism when compared to dry nonliving yeasts which did not usually accumulate more than 20 mg Cd/g. The uptake of cadmium was relatively fast, 75% of the sorption completed in less than 5 min.

  7. The effects of microgravity on induced mutation in Escherichia coli and Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Takahashi, A.; Ohnishi, K.; Takahashi, S.; Masukawa, M.; Sekikawa, K.; Amano, T.; Nakano, T.; Nagaoka, S.; Ohnishi, T.

    2001-01-01

    We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91). We prepared dried samples of repair-deficient strains and parental strains of Escherichia ( E.) coli and Saccharomyces ( S.) cerevisiae given DNA damage treatment. After culture in space, we measured the induced-mutation frequencies and SOS-responses under microgravity. The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples. It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair. In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E. coli and S. cerevisiae cells.

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

    PubMed Central

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-01-01

    Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min−1•mg−1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation. PMID:26781725

  9. Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway

    PubMed Central

    Bettiga, Maurizio; Bengtsson, Oskar; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2009-01-01

    Background Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation. Results A new recombinant S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized. Conclusion Pentose fermenting recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar. PMID:19630951

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

    PubMed

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D; Mukhopadhyay, Aindrila

    2016-01-01

    Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min(-1)•mg(-1)) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation. PMID:26781725

  11. L-Histidine Inhibits Biofilm Formation and FLO11-Associated Phenotypes in Saccharomyces cerevisiae Flor Yeasts

    PubMed Central

    Bou Zeidan, Marc; Zara, Giacomo; Viti, Carlo; Decorosi, Francesca; Mannazzu, Ilaria; Budroni, Marilena; Giovannetti, Luciana; Zara, Severino

    2014-01-01

    Flor yeasts of Saccharomyces cerevisiae have an innate diversity of FLO11 which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling FLO11 alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce FLO11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to FLO11 expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air–liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the FLO11 gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts. PMID:25369456

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

    DOE PAGESBeta

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D.; Mukhopadhyay, Aindrila

    2016-01-19

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

  13. Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts

    PubMed Central

    2013-01-01

    Background Cellular senescence can be induced by a variety of extrinsic stimuli, and sustained exposure to sunlight is a key factor in photoaging of the skin. Accordingly, irradiation of skin fibroblasts by UVB light triggers cellular senescence, which is thought to contribute to extrinsic skin aging, although molecular mechanisms are incompletely understood. Here, we addressed molecular mechanisms underlying UVB induced senescence of human diploid fibroblasts. Results We observed a parallel activation of the p53/p21WAF1 and p16INK4a/pRb pathways. Using genome-wide transcriptome analysis, we identified a transcriptional signature of UVB-induced senescence that was conserved in three independent strains of human diploid fibroblasts (HDF) from skin. In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process. Bioinformatic analysis of miRNA-mRNA networks was performed to identify new functional mRNA targets with high confidence for miR-15a, miR-20a, miR-20b, miR-93, and miR-101. Already known targets of these miRNAs were identified in each case, validating the approach. Several new targets were identified for all of these miRNAs, with the potential to provide new insight in the process of UVB-induced senescence at a genome-wide level. Subsequent analysis was focused on miR-101 and its putative target gene Ezh2. We confirmed that Ezh2 is regulated by miR-101 in human fibroblasts, and found that both overexpression of miR-101 and downregulation of Ezh2 independently induce senescence in the absence of UVB irradiation. However, the downregulation of miR-101 was not sufficient to block the phenotype of UVB-induced senescence, suggesting that other UVB-induced processes induce the senescence response in a pathway redundant with upregulation of miR-101. Conclusion We performed a comprehensive screen for UVB-regulated microRNAs in human diploid

  14. Obstruction of adaptation in diploids by recessive, strongly deleterious alleles

    PubMed Central

    Assaf, Zoe June; Petrov, Dmitri A.; Blundell, Jamie R.

    2015-01-01

    Recessive deleterious mutations are common, causing many genetic disorders in humans and producing inbreeding depression in the majority of sexually reproducing diploids. The abundance of recessive deleterious mutations in natural populations suggests they are likely to be present on a chromosome when a new adaptive mutation occurs, yet the dynamics of recessive deleterious hitchhikers and their impact on adaptation remains poorly understood. Here we model how a recessive deleterious mutation impacts the fate of a genetically linked dominant beneficial mutation. The frequency trajectory of the adaptive mutation in this case is dramatically altered and results in what we have termed a “staggered sweep.” It is named for its three-phased trajectory: (i) Initially, the two linked mutations have a selective advantage while rare and will increase in frequency together, then (ii), at higher frequencies, the recessive hitchhiker is exposed to selection and can cause a balanced state via heterozygote advantage (the staggered phase), and (iii) finally, if recombination unlinks the two mutations, then the beneficial mutation can complete the sweep to fixation. Using both analytics and simulations, we show that strongly deleterious recessive mutations can substantially decrease the probability of fixation for nearby beneficial mutations, thus creating zones in the genome where adaptation is suppressed. These mutations can also significantly prolong the number of generations a beneficial mutation takes to sweep to fixation, and cause the genomic signature of selection to resemble that of soft or partial sweeps. We show that recessive deleterious variation could impact adaptation in humans and Drosophila. PMID:25941393

  15. AGAPE (Automated Genome Analysis PipelinE) for Pan-Genome Analysis of Saccharomyces cerevisiae

    PubMed Central

    Song, Giltae; Dickins, Benjamin J. A.; Demeter, Janos; Engel, Stacia; Dunn, Barbara; Cherry, J. Michael

    2015-01-01

    The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which integrates the steps of assembly, annotation, and variation calling. To assign strain-specific functional annotations, we identified genes that were not present in the reference genome. We classified these according to their presence or absence across strains and characterized each group of genes with known functional and phenotypic features. The functional roles of novel genes not found in the reference genome and associated with strains or groups of strains appear to be consistent with anticipated adaptations in specific lineages. As more S. cerevisiae strain genomes are released, our analysis can be used to collate genome data and relate it to lineage-specific patterns of genome evolution. Our new tool set will enhance our understanding of genomic and functional evolution in S. cerevisiae, and will be available to the yeast genetics and molecular biology community. PMID:25781462

  16. Mating-type Gene Switching in Saccharomyces cerevisiae.

    PubMed

    Lee, Cheng-Sheng; Haber, James E

    2015-04-01

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

  17. Gluconobacter cerevisiae sp. nov., isolated from the brewery environment.

    PubMed

    Spitaels, Freek; Wieme, Anneleen; Balzarini, Tom; Cleenwerck, Ilse; Van Landschoot, Anita; De Vuyst, Luc; Vandamme, Peter

