Brown rot fungi show a two-step wood degradation mechanism comprising oxidative radical-based and enzymatic saccharification systems. Recent studies have demonstrated that the brown rot fungus Rhodonia placenta expresses oxidoreductase genes ahead of glycoside hydrolase genes and spatially protects the saccharification enzymes from oxidative damage of the oxidoreductase reactions. This study aimed to assess the generality of the spatial gene regulation of these genes in other brown rot fungi and examine the effects of carbon source on the gene regulation. Gene expression analysis was performed on 14 oxidoreductase and glycoside hydrolase genes in the brown rot fungus Gloeophyllum trabeum, directionally grown on wood, sawdust-agar, and glucose-agar wafers. In G. trabeum, both oxidoreductase and glycoside hydrolase genes were expressed at higher levels in sections behind the wafers. The upregulation of glycoside hydrolase genes was significantly higher in woody substrates than in glucose, whereas the oxidoreductase gene expression was not affected by substrates.

We assessed the efficacy of a discontinuous soil treatment using a diluent of fipronil suspension concentrate in controlling colonies of Coptotermes formosanus and Reticulitermes speratus. In-ground monitoring stations were installed at Isogi Park and Kindai University, and individual termites inhabiting the stations were collected for four or six years to determine the numbers and locations of colonies present in test areas before and after the discontinuous soil treatment. Microsatellite genotyping indicated that two C. formosanus and two R. speratus colonies in the test area at Isogi Park and five R. speratus colonies in the test area at Kindai University were active and that their territories fluctuated every year. One of the two C. formosanus colonies at Isogi Park and one of the five R. speratus colonies at Kindai University were subjected to discontinuous soil treatments with fipronil and were strongly affected by the treatment at the colony level, resulting in the suppression and possible elimination of colonies. Termite activity of the fipronil-treated colony of C. formosanus was detected within one week after the discontinuous soil treatment and was not found for more than two years (28 months), while termite activity of the fipronil-treated colony of R. speratus was detected within four days and three weeks after the discontinuous soil treatment and was not detected thereafter for three years. Fipronil residue analysis showed that workers of C. formosanus moved at least 28 m and that workers of R. speratus moved 6 m from the treated soil locations for up to three weeks.

Brown rot fungi have great potential in biorefinery wood conversion systems because they are the primary wood decomposers in coniferous forests and have an efficient lignocellulose degrading system. Their initial wood degradation mechanism is thought to consist of an oxidative radical-based system that acts sequentially with an enzymatic saccharification system, but the complete molecular mechanism of this system has not yet been elucidated. Some studies have shown that wood degradation mechanisms of brown rot fungi have diversity in their substrate selectivity. Gloeophyllum trabeum, one of the most studied brown rot species, has broad substrate selectivity and even can degrade some grasses. However, the basis for this broad substrate specificity is poorly understood. In this study, we performed RNA-seq analyses on G. trabeum grown on media containing glucose, cellulose, or Japanese cedar (Cryptomeria japonica) as the sole carbon source. Comparison to the gene expression on glucose, 1,129 genes were upregulated on cellulose and 1,516 genes were upregulated on cedar. Carbohydrate Active enZyme (CAZyme) genes upregulated on cellulose and cedar media by G. trabeum included glycoside hyrolase family 12 (GH12), GH131, carbohydrate esterase family 1 (CE1), auxiliary activities family 3 subfamily 1 (AA3_1), AA3_2, AA3_4 and AA9, which is a newly reported expression pattern for brown rot fungi. The upregulation of both terpene synthase and cytochrome P450 genes on cedar media suggests the potential importance of these gene products in the production of secondary metabolites associated with the chelator-mediated Fenton reaction. These results provide new insights into the inherent wood degradation mechanism of G. trabeum and the diversity of brown rot mechanisms.

