UWCScholar :: Browsing by Author "den Haan, Riaan"

Browsing by Author "den Haan, Riaan"

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Adapting the yeast consolidated bioprocessing paradigm for biorefineries
(Green Wave Publishing of Canada, 2018) den Haan, Riaan
Despite decades long development, no natural or engineered organism has been isolated that can produce commodity products at the rates and yields required by industry via direct microbial conversion. However, new genomic editing tools and systems level knowledge of metabolism provides opportunities to develop yeast strains for second-generation biorefineries.
Comparison of multi-gene integration strategies in CRISPR-based transformation of Saccharomyces cerevisiae
(University of Western Cape, 2021) Jacob, Odwa; den Haan, Riaan
Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method.
Crispr-based multi-gene integration strategies to create saccharomyces cerevisiae strains for consolidated bioprocessing
(MDPI, 2022) Jacob, Odwa; van Lill, Gert Rutger; den Haan, Riaan
Significant engineering of Saccharomyces cerevisiae is required to enable consolidated bioprocessing (CBP) of lignocellulose to ethanol. Genome modification in S. cerevisiae has been successful partly due to its efficient homology-directed DNA repair machinery, and CRISPR technology has made multi-gene editing even more accessible. Here, we tested the integration of cellulase encoding genes to various sites on the yeast genome to inform the best strategy for creating cellulolytic strains for CBP. We targeted endoglucanase (EG) or cellobiohydrolase (CBH) encoding genes to discreet chromosomal sites for single-copy integration or to the repeated delta sites for multi-copy integration. CBH1 activity was significantly higher when the gene was targeted to the delta sequences compared to single gene integration loci. EG production was comparable, though lower when the gene was targeted to a chromosome 10 site. We subsequently used the information to construct a strain containing three cellulase encoding genes. While individual cellulase activities could be assayed and cellulose conversion demonstrated, it was shown that targeting specific genes to specific loci had dramatic effects on strain efficiency. Since marker-containing plasmids could be cured from these strains, additional genetic changes can subsequently be made to optimize strains for CBP conversion of lignocellulose.
The effect of blocking selected endocytic mechanisms on heterologous protein secretion in the yeast saccharomyces cerevisiae
(University of the Western Cape, 2018) Freeman, Kim; den Haan, Riaan; van Zyl, Emile
The yeast Saccharomyces cerevisiae is considered a good host used for heterologous protein production due to the organism’s microbial safety, rapid growth and eukaryotic post- translational processing. As a fermentative organism, S. cerevisiae is thus not only a useful platform for the production of biopharmaceuticals and industrial enzymes, but also a promising organism for second-generation biofuel production. Substantial effort has been focused on alleviating the many bottlenecks in recombinant gene expression, as well as in the secretory pathway to enhance heterologous protein titres. It was recently shown that highly active endocytosis could decrease the overall secreted protein titre in the supernatant. In this study, we aimed to block endocytotic and vacuolar complexes to ultimately disrupt, or impair, the endocytotic and vacuolar mechanisms of proteolysis and test the effect that this would have on secreted heterologous protein titres. This was accomplished by knocking out various genes involved in endocytosis and transforming the strains with genes encoding various hydrolases including β-glucosidase (Bgl), xylanase (Xyn2), endoglucanase (Eg2) and cellobiohydrylase (Cbh1). Our study demonstrated that genetic blocking of endocytotic mechanisms as well as vacuolar complexes could theoretically improve heterologous protein secretion in S. cerevisiae. Endoglucanase (Eg2) titres displayed improvement of 26% and 30% in strains which had the RVS161 and VRP1 genes deleted and xylanase titres displayed an improvement of 71% and 143% in strains with the END3 and SSA4 gene deletions. Several of the gene knockouts tested improved Xyn2 and Eg2 titres but the effect of the different gene targets varied widely. A double knock-out strain with deletions in CLC1 and RVS161 secreted 104% more Eg2 than its parental control strain on a per dry cell weight basis, a significant synergistic improvement. Other double knock-out strains displayed additive or similar activities when compared to their controls. Cbh1 secretion could not be improved through the gene deletions tested in our study and Bgl activity could not be measured in our transformants. These results demonstrate the different relationships of various heterologous proteins with various components of the secretion machinery and may also imply how endocytosis as well as vacuolar complexes affect the level of secreted protein.
