Doctoral Degrees (Microbiology)
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Browsing Doctoral Degrees (Microbiology) by browse.metadata.advisor "Den Haan, Riaan"
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- ItemThe effects of native Saccharomyces cerevisiae snare gene over expression on Heterologous cellulase secretion(Stellenbosch : Stellenbosch University, 2015-12) Van Zyl, J. H. D.; Van Zyl, Willem Heber; Den Haan, Riaan; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The budding yeast Saccharomyces cerevisiae has been successfully utilized in several industrial sectors and has over the last decade emerged as a promising host for the production of valuable heterologous proteins. As with the development of most biologicallybased production systems, there are invariably hurdles to overcome, the most pressing being the sub-optimal production yields for many heterologous proteins. The low protein secretion capacity of S. cerevisiae has been attributed to a great number of factors including various unknown secretory bottlenecks within the secretion pathway that collectively result in secretory titers that are often lower than 1% of the theoretical estimates. Increased secretory titers for the industrially significant fungal cellulases in the S. cerevisiae protein production host would greatly contribute to the economic feasibility of second generation bioethanol production. Improved titers will also benefit the production of commercially important biopharmaceutical proteins. SNAREs (Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment REceptor proteins) represent a class of membrane proteins that are required for the majority of membrane fusion events in the cell, including fusion of the protein secretory vesicles with the cis-Golgi and the plasma membrane. In this study, we attempted to elucidate whether the overproduction of some of these SNARE components at the cis-Golgi interface (BOS1, BET1, SEC22 and SED5) and at the plasma membrane (SNC1, SNC2, SSO1, SSO2 and SEC9) could increase the efficiency of the protein secretion process in S. cerevisiae for two industrially significant fungal cellulases – the Saccharomycopsis fibuligera Cel3A (β-glucosidase) and the Talaromyces emersonii Cel7A (cellobiohydrolase I). Our investigation further attempted to elucidate other physiological effects that these genetic modifications could bring about, both in terms of growth vigor and response to secretory stress. The exocytic t-SNARE Sso1p yielded the most improved secretory phenotype for Sf-Cel3A, with an improvement of approximately 43%, whilst the Snc1p v-SNARE component yielded the largest improvement in Te-Cel7A secretion of 71% (relative to the parental strain). The improvements for this reporter protein could be semi-quantitatively illustrated using SDSPAGE and densitometry analysis. Simultaneous overexpression of exocytic SNARE genes led to a moderate improvement of 52% and 48% for the secretion of Te-Cel7A and Sf-Cel3A, respectively, whilst simultaneous SNARE-overexpression in the strains producing the Sf-Cel3A led to measurable decreases in ethanol and osmotolerance, as well as a decreased growth vigor. For the Endoplasmic Reticulum (ER)-to-Golgi SNAREs, it was the t-SNARE Sed5p that yielded the biggest improvements in the secretion of Sf-Cel3A (22%) and Te-Cel7A (68%). However, overexpression of Sed5p did lead to decreases in ethanol and osmotolerance for strains harboring either of the heterologous cellulases expressed on episomal plasmids, in addition to slight decreases in growth vigor. Simultaneous ER-to-Golgi SNARE overexpression led to less significant secretory improvements for Te-Cel7A and decreased secretory titers for Sf-Cel3A, whilst the yeast could not maintain cell viability upon simultaneous overexpression of the ER-to-Golgi SNAREs in the presence of the beforementioned reporter protein. Co-overexpression of the most promising ER-to-Golgi and exocytic SNARE components identified for the improvement of Sf-Cel3A secretion (SED5 and SSO1, respectively) led to a significant improvement in extracellular activity of 130%. The production of Sf-Cel3A led to a measurably increased unfolded protein response (UPR), a mechanism proportionately induced by the buildup of folded and misfolded proteins in the ER. When Sed5p, which led to an improved secretion phenotype for Sf-Cel3A, was overexpressed in conjunction with the aforementioned reporter protein, the UPR activation was notably diminished. This suggests that a higher dosage of Sed5p may improve ER-to- Golgi protein transport to such an extent that the UPR response diminished. Overexpression of the exocytic SNAREs proved more effective for the improvement of native invertase secretion, with Sso1p and Snc1p leading to improvements of 53% and 32%, respectively. However, Sed5p only yielded a 15% improvement. This study suggests that SNAREs fulfill a prominent role within a larger cascade of secretory pathway components that hold potential as secretory-enhancing factors for the S. cerevisiae heterologous protein production host. The positive effects that overexpression of SNAREs introduced for the secretion of heterologous and native proteins (such as invertase) indicate that these components may be implicated in secretory bottlenecks at the cis-Golgi and/or plasma membrane interface.
