Heterologous expression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in Aspergillus spp. and characterization of the recombinant enzyme

Pyranose dehydrogenase (PDH) is a flavin-dependant sugar oxidoreductase found in the family Agaricaceae, basidiomycetes that degrade lignocellulose-rich forest litter, and is catalytically related to the fungal enzymes pyranose 2-oxidase and cellobiose dehydrogenase. It has broad substrate specificity and displays similar activities with most sugar constituents of lignocellulose including disaccharides and oligosaccharides, a number of (substituted) quinones, and metal ions are suitable electron acceptors rather than molecular oxygen. In contrast to pyranose 2-oxidase and cellobiose dehydrogenase, which oxidize regioselectively at C-2 and C-1, respectively, PDH is capable of oxidation on C-1 to C-4 as well as double oxidations, depending on the nature of the substrate. This makes it a very interesting enzyme for biocatalytic applications, as many of the reaction products are otherwise unaccessible by chemical or enzymatic means. PDH was characterized in detail in a limited number of fungi, and the first encoding genes were isolated only recently. We report here, for the first time, the heterologous expression of one of these genes, encoding the major PDH protein in Agaricus meleagris, in the filamentous fungi Aspergillus nidulans, and Aspergillus niger.     

Screening of basidiomycete fungi for the quinone-dependent sugar C-2/C-3 oxidoreductase, pyranose dehydrogenase, and properties of the enzyme from Macrolepiota rhacodes

Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O(2) as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml(-1) in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K(m)=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.     

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe3? (pathway 1) and reduction of ferric ions to Fe2? reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed.     

Structural requirements for binding to the sugar-transport system of the human erythrocyte

The structural requirements for binding to the glucose/sorbose-transport system in the human erythrocyte were explored by measuring the inhibition constants, K(i), for specifically substituted analogues of d-glucose when l-sorbose was the penetrating sugar. Derivatives in which a hydroxyl group in the d-gluco configuration was inverted, or replaced by a hydrogen atom, at C-1, C-2, C-3, C-4 or C-6 of the d-glucose molecule, all bound to the carrier, confirming that no single hydroxyl group is essential for binding to the carrier. The binding and transport of 1-deoxy-d-glucose confirmed that the sugars bind in the pyranose form. The relative inhibition constants of d-glucose and its deoxy, epimeric and fluorinated analogues are consistent with the combination of beta-d-glucopyranose with the carrier by hydrogen bonds at C-1, C-3, probably C-4, and possibly C-6 of the sugar. Both polar and non-polar substituents at C-6 enhance the affinity of d-glucose derivatives relative to d-xylose, and d-galactose derivatives relative to l-arabinose, and it is suggested that the carrier region around C-6 of the sugar may contain both hydrophobic and polar binding groups. The spatial requirements at C-1, C-2, C-3, C-4 and C-6 were explored by comparing the relative binding of d-glucose and its halogeno and O-alkyl substituents. The carrier protein closely approaches the sugar except at C-3 in the d-gluco configuration, C-4 and C-6. d-Glucal was a good inhibitor, showing that a strict chair form is not essential for binding. 3-O-(2',3'-Epoxypropyl)-d-glucose, a potential substrate-directed alkylating agent, bound to the carrier, but did not inactivate it.     

Purification, characterization, and molecular cloning of a pyranose oxidase from the fruit body of the basidiomycete, Tricholoma matsutake

A new H(2)O(2)-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H(2)O(2) and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V(max), K(m), and k(cat) for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50 degrees C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.     

Triazole and Imidazole Reversed Nucleosides of L-Idose and D-Glucose

Abstract

6-azido-6-deoxy-gluco-(galacto)pyranose and 5-azido-5-deoxy-glucofuranose derivatives were used to obtain reversed nucleoside analogues with either the 5-aminoimidazole-4-carboxamide or 5-amino-1,2,3-triazol-4-carboxamide groups attached, through the N-1 site, to the C-6′ (C-5′) site of the sugar. When deprotected some of these compounds cyclised spontaneously to form a bond between the exocylic nitrogen and the anomeric carbon of the sugar.     

