Brown-rot Basidiomycete Research Articles

Biocalorimetry-aided monitoring of fungal pretreatment of lignocellulosic agricultural residues

The present study aimed to investigate whether and how non-invasive biocalorimetric measurements could serve for process monitoring of fungal pretreatment during solid-state fermentation (SSF) of lignocellulosic agricultural residues such as wheat straw. Seven filamentous fungi representing different lignocellulose decay types were employed. Water-soluble sugars being immediately available after fungal pretreatment and those becoming water-extractable after enzymatic digestion of pretreated wheat straw with hydrolysing (hemi)cellulases were considered to constitute the total bioaccessible sugar fraction. The latter was used to indicate the success of pretreatments and linked to corresponding species-specific metabolic heat yield coefficients (YQ/X) derived from metabolic heat flux measurements during fungal wheat straw colonisation. An YQ/X range of about 120 to 140 kJ/g was seemingly optimal for pretreatment upon consideration of all investigated fungi and application of a non-linear Gaussian fitting model. Upon exclusion from analysis of the brown-rot basidiomycete Gloeophyllum trabeum, which differs from all other here investigated fungi in employing extracellular Fenton chemistry for lignocellulose decomposition, a linear relationship where amounts of total bioaccessible sugars were suggested to increase with increasing YQ/X values was obtained. It remains to be elucidated whether an YQ/X range being optimal for fungal pretreatment could firmly be established, or if the sugar accessibility for post-treatment generally increases with increasing YQ/X values as long as “conventional” enzymatic, i.e. (hemi)cellulase-based, lignocellulose decomposition mechanisms are operative. In any case, metabolic heat measurement–derived parameters such as YQ/X values may become very valuable tools supporting the assessment of the suitability of different fungal species for pretreatment of lignocellulosic substrates.Key points• Biocalorimetry was used to monitor wheat straw pretreatment with seven filamentous fungi.• Metabolic heat yield coefficients (YQ/X) seem to indicate pretreatment success.• YQ/X values may support the selection of suitable fungal strains for pretreatment.

Bark beetle outbreaks in Picea abies stands are associated with changes in wood-inhabiting fungal communities and seedling establishment on logs

In recent decades bark beetle outbreaks have caused high mortality in natural mountain Picea abies forests in Central Europe. This study evaluated factors affecting seedling establishment of P. abies by focusing on the role of fungal communities in decaying logs, which is an important regeneration microsite. At the control site, which was affected by lower severity disturbance, well decayed logs with moss and vegetation cover hosted many seedlings. At the disturbed site, which experienced high mortality by bark beetles, greater canopy openness suppressed vegetation on logs and lowered seedling density. Additionally, the presence of a white rot basidiomycete Phellopilus nigrolimitatus was positively associated with seedling density. In contrast, the presence of a brown rot basidiomycete Fomitopsis pinicola was negatively associated with seedling density. The relationships between these decomposer fungi and seedling density might be partly attributed to changes in wood chemical properties and associated mycorrhizal and pathogenic fungi.

Fungal outbreak in the Catacombs of SS. Marcellino and Pietro Rome (Italy): From diagnosis to an emergency treatment.

The present study reports a sudden fungal outbreak that occurred in the corridor near the entrance of the Catacombs of SS. Marcellino and Pietro in Rome (Italy) observed after 1 year of a restoration treatment that interested the walls of the entrance of the Catacombs and some artifacts placed in situ. The colonization was observed on the vault at the entrance and in correspondence with the restored marble pieces displayed on the left side of the corridor. No growth was observed on the right side where similarly treated marble slabs were placed. Samples taken in correspondence with fungal biofilm were analyzed through the combined use of microscopical, cultural, and molecular tools and showed that the vault and the left side of the corridor entrance were colonized by a complex fungal biofilm consisting mainly of Coniophora sp. and other genera, such as Hypomyces, Purpureocillium, Acremonium, Penicillium, and Alternaria, many of which are well known as responsible of biodeterioration of stone surfaces. Regarding the brown-rot basidiomycete Coniophora, it was able to form very large colonies on the substrata with a diameter of up to 57 cm. Although the direct observation under a light microscope evidenced the presence of abundant brown fungal conidia, several attempts to cultivate the microorganism failed, therefore only through DNA sequencing analyses, it was possible to identify and characterize this fungus. There is very little literature on the genus Coniophora which is reported as one of the causes of wet-rot decay of wood in buildings. A connection with calcium-containing materials such as bricks and mortars was demonstrated, but no data were available about the possible role of this species in the biodeterioration of stones. This study features the first finding of a strain related to the basidiomycetous genus of Coniophora in the order Boletales in association with evident phenomena of biodeterioration.

