Draft Genome Sequence of the White-Rot Fungus Obba rivulosa 3A-2.

We report here the first genome sequence of the white-rot fungus Obba rivulosa (Polyporales, Basidiomycota), a polypore known for its lignin-decomposing ability. The genome is based on the homokaryon 3A-2 originating in Finland. The genome is typical in size and carbohydrate active enzyme (CAZy) content for wood-decomposing basidiomycetes.

Cell-based bioreporter assay coupled to HPLC micro-fractionation in the evaluation of antimicrobial properties of the basidiomycete fungus Pycnoporus cinnabarinus.

CONTEXT: Identification of bioactive components from complex natural product extracts can be a tedious process that aggravates the use of natural products in drug discovery campaigns. OBJECTIVE: This study presents a new approach for screening antimicrobial potential of natural product extracts by employing a bioreporter assay amenable to HPLC-based activity profiling. MATERIALS AND METHODS: A library of 116 crude extracts was prepared from fungal culture filtrates by liquid-liquid extraction with ethyl acetate, lyophilised, and screened against Escherichia coli using TLC bioautography. Active extracts were studied further with a broth microdilution assay, which was, however, too insensitive for identifying the active microfractions after HPLC separation. Therefore, an assay based on bioluminescent E. coli K-12 (pTetLux1) strain was coupled with HPLC micro-fractionation. RESULTS: Preliminary screening yielded six fungal extracts with potential antimicrobial activity. A crude extract from a culture filtrate of the wood-rotting fungus, Pycnoporus cinnabarinus (Jacq.) P. Karst. (Polyporaceae), was selected for evaluating the functionality of the bioreporter assay in HPLC-based activity profiling. In the bioreporter assay, the IC50 value for the crude extract was 0.10 mg/mL. By integrating the bioreporter assay with HPLC micro-fractionation, the antimicrobial activity was linked to LC-UV peak of a compound in the chromatogram of the extract. This compound was isolated and identified as a fungal pigment phlebiarubrone. DISCUSSION AND CONCLUSION: HPLC-based activity profiling using the bioreporter-based approach is a valuable tool for identifying antimicrobial compound(s) from complex crude extracts, and offers improved sensitivity and speed compared with traditional antimicrobial assays, such as the turbidimetric measurement.

Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens.

White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification.

Aromatic metabolism of filamentous fungi in relation to the presence of aromatic compounds in plant biomass.

The biological conversion of plant lignocellulose plays an essential role not only in carbon cycling in terrestrial ecosystems but also is an important part of the production of second generation biofuels and biochemicals. The presence of the recalcitrant aromatic polymer lignin is one of the major obstacles in the biofuel/biochemical production process and therefore microbial degradation of lignin is receiving a great deal of attention. Fungi are the main degraders of plant biomass, and in particular the basidiomycete white rot fungi are of major importance in converting plant aromatics due to their ability to degrade lignin. However, the aromatic monomers that are released from lignin and other aromatic compounds of plant biomass are toxic for most fungi already at low levels, and therefore conversion of these compounds to less toxic metabolites is essential for fungi. Although the release of aromatic compounds from plant biomass by fungi has been studied extensively, relatively little attention has been given to the metabolic pathways that convert the resulting aromatic monomers. In this review we provide an overview of the aromatic components of plant biomass, and their release and conversion by fungi. Finally, we will summarize the applications of fungal systems related to plant aromatics.

Production of manganese peroxidase and laccase in a solid-state bioreactor and modeling of enzyme production kinetics.

Lignin-modifying enzymes have various promising applications such as biobleaching, biopulping, the functionalization of lignocellulosic materials, the modification of wood fibers, the remediation of contaminated soil and effluents, as well as improvement of the enzymatic hydrolysis of lignocellulosic substrates. In this study, the production of laccase and manganese peroxidase (MnP) in solid-state cultivation was examined. Oat husks were used as an inexpensive substrate for the white-rot fungus Cerrena unicolor PM170798 (FBCC 387). The addition of a fines fraction (consisting of oat flour and finely ground husks) enhanced MnP production fivefold and laccase production almost threefold. The enzyme production was studied first on a 100 g scale, and the cultivation experiments were then repeated at a larger laboratory-scale (4 kg) in a solid-state bioreactor. High enzyme activity levels were obtained (MnP: 340 nkat g(-1) DM, laccase: 470 nkat g(-1) DM). In addition, the correlation between the CO2 evolution rate and enzyme production was mathematically modeled from the bioreactor experimental data. The model parameters could be used to predict enzyme production.

