Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Author "Brandt, Bianca Anina"

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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.