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Bernd Nidetzky - One of the best experts on this subject based on the ideXlab platform.
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systematic strain construction and process development xylitol production by saccharomyces cerevisiae expressing candida tenuis Xylose Reductase in wild type or mutant form
Bioresource Technology, 2015Co-Authors: S M Pratter, Thomas Eixelsberger, Bernd NidetzkyAbstract:A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis Xylose Reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited Xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) Xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for Xylose reduction and point out parameters to improve "green" xylitol production.
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fermentation of mixed glucose Xylose substrates by engineered strains of saccharomyces cerevisiae role of the coenzyme specificity of Xylose Reductase and effect of glucose on Xylose utilization
Microbial Cell Factories, 2010Co-Authors: Stefan Krahulec, Barbara Petschacher, Karin Longus, Michael Wallner, Mario Klimacek, Bernd NidetzkyAbstract:Background In spite of the substantial metabolic engineering effort previously devoted to the development of Saccharomyces cerevisiae strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, Xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for Xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from Xylose. The previously reported yeast strain BP10001, which expresses heterologous Xylose Reductase from Candida tenuis in mutated (NADH-preferring) form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L Xylose as sole source of carbon, BP10001 displayed a low specific uptake rate qXylose (g Xylose/g dry cell weight/h) of 0.08. The study presented herein was performed with the aim of analysing (external) factors that limit qXylose of BP10001 under Xylose-only and mixed glucose-Xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for Xylose reduction during co-fermentation of glucose and Xylose.
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the catalytic mechanism of nadh dependent reduction of 9 10 phenanthrenequinone by candida tenuis Xylose Reductase reveals plasticity in an aldo keto Reductase active site
Biochemical Journal, 2009Co-Authors: Simone L Pival, Mario Klimacek, Bernd NidetzkyAbstract:Despite their widely varying physiological functions in carbonyl metabolism, AKR2B5 ( Candida tenuis Xylose Reductase) and many related enzymes of the aldo-keto Reductase protein superfamily utilise PQ (9,10-phenanthrenequinone) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr 51 , Lys 80 and His 113 ) of AKR2B5 in the conversion of the reactive α-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr 51 , Lys 80 and His 113 individually replaced by alanine) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 10 3 –10 4 -fold decrease in the rate constant for hydride transfer from NADH to PQ, whose value in the wild-type enzyme was determined as ∼8×10 2 s −1 . The data presented support an enzymic mechanism in which a catalytic proton bridge from the protonated side chain of Lys 80 (p K =8.6±0.1) to the carbonyl group adjacent to the hydride acceptor carbonyl facilitates the chemical reaction step. His 113 contributes to positioning of the PQ substrate for catalysis. Contrasting its role as catalytic general acid for conversion of the physiological substrate Xylose, Tyr 51 controls release of the hydroquinone product. The proposed chemistry of AKR2B5 action involves delivery of both hydrogens required for reduction of the α-dicarbonyl substrate to the carbonyl group undergoing (stereoselective) transformation. Hydride transfer from NADH probably precedes the transfer of a proton from Tyr 51 whose p K of 7.3±0.3 in the NAD + -bound enzyme appears suitable for protonation of a hydroquinone anion (p K =8.8). These results show that the mechanism of AKR2B5 is unusually plastic in the exploitation of the active-site residues, for the catalytic assistance provided to carbonyl group reduction in α-dicarbonyls differs from that utilized in the conversion of Xylose.