Objectives: The aim of the study was to obtain information about the enzymatic properties of aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea (rCcAAO), which is classified into the auxiliary activities family 3 subfamily 2 (AA3_2). Results: The gene encoding AAO from the plant saprophytic basidiomycete Coprinopsis cinerea (CcAAO) was cloned, and the recombinant CcAAO (rCcAAO) was heterologously expressed in the methylotrophic yeast Pichia pastoris. The purified rCcAAO showed significant activity not only against trans,trans-2,4-hexadien-1-ol but also against a broad range of aromatic alcohols including aromatic compounds that were reported to be poor substrates for known AAOs. Moreover, site-directed mutagenesis analysis demonstrated that mutants with substitutions from leucine to phenylalanine and tryptophan at position 416 exhibited decreases of activity for aromatic alcohols but still maintained the activity for trans,trans-2,4-hexadien-1-ol. Conclusions: Leucine 416 in CcAAO contributes to the broad substrate specificity against various aromatic alcohols, which is useful for the production of hydrogen peroxide using this enzyme.

Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs catalyze the oxidative cleavage of glycosidic bonds after activation by an external electron donor. So far, only flavin-dependent oxidoreductases (from the auxiliary activity [AA] family AA3) have been shown to activate LPMOs. Here, we present LPMO activation by a pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase (PDH) from Coprinopsis cinerea, CcPDH, the founding member of the recently discovered auxiliary activity family AA12. CcPDH contains a C-terminal family 1 carbohydrate binding module (CBM1), an N-terminal family AA8 cytochrome domain, and a central AA12 dehydrogenase domain. We have studied the ability of full-length CcPDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs. The results show that CcPDH indeed can activate the C-1-oxidizing N. crassa LPMO 9F (NcLPMO9F) and the C-4-oxidizing Neurospora crassa LPMO 9C (NcLPMO9C), that this activation depends on the cytochrome domain, and that the dehydrogenase and the LPMO reactions are strongly coupled. The two tested CcPDH-LPMO systems showed quite different efficiencies, and this difference disappeared upon the addition of free PQQ acting as a diphenol/quinone redox mediator, showing that LPMOs differ when it comes to their direct interactions with the cytochrome domain. Surprisingly, removal of the CBM domain from CcPDH had a considerable negative impact on the efficiency of the CcPDH-LPMO systems, suggesting that electron transfer in the vicinity of the substrate is beneficial. CcPDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool for studying cellulose-oxidizing LPMOs.

A total of 114 microRNAs (miRNAs) were identified for Coptotermes formosanus workers, while a total of 97 miRNAs were identified for Reticulitermes speratus workers, of which 91 were common in C. formosanus and R. speratus. While the relationship between miRNA expression levels in C. formosanus and R. speratus workers had a strong positive correlation, considerable difference in miRNA expressions between C. formosanus and R. speratus was observed. Among the miRNAs up-regulated in C. formosanus workers, miR-11-3p showed the highest expression increase of 2.30 fold, while miR-13b-3p showed the highest expression increase of 5.16 fold among the up-regulated miRNAs in R. speratus workers. Workers of C. formosanus and R. speratus seem to use different miRNAs, miR-11-3p in C. formosanus or miR-13b-3p in R. speratus, to regulate homeodomain transcription factor genes, araucan, caupolican, and nubbin, basic helix-loop-helix (bHLH) transcription factor genes, spineless and E(spl)m3-HLH, and zinc finger transcription factor gene, castor.

The diversity and community structures of wood-inhabiting fungi in 16 decayed wood samples from ten wooden houses in Japan were analyzed using a next-generation sequencing (NGS) to determine the fungi responsible for wood decay. DNA of fungi in decayed wood was extracted directly, the internal transcribed spacer (ITS) region in ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR), and then, sequences of tagged ITS fragments were analyzed by NGS. Results of sequencing indicated that 68 species of ascomycetes, 37 species of basidiomycetes, and one fungus each from Mortierellales and Mucoromycetes were detected. The fungal community structures showed diversity and included various species of ascomycetes. A microscopic examination of cell wall structure in decayed wood samples suggested that some ascomycetes were soft-rot fungi. Heat map analysis indicated that the similarity in the structures of fungal communities was influenced to a greater extent by the wood species of samples than where they were used as a component.

Microbial volatile organic compounds (MVOCs) produced by the brown-rot fungus Fomitopsis palustris and white-rot fungus Trametes versicolor grown on wood chip and potato dextrose agar were analyzed by GC-MS. In total, 110 organic compounds were identified as MVOCs. Among them, only 23 were MVOCs commonly observed in both types of fungi, indicating that the fungi have differential MVOC expression profiles. In addition, F. palustris and T. versicolor produced 38 and 22 MVOCs, respectively, which were detected only after cultivation on wood chip. This suggests that the fungi specifically released these MVOCs when degrading the cell-wall structure of the wood. Time course analysis of MVOC emission showed that both types of fungi produced the majority of MVOCs during the active phase of wood degradation. As both fungi produced specific MVOCs in the course of wood degradation indicates the possibility of the application of MVOCs as detection markers for wood-decay fungus existing in woody materials.

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-D-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. 1H nuclear magnetic resonance (H-1-NMR) analysis of reaction products revealed 2-keto-D-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.

The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.

Pyrroloquinoline quinone (PQQ) is a redox cofactor utilized by a number of prokaryotic dehydrogenases. Not all prokaryotic organisms are capable of synthesizing PQQ, even though it plays important roles in the growth and development of many organisms, including humans. The existence of PQQ-dependent enzymes in eukaryotes has been suggested based on homology studies or the presence of PQQ-binding motifs, but there has been no evidence that such enzymes utilize PQQ as a redox cofactor. However, during our studies of hemoproteins, we fortuitously discovered a novel PQQ-dependent sugar oxidoreductase in a mushroom, the basidiomycete Coprinopsis cinerea. The enzyme protein has a signal peptide for extracellular secretion and a domain for adsorption on cellulose, in addition to the PQQ-dependent sugar dehydrogenase and cytochrome domains. Although this enzyme shows low amino acid sequence homology with known PQQ-dependent enzymes, it strongly binds PQQ and shows PQQ-dependent activity. BLAST search uncovered the existence of many genes encoding homologous proteins in bacteria, archaea, amoebozoa, and fungi, and phylogenetic analysis suggested that these quinoproteins may be members of a new family that is widely distributed not only in prokaryotes, but also in eukaryotes.

The crystal structure of the N-terminal putative catalytic domain of a glycoside hydrolase family 131 protein from Coprinopsis cinerea (CcGH131A) was determined. The structure of CcGH131A was found to be composed of a beta-jelly roll fold and mainly consisted of two beta-sheets, sheet-A and sheet-B. A concave of sheet-B, the possible active site, was wide and shallow, and three glycerol molecules were present in the concave. Arg96, Glu98, Glu138, and His218 are likely to be catalytically critical residues, and it was suggested that the catalytic mechanism of CcGH131A is different from that of typical glycosidases. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

褐色腐朽菌は針葉樹の主要な分解生物であり、木造建築物の腐朽被害および木質バイオマスの糖化技術への応用の観点から極めて重要な生物である。褐色腐朽菌は木材細胞壁の主成分であるセルロースを、非酵素的な酸化的分解反応により低分子化し、非晶性セルロース分解酵素により単糖にまで分解するという、二段階の反応で分解すると考えられてきた。しかし、本菌は他の糸状菌で知られる結晶性セルロース分解酵素を持たないことから、結晶性セルロースをどのように分解しているのか、その分解機構は明らかになっていない。近年、申請者らは褐色腐朽菌Gloeophyllum trabeumにおいて網羅的遺伝子発現解析から、ファミリー9溶解性多糖モノオキシゲナーゼ（LPMO9）、ファミリー14 LPMO（LPMO14）、エクスパンシン様タンパク質の3種類の結晶性セルロース分解への関与が予想されるタンパク質の遺伝子が、木質基質により顕著に発現増加することを見いだした。本研究では、これら3種類のタンパク質の機能解析を行い、これらのタンパク質の褐色腐朽菌における結晶性セルロース分解への関与を明らかにすることを目指している。本年度は、これら3種類のタンパク質の遺伝子を酵母菌Pichia pastorisに導入し、組換えタンパク質として発現させた。エクスパンシン様タンパク質の機能解析から、本タンパク質の糖質結合能が明らかになってきた。次年度の詳細な機能解析により、これら3種のタンパク質の生理的な機能が明らかになることが期待できる。