Identification of superior cellulase secretion phenotypes in haploids derived from natural Saccharomyces cerevisiae isolates
(Oxford University Press, 2019) Davison, Steffi A.; den Haan, Riaan; van Zyl, Willem Heber
The yeast Saccharomyces cerevisiae is considered an important host for consolidated bioprocessing and the production of high titres of recombinant cellulases is required for efficient hydrolysis of lignocellulosic substrates to fermentable sugars. Since recombinant protein secretion profiles vary highly among different strain backgrounds, careful selection of robust strains with optimal secretion profiles is of crucial importance. Here, we construct and screen sets of haploid derivatives, derived from natural strain isolates YI13, FINI and YI59, for improved general cellulase secretion. This report details a novel approach that combines secretion profiles of strains and phenotypic responses to stresses known to influence the secretion pathway for the development of a phenotypic screen to isolate strains with improved secretory capacities. A clear distinction was observed between the YI13 haploid derivatives and industrial and laboratory counterparts, Ethanol Red and S288c, respectively. By using sub-lethal concentrations of the secretion stressor tunicamycin and cell wall stressor Congo Red, YI13 haploid derivative strains demonstrated tolerance profiles related to their heterologous secretion profiles. Our results demonstrated that a new screening technique combined with a targeted mating approach could produce a pool of novel strains capable of high cellulase secretion.
Improved cellulase expression in diploid yeast strains enhanced consolidated bioprocessing of pretreated corn residues
(ScienceDirect, 2019) den Haan, Riaan
In an effort to find a suitable genetic background for efficient cellulolytic secretion, genetically diverse strains were transformed to produce core fungal cellulases namely, β-glucosidase (BGLI), endoglucanase (EGII) and cellobiohydrolase (CBHI) in various combinations and expression configurations. The secreted enzyme activity levels, gene copy number, substrate specificities, as well as hydrolysis and fermentation yields of the transformants were analysed. The effectiveness of the partially cellulolytic yeast transformants to convert two different pre-treated corn residues, namely corn cob and corn husk was then explored.
The in vitro detection and measurement of the unfolded protein response in Saccharomyces cerevisiae
(University of the Western Cape, 2018) Cedras, Gillian; den Haan, Riaan; van Zyl, Willem H.
Bioethanol is currently the most widely used biofuel and can be used as a direct replacement for current fossil fuel based transportation fuels. Consolidated bioprocessing (CBP), in which bioethanol is produced in a single reactor by a single microorganism, is a cost-effective way of producing bioethanol in a second generation process using lignocellulosic biomass as feedstock. The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and is able to produce heterologous cellulases, which are required for CBP. However, S. cerevisiae produces low titres of cellulases and one suspected reason for this is the stress caused by the heterologous proteins that induce the unfolded protein response (UPR). The UPR is a stress response pathway that will either lead to increased folding capacity within the ER or to degradation of these proteins and possibly apoptosis of the cell. It is thus beneficial to be able to determine when and to what extent the UPR is active during CBP organism development. Current methods of measuring the UPR include RNA and reverse transcriptase qPCR (r.t.qPCR) measurements, which can be cumbersome and expensive. The purpose of this study was to develop a vector based biosensor that will detect and quantify UPR activation. The vector consisted of either the T.r.xyn2 or eGFP reporter genes under the control of the S. cerevisiae HAC1p or KAR2p promoters. HAC1 and KAR2 are important regulators of UPR as their activation allows the UPR to achieve its function. The eGFP reporter under the transcriptional control of KAR2p was shown to be the superior combination due to the improved dynamic range when the UPR was induced in transformed S. cerevisiae strains by the stress inducer, tunicamycin. This UPR biosensor also proved to be more sensitive when measuring UPR induction in cellulase producing strains, depicting significant differences, compared to previous r.t.qPCR based tests which were unable to detect these differences. We thus developed a UPR biosensor that has greater sensitivity for changes in UPR induction compared to RNA based methods and the first KAR2p based UPR biosensor plasmid that allowed for more accurate detection and measurement of the UPR in cellulase secreting S. cerevisiae strains. The ability to quantify UPR induction will assist in identifying candidate cellulase genes that do not greatly induce the UPR, making them ideal to use in developing CBP yeasts.
