The pyrroloquinoline-quinone (PQQ)-dependent quinohemoprotein pyranose dehydrogenase from Coprinopsis cinerea (CcPDH), belonging to the AA12 family, drives lytic polysaccharide monooxygenase (LPMO) action

Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs accelerate the decomposition of cellulose by cellulases by catalyzing the oxidative cleavage of glycosidic bonds after activation by an external electron donor (1-3). LPMOs procure electrons from non-enzymatic electron donors, such as ascorbic acid, lignin and other plant biomass-derived phenols (1-3), or they can be activated by flavin-dependent oxidoreductases, directly or through plant-derived diphenols and quinones acting as redox mediators (3-7). Cellobiose dehydrogenase, in particular, efficiently transfers electrons from its AA3_1 dehydrogenase domain to LPMOs via an appended AA8 cytochrome domain (7). Here we show that LPMOs can be activated by a quinohemoprotein, namely the pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase CcPDH from Coprinopsis cinerea, the founding member of the recently discovered AA12 family (8). CcPDH has a domain composition similar to that of cellobiose dehydrogenases (CDHs) but contains a central catalytic AA12 dehydrogenase domain, rather than an AA3_1 domain. We have studied the ability of full length CcPDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs, NcLPMO9F and NcLPMO9C. Our study shows that both the AA8 and CBM1 domains of CcPDH have a positive effect on the CcPDH-NcLPMO system. The interplay between the PDH and LPMOs seemed also to depend on whether the LPMO contained a CBM. Unlike the single dehydrogenase domain of MtCDH from Myriococcum thermophylum, the AA12 dehydrogenase domain of CcPDH could drive the LPMO reaction, which is due to the non-covalently bound PQQ co-factor acting as a diphenol/quinone redox mediator. CcPDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool in future studies of LPMOs and redox enzyme systems involved in cellulose degradation. References: [1] Vaaje-Kolstad, G. et al. (2010) Science 330, 219-222. [2] Hemsworth, G. R., et al. (2015) Trends Biotechnol 33:747-761. [3] Kracher, D. et al. (2016) Science 352, 1098-1101. [4] Westereng, B. et al. (2015) Sci Rep 5:18561. [5] Langston, J.A. et al. (2011) Appl Environ Microbiol 77:7007-7015. [6] Garajova, S. et al. (2016) Sci Rep 6:28276. [7] Tan, T.C. et al. (2015) Nat Commun 6:7542. [8] Takeda, K. et al. (2015) PLoS One 10:e0115722

Transcriptome analysis of the brown rot fungus \u3cem\u3eGloeophyllum trabeum\u3c/em\u3e during lignocellulose degradation

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

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea

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

Exploration of key enzymes for crystalline cellulose degradation in brown rot fungi

褐色腐朽菌における結晶性セルロースの分解機構を明らかにすべく、申請者らが網羅的遺伝子発現解析から見いだした、木質基質により顕著に発現増加する3種類のタンパク質である、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機能解析を行った。本研究では、これらの3種類のタンパク質を組換えタンパク質として発現させ、その糖質加水分解酵素との相乗作用および糖質結合能の調査を進めることで、その機能の解明につながる新たな知見を得ることができた。To clarify the degradation mechanism of crystalline cellulose in brown rot fungi, the functions of three proteins, a family 9 lytic polysaccharide monooxygenase (LPMO9), a family 14 lytic polysaccharide monooxygenase (LPMO14) and an expansin-like protein, which significantly up-regulated by woody substrates were analysed. These three proteins were expressed as recombinant proteins and their synergistic effects with glycoside hydrolases and their carbohydrate-binding ability were investigated. These results provide new insights into their physiological functions in brown rot fungi.研究分野：木材腐朽菌

Colony Suppression and Possible Colony Elimination of the Subterranean Termites Coptotermes formosanus and Reticulitermes speratus by Discontinuous Soil Treatment Using a Diluent of Fipronil Suspension Concentrate

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

Transcriptome analysis of the brown rot fungus Gloeophyllum trabeum during lignocellulose degradation.

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

Discovery of a eukaryotic pyrroloquinoline quinone-dependent oxidoreductase belonging to a new auxiliary activity family in the database of carbohydrate-active enzymes.

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

3D structure model of <i>Cc</i>SDH showing its three domains the N-terminal AA family 8 cytochrome domain (<i>orange</i>), the PQQ-dependent dehydrogenase domain (<i>magenta</i>), and the C-terminal cellulose-binding domain belonging to CBM family 1 (<i>green</i>).

<p>PQQ of sGDH from <i>Acinetobacter calcoaceticus</i> and heme in the cytochrome domain of <i>P. chrysosporium</i> CDH (PDBID 1CRU and 1D7C, respectively) were superimposed to the model.</p

Phylogenetic tree of prokaryotic and eukaryotic quinoproteins.

<p>The phylogenetic tree was generated from the amino acid sequences of <i>Cc</i>SDH, proteins homologous to <i>Cc</i>SDH, and known quinoproteins using ClustalX (ver. 2.1) with the neighbor-joining method.</p

Alignment of the amino acid sequences of the catalytic domain of <i>Cc</i>SDH and known structure of six-bladed quinoproteins.

<p>Perfect matches are enclosed in boxes with a black background. Boxes with a magenta background indicate the amino acid residues interacting with PQQ via direct hydrogen bonds in known structures. The filled arrowhead indicates the position of the proposed catalytic histidine in bacterial quinoproteins. Conserved residues involved in PQQ-binding in known quinoproteins are indicated by open arrowheads if also conserved in <i>Cc</i>SDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104851#pone.0104851-Oubrie3" target="_blank">[32]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104851#pone.0104851-Southall1" target="_blank">[34]</a>.</p