Browsing 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.
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    Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains
    (Stellenbosch : Stellenbosch University, 2016-03) Davison, Steffi Angela; Van Zyl, Willem Heber; Den Haan, Riaan; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae is regularly chosen for the heterologous production of industrial and medically relevant proteins, due to its rapid growth rate, high cell density fermentation capabilities, microbial safety and eukaryotic post-translational processing. Identifying strains with superior secretion and production of recombinant proteins, whether for pharmaceutical, agricultural or industrial processes, has the benefit of lowering production costs. This holds true for second generation (2G) cellulosic bioethanol production, where high titers of key cellulolytic enzymes are needed to break down complex lignocellulosic substrates. While several secretion-enhancing strategies have been attempted in heterologous production hosts, these strategies were limited by bottlenecks in the secretory pathway. Although protein characteristics and host restrictions are likely to contribute to these bottlenecks, these limitations are poorly understood. Exploiting naturally occurring yeast variants has shown great potential to identifying strains with varying fermentation profiles and tolerance to industrial stresses. The same variation is expected in the secreted and total heterologous cellulolytic activity levels between natural S. cerevisiae strains. Many natural yeast strains may not be suitable for direct industrial fermentation, however industrially relevant traits could be transferred to industrial strains, thereby creating a novel yeast strains with extra beneficial features. In this study, the potential of natural S. cerevisiae strains with regards to superior cellulolytic activity levels, robustness and other ideal characteristics for 2G cellulosic bioethanol production were evaluated. Preliminary screening of thirty natural strains for the production of Saccharomycopsis fibuligera Cel3A (S.f.Cel3A) activity demonstrated variation in secreted cellulase activity levels, allowing us to select seven strains with promising phenotypes. After cellulase genes were expressed on episomal and delta integrative plasmids in S .cerevisiae strains, the secreted activity yields of episomally produced Trichoderma reesei Cel5A (T.r.Cel5A) and Talaromyces emersonii Cel7A (T.e.Cel7A) were 3.5- and 3.7-fold higher in natural strain YI13 compared to reference strain S288c. However, no single strain had highest secreted activity for all three enzymes, suggesting cell specific activity levels is dependent on the genetic background of the host and properties of the protein. Nevertheless, YI13 was identified to be highly tolerant to secretion and cell wall stresses (predicted to result in higher cell specific activities). After evaluating other industrially relevant characteristics including growth vigour, fermentation vigour and tolerance to industrial stressors, natural strains were identified to have promising features for 2G cellulosic ethanol production. Variation in the fermentative (YP-glucose and Avicel cellulose) profiles of S. cerevisiae strains are observed, with the natural strains producing similar titers of ethanol (9.0 g/L) compared with the benchmark MH1000 strain in YP-glucose fermentation conditions. Multi-tolerance traits to industrial stresses were demonstrated by the YI13 strain including high ethanol tolerance (10% w/v), high temperature tolerance (37oC and 40oC), and tolerance to a cocktail of inhibitory compounds found in lignocellulosic hydrolysates, suggesting that this strain has a balance between an effective secretion pathway and robustness to withstand environmental conditions. These strains are a significant step toward creating an efficient cellulase secreting yeast for 2G bioethanol production.