Browsing by Subject "Yeast"
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Item Alternative feeds or feed additives in feedlot diets(2014-03) Compart, Devan Marie PaulusThree experiments were conducted to determine effects of feeding alternative feeds or feed additives to cattle consuming feedlot diets on diet digestibility, rumen fermentation, growth performance, and carcass characteristics. In the first experiment, effects of adding a Saccharomyces cerevisiae product (SC) to cattle fed feedlot diets on diet digestibility and rumen fermentation were examined. Results of the first experiment suggest that feeding 1.0 g SC/hd daily may result in improved rumen acetate: propionate ratio. However, feeding 1.0 g SC/hd daily reduced rumen VFA concentrations, NH3-N concentration, and pH. In the second experiment, effects of partially replacing steam flaked corn with soy glycerin and distillers grains on diet digestibility and rumen fermentation in cattle were examined. Feeding distillers grains resulted in increased rumen propionate, rumen branched-chain VFA, and total rumen VFA. Feeding glycerin resulted in increased rumen pH and rumen propionate, and decreased rumen acetate. Feeding distillers grains or glycerin caused a reduction in rumen acetate: propionate ratio. In the third experiment, effects of replacing dry rolled corn with either 20% full-fat distillers grains, or 20% or 47% reduced-fat distillers grains (equal fat concentration as inclusion of 20% full-fat distillers grains) on feedlot cattle growth performance and carcass characteristics were examined. Results from this experiment indicated that utilizing reduced-fat distillers grains in place of full-fat distillers grains or dry rolled corn does not impact animal growth performance or carcass characteristics.Item Computational analysis of genetic interaction network structures and gene properties(2017-07) Koch, ElizabethCellular systems are responsible for many complex tasks, such as carrying out cell cycle phases, responding to intra- and extra-cellular conditions, and resolving errors. Through analysis of biological networks, researchers have begun to describe how cells coordinate these processes by means of modularity and between-process connections. However, descriptions of this network-based cellular organization often do not incorporate the diverse characteristics and individual behaviors of the genes that compose it. Knowledge of gene properties and their relationships with biological network evolution is crucial for a complete understanding of cellular function, and investigation in this area can lead to general principles of biology that apply to many species. This dissertation will describe analyses of the Saccharomyces cerevisiae (baker’s yeast) genetic interaction network that connect gene topological behavior with various physical, functional, and evolutionary properties of genes. Genetic interactions occur between paired genes whose simultaneous mutations produce unexpected double-mutant phenotypes, which are indicative of a range of functional relationships. Because genetic interactions can be identified genome-wide in high-throughput experiments, their networks are comprehensive and unbiased representations of function to which we can apply computational methods that search for structure-function relationships. We begin by exploring the association between a set of gene properties and gene genetic interaction (GI) degree. Here, we build a decision tree model that sorts genes based on a set of properties, each of which has a correlation with GI degree, and accurately predicts GI degree. We show that our model, trained on S. cerevisiae, is also accurate for a very distant yeast species, Schizosaccharomyces pombe, demonstrating that the rules governing gene connectivity are well conserved. Finally, we used predictions from the model to identify gene modules that differ between the two yeast species. Next, we further characterize hub genes through an investigation of pleiotropy, the phenomenon of a single genetic locus with multiple phenotypic effects. Pleiotropy has typically been described by counting organism-level phenotypes, but a characterization based on genetic interactions can capture details about cellular processes that are buffered by the cell and never manifest in single mutant cellular phenotypes. For this analysis, we use frequent item set mining to discover GI modules, which we annotate with high-level processes, and use entropy to measure the functional diversity of each gene’s set of containing modules, thus distinguishing between genes whose functional influence is limited to very few bioprocesses and those whose roles are important for varied cellular functions. We identified a number of gene and protein characteristics that differed between genes with high and low pleiotropy and discuss the implications of these results regarding the nature and evolution of pleiotropy.Item Effect of yeast, protected minerals and bismuth subsalicylate on in vitro fermentation by rumen microbes.