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whole cell bioreduction of aromatic α keto esters using candida tenuis Xylose Reductase and candida boidinii formate dehydrogenase co expressed in escherichia coli
Microbial Cell Factories, 2008Co-Authors: Regina Kratzer, Matej Pukl, Sigrid Egger, Bernd NidetzkyAbstract:Background: Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful Reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that Reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP + or NAD + dependent dehydrogenase that is co-expressed. Candida tenuis Xylose Reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain coexpressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR. Results: Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular CtXR and CbFDH activity per gram of dry E. coli cell mass (gCDW). The maximum conversion rate (r S ) for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphatepermeabilized cells was similar (2 mmol/gCDWh), suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH in the presence of the α-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 g CDW /L), we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e.e.) of 97.2% in a yield of 82%. By increasing the substrate concentration to 500 mM, the e.e. could be enhanced to ≅100%, however, at the cost of a 3-fold decreased yield. A recombinant strain of S. cerevisiae converted 100 mM substrate to 45 mM ethyl R-4-cyanomandelate with an e.e. of ≥ 99.9%. Modifications to the recombinant E. coli (cell permeabilisation; addition of exogenous NAD+) and addition of a water immiscible solvent (e.g. hexane or 1-butyl-3-methylimidazolium hexafluorophosphate) were not useful. To enhance the overall capacity for NADH regeneration in the system, we supplemented the original biocatalyst
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whole cell bioreduction of aromatic α keto esters using candida tenuis Xylose Reductase and candida boidinii formate dehydrogenase co expressed in escherichia coli
Microbial Cell Factories, 2008Co-Authors: Regina Kratzer, Matej Pukl, Sigrid Egger, Bernd NidetzkyAbstract:Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful Reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that Reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP+ or NAD+ dependent dehydrogenase that is co-expressed. Candida tenuis Xylose Reductase (Ct XR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing Ct XR and formate dehydrogenase from Candida boidinii (Cb FDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing Ct XR. Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular Ct XR and Cb FDH activity per gram of dry E. coli cell mass (gCDW). The maximum conversion rate (rS) for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/gCDWh), suggesting that the activity of Cb FDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of Ct XR and Cb FDH in the presence of the α-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 gCDW/L), we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e.e.) of 97.2% in a yield of 82%. By increasing the substrate concentration to 500 mM, the e.e. could be enhanced to ≅100%, however, at the cost of a 3-fold decreased yield. A recombinant strain of S. cerevisiae converted 100 mM substrate to 45 mM ethyl R-4-cyanomandelate with an e.e. of ≥ 99.9%. Modifications to the recombinant E. coli (cell permeabilisation; addition of exogenous NAD+) and addition of a water immiscible solvent (e.g. hexane or 1-butyl-3-methylimidazolium hexafluorophosphate) were not useful. To enhance the overall capacity for NADH regeneration in the system, we supplemented the original biocatalyst after permeabilisation with also permeabilised E. coli cells that expressed solely Cb FDH (410 U/gCDW). The positive effect on yield (18% → 62%; 100 mM substrate) caused by a change in the ratio of FDH to XR activity from 2 to 20 was invalidated by a corresponding loss in product enantiomeric purity from 86% to only 71%. A whole-cell system based on E. coli co-expressing Ct XR and Cb FDH is a powerful and surprisingly robust biocatalyst for the synthesis of ethyl R-4-cyanomandelate in high optical purity and yield. A clear requirement for further optimization of the specific productivity of the biocatalyst is to remove the kinetic bottleneck of NADH regeneration through enhancement (≥ 10-fold) of the intracellular level of FDH activity.
Barbel Hahnhagerdal - One of the best experts on this subject based on the ideXlab platform.
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Xylose Reductase from pichia stipitis with altered coenzyme preference improves ethanolic Xylose fermentation by recombinant saccharomyces cerevisiae
Biotechnology for Biofuels, 2009Co-Authors: Oskar Bengtsson, Barbel Hahnhagerdal, Mariefrancoise GorwagrauslundAbstract:Background Xylose Reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for Xylose utilization. The availability of NAD+ for XDH is limited during anaerobic Xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference.
