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Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: L-carnitine is a compound with a long history in biochemistry. It plays an important role in mammals, where many functions have been attributed to it. Those functions include the p-oxidation of long-chain fatty acids, the regulation of the free CoASH/ Acyl-CoA ratio and the translocation of acetyl units into mitochondria. Carnitine is also found in lower eukaryotic organisms. However, in contrast to the multiple roles it plays in mammalian cells, its action appears to be restricted to the transport of activated acyl residues across intracellular membranes in the lower eukaryotes. In the yeast Saccharomyces cere visiae , the role of carnitine consists mainly of the transfer of activated acetyl residues from the peroxisome and cytoplasm to the mitochondria. This process is referred to as the carnitine shuttle. This system involves the transfer of the acetyl moiety of acetyl-CoA, which cannot cross organellar membranes, to a molecule of carnitine. Subsequently, the acetylcarnitine is transported across membranes into the mitochondria, where the reverse transfer of the acetyl group to a molecule of free CoA occurs for further metabolism. Carnitine acetyl transferases (CATs) are the enzymes responsible for catalysing the transfer of the activated acetyl group of acetyl-CoA to carnitine as well as for the reverse reaction. In the yeast S. cerevisiae, three CAT enzymes, encoded by the genes CAT2, YAT1 and YAT2, have been identified. Genetic data suggest, that despite the high sequence similarity, each of the genes encodes for a highly specific activity that is part of the carnitine shuttle. So far, the specific function of any of the three CAT enzymes has been elucidated only partially. The literature review focuses mainly on the importance of the carnitine system in mammals. After discussing the discovery and biosyntheses of carnitine, the enzymatic background of and molecular studies on the carnitine acyltransferases are described. The experimental section focuses on elucidating the physiological roles and cellular localisation of the three carnitine acetyltransferase of S. cere visia e. We developed a novel enzymatic assay to study CAT activity in vivo. By C-terminal tagging with a green fluorescent protein, we localised the three CAT enzymes. However, all our genetic attempts to reveal specific roles for and functions of these enzymes were unsuccessful. The overexpression of any of the CAT genes could not cross-complement the growth defect of other CAT mutant strains. No phenotypical difference could be observed between strains carrying single, double and triple deletions of the CAT genes. Furthermore, the expression of the Schizosaccharomyces pombe dicarboxylic acid transporter can complement the deletion of the peroxisomal citrate synthase, but has no effect on the carnitine shuttle per se. Our data nevertheless suggest that Cat2p is the enzyme mainly responsible for the forward reaction, e.g. the formation of acetylcarnitine and free CoA-SH from acetyl-CoA and carnitine, whereas Yat1 pand Yat2p may be required mainly for the reverse reaction. / AFRIKAANSE OPSOMMING: L-karnitien is 'n verbinding met 'n lang geskiedenis in die biochemie-veld. Dit speel 'n belangrike rol in soogdiere, waar verskeie funksies daaraan toegeskryf word. Dié funksies sluit in die p-oksidasie van lang-ketting-vetsure, die regulering van die vrye KoA-SH-tot-asiel-KoA-verhouding en die oordrag van asetieleenhede na die mitochondria. Karnitien word ook in laer eukariotiese organismes gevind. In teenstelling met die verskeidenheid rolle wat dit in soogdierselle vervul, is die funksie in laer eukariote tot die transport van geaktiveerde asetielderivate oor intrasellulêre membrane beperk. In die gis Saccharomyces cerevisiae is die funksie van karnitien meestal beperk tot die vervoer van geaktiveerde asetielresidu's vanaf die sitoplasma en piroksisome na mitochondria, 'n proses wat as die "karnitiensiklus" bekend staan. Die proses behels die oordrag van die asetielgedeelte van asetiel-KoA, wat nie oor organelmembrane kan beweeg nie, na 'n molekuul van karnitien. Gevolglik word die asetielkarnitien oor die membraan na die mitochondria vervoer, waar - met die oog op verdere metabolisme - die omgekeerde oordrag van die asetielgroep na 'n vrye molekuul van KoA plaasvind. Karnitienasetiel-transferases (KAT's) is die ensieme wat verantwoordelik is vir die katalisering van die oordrag van die geaktiveerde asetielgroepe van asetiel-KoA na karnitien, sowel as vir die omgekeerde reaksie. In die gis S. cerevisiae is drie KAT-ensieme geïdentifiseer wat deur die gene CAT2, YAT1 en YAT2 gekodeer word. Genetiese data dui daarop dat, ten spyte van die hoë mate van homologie van die DNA-volgordes, elke geen vir 'n hoogs spesifieke aktiwiteit, wat deel van die karnitiensiklus is, kodeer. Tot dusver is die spesifieke funksie van die drie individuele KAT-ensieme net gedeeltelik ontrafel. Die literatuurstudie fokus hoofsaaklik op die belangrikheid van karnitiensisteme in soogdiere. Na 'n bespreking van die ontdekking en biosintese van karnitien, word die ensimatiese agtergrond en molekulêre studies van KAT's beskryf. Die eksperimentele deel konsentreer op die ontrafelling van die fisiologiese rol en intrasellulêre lokalisering van die drie KAT-ensieme van S. cerevisiae. Eerstens is 'n nuwe ensimatiese toets ontwikkel om KAT-aktiwiteit in vivo te bestudeer. Deur C-terminale aanhegting van 'n groen fluoreserende proteïen kon die drie KATensieme gelokaliseer word. Daar kon egter nie met behulp van genetiese studies verder lig gewerp word op die spesifieke rolle en funksies van hierdie KAT-ensieme nie. Die ooruitdrukking van enige van die KAT-gene kon nie die groeidefek van ander KAT-mutantrasse kruiskomplementeer nie. Geen fenotipiese verskil tussen rasse wat 'n enkel, dubbel of trippel delesie van die KAT-gene bevat, kon waargeneem word nie. Verder kon die uitdrukking van Schizosaccharomyces pombe se dikarboksielsuurtransporter die delesie van die peroksisomale sitraatsintetase komplementeer, maar het dit as sulks geen effek op die karnitiensiklus gehad nie. Die data wat deur hierdie studie verkry is, dui nogtans daarop dat Cat2p die ensiem is wat hoofsaaklik verantwoordelik is vir die voorwaartse reaksie, met ander woorde die vorming van asetielkarnitien en vrye KoH-SH van asetiel-KoA en karnitien, terwyl Yat1 p en Yat2p hoofsaaklik vir die omgekeerde reaksie benodig word.