(2012-04) Moreno, Martín RuizThree experiments were conducted using a dual flow continuous culture fermenter system. In Experiment I, two levels of active dry yeast at 0 or 2 mg/fermenter/day (NY and YS, respectively) were infused twice daily to fermenters in a completely randomized arrangement of treatments. Apparent and true OM digestion was not affected by yeast. No differences were obtained in NDF and ADF digestion. Total VFA concentrations were not affected by treatments. Addition of yeast did not affect VFA molar proportions or estimated CH4S production but resulted in a trend for a lower A:P ratio. Addition of yeast decreased NH3-N concentration and NH3-N daily flow, without affecting crude protein digestion and efficiency of microbial protein synthesis. Mean and minimum pH of fermenters did not differ between treatments but a trend for a lower maximum pH was obtained with yeast. In conclusion, a low dose of active dry yeast decreased NH3-N concentration and daily flow, without affecting any other of the in vitro rumen fermentation characteristics measured in this study. In Experiment II, effects of two levels of lignosulfonate and two sources of minerals (protected and unprotected) on rumen fermentation were evaluated using a 2 x 2 factorial arrangement of treatments. Addition of lignosulfonate tended to decrease daily flow of non NH3-N, efficiency of microbial protein synthesis, total VFA concentration and molar proportion of acetate, but increased molar proportion of propionate, valerate and caproate. Protected minerals decreased molar proportion of propionate. Addition of lignosulfonate increased ruminally soluble Cu and Mn, whereas protected minerals reduced ruminally soluble Cu. Concentrations of bacterial Cu and Zn increased with protected minerals in absence of lignosulfonate. Concentration of Mn was not affected by treatments. Addition of lignosulfonate resulted in higher enzymatic release of Zn from solids outflow but lower from bacterial pellets. Mean, minimum and maximum fermentation pH was higher with lignosulfonate, and not affected by mineral source. Addition of lignosulfonate induced major changes in ruminal fermentation. Protection of minerals decreased rumen soluble Cu and increased bacterial Cu and Zn without affecting postruminal release of minerals. In Experiment III, addition of bismuth subsalicylate (BSS) at 1% of DM and monensin (MON; 5 ppm) were used to assess their effects on rumen metabolism and H2S release by rumen microbes in a 2 x 2 factorial arrangement of treatments. Addition of BSS increased digestion of OM, NDF and ADF but decreased that of NFC and total VFA concentrations. Molar proportions of acetate and propionate increased with BSS in the diet, while that of butyrate decreased. Monensin decreased ADF digestion and A:P ratio, without affecting molar proportions of major VFA. Regarding nitrogen metabolism, MON increased non NH3-N outflow without affecting other measurements. Addition of BSS to the diet increased NH3-N concentration, NH3-N flow and dietary-N flow, while decreasing microbial-N outflow, CP digestion, and efficiency of microbial protein synthesis. Headspace H2S was reduced by 99% with BSS treatment but was not affected by MON. Only minor changes in fermentation pH were found with MON, but an increase in mean, minimum and maximum fermentation pH were found following addition of BSS. Results indicate that BSS can markedly reduce H2S production in short term and long term in vitro rumen incubations.Item Methods of Enhancing Triterpenoid Production in Yeast(2023-07) Scott, SamuelMicrobial cell factories, particularly those using eukaryotic yeasts, are ideal platforms for producing plant secondary metabolites, including flavonoids, alkaloids, and terpenoids. Accordingly, this study aimed to increase triterpenoid production in the Saccharomyces cerevisiae strain BY4743 through CRISPR/Cas9 and cultivation engineering. The ROX1, DGK1, and PAH1 genes were targeted for knockout experiments. Sanger sequencing showed all three targets were successfully mutated; however, only the DGK1 knockout strain had a significant change in triterpenoid production at 130% compared to the wild-type. Various cultivation strategies were also explored, but none increased triterpenoid production significantly. Additionally, to illustrate the potential applications of engineered yeast, five uncharacterized oxidosqualene cyclases (OSCs) from Erysimum cheiranthioides were tested in the ROX1 knockout strain, revealing one responsible for producing the steroid core of medicinal cardenolides. In summary, this thesis provides engineered yeast strains with improved MVA pathway derivative production potential and comprehensive CRISPR/Cas9 methodologies for S. cerevisiae.Item Modeling and analysis of microtubule-mediated chromosome transport during mitosis.(2008-08) Gardner, Melissa KleinDuring mitosis, dynamic arrays of kinetochore-associated microtubules (kMTs) and molecular motors are organized into a mitotic spindle that serves to accurately segregate chromosomes into daughter cells. Understanding the dynamics and organization of mitotic spindle components could ultimately apply to clinical applications, such as in cancer treatment, because of the centrality of the mitotic spindle in mediating cell mitosis. Computer simulation can provide a bridge between mitotic spindle phenotypes and the individual dynamic spindle components that produce these phenotypes. I have found that by simulating the dynamics of kMTs mediating chromosome segregation during mitosis, it is possible to build a model for their regulation which results in specific predictions for molecular functions within the mitotic spindle. Specifically, by simulating the dynamics of molecular motors and chromosomes relative to kMT dynamics, and by comparing these simulations to experiments using fluorescent proteins and cryo-electron tomography, major mechanisms regulating proper chromosome congression in yeast have been uncovered. I have shown (1) that tension generated via the stretch of chromosomes between sister kinetochores is important in regulating the