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comparing the Xylose Reductase xylitol dehydrogenase and Xylose isomerase pathways in arabinose and Xylose fermenting saccharomyces cerevisiae strains
Biotechnology for Biofuels, 2008Co-Authors: Maurizio Bettiga, Barbel Hahnhagerdal, Mariefrancoise GorwagrauslundAbstract:Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and Xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and Xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different Xylose utilisation pathways: the Xylose Reductase/xylitol dehydrogenase and Xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and Xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and Xylose.
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comparison of the Xylose Reductase xylitol dehydrogenase and the Xylose isomerase pathways for Xylose fermentation by recombinant saccharomyces cerevisiae
Microbial Cell Factories, 2007Co-Authors: Kaisa Karhumaa, Barbel Hahnhagerdal, Rosa Garcia Sanchez, Mariefrancoise GorwagrauslundAbstract:Two heterologous pathways have been used to construct recombinant Xylose-fermenting Saccharomyces cerevisiae strains: i) the Xylose Reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the Xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve Xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose Reductase gene GRE3). The two isogenic strains and the industrial Xylose-fermenting strain TMB 3400 were studied regarding their Xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. In defined mineral medium, the Xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Despite by-product formation, the XR-XDH Xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed.
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high activity of Xylose Reductase and xylitol dehydrogenase improves Xylose fermentation by recombinant saccharomyces cerevisiae
Applied Microbiology and Biotechnology, 2007Co-Authors: Kaisa Karhumaa, Barbel Hahnhagerdal, Romain Fromanger, Mariefrancoise GorwagrauslundAbstract:Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high Xylose Reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose Reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on Xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the Xylose consumption rate. The results indicate that ethanolic Xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which Xylose is introduced in the central metabolism.
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xylulokinase overexpression in two strains of saccharomyces cerevisiae also expressing Xylose Reductase and xylitol dehydrogenase and its effect on fermentation of Xylose and lignocellulosic hydrolysate
Applied and Environmental Microbiology, 2001Co-Authors: Björn Johansson, Camilla Christensson, Timothy John Hobley, Barbel HahnhagerdalAbstract:Fermentation of the pentose sugar Xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize Xylose only when expressing the heterologous genes XYL1 (Xylose Reductase) and XYL2 (xylitol dehydrogenase). Xylose Reductase and xylitol dehydrogenase convert Xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant Xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total Xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more Xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed Xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient Xylose-fermenting recombinant strain of S. cerevisiae.
Mariefrancoise Gorwagrauslund - One of the best experts on this subject based on the ideXlab platform.
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Xylose Reductase from pichia stipitis with altered coenzyme preference improves ethanolic Xylose fermentation by recombinant saccharomyces cerevisiae
Biotechnology for Biofuels, 2009Co-Authors: Oskar Bengtsson, Barbel Hahnhagerdal, Mariefrancoise GorwagrauslundAbstract:Background Xylose Reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for Xylose utilization. The availability of NAD+ for XDH is limited during anaerobic Xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference.
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comparing the Xylose Reductase xylitol dehydrogenase and Xylose isomerase pathways in arabinose and Xylose fermenting saccharomyces cerevisiae strains
Biotechnology for Biofuels, 2008Co-Authors: Maurizio Bettiga, Barbel Hahnhagerdal, Mariefrancoise GorwagrauslundAbstract:Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and Xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and Xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different Xylose utilisation pathways: the Xylose Reductase/xylitol dehydrogenase and Xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and Xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and Xylose.
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comparison of the Xylose Reductase xylitol dehydrogenase and the Xylose isomerase pathways for Xylose fermentation by recombinant saccharomyces cerevisiae
Microbial Cell Factories, 2007Co-Authors: Kaisa Karhumaa, Barbel Hahnhagerdal, Rosa Garcia Sanchez, Mariefrancoise GorwagrauslundAbstract:Two heterologous pathways have been used to construct recombinant Xylose-fermenting Saccharomyces cerevisiae strains: i) the Xylose Reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the Xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve Xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose Reductase gene GRE3). The two isogenic strains and the industrial Xylose-fermenting strain TMB 3400 were studied regarding their Xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. In defined mineral medium, the Xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Despite by-product formation, the XR-XDH Xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed.
