Molecular evolutionary insight of structural zinc atom in yeast xylitol dehydrogenases and its application in bioethanol production by lignocellulosic biomass.

Xylitol dehydrogenase (XDH) catalyzes the NAD+-dependent oxidization of xylitol into D-xylulose, and belongs to a zinc-dependent medium-chain dehydrogenase/reductase family. This protein family consists of enzymes with one or two zinc atoms per subunit, among which catalytic zinc is necessary for the activity. Among many XDHs from yeast and fungi, XDH from Pichia stipitis is one of the key enzymes for bioethanol production by lignocellulosic biomass, and possesses only a catalytic zinc atom. Despite its importance in bioindustry, a structural data of XDH has not yet been available, and little insight into the role of a second zinc atom in this protein family is known. We herein report the crystal structure of XDH from P. stipitis using a thermostabilized mutant. In the refined structure, a second zinc atom clearly coordinated with four artificially introduced cysteine ligands. Homologous mutations in XDH from Saccharomyces cerevisiae also stabilized and enhanced activity. The substitution of each of the four cysteine ligands with an aspartate in XDH from Schizosaccharomyces pombe contributed to the significantly better maintenance of activity and thermostability than their substitution with a serine, providing a novel hypothesis for how this zinc atom was eliminated.

Conditional dependence of enzyme cascade reaction efficiency on the inter-enzyme distance.

A dual-enzyme cascade, xylitol dehydrogenase and xylulose kinase, derived from the xylose metabolic pathway, was constructed on a three-dimensional DNA scaffold which exhibited a dynamic shape transition from an open state to a closed hexagonal prism. Evaluation of the cascade reaction efficiencies in the open and closed states revealed little to no inter-enzyme distance dependence, presumably due to the far larger catalytic constant of the downstream enzyme. The inter-enzyme distance was not the dominant factor for cascade efficiency when the kinetic parameters of the cascade enzymes were imbalanced with the highly efficient downstream enzyme.

Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes.

The catalytic enhancements of enzymes loaded on DNA nanostructures have been attributed to the characteristics provided by highly negative charges on the surface of the DNA scaffold, such as the modulation of the local pH near enzymes. In this study, two types of enzymes with optimal activity at pH 6 and 8 equally displayed significant catalytic enhancements on the DNA scaffold surface. By using a ratiometric pH indicator, a lower local pH shift of 0.8 was observed near the DNA scaffold surface. The postulated local pH change near the DNA scaffold surface is unlikely to play a general role in enhancing the activity of the scaffolded enzymes.

Valorization of apple pomace using bio-based technology for the production of xylitol and 2G ethanol.

Apple pomace was studied as a raw material for the production of xylitol and 2G ethanol, since this agroindustrial residue has a high concentration of carbohydrate macromolecules, but is still poorly studied for the production of fermentation bioproducts, such as polyols. The dry biomass was subjected to dilute-acid hydrolysis with H2SO4 to obtain the hemicellulosic hydrolysate, which was concentrated, detoxified and fermented. The hydrolyzate after characterization was submitted to submerged fermentations, which were carried out in Erlenmeyer flasks using, separately, the yeasts Candida guilliermondii and Kluyveromyces marxianus. High cellulose (32.62%) and hemicellulose (23.60%) contents were found in this biomass, and the chemical hydrolysis yielded appreciable quantities of fermentable sugars, especially xylose. Both yeasts were able to metabolize xylose, but Candida guilliermondii produced only xylitol (9.35 g L-1 in 96 h), while K. marxianus produced ethanol as the main product (10.47 g L-1 in 24 h) and xylitol as byproduct (9.10 g L-1 xylitol in 96 h). Maximum activities of xylose reductase and xylitol dehydrogenase were verified after 24 h of fermentation with C. guilliermondii (0.23 and 0.53 U/mgprot, respectively) and with K. marxianus (0.08 e 0.08 U/mgprot, respectively). Apple pomace has shown potential as a raw material for the fermentation process, and the development of a biotechnological platform for the integrated use of both the hemicellulosic and cellulosic fraction could add value to this residue and the apple production chain.

Cloning and functional characterization of xylitol dehydrogenase genes from Issatchenkia orientalis and Torulaspora delbrueckii.