Fomitopsis meliae CFA 2, a novel brown rot for endoglucanase: emphasis towards enhanced endoglucanase production by statistical approach

ABSTRACT Brown rot basidiomycetes are a principal group of wood-decaying fungi which degrade wood cellulose and hemicellulose by the combination of carbohydrate active enzymes and non-enzymatic oxidation reactions. Very scant information is available on carbohydrate active enzymes of brown rot fungi. In this context, present study focused on the production of cellulolytic–hemicellulolytic enzymes from newly isolated brown rot Fomitopsis meliae CFA 2. Under solid-state fermentation using wheat bran as the substrate Fomitopsis meliae CFA 2 was able to produce a maximum of 1391.12 ± 21.13 U/g of endoglucanase along with other cellulolytic and hemicellulolytic enzymes. Various fermentation parameters were optimised for enhanced production of endoglucanase by employing Plackett-Burman design followed by Box-Behnken design. A well-fitted regression equation with R2 value of 98.91% was attained for endoglucanase. The yield of endoglucanase was enhanced by 1.83-fold after executing statistical optimisation of various fermentative parameters. The newly isolated Fomitopsis meliae CFA 2 was found to be a potential producer of endoglucanase. Enzymatic saccharification of alkali-treated wheat straw and rice straw resulted in release of 190.8 and 318.8 mg/g of reducing sugars, respectively.

Gene Expression Analysis of Three Putative Copper-Transporting ATPases in Copper-Tolerant Fibroporia radiculosa.

Copper tolerance of brown-rot basidiomycete decay fungi can lessen the efficacy of copper-containing wood preservatives for wood products in-service. The purpose of this study was to evaluate wood mass loss and differential expression of three genes that have putative annotations for copper-transporting ATPase pumps (FIBRA_00974, FIBRA_04716, and FIBRA_01430). Untreated southern pine (SP) and SP treated with three concentrations of ammoniacal copper citrate (CC, 0.6, 1.2, and 2.4%) were exposed to two copper-tolerant Fibroporia radiculosa isolates (FP-90848-T and L-9414-SP) and copper-sensitive Gloeophyllum trabeum isolate (MAD 617) in a 4-week-long standard decay test (AWPA E10-19). Decay of copper-treated wood was inhibited by G. trabeum (p = 0.001); however, there was no inhibition of decay with increasing copper concentrations by both F. radiculosa isolates. Initially, G. trabeum and one F. radiculosa isolate (L-9414-SP) highly upregulated FIBRA_00974 and FIBRA_04716 on copper-treated wood at week 1 (p = 0.005), but subsequent expression was either not detected or was similar to expression on untreated wood (p = 0.471). The other F. radiculosa isolate (FP-90848-T) downregulated FIBRA_00974 (p = 0.301) and FIBRA_04716 (p = 0.004) on copper-treated wood. FIBRA_01430 expression by G. trabeum was not detected, but was upregulated by both F. radiculosa FP-90848-T (p = 0.481) and L-9414-SP (p = 0.392). Results from this study suggest that all three test fungi utilized different mechanisms when decaying copper-treated wood. Additionally, results from this study do not provide support for the involvement of these putative gene annotations for copper-transporting ATPase pumps in the mechanism of copper-tolerance.

