Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by browse.metadata.advisor "Bloom, M."

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    Characterisation of L-malic acid metabolism in strains of Saccharomyces and the development of a commercial wine yeast strain with an efficient malo-ethanolic pathway
    (Stellenbosch : Stellenbosch University, 2002-12) Volschenk, Heinrich; Van Vuuren, H. J. J.; Bloom, M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: L-Malic and tartaric acid are the most prominent organic acids in wine and playa crucial role in winemaking processes and wine quality, including the organoleptic quality and the physical, biochemical and microbial stability of wine. The production of premium wines depends on the oenologist's skill to accurately adjust wine acidity to obtain the optimum balance with other wine components to produce wine with optimum colour and flavour. Strains of Saccharomyces, in general, rarely degrade L-malic acid completely in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. The degree of L-malic acid degradation, however, varies from strain to strain. Some strains of Saccharomyces are known to be able to degrade a higher percentage of L-malic acid, but the underlying reason for this phenomenon is unknown. The underlying mechanisms of this phenomenon have been partially revealed during preliminary transcriptional regulation research during this study. In contrast, S. pombe cells can effectively degrade up to 29 gil L-malic acid via the malo-ethanolic pathway that converts L-malic acid to pyruvate and CO2, and ultimately to ethanol under fermentative conditions. A number of reasons for the weak degradation of L-malic acid in Saccharomyces cerevisiae have been postulated. Firstly, S. cerevisiae lacks the machinery for the active transport of L-malic acid found in S. pombe and relies on rate-limiting simple diffusion for the uptake of extracellular L-malic acid. Secondly, the malic enzyme of S. cerevisiae has a significantly lower substrate affinity for L-malic acid (Km = 50 mM) than that of S. pombe (Km = 3.2 mM), which contributes to the weaker degradation of L-malic acid in S. cerevisiae. Lastly, the mitochondrial location of the malic enzyme of S. cerevisiae, in contrast to the cytosolic S. pombe malic enzyme, suggests that the S. cerevisiae malic enzyme is inherently subject to the regulatory effects of fermentative metabolism. The malate permease gene tmael) and the malic enzyme gene (mae2) of S. pombe was therefore cloned and co-expressed in single or multi-copy under regulation of the constitutive S. cerevisiae 3-phosphoglycerate kinase (PGK1) promoter and terminator sequences in a laboratory strain of S. cerevisiae. This introduced a strong malo-ethanolic phenotype in S. cerevisiae where L-malic acid was rapidly and efficiently degraded in synthetic and Chardonnay grape must with the concurrent production of higher levels of ethanol. Functional expression of the malo-ethanolic pathway genes of S. pombe in a laboratory strain of S. cerevisiae paved the way for the genetic modification of industrial wine yeast strains of Saccharomyces for commercial winemaking. A prerequisite for becoming an inherited component of yeast is the stable integration of the malo-ethanolic genes into the genome of industrial wine yeast strains. Genetic engineering of wine yeasts strains of Saccharomyces is, however, complicated by the homothallic, multiple ploidy and prototrophic nature of industrial strains of Saccharomyces. Transformation and integration of heterologous genes into industrial strains of Saccharomyces require the use of dominant selectable markers, i.e. antibiotic or toxic compound resistance markers. Integration of these markers into the yeast genome is, however, not acceptable for commercial application due to the absence of long-term risk assessment and consumer resistance. A unique strategy for the integration of the S. pombe mae} and mae2 expression cassettes without the incorporation of any non-yeast derived DNA sequences was. The malo-ethanolic cassette, containing the S. cerevisiae PGK} promoter and terminator regions together with the S. pombe mae] and mae2 open reading frames, was integrated into the VRA3 locus of an industrial strain of Saccharomyces bayanus EC 1118 during co-transformation with a phleomycin-resistance plasmid, pUT332. After initial screening for phleomycin resistance, S. bayanus EC1118 transformants were cured of the phleomycin-resistance plasmid, resulting in the loss of non-yeast derived DNA sequences. After correct integration of the mae] and mae2 expression cassettes was verified, small-scale vinification in synthetic and Chardonnay grape must with stable transformants resulted in rapid and complete degradation of L-malic acid during the early stages of alcoholic fermentation. Integration and expression of the malo-ethanolic genes in S. bayanus ECll18 had no adverse effect on the fermentation ability of the yeast, while sensory evaluation and chemical analysis of the Chardonnay wines indicated an improvement in wine flavour compared to the control wines, without the production of any off-flavours.
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    Characterisation of the malate transporter and malic enzyme from Candida utilis
    (Stellenbosch : University of Stellenbosch, 2011-10) Saayman, Maryna; Bloom, M.; Van Zyl, Willem Heber; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. The fission yeast Schizosaccharomyces pombe effectively degrades L-malate, but only in the presence of an assimilable carbon source. In contrast, the yeast Saccharomyces cerevisiae is unable to effectively degrade L-malate, which is ascribed to the slow uptake of L-malate by diffusion. In contrast, the yeast Candida utilis can utilise L-malate as the only source of carbon and energy, but this is subject to substrate induction and catabolite repression. Very little research has been done on a molecular level in C. utilis and only a few of its genes have been studied. In this study, we have shown that the yeast C. utilis effectively degraded extracellular L-malate and fumarate, but in the presence of glucose or other assimilable carbon sources, the transport and degradation of these dicarboxylic acids was repressed. The transport of both dicarboxylic acids was shown to be strongly inducible by either L-malate or fumarate and kinetic studies suggest that the same transporter protein transports the two dicarboxylic acids. In contrast, S. pombe effectively degraded extracellular L-malate, but not fumarate, only in the presence of glucose or other assimilable carbon sources. The S. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of L-malate. In order to clone the C. utilis dicarboxylic acid transporter, a cDNA library from C. utilis was constructed using a number of strategies to ensure representativeness and high transformation frequencies. The cDNA library was transformed in a S. cerevisiae strain carrying a plasmid containing the S. pombe malic enzyme gene (mae2) to allow screening for a malate-degrading S. cerevisiae clone. However, no positive clones that would indicate the successful cloning of the C. utilis malate transporter were obtained. The C. utilis malic enzyme gene, CuME, was subsequently isolated from the cDNA library based on conserved sequence homologies with the genes of S. cerevisiae and S. pombe, and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of 70.2 kDa. The optimum temperature for the C. utilis malic enzyme was 52°C and the enzyme was stable at 50°C for 2 hours. The inferred amino acid sequence showed significant homology with the malic enzymes of S. pombe and S. cerevisiae. Expression of the CuME gene is subject to glucose repression and substrate induction, as was observed for the dicarboxylic acid transporter from C. utilis. The CuME gene was successfully coexpressed with the S. pombe malate permease gene (mae1), resulting in a recombinant strain of S. cerevisiae able to effectively degrade L-malate.