Saccharomyces cerevisiae can obtain xylose utilization capacity via integration of heterogeneous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes into its metabolic pathway, and XYL2 which encodes the XDH plays an essential role in this process. Herein, we reported that two hypothetical XYL2 genes from the multistress-tolerant yeasts of Issatchenkia orientalis and Torulaspora delbrueckii were cloned, and they encoded two XDHs, IoXyl2p and TdXyl2p, respectively, with the activities for oxidation of xylitol to xylulose. Comparative studies demonstrated that IoXyl2p and TdXyl2p, like the SsXyl2p from Scheffersomyces stipitis, were probably localized to the cytoplasm and strictly dependent on NAD+ rather than NADP+ as the cofactor for catalyzing the oxidation reaction of xylitol. IoXyl2p had the highest specific activity, maximum velocity (Vmax), affinity to xylitol (Km), and catalytic efficiency (kcat/Km) among the three XDHs. The optimum temperature for oxidation of xylitol were at 45 °C by IoXyl2p and at 35 °C by TdXyl2p and SsXyl2p, and the optimum pH of IoXyl2p, TdXyl2p and SsXyl2p for oxidation of xylitol was 8.0, 8.5 and 7.5, respectively. Mg2+ promoted the activities of IoXyl2p and TdXyl2p, but slightly inhibited the activity of SsXyl2p. Most metal ions had much weaker inhibition effects on IoXyl2p and TdXyl2p than SsXyl2p. IoXyl2p displayed the strongest salt resistance among the three XDHs. To summarize, IoXyl2p from I. orientalis and TdXyl2p from T. delbrueckii characterized in this study are considered to be the attractive candidates for the construction of genetically engineered S. cerevisiae for efficiently fermentation of carbohydrate in lignocellulosic hydrolysate.

High-temperature ethanol production by a series of recombinant xylose-fermenting Kluyveromyces marxianus strains.

Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 and DMB13, that express xylose reductase (XR), NAD+- or protein-engineered NADP+-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK) from K. marxianus. These strains, together with previously reported strain DMB3-7, which expresses Scheffersomyces stipitis XR and NAD+-dependent XDH and Saccharomyces cerevisiae XK, were compared to evaluate enzymatic activities and ethanol productivities at 30 °C and 40 °C. Unlike the activities of xylose metabolic enzymes in DMB3-7, enzymatic activities of XR, XDH, and XK in both DMB5 and DMB13 hardly decreased even at 40 °C, suggesting that these enzymes from K. marxianus are highly thermostable. The most efficient glucose/xylose co-fermentation at 40 °C was found in DMB13; namely, DMB13 rapidly converted xylose to ethanol, especially after glucose depletion, and showed the highest ethanol yield (0.402 g/g). These findings support the view that alteration of coenzyme specificity of XDH expressed in K. marxianus will be efficacious for high-temperature ethanol production from mixed sugars containing xylose.

Comparison of two models of surface display of xylose reductase in the Saccharomyces cerevisiae cell wall.

In order to display xylose reductase at the surface of S. cerevisiae cells two different gene constructs have been prepared. In the first, xylose reductase gene GRE3 was fused with two parts of the CCW12 gene, the N-terminal one coding for the secretion signal sequence, and the C-terminal coding for the glycosylphosphatidylinositol anchoring signal. Transformed cells synthesized xylose reductase and incorporated it in the cell wall through the remnant of the glycosylphosphatidylinositol anchor. The other construct was prepared by fusing the GRE3 with the PIR4 gene coding for one of the proteins of the Pir-family containing the characteristic N-terminal repetitive sequence that anchors Pir proteins to ß-1,3-glucan. In this way xylose reductase was covalently attached to glucan through its N-terminus. For the expression of the constructs either the GAL1, or the PHO5 promoters have been used. Both strains displayed active xylose reductases and their enzyme properties were compared with the control enzyme bearing the secretion signal sequence but no anchoring signals, thus secreted into the medium. The enzyme displayed through the N-terminal fusion with PIR4 had higher affinity for xylose than the other construct, but they both expressed somewhat lower affinity than the control enzyme. Similarly, the Km values for NADPH of both immobilized enzymes were somewhat higher than the Km of the control XR. Both displayed enzymes, especially the one fused with Pir4, had higher thermal and pH stability than the control, while other enzymatic properties were not significantly impaired by surface immobilization.

Enhanced xylitol production: Expression of xylitol dehydrogenase from Gluconobacter oxydans and mixed culture of resting cell.