    2014-04-01

    Three strains, LMG 27748(T), LMG 27749 and LMG 27882 with identical MALDI-TOF mass spectra were isolated from samples taken from the brewery environment. Analysis of the 16S rRNA gene sequence of strain LMG 27748(T) revealed that the taxon it represents was closely related to type strains of the species Gluconobacter albidus (100 % sequence similarity), Gluconobacter kondonii (99.9 %), Gluconobacter sphaericus (99.9 %) and Gluconobacter kanchanaburiensis (99.5 %). DNA-DNA hybridization experiments on the type strains of these species revealed moderate DNA relatedness values (39-65 %). The three strains used d-fructose, d-sorbitol, meso-erythritol, glycerol, l-sorbose, ethanol (weakly), sucrose and raffinose as a sole carbon source for growth (weak growth on the latter two carbon sources was obtained for strains LMG 27748(T) and LMG 27882). The strains were unable to grow on glucose-yeast extract medium at 37 °C. They produced acid from meso-erythritol and sucrose, but not from raffinose. d-Gluconic acid, 2-keto-d-gluconic acid and 5-keto-d-gluconic acid were produced from d-glucose, but not 2,5-diketo-d-gluconic acid. These genotypic and phenotypic characteristics distinguish strains LMG 27748(T), LMG 27749 and LMG 27882 from species of the genus Gluconobacter with validly published names and, therefore, we propose classifying them formally as representatives of a novel species, Gluconobacter cerevisiae sp. nov., with LMG 27748(T) ( = DSM 27644(T)) as the type strain. PMID:24368694

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

    PubMed Central

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

    2012-01-01

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

  19. Ecological success of a group of Saccharomyces cerevisiae/Saccharomyces kudriavzevii hybrids in the northern european wine-making environment.

    PubMed

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

    2012-05-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-02-01

    Baker's yeast (Saccharomyces cerevisiae) is the common yeast used in the fields of bread making, brewing, and bioethanol production. Growth rate, stress tolerance, ethanol titer, and byproducts yields are some of the most important agronomic traits of S. cerevisiae for industrial applications. Here, we developed a novel method of constructing S. cerevisiae strains for co-producing bioethanol and ergosterol. The genome of an industrial S. cerevisiae strain, ZTW1, was first reconstructed through treatment with an antimitotic drug followed by sporulation and hybridization. A total of 140 mutants were selected for ethanol fermentation testing, and a significant positive correlation between ergosterol content and ethanol production was observed. The highest performing mutant, ZG27, produced 7.9 % more ethanol and 43.2 % more ergosterol than ZTW1 at the end of fermentation. Chromosomal karyotyping and proteome analysis of ZG27 and ZTW1 suggested that this breeding strategy caused large-scale genome structural variations and global gene expression diversities in the mutants. Genetic manipulation further demonstrated that the altered expression activity of some genes (such as ERG1, ERG9, and ERG11) involved in ergosterol synthesis partly explained the trait improvement in ZG27. PMID:25475753

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

    PubMed

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

    2015-07-01

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

  3. In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

    PubMed

    Ravandi, E Ghotbi; Rezanejad, F; Dehghan, E

    2014-01-01

    Polyploidy has played a significant role in the evolutionary history of plants and is a valuable tool for obtaining useful characteristics. Because of the novelty of polyploids, comparison of their in vitro culture response with diploids would be notable. In this study, leaf explants from diploid, autotetraploid and mixoploid plants of Cichorium intybus L. were cultured in vitro on the similar media and under same conditions. The ploidy level of the obtained calluses and regenerants were determined by flow cytometry analysis. The callogenic response of leaf explants cultured on the callus induction medium did not depend on the ploidy level of their parental plants. According to the flow cytometry analysis, the increased ploidy levels (4x) and (8x) were observed in the callus cultures with diploid and tetraploid origin, respectively. A considerable difference was observed between the ploidy level of mixoploid plants and their calluses, indicating the dominance of diploid cells in the callus tissue. The results showed that polyploidy led to the loss of organogenic potential as the tetraploid origin calluses failed to regenerate, while the diploid origin calluses successfully regenerated to whole plants. PMID:25016827

  4. Comparison of Leaf Proteomes of Cassava (Manihot esculenta Crantz) Cultivar NZ199 Diploid and Autotetraploid Genotypes

    PubMed Central

    An, Feifei; Fan, Jie; Li, Jun; Li, Qing X.; Li, Kaimian; Zhu, Wenli; Wen, Feng; Carvalho, Luiz J. C. B.; Chen, Songbi

    2014-01-01

    Cassava polyploid breeding has drastically improved our knowledge on increasing root yield and its significant tolerance to stresses. In polyploid cassava plants, increases in DNA content highly affect cell volumes and anatomical structures. However, the mechanism of this effect is poorly understood. The purpose of the present study was to compare and validate the changes between cassava cultivar NZ199 diploid and autotetraploid at proteomic levels. The results showed that leaf proteome of cassava cultivar NZ199 diploid was clearly differentiated from its autotetraploid genotype using 2-DE combined MS technique. Sixty-five differential protein spots were seen in 2-DE image of autotetraploid genotype in comparison with that of diploid. Fifty-two proteins were identified by MALDI-TOF-MS/MS, of which 47 were up-regulated and 5 were down-regulated in autotetraploid genotype compared with diploid genotype. The classified functions of 32 up-regulated proteins were associated with photosynthesis, defense system, hydrocyanic acid (HCN) metabolism, protein biosynthesis, chaperones, amino acid metabolism and signal transduction. The remarkable variation in photosynthetic activity, HCN content and resistance to salt stress between diploid and autotetraploid genotypes is closely linked with expression levels of proteomic profiles. The analysis of protein interaction networks indicated there are direct interactions between the 15 up-regulation proteins involved in the pathways described above. This work provides an insight into understanding the protein regulation mechanism of cassava polyploid genotype, and gives a clue to improve cassava polyploidy breeding in increasing photosynthesis and resistance efficiencies. PMID:24727655

  5. Comparison of leaf proteomes of cassava (Manihot esculenta Crantz) cultivar NZ199 diploid and autotetraploid genotypes.

    PubMed

    An, Feifei; Fan, Jie; Li, Jun; Li, Qing X; Li, Kaimian; Zhu, Wenli; Wen, Feng; Carvalho, Luiz J C B; Chen, Songbi

    2014-01-01

    Cassava polyploid breeding has drastically improved our knowledge on increasing root yield and its significant tolerance to stresses. In polyploid cassava plants, increases in DNA content highly affect cell volumes and anatomical structures. However, the mechanism of this effect is poorly understood. The purpose of the present study was to compare and validate the changes between cassava cultivar NZ199 diploid and autotetraploid at proteomic levels. The results showed that leaf proteome of cassava cultivar NZ199 diploid was clearly differentiated from its autotetraploid genotype using 2-DE combined MS technique. Sixty-five differential protein spots were seen in 2-DE image of autotetraploid genotype in comparison with that of diploid. Fifty-two proteins were identified by MALDI-TOF-MS/MS, of which 47 were up-regulated and 5 were down-regulated in autotetraploid genotype compared with diploid genotype. The classified functions of 32 up-regulated proteins were associated with photosynthesis, defense system, hydrocyanic acid (HCN) metabolism, protein biosynthesis, chaperones, amino acid metabolism and signal transduction. The remarkable variation in photosynthetic activity, HCN content and resistance to salt stress between diploid and autotetraploid genotypes is closely linked with expression levels of proteomic profiles. The analysis of protein interaction networks indicated there are direct interactions between the 15 up-regulation proteins involved in the pathways described above. This work provides an insight into understanding the protein regulation mechanism of cassava polyploid genotype, and gives a clue to improve cassava polyploidy breeding in increasing photosynthesis and resistance efficiencies. PMID:24727655

  6. Engineering of Saccharomyces cerevisiae to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase, xylosidase and a bacterial xylose isomerase.

