Browsing by Author "Van Zyl, Willem H."
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- ItemBioenergy and African transformation(BioMed Central, 2015-02) Lynd, Lee R.; Sow, Mariam; Chimphango, Annie F. A.; Cortez, Luis A. B.; Brito Cruz, Carlos H.; Elmissiry, Mosad; Laser, Mark; Mayaki, Ibrahim A.; Moraes, Marcia A. F. D.; Nogueira, Luiz A. H.; Wolfaardt, Gideon M.; Woods, Jeremy; Van Zyl, Willem H.Among the world’s continents, Africa has the highest incidence of food insecurity and poverty and the highest rates of population growth. Yet Africa also has the most arable land, the lowest crop yields, and by far the most plentiful land resources relative to energy demand. It is thus of interest to examine the potential of expanded modern bioenergy production in Africa. Here we consider bioenergy as an enabler for development, and provide an overview of modern bioenergy technologies with a comment on application in an Africa context. Experience with bioenergy in Africa offers evidence of social benefits and also some important lessons. In Brazil, social development, agricultural development and food security, and bioenergy development have been synergistic rather than antagonistic. Realizing similar success in African countries will require clear vision, good governance, and adaptation of technologies, knowledge, and business models to myriad local circumstances. Strategies for integrated production of food crops, livestock, and bioenergy are potentially attractive and offer an alternative to an agricultural model featuring specialized land use. If done thoughtfully, there is considerable evidence that food security and economic development in Africa can be addressed more effectively with modern bioenergy than without it. Modern bioenergy can be an agent of African transformation, with potential social benefits accruing to multiple sectors and extending well beyond energy supply per se. Potential negative impacts also cut across sectors. Thus, institutionally inclusive multi-sector legislative structures will be more effective at maximizing the social benefits of bioenergy compared to institutionally exclusive, single-sector structures.
- ItemBiologically based methods for control of fumonisin-producing fusarium species and reduction of the fumonisins(Frontiers Media, 2016) Alberts, Johanna F.; Van Zyl, Willem H.; Gelderblom, Wentzel C. A.Infection by the fumonisin-producing Fusarium spp. and subsequent fumonisin contamination of maize adversely affect international trade and economy with deleterious effects on human and animal health. In developed countries high standards of the major food suppliers and retailers are upheld and regulatory controls deter the importation and local marketing of fumonisin-contaminated food products. In developing countries regulatory measures are either lacking or poorly enforced, due to food insecurity, resulting in an increased mycotoxin exposure. The lack and poor accessibility of effective and environmentally safe control methods have led to an increased interest in practical and biological alternatives to reduce fumonisin intake. These include the application of natural resources, including plants, microbial cultures, genetic material thereof, or clay minerals pre- and post-harvest. Pre-harvest approaches include breeding for resistant maize cultivars, introduction of biocontrol microorganisms, application of phenolic plant extracts, and expression of antifungal proteins and fumonisin degrading enzymes in transgenic maize cultivars. Post-harvest approaches include the removal of fumonisins by natural clay adsorbents and enzymatic degradation of fumonisins through decarboxylation and deamination by recombinant carboxylesterase and aminotransferase enzymes. Although, the knowledge base on biological control methods has expanded, only a limited number of authorized decontamination products and methods are commercially available. As many studies detailed the use of natural compounds in vitro, concepts in reducing fumonisin contamination should be developed further for application in planta and in the field pre-harvest, post-harvest, and during storage and food-processing. In developed countries an integrated approach, involving good agricultural management practices, hazard analysis and critical control point (HACCP) production, and storage management, together with selected biologically based treatments, mild chemical and physical treatments could reduce fumonisin contamination effectively. In rural subsistence farming communities, simple, practical, and culturally acceptable hand-sorting, maize kernel washing, and dehulling intervention methods proved to be effective as a last line of defense for reducing fumonisin exposure. Biologically based methods for control of fumonisin-producing Fusarium spp. and decontamination of the fumonisins could have potential commercial application, while simple and practical intervention strategies could also impact positively on food safety and security, especially in rural populations reliant on maize as a dietary staple.
- ItemA Chromogenic substrate for a β-xylosidase-coupled assay of α-glucuronidase(Elsevier, 2000-08) Biely, Peter; Hirsch, Jan; La Grange, Daniel C.; Van Zyl, Willem H.; Prior, Bernard A.-Nitrophenyl 2-(4-O-methyl-α- -glucopyranuronosyl)-β- -xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-α- -glucopyranosyluronate)-β- -xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145–149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic α-glucuronidase activity. A new precise α-glucuronidase assay was developed by coupling the α-glucuronidase-catalyzed formation of 4-nitrophenyl β- -xylopyranoside with its efficient hydrolysis by β-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the β-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of β-xylosidase. The activity values of β-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the α-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions.
- ItemCloning of the Bacillus pumilus beta-xylosidase gene (xynB ) and its expression in Saccharomyces cerevisiae(Springer-Verlag, 1997-07) La Grange, Daniel C.; Pretorius, Isak S.; Van Zyl, Willem H.A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the beta-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone a-factor (MFa1S) before insertion between the ADH2P and ADH2T sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2P-MFa1SxynB-ADH2T was designated XLO1. Both plasmids pDLG11 and pDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional beta-xylosidase. The total beta-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45±50 °C and pH 6.6 respectively. The enzyme was thermostable at 45 °C; however, at 60 °C most of the Xlo1 was inactive after 5 min.
- ItemCoexpression of the Bacillus pumilus beta-xylosidase (xynB) gene with the Trichoderma reesei beta xylanase 2 (xyn2) gene in the yeast Saccharomyces cerevisiae(Springer-Verlag, 1999-12) La Grange, Daniel C.; Claeyssens, Mark; Pretorius, Isak S.; Van Zyl, Willem H.The xynB gene encoding the Bacillus pumilus beta-xylosidase was expressed separately and jointly with the Trichoderma reesei beta-xylanase (xyn2) gene in the yeast Saccharomyces cerevisiae. Both genes were placed under the transcriptional control of the glucose-derepressible alcohol dehydrogenase 2 promoter (ADH2p) and terminator (ADH2T) sequences. The xynB gene was fused in frame to the yeast mating factor alpha1 secretion sequence (MFalpha1s) to effect secretion in S. cerevisiae. The fusion protein was designated Xlo1. Xlo1 produced in S. cerevisiae exhibited low affinity for xylobiose, but eventually hydrolyzed xylobiose and xylotriose to the monomeric constituent, D-xylose. Coproduction of Xyn2 and Xlo1 by S. cerevisiae led to a 25% increase in the amount of reducing sugars released from birchwood xylan compared to S. cerevisiae producing only the Xyn2 beta-xylanase. However, no D-xylose was produced from birchwood xylan, presumably due to very low Xlo1 beta-xylosidase activity and its low affinity for xylobiose.
- 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.
- ItemConstruction of cellobiose-growing and fermenting Saccharomyces cerevisiae strains(Elsevier, 2004-12) Van Rooyen, Ronel; Hahn-Hagerdal, Barbel; La Grange, Daniel C.; Van Zyl, Willem H.β-Glucosidase genes of fungal origins were isolated and heterologously expressed in Saccharomyces cerevisiae to enable growth on the disaccharide, cellobiose. To promote secretion of the β-glucosidases, the genes were fused to the secretion signal of the Trichoderma reesei xyn2 gene and constitutively expressed from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The two most promising enzymes, BGL1 from Saccharomycopsis fibuligera and BglA from Aspergillus kawachii, were anchored to the yeast cell surface by fusing the mature proteins to the α-agglutinin (AGα1) or cell wall protein 2 (Cwp2) peptides. The maximum specific growth rates (μmax) of the recombinant S. cerevisiae strains were determined in batch cultivation. S. cerevisiae secreting the recombinant S. fibuligera BGL1 enzyme sustained growth aerobically and anaerobically, in minimal medium containing 5 g L−1 cellobiose at 0.23 h−1 (compared to 0.29 h−1 on glucose) and 0.18 h−1 (compared to 0.25 h−1 on glucose), respectively. Substrate consumption and product formation were determined to evaluate product yields in glucose and cellobiose.
- ItemConstruction of industrial Saccharomyces cerevisiae strains for the efficient consolidated bioprocessing of raw starch(BMC (part of Springer Nature), 2019-08-20) Cripwell, Rosemary A.; Rose, Shaunita H.; Favaro, Lorenzo; Van Zyl, Willem H.Background: Consolidated bioprocessing (CBP) combines enzyme production, saccharification and fermentation into a one-step process. This strategy represents a promising alternative for economic ethanol production from starchy biomass with the use of amylolytic industrial yeast strains. Results: Recombinant Saccharomyces cerevisiae Y294 laboratory strains simultaneously expressing an α-amylase and glucoamylase gene were screened to identify the best enzyme combination for raw starch hydrolysis. The codon optimised Talaromyces emersonii glucoamylase encoding gene (temG_Opt) and the native T. emersonii α-amylase encoding gene (temA) were selected for expression in two industrial S. cerevisiae yeast strains, namely Ethanol Red™ (hereafter referred to as the ER) and M2n. Two δ-integration gene cassettes were constructed to allow for the simultaneous multiple integrations of the temG_Opt and temA genes into the yeasts’ genomes. During the fermentation of 200 g l−1 raw corn starch, the amylolytic industrial strains were able to ferment raw corn starch to ethanol in a single step with high ethanol yields. After 192 h at 30 °C, the S. cerevisiae ER T12 and M2n T1 strains (containing integrated temA and temG_Opt gene cassettes) produced 89.35 and 98.13 g l−1 ethanol, respectively, corresponding to estimated carbon conversions of 87 and 94%, respectively. The addition of a commercial granular starch enzyme cocktail in combination with the amylolytic yeast allowed for a 90% reduction in exogenous enzyme dosage, compared to the conventional simultaneous saccharification and fermentation (SSF) control experiment with the parental industrial host strains. Conclusions: A novel amylolytic enzyme combination has been produced by two industrial S. cerevisiae strains. These recombinant strains represent potential drop-in CBP yeast substitutes for the existing conventional and raw starch fermentation processes.
- ItemDegradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger β-xylosidase (xlnD) and the Trichoderma reesei Xylanase II (xyn2) genes(American Society of Microbiology, 2001-12) La Grange, Daniel C.; Pretorius, Isak S.; Claeyssens, Mark; Van Zyl, Willem H.The β-xylosidase-encoding xlnD gene of Aspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor α1 signal peptide (MFα1s) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant β-xylosidase showed optimum activity at 60°C and pH 3.2 and optimum stability at 50°C. The Ki(app) value for D-xylose and xylobiose for the recombinant β-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The XLO2 fusion gene and the XYN2 β-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of β-xylanase activity when expressing only the β-xylanase and 860 nkat/ml when coexpressing the β-xylanase with the β-xylosidase. The maximum β-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the β-xylanase. Coproduction of the β-xylanase and β-xylosidase enabled S. cerevisiae to degrade birchwood xylan to D-xylose.
- 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.
- ItemEnzyme-coupled assay of acetylxylan esterases on monoacetylated 4-nitrophenyl beta-D-xylopyranoside(Elsevier, 2004-03) Biely, Peter; Mastihubova, Maria; La Grange, Daniel C.; Van Zyl, Willem H.; Prior, Bernard A.Three different monoacetates of 4-nitrophenyl beta-D-xylopyranoside were tested as substrates for beta -xylosidase and for microbial carbohydrate esterases and a series of non-hemicellulolytic esterases. The acetyl group in 2-O-acetyl, 3-O-acetyl, and 4-O-acetyl 4-nitrophenyl beta-D-xylopyranoside makes the glycoside resistant to the action of beta-xylosidase (EC 3.2.1.37). This fact was explored to introduce a new enzyme-coupled assay of acetylxylan esterases (EC 3.1.1.72) and other carbohydrate-deacetylating enzymes. The deacetylation converts the monoacetates into the substrate of beta -xylosidase, the auxiliary enzyme. The eVect of the acetyl group migration along the xylopyranoid ring in aqueous media can be avoided by shortening the assay duration. The assay enables an easy examination of the positional specificity of the enzymes, which is important for classification of acetylxylan esterases and for elucidation of the structure–function relationship among carbohydrate esterases in general. Non-hemicellulolytic esterases showed different positional specificity of deacetylation than did acetylxylan esterases.
- ItemExploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production(BioMed Central, 2013-11) Favaro, Lorenzo; Basaglia, Marina; Trento, Alberto; Van Rensburg, Eugene; Garcia-Aparicio, Maria; Van Zyl, Willem H.; Casella, SergioBackground Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance. Results Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content. Forty newly isolated Saccharomyces cerevisiae strains gave high ethanol yields at 40°C when inoculated in minimal media at high sugar concentrations of up to 200 g/l glucose. In addition, the isolates displayed distinct inhibitor tolerance in defined broth supplemented with increasing levels of single inhibitors or with a cocktail containing several inhibitory compounds. Both the fermentation ability and inhibitor resistance of these strains were greater than those of established industrial and commercial S. cerevisiae yeasts used as control strains in this study. Liquor from steam-pretreated sugarcane bagasse was used as a key selective condition during the isolation of robust yeasts for industrial ethanol production, thus simulating the industrial environment. The isolate Fm17 produced the highest ethanol concentration (43.4 g/l) from the hydrolysate, despite relatively high concentrations of weak acids, furans, and phenolics. This strain also exhibited a significantly greater conversion rate of inhibitory furaldehydes compared with the reference strain S. cerevisiae 27P. To our knowledge, this is the first report describing a strain of S. cerevisiae able to produce an ethanol yield equal to 89% of theoretical maximum yield in the presence of high concentrations of inhibitors from sugarcane bagasse. Conclusions This study showed that yeasts with high tolerance to multiple stress factors can be obtained from unconventional ecological niches. Grape marc appeared to be an unexplored and promising substrate for the isolation of S. cerevisiae strains showing enhanced inhibitor, temperature, and osmotic tolerance compared with established industrial strains. This integrated approach of selecting multiple resistant yeasts from a single source demonstrates the potential of obtaining yeasts that are able to withstand a number of fermentation-related stresses. The yeast strains isolated and selected in this study represent strong candidates for bioethanol production from lignocellulosic hydrolysates.
- ItemExpression of a Trichoderma reesei beta-xylanase gene (XYN2) in Saccharomyces cerevisiae(American Society for Microbiology, 1995-04) La Grange, Daniel C.; Pretorius, Isak S.; Van Zyl, Willem H.The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-beta-1,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 699-bp open reading frame that encodes a 223-amino-acid propeptide. The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively. The 33-amino-acid leader peptide of the Xyn2 beta-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous beta-xylanase. The molecular mass of the recombinant beta-xylanase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 27 kDa. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of the URA3-based XYN2 shuttle vectors in nonselective complex medium. These autoselective S. cerevisiae strains produced 1,200 and 160 nkat of beta-xylanase activity per ml under the control of the ADH2 and PGK1 promoters in rich medium, respectively. The recombinant enzyme showed highest activity at pH 6 and 60 degrees C and retained more than 90% of its activity after 60 min at 50 degrees C.
- 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.
- ItemMetabolomic alterations do not induce metabolic burden in the industrial yeast M2n[pBKD2-Pccbgl1]-C1 engineered by multiple δ-integration of a fungal β-glucosidase gene(Frontiers Media, 2019-11-28) Favaro, Lorenzo; Cagnin, Lorenzo; Corte, Laura; Roscini, Luca; De Pascale, Fabio; Treu, Laura; Campanaro, Stefano; Basaglia, Marina; Van Zyl, Willem H.; Casella, Sergio; Cardinali, GianluigiIn the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple d-integration of the b-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the d-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple d-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.
- ItemRational engineering of Saccharomyces cerevisiae towards improved tolerance to multiple inhibitors in lignocellulose fermentations(BMC (part of Springer Nature), 2021-08-28) Brandt, Bianca A.; Garcia‑Aparicio, Maria D. P.; Gorgens, Johann F.; Van Zyl, Willem H.Background: The fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable Saccharomyces cerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S. cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability. The strategy consisted of multiple sequential deltaintegrations of double-gene cassettes containing one gene conferring broad inhibitor tolerance (ARI1, PAD1 or TAL1) coupled with an inhibitor-specific gene (ADH6, FDH1 or ICT1). The performances of the transformants were compared with the parental strain in terms of biomass growth, ethanol yields and productivity, as well as detoxification capacities in a synthetic inhibitor cocktail, sugarcane bagasse hydrolysate as well as hardwood spent sulphite liquor. Results: The first and second round of delta-integrated transformants exhibited a trade-off between biomass and ethanol yield. Transformants showed increased inhibitor resistance phenotypes relative to parental controls specifically in fermentations with concentrated spent sulphite liquors at 40% and 80% v/v concentrations in 2% SC media. Unexpectedly, the xylose fermentation capacity of the transformants was reduced compared to the parental control, but certain combinations of genes had a minor impact (e.g. TAL1 + FDH1). The TAL1 + ICT1 combination negatively impacted on both biomass growth and ethanol yield, which could be linked to the ICT1 protein increasing transformant susceptibility to weak acids and temperature due to cell membrane changes. Conclusions: The integration of the selected genes was proven to increase tolerance to pretreatment inhibitors in synthetic or industrial hydrolysates, but they were limited to the fermentation of glucose. However, some gene combination sequences had a reduced impact on xylose conversion.
- ItemRaw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases(BioMed Central, 2013-11) Viktor, Marko J.; Rose, Shaunita H.; Van Zyl, Willem H.; Viljoen-Bloom, MarindaBackground: Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol. Results: The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l⁻¹ raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l⁻¹ (0.038 and 0.028 g l⁻¹ h⁻¹), respectively, after 10 days. With a substrate load of 200 g l⁻¹ raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l⁻¹ ethanol (0.58 g l⁻¹ h⁻¹) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l-1 ethanol (0.36 g l⁻¹ h⁻¹). Conclusions: In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l⁻¹ raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.
- ItemRole of cultivation media in the development of yeast strains for large scale industrial use(BioMed Central, 2005-11) Hahn-Hagerdal, Barbel; Karhumaa, Kaisa; Larsson, Christer U.; Gorwa-Grauslund, Marie; Gorgens, Johann; Van Zyl, Willem H.Abstract: The composition of cultivation media in relation to strain development for industrial application is reviewed. Heterologous protein production and pentose utilization by Saccharomyces cerevisiae are used to illustrate the influence of media composition at different stages of strain construction and strain development. The effects of complex, defined and industrial media are compared. Auxotrophic strains and strain stability are discussed. Media for heterologous protein production and for bulk bio-commodity production are summarized.