Plant-polysaccharide-degrading enzymes from Basidiomycetes.

SUMMARY: Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation.

Phenolic mediators enhance the manganese peroxidase catalyzed oxidation of recalcitrant lignin model compounds and synthetic lignin.

Fungal oxidative enzymes, such as peroxidases and laccases, are the key catalysts in lignin biodegradation in vivo, and consequently provide an important source for industrial ligninolytic biocatalysts. Recently, it has been shown that some syringyl-type phenolics have potential as industrial co-oxidants or mediators, in laccase-catalyzed modification of lignocellulosic material. We have now studied the effect of such mediators with ligninolytic peroxidases on oxidation of the most recalcitrant lignin model compounds. We found that they are able to enhance the manganese peroxidase (MnP) catalyzed oxidation reactions of small non-phenolic compounds, veratryl alcohol and veratrylglycerol ß-guaiacyl ether (adlerol), which are not usually oxidized by manganese peroxidases alone. In these experiments we compared two peroxidases from white-rot fungi, MnP from Phlebia sp. Nf b19 and versatile peroxidase (VP) from Bjerkandera adusta under two oxidation conditions: (i) the Mn(III) initiated mediated oxidation by syringyl compounds and (ii) the system involving MnP-dependent lipid peroxidation, both with production of (hydrogen) peroxides in situ to maintain the peroxidase catalytic cycle. It was found that both peroxidases produced α-carbonyl oxidation product of veratryl alcohol in clearly higher yields in reactions mediated by phenoxy radicals than in lipid-peroxyl radical system. The oxidation of adlerol, on the other hand, was more efficient in lipid-peroxidation-system. VP was more efficient than MnP in the oxidation of veratryl alcohol and showed its lignin peroxidase type activity in the reaction conditions indicated by some cleavage of Cα-Cß-bond of adlerol. Finally, the mediator assisted oxidation conditions were applied in the oxidation of synthetic lignin (DHP) and the structural analysis of the oxidized polymers showed clear modifications in the polymer outcome, e.g. the oxidation resulted in reduced amount of aliphatic hydroxyls indicated by (31)P NMR.

Carbohydrate-binding modules of fungal cellulases: occurrence in nature, function, and relevance in industrial biomass conversion.

In this review, the present knowledge on the occurrence of cellulases, with a special emphasis on the presence of carbohydrate-binding modules (CBMs) in various fungal strains, has been summarized. The importance of efficient fungal cellulases is growing due to their potential uses in biorefinery processes where lignocellulosic biomasses are converted to platform sugars and further to biofuels and chemicals. Most secreted cellulases studied in detail have a bimodular structure containing an active core domain attached to a CBM. CBMs are traditionally been considered as essential parts in cellulases, especially in cellobiohydrolases. However, presently available genome data indicate that many cellulases lack the binding domains in cellulose-degrading organisms. Recent data also demonstrate that CBMs are not necessary for the action of cellulases and they solely increase the concentration of enzymes on the substrate surfaces. On the other hand, in practical industrial processes where high substrate concentrations with low amounts of water are employed, the enzymes have been shown to act equally efficiently with and without CBM. Furthermore, available kinetic data show that enzymes without CBMs can desorb more readily from the often lignaceous substrates, that is, they are not stuck on the substrates and are thus available for new actions. In this review, the available data on the natural habitats of different wood-degrading organisms (with emphasis on the amount of water present during wood degradation) and occurrence of cellulose-binding domains in their genome have been assessed in order to identify evolutionary advantages for the development of CBM-less cellulases in nature.

Biochemical and molecular characterization of an atypical manganese peroxidase of the litter-decomposing fungus Agrocybe praecox.

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer.

Transcriptional analysis of selected cellulose-acting enzymes encoding genes of the white-rot fungus Dichomitus squalens on spruce wood and microcrystalline cellulose.