    PubMed

    Mert, Marlin John; la Grange, Daniël Coenrad; Rose, Shaunita Hellouise; van Zyl, Willem Heber

    2016-04-01

    Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes. PMID:26749525

  7. Molecular Epidemiology of Entamoeba spp.: Evidence of a Bottleneck (Demographic Sweep) and Transcontinental Spread of Diploid Parasites

    PubMed Central

    Ghosh, Sudip; Frisardi, Marta; Ramirez-Avila, Lynn; Descoteaux, Steven; Sturm-Ramirez, Katherine; Newton-Sanchez, Oscar Alberto; Santos-Preciado, Jose Ignacio; Ganguly, Chaiti; Lohia, Anuradha; Reed, Sharon; Samuelson, John

    2000-01-01

    Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico and India. Entamoeba dispar is morphologically identical but is not associated with disease. Here we determined the ploidy of E. histolytica and developed PCR-based methods for distinguishing field isolates of E. histolytica or E. dispar. Fluorescence in situ hybridization showed that E. histolytica trophozoites are diploid for five “single-copy” probes tested. Intergenic sequences between superoxide dismutase and actin 3 genes of clinical isolates of E. histolytica from the New and Old Worlds were identical, as were those of E. dispar. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast, E. histolytica and E. dispar genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic. chitinase alleles suggested that the early axenized strains of E. histolytica, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similar E. dispar parasites can be identified in both the New and Old Worlds. Ser-rich protein alleles, which suggested the presence of the HM-1 strain in Mexico City, included some E. histolytica genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection at chitinase and Ser-rich protein loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of E. histolytica and E. dispar. PMID:11015408

  8. Glucose repression in Saccharomyces cerevisiae.

    PubMed

    Kayikci, Ömur; Nielsen, Jens

    2015-09-01

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

  9. Postreplication repair in Saccharomyces cerevisiae

    SciTech Connect

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

    1981-04-01

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

  10. Glucose repression in Saccharomyces cerevisiae

    PubMed Central

    Kayikci, Ömur; Nielsen, Jens

    2015-01-01

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

  11. Development and characterization of microsatellite loci for the haploid–diploid red seaweed Gracilaria vermiculophylla

    PubMed Central

    Byers, James E.; Greig, Thomas W.; Strand, Allan E.; Weinberger, Florian

    2015-01-01

    Microsatellite loci are popular molecular markers due to their resolution in distinguishing individual genotypes. However, they have rarely been used to explore the population dynamics in species with biphasic life cycles in which both haploid and diploid stages develop into independent, functional organisms. We developed microsatellite loci for the haploid–diploid red seaweed Gracilaria vermiculophylla, a widespread non-native species in coastal estuaries of the Northern hemisphere. Forty-two loci were screened for amplification and polymorphism. Nine of these loci were polymorphic across four populations of the extant range with two to eleven alleles observed. Mean observed and expected heterozygosities ranged from 0.265 to 0.527 and 0.317 to 0.387, respectively. Overall, these markers will aid in the study of the invasive history of this seaweed and further studies on the population dynamics of this important haploid–diploid primary producer. PMID:26339541

  12. Multiple Gene Mediated NAD(P)H-Dependent Aldehyde Reduction is a Mechanism of in situ Detoxification of Furfural and HMF by Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furfural and 5-hydroxymethylfurfural (HMF) are representative inhibitors to ethanologenic yeast generated from biomass pretreatment using dilute acid hydrolysis. Few yeast strains tolerant to inhibitors are available. In this study, we report a tolerant strain 12HF10 of Saccharomyces cerevisiae ha...

  13. Heterofertilization exhibited by trifluralin-induced bicellular pollen on diploid and tetraploid maize crosses.

    PubMed

    Kato, A

    2001-12-01

    The heterofertilization rates and fertility of trifluralin-induced bicellular pollen were investigated in maize (Zea mays L.). A diploid inbred line, Oh43 (r1/r1), and a tetraploid line, Q28-1 (r1/r1/r1/r1), were pollinated with a trifluralin treated diploid stock heterozygous for R1-scm2. The gene R1-scm2 conditions purple pigmentation in both the embryo and the aleurone layer. Heterofertilized kernels were detected as discordant kernels, i.e., yellow kernel with purple embryo or purple kernel with white embryo. The diploid-diploid crosses treated with 0.2-0.3% Trefanocide solution (0.09-0.13% trifluralin) resulted in incidences of discordant kernels (3.7-4.8%) that were significantly higher than the control (2.3%). Most of the seedlings (86%) of the discordant kernels in the 0.3% treatment were triploids or triploid-class aneuploids. In tetraploid-diploid crosses, trifluralin treatments increased the number of plump kernels on the tetraploid ears. In the 0.3% treatment, 5.2% of ovaries produced plump kernels on the ears and most of the seedlings (92%) were tetraploids or tetraploid-class aneuploids, whereas in the control, only 1.5% ovaries produced plump kernels and most of the seedlings (98%) were triploids or triploid-class aneuploids. A high rate of discordance was observed among the plump kernels both in the treated plots (36.1-48.0%) and in the control (33.3%). Consequently, almost all of the plump kernels from the tetraploid-diploid crosses were considered to be the results of heterofertilization. PMID:11768215

  14. Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

    PubMed

    Quarterman, Josh; Skerker, Jeffrey M; Feng, Xueyang; Liu, Ian Y; Zhao, Huimin; Arkin, Adam P; Jin, Yong-Su

    2016-07-10

    In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without

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

    PubMed

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

    2013-04-01

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

  16. Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae

    PubMed Central

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

    2002-01-01

    Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1Δ nde1Δ nde2Δ gut2Δ quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h−1 at 100 g of glucose · liter−1). In aerated batch cultures grown on 400 g of glucose · liter−1, this engineered S. cerevisiae strain produced over 200 g of glycerol · liter−1, corresponding to a molar yield of glycerol on glucose close to unity. PMID:12039737

  17. Production of Yarrowia lipolytica Nha2 Na+/H+ antiporter improves the salt tolerance of Saccharomyces cerevisiae.

    PubMed

    Papousková, K; Sychrová, H

    2007-01-01

    Yarrowia lipolytica plasma-membrane Na+/H+ antiporter, encoded by the YlNHA2 gene, is a very efficient exporter of surplus sodium from the cytosol. Its heterologous expression in Saccharomyces cerevisiae wild-type laboratory strains increased their sodium tolerance more efficiently than the expression of ZrSod2-22 antiporter from the osmotolerant yeast Zygosaccharomvces rouxii. PMID:18450222

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  20. ISOLATION OF THE CANDIDA TROPICALIS GENE FOR P450 LANOSTEROL DEMETHYLASE AND ITS EXPRESSION IN SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have isolated the gene for cytochrome P450 lanosterol 14a-demethylase (14DM) from the yeast Candida tropicalis. his was accomplished by screening genomic libraries of strain ATCC750 in E. coli using a DNA fragment containing the yeast Saccharomyces cerevisiae 14DM gene. dentit...