Xylitol has numerous applications in food and pharmaceutical industry, and it can be biosynthesized by microorganisms. In the present study, xdh gene, encoding xylitol dehydrogenase (XDH), was cloned from the genome of Gluconobacter oxydans CGMCC 1.49 and overexpressed in Escherichia coli BL21. Sequence analysis revealed that XDH has a TGXXGXXG NAD(H)-binding motif and a YXXXK active site motif, and belongs to the short-chain dehydrogenase/reductase family. And then, the enzymatic properties and kinetic parameter of purified recombinant XDH were investigated. Subsequently, transformations of xylitol from d-xylulose and d-arabitol, respectively, were studied through mixed culture of resting cells of G. oxydans wild-type strain and recombinant strain BL21-xdh. We obtained 28.80 g/L xylitol by mixed culture from 30 g/L d-xylulose in 28 h. The production was increased by more than three times as compared with that of wild-type strain. Furthermore, 25.10 g/L xylitol was produced by the mixed culture from 30 g/L d-arabitol in 30 h with a yield of 0.837 g/g, and the max volumetric productivity of 0.990 g/L h was obtained at 22 h. These contrast to the fact that wild-type strain G. oxydans only produced 8.10 g/L xylitol in 30 h with a yield of 0.270 g/g. To our knowledge, these values are the highest among the reported yields and productivity efficiencies of xylitol from d-arabitol with engineering strains.

Spatially Organized Enzymes Drive Cofactor-Coupled Cascade Reactions.

We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates' diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD(+) likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes.

Enhanced Xylitol Production by Mutant Kluyveromyces marxianus 36907-FMEL1 Due to Improved Xylose Reductase Activity.

A directed evolution and random mutagenesis were carried out with thermotolerant yeast Kluyveromyces marxianus ATCC 36907 for efficient xylitol production. The final selected strain, K. marxianus 36907-FMEL1, exhibited 120 and 39 % improvements of xylitol concentration and xylitol yield, respectively, as compared to the parental strain, K. marxianus ATCC 36907. According to enzymatic assays for xylose reductase (XR) activities, XR activity from K. marxianus 36907-FMEL1 was around twofold higher than that from the parental strain. Interestingly, the ratios of NADH-linked and NADPH-linked XR activities were highly changed from 1.92 to 1.30 when K. marxianus ATCC 36907 and K. marxianus 36907-FMEL1 were compared. As results of KmXYL1 genes sequencing, it was found that cysteine was substituted to tyrosine at position 36 after strain development which might cause enhanced XR activity from K. marxianus 36907-FMEL1.

Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii.

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.

Identification of a xylitol dehydrogenase gene from Kluyveromyces marxianus NBRC1777.

Xylitol dehydrogenase (XDH) (EC 1.1.1.9) is one of the key enzymes in the xylose fermentation pathway in yeast and fungi. A xylitol dehydrogenase gene (XYL2) encoding a XDH was cloned from Kluyveromyces marxianus NBRC 1777, and the in vivo function was validated by disruption and complementation analysis. The highest activity of KmXDH could be observed at pH 9.5 during 55°C. The values of k(cat)/K(m) indicate that KmXDH prefers NAD(+) to NADP(+) (k(cat)/K(m NAD)(+) 3681/min mM and k(cat)/K(m NADP)(+) 1361/min mM). The different coenzyme preference between KmXR and KmXDH caused an accumulation of NADH in the xylose utilization pathway. The redox imbalance may be one of the reasons to cause the poor xylose fermentation under oxygen-limited conditions in K. marxianus NBRC1777.

Co-immobilization of glucose oxidase and xylose dehydrogenase displayed whole cell on multiwalled carbon nanotube nanocomposite films modified electrode for simultaneous voltammetric detection of D-glucose and D-xylose.

In this paper, we first report the construction of Nafion/glucose oxidase (GOD)/xylose dehydrogenase displayed bacteria (XDH-bacteria)/multiwalled carbon nanotubes (MWNTs) modified electrode for simultaneous voltammetric determination of D-glucose and D-xylose. The optimal conditions for the immobilized enzymes were established. Both enzymes retained their good stability and activities. In the mixture solution of D-glucose and D-xylose containing coenzyme NAD⁺ (the oxidized form of nicotinamide adenine dinucleotide), the Nafion/GOD/XDH-bacteria/MWNTs modified electrode exhibited quasi-reversible oxidation-reduction peak at -0.5 V (vs. saturated calomel electrode, SCE) originating from the catalytic oxidation of D-glucose, and oxidation peak at +0.55 V(vs. SCE) responding to the oxidation of NADH (the reduced form of nicotinamide adenine dinucleotide) by the carbon nanotubes, where NADH is the resultant product of coenzyme NAD⁺ involved in the catalysis of D-xylose by XDH-displayed bacteria. For the proposed biosensor, cathodic peak current at -0.5 V was linear with the concentration of D-glucose within the range of 0.25-6 mM with a low detection limit of 0.1 mM D-glucose (S/N=3), and the anodic peak current at +0.55 V was linear with the concentration of d-xylose in the range of 0.25∼4 mM with a low detection limit of 0.1 mM D-xylose (S/N=3). Further, D-xylose and D-glucose did not interfere with each other. 300-fold excess saccharides including D-maltose, D-galactose, D-mannose, D-sucrose, D-fructose, D-cellobiose, and 60-fold excess L-arabinose, and common interfering substances (100-fold excess ascorbic acid, dopamine, uric acid) as well as 300-fold excess D-xylitol did not affect the detection of D-glucose and D-xylose (both 1 mM). Therefore, the proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective, which holds great potential in real applications.