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Bibliography: leaves 182-190. / xv, 190 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Develops a suitable approach to determining the fundamental cell-wall mechanical properties of single biological cells, by combining the single-cell compression experiment with a mechanical model to determine the cell-wall mechanical properties of the yeast Saccharomyces cerevisiae / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1999

Being at the center of most metabolic pathways and also one of the best known pathways, the glycolytic pathway has been of interest to modeling studies. This study is composed of our attempts to model ethanolic fermentation by yeast through kinetic equations of glycolytic steps and its branches. Model was based totally on experimentally measured kinetics of enzymes and transport steps, either obtained in this study or from the literature. Effect of ethanol on enzyme activities was tested in the range of ethanol 0 to 20% (v/v) in assay mixture. All enzymes were inhibited by ethanol to some degree and these inhibitions started at different ethanol concentrations, the least affected being the pyruvate kinase and the most inhibited ones being glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphogluco kinase, and alcohol dehydrogenase (forward). Effect of temperature on the activities of enzymes was tested within 10-30 &deg / C with five degrees of increments. Activation energies of enzymes were calculated using the Arrhenius equation. Activation energies of upper part of the glycolysis and the glycerol branch (glycerol-3-phosphate dehydrogenase) were relatively higher than that of lower part enzymes as well as the ethanol branch (alcohol dehydrogenase). Results obtained from these in vitro studies were incorporated into the model as mathematical relations. Model output thus obtained was compared with results of experiments conducted at several temperatures and initial ethanol concentrations. Model could estimate general trend in ethanolic fermentation that fermentation is inhibited by increasing concentrations of ethanol. Decrease in glycerol yields at lower temperatures was also estimated by the model. However, model did not fit exactly to experimental results, especially at low temperature and high ethanol concentrations. This could be attributed to stress responses of cells under these conditions, which are not considered in the model.

Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--Stellenbosch University, 2008. / The occurrence of sluggish and stuck fermentations continues to be a serious problem in the global wine industry, leading to loss of product, low quality wines, cellar management problems and consequently to significant financial losses. Comprehensive research has shown that many different factors can act either in isolation, or more commonly synergistically, to negatively affect fermentative activity of wine yeast strains of the species Saccharomyces cerevisiae. The individual factors most commonly referred to in the literature are various nutrient and oxygen limitations. However, other factors have been shown to contribute to the problem. Because of the mostly synergistic nature of the impacts, no single factor can usually be identified as the primary cause of stuck fermentation. In this study, several strategies to evolutionarily engineer wine yeast strains that are expected to reduce the occurrence of stuck and sluggish fermentations are investigated. In particular, the investigations focus on improving the ability of wine yeast to better respond to two of the factors that commonly contribute to the occurrence of such fermentations, nitrogen limitation and the development of an unfavorable ratio of glucose and fructose during fermentation. The evolutionary engineering strategies relied on mass-mating or mutagenesis of successful commercial wine yeast strains to generate yeast populations of diverse genetic backgrounds. These culture populations were then exposed to enrichment procedures either in continuous or sequential batch cultivation conditions while applying specific evolutionary selection pressures. In one of the stragegies, yeast populations were subjected to continuous cultivation under hexose, and especially fructose, limitation. The data show that the strains selected after this procedure were usually able to out-compete the parental strains in these selective conditions. However, the improved phenotype was not detectable when strains were evaluated in laboratory scale wine fermentations. In contrast, the selection procedure in continuous cultivation in nitrogen limiting conditions proved to be highly efficient for the generation of yeast strains with higher total fermentative capacity in low nitrogen musts. Furthermore, yeast strains selected after mutagenesis and sequential batch cultivation in synthetic musts with a very low glucose on fructose ratio showed a fructose specific improvement in fermentative capacity. This phenotype, which corresponds to the desired outcome, was also present in laboratory scale wine fermentations, where the discrepancy between glucose and fructose utilization of the selected strains was significantly reduced when compared to the parents. Finally, a novel strategy for the rectification of stuck fermentations was adjusted to industrial conditions. The strategy is based on the use of a natural isolate of the yeast species Zygosaccharomyces bailii, which is known for its preference of fructose. This process was successfully established and implemented in the wine industry.