- ItemExploring 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.
- ItemExpression and characterization of exoglucanases in Saccharomyces cerevisiae(Stellenbosch : University of Stellenbosch, 2010-03) Van Wyk, Niel; Van Zyl, Willem Heber; Den Haan, Riaan; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Currently a world-wide tendency exists to shift away from relying on fossil fuels as a primary energy source and to focus on sustainable, environmentally-friendly alternatives. Ethanol is one such alternative and shows potential to replace petroleum as a transport fuel. Plant biomass, deemed a renewable energy source, can be converted to ethanol. The process of conversion via biologicallymediated events is problematic mainly due to the recalcitrance of the chief components of plant biomass namely cellulose, hemicellulose and lignin towards enzymatic degradation. A concept of consolidated bioprocessing (CBP) aims to make the process of bioconversion of plant biomass to ethanol cost-effective. For such a bioconversion, a biocatalyst is needed which can depolymerize the complex carbohydrates i.e. the cellulose and hemicellulose to their respective monomers for concurrent fermentation to ethanol. Saccharomyces cerevisiae shows potential as a candidate CBPbiocatalyst due to its high ethanol productivity, general robustness and amenability to genetic manipulation. However, S. cerevisiae does not possess the ability to break down the abovementioned carbohydrates. This study attempted to address certain aspects of yeast strain development for CBP. Genes encoding cellulases responsible for major crystalline cellulose hydrolysis i.e. exoglucanases were expressed in S. cerevisiae and the recombinant proteins were characterized. Further work involved exploring different ways of increasing the cellulolytic capability of recombinant S. cerevisiae. Both the cel9A of Thermobifida fusca and Npcel6A of Neocallimastix patriciarum were functionally expressed in S. cerevisiae. Expression of cel9A enabled S. cerevisiae to grow on phosphoric acid swollen cellulose reaching an aerobic growth rate (mMAX) of 0.088 h-1. This is the first report of S. cerevisiae growing on such a substrate while producing only one heterologous protein. An increase in the cellulolytic capability of recombinant S. cerevisiae was observed when cel9A was coexpressed with Trcel6A, cel7A and cel7B of Trichoderma reesei. These results proved that the synergy between cellulases can contribute towards increasing the cellulolytic capability of recombinant S. cerevisiae. NpCel6A has the highest reported individual activity on a crystalline cellulose substrate. However, expression of Npcel6A by S. cerevisiae resulted in lower levels of exoglucanase activity on Avicel of 0.540±0.062 mU/gDCW compared to the recombinant S. cerevisiae strains that produces Cel6A of T. reesei (4.101±0.243 mU/gDCW). This observation could be ascribed to glycosylation of the catalytic domain of NpCel6A. The replacement of the carbohydrate-binding module (CBM) and asparagine-rich linker of NpCel6A with the CBM and serine/threonine-rich linker of TrCel6A resulted in a decrease in recombinant cellulolytic activity produced by S. cerevisiae. In contrast, when the CBM and linker of NpCel6A were appended to the N-terminus of the catalytic domain of TrCel6A, significantly higher levels of cellulase activity were observed when produced by S. cerevisiae. This observation was largely attributed to the difference in glycosylation of the linkers. These results showed the value of domain swapping for obtaining increased cellulase secretion by S. cerevisiae. The native S. cerevisiae genes PSE1 and SOD1, were individually overexpressed in the S. cerevisiae strain producing NpCel6A, Cel3A of Saccharomycopsis fibuligera and Cel7B of Trichoderma reesei. The DDI1 gene of S. cerevisiae was also disrupted in the strain producing NpCel6A. In all cases, transformants were identified which displayed higher levels of cellulase activity compared to the original strain. This demonstrated the potential of S. cerevisiae to be considered as a “chassis”- strain that can, with the help of metabolic engineering, produce more recombinant cellulases. The swelling factor protein called swollenin, a contributor in the disruption of the crystallinity of cellulose, was co-expressed with cel9A and Npcel6A individually in S. cerevisiae. Even though functionality of swollenin was confirmed, no noteworthy increase in the levels of cellulase activity was observed for recombinant strains. The recombinant yeast strains generated during this study represent significant progress towards developing S. cerevisiae as a CBP organism.