proper separation of sister kinetochores during metaphase, and (2) that a molecular motor, specifically the Kinesin-5 molecular motor Cin8p, is responsible for mediating a gradient in kMT catastrophe frequency that is required for proper chromosome congression. Dynamic microtubule plus-ends are responsible for the proper segregation of chromosomes during mitosis, as well as for other critical cellular functions. By performing molecular-level Monte Carlo simulations of microtubule assembly and comparing these simulations to in vitro measurements of microtubule assembly, I have found that microtubule assembly at the nanoscale is highly variable. This result supports a model for microtubule dynamic instability in which there is exists a substantial and dynamic GTP-cap during microtubule assembly that is critical for microtubule growth.Item New experimental approaches to the population balance equation:Eulerian and Lagrangian viewpoints(2011-06) Sitton, Gregory WalterCell-to-cell variability in an asynchronous population of cells can be generally related to genetic differences, to different positions in the cell cycle, to the exposure of a heterogeneous environment, or to stochastic variations due to the low number of molecules in individual cells. To experimentally measure this cell to cell variability in response to different extra-cellular environments, one must measure the cells' phenotype using either an Eulerian or Lagrangian reference frame. In the Eulerian reference frame one measures the state of an entire cell population at discrete time points. In order to extract the single-cell dynamics from a time series of such measurements it is necessary to solve an inverse problem that extracts single cell behavior from the population data. This requires assumptions about cell behavior that may not be accurate in all cases. In contrast, in the Lagrangian reference frame one tracks individual cells over time and the dynamic properties of cells directly result from the observations. The properties of the entire cell population are then obtained as the sum of contributions of the individual components. Experimental data generated with the Eulerian viewpoint is primarily generated using flow cytometry. This instrument yields the cellular property distribution as a snapshot in time and cells are discarded after the measurement. Automated flow cytometry was developed to obtain high frequency snapshots of the cellular property distribution over time. This technique was used in this thesis to both quantitatively and qualitatively describe the cell cycle dynamics of CHO cells, transient gene expression in CHO cells, and to develop a fed-batch control strategy for CHO cells. To evaluate the single cell variability using the Lagrangian reference frame we have developed a novel flow cytometry instrument that is able to track individual, suspended cells in time. Individual cells can be repeatedly measured as they grow and express different proteins or as they respond to specific external stimuli of the growth environment. The measurement approach takes advantage of the Segre Silberberg effect that applies when dilute particles are subjected to Poiseuille flow in a capillary. Under such conditions particles of a given size and shape self-organize on the same streamline and keep their relative position in an oscillatory flow regime. We demonstrate that tens and perhaps hundreds of suspended cells can be tracked over hours with this device. With the developed instrument we have followed the Gfp expression modulated by variation in growth temperature as well as the induction kinetics of Gfp in individual yeast and CHO cells over extended periods of time. The data indicate a large variability of the kinetic response of individual cells that is not apparent if the Eulerian reference frame is used with conventional flow cytometry. Thus, the instrument permits evaluation of suspended cell populations at a level of detail that can not be achieved by existing instrumentation. The developed approach will be useful in the study of individual cell behavior and helpful in the rapid development of new drugs.Item Patulin degradation by yeast protein extract(2014-05) Folger, Brian CharlesThe mycotoxin patulin, produced by a number of fungi, most prominently Penicillium expansum, has proven problematic for the apple industry due to contamination of apple juice and apple cider. Presently, techniques to control patulin accumulation have proven increasingly ineffective due to the presence of antifungal resistant strains of mold, stability of patulin during thermal processing, and conflicting data on the efficacy of other treatments. However, fermented apple products such as hard ciders and apple cider vinegars are devoid of patulin. Fermentation with yeast resulted in complete degradation of patulin, possibly due to enzymatic degradation by yeast enzymes. Patulin has also been shown to be susceptible to adduct formation with free thiol containing molecules such as glutathione, which is naturally present in yeast cells. Limited studies have also looked at patulin adsorptivity onto the call walls of yeast. Degradation of patulin is, therefore, hypothesized to be caused by multiple mechanisms mainly caused by yeast proteins/enzymes.To assess the loss of patulin by protein extracted from yeast (Rhodosporidium kratochvilovae strain 62-121), patulin extraction methods were compared to determine the optimal method for patulin extraction from protein rich environments. The effect of boiling to halt any possible enzymatic degradation on total patulin