  1. Expression of a lipid-inducible, self-regulating form of Yarrowia lipolytica lipase LIP2 in Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Yarrowia lipolytica lipase 2 gene (YlLIP2) was cloned into galactose- and fatty acid-inducible Saccharomyces cerevisiae expression vectors and used to generate yeast strains that secrete active LIP2 enzyme activity, as evidenced by results from gene expression analysis and tributyrin turbidity c...

  2. ISOLATION OF THE CANDIDA TROPICALIS GENE FOR P450 LANOSTEROL DEMETHYLASE AND ITS EXPRESSION IN SACCAROMYCES CEREVISIAE

    EPA Science Inventory

    We have isolated the gene for cytochrome P450 lanosterol 14-demethylase (14DM) from the yeast Candida tropicalis. This was accomplished by screening genomic libraries of strain ATCC750 in E. coli using a DNA fragment containing the yeast Saccharomyces cerevisiae 14DM gene. Identi...

  3. [Differentiation of haploid and diploid rape plants at the cytological and morphological levels].

    PubMed

    Soroka, A I

    2013-01-01

    Some cytological and morphological characteristics of haploid and dihaploid plants of winter rape obtained via anther culture were studied. It was shown that in haploid plants the number of chloroplasts in stomata guard cells and the size of the stomata guard cells themselves were much smaller, and the number of stomata per unit area was greater than in doubled haploids and diploids. Haploids were also characterized by a smaller size of petals and anthers, and in general, a smaller flower compared to dihaploids and diploids. PMID:23745361

  4. Fermentation studies using Saccharomyces diastaticus yeast strains

    SciTech Connect

    Erratt, J.A.; Stewart, G.G.

    1981-01-01

    The yeast species, Saccharomyces diastaticus, has the ability to ferment starch and dextrin, because of the extracellular enzyme, glucoamylase, which hydrolyzes the starch/dextrin to glucose. A number of nonallelic genes--DEX 1, DEX 2, and dextrinase B which is allelic to STA 3--have been isolated, which impart to the yeast the ability to ferment dextrin. Various diploid yeast strains were constructed, each being either heterozygous or homozygous for the individual dextrinase genes. Using 12 (sup 0) plato hopped wort (30% corn adjunct) under agitated conditions, the fermentation rates of the various diploid yeast strains were monitored. A gene-dosage effect was exhibited by yeast strains containing DEX 1 or DEX 2, however, not with yeast strains containing dextrinase B (STA 3). The fermentation and growth rates and extents were determined under static conditions at 14.4 C and 21 C. With all yeast strains containing the dextrinase genes, both fermentation and growth were increased at the higher incubation temperature. Using 30-liter fermentors, beer was produced with the various yeast strains containing the dextrinase genes and the physical and organoleptic characteristics of the products were determined. The concentration of glucose in the beer was found to increase during a 3-mo storage period at 21 C, indicating that the glucoamylase from Saccharomyces diastaticus is not inactivated by pasteurization. (Refs. 36).

  5. Quantitative trait loci for resistance to common scab and cold-induced sweetening from the diploid potato Solanum chacoense

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wild diploid potato has historically been an important source of genetic variation for variety development in S. tuberosum. In previous research, the diploid line S. chacoense 524-8 had consistently displayed resistance to common scab, a widespread disease caused by the soil-borne bacterial pathogen...

  6. Ethanol production characteristics for a respiratory deficient mutant yeast strain

    SciTech Connect

    Garcia, A. III; Grilione, P.

    1982-01-01

    Barley was fermented with a defined strain of Saccharomyces cerevisiae and a chemical induced respiratory deficient mutant RD, specific gravity, pH, CO/sub 2/ production and ethanol production rates and yield were compared. RD fermentation were slower but yielded slightly more ethanol after a considerable time. Partial reversion to a respiratory capable strain occurred.

  7. Enhanced resistance of Saccharomyces cerevisiae to vanillin by expression of lacA from Trametes sp. AH28-2.

    PubMed

    Ji, Lei; Shen, Yu; Xu, Lili; Peng, Bingyin; Xiao, Yazhong; Bao, Xiaoming

    2011-09-01

    Saccharomyces cerevisiae is affected by the presence of certain phenolic compounds such as vanillin during fermentation of pretreated lignocellulosic hydrolysates. Since vanillin can be polymerized in the presence of laccase into compounds with lower toxicity, the laccase gene, lacA, from Trametes sp. AH28-2 was fused to the α-factor signal sequence and transferred into S. cerevisiae CEN.PK strains for secretory expression. Furthermore, the chaperone gene, KAR2, was overexpressed to promote the translocation of laccase. In the presence of 8 mmol/L vanillin, a shorter lag phase was observed in the lacA gene expressing strains. The vanillin-specific conversion rate of the lacA-expressing strain BSJX0A2 was 0.069 g g(-1)biomass h(-1), while it was 0.065 g g(-1)biomass h(-1) in the reference strain. PMID:21727001

  8. Mutagenic Inverted Repeats Assisted Genome Engineering (MIRAGE) in Saccharomyces cerevisiae: deletion of gal7.

    PubMed

    Nair, Nikhil U; Zhao, Huimin

    2012-01-01

    MIRAGE is a unique in vivo genome editing technique that exploits the inherent instability of inverted repeats (palindromes) in the Saccharomyces cerevisiae chromosome. As a technique able to quickly create deletions as well as precise point mutations, it is valuable in applications that require creation of designer strains of this yeast. In particular, it has various potential applications in metabolic engineering, systems biology, synthetic biology, and molecular genetics. PMID:22144353

  9. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. 866.5785 Section 866.5785 Food and Drugs FOOD AND DRUG ADMINISTRATION... controls). The special control is FDA's “Guidance for Industry and FDA Reviewers: Class II Special...