Pyranose 2-dehydrogenase, a novel sugar oxidoreductase from the basidiomycete fungus Agaricus bisporus

A novel C-2-specific sugar oxidoreductase, tentatively designated as pyranose 2-dehydrogenase, was purified 68-fold to apparent homogeneity (16.4 U/mg protein) from the mycelia of Agaricus bisporus, which expressed maximum activity of the enzyme during idiophasic growth in liquid media. Using 1,4-benzoquinone as an electron acceptor, pyranose 2-dehydrogenase oxidized d-glucose to d-arabino-2-hexosulose (2-dehydroglucose, 2-ketoglucose), which was identified spectroscopically through its N,N-diphenylhydrazone. The enzyme is highly nonspecific. d-,l-Arabinose, d-ribose, d-xylose, d-galactose, and several oligosaccharides and glycopyranosides were all converted to the corresponding 2-aldoketoses (aldosuloses) as indicated by TLC. d-Glucono-1,5-lactone, d-arabino-2-hexosulose, and l-sorbose were also oxidized at significant rates. UV/VIS spectrum of the native enzyme (λ_max 274, 362, and 465 nm) was consistent with a flavin prosthetic group. In contrast to oligomeric intracellular pyranose 2-oxidase (EC 1.1.3.10), pyranose 2-dehydrogenase is a monomeric glycoprotein (pI 4.2) incapable of reducing O₂ to H₂O₂ (> 5 × 10⁴-fold lower rate using a standard pyranose oxidase assay); pyranose 2-dehydrogenase is actively secreted into the extracellular fluid (up to 0.5 U/ml culture filtrate). The dehydrogenase has a native molecular mass of ∼79 kDa as determined by gel filtration; its subunit molecular mass is ∼75 kDa as estimated by SDS-PAGE. Two pH optima of the enzyme were found, one alkaline at pH 9 (phosphate buffer) and the other acidic at pH 4 (acetate buffer). Ag⁺, Hg²⁺, Cu²⁺, and CN^– (10 mM) were inhibitory, while 50 mM acetate had an activating effect. Received: 19 August 1996 / Accepted: 21 November 1996     

Characterization of pyranose dehydrogenase from Agaricus meleagris and its application in the C-2 specific conversion of D-galactose

Pyranose dehydrogenase (PDH) of the mushroom Agaricus meleagris was purified from mycelial culture media to substantial homogeneity using ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric polypeptide with a molecular mass of 66,547Da as determined by matrix-assisted laser desorption/ionisation mass spectrometry containing approximately 7% carbohydrate and covalently bound flavin adenine dinucleotide. The enzyme exhibited a broad sugar substrate tolerance, oxidizing different aldopyranoses to the corresponding C-2 or C-2,3 (di)dehydro sugars. Preferred electron donors with the highest k(cat)/K(m) values were major sugar constituents of cellulose and hemicellulose, namely d-glucose, D-galactose, l-arabinose, D-xylose and cellobiose. This indicates a possible physiological role of the enzyme in lignocellulose breakdown. PDH showed no detectable activity with oxygen, and its reactivity towards electron acceptors was limited to various substituted benzoquinones and complexed metal ions, with the ferricenium ion and the benzoquinone imine 2,6-dichloroindophenole displaying the highest k(cat)/K(m). The enzyme catalyzed in up to 95% yields the regiospecific conversion of D-galactose to 2-dehydro-D-galactose, an intermediate in a possible biotechnologically interesting process for redox isomerization of D-galactose to the prebiotic sugar D-tagatose.     

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.     

Continuous Enzymatic Regeneration of Electron Acceptors Used by Flavoenzymes: Cellobiose Dehydrogenase-Catalyzed Production of Lactobionic Acid as an Example

An efficient enzymatic bioprocess is described in which lactose, an abundant renewable resource produced by the dairy industry, is completely and efficiently converted with a specific productivity of up to 32 g (kU h)^-1 into lactobionic acid, without the formation of any by-products. The key biocatalyst of this new process is the fungal enzyme cellobiose dehydrogenase which oxidizes several β-1,4-linked disaccharides including lactose specifically at position C-1 of the reducing sugar moiety to the corresponding lactones. The electron acceptor employed in this reaction is continuously regenerated with the help of laccase, a H₂O-producing, copper-containing oxidase, and therefore has to be added in low, catalytic amounts only. Redox mediators that were successfully employed in this novel process and hence are compatible with the laccase regeneration system include benzoquinone, ABTS, ferricyanide, or ferrocene, amongst others. Factors affecting operational stability of the biocatalysts employed in this process include the redox mediator used, the temperature, and importantly the volumetric gas flow necessary for maintaining the dissolved oxygen tension. Lactobionic acid is a mild and sweet tasting acid with excellent chelating properties. These useful characteristics have lead to a growing number of patents for diverse applications in the food, pharmaceutical and detergent industries.     