- ItemImpact of inhibitors associated with lignocellulosic hydrolysates on recombinant cellulolytic enzymes(Stellenbosch : Stellenbosch University, 2017-03) Mhlongo, Sizwe Innocent; Van Zyl, Willem Heber; Viljoen-Bloom, Marinda; Den Haan, Riaan; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Enzymatic hydrolysis contributes a significant cost towards the production of bioethanol and is estimated to comprise 15% of the minimum ethanol selling price. One of the areas of concern during the enzymatic hydrolysis is the non-productive adsorption of enzymes by pretreatment by-products that may lead to the inhibition/deactivation of cellulases. Nonproductive adsorption of cellulases onto lignin is mainly driven by hydrophobic interactions and the extent of adsorption varies depending on the hydrophobicity of the lignin. Most fungal cellulases are bimodular with a catalytic domain and a carbohydrate binding domain (CBM) connected by a flexible linker. To achieve high yields of fermentable sugars for subsequent conversion to ethanol, it is desirable to include sugars from both cellulosic- and hemicelluloserich fractions, which implies the presence of inhibitory degradation compounds during enzymatic hydrolysis. To reduce the enzyme loading for hydrolysis, the inhibitor compounds in lignocellulosic biomass should be reduced to below toxic levels or be removed from hydrolysates. The first aim of the study was to investigate the role of individual lignocellulose-associated compounds in the inhibition and/or deactivation of the Talaromyces emersonii cellobiohydrolase (TeCel7A) fused to the Trichoderma reesei carbohydrate binding domain (TrCBM), Trichoderma reesei endoglucanase TrCel5A and Saccharomycopsis fibuligera β- glucosidase (SfCel3A) cellulases. The second aim was to explore detoxification strategies in the alleviation of the cellulose inhibition. The final aim was to investigate the mechanism(s) involved in the inhibition of cellulases. The impact of selected inhibitor compounds on the hydrolysis of Avicel was also investigated using a combination of TeCel7A-TrCBM and TrCel5A in the presence of Novozyme 188 Cel3A to prevent feedback inhibition by cellobiose. The study revealed that polymeric phenols, such as tannic acid, are strong inhibitors of cellulases, whereas monomeric phenols with aldehyde groups showed a strong inhibition of cellulose with increased contact time. This further confirmed that compounds with increased surface hydrophobicity have a strong inhibition effect. TrCel7A was shown to be quite resistant to inhibition and only hydroxymethyl furfural (HMF) strongly inhibited this cellobiohydrolase. This selective inhibition of retaining cellulases (TrCel7A), but not inverting cellulases (TrCel5A), was also observed with acetic and formic acid. This suggests that the non-processive nature and groove-shaped active site of TrCel5A allows it to escape non-productive binding to inhibitor compounds through the same mechanism it employs during cellulose hydrolysis. Further investigation revealed that increasing inhibition was not linked to contact time, but rather ascribed to increased concentration of inhibitor compounds. Detoxification strategies were explored as enhancers of enzymatic hydrolysis and tools to alleviate inhibition in biomass conversion processes. The results indicated that reducing agents (sodium dithionite and sodium sulfite) strongly reacted with coniferyl aldehyde and syringaldehyde, but not tannic acid. The addition of reducing agents substantially increased the hydrolysis of Avicel containing 10% bagasse pretreatment liquid. Application of the differential scanning fluorimeter (DSF) technique showed that increased concentrations of furans and acetic acid sharply increased unfolding of TeCel7A. This study showed that DSF could be developed as a tool to study cellulase binding, but this will depend on the development of dyes not based on hydrophobic interactions.