Aspects Determining the Dominance of Fomitopsis pinicola in the Colonization of Deadwood and the Role of the Pathogenicity Factor Oxalate

Carbon and mineral cycling in sustainable forest systems depends on a microbiome of basidiomycetes, ascomycetes, litter-degrading saprobes, ectomycorrhizal, and mycoparasitic fungi that constitute a deadwood degrading consortium. The brown rot basidiomycete Fomitopsis pinicola (Swartz: Fr.) P. Karsten (Fp), as an oxalate-producing facultative pathogen, is an early colonizer of wounded trees and fresh deadwood. It replaces basidiomycetous white rot fungi and non-basidiomycetous fungal phyla in the presence of its volatilome, but poorly in its absence. With the goal of determining its dominance over the most competitive basidiomycetes and its role in fungal successions within the forest microbiome in general, Fp was exposed to the white rot fungus Kuehneromyces mutabilis (Schaeff.: Fr.) Singer & Smith (Km) in aseptic dual culture established on fertilized 100 mm-long wood dust columns in glass tubes with the inclusion of their volatilomes. For the mycelia approaching from the opposite ends of the wood dust columns, the energy-generating systems of laccase and manganese peroxidase (MnP), the virulence factor oxalate, and the exhalation of terpenes were determined by spectrophotometry, High Pressure Liquid Chromatography (HPLC), and Gas Chromatography-Mass Spectrometry (GC-MS). Km mycelia perceived the approaching Fp over 20 mm of non-colonized wood dust, reduced the laccase activity to 25%, and raised MnP to 275%–500% by gaining energy and presumably by controlling oxalate, H2O2, and the dropping substrate pH caused by Fp. On mycelial contact, Km stopped Fp, secured its substrate sector with 4 mm of an impermeable barrier region during an eruption of antimicrobial bisabolenes, and dropped from the invasion mode of substrate colonization into the steady state mode of low metabolic and defensive activity. The approaching Fp raised the oxalate production throughout to >20 g kg−1 to inactivate laccase and caused, with pH 1.4–1.7, lethal conditions in its substrate sector whose physiological effects on Km could be reproduced with acidity conditions incited by HCl. After a mean lag phase of 11 days, Fp persisting in a state of high metabolic activity overgrew and digested the debilitated Km thallus and terminated the production of oxalate. It is concluded that the factors contributing to the competitive advantage of F. pinicola in the colonization of wounded trees and pre-infected deadwood are the drastic long-term acidification of the timber substrate, its own insensitivity to extremely low pH conditions, its efficient control of the volatile mono- and sesquiterpenes of timber and microbial origin, and the action of a undefined blend of terpenes and allelopathic substances.

Insight into metabolic diversity of the brown-rot basidiomycete Postia placenta responsible for sesquiterpene biosynthesis: semi-comprehensive screening of cytochrome P450 monooxygenase involved in protoilludene metabolism.

SummaryA wide variety of sesquiterpenoids have been isolated from basidiomycetes, and their bioactive properties have attracted significant attention in an effort to understand biosynthetic machineries. As both sesquiterpene synthases and cytochrome P450 monooxygenases play key roles in the diversification of sesquiterpenoids, it is important to widely and mutually understand their biochemical properties. In this study, we performed genome‐wide annotation and functional characterization of sesquiterpene synthases from the brown‐rot basidiomycete Postia placenta. Using RT‐PCR, we isolated 16 sesquiterpene synthases genes as full‐length cDNAs. Heterologous expression revealed that the sesquiterpene synthases could produce a series of sesquiterpene scaffolds with distinct metabolic profiles. Based on metabolic studies, we identified 25 sesquiterpene scaffolds including Δ6‐protoilludene produced by the sesquiterpene synthases. In particular, a protoilludene synthase from the brown‐rot basidiomycete was characterized for the first time. Furthermore, we conducted a semi‐comprehensive functional screening of cytochrome P450 monooxygenases from P. placenta to elucidate biosynthetic machineries involved in metabolisms of Δ6‐protoilludene. Coexpression of protoilludene synthase and 184 isoforms of cytochrome P450 monooxygenases enabled the identification of CYP5344B1, CYP5348E1 and CYP5348J3, which catalysed the hydroxylation reaction of Δ6‐protoilludene to produce Δ6‐protoilludene‐8‐ol and Δ6‐protoilludene‐5‐ol. Furthermore, structural isomers of Δ7‐protoilludene‐6‐ol were obtained from incubation of Δ6‐protoilludene‐8‐ol in acidic culture medium.