Supplementation of recombinant cellulases with LPMOs and CDHs improves consolidated bioprocessing of cellulose
(Elsevier, 2023) Smuts, Ivy E.; den Haan, Riaan; Blakeway, Nicole J.
The increased demand for energy has sparked a global search for renewable energy sources that could partly replace fossil fuel resources and help mitigate climate change. Cellulosic biomass is an ideal feedstock for renewable bioethanol production, but the process is not currently economically feasible due to the high cost of pretreatment and enzyme cocktails to release fermentable sugars. Lytic polysaccharide monooxygenases (LPMOs) and cellobiose dehydrogenases (CDHs) are auxiliary enzymes that can enhance cellulose hydrolysis. In this study, four LPMO and two CDH genes were subcloned and expressed in the Saccharomyces cerevisiae Y294 laboratory strain. SDS-PAGE analysis confirmed the extracellular production of the LPMOs and CDHs in the laboratory S. cerevisiae Y294 strain. A rudimentary cellulase cocktail (cellobiohydrolase 1 and 2, endoglucanase and β-glucosidase) was expressed in the commercial CelluX™ 4 strain and extracellular production of the individual cellulases was confirmed by SDS-PAGE analysis. In vitro cooperation of the CDHs and LPMOs with the rudimentary cellulases produced by strain CelluX™ 4[F4–1] was demonstrated on Whatman filter paper. The significant levels of soluble sugars released from this crystalline cellulose substrate indicated that these auxiliary enzymes could be important components of the CBP yeast cellulolytic system.
Using CRISPR/Cas9 to construct consolidated bioprocessing strains from natural isolates of Saccharomyces cerevisiae
(University of the Western Cape, 2022) Minnaar, Letitia; den Haan, Riaan
Saccharomyces cerevisiae has gained much attention as a host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high ethanol producing titres, heterologous protein producing capabilities, and tolerance to various industry-relevant stresses. Since the secretion profiles of heterologous proteins are relatively low in industrial and laboratory strains of S. cerevisiae, natural isolates may offer a more diverse genetic background with increased robustness to allow for improved heterologous protein secretion. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core cellulase enzyme complex (CBHI, CBHII, EG and BGL), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated.
Valorisation of brewers’ spent grain: enzymatic hydrolysis in the production of xylooligosaccharides
(University of the Western Cape, 2023) Arries, Chelsey; den Haan, Riaan
The beverage industry constitutes approximately 26% of all food wastes, making it one of the largest contributors in this waste segment. By utilising waste or by-products from agriculture and food production in manufacturing value added compounds, the concepts of waste mitigation and green chemistry can contribute to establishing a circular bio-economy. In a biorefinery, bio-catalytic, thermal, chemical and physical techniques are used to extract valuable compounds from food and agricultural wastes. Brewers’ spent grain (BSG) is an ideal candidate for such a biorefinery approach. This high moisture, nutrient-rich by-product from beer production is either disposed of in landfills or used as an animal feed. However, high value products, such as xylooligosaccharides (XOS) can be extracted from BSG, thereby valorising this brewery waste. Xylooligosaccharides are sought after for their ability to function as a low caloric sweetener while exhibiting prebiotic effects in stimulating the growth of probiotic bacteria in the mammalian gut.

Browsing by Author "den Haan, Riaan"

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