- ItemImproving the protein secretion capacity of Saccharomyces cerevisiae with strain engineering(Stellenbosch : Stellenbosch University, 2015-03) Kroukamp, Heinrich; Van Zyl, Willem Heber; Den Haan, Riaan; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae is frequently chosen for the production of industrial and pharmaceutical proteins, due to its rapid growth, microbial safety, eukaryotic post-translational processing and high-density fermentation capability. With the development of recombinant DNA technologies and efficient expression systems, this yeast also gained a prominent new role as a protein production host, due to its ease of genetic manipulation. Improving the production and secretion of recombinant proteins, whether for pharmaceutical, agricultural or industrial application, has the benefit of reducing the production costs and promoting accessibility to these technologies. This also holds true for the second generation biofuel production, where high levels of hydrolytic enzymes are required to break down the complex carbohydrates in lignocellulose. While high copy number expression vector systems, strong promoters and efficient secretion signals resulted in significant enhancement of protein production yields, these strategies are often limited by bottlenecks in the yeast secretion pathway. Although protein characteristics and host restrictions are likely to contribute to these bottlenecks, these factors are still poorly understood. Nonetheless, strain engineering approaches have shown great potential as a means to relieve these protein secretion bottlenecks and advancing recombinant protein production to new levels. In this study, the potential of strain engineering, with regards to enhancing cellulase secretion for second generation bioethanol production, was evaluated. Further work involved the identification of novel genetic elements enhancing protein secretion and the elucidation of possible mechanisms involved in high cellulase secretion by S. cerevisiae. When the native PSEI gene was expressed under the transcriptional control of the PGK1 promoter in S. cerevisiae, the secreted yields of recombinantly produced Neocallimastix patriciarum Cel6A, Trichoderma reesei Cel7B and Saccharomycopsis fibuligera Cel3A were increased 1.15, 1.25 and 3.70 fold, respectively. The overexpression of SOD1 did not increase any of the above mentioned cellulases. When SOD1 was overexpressed in combination with PSE1, a synergistic enhancement in secreted Cel3A was obtained. To our knowledge, this is the first reported case where SOD1 overexpression in S. cerevisiae resulted in higher heterologous protein secretion. The effect of disrupting protein N-glycosylation elongation at various steps, on the secretion of heterologously produced Neosartorya fischeri Cel12A (lacking N-glycosylation sites) and the S. fibuligera Cel3A, was investigated. The deletion of the MNN2 gene was shown to increase the extracellular Cel12A by 1.30 fold, while the deletion of MNN11 resulted in a 1.26 fold increase in extracellular Cel3A. These results implicate the cell wall as a possible barrier to protein secretion. It was also found that Cel3A with shorter N-glycosylation chains had reduced cell wall retention, compared to enzymes resembling the native glycosylation pattern. The removal of the PMR1 gene product (predicted to result in a general decrease in Golgi mannosyltranferase activities) was the only modification which enhanced the cell specific activities of both reporter cellulases, although this mutant's poor growth makes it an unlikely candidate for industrial application. The S. cerevisiae M0341 strain that secretes high levels of recombinant Talaromyces emersonii Cel7A, was mated to the Y294 control strain to produce the H3 hybrid strain. Several high secreting progeny were selected after sporulating the H3 strain. Through genome shuffling and single nucleotide polymorphism (SNP) analysis of pooled segregants, five genomic regions of the M0341 strain were identified that contain putative alleles that are beneficial to Cel7A secretion. Identifying these alleles proved problematic due to strain instability. When the T. emersonii CEL7A, N. fischeri CEL12A and S. fibuligera CEL3A were expressed in selected H3 progeny on episomal plasmids, these strains had up to ~3.5 fold increased Cel7A secretion compared to the M0341 parental strain, but no increase were observed for the other cellulase. It was also shown that cell flocculation could improve secretion. The strains constructed in this study represent a step toward efficient cellulase secreting yeasts for second generation biofuel production and presents a novel strategy to identify secretion enhancing elements for the protein production industry.