  10. Phenotypic and metabolic traits of commercial Saccharomyces cerevisiae yeasts

    PubMed Central

    2014-01-01

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

  11. CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

    PubMed

    Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D

    2016-01-01

    This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory. PMID:27250940

  12. Reciprocal translocations in Saccharomyces cerevisiae formed by nonhomologous end joining.

    PubMed

    Yu, Xin; Gabriel, Abram

    2004-02-01

    Reciprocal translocations are common in cancer cells, but their creation is poorly understood. We have developed an assay system in Saccharomyces cerevisiae to study reciprocal translocation formation in the absence of homology. We induce two specific double-strand breaks (DSBs) simultaneously on separate chromosomes with HO endonuclease and analyze the subsequent chromosomal rearrangements among surviving cells. Under these conditions, reciprocal translocations via nonhomologous end joining (NHEJ) occur at frequencies of approximately 2-7 x 10(-5)/cell exposed to the DSBs. Yku80p is a component of the cell's NHEJ machinery. In its absence, reciprocal translocations still occur, but the junctions are associated with deletions and extended overlapping sequences. After induction of a single DSB, translocations and inversions are recovered in wild-type and rad52 strains. In these rearrangements, a nonrandom assortment of sites have fused to the DSB, and their junctions show typical signs of NHEJ. The sites tend to be between open reading frames or within Ty1 LTRs. In some cases the translocation partner is formed by a break at a cryptic HO recognition site. Our results demonstrate that NHEJ-mediated reciprocal translocations can form in S. cerevisiae as a consequence of DSB repair. PMID:15020464

  13. Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics.

    PubMed Central

    Ernst, J F; Chan, R K

    1985-01-01

    We describe mutants of Saccharomyces cerevisiae that are more sensitive than the wild type to the aminoglycoside antibiotics G418, hygromycin B, destomycin A, and gentamicin X2. In addition, the mutants are sensitive to apramycin, kanamycin B, lividomycin A, neamine, neomycin, paromomycin, and tobramycin--antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is caused by mutations in at least three genes, denoted AGS1, AGS2, and AGS3 (for aminoglycoside antibiotic sensitivity). Mutations in all three genes are required for highest antibiotic sensitivity; ags1 ags2 double mutants have intermediate antibiotic sensitivity. AGS1 was mapped 8 centimorgans distal from LEU2 on chromosome III. Analyses of yeast strains transformed with vectors carrying antibiotic resistance genes revealed that G418, gentamicin X2, kanamycin B, lividomycin A, neamine, and paromomycin are inactivated by the Tn903 phosphotransferase and that destomycin A is inactivated by the hygromycin B phosphotransferase. ags strains are improved host strains for vectors carrying the phosphotransferase genes because a wide spectrum of aminoglycoside antibiotics can be used to select for plasmid maintenance. PMID:2989254

  14. Comparative Proteomics Analysis of Engineered Saccharomyces cerevisiae with Enhanced Biofuel Precursor Production

    PubMed Central

    Tang, Xiaoling; Feng, Huixing; Zhang, Jianhua; Chen, Wei Ning

    2013-01-01

    The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production. PMID:24376832

  15. Decreased fluidity of cell membranes causes a metal ion deficiency in recombinant Saccharomyces cerevisiae producing carotenoids.

    PubMed

    Liu, Peitong; Sun, Liang; Sun, Yuxia; Shang, Fei; Yan, Guoliang

    2016-04-01

    The genome-wide transcriptional responses of S. cerevisiae to heterologous carotenoid biosynthesis were investigated using DNA microarray analysis. The results show that the genes involved in metal ion transport were specifically up-regulated in the recombinant strain, and metal ions, including Cu(2+), Fe(2+), Mn(2+), and Mg(2+), were deficient in the recombinant strain compared to the ion content of the parent strain. The decrease in metal ions was ascribed to a decrease in cell membrane (CM) fluidity caused by lower levels of unsaturated fatty acids and ergosterol. This was confirmed by the observation that metal ion levels were restored when CM fluidity was increased by supplying linoleic acid. In addition, a 24.3 % increase in the β-carotene concentration was observed. Collectively, our results suggest that heterologous production of carotenoids in S. cerevisiae can induce cellular stress by rigidifying the CM, which can lead to a deficiency in metal ions. Due to the importance of CM fluidity in cellular physiology, maintaining normal CM fluidity might be a potential approach to improving carotenoid production in genetically engineered S. cerevisiae. PMID:26749524

  16. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.

    PubMed

    Runguphan, Weerawat; Keasling, Jay D

    2014-01-01

    As the serious effects of global climate change become apparent and access to fossil fuels becomes more limited, metabolic engineers and synthetic biologists are looking towards greener sources for transportation fuels. In recent years, microbial production of high-energy fuels by economically efficient bioprocesses has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fatty acid-derived biofuels and chemicals from simple sugars. Specifically, we overexpressed all three fatty acid biosynthesis genes, namely acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1) and fatty acid synthase 2 (FAS2), in S. cerevisiae. When coupled to triacylglycerol (TAG) production, the engineered strain accumulated lipid to more than 17% of its dry cell weight, a four-fold improvement over the control strain. Understanding that TAG cannot be used directly as fuels, we also engineered S. cerevisiae to produce drop-in fuels and chemicals. Altering the terminal "converting enzyme" in the engineered strain led to the production of free fatty acids at a titer of approximately 400 mg/L, fatty alcohols at approximately 100mg/L and fatty acid ethyl esters (biodiesel) at approximately 5 mg/L directly from simple sugars. We envision that our approach will provide a scalable, controllable and economic route to this important class of chemicals. PMID:23899824

  17. Management of 41 persons exposed to a rabid dog: unplanned experience with human diploid vaccine.

    PubMed Central

    Dempster, G.; Stead, S.; Zbitnew, A.; Rhodes, A. J.; Zalan, E.

    1979-01-01

    Thirty-six persons -- veterinarians, technicians and students at a veterinary clinic -- were unwittingly exposed to a rabid dog over a period of 21/2 days. One veterinarian received a penetrating bite, two other individuals were grabbed by the dog but the skin was not penetrated, and many were exposed to saliva or urine or both. In addition, the owner of the dog and his wife and three children, while not bitten, were exposed to saliva. The diagnosis was made post mortem when specimens of the dog's brain were examined by indirect fluorescent antibody testing. All but one of the students had been vaccinated against rabies with hamster kidney vaccine, but eight members of the veterinary college's staff had not been so vaccinated. Treatment started with duck embryo vaccine; if necessary, rabies (human) immune globulin was also given. When one student reacted severely to the first dose of duck embryo vaccine permission was sought to bring a human diploid vaccine into Canada. In five patients the human diploid vaccine was substituted for the duck embryo vaccine because of severe reactions to the latter. Twenty-five staff members and the family of five received both vaccines. Reactions to the human diploid vaccine were minor and transient. Recommendations include the early licensing of the human diploid vaccine in Canada. PMID:445300

  18. De Novo Transcriptome Assembly and Analyses of Gene Expression during Photomorphogenesis in Diploid Wheat Triticum monococcum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Triticum monococcum (2n), a close ancestor of the A-genome progenitor of cultivated hexaploid wheat, was used as a model to study components regulating photomorphogenesis in diploid wheat. Constructed were genome-wide transcriptomes of two Triticum monococcum subspecies, the wild winter wheat T. mo...