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation.

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans-a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxidative cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications.

Characterization of a GH12 endoglucanase from Volvariella volvacea exhibiting broad substrate specificity and potential synergy with crude cellulase

Fungal glycoside hydrolase family GH12 has a single catalytic domain, exhibiting a great diversity of properties and application potentials in biomass biorefinery, feed, and textile industries. To discover new GH12 enzymes from white- and brown-rot basidiomycetes for application in the saccharification of lignocelluloses, two putative genes, VvGH12A and VvGH12B, were identified from the Volvariella volvacea genome and classified into basidiomycetous subfamily GH12-1 and GH12-2, respectively. One enzyme VvGH12A was successfully expressed in Pichia pastoris, and characterized. VvGH12A was the most active on CMC but with broad substrate specificities on polysaccharides with b-1,4 linked and b-1,3-1,4-mixed glucans. Furthermore, VvGH12A was also active on xylan and mannan. Unlike other fungal GH12 endoglucanases, VvGH12A showed a weak processivity independent of the carbohydrate-binding module (CBM) due to both “endo” and “exo” types of enzyme activity. The pH-optimum was significantly affected by the acidity and basicity of amino acid at site 98. The enzyme optimum pH was engineered to a higher neutral or alkaline pH (from pH 6.5 to pH 7.0-8.0) when Asp98 was replaced with nonpolar or neutral or amide residue. VvGH12A exhibited synergistic action with crude cellulase from Trichoderma reesei D-86271 (Rut C-30) in saccharification of delignified wheat straw, suggesting that VvGH12A plays a functional role in efficiently hydrolyzing plant cell wall polysaccharides.

The complete genome sequence and phylogenetic analysis of the mitochondrial DNA of the wood-decaying fungus Fomitopsis palustris

The complete mitochondrial genome sequence of the wood-decaying fungus Fomitopsis palustris (Basidiomycete, Agaricomycotina) was determined by next-generation sequencing technology. The complete sequence of the circular mitochondrial DNA of F. palustris was 63,479 bp in length with a 75.98% AT content. The mitochondrial genome encoded 14 conserved proteins, 2 ribosomal RNAs, 26 transfer RNAs, and 19 additional open reading frames. The coxI and cob genes contained six and one group I introns, respectively, and encoded eight open reading frames, including seven intron-encoded endonucleases. The complete mitochondrial genome of F. palustris presented herein represents the first such report for brown rot basidiomycetes. In addition, the BLAST score ratio and phylogenetic analysis may open new avenues to understanding the evolutionary status of this fungus .

Effect of Trichoderma-enriched organic charcoal in the integrated wood protection strategy.

The gradual elimination of chromium from wood preservative formulations results in higher Cu leaching and increased susceptibility to wood decay fungi. Finding a sustainable strategy in wood protection has become of great interest among researchers. The objective of these in vitro studies was to demonstrate the effect of T-720-enriched organic charcoal (biochar) against five wood decay basidiomycetes isolated from strongly damaged poles. For this purpose, the antagonistic potential of Trichoderma harzianum (strain T-720) was confirmed among other four Trichoderma spp. against five brown-rot basidiomycetes in dual culture tests. T-720 was genetically transformed and tagged with the green fluorescent protein (GFP) in order to study its antagonistic mechanism against wood decay basidiomycetes. It was also demonstrated that T-720 inhibits the oxalic acid production by basidiomycetes, a well-known mechanism used by brown-rot fungi to detoxify Cu from impregnated wood. Additionally, this study evaluated the effect of biochar, alone or in combination with T-720, on Cu leaching by different preservatives, pH stabilization and prevention of wood decay caused by five basidiomycetes. Addition of biochar resulted in a significant Cu binding released from impregnated wood specimens. T-720-enriched biochar showed a significant reduction of wood decay caused by four basidiomycetes. The addition of T-720-enriched biochar to the soil into which utility poles are placed may improve the efficiency of Cr-free wood preservatives.