The recent discovery of oxidative cellulose degradation enhancing enzymes has considerably changed the traditional concept of hydrolytic cellulose degradation. The relative expression levels of ten cellulose-acting enzyme encoding genes of the white-rot fungus Dichomitus squalens were studied on solid-state spruce wood and in microcrystalline Avicel cellulose cultures. From the cellobiohydrolase encoding genes, cel7c was detected at the highest level and showed constitutive expression whereas variable transcript levels were detected for cel7a, cel7b and cel6 in the course of four-week spruce cultivation. The cellulolytic enzyme activities detected in the liquid cultures were consistent with the transcript levels. Interestingly, the selected lytic polysaccharide monooxygenase (LPMO) encoding genes were expressed in both cultures, but showed different transcription patterns on wood compared to those in submerged microcrystalline cellulose cultures. On spruce wood, higher transcript levels were detected for the lpmos carrying cellulose binding module (CBM) than for the lpmos without CBMs. In both cultures, the expression levels of the lpmo genes were generally higher than the levels of cellobiose dehydrogenase (CDH) encoding genes. Based on the results of this work, the oxidative cellulose cleaving enzymes of D. squalens have essential role in cellulose degrading machinery of the fungus.

Mycoremediation of wood and soil from an old sawmill area contaminated for decades.

We investigated the potential of white-rot and litter-decomposing fungi for the treatment of soil and wood from a sawmill area contaminated with aged chlorinated phenols, dibenzo-p-dioxins and furans (PCDD/F). Eight screening assays with emphasis on application of non-sterile conditions were carried out in order to select the strains with capability to withstand indigenous microbes and contamination. Nine fungi were then selected for degrading pentachlorophenol (PCP), and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP) and mineralizing radiolabelled pentachlorophenol ((14)C-PCP) in non-sterile soil or wood during 15 weeks of incubation. Soil indigenous microbes and fungal inoculated soil (fungal inoculum+indigenous microbes) achieved similar degradation of PCP and 2,3,4,6-TeCP and mineralization of (14)C-PCP. However, the mineralization rate of (14)C-PCP by indigenous microbes was much slower than that boosted by fungal inoculum. The litter-decomposing fungus (LDF) Stropharia rugosoannulata proved to be a suitable fungus for soil treatment. This fungus mineralized 26% of (14)C-PCP and degraded 43% of 2,3,4,6-TeCP and 73% of PCP. Furthermore, S. rugosoannulata attained 13% degradation of PCDD/F (expressed as WHO-Toxic Equivalent). In wood, white-rot fungi grew and degraded chlorophenols better than LDF. No efficient indigenous degraders were present in wood. Interestingly, production of toxic chlorinated organic metabolites (anisoles and veratroles) by LDF in wood was negligible.

Bioluminescent yeast assay for detection of organotin compounds.

Organotin compounds are toxic and endocrine disrupting compounds, which have been intensively used as antifouling paints for ship hulls and thus are widely spread in the environment. They are suspected to cause imposex, the formation of male characteristics in female gastropods, because of the activation of retinoid X receptor (RXR) at very low environmental concentrations. Here we report the development and optimization of a bioluminescent yeast assay for the detection of organotin compounds based on the interaction with a hybrid RXR and subsequent expression of a reporter luciferase gene. This assay is highly specific toward organotin compounds and natural ligands of the RXR. It detects tributyltin and triphenyltin in nanomolar concentrations (detection limits were found to be 30 nM and 110 nM, respectively) and allows small-scale high-throughput analyses. Furthermore it was possible to measure tributyltin directly in untreated spiked sediments. Thus, the results provided within one working day can be used for the assessment of bioavailability and mixture effect of organotin compounds in environmental samples.

Effect of copper, nutrient nitrogen, and wood-supplement on the production of lignin-modifying enzymes by the white-rot fungus Phlebia radiata.