  19. Differential gene expression and alternative splicing between diploid and tetraploid watermelon.

    PubMed

    Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A; Vajja, Venkata G; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K; Levi, Amnon; Wehner, Todd; Reddy, Umesh K

    2015-03-01

    The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits. PMID:25520388

  20. Phenotypic and Transcriptomic Analyses of Autotetraploid and Diploid Mulberry (Morus alba L.)

    PubMed Central

    Dai, Fanwei; Wang, Zhenjiang; Luo, Guoqing; Tang, Cuiming

    2015-01-01

    Autopolyploid plants and their organs are often larger than their diploid counterparts, which makes them attractive to plant breeders. Mulberry (Morus alba L.) is an important commercial woody plant in many tropical and subtropical areas. In this study, we obtained a series of autotetraploid mulberry plants resulting from a colchicine treatment. To evaluate the effects of genome duplications in mulberry, we compared the phenotypes and transcriptomes of autotetraploid and diploid mulberry trees. In the autotetraploids, the height, breast-height diameter, leaf size, and fruit size were larger than those of diploids. Transcriptome data revealed that of 21,229 expressed genes only 609 (2.87%) were differentially expressed between diploids and autotetraploids. Among them, 30 genes were associated with the biosynthesis and signal transduction of plant hormones, including cytokinin, gibberellins, ethylene, and auxin. In addition, 41 differentially expressed genes were involved in photosynthesis. These results enhance our understanding of the variations that occur in mulberry autotetraploids and will benefit future breeding work. PMID:26402678

  1. Meiotic recombination in sexual diploid and apomictic triploid dandelions (Taraxacum officinale L.).

    PubMed

    van Baarlen, P; van Dijk, P J; Hoekstra, R F; de Jong, J H

    2000-10-01

    Taraxacum officinale L. (dandelion) is a vigorous weed in Europe with diploid sexual populations in the southern regions and partially overlapping populations of diploid sexuals and triploid or tetraploid apomicts in the central and northern regions. Previous studies have demonstrated unexpectedly high levels of genetic variation in the apomictic populations, suggesting the occurrence of genetic segregation in the apomicts and (or) hybridization between sexual and apomictic individuals. In this study we analysed meiosis in both sexual diploid and apomictic triploid plants to find mechanisms that could account for the high levels of genetic variation in the apomicts. Microscopic study of microsporocytes in the triploid apomicts revealed that the levels of chromosome pairing and chiasma formation at meiotic prophase I were lower than in that of the sexual diploids, but still sufficient to assume recombination between the homologues. Nomarski DIC (differential interference contrast) microscopy of optically cleared megasporocytes in the apomicts demonstrated incidental formation of tetrads, which suggests that hybridization can occur in triploid apomicts. PMID:11081973

  2. Phenotypic and Transcriptomic Analyses of Autotetraploid and Diploid Mulberry (Morus alba L.).

    PubMed

    Dai, Fanwei; Wang, Zhenjiang; Luo, Guoqing; Tang, Cuiming

    2015-01-01

    Autopolyploid plants and their organs are often larger than their diploid counterparts, which makes them attractive to plant breeders. Mulberry (Morus alba L.) is an important commercial woody plant in many tropical and subtropical areas. In this study, we obtained a series of autotetraploid mulberry plants resulting from a colchicine treatment. To evaluate the effects of genome duplications in mulberry, we compared the phenotypes and transcriptomes of autotetraploid and diploid mulberry trees. In the autotetraploids, the height, breast-height diameter, leaf size, and fruit size were larger than those of diploids. Transcriptome data revealed that of 21,229 expressed genes only 609 (2.87%) were differentially expressed between diploids and autotetraploids. Among them, 30 genes were associated with the biosynthesis and signal transduction of plant hormones, including cytokinin, gibberellins, ethylene, and auxin. In addition, 41 differentially expressed genes were involved in photosynthesis. These results enhance our understanding of the variations that occur in mulberry autotetraploids and will benefit future breeding work. PMID:26402678

  3. Karyotype Analysis in Wild Diploid, Tetraploid, and Hexaploid Strawberries, Fragaria (Rosaceae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Strawberry, genus Fragaria (Rosaceae) has a basic chromosome count of x = 7, and is comprised of 20 wild species having an euploid series from diploid (2n = 2x = 14) through decaploid (2n = 10x = 70). Few karyotypes of species in this genus have been reported. The objective of this research was ...

  4. M6: A diploid potato inbred line for use in breeding and genetics research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    M6 is a vigorous, homozygous breeding line derived by self-pollinating the diploid wild potato relative Solanum chacoense for seven generations. While most wild Solanum species are self-incompatible, this clone is homozygous for the dominant self-incompatibility inhibitor gene Sli. It is homozygous ...

  5. Synthesis of trigeneric hybrids of hexaploid wheat with diploid wheatgrasses: Specificity of chromosome pairing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wild grasses in the tribe Triticeae are excellent sources of genes for superior traits, including resistance to various diseases. Diploid wheatgrasses – Lophopyrum elongatum (Host) Á. Löve (2n = 2x = 14; EE genome) and Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve (2n = 2x = 14; JJ genome) – are...

  6. Genotyping-by-sequencing of a diploid potato F2 population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genotyping-by-sequencing, or GBS, is an attractive technology for genome-wide markers because of its low per-sample cost and lack of ascertainment bias. To investigate its feasibility for potato, we used GBS at 96-plex to genotype a diploid F2 population created from the inbred lines DM (S. tuberosu...

  7. Differential gene expression and alternative splicing between diploid and tetraploid watermelon

    PubMed Central

    Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A.; Vajja, Venkata G.; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K.; Levi, Amnon; Wehner, Todd; Reddy, Umesh K.

    2015-01-01

    The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits. PMID:25520388

  8. IDENTIFICATION OF RB-ORTHOLOGOUS GENES FROM LATE BLIGHT RESISTANT DIPLOID POTATO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Late blight, caused by the oomycete pathogen, Phytophthora infestans, is a devastating disease of potatoes and tomatoes. A gene RB, cloned from the Mexican diploid potato species Solanum bulbocastanum, confers broad spectrum resistance to potato late blight. To identify RB-like genes and understand ...

  9. FveGD: an online resource for diploid strawberry (fragaria vesca) genomics data

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fragaria vesca, a diploid strawberry species commonly known as the alpine or woodland strawberry, is a versatile experimental plant system that is an emerging model for the Rosaceae family. An ancestral F. vesca genome contributed to the genome of the octoploid dessert strawberry (F. xananassa) and...

  10. Induced Polyploidy in Diploid Ornamental Ginger (Hedychium muluense) Using Colchicine and Oryzalin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ploidy level of H. muluense, a diploid (2n = 2x = 34) and dwarf ornamental ginger species, has been determined and is reported for the first time. Oryzalin and colchicine were successfully used to induce polyploidy in Hedychium muluense in vitro. Embryogenic cell lines were treated with oryzalin...

  11. Length variations of i-type low-molecular-weight glutenin subunit genes in diploid wheats.

    PubMed

    Long, H; Huang, Z; Wei, Y-M; Yan, Z-H; Ma, Z-C; Zheng, Y-L

    2008-04-01

    Allelic variation of the low-molecular-weight glutenin subunit (LMW-GS) is associated with the significant differences of dough quality in bread and durum wheat, and has been widely evaluated at protein level in wheat and its relatives. In this study, a PCR primer set, targeting the high variable repetitive domains, was employed to assay the length variation of i-type LMW-GS genes in the A-genomes of diploid wheats, the diploid progenitors of tetraploid and hexaploid wheat. A total of 71 accessions of diploid wheats, belonging to two wild and one cultivated species, were investigated. The higher variations of repetitive length in i-type LMW-GS genes were found in diploid wheats with Nei's genetic variation index (H) of 0.834. The two wild species, T. boeoticum and T. urartu, were found to possess the similar degree of variability, with the Nei's genetic variation index of 0.806 and 0.783, respectively. Less variations were detected in T. monococcum (H = 0.680), a cultivated species domesticated from T. boeoticum. The sufficient variations found in this study could be used as valuable sources for the enrichment of the genetic variations and the alteration of flour-processing properties of the cultivated wheat. To our knowledge, it was the first time that an analysis of length variation targeting a particular group of genes of LMW-GS complex multigene families was conducted. PMID:18666554

  12. Flower and early fruit development in a diploid strawberry, Fragaria vesca

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diploid woodland strawberry Fragaria vesca is being recognized as an ideal model for the more complex octoploid commercial strawberry (Fragaria ' ananassa) due to the recent completion of a draft genome sequence of F. vesca, short seed to seed cycle, and facile transformation. These features pot...

  13. Profiling polyphenols of two diploid strawberry (Fragaria vesca) inbred lines using UHPLC-HRMSn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenolic compounds in the fruits of two diploid strawberries (Fragaria vesca f. semperflorens) inbred lines-Ruegen F7-4 (a red fruited genotype) and YW5AF7 (a yellow fruited genotype) were characterized using ultra high-performance liquid chromatography in tandem with high resolution mass spectromet...

  14. Genetic diversity of diploid Japanese strawberry species based on microsatellite markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The United States Department of Agriculture (USDA) - Agricultural Research Service (ARS) - National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, is a genebank that preserves strawberry genetic resources. In 2004, representatives of two Japanese diploid species, F. iinumae Makino and F. n...

  15. The Effect of Prolonged Culture of Chromosomally Abnormal Human Embryos on The Rate of Diploid Cells

    PubMed Central

    Bazrgar, Masood; Gourabi, Hamid; Eftekhari-Yazdi, Poopak; Vazirinasab, Hamed; Fakhri, Mostafa; Hassani, Fatemeh; Chehrazi, Mohamad; Valojerdi, Mojtaba Rezazadeh

    2016-01-01

    Background A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies. Materials and Methods In this cohort study, we used fluorescence in situ hybridi- zation (FISH) to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis (PGD) on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization. Results Of the 30 analyzed blastocysts, mosaicism was observed in 26(86.6%), while 2(6.7%) were diploid, and 2(6.7%) were triploid. Of those with mosaicism, 23(88.5%) were determined to be diploid-aneuploid and 3(11.5%) were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 (P<0.05); however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality. Conclusion Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells. PMID:26985346

  16. Inheritance of anthocyanin content in the ripe berries of a tetraploid × diploid grape cross population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Variation patterns and inheritance of anthocyanin content in the ripe berries of a tetraploid × diploid table grape cross population were investigated in two successive years. The population segregated for three different ploidy levels: dipolids, triploids, and tetraploids. A total of 28 different a...

  17. The diploid D genome cottons (Gossypium spp.) of the new world

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The diploid D genome cottons (Gossypium spp.) of the New World are part of a great reservoir of important genes for improving fiber quality, pest and disease resistance, and drought and salt tolerance in the modern cultivated Upland/Acala (G. hirsutum) and Pima [also known as Sea Island or Egyptian ...

  18. Transfer of crown rust resistance from diploid oat Avena strigosa into hexaploid cultivated oat A. sativa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New sources of resistance to crown rust, Puccinia coronata f. sp. avenae (Eriks.), the major fungal disease of cultivated oat, Avena sativa L. (2n = 6x = 42), are constantly needed due to frequent, rapid shifts in the virulence pattern of the pathogen. Crown rust resistance identified in the diploid...

  19. Diverse functions of KNOX transcription factors in the diploid body plan of plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    KNOX genes were initially found as shoot meristem regulators in angiosperms. Recent studies in diverse plant lineages however, have revealed the divergence of KNOX gene function during the evolution of diploid body plans. Using genomic approaches, class I KNOX transcription factors have been shown t...

  20. Complex karyotypes in flow cytometrically DNA-diploid squamous cell carcinomas of the head and neck.

    PubMed Central

    Akervall, J.; Jin, Y.; Baldetorp, B.; Mertens, F.; Wennerberg, J.

    1998-01-01

    In squamous cell carcinoma of the head and neck (SCCHN), DNA ploidy as determined by flow cytometry (FCM) has been found to yield prognostic information but only for tumours at oral sites. Cytogenetic findings have indicated complex karyotype to be a correlate of poor clinical outcome. In the present study, 73 SCCHN were investigated with the two techniques. Aneuploid cell populations were identified in 49 (67%) cases by FCM but in only 21 (29%) cases by cytogenetic analysis. The chromosome index (CI), calculated as the mean chromosome number divided by 46, was compared with the respective DNA index (DI) obtained by FCM in 15 tumours, non-diploid according to both techniques, DI being systematically 12% higher than CI in this subgroup. Eight (33%) of the 24 tumours diploid according to FCM had complex karyotypes, three of the tumours being cytogenetically hypodiploid, three diploid and two non-diploid. The findings in the present study may partly explain the low prognostic value of ploidy status as assessed by FCM that has been observed in SCCHN. In addition, we conclude that FCM yields information of the genetic changes that is too unspecific, and that cytogenetic analysis shows a high rate of unsuccessful investigations, thus diminishing the value of the two methods as prognostic factors in SCCHN. Images Figure 1 PMID:9569043

  1. Proteomic analysis of skeletal deformity in diploid and triploid rainbow trout (Oncorhynchus mykiss) larvae.

    PubMed

    Babaheydari, Samad Bahrami; Keyvanshokooh, Saeed; Dorafshan, Salar; Johari, Seyed Ali

    2016-09-01

    A proteomic screening approach was employed to achieve a better understanding of the changes that occur in protein expression patterns associated with skeletal deformities in both diploid and triploid rainbow trout larvae. Triploidy was induced through the application of heat shock of 28°C for 10min to eggs 10-min post fertilization in an aquarium equipped with a heater. Percentage of skeletal deformity in heat-shocked larvae (2.88±0.30, mean±S.E.) was significantly (P<0.05) greater than that of the diploids (0.55±0.24). At five days after hatching, proteins of normal and deformed specimens of deyolked larvae were subjected to proteomic analysis using two-dimensional electrophoresis and mass spectrometry. Among the identified protein spots from diploids, creatine kinase was found to be increased in larvae with skeletal deformities, while apolipoprotein A-I-2, apolipoprotein A-II and calmodulin were found to be decreased in deformed fish. Among the five protein spots that were identified in heat-shocked fish, apolipoprotein A-I-2, apolipoprotein A-II, parvalbumin, myosin light chain 1-1 and nucleoside diphosphate kinase were found to be decreased in deformed larvae. The identification of nine protein spots showing altered expression in deformed fish allows us to reach a preliminary view of the molecular mechanisms that are involved in the development of skeletal malformations in diploid and triploid fish. PMID:27219664

  2. Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids.

    PubMed

    Yu, Ai-Qun; Juwono, Nina Kurniasih Pratomo; Foo, Jee Loon; Leong, Susanna Su Jan; Chang, Matthew Wook

    2016-03-01

    Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement. PMID:26721212

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

    PubMed

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

    2013-11-01

    The SSU1 gene encoding a membrane sulfite pump is a main facilitator invovled in sulfite efflux. In Saccharomyce cerevisiae, various range of resistance to sulfite was observed among strains. To explore the evolution traits of SSU1 gene, the population data of S. cerevisiae were collected and analyzed. The phylogenetic analysis indicated that S. cerevisiae population can be classified into three sub-populations, and the positive selection was detected in population by McDonald-Kreitman test. The anaylsis of Ka/Ks ratios further showed that S. cerevisiae sub-population was undergoing positive selection. This finding was also supported by PAML branch model. Nine potential positive selection sites were predicted by branch-site model, and four sites exclusively belong to the sub-population under positive seletion. The data from ssulp protein structure demonstrated that three sites are substitutions between polar and hydrophobic amino acids, and only one site of substitutaion from basic amino acid to basic amino acid (345R/K). Because amino acid pKa values are crucial for sulfite pump to maintain their routine function, positive selection of these amino acid substitutions might affect sulfite efflux efficient. PMID:24579315

  4. Identification of Novel Knockout Targets for Improving Terpenoids Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Li, Jing; Wang, Jianfeng; Li, Qian; Wang, Yong; Zhang, Yansheng

    2014-01-01

    Many terpenoids have important pharmacological activity and commercial value; however, application of these terpenoids is often limited by problems associated with the production of sufficient amounts of these molecules. The use of Saccharomyces cerevisiae (S. cerevisiae) for the production of heterologous terpenoids has achieved some success. The objective of this study was to identify S. cerevisiae knockout targets for improving the synthesis of heterologous terpeniods. On the basis of computational analysis of the S. cerevisiae metabolic network, we identified the knockout sites with the potential to promote terpenoid production and the corresponding single mutant was constructed by molecular manipulations. The growth rates of these strains were measured and the results indicated that the gene deletion had no adverse effects. Using the expression of amorphadiene biosynthesis as a testing model, the gene deletion was assessed for its effect on the production of exogenous terpenoids. The results showed that the dysfunction of most genes led to increased production of amorphadiene. The yield of amorphadiene produced by most single mutants was 8–10-fold greater compared to the wild type, indicating that the knockout sites can be engineered to promote the synthesis of exogenous terpenoids. PMID:25386654

  5. Proteomics of Saccharomyces cerevisiae Organelles*

    PubMed Central

    Wiederhold, Elena; Veenhoff, Liesbeth M.; Poolman, Bert; Slotboom, Dirk Jan

    2010-01-01

    Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them with other large scale studies on protein localization and abundance. We evaluate the completeness and reliability of the organelle proteomics studies. Reliability depends on the purity of the organelle preparations, which unavoidably contain (small) amounts of contaminants from different locations. Quantitative proteomics methods can be used to distinguish between true organellar constituents and contaminants. Completeness is compromised when loosely or dynamically associated proteins are lost during organelle preparation and also depends on the sensitivity of the analytical methods for protein detection. There is a clear trend in the data from the 18 organelle proteomics studies showing that proteins of low abundance frequently escape detection. Proteins with unknown function or cellular abundance are also infrequently detected, indicating that these proteins may not be expressed under the conditions used. We discuss that the yeast organelle proteomics studies provide powerful lead data for further detailed studies and that methodological advances in organelle preparation and in protein detection may help to improve the completeness and reliability of the data. PMID:19955081

  6. The inheritance of mtDNA in lager brewing strains.

    PubMed

    Rainieri, Sandra; Kodama, Yukiko; Nakao, Yoshihiro; Pulvirenti, Andrea; Giudici, Paolo

    2008-06-01

    In this work, we compared the mtDNA of a number of interspecific Saccharomyces hybrids (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces bayanus) to the mtDNA of 22 lager brewing strains that are thought to be the result of a natural hybridization between S. cerevisiae and another Saccharomyces yeast, possibly belonging to the species S. bayanus. We detected that in hybrids constructed in vitro, the mtDNA could be inherited from either parental strain. Conversely, in the lager strains tested, the mtDNA was never of the S. cerevisiae type. Moreover, the nucleotide sequence of lager brewing strains COXII gene was identical to S. bayanus strain NBRC 1948 COXII gene. MtDNA restriction analysis carried out with three enzymes confirmed this finding. However, restriction analysis with a fourth enzyme (AvaI) provided restriction patterns for lager strains that differed from those of S. bayanus strain NBRC 1948. Our results raise the hypothesis that the human-driven selection carried out on existing lager yeasts has favored only those bearing optimal fermentation characteristics at low temperatures, which harbor the mtDNA of S. bayanus. PMID:18318709

  7. Detection and sequencing of Okazaki fragments in S. cerevisiae

    PubMed Central

    Smith, Duncan J.; Yadav, Tejas; Whitehouse, Iestyn

    2016-01-01

    SUMMARY We have previously demonstrated that lagging-strand synthesis in budding yeast is coupled with chromatin assembly on newly synthesized DNA. Using a strain of S. cerevisiae in which DNA ligase I can be conditionally depleted, we can enrich and purify Okazaki fragments. We delineate a method to extract, end-label and visualize Okazaki fragments using denaturing agarose gel electrophoresis. Furthermore, we describe an ion-exchange chromatographic method for purification of fragments and preparation of strand-specific sequencing libraries. Deep sequencing of Okazaki fragments generates a comprehensive, genomic map of DNA synthesis, starting from a single asynchronous culture. Altogether this approach represents a tractable system to investigate key aspects o