Rare sugars and sugar-based synthons by chemo-enzymatic synthesis

The unique catalytic potential of the fungal enzyme pyranose oxidase was demonstrated by preparative conversions of a variety of carbohydrates, and by extensive chemical characterization of the reaction products with NMR spectroscopy. The studies revealed that POx not only oxidizes most substrates very efficiently but also that POx possesses a glycosyl-transfer potential, producing disaccharides from beta-glycosides of higher alcohols. Although most substrates are oxidized by POx at the C-2 position, several substrates are converted into the 3-keto-derivatives. On the basis of these products, strategies are developed for the convenient production of sugar-derived synthons, rare sugars and fine chemicals by combining biotechnical and chemical methods.     

Purification and characterization of pyranose oxidase from the white rot fungus Trametes multicolor

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are D-glucose, D-xylose, and L-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k(cat)/K(m)), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H(2)O(2). An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin.     

Continuous Enzymatic Regeneration of Electron Acceptors Used by Flavoenzymes: Cellobiose Dehydrogenase-Catalyzed Production of Lactobionic Acid as an Example

An efficient enzymatic bioprocess is described in which lactose, an abundant renewable resource produced by the dairy industry, is completely and efficiently converted with a specific productivity of up to 32 g (kU h)^?1 into lactobionic acid, without the formation of any by-products. The key biocatalyst of this new process is the fungal enzyme cellobiose dehydrogenase which oxidizes several β-1,4-linked disaccharides including lactose specifically at position C-1 of the reducing sugar moiety to the corresponding lactones. The electron acceptor employed in this reaction is continuously regenerated with the help of laccase, a H₂O-producing, copper-containing oxidase, and therefore has to be added in low, catalytic amounts only. Redox mediators that were successfully employed in this novel process and hence are compatible with the laccase regeneration system include benzoquinone, ABTS, ferricyanide, or ferrocene, amongst others. Factors affecting operational stability of the biocatalysts employed in this process include the redox mediator used, the temperature, and importantly the volumetric gas flow necessary for maintaining the dissolved oxygen tension. Lactobionic acid is a mild and sweet tasting acid with excellent chelating properties. These useful characteristics have lead to a growing number of patents for diverse applications in the food, pharmaceutical and detergent industries.     

Screening of sugar converting enzymes using quantitative MALDI-ToF mass spectrometry

Quantitative matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) was applied for the screening of ten pyranose oxidase variants. Quantitative MALDI-ToF MS using isotopic labeled internal standards and ionic liquid matrices was performed using aliquots of enzyme reaction mixtures without prepurification steps. The results obtained were in good agreement with HPLC measurements. Analysis time was approx. 3.5 min for a five-fold determination. Thus, quantitative MALDI-ToF MS can be used as a tool for screening of sugar converting enzymes.     

Novel scheme for biosynthesis of aryl metabolites from L-phenylalanine in the fungus Bjerkandera adusta

Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with L-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors ((14)C- and (13)C-labelled L-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that L-phenylalanine was deaminated to trans-cinnamic acid by a phenylalanine ammonia lyase and trans-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from L-phenylalanine to aryl metabolites. Additionally, trans-cinnamic acid can also be transformed via beta-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a beta-oxidation degradation intermediate. To our knowledge, this is the first time that a beta-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from L-phenylalanine is proposed.     

A coupled enzyme assay for aldose 1-epimerase

Aldose 1-epimerase (mutarotase) EC 5.1.3.3. catalyzes the mutarotation of selected pyranose sugars (1). The enzyme has been implicated as a component of the sugar transport system in kidney and intestine (2,3,4). Conventional analytical methods for monitoring catalytic activity involve relatively long, finite-interval polarimetric or spectrophotometric measurements of mutarotation rates employing α-d-glucose as the substrate (5). We have found this method to be somewhat cumbersome and time consuming, as α-d-glucose solutions spontaneously epimerize at rates requiring their individual preparation for each experiment. We report here a kinetic assay method for aldose 1-epimerase based upon fastin situ generation of α-d-glucose employing hydrolysis of sucrose by β-fructofuranosidase and a subsequent reporter reaction involving the aerobic oxidation of β-d-glucose via glucose oxidase. Analytical monitoring of the rate limiting epimerization step in the three-enzyme system is achieved by measurement of oxygen depletion in solution employing a conventional Clark electrode assembly.     