loss was assessed by comparing patulin recovery to that of samples placed on ice after the assay. Yeast growth was optimized for the production of patulin-degrading protein extracts by surveying days of growth and subsequent storage at 4°C. Additionally, free thiol group reactivity with patulin was assessed upon incubation with protein extract, cysteine, and glutathione. Liquid chromatography and mass spectrometry (LC/MS) was used to detect patulin degradation products. Potential enzymatic activity was assessed by comparing the degradation activity of different protein extracts from yeast. Finally, patulin loss due to adsorption to inactivated yeast cell walls was determined. The use of acid and salt to precipitate the protein before patulin extraction resulted in the best patulin recovery from protein rich media, and an additional extraction following a modified AOAC method allowed for removal of excess salt without sacrificing patulin recovery. The use of boiling to denature the protein after the assay resulted in 10% higher patulin loss than when the samples were placed on ice, presumably due to adduct formation with thiol groups. Growing yeast for 6 days at room temperature was deemed adequate to obtain optimal patulin degradation; and subsequent incubation of the yeast at 4°C did not impair the patulin degradation activity. Yeast protein extracts were found to be inconsistent with respect to patulin degradation activity, nevertheless patulin degradation activity (up to 100% patulin) was observed in several batches. Patulin incubated with cysteine showed signs of free thiol blockage in both samples of protein extract and pure cysteine. Patulin incubated with glutathione was degraded at both pH 7 and 3.7, and one patulin-glutathione adduct (462 m/z) was identified via LC/MS. Lyophilized yeast cells demonstrated patulin adsorption capabilities after incubation at 30°C for 20 min. Observed results confirm that patulin can be degraded by the protein extract from yeast. The exact mechanism of patulin degradation by protein extracts remains unclear, yet it appears to be either enzymatic or chemical through thiol adduct formation. Our results indicated that the mechanism is a combination of the two. This research offers insight into possible patulin degradation mechanisms, and can give direction in applying this new method of patulin control in an industrial setting.Item Understanding the genetic requirements for Saccharomyces cerevisiae to survive at low temperatures.(2012-05) Haas, Kelaine C. ZimmermanMost organisms must possess molecular mechanisms that enable them to respond to both long-term and transient temperature fluctuations in their environment. As a first step in understanding these mechanisms, we focused on identifying molecular mechanisms that allow the budding yeast, Saccharomyces cerevisiae, to respond to low temperature. The goal of this study was to analyze the growth rate of S. cerevisiae mutant strains to identify genes required for growth at 10˚C. Within the S. cerevisiae homozygous deletion collection, we identified cold-sensitive strains representing 790 genes. The CS790 genes encode proteins that function in a wide range of biological processes, suggesting that genes required for S. cerevisiae growth at low temperatures represent complex networks of cellular and molecular functions. Two processes were statistically enriched among these 790 genes: genes required for threonine and tryptophan biosynthesis, and genes encoding components of the dynein/dynactin complex. These results provide a framework for future studies to identify the specific molecular mechanisms responsible for cold adaptation and growth of organisms. In addition to these enriched categories, previous work in our lab has also shown that three S. cerevisiae null mutants, ubc7∆, cue1∆, and doa10∆, are cold sensitive at 10˚C. UBC7, CUE1, and DOA10 encode proteins that function in the Ubc7-dependent ERAD pathway, which targets aberrant proteins in the ER lumen or membrane for ubiquitin-mediated proteasomal degradation. By introducing point mutations in the Ubc7 active site C89 residue, we created a catalytically inactive Ubc7 protein, and confirmed that the ubc7∆ strain is cold sensitive due to the loss of Ubc7 enzymatic activity. Therefore, the ability of Ubc7 to ubiquitinate appropriate target substrates is required for yeast growth at 10˚C. In an effort to identify relevant targets of this ERAD pathway, we used the Synthetic Genetic Array (SGA) protocol to create a complete set of haploid double mutants that carry both a deletion of UBC7 and a deletion of another gene. Genetic suppressors were identified by double mutant strains that no longer exhibited the cold-sensitive phenotype seen in ubc7∆ strains. This analysis identified 10 deletions that suppress the cold-sensitivity of ubc7∆, cue1∆, and doa10∆ cold-sensitivity, indicating that the suppression is not limited to the UBC7 gene, but instead affects the entire Ubc7-dependent ERAD pathway. Therefore, the proteins represented by these 10 deletion mutants are potential Ubc7-ERAD targets. Interestingly, four of these suppressors, PHO84, PHO81, SPL2 and YML122C, are members of the high-affinity phosphate transport pathway. Additional analysis of S. cerevisiae triple mutant strains with deletions in UBC7, SPL2, and the high-affinity Pi transporters, PHO84 or PHO89, shows that UBC7 may be working in a parallel pathway to SPL2. These results suggest that Ubc7 may directly or indirectly regulate the low-affinity branch of the phosphate transport pathway, via Pho87 and Pho90.