Extracellular redox cycling and hydroxyl radical production occurs widely in lichenized Ascomycetes

Some free-living Ascomycetes and white and brown rot Basidiomycetes can generate hydroxyl radicals using extracellular redox cycling. However, the mechanisms of hydroxyl radical production differ between white and brown rot Basidiomycetes, and are unknown for Ascomycetes. Here, we present a survey of extracellular hydroxyl radical production by a range of lichenized Ascomycetes. Results show that given a quinone and chelated ferric ions, many lichens can readily produce hydroxyl radicals, and this is accompanied by the reduction of Fe3+ to Fe2+. In white rot fungi, extracellular redox enzymes have been proposed to be involved in hydroxyl radical generation. However, a survey of a wide range of lichens suggests that in these fungi hydroxyl radical production does not directly correlate with the activity of laccases and peroxidases. Rather, radicals are probably produced by a mechanism like that proposed for brown rot fungi. Potential roles of hydroxyl radicals produced by lichens include the breakdown of lignocellulosic residues in the soil which may allow lichens to live a partially saprotrophic existence, the breakdown of toxic soil chemicals and the formation of an ‘oxidative burst’ to deter potential pathogens.

Insights into the mechanism of copper-tolerance in Fibroporia radiculosa: The biosynthesis of oxalate

Copper is currently used as the key component in wood preservatives despite the known tolerance of many brown-rot Basidiomycetes. Copper-tolerant fungi, like Fibroporia radiculosa, produce and accumulate high levels of oxalate when exposed to copper. To gain insight into the mechanism of oxalate production, four F. radiculosa isolates decaying untreated and 1.2% ammoniacal copper citrate-treated wood were evaluated for the differential expression of citrate synthase, isocitrate lyase, glyoxylate dehydrogenase, a succinate/fumarate antiporter, and a copper resistance-associated ATPase pump. Samples were analyzed at 2, 4, 6, and 8 weeks for oxalate production and gene expression. ATPase pump expression increased in the presence of copper when initial oxalate concentrations were low, suggesting it functions in helping the fungus adapt to the copper-rich environment by pumping toxic copper ions out of the cell. A connection in expression levels between citrate synthase, the succinate/fumarate antiporter isocitrate lyase, and glyoxylate dehydrogenase for the four isolates was found suggesting the production of oxalate originates in the mitochondrial TCA cycle via citrate synthase, shunts to the glyoxysomal glyoxylate cycle via the succinate/fumarate antiporter, moves through a portion of the glyoxylate cycle (isocitrate lyase), and ultimately is made in the cytoplasm (gyloxylate dehydrogenase).

Potential of Wood-Rotting Fungi to Attack Polystyrene Sulfonate and Its Depolymerisation by Gloeophyllum trabeum via Hydroquinone-Driven Fenton Chemistry.