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high activity of Xylose Reductase and xylitol dehydrogenase improves Xylose fermentation by recombinant saccharomyces cerevisiae
Applied Microbiology and Biotechnology, 2007Co-Authors: Kaisa Karhumaa, Barbel Hahnhagerdal, Romain Fromanger, Mariefrancoise GorwagrauslundAbstract:Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high Xylose Reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose Reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on Xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the Xylose consumption rate. The results indicate that ethanolic Xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which Xylose is introduced in the central metabolism.
David K. Wilson - One of the best experts on this subject based on the ideXlab platform.
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catalytic mechanism and substrate selectivity of aldo keto Reductases insights from structure function studies of candida tenuis Xylose Reductase
Iubmb Life, 2006Co-Authors: Regina Kratzer, David K. Wilson, Bernd NidetzkyAbstract:Aldo-keto Reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)-dependent oxidoReductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure-function relationship studies for Xylose Reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the epsilon-NH3+ group of Lys is a key source of catalytic power of Xylose Reductase. A molecular-level analysis of the substrate binding pocket of Xylose Reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel Reductases with improved specificities for biocatalytic applications.
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fine tuning of coenzyme specificity in family 2 aldo keto Reductases revealed by crystal structures of the lys 274 arg mutant of candida tenuis Xylose Reductase akr2b5 bound to nad and nadp
FEBS Letters, 2005Co-Authors: Barbara Petschacher, David K. Wilson, Stefan Leitgeb, Bernd NidetzkyAbstract:Aldo-keto Reductases of family 2 employ single site replacement Lys fi Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys- 274 fi Arg mutant of Candida tenuis Xylose Reductase (AKR2B5) bound to NAD + and NADP + were determined at a resolution of 2.4 and 2.3 A ˚ , respectively. Due to steric conflicts in the NADP + -bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2 0 -phosphate and 3 0 -hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P) + in the wild-type remains partly disordered in the NADP + -bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type. � 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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the coenzyme specificity of candida tenuis Xylose Reductase akr2b5 explored by site directed mutagenesis and x ray crystallography
Biochemical Journal, 2005Co-Authors: Barbara Petschacher, Kathryn L. Kavanagh, David K. Wilson, Stefan Leitgeb, Bernd NidetzkyAbstract:CtXR (Xylose Reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-Xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274-->Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R-N276D were characterized by steady-state kinetic analysis of enzymic D-Xylose reductions with NADH and NADPH at 25 degrees C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked Xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 A resolution.
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structure of Xylose Reductase bound to nad and the basis for single and dual co substrate specificity in family 2 aldo keto Reductases
Biochemical Journal, 2003Co-Authors: Kathryn L. Kavanagh, Mario Klimacek, Bernd Nidetzky, David K. WilsonAbstract:The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose Reductase (XR; AKR2B5) is an unusual member of aldo-keto Reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting Xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto Reductases. The dual specificity found in XR is also an important consideration in designing a high-flux Xylose metabolic pathway, which may be improved with an enzyme specific for NADH.
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The Structure of Apo and Holo Forms of Xylose Reductase, a Dimeric Aldo-Keto Reductase from Candida tenuis†,‡
Biochemistry, 2002Co-Authors: Kathryn L. Kavanagh, Mario Klimacek, Bernd Nidetzky, David K. WilsonAbstract:Xylose Reductase is a homodimeric oxidoReductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto Reductase superfamily of proteins. It catalyzes the first step in the assimilation of Xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain Xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a Xylose Reductase, a dimeric aldo-keto Reductase or a family 2 aldo-keto Reductase. The structures of the Candida tenuis Xylose Reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments wi...
Barbara Petschacher - One of the best experts on this subject based on the ideXlab platform.
