Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)
Permanent URI for this collection
Browse
Browsing Doctoral Degrees (Microbial, Biochemical and Food Biotechnology) by Advisor "Du Preez, J. C."
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Open Access A combined systems biology and genomics approach to the study of metabolism in Kluyveromyces marxianus(University of the Free State, 2016-10) Schabort, Du Toit Willem Petrus; Du Preez, J. C.; Kilian, S. G.English: The yeast Kluyveromyces marxianus has become an important micro-organism for industrial applications, as have other non-conventional yeasts. It has the advantages over Saccharomyces cerevisiae (baker’s yeast) in that it is more thermotolerant, has a much higher growth rate and can utilise a wider range of sugars, including the pentose D-xylose, which is found abundantly in lignocellulosic biomass. Although considerable advances have been made in engineering S. cerevisiae strains to ferment pentose sugars, their performance in this respect still does not approach that of glucose fermentation. S. cerevisiae is the model Crabtree positive yeast, meaning that it naturally ferments glucose even if oxygen is present at a high level. Crabtree negative yeasts, such as K. marxianus, have to be forced into a fermentative metabolism by imposing oxygen-limited conditions, which is impractical on industrial scale. Thus, a tremendous amount of knowledge needs to be gained regarding the regulation of metabolism in this non-conventional yeast before success could be expected in the re-programming of K. marxianus strains into xylose fermenting, Crabtree positive strains. The challenge of bringing a non-model species such as K. marxianus to the point of identifying key regulators affecting central metabolic pathways seems formidable. The aims of this work was to firstly harness the new technology of next-generation sequencing (NGS) to create a first draft genome for K. marxianus strain UFS-Y2791 and to generate high-quality RNA-seq differential transcriptome datasets, simultaneously capturing a tremendous amount of information. Efficient analytical methods and software implementations were also developed to explore these large datasets in an efficient manner, revealing new insights into the response of this species to glucose and xylose as carbon sources. RNA-seq data revealed a striking resemblance with the pattern of glucose derepression in the xylose medium, with up-regulation of genes for alternative carbon source utilisation, especially in the peroxisomes. Subsequently, two independent approaches were taken to identify differentially active transcription factors regulating the response. The first was the enumerative method of heptamer frequency comparisons, revealing the most likely regulators of differentially expressed genes. Secondly, a likelihood statistical approach was designed that employs multiple sources of evidence, which resulted in the construction of the first genome-wide gene regulatory network for K. marxianus. The method bridges the gap between the new NGS-based methods, which can rapidly generate data on any non-model species, and the wealth of experimental data that exist for a model species such as S. cerevisiae. Gene set enrichment statistics of the transcription factor target sets showed a general pattern that the activities of differentially active transcription factors were regulated primarily by post translational modifications instead of gene regulation. The use of RNA-seq was further expanded to the elucidation of the kinases that regulate transcription factors. The chromosomal context of differential gene expression was also investigated. Clusters of genes were identified, similar to the sub-telomeric regions previously identified in S. cerevisiae, but not close to telomeres. These regions contain industrially important enzymes and the potential binding sites for differentially active transcription factors. Finally, the possible roles of cofactor balances were investigated. Flux balance analysis was demonstrated here in rationalising the genetic response observed in RNA-seq transcriptomics and to understand the complex interplay between ATP, NADPH and NADH, the cofactor specificity of the oxidative pentose phosphate pathway, as well as the role of fructose-1,6-bisphosphatase. New roles are proposed for the latter enzyme, which differs from the currently accepted norm. A strategy for the metabolic engineering of a future xylose fermenting K. marxianus strain is also presented. The integrated analysis presented here expands our knowledge base of this yeast species, which is set to become increasingly important in a future bio-economy.Item Open Access Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism Saccharomyces cerevisiae(University of the Free State, 2007-05) De Smidt, Olga; Albertyn, J.; Du Preez, J. C.English: When Saccharomyces cerevisiae is grown on a fermentable carbon source such as glucose, the fermentative alcohol dehydrogenase, ADH I , catalyses the regeneration of NAD+ from NADH and produces ethanol from acetaldehyde. When the fermentable carbon source is depleted, a variety of other enzymes are derepressed in order to utilise the previously excreted ethanol via oxidative respiration and gluconeogenesis . To provide both the carbon source and energy for this system, the yeast cell requires an efficient method for oxidising this previously excreted ethanol. ADH II is a catabolite repressible isoenzyme which primarily functions in the cell to oxidise ethanol to acetaldehyde, which can be metabolised via the tricarboxylic acid cycle or act as intermediate product in gluconeogenesis. ADH III is a mitochondrial isoenzyme participating in the respiratory metabolism by forming part of the ethanol-acetaldehyde shuttle that is important for shuttling mitochondrial reducing equivalents to the cytosol under anaerobic conditions. The physiological roles and regulation of ADH1, ADH2, ADH3, ADH4 and ADH5 were investigated by monitoring transcription levels in chemostat and batch cultivations with Northern blotting and real-time RT-PCR. ADH I was shown to be the key enzyme in the reduction of acetaldehyde to ethanol and also demonstrated ample ability to oxidise ethanol. ADH2 transcription was inhibited by glucose and ethanol in chemostat cultures pulsed with both these carbon sources, but only glucose repression was evident in batch cultures. Northern blot analysis showed that the ADH3 gene was induced during the ethanol phase of the pulses suggested that the mitochondrial ADH III enzyme could also be involved in the first step in ethanol utilisation. The growth kinetics of a strain expressing only ADH III demonstrated that the ADH3 gene product could fulfil the same function as ADH II. ADH4 transcription was detected for the first time in batch cultures and was shown not to be involved in the production or assimilation of ethanol. ADH5 transcription was also demonstrated for the first time and data suggest that ADH V is not involved in ethanol production in a adh1-adh4 deletion mutant.