Synthetic polymers often pose environmental hazards due to low biodegradation rates and resulting accumulation. In this study, a selection of wood-rotting fungi representing different lignocellulose decay types was screened for oxidative biodegradation of the polymer polystyrene sulfonate (PSS). Brown-rot basidiomycetes showed PSS depolymerisation of up to 50 % reduction in number-average molecular mass (Mn) within 20 days. In-depth investigations with the most efficient depolymeriser, a Gloeophyllum trabeum strain, pointed at extracellular hydroquinone-driven Fenton chemistry responsible for depolymerisation. Detection of hydroxyl radicals present in the culture supernatants showed good compliance with depolymerisation over the time course of PSS degradation. 2,5-Dimethoxy-1,4-hydroquinone (2,5-DMHQ), which was detected in supernatants of active cultures via liquid chromatography and mass spectrometry, was demonstrated to drive the Fenton processes in G. trabeum cultures. Up to 80% reduction in Mn of PSS where observed when fungal cultures were additionally supplemented with 2,5-dimethoxy benzoquinone, the oxidized from of 2,5-DMHQ. Furthermore, 2,5-DMHQ could initiate the Fenton's reagent-mediated PSS depolymerisation in cell-free systems. In contrast, white-rot fungi were unable to cause substantial depolymerising effects despite the expression of lignin-modifying exo-enzymes. Detailed investigations with laccase from Trametes versicolor revealed that only in presence of certain redox mediators limited PSS depolymerisation occurred. Our results indicate that brown-rot fungi might be suitable organisms for the biodegradation of recalcitrant synthetic polymeric pollutants.

Comparative analysis of secretomes in basidiomycete fungi

Lignocellulose, the major reservoir of organic carbon on Earth, is recalcitrant to turnover and resistant to microbial and enzymatic attack because of the protective action of lignin and the crystalline structure of cellulose. Basidiomycetes are the only organisms known to degrade lignocellulose using two alternative degradation strategies: white rot basidiomycetes break down the lignin moiety extensively before attacking the cellulose, whereas brown rot basidiomycetes cause limited lignin alteration while primarily degrading cellulose. These strategies depend on complex portfolios of extracellular enzymes whose production and export are environment and substrate dependent. More than 265 fungal genomes (more than 90 corresponding to basidiomycetes) have been sequenced. These sequences can be bioinformatically screened to identify presumptive secreted proteins that, however, differ from the experimental data, stressing the importance of using complementary approaches for analyzing these complex systems. The study of fungal secretomes from the scope of their different lignin degradation strategies and lifestyles would facilitate their use in the treatment of lignocellulose as carbon feedstock for biofuel production and further biorefinery processes. In this article, we carry out a comparative review of the analytical tools and strategies used for studying basidiomycetes' secretomes and compare the available results from these two points of view. In this article, we review the tools and strategies used in the analysis of basidiomycetes' secretomes and we compare the secretome data available for this type of fungi with a special emphasis on the enzymes involved in lignocellulose degradation. This comparative study provides a basis for distinguishing different lifestyles (saprotrophs, symbionts and parasites) on the basis of their respective secretomes.

Cu(II)-induced molecular and physiological responses in the brown-rot basidiomycete Polyporales sp. KUC9061

A potentially safe disposal method for copper-containing waste wood is bioremediation using brown-rot fungi. However, the mechanisms regulating brown-rot fungi copper tolerance are poorly understood. The objective of this study was to better understand the molecular and physiological changes in Polyporales sp. KUC9061 in response to Cu(II) using GeneFishing technology. The presence of Cu(II) in the malt extract agar (MEA) media decreased the brown-rot fungi's growth rate in a concentration-dependent manner, but the fungal biomass was significantly increased in part for the biosorption of Cu(II). Increased expression of the genes encoding for the GIS2 DNA-binding protein and the 40S ribosomal protein S3A appears to be involved in this process. Oxalic acid is not used as a defence mechanism against high copper exposure, and ATP citrate lyase is not directly involved in oxalic acid production in this fungus. Several Cu(II)-sensitive proteins showed stable gene expression, suggesting that mechanisms that do not rely on these genes are responsible for the Cu(II) tolerance of the fungus. Polyporales sp. KUC9061 does not use oxalic acid to chelate excess Cu(II) and potentially has other mechanisms, including the increased production of mycelia, to regulate Cu(II) biosorption. This study is the first effort to examine Cu(II)-induced differential gene expression and the related physiological changes in the brown-rot fungus, a potential degrader of copper-containing waste wood. The results of this study will help with using this fungus to safely dispose of waste wood safe.

Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta

We explored the molecular diversity and functional capabilities of cytochrome P450 monooxygenases (P450s) from the brown-rot basidiomycete Postia placenta. Using bioinformatic and experimental data, we found 250 genes of P450s in the whole genome, including 60 putative allelic variants. Phylogenetic analysis revealed the presence of 42 families, including 18 novel families. Comparative phylogenetic analysis of P450s from P. placenta and the white-rot basidiomycete Phanerochaete chrysosporium suggested that vigorous gene duplication and molecular evolution occurred after speciation of basidiomycetes. Among the 250 gene models, 184 were isolated as full-length cDNA and transformed into Saccharomyces cerevisiae to construct a functional library in which recombinant P450s were co-expressed with yeast NADPH-P450 oxidoreductase. Using this library, the catalytic potentials of P450s against a wide variety of compounds were investigated. A functionomic survey allowed the discovery of novel catalytic properties of P. placenta P450s. The phylogenetic diversity of the CYP53 family in P. placenta was clear, and CYP53D2 is capable of converting stilbene derivatives. This is the first report of this peculiar function of the CYP53 family. Our increased understanding of the molecular and functional diversity of P450s in this fungus will facilitate comprehension of metabolic diversity in basidiomycetes and has future biotechnology applications.

Analysis of Expressed Sequence Tags from the Wood-Decaying Fungus Fomitopsis palustris and Identification of Potential Genes Involved in the Decay Process

Fomitopsis palustris, a brown-rot basidiomycete, causes the most destructive type of decay in wooden structures. In spite of its great economic importance, very little information is available at the molecular level regarding its complex decay process. To address this, we generated over 3,000 expressed sequence tags (ESTs) from a cDNA library constructed from F. palustris. Clustering of 3,095 high-quality ESTs resulted in a set of 1,403 putative unigenes comprising 485 contigs and 918 singlets. Homology searches based on BlastX analysis revealed that 78% of the F. palustris unigenes had a significant match to proteins deposited in the nonredundant databases. A subset of F. palustris unigenes showed similarity to the carbohydrateactive enzymes (CAZymes), including a range of glycosyl hydrolase (GH) family proteins. Some of these CAZymeencoded genes were previously undescribed for F. palustris but predicted to have potential roles in biodegradation of wood. Among them, we identified and characterized a gene (FpCel45A) encoding the GH family 45 endoglucanase. Moreover, we also provided functional classification of 473 (34%) of F. palustris unigenes using the Gene Ontology hierarchy. The annotated EST data sets and related analysis may be useful in providing an initial insight into the genetic background of F. palustris.

Multidimensional NMR analysis reveals truncated lignin structures in wood decayed by the brown rot basidiomycete Postia placenta

Lignocellulose biodegradation, an essential step in terrestrial carbon cycling, generally involves removal of the recalcitrant lignin barrier that otherwise prevents infiltration by microbial polysaccharide hydrolases. However, fungi that cause brown rot of wood, a major route for biomass recycling in coniferous forests, utilize wood polysaccharides efficiently while removing little of the lignin. The mechanism by which these basidiomycetes breach the lignin remains unclear. We used recently developed methods for solubilization and multidimensional (1) H-(13) C solution-state NMR spectroscopy of ball-milled lignocellulose to analyse aspen wood degraded by Postia placenta. The results showed that decay decreased the content of the principal arylglycerol-β-aryl ether interunit linkage in the lignin by more than half, while increasing the frequency of several truncated lignin structures roughly fourfold over the level found in sound aspen. These new end-groups, consisting of benzaldehydes, benzoic acids and phenylglycerols, accounted for 6-7% of all original lignin subunits. Our results provide evidence that brown rot by P. placenta results in significant ligninolysis, which might enable infiltration of the wood by polysaccharide hydrolases even though the partially degraded lignin remains in situ. Recent work has revealed that the P. placenta genome encodes no ligninolytic peroxidases, but has also shown that this fungus produces an extracellular Fenton system. It is accordingly likely that P. placenta employs electrophilic reactive oxygen species such as hydroxyl radicals to disrupt lignin in wood.

Identification and functional analysis of a gene encoding β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes.