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Fungi defend their ecological niche against antagonists by producing antibiosis molecules. Some of these molecules are only produced upon confrontation with the antagonist. The basidiomycete Coprinopsis cinerea induces the expression of the sesquiterpene synthase-encoding gene cop6 and its two neighboring genes coding for cytochrome P450 monooxygenases in response to bacteria. We further investigated this regulation of cop6 and examined if the gene product is involved in the production of antibacterials. Cell-free supernatants of axenic cultures of the Gram-positive bacterium Bacillus subtilis were sufficient to induce cop6 transcription assessed using a fluorescent reporter strain. Use of this strain in a microfluidic device revealed that the cop6 gene was induced in all hyphae directly exposed to the supernatant and that induction occurred within less than one hour. Targeted replacement of the cop6 gene demonstrated the requirement of the encoded synthase for the biosynthesis of the sesquiterpene lagopodin B, a previously reported antibacterial compound from related species. Accordingly, lagopodin B from C. cinerea inhibited the growth of several Gram-positive bacteria including B. subtilis but not Gram-negative bacteria. Our results demonstrate that the C. cinerea vegetative mycelium responds to soluble compounds of a bacterial culture supernatant by local production of an antibacterial secondary metabolite.

Hexarates are platform chemicals. Methods to produce d-glucarate and d-mannarate are desirable because these hexarates can be gained by oxidation of the corresponding hexuronates, which are abundantly found in algae and plants as the units of polyuronates. Oxidative production of the hexarates can be combined with a reductive reaction to coproduce electricity. An enzymatic biofuel cell is a device that enables this coproduction. To construct the cell, it is necessary to find enzymes that catalyze platform chemical production and are also suitable as anode catalysts. Here, we show the production of d-glucarate and d-mannarate from d-glucuronate and d-mannuronate, with both reactions catalyzed by pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase, in addition to the production of d-glucarate from l-guluronate by the PQQ domain of pyranose dehydrogenase from Coprinopsis cinerea. The enzymes are suitable as anode catalysts in biofuel cells that coproduce these hexarates and electricity.

In this study, trends in synonymous codons usage of Volvariella volvecea have been first examined by analysis of complete coding sequences and gene chip data. The results showed that GC content at three codon positions are obviously different and there were several factors shaping the codon usage of V. volvacea genes, including base composition. The comparison of codon usage among four edible fungi such as V. volvacea, Agaricus bisporus, Coprinopsis cinerea, and Pleurotus ostreatus indicated that the similar codon usage pattern was used among V. volvacea, A. bisporus and P. ostreatus, but there was significantly different codon usage pattern of C. cinerea. Two arrays of optimal codons were determined by effective number of codons (ENC) values and gene chip database separately, resulting that most of the ENC-predicted optimal codons were included in the array of gene chip resulted optimal codons. This study can provide useful information for codon usage pattern analysis and gene transformation of V. volvacea.

Galactoseβ1-4fucose (Galβ1-4Fuc), a unique disaccharide unit found only on the N-glycans of Protostomia, has been intensively studied, particularly in Nematoda. Galβ1-4Fuc attached to the 6-OH of the innermost GlcNAc of N-glycans has been identified as an endogenous target recognized by Caenorhabditis elegans galectin LEC-6 and might function as an endogenous ligand for other galectins as well. Interactions between galectins and N-glycans might be subject to fine-tuning through modifications of the penultimate GlcNAc and the Galβ1-4Fuc unit. Similar fine-tuning is also observable in vertebrate galectins, although their major recognition unit is a Galβ1-4GlcNAc. In Protostomia, it can be postulated that glycan-binding proteins and their ligands have coevolved; however, epitopes such as Galβ1-4Fuc were then hijacked as targets by other organisms. Fungal (Coprinopsis cinerea) galectin 2, CGL2, binds the Galβ1-4Fuc on C. elegans glycans to exert its nematotoxicity. Some human and mouse galectins bind to synthesized Galβ1-4Fuc; as some parasitic nematodes express this motif, its recognition by mammalian galectins could hypothetically be involved in host defense, similar to fungal CGL2. In this review, we discuss the Galβ1-4Fuc unit in Protostomia as a possible equivalent for the Galβ1-4GlcNAc unit in vertebrates and a potential non-self glycomarker useful for pathogen recognition.

Monohydroxylated metabolites of vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol), generically known as 25-hydroxycalciferol, are better for several diseases, and other applications, than vitamin D (calciferol). This work describes a novel biotechnological approach for the preparation of 25-hydroxycalciferols, starting from readily available cholecalciferol and ergocalciferol. This approach enables the regioselective (100 %) hydroxylation of these compounds (at the C-25 position) under mild and environmentally friendly conditions by using a peroxidase from the fungus Coprinopsis cinerea (gene model

(E, E, E)-Geranylgeraniol (GGOH) is a valuable ingredient of many perfumes and a valuable precursor for synthesizing pharmaceuticals. In an attempt to increase the GGOH concentration in Coprinopsis cinerea, we demonstrated that the expression of geranylgeranyl diphosphate synthase (ggpps) gene isolated from Taxus x media could promote GGOH production. Furthermore, the concentrations of squalene and ergosterol were measured in the engineered strains. Expectedly, significant decreases of squalene and ergosterol levels were observed in those strains transformed with ggpps gene. This could be explained by the partial redirection of metabolic flux from squalene to GGOH, whose biosynthesis competes for the same precursor with squalene. This work suggested that the expression of ggpps in higher fungi was an effective method for bio-production of GGOH.

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 β-jelly roll fold and mainly consisted of two β-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.

The basidiomycete Coprinopsis cinerea produces five cellobiohydrolases belonging to glycoside hydrolase family 6 (GH6). Among these enzymes, C. cinerea cellulase 6C (CcCel6C), but not C. cinerea cellulase 6A (CcCel6A), can efficiently hydrolyze carboxymethyl cellulose and is constitutively expressed in C. cinerea. In contrast, CcCel6A possesses a cellulose-binding domain, and is strongly induced by cellobiose. Here, we determined the crystal structures of the CcCel6A catalytic domain complexed with a Hepes buffer molecule, with cellobiose, and with p-nitrophenyl β-D-cellotrioside (pNPG3). A notable feature of the GH6 cellobiohydrolases is that the active site is enclosed by two loops to form a tunnel, and the loops have been demonstrated to open and close in response to ligand binding. The enclosed tunnel of CcCel6A-Hepes is seen as the open form, whereas the tunnels of CcCel6A-cellobiose and CcCel6A-pNPG3 adopt the closed form. pNPG3 was not hydrolyzed by CcCel6A, and bound in subsites +1 to +4. On the basis of this observation, we constructed two mutants, CcCel6A D164A and CcCel6C D102A. Neither CcCel6A D164A nor CcCel6C D102A hydrolyze phosphoric acid-swollen cellulose. We have previously determined the crystal structures of CcCel6C unbound and in complex with ligand, both of which adopt the open form. In the present study, both CcCel6A and CcCel6C mutants were identified as the closed form. However, the motion angle of CcCel6C was more than 10-fold greater than that of CcCel6A. The width of the active site cleft of CcCel6C was narrowed, owing to a tweezer-like motion.

Fruiting body lectins are ubiquitous in higher fungi and characterized by being synthesized in the cytoplasm and up-regulated during sexual development. The function of these lectins is unclear. A lack of phenotype in sexual development upon inactivation of the respective genes argues against a function in this process. We tested a series of characterized fruiting body lectins from different fungi for toxicity towards the nematode Caenorhabditis elegans, the mosquito Aedes aegypti and the amoeba Acanthamoeba castellanii. Most of the fungal lectins were found to be toxic towards at least one of the three target organisms. By altering either the fungal lectin or the glycans of the target organisms, or by including soluble carbohydrate ligands as competitors, we demonstrate that the observed toxicity is dependent on the interaction between the fungal lectins and specific glycans in the target organisms. The toxicity was found to be dose-dependent such that low levels of lectin were no longer toxic but still led to food avoidance by C. elegans. Finally, we show, in an ecologically more relevant scenario, that challenging the vegetative mycelium of Coprinopsis cinerea with the fungal-feeding nematode Aphelenchus avenae induces the expression of the nematotoxic fruiting body lectins CGL1 and CGL2. Based on these findings, we propose that filamentous fungi possess an inducible resistance against predators and parasites mediated by lectins that are specific for glycans of these antagonists.

The basidiomycete Coprinopsis cinerea produces the glycoside hydrolase family 6 enzyme CcCel6C at low and constitutive levels. CcCel6C exhibits unusual cellobiohydrolase activity; it hydrolyses carboxymethyl cellulose, which is a poor substrate for typical cellobiohydrolases. Here, we determined the crystal structures of CcCel6C unbound and in complex with p-nitrophenyl β-d-cellotrioside and cellobiose. CcCel6C consists of a distorted seven-stranded β/α barrel and has an enclosed tunnel, which is observed in other cellobiohydrolases from ascomecetes Hypocrea jecorina (HjeCel6A) and Humicola insolens (HinCel6A). In HjeCel6A and HinCel6A, ligand binding produces a conformational change that narrows this tunnel. In contrast, the tunnel remains wide in CcCel6C and the conformational change appears to be less favourable than in HjeCel6A and HinCel6A. The ligand binding cleft for subsite −3 of CcCel6C is also wide and is rather similar to that of endoglucanase. These results suggest that the open tunnel and the wide cleft are suitable for the hydrolysis of carboxymethyl cellulose.

Basidiomycetes inhabiting lignocellulose comprise an important class of filamentous fungi with ecological and biotechnological relevance. Extracellular enzymes of wood-degrading fungi such as laccases, manganese-dependent (or independent) peroxidases, cellulases and xylanases are of considerable interest for biotechnological applications. Still, proteomic studies of fungal secretomes based on 2-DE can be very challenging due to low protein concentrations and variable amounts of fungal metabolites. Comparison of different standard methods for protein precipitation has demonstrated their limited applicability to fungal secretomes. The extracellular metabolites impair standard methods for protein quantification and can result in a strong overestimation of total protein. We have developed an optimized protocol for the precipitation of extracellular proteins from liquid cultures of Coprinopsis cinerea growing in an exponential phase on a complex media. We found that a considerable amount of gelatinous material can be removed from the liquid culture supernatants by high-speed centrifugation. Fungal proteins can be effectively enriched by TCA precipitation and coprecipitated metabolites hampering 2-DE can be removed through the application of Tris/acetone. Following our protocol makes it possible to concentrate proteins from culture supernatants and to simultaneously remove most of the impeding compounds from a given sample. We have applied this procedure in the 2-DE of secretomes from the model organism C. cinerea as well as other basidiomycetes such as Pleurotus ostreatus, Phanerochaete chrysosporium, Polyporus brumalis and Schizophyllum commune.

International audience; In forest soils, ectomycorrhizal and saprotrophic Agaricales differ in their strategies for carbon acquisition, but share common gene families encoding multi-copper oxidases (MCOs). These enzymes are involved in the oxidation of a variety of soil organic compounds. The MCO gene family of the ectomycorrhizal fungus Laccaria bicolor is composed of 11 genes divided into two distinct subfamilies corresponding to laccases (lcc) sensu stricto (lcc1 to lcc9), sharing a high sequence homology with the coprophilic Coprinopsis cinerea laccase genes, and to ferroxidases (lcc10 and lcc11) that are not present in C. cinerea. The fet3-like ferroxidase genes lcc10 and lcc11 in L. bicolor are each arranged in a mirrored tandem orientation with an ftr gene coding for an iron permease. Unlike C. cinerea, L. bicolor has no sid1/sidA gene for siderophore biosynthesis. Transcript profiling using whole-genome expression arrays and quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) revealed that some transcripts were very abundant in ectomycorrhizas (lcc3 and lcc8), in fruiting bodies (lcc7) or in the free-living mycelium grown on agar medium (lcc9 and lcc10), suggesting a specific function of these MCOs. The amino acid composition of the MCO substrate binding sites suggests that L. bicolor MCOs interact with substrates different from those of saprotrophic fungi.

Sexual development in the mushroom Coprinus cinereus is under the control of two mating type loci, A and B. When two haploid homokaryons with compatible alleles at both A and B loci are mated, the coordinated activities of A- and B-regulated pathways lead to formation of a mycelium termed the dikaryon, in which the two nuclei from the mating partners pair in each cell without fusing. The dikaryon is a prolonged mycelial stage that can be induced to develop a multicellular structure, the mushroom, under proper environmental conditions. The two nuclei fuse in specialized cells on the mushroom and immediately undergo meiosis to complete the sexual life cycle. It has been established recently that the A genes encode two classes of homeodomain proteins while the B genes encode pheromones and their receptors. More recently, molecular genetics has been used to reveal genes that work downstream of the mating type genes to regulate dikaryon formation, mushroom morphogenesis, and meiosis.