Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Author "Davison, Steffi Angela"

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    Exploring phenotypic and genetic diversity of natural Saccharomyces cerevisiae strains for improved recombinant cellulase secretion
    (Stellenbosch : Stellenbosch University, 2019-12) Davison, Steffi Angela; Van Zyl, Willem Heber; Den Haan, Riaan; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH SUMMARY: The yeast Saccharomyces cerevisiae is considered an important host for the consolidated bioprocessing (CBP) of plant biomass to fuels and commodity products, but the production of high titres of recombinant cellulases is required for efficient hydrolysis of heterogonous lignocellulosic substrates to fermentable sugars. Recently, it was shown that S. cerevisiae strain diversity represents a treasure trove of genetic determinants for industrially relevant traits, including secretory capacity for recombinant cellulases. Since recombinant protein secretion profiles vary significantly among different strain backgrounds, careful selection of robust strains with optimal secretion profiles is crucial. This dissertation addresses numerous central challenges surrounding S. cerevisiae CBP namely, (1) improving the yeast’s low secretion capacity for recombinant cellulase through the construction and screening of hybrids of natural and industrial strains; (2) the evaluation of different cellulolytic yeast strain configurations to handle the heterogeneity of lignocellulosic substrates; and (3) the identification of genetic elements associated with the complex, polygenic trait of heterologous cellulase production and secretion through whole genome sequencing of selected yeast strains. We detail a novel approach, which combines cellulase secretion profiles and phenotypic responses of strains to stresses known to influence the secretion pathway, for the development of a phenotypic screen. The construction and screening of haploids derived from natural strain isolates YI13, FINI and YI59, consequently yielded several haploid strains with enhanced general cellulase secretion. A clear distinction was observed between the YI13 haploid derivatives and industrial and laboratory counterparts, Ethanol Red and S288c, respectively. Our results demonstrated that a new screening technique combined with a targeted mating approach could produce a pool of novel strains capable of improved cellulase secretion. In an effort to find a suitable genetic background for efficient cellulase secretion, genetically diverse strains were created to produce core sets of fungal cellulases, namely, β-glucosidase, endoglucanase and cellobiohydrolase, in various combinations. Higher secretion titers were achieved by cellulolytic strains with the YI13 genetic background and cellulolytic transformants released up to 1.34-fold higher glucose concentrations (g/L) than a control mixture composed of equal amounts of each enzyme type. The transformant co-producing BGLI and EGII in a secreted cellulase activity ratio of 1:15 (unit per gram dry cell weight) converted 56.5% of the cellulose present in corn cob to glucose in hydrolysis experiments, and yielded 4.05 g/L ethanol in fermentations. Finally, by performing pooled-segregant whole genome sequence analysis with subsequent quantitative trait loci mapping of an industrial strain (Ethanol Red) and a natural strain (YI13), we identified a large list of potential causative gene candidates linked to the high secretion phenotype. Some of these gene candidates were previously demonstrated to be active at different phases of secretion, ranging from the initiation of transcription, translation, post- translational modification to protein folding. Furthermore, we have identified several targets for future yeast strain improvement strategies. The yeast strains developed in this study therefore represent a new step towards efficient cellulase secretion for CBP bioethanol production.