Production of the oxidoreductive lignin-modifying enzymes - lignin and manganese peroxidases (MnPs), and laccase - of the white-rot basidiomycete Phlebia radiata was investigated in semi-solid cultures supplemented with milled grey alder or Norway spruce and charcoal. Concentrations of nutrient nitrogen and Cu-supplement varied also in the cultures. According to extracellular activities, production of both lignin peroxidase (LiP) and MnP was significantly promoted with wood as carbon source, with milled alder (MA) and low nitrogen (LN) resulting with the maximal LiP activities (550 nkat l(-1)) and noticeable levels of MnP (3 µkat l(-1)). Activities of LiP and MnP were also elevated on high nitrogen (HN) complex medium when supplemented with spruce and charcoal. Maximal laccase activities (22 and 29 µkat l(-1)) were obtained in extra high nitrogen (eHN) containing defined and complex media supplemented with 1.5 mM Cu(2+). However, the nitrogen source, either peptone or ammonium nitrate and asparagine, caused no stimulation on laccase production without Cu-supplement. This is also the first report to demonstrate a new, on high Cu(2+) amended medium produced extracellular laccase of P. radiata with pI value of 4.9, thereby complementing our previous findings on gene expression, and cloning of a second laccase of this fungus.

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus.

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 µkat/µg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.

Comparative evaluation of manganese peroxidase- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids by different methods.

The peroxidation of C18 unsaturated fatty acids by fungal manganese peroxidase (MnP)/Mn(II) and by chelated Mn(III) was studied with application of three different methods: by monitoring oxygen consumption, by measuring conjugated dienes and by thiobarbituric acid-reactive substances (TBARS) formation. All tested polyunsaturated fatty acids (PUFAs) were oxidized by MnP in the presence of Mn(II) ions but the rate of their oxidation was not directly related to degree of their unsaturation. As it has been shown by monitoring oxygen consumption and conjugated dienes formation the linoleic acid was the most easily oxidizable fatty acid for MnP/Mn(II) and chelated Mn(III). However, when the lipid peroxidation (LPO) activity was monitored by TBARS formation the linolenic acid gave the highest results. High accumulation of TBARS was also recorded during peroxidation of linoleic acid initiated by MnP/Mn(II). Action of Mn(III)-tartrate on the PUFAs mimics action of MnP in the presence of Mn(II) indicating that Mn(III) ions are involved in LPO initiation. Although in our experiments Mn(III) tartrate gave faster than MnP/Mn(II) initial oxidation of the unsaturated fatty acids with consumption of O(2) and formation of conjugated dienes the process was not productive and did not support further development of LPO. The higher effectiveness of MnP/Mn(II)-initiated LPO system depends on the turnover of manganese provided by MnP. It is proposed that the oxygen consumption assay is the best express method for evaluation of MnP- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids.

Fate of bisphenol A during treatment with the litter-decomposing fungi Stropharia rugosoannulata and Stropharia coronilla.

Bisphenol A is an endocrine disrupting compound, which is ubiquitous in the environment due to its wide use in plastic and resin production. Seven day old cultures of the litter-decomposing fungus Stropharia coronilla removed the estrogenic activity of bisphenol A (BPA) rapidly and enduringly. Treatment of BPA with purified neutral manganese peroxidase (MnP) from this fungus also resulted in 100% reduction of estrogenic activity, as analyzed using a bioluminescent yeast assay, and in the formation of polymeric compounds. In cultures of Stropharia rugosoannulata, estrogenic activity also quickly disappeared but temporarily re-emerged in the further course of cultivation. LC-MS analysis of the extracted estrogenic culture liquid revealed [M-H](-) ions with m/z values of 219 and 235. We hypothesize that these compounds are ring fission products of BPA, which still exhibit one intact hydroxyphenyl group to interact with estrogen receptors displayed by the yeast.

Potency determinations of acetylcholinesterase inhibitors using Ellman's reaction-based assay in screening: Effect of assay variants.

In primary drug discovery screenings and potency determinations of active acetylcholinesterase (AChE) inhibitors, different variations of the Ellman's reaction-based assay are extensively applied. However, these are prone to produce variable results. Here we studied how assay variants differing in the order of reagent addition and substrate concentrations influence potency measurements of AChE inhibitors. Three model compounds were used: tacrine, physostigmine, and a newly reported inhibitor, 1-[5-[4-[(hexahydro-1H-azepin-1-yl)carbonyl]-1-piperidinyl]-1,3,4-thiadiazol-2-yl]-2-pyrrolidinone. Different patterns of potency changes related to the different inhibition mechanisms of the compounds were detected. Recognizing this, better judgment can be applied when publishing results and selecting optimal screening assays.