Cloning and characterization of a novel NAD+ -dependent xylitol dehydrogenase from Gluconobacter oxydans CGMCC 1. 637.

OBJECTIVE: To clone the xylitol dehydrogenase gene from Gluconobacter oxydans CGMCC 1.637, to characterize enigmatic properties of xylitol dehydrogenase and to investigate the induction abilities of various carbon sources on the oxidative activity of xylitol dehydrogenase and the effect of various carbon sources on the bioconversion of d-xylulose to xylitol in G. oxydans CGMCC 1.637. METHODS: Touch-down polymerase chain reaction (PCR) was applied to clone the xylitol dehydrogenase gene from chromosomal DNA of G. oxydans CGMCC 1.637. RESULTS: The 798-bp open reading frame of xylitol dehydrogenase encoded a protein of 265 amino acids, with the molecular mass of 27.95 kDa. Sequence analysis of the putative protein revealed it to be a member of short-chain dehydrogenase/reductase family. Xylitol dehydrogenase showed oxidative activity with xylitol and sorbitol and no activity with other polyols, such as d-arabitol. K(m) and V(max) with xylitol was 78.97 mmol/L and 40.17 U/mg, respectively. The highest oxidative activity of xylitol dehydrogenase for xylitol was only 23.27 U/mg under optimum conditions (pH 10.0, 35 degrees C). However, the activity of its reverse reaction, d-xylulose reduction, reached 255.55 U/mg under optimum conditions (pH 6.0, 30 degrees C), 10-times higher than that of xylitol oxidation. Oxidative activity of xylitol dehydrogenase was induced when G. oxydans CGMCC 1.637 was cultivated on d-sorbitol. D-arabitol, which supported a high cell growth, inhibited the oxidative activity of xylitol dehydrogenase and the bioconversion ability of G. oxydans CGMCC 1.637. CONCLUSIONS: The obtained gene from G. oxydans CGMCC 1.637 was a novel gene encoding xylitol dehydrogenase. Oxidative activity of xylitol dehydrogenase in G. oxydans CGMCC 1.637 and the bioconversion ability of G. oxydans CGMCC 1.637 after grown on d-arabitol were inhibited, which provided a valuable clue for further study to increase xylitol yield from d-arabitol.

CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes.

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30°C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4±15.7U/g(cells)) > freezing-thawing (54.3 ± 1.9U/g(cells))>Triton X-100 (23.5 ± 0.0U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 ± 0.1 U/g(cells).

[Homology modeling and molecular docking of xylitol dehydrogenase from Aspergillus Oryzae].

OBJECTIVE: We investigated the structure model and function of xylitol dehydrogenase from Aspergillus oryzae. METHODS: Xylitol dehydrogenase (XDH) gene from Aspergillus oryzae was cloned and sequenced. We constructed four tertiary structure models of XDH by homology modeling with Swiss-MODEL and Modeller and obtained the best quality model by evaluation of PROCHECK and Prosa2003. The dockings of NAD+, Zn2+ and xylitol with XDH were performed by Molsoft program. RESULTS: Structure analysis suggested that XDH was a member of medium-chain dehydrogenase/reductase (MDR) family. This was supported by the presence of the zinc-containing alcohol dehydrogenase signature and a typical alcohol dehydrogenase Rossmann fold pattern composed by NAD+ binding domain present in MDR superfamily. The molecular docking indicated that amino acid residues Asp206, Arg211, Ser255, Ser301 and Arg303 in XDH binding domain had hydrogen bonding with NAD+, His72 and Glu73 in catalytic domain had hydrogen bonding with Zn2+, Ile46, Ile349, Lys350 and Thr351 in catalytic domain had hydrogen bonding with xylitol. CONCLUSION: These key amino acid residues might play a vital role in the XDH catalytic reaction and can instruct the further directed modification of XDH.

