Browsing by Author "Brandt, Bianca Anina"

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    Generating lower ethanol yields in fermentations by Saccharomyces cerevisiae via diversion of carbon flux towards the production of fructo-oligosaccharides
    (Stellenbosch : Stellenbosch University, 2013-03) Brandt, Bianca Anina; Nieuwoudt, Helene; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: There is a growing international consumer demand for the production of lower ethanol wines. This can be attributed to various qualitative, social, economic and health concerns that are associated with high ethanol wines (Kutyna et al., 2010; Varela et al., 2012). There is continuous development and research into methods and technologies to lower the ethanol concentration in wine. However, in addition to the added cost and complexity these technologies all have various shortcomings. The development of yeast strains with lower ethanol productivity, yet desirable organoleptic and fermentation capacity, therefore remains a highly sought after research and development target in the wine industry. Biologically based approaches aim to generate yeast strains with the capacity to divert carbon from ethanol production towards targeted metabolic endpoints (Kutyna et al., 2010). This should ultimately be achieved without the production of unwanted metabolites that can negatively affect wine characteristics. In the context of these challenges, this study aimed to investigate the use of fructans as carbon sinks during fermentation to divert fructose from glycolysis and ethanol production toward intracellular fructan production by generating levan producing strains. In addition, the impact of fructan production on metabolic carbon flux during fermentation by these strains was analyzed. This was the first attempt to analyze intracellular fructan production in Saccharomyces cerevisiae under fermentative conditions with fructans acting as carbon sinks. Fructans are fructose polymers that act as storage molecules in certain plants and function as part of the extracellular matrix in microbial biofilms, and are intensively studied due to their economic interest. Here we undertook the heterologous expression of a levansucrase (LS) M1FT from Leuconostoc mesenteroides, an enzyme producing β(2-6) levan-type fructans, in the S. cerevisiae BY4742Δsuc2 strains without invertase activity (encoded by SUC2). Levansucrases indeed utilize sucrose as both fructose donor and initial polymerization substrate, and the sucrose concentration is of import to maintain transfructosylation activity of enzyme. High intracellular sucrose accumulation was achieved by the heterologous expression of either a sucrose synthase (Susy; cloned from potato) or by growing strains expressing the spinach sucrose transporter (SUT) in sucrose containing media. Endogenous sucrose synthesis was of specific interest to the overall goal of the project, which was to reroute carbon flux away from glycolysis in grape must containing only hexoses as carbon source. In addition, this approach of combining intracellular sucrose production with intracellular levan production could be used in various applications to limit the need for sucrose in media as both carbon source and LS substrate. The extracellular LS M1FT was introduced into Susy and SUT strains as either the complete gene (M1FT) or 50bp truncation (M1FTΔsp) without the predicted signal peptide. The data show that intracellular levan accumulation occurred in aerobic, but not anaerobic conditions. The data also suggest that the production of levan did not impact negatively on general yeast physiology or metabolism in these conditions. However, no significant reduction in ethanol yields were observed, suggesting that further optimisation of the expression system is required. This is the first report of levan synthesis by S. cerevisiae, and contributes towards expanding the possibilities for further industrial applications of these compounds.
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    Hardening yeast for cellulosic ethanol production
    (Stellenbosch : Stellenbosch University, 2019-04) Brandt, Bianca Anina; Van Zyl, Willem Heber; Gorgens, Johann F.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The physico-chemical pretreatment of lignocellulose biomass is required to provide substrate access to enzymes responsible for the release fermentable sugars, but also releases microbial inhibitors such as furan aldehydes, weak acids, and various lignin-derived phenolic compounds, that detrimentally effect the subsequent bioconversion by Saccharomyces cerevisiae. Sustainable and cost effective lignocellulose bioconversion therefore relies heavily on the generation and optimization of robust S. cerevisiae strains with resistance to multiple inhibitors. In this context, the study aimed to improve the lignocellulose-hydrolysate fermentation efficiency of S. cerevisiae strains previously engineered for xylose fermentation capacity (introduced xylose isomerase - XI), using a combination of rational engineering and evolutionary adaptation to generate “hardened” strains exhibiting multi-inhibitor resistance phenotypes. A xylose capable S. cerevisiae was used as parental and industrial control strain for the rational engineering of seven genes identified by literature to address furan aldehyde, weak acid and phenolic-induced stresses. These genes included the TAL1, FDH1, ARI1, ADH6, and PAD1 genes that have been characterized to contribute to in situ inhibitor detoxification phenotypes. Enzymatic in situ detoxification was combined with plasma membrane modification whereby the FPS1 gene (membrane pore) was partially deleted, and the ICT1 gene (membrane modifier) overexpressed. The overexpression genes were integrated in novel combinations into a partial FPS1 deletion background, to identify gene combinations that confer multi-inhibitor resistance phenotypes, yet limit the impact on xylose metabolism. The study yielded transformants with novel gene combinations that conferred multi-inhibitor resistance phenotypes based on the sequence of integration, and increased both biomass and ethanol yield. Xylose utilization was negatively impacted by the overexpression of the gene combinations, however, transformants with specific gene combinations (TAL1-FDH1 + ARI1- ADH6) showed a limited impact. The resultant transformants were adapted in concentrated un-detoxified spent sulphite liquor (SSL) to further strengthen the introduced multi-inhibitor resistance phenotype and to include inhibitors typical to SSL waste streams. The adaptation followed a serial transfer in shake flask methodology, with increasing concentrations of concentrated SSL as selective pressure. The adapted isolates exhibited stable increased resistance phenotypes and increased ethanol yields relative to parental control, in 60% and 80% v/v SSL fermentations, however, there appeared to be a trade-off between the expressed phenotype and ethanol productivity. The adapted isolates exhibited an increased robust phenotypes, however, a regression on ethanol productivity was observed as adaptation progressed. Interestingly, the xylose utilization of the adapted isolates was only similar to that of the parental strain under stressed conditions, versus nonselective growth. Taken together, this study undertook the development of multi-inhibitor resistance phenotypes in a xylose capable S. cerevisiae and elaborated on its implications under industrially relevant conditions. The study highlighted novel gene combinations TF, AA and PI that can be used to “harden” yeast to generate robust biocatalyst strains, with limited impact on xylose utilization. This contributes towards furthering the understanding of inhibitor phenotypes and possible links to carbon metabolism. In addition, this study illustrated the use of rational engineering in combination with evolutionary adaptation to manipulate and improve a complex phenotype in an industrial strain. This paves the way forward towards developing inhibitor resistant xylose capable industrial strains for efficient cellulosic ethanol production.