Miniaturization and validation of the Ellman's reaction based acetylcholinesterase inhibitory assay into 384-well plate format and screening of a chemical library.

The aim of this study was to screen for acetylcholinesterase (AChE) inhibitors from a large chemical library of commercially available compounds. For this purpose, the Ellman's reaction based assay was miniaturized into 384-well plate format, and two modifications of the kinetic protocol were studied with the aim of developing a rapid screening platform that could ensure high efficiency in finding true hits. It was proven that when starting the kinetic reaction by addition of the substrate, better assay performance was achieved and more practical benefits obtained. Using the optimized automated protocol, a chemical library of 56,320 compounds was screened. A total of 350 positive hits were identified and their IC50 calculated. Three highly active compounds were identified with IC50 values close or even lower to physostigmine (< 0.1 microM). The activity towards butyrylcholinesterase (BChE) of these three most active hits was also evaluated. The most active hit (IC(50(AChE)) = 0.019 microM), was identified as a new inhibitor, belonging to ChemDiv chemical library: (N-[3-(3,5-dimethyl-1-piperidinyl)propyl]-5-ethyl-2-methyl-8-oxo-thieno[2',3':4,5]pyrrolo[1,2-d] [1,2,4] triazine-7(8H)-acetamid), with no other biological activities reported until now. The interactions of this hit with both cholinesterases were further analyzed using computational docking studies. To our knowledge, this is the largest published screening campaign of commercially available compounds that has focused on finding new AChE inhibitors. The miniaturized 384-well plate format of the Ellman's method was proven to be robust and to perform reliably.

Scots pine (Pinus sylvestris) bark composition and degradation by fungi: potential substrate for bioremediation.

The composition of Scots pine bark, its degradation, and the production of hydrolytic and ligninolytic enzymes were evaluated during 90 days of incubation with Phanerochaete velutina and Stropharia rugosoannulata. The aim was to evaluate if pine bark can be a suitable fungal substrate for bioremediation applications. The original pine bark contained 45% lignin, 25% cellulose, and 15% hemicellulose. Resin acids were the most predominant lipophilic extractives, followed by sitosterol and unsaturated fatty acids, such as linoleic and oleic acids. Both fungi degraded all main components of bark, specially cellulose (79% loss by P. velutina). During cultivation on pine bark, fungi also degraded sitosterol, produced malic acid, and oxidated unsaturated fatty acids. The most predominant enzymes produced by both fungi were cellulase and manganese peroxidase. The results indicate that Scots pine bark supports enzyme production and provides nutrients to fungi, thus pine bark may be suitable fungal substrate for bioremediation.

Oxalate decarboxylase of the white-rot fungus Dichomitus squalens demonstrates a novel enzyme primary structure and non-induced expression on wood and in liquid cultures.

Oxalate decarboxylase (ODC) catalyses the conversion of oxalic acid to formic acid and CO(2) in bacteria and fungi. In wood-decaying fungi the enzyme has been linked to the regulation of intra- and extracellular quantities of oxalic acid, which is one of the key components in biological decomposition of wood. ODC enzymes are biotechnologically interesting for their potential in diagnostics, agriculture and environmental applications, e.g. removal of oxalic acid from industrial wastewaters. We identified a novel ODC in mycelial extracts of two wild-type isolates of Dichomitus squalens, and cloned the corresponding Ds-odc gene. The primary structure of the Ds-ODC protein contains two conserved Mn-binding cupin motifs, but at the N-terminus, a unique, approximately 60 aa alanine-serine-rich region is found. Real-time quantitative RT-PCR analysis confirmed gene expression when the fungus was cultivated on wood and in liquid medium. However, addition of oxalic acid in liquid cultures caused no increase in transcript amounts, thereby indicating a constitutive rather than inducible expression of Ds-odc. The detected stimulation of ODC activity by oxalic acid is more likely due to enzyme activation than to transcriptional upregulation of the Ds-odc gene. Our results support involvement of ODC in primary rather than secondary metabolism in fungi.