Browsing by Author "Den Haan, Riaan"
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- ItemCombined cell‑surface displayand secretion‑based strategies for production of cellulosic ethanol with Saccharomyces cerevisiae(BioMed Central, 2015-09-26) Liu, Zhuo; Inokuma, Kentaro; Ho, Shih‑Hsin; Den Haan, Riaan; Hasunuma, Tomohisa; Van Zyl, Willem H.; Kondo, AkihikoBackground: Engineering Saccharomyces cerevisiae to produce heterologous cellulases is considered as a promising strategy for production of bioethanol from lignocellulose. The production of cellulase is usually pursued by one of the two strategies: displaying enzyme on the cell surface or secreting enzyme into the medium. However, to our knowledge, the combination of the two strategies in a yeast strain has not been employed. Results: In this study, heterologous endoglucanase (EG) and cellobiohydrolase I (CBHI) were produced in a β-glucosidase displaying S. cerevisiae strain using cell-surface display, secretion, or a combined strategy. Strains EGD- CBHI-D and EG-S-CBHI-S (with both enzymes displayed on the cell surface or with both enzymes secreted to the surrounding medium) showed higher ethanol production (2.9 and 2.6 g/L from 10 g/L phosphoric acid swollen cellulose, respectively), than strains EG-D-CBHI-S and EG-S-CBHI-D (with EG displayed on cell surface and CBHI secreted, or vice versa). After 3-cycle repeated-batch fermentation, the cellulose degradation ability of strain EG-D-CBHI-D remained 60 % of the 1st batch, at a level that was 1.7-fold higher than that of strain EG-S-CBHI-S. Conclusions: This work demonstrated that placing EG and CBHI in the same space (on the cell surface or in the medium) was favorable for amorphous cellulose-based ethanol fermentation. In addition, the cellulolytic yeast strain that produced enzymes by the cell-surface display strategy performed better in cell-recycle batch fermentation compared to strains producing enzymes via the secretion strategy.
- ItemEngineering cellulolytic ability into bioprocessing organisms(Springer-Verlag, 2010-03) La Grange, Daniel C.; Den Haan, Riaan; Van Zyl, Willem H.Lignocellulosic biomass is an abundant renewable feedstock for sustainable production of commodities such as biofuels. The main technological barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose. Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies. This would allow steps that are currently accomplished in different reactors, often by different organisms, to be combined in a consolidated bioprocess (CBP). The development of such organisms has focused on engineering naturally cellulolytic microorganisms to improve product-related properties or engineering non-cellulolytic organisms with high product yields to become cellulolytic. The latter is the focus of this review. While there is still no ideal organism to use in one-step biomass conversion, several candidates have been identified. These candidates are in various stages of development for establishment of a cellulolytic system or improvement of product-forming attributes. This review assesses the current state of the art for enabling non-cellulolytic organisms to grow on cellulosic substrates.
- ItemEngineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production(Springer Nature, 2016) Liu, Zhuo; Ho, Shih-Hsin; Sasaki, Kengo; Den Haan, Riaan; Inokuma, Kentaro; Ogino, Chiaki; Van Zyl, Willem H.; Hasunuma, Tomohisa; Kondo, AkihikoCellulosic biofuel is the subject of increasing attention. The main obstacle toward its economic feasibility is the recalcitrance of lignocellulose requiring large amount of enzyme to break. Several engineered yeast strains have been developed with cellulolytic activities to reduce the need for enzyme addition, but exhibiting limited effect. Here, we report the successful engineering of a cellulose-adherent Saccharomyces cerevisiae displaying four different synergistic cellulases on the cell surface. The cellulase-displaying yeast strain exhibited clear cell-to-cellulose adhesion and a “tearing” cellulose degradation pattern; the adhesion ability correlated with enhanced surface area and roughness of the target cellulose fibers, resulting in higher hydrolysis efficiency. The engineered yeast directly produced ethanol from rice straw despite a more than 40% decrease in the required enzyme dosage for high-density fermentation. Thus, improved cell-to-cellulose interactions provided a novel strategy for increasing cellulose hydrolysis, suggesting a mechanism for promoting the feasibility of cellulosic biofuel production.
- ItemEngineering of Pichia stipitis for enhanced xylan utilization(Stellenbosch : Stellenbosch University, 2003-12) Den Haan, Riaan; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH ABSTRACT: Plant biomass, the most abundant renewable resource in nature, consists of matrices of mainly lignin, cellulose, hemicellulose as well as inorganic components. Xylan, the major hemicellulose component in plant cell walls, is the most abundant polysaccharide after cellulose. This makes the main constituent sugar of xylan, D-xylose, the second most abundant renewable monosaccharide in nature. Very few hemicelluloses are either homopolymeric or entirely linear. Therefore, the variety of enzymes involved in their hydrolysis is more complex than the enzyme group responsible for the hydrolysis of cellulose. Although the ability to degrade xylan is common among bacteria and filamentous fungi, this trait is relatively rare among yeasts. However, some strains of the yeast Pichia stipitis are, amongst others, able to degrade xylan. As P. stipitis is also one of the best D-xylose fermenting yeasts thus far described, this yeast has the potential of fermenting polymeric xylan directly to ethanol. However, it was shown that the natural xylanolytic ability of this yeast is very weak. In this study, xylanolytic genes were expressed in P. stipitis to test the ability of the yeast to produce heterologous proteins, and to determine the enhancement of xylan utilisation by the recombinant strain. The native xylose reductase gene (XYLl) and transketolase gene (TKL) and the heterologous Saccharomyces cerevisiae phosphoglycerate kinase (PGKl) gene promoter were cloned into P. stipitis transformation vectors and used to express the Trichoderma reesei ~-xylanase encoding gene (xyn2) as reporter gene. It was shown that the XYLl promoter was induced in the presence of D-xylose and that the TKL promoter was constitutively expressed. The PGKl promoter of S. cerevisiae did not function in P. stipitis . When the T reesei xyn2 gene and the Aspergillus kawachii ~-xylanase encoding gene (xynC) were expressed under control of the XYLl promoter, extracellular ~-xylanase activity of up to 136 nkat/ml and 171 nkatlml was observed, respectively. This activity declined over time due to the presence of extracellular proteases, secreted by P. stipitis. Growing the cultures in a fermentor and controlling the pH level to pH 6 did not alleviate the reduction of heterologous l3-xylanase activity. When the Aspergillus niger l3-xylosidase encoding gene (xlnD) was expressed as a fusion gene (designated XL02) with the S. cerevisiae mating factor secretion signal (MFal) under control of the P. stipitis TKL promoter, extracellular l3-xylosidase activity of 0.132 nkatlml was observed. Co-expression of the xyn2 and XL02 genes led to B-xylanase and l3-xylosidase activities of 128 nkatlml and 0.113 nkat/ml, respectively. Co-expression of the xynC and XL02 genes led to l3-xylanase and l3-xylosidase activities of 165 nkat/ml and 0.124 nkatlml, respectively. The expression of the fungal xylanolytic genes in P. stipitis also led to an increased biomass yield when the recombinant strains were cultured on birchwood xylan as sole carbon source. The strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding genes was the most successful, yielding a 3.2-fold higher biomass level than the control strain. Biomass levels of the recombinant strains were further improved on average by 85% by growing them in a fermentor under conditions of high oxygenation. The strains were also tested for direct conversion of xylan to ethanol and the strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding genes produced 1.35 giL ethanol, which represents a 3.6-fold increase in ethanol yield over the reference strain. These strains represent a step towards the efficient degradation and utilisation of hemicellulosic materials by ethanol-producing yeasts.
- ItemFunctional expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol(Elsevier, 2006-09) Den Haan, Riaan; Mcbride, John E.; La Grange, Daniel C.; Lynd, Lee R.; Van Zyl, Willem H.To investigate the possible use of Saccharomyces cerevisiae as a candidate for consolidated bioprocessing (CBP) of cellulose to ethanol, four fungal cellobiohydrolase (CBH) encoding genes (Trichoderma reesei cbh1 and cbh2, Aspergillus niger cbhB and Phanerochaete chrysosporium cbh1–4) were expressed in this yeast. All four CBHs were successfully expressed and similar extracellular activity was demonstrated on phosphoric acid swollen cellulose (PASC) and bacterial microcrystalline cellulose (BMCC) using a modified affinity digestion procedure. Our results suggest that although heterologous CBHs can be produced in S. cerevisiae the titers of functionally secreted CBH are relatively low. However, the specific activity of recombinant CBH1 on PASC and BMCC, determined by a sensitive ELISA-based technique, was found not to differ significantly from that of the native T. reesei enzyme. Given this similarity in specific activity, but the disparity between current levels of CBH expression relative to expression levels required to enable growth on crystalline cellulosic substrates with concomitant ethanol production, future studies should aim to increase the expression levels of CBHs.
- ItemHigh level secretion of cellobiohydrolases by Saccharomyces cerevisiae(BioMed Central, 2011-09) Ilmen, Marja; Den Haan, Riaan; Brevnova, Elena; McBride, John; Wiswall, Erin; Froehlich, Allan; Koivula, Anu; Voutilainen, Sanni P.; Siika-aho, Matti; LaGrange, Daniel C.; Thorngren, Naomi; Ahlgren, Simon; Mellon, Mark; Deleault, Kristen; Rajgarhia, Vineet; Van Zyl, Willem H.; Penttila, MerjaBACKGROUND: The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. RESULTS: We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. CONCLUSIONS: Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.