469 results on '"Coprinopsis cinerea"'
Search Results
202. Overproduction of geranylgeraniol inCoprinopsis cinereaby the expression of geranylgeranyl diphosphate synthase gene
- Author
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Jun-Fang Lin, Li-Qiong Guo, Tao Ren, Lin-Feng You, and Jian-Rong Wang
- Subjects
Ergosterol ,Farnesyltranstransferase ,biology ,General Medicine ,biology.organism_classification ,Applied Microbiology and Biotechnology ,chemistry.chemical_compound ,Coprinopsis cinerea ,Squalene ,Taxus ,chemistry ,Biochemistry ,Biosynthesis ,Geranylgeraniol ,Overproduction - Abstract
(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.
- Published
- 2014
203. An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase activity
- Author
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Manfred Nimtz, Nicole Lehnert, Diana Linke, and Ralf G. Berger
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Molecular Sequence Data ,Bioengineering ,Phanerochaete ,Applied Microbiology and Biotechnology ,Biochemistry ,Substrate Specificity ,Fungal Proteins ,chemistry.chemical_compound ,Glycerol ,Amino Acid Sequence ,DNA, Fungal ,chemistry.chemical_classification ,Base Sequence ,Sequence Homology, Amino Acid ,biology ,Hydrogen-Ion Concentration ,biology.organism_classification ,Enzyme assay ,Alcohol oxidase ,Alcohol Oxidoreductases ,Coprinopsis cinerea ,Enzyme ,chemistry ,biology.protein ,Gloeophyllum trabeum ,Methanol ,Sugar Alcohol Dehydrogenases ,Biotechnology - Abstract
An intracellular alcohol oxidase (AOX) was isolated from the white-rot basidiomycete Phanerochaete chrysosporium (Pch), grown on l-lactate induction medium, and purified to electrophoretic homogeneity. The dimeric protein consisted of two identical 75kDa subunits. The open reading frame of 1,956bp resulted in a monomer consisting of 651 amino acids. The enzyme showed a pI at 5.4, a pH optimum of 9, a temperature optimum at 50°C, possessed putative conserved domains of the GMC superfamily, a FAD binding domain, and showed up to 86% homology to alcohol oxidase sequences of Gloeophyllum trabeum and Coprinopsis cinerea. As was shown for the first time for an AOX from a basidiomycete, not only methanol, but also lower primary alcohols and glycerol were accepted as substrates. An assay based on aldehyde dehydrogenase confirmed d-glyceraldehyde as the product of the reaction. A bioprocess based on this enzyme could alleviate the problems associated with the huge side-stream of glycerol occurring during the manufacture of biodiesel, yielding the green oxidant hydrogen peroxide.
- Published
- 2014
204. Induction of a laccase Lcc9 from Coprinopsis cinerea by fungal coculture and its application on indigo dye decolorization
- Author
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Wei Fang, Zemin Fang, Qiang Yin, Xiaolan Xu, Tianwei Zhang, Yuzhi Hong, Yazhong Xiao, Kai Pan, and Nannan Zhao
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Environmental Engineering ,Nitrogen ,Color ,Bioengineering ,Gongronella sp ,Indigo Carmine ,Indigo ,Substrate Specificity ,Microbiology ,Coprinus ,chemistry.chemical_compound ,Enzyme Stability ,High activity ,Organic Chemicals ,Coloring Agents ,Waste Management and Disposal ,Incubation ,Laccase ,chemistry.chemical_classification ,biology ,Renewable Energy, Sustainability and the Environment ,Temperature ,Indigo dye ,General Medicine ,Hydrogen-Ion Concentration ,biology.organism_classification ,Carbon ,Coculture Techniques ,Native Polyacrylamide Gel Electrophoresis ,Coprinopsis cinerea ,Biodegradation, Environmental ,Enzyme ,chemistry ,Enzyme Induction ,Solvents ,Extracellular Space ,Nuclear chemistry - Abstract
A fungal coculture system comprised of Coprinopsis cinerea Okayama 7 (#130) and Gongronella sp. w5 produced 900 times higher laccase activity than that in pure culture. A fungal laccase named Lcc9 was induced from C. cinerea for the first time by coculture. Lcc9 was purified, characterized, and found to have high activity toward phenolic substrates at the optimum pH of 6.5 and temperature of 60°C. The laccase was stable at alkaline pH values, and its activity was not significantly affected by cations and organic solvents. Lcc9 showed decolorization capability toward indigo dye in the presence of 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate), with 75% of indigo was decolorized by 50 U/L enzyme after 1h of incubation under optimal catalytic conditions. These results showed that fungal coculture could active silent laccase gene, and the unusual properties make Lcc9 a candidate for specific industrial and environmental applications.
- Published
- 2014
205. Phenol oxidation by DyP-type peroxidases in comparison to fungal and plant peroxidases
- Author
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Holger Zorn, Dietmar A. Plattner, Eric Strittmatter, René Ullrich, Klaus Piontek, Elizabet Aranda, Christiane Liers, and Martin Hofrichter
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chemistry.chemical_classification ,biology ,Compound II/resting state ,Process Chemistry and Technology ,Bioengineering ,Lignin peroxidase ,biology.organism_classification ,Redox ,Biochemistry ,Catalysis ,Coprinopsis cinerea ,chemistry.chemical_compound ,Dye-decolorizing peroxidases ,Enzyme ,chemistry ,biology.protein ,Phanerochaete ,Lignin ,Organic chemistry ,Phenols ,Redox potential ,Peroxidase - Abstract
Over the last years, novel peroxidases secreted by lignocellulose-degrading agaricomycetes have been discovered. Among them, the so-called DyP-type peroxidases (DyPs) that are secreted under conditions close to nature (i.e. in wood cultures) are of particular interest, since they are able to oxidize diverse substrates including veratryl alcohol, non-phenolic lignin model dimers as well as recalcitrant phenols and dyes. In spite of their unique protein structure and their catalytic versatility, the estimation of the redox potential of this new peroxidase group is still pending. To solve this problem, we used a catalytic approach developed by Ayala et al., 2007 [21] , which is based on the Marcus equation and the determination of the redox thermodynamics between heme-peroxidase compound II and the resting state enzyme. Five fungal DyPs (among them four wild-type enzymes and one recombinant protein) were tested regarding phenol oxidation in comparison to other well-studied plant and fungal peroxidases (soybean peroxidase, SBP, Coprinopsis cinerea peroxidase, CiP, lignin peroxidase of Phanerochaete chrysosporium , LiP). DyP-type peroxidases have a high affinity for phenols and can oxidize even recalcitrant representatives such as p -nitrophenol. Based on this “phenol oxidation method”, their redox potential was estimated to range between 1.10 ± 0.02 and 1.20 ± 0.1 V, which is between the values calculated for high-redox potential LiP (1.26 ± 0.17 V) and low-redox potential, phenol-oxidizing plant (0.93 ± 0.04 V for SBP) and fungal (1.06 ± 0.07 V for CiP) peroxidases.
- Published
- 2014
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206. Phanerochaete chrysosporium inoculation shapes the indigenous fungal communities during agricultural waste composting
- