Isoleucyl-tRNA synthetase genes from the yeast Saccharomyces cerevisiae and the ciliated protozoan Tetrahymena thermophila were sequenced. The intronless S. cerevisiae gene (ILS1) encodes a putative polypeptide of 1072 amino acids. Two putative promoter elements were identified, one for general amino acid control and one for constitutive transcription. A heat shock protein gene lies upstream of ILS1. The T. thermophila isoleucyl-tRNA synthetase gene (ilsA: formerly cupC) has eight introns, four transcription start sites, and codes for a putative polypeptide of 1081 amino acids with two leucine-zippers. These eukaryotic isoleucyl-tRNA synthetases are 47% identical. They are compared to homologous enzymes from Escherichia coli and an archaebacterium, and to other aminoacyl-tRNA synthetases. / Intron sequences and junctions from T. thermophila and other eukaryotes were analyzed and all but yeast and mammalian introns were found to be A + T enriched. T. thermophila transcription start sites were analyzed and occur at a T or an A within the consensus sequence (A/T)$ sb{ rm n}$ T A A (A)$ sb{ rm n}.$

The wine yeast Saccharomyces cerevisiae experiences a range of stress conditions during wine fermentation. These stresses include osmotic stress, hypoxia, nutrient starvation, cold stress and increasing ethanol stress as fermentable sugars are converted to ethanol. These various stresses affect the functionality of the plasma membrane, cell wall and subsequently the yeast’s efficiency during fermentation. Etp1, a poorly characterized protein has been shown to have several different fermentation related phenotypes; it is needed for the turnover of the hexose transporter Hxt3 upon a shift from glucose to ethanol, and the transcriptional activation of the stress response genes HSP12 and HSP26 under ethanol stress. The molecular function of Etp1 during fermentation is not understood. This research aims to understand the molecular mechanism of Etp1 function and its involvement in Chardonnay fermentation using the S. cerevisiae M2 wine yeast strain. / OMAFRA, Genome Canada

No description available.

Ethanol production through biomass fermentation is one of the major technologies available to produce liquid fuel from renewable energy sources. A major problem associated with the production of ethanol through fermentation remains the inhibition of the yeast Saccharomyces cerevisiae by the produced ethanol. Currently high water dilution rates are used to keep the ethanol concentrations in the fermentation broth at low concentrations, resulting in low yields and increased downstream processing to remove the excess water. Yeast strains that have a high tolerance for ethanol have been isolated but the time and cost associated with doing so poses a challenge. The fermentation process can be combined with pervaporation, thereby continuously removing ethanol while it is being formed. In this study a mathematical model for ethanol fermentation with yeast, Saccharomyces cerevisiae, coupled with pervaporation was developed. The fermentation of glucose was optimised in the first part of the study and experimental data were obtained to find a kinetic model for fermentation. It was found that an optimum ethanol yield can be obtained with an initial glucose concentration of 15wt%, a yeast concentration of 10 g.L–1, and a pH between 3.5 and 6. The maximum ethanol yield obtained in this study was 0.441g.g–1 (86% of the theoretical maximum) using 15wt% glucose, 10g/L yeast and a pH of 3.5. Two kinetic models for fermentation were developed based on the Monod model. The substrate–limiting model, predicted fermentation very accurately when the initial glucose concentration was below 20wt%. The second model, the substrate–inhibition model, predicted fermentation very well when high initial glucose concentrations were used but at low glucose concentrations, the substrate–limiting model was more accurate. The parameters for both models were determined by non–linear regression using the simplex optimisation method combined with the Runge–Kutta method. The PERVAP®4060 membrane was identified as a suitable membrane in this study. The effect of the ethanol content in the feed as well as the influence of the glucose content was investigated. The total pervaporation flux varied with ethanol content of the feed and the highest total flux of 0.853 kg/m2h was obtained at a feed with 20wt% ethanol. The addition of glucose had almost no effect on the ethanol flux but it lowered the water flux, thereby increasing the enrichment factor of the membrane. The mass transport through the PERVAP®4060 membrane was modelled using the solution–diffusion model and Greenlaw’s model for diffusion coefficients was used. The limiting diffusion coefficient (Di0) and plasticisation coefficients (Bij) were determined by using the Nelder–Mead simplex optimisation method. The theoretical values predicted with the model showed good agreement with the measured experimental values with R2 values above 0.998. In the third part of this investigation, the kinetic model developed for fermentation was combined with the transport model developed for pervaporation. The combined kinetic model was compared to experimental data and it was found that it could accurately predict fermentation when coupled with pervaporation. This model can be used to describe and better understand the process when fermentation is coupled with pervaporation. / Thesis (M.Ing. (Chemical Engineering))--North-West University, Potchefstroom Campus, 2012.

Ethanol production through biomass fermentation is one of the major technologies available to produce liquid fuel from renewable energy sources. A major problem associated with the production of ethanol through fermentation remains the inhibition of the yeast Saccharomyces cerevisiae by the produced ethanol. Currently high water dilution rates are used to keep the ethanol concentrations in the fermentation broth at low concentrations, resulting in low yields and increased downstream processing to remove the excess water. Yeast strains that have a high tolerance for ethanol have been isolated but the time and cost associated with doing so poses a challenge. The fermentation process can be combined with pervaporation, thereby continuously removing ethanol while it is being formed. In this study a mathematical model for ethanol fermentation with yeast, Saccharomyces cerevisiae, coupled with pervaporation was developed. The fermentation of glucose was optimised in the first part of the study and experimental data were obtained to find a kinetic model for fermentation. It was found that an optimum ethanol yield can be obtained with an initial glucose concentration of 15wt%, a yeast concentration of 10 g.L–1, and a pH between 3.5 and 6. The maximum ethanol yield obtained in this study was 0.441g.g–1 (86% of the theoretical maximum) using 15wt% glucose, 10g/L yeast and a pH of 3.5. Two kinetic models for fermentation were developed based on the Monod model. The substrate–limiting model, predicted fermentation very accurately when the initial glucose concentration was below 20wt%. The second model, the substrate–inhibition model, predicted fermentation very well when high initial glucose concentrations were used but at low glucose concentrations, the substrate–limiting model was more accurate. The parameters for both models were determined by non–linear regression using the simplex optimisation method combined with the Runge–Kutta method. The PERVAP®4060 membrane was identified as a suitable membrane in this study. The effect of the ethanol content in the feed as well as the influence of the glucose content was investigated. The total pervaporation flux varied with ethanol content of the feed and the highest total flux of 0.853 kg/m2h was obtained at a feed with 20wt% ethanol. The addition of glucose had almost no effect on the ethanol flux but it lowered the water flux, thereby increasing the enrichment factor of the membrane. The mass transport through the PERVAP®4060 membrane was modelled using the solution–diffusion model and Greenlaw’s model for diffusion coefficients was used. The limiting diffusion coefficient (Di0) and plasticisation coefficients (Bij) were determined by using the Nelder–Mead simplex optimisation method. The theoretical values predicted with the model showed good agreement with the measured experimental values with R2 values above 0.998. In the third part of this investigation, the kinetic model developed for fermentation was combined with the transport model developed for pervaporation. The combined kinetic model was compared to experimental data and it was found that it could accurately predict fermentation when coupled with pervaporation. This model can be used to describe and better understand the process when fermentation is coupled with pervaporation. / Thesis (M.Ing. (Chemical Engineering))--North-West University, Potchefstroom Campus, 2012.

A selection system for yeast cells expressing mutated invertase (EC 3.2.1.26, SUC2) with altered fructotransferase activity was developed based on the survival of a fructose-intolerant strain in the presence of suitable acceptor substrates and sucrose. Cells of such a strain expressing a wild-type hydrolase activity will not grow due to the release of free fructose from sucrose. Cells expressing an inactive invertase mutant will not grow since they cannot cleave the sucrose, the sole carbon source. Only cells expressing sucrose fructosyl-transferase activity will thrive, growing on the released glucose, the fructosyl moiety not being released. A strain of Saccharomyces cerevisiae was engineered to be intolerant of the presence of fructose in its growth media. This was achieved by inducing a condition in yeast similar to liver cells of humans suffering from hereditary fructose intolerance (MIM 22960). This disorder results from a deficiency of aldolase B (EC 4.1.2.13), and phosphorylation of fructose by ketohexokinase (EC 2.7.1.3) results in an accumulation of fructose 1-phosphate, with a consequent depletion of cytoplasmic phosphate and ATP. Thus, cells in which ketohexokinase phosphorylates fructose, but which lack aldolase B, are intolerant of fructose. Yeast possess neither of these enzymes, and so expression of ketohexokinase in yeast would result in fructose-intolerance. A strain of yeast, for ketohexokinase expression, was initially bred to be unable to metabolize sucrose or fructose, yet remain capable of utilizing glucose, as well as lacking non-specific phosphatases, to prevent remobilization of sequestered fructose 1-phosphate. Rat liver ketohexokinase was purified to heterogeneity, and the partial amino acid sequence subsequently generated exploited to amplify a region of the ketohexokinase cDNA by PCR. This was used to probe a cDNA library, yielding clones encoding the entire ketohexokinase coding region. This was cloned into pMA91, and subsequent expression in yeast resulted in a strain intolerant of fructose in its growth medium, although still capable of growing on glucose. In order to produce a stable fructose-intolerant selection strain, a vector (pIADl) was constructed that allowed multiple integration of an expression cassette containing ketohexokinase cDNA into the rDNA locus of yeast chromosome XII. Expression of wild type invertase from the episomal plasmid pIAD3 in this strain resulted in sucrose-intolerance. A preliminary programme of mutagenesis of the SUC2 gene yielded eight libraries of about one hundred clones each. None of these contained any mutants showing solely sucrose fructosyl-transferase activity, although this system would clearly provide an ideal selection for such mutants from a much larger library.

Inorganic phosphate is an essential nutrient for all organisms. It is required for many cellular components as nucleic acids and phospholipids, and as energy-carrying compounds such as ATP. Thus, a regulated uptake of this pivotal nutrient is of outermost importance. Depending of the availability of phosphate in the surroundings the yeast Saccharomyces cerevisiae make use of two different systems for transporting phosphate into the interior of the cell: a low-affinity system that is active during surplus phosphate conditions and a high-affinity system that is active when the availability becomes limited. This thesis focuses on the high-affinity system, which is comprised of the Pho84 and Pho89 transporters. Of the two transporters, Pho84 is the predominant one, responsible for almost all phosphate uptake during low phosphate conditions, and the contribution of Pho89 is of minor importance. Hence Pho84 is by far the most well characterized phosphate transporter. Even though much is known about phosphate transporters in yeast little in known about how phosphate is transported. The work in this thesis aims to broaden the knowledge about the transport mechanism by the means of site-directed mutagenesis and functional characterization. Also the similarity of Pho84 to glucose sensors and the potential role of conserved residues in phosphate signaling are investigated. By the use of a high-affinity system deletion strain (∆Pho84 ∆Pho89), we also managed to investigate the functional importance of well conserved residues in Pho89. In summary: the work presented in this thesis has contributed to increase the knowledge about transport mechanisms in phosphate transporters.

S. cerevisiae Sup35p inhabits two metastable states: functional translation termination factor; and prion-like aggregate [PSI+], which propagates by converting soluble Sup35p to its own misfolded form. Once initiated, Sup35p polymerization in [PSI+] cells is spontaneous, but [PSI+] prion inheritance depends on the Hsp104p disaggregase. To identify Hsp104-interacting sequences, Sup35p was subjected to a systematic deletion screen. [PSI+] maintenance by mutant Sup35p was assessed in both presence and absence of plasmid-encoded WT Sup35p in haploid sup35 cells. Large deletions abolished [PSI+], implying perturbations of prion structure, while others imparted [PSI+]-dependent toxicity. Removal of a single 25aa segment destabilised [PSI+] inheritance, resulting in enhanced rates of prion loss. This is consistent with the expected prion propagation defect in response to reduced Hsp104p interaction. However, several mutants containing this 25aa segment share the destabilised prion phenotype, suggesting chaperone/prion interactions are strongly context-dependent, and no one Sup35p region is solely responsible for Hsp104p recognition.

S. cerevisiae Sup35p inhabits two metastable states: functional translation termination factor; and prion-like aggregate [PSI+], which propagates by converting soluble Sup35p to its own misfolded form. Once initiated, Sup35p polymerization in [PSI+] cells is spontaneous, but [PSI+] prion inheritance depends on the Hsp104p disaggregase. To identify Hsp104-interacting sequences, Sup35p was subjected to a systematic deletion screen. [PSI+] maintenance by mutant Sup35p was assessed in both presence and absence of plasmid-encoded WT Sup35p in haploid sup35 cells. Large deletions abolished [PSI+], implying perturbations of prion structure, while others imparted [PSI+]-dependent toxicity. Removal of a single 25aa segment destabilised [PSI+] inheritance, resulting in enhanced rates of prion loss. This is consistent with the expected prion propagation defect in response to reduced Hsp104p interaction. However, several mutants containing this 25aa segment share the destabilised prion phenotype, suggesting chaperone/prion interactions are strongly context-dependent, and no one Sup35p region is solely responsible for Hsp104p recognition.

Selenium (Se) is an essential element. Supplementation of Se as yeast-Se in animal and human diets has been proven to have beneficial health effects. The goal of this study was to add a maximum amount of Se in yeast metabolism in order to optimize its incorporation in amino acids. A bakery yeast strain of Saccharomyces cerevisiae (S. cerevisiae) was studied for its tolerance to Se when the latter was incorporated at different levels. Fermentations were run at 27°C for 8 h and 24 h. The maximum Se incorporation was achieved when 12.6 mmol of Se, as sodium selenite salt, was added to the culture medium and fermented for 24 h. A final Se concentration of 1550 +/- 35 mug/g yeast was obtained by this treatment. / Different yeast strains of S. cerevisiae were also studied for their capacity to incorporate Se. Five yeast strains of wine and four yeast strains of beer were fermented for 24 h and tested for their capacity to incorporate Se. The amount of 12.6 mmol Se was added in the growth medium. A maximum of 642.6 +/- 3.6 mug Se/g yeast was found to be incorporated in Uvaferm BC wine strain. Uvaferm windsor of beer strain was able to incorporate a maximum of 826.8 +/- 10.4 mug Se/g yeast. These yeast strains could be used as alternatives for Se supplementation. / Se speciation was carried out on the bakery yeast strain containing 1550 +/- 35 mug Se/g yeast, using Fast Phase Liquid Chromatography (FPLC) and amino acid analysis. Out of 1550 +/- 35 mug Se/g yeast, 57.5% Se was present as selenoaminoacids. The yeast extract contained 147 +/- 14 mug/g of SeCys, 248 +/- 13 mug/g of SeCyst and 295 +/- 17 mug/g of SeMet. Yeast cell walls contained 65 +/- 8 mug/g of SeMet; 69 +/- 5 mug/g of SeCyst and 67 +/- 9 mug/g of SeCys. These selenoaminoacids are known for their beneficial health effects. The produced Se-enriched bakery yeast could be used, after evaluated to be toxicologically safe, as an efficient dietary supplement.