Evaluation of different expression systems for the heterologous expression of pyranose 2-oxidase from <Emphasis Type="Italic">Trametes multicolor</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The heterologous production of the industrially relevant fungal enzyme pyranose 2-oxidase in the prokaryotic host E. coli was investigated using 3 different expression systems, i.e. the well-studied T7 RNA polymerase based pET21d⁺, the L-arabinose inducible pBAD and the pCOLD system. Preliminary experiments were done in shaking flasks at 25°C and optimized induction conditions to compare the productivity levels of the different expression systems. The pET21d⁺ and the pCOLD system gave 29 U/L·h and 14 U/L·h of active pyranose 2-oxidase, respectively, whereas the pBAD system only produced 6 U/L·h. Process conditions for batch fermentations were optimized for the pET21d⁺ and the pCOLD systems in order to reduce the formation of inactive inclusion bodies. The highest productivity rate with the pET21d⁺ expression system in batch fermentations was determined at 25°C with 32 U/L·h. The pCOLD system showed the highest productivity rate (19 U/L·h) at 25°C and induction from the start of the cultivation. Using the pCOLD system in a fed batch fermentation at 25°C with a specific growth rate of μ = 0.15 h^-1resulted in the highest productivity rate of active pyranose oxidase with 206 U/L·h.     

Bubble-free oxygenation of a bi-enzymatic system: effect on biocatalyst stability

The effect of bubble-free oxygenation on the stability of a bi-enzymatic system with redox mediator regeneration for the conversion of lactose to lactobionic acid was investigated in a miniaturized reactor with bubbleless oxygenation. Earlier investigations of this biocatalytic oxidation have shown that the dispersive addition of oxygen can cause significant enzyme inactivation. In the process studied, the enzyme cellobiose dehydrogenase (CDH) oxidizes lactose at the C-1 position of the reducing sugar moiety to lactobionolactone, which spontaneously hydrolyzes to lactobionic acid. 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was used as electron acceptor for CDH and was continuously regenerated (reoxidized) by laccase, a blue multi-copper oxidase. Oxygen served as the terminal electron acceptor of the reaction and was fully reduced to water by laccase. The overall mass transfer coefficient of the miniaturized reactor was determined at 30 and 45 degrees C; conversions were conducted both in the reaction-limited and diffusion-limited regime to study catalyst inactivation. The bubbleless oxygenation was successful in avoiding gas/liquid interface inactivation. It was also shown that the oxidized redox mediator plays a key role in the inactivation mechanism of the biocatalysts unobserved during previous studies.     

iTRAQ-based quantitative secretome analysis of Phanerochaete chrysosporium

The basidiomycete fungi such as Phanerochaete chrysosporium secrete large amount of hydrolytic and oxidative enzymes and degrade lignocellulosic biomass. The lignin depolymerizing proteins were extensively studied, but cellulose, hemicellulose and pectin hydrolyzing enzymes were poorly explored. In this study P. chrysosporium was grown in cellulose, lignin and mixture of cellulose and lignin, and secretory proteins were quantified by isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS). An iTRAQ quantified 117 enzymes comprising cellulose hydrolyzing endoglucanases, exoglucanases, beta-glucosidases; hemicelluloses hydrolyzing xylanases, acetylxylan esterases, mannosidases, mannanases; pectin-degrading enzymes polygalacturonase, rhamnogalacturonase, arabinose and lignin degrading protein belonging to oxidoreductase family. Under cellulose and cellulose with lignin culture conditions, enzymes such as endoglucanases, exoglucanases, β-glucosidases and cellobiose dehydrogenase were significantly upregulated and iTRAQ data suggested hydrolytic and oxidative cellulose degradation. When lignin was used as a major carbon source, enzymes such as copper radical oxidase, isoamyl oxidase, glutathione S-transferase, thioredoxin peroxidase, quinone oxidoreductase, aryl alcohol oxidase, pyranose 2-oxidase, aldehyde dehydrogenase, and alcohol dehydrogenase were expressed and significantly regulated. This study explored cellulose, hemicellulose, pectin and lignin degrading enzymes of P. chrysosporium that are valuable for lignocellulosic bioenergy.