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fermentation of mixed glucose Xylose substrates by engineered strains of saccharomyces cerevisiae role of the coenzyme specificity of Xylose Reductase and effect of glucose on Xylose utilization
Microbial Cell Factories, 2010Co-Authors: Stefan Krahulec, Barbara Petschacher, Karin Longus, Michael Wallner, Mario Klimacek, Bernd NidetzkyAbstract:Background In spite of the substantial metabolic engineering effort previously devoted to the development of Saccharomyces cerevisiae strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, Xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for Xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from Xylose. The previously reported yeast strain BP10001, which expresses heterologous Xylose Reductase from Candida tenuis in mutated (NADH-preferring) form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L Xylose as sole source of carbon, BP10001 displayed a low specific uptake rate qXylose (g Xylose/g dry cell weight/h) of 0.08. The study presented herein was performed with the aim of analysing (external) factors that limit qXylose of BP10001 under Xylose-only and mixed glucose-Xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for Xylose reduction during co-fermentation of glucose and Xylose.
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altering the coenzyme preference of Xylose Reductase to favor utilization of nadh enhances ethanol yield from Xylose in a metabolically engineered strain of saccharomyces cerevisiae
Microbial Cell Factories, 2008Co-Authors: Barbara Petschacher, Bernd NidetzkyAbstract:Metabolic engineering of Saccharomyces cerevisiae for Xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of Xylose Reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of Xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on Xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274→Arg Asn276→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of Xylose by NADH. This work was carried out to assess physiological consequences in Xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity. An isogenic pair of yeast strains was derived from S. cerevisiae Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for C. tenuis XR in wild-type or double mutant form, XDH from Galactocandida mastotermitis, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of Xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The Xylose uptake rate per gram of cell dry weight was identical (0.07 ± 0.02 h-1) in both strains. Integration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of Xylose results in notably improved fermentation capabilities of recombinant S. cerevisiae.
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engineering candida tenuis Xylose Reductase for improved utilization of nadh antagonistic effects of multiple side chain replacements and performance of site directed mutants under simulated in vivo conditions
Applied and Environmental Microbiology, 2005Co-Authors: Barbara Petschacher, Bernd NidetzkyAbstract:Six single- and multiple-site variants of Candida tenuis Xylose Reductase that were engineered to have side chain replacements in the coenzyme 2′-phosphate binding pocket were tested for NADPH versus NADH selectivity (Rsel) in the presence of physiological reactant concentrations. The experimental Rsel values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274→Arg and Arg-280→His substitutions, which individually improved wild-type Rsel 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.
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fine tuning of coenzyme specificity in family 2 aldo keto Reductases revealed by crystal structures of the lys 274 arg mutant of candida tenuis Xylose Reductase akr2b5 bound to nad and nadp
FEBS Letters, 2005Co-Authors: Barbara Petschacher, David K. Wilson, Stefan Leitgeb, Bernd NidetzkyAbstract:Aldo-keto Reductases of family 2 employ single site replacement Lys fi Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys- 274 fi Arg mutant of Candida tenuis Xylose Reductase (AKR2B5) bound to NAD + and NADP + were determined at a resolution of 2.4 and 2.3 A ˚ , respectively. Due to steric conflicts in the NADP + -bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2 0 -phosphate and 3 0 -hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P) + in the wild-type remains partly disordered in the NADP + -bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type. � 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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the coenzyme specificity of candida tenuis Xylose Reductase akr2b5 explored by site directed mutagenesis and x ray crystallography
Biochemical Journal, 2005Co-Authors: Barbara Petschacher, Kathryn L. Kavanagh, David K. Wilson, Stefan Leitgeb, Bernd NidetzkyAbstract:CtXR (Xylose Reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-Xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2'-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274-->Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R-N276D were characterized by steady-state kinetic analysis of enzymic D-Xylose reductions with NADH and NADPH at 25 degrees C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked Xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 A resolution.