Covalent immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase onto modified silica nanoparticles.

Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to give different functional groups, the silica nanoparticle derivatized with epoxy groups showed the highest immobilization efficiency (92%). The thermostability of ReXDH was improved more than tenfold by immobilization on epoxy-silica nanoparticles; the t(1/2) of the ReXDH was enhanced from 120 min to 1,410 min at 40 °C and from 30 min to 450 min at 50 °C. The K(m) of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into L-xylulose. A∼71% conversion and a productivity of 10.7 g h(-1)l(-1) were achieved when the immobilized ReXDH was employed to catalyze the biotransformation of xylitol to L-xylulose, a sugar that has been used in medicine and in the diagnosis of hepatitis. These results suggest that immobilization of ReXDH onto epoxy-silica nanoparticles has potential industrial application in rare sugar production.

Cloning and characterization of thermotolerant xylitol dehydrogenases from yeast Pichia angusta.

Pichia angusta (syn. Hansenula polymorpha) represents one of the rare yeast that can grow and ferment both xylose and glucose at higher temperature (50°C). However, little is known about the enzymes involved in xylose utilization from this species. Previous studies indicated the presence of one xylose reductase and two xylitol dehydrogenase genes in P. angusta. In this study, we have expressed both xylitol dehydrogenases (PaXdh1p and PaXdh2p) in Escherichia coli and purified them as 6X-Histidine-tagged proteins. Biochemical characterization of the recombinant proteins reveals that both PaXdh1p and PaXdh2p are thermotolerant enzymes. PaXdh2p contains a catalytic and a structural Zn atom. However, the structural Zn atom is not present in PaXdh1p. Both enzymes also differ in their affinity for the substrate as well as in the catalytic efficiency. Through mutagenesis and modeling approaches, we have also identified residues important for catalysis and substrate binding.

Cloning and characterization of a thermostable xylitol dehydrogenase from Rhizobium etli CFN42.

An NAD(+)-dependent xylitol dehydrogenase from Rhizobium etli CFN42 (ReXDH) was cloned and overexpressed in Escherichia coli. The DNA sequence analysis revealed an open reading frame of 1,044 bp, capable of encoding a polypeptide of 347 amino acid residues with a calculated molecular mass of 35,858 Da. The ReXDH protein was purified as an active soluble form using GST affinity chromatography. The molecular mass of the purified enzyme was estimated to be approximately 34 kDa by sodium dodecyl sulfate-polyacrylamide gel and approximately 135 kDa with gel filtration chromatography, suggesting that the enzyme is a homotetramer. Among various polyols, xylitol was the preferred substrate of ReXDH with a K (m) = 17.9 mM and k(cat) /K (m) = 0.5 mM(-1) s(-1) for xylitol. The enzyme had an optimal pH and temperature of 9.5 and 70 degrees C, respectively. Heat inactivation studies revealed a half life of the ReXDH at 40 degrees C of 120 min and a half denaturation temperature (T (1/2)) of 53.1 degrees C. ReXDH showed the highest optimum temperature and thermal stability among the known XDHs. Homology modeling and sequence analysis of ReXDH shed light on the factors contributing to the high thermostability of ReXDH. Although XDHs have been characterized from several other sources, ReXDH is distinguished from other XDHs by its high thermostability.

Cloning, heterologous expression, and characterization of the xylitol and L-arabitol dehydrogenase genes, Texdh and Telad, from the thermophilic fungus Talaromyces emersonii.

The genes encoding xylitol dehydrogenase (Texdh) and L: -arabitol dehydrogenase (Telad) are involved in the fungal pentose pathway and were isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli, and the products purified to homogeneity. TeXDH showed activity toward xylitol and D: -sorbitol. TeLAD was active with L: -arabitol, xylitol, and D: -sorbitol. Phylogenetic analysis showed TeLAD has evolved from D: -sorbitol dehydrogenase as a result of environmental adaptation. Substrate specificity studies indicate that TeXDH is likely to have evolved from the more broadly acting TeLAD. Texdh and Telad expression was inducible by the same carbon sources responsible for induction of genes involved in biomass degradation, suggesting for the first time a coordinated regulatory control mechanism for expression of genes encoding extracellular hydrolases and intracellular metabolic genes in the pentose utilization pathways of T. emersonii. These data also suggest that TeXDH and TeLAD may be valuable in the production of xylitol, L: -arabitol, and ethanol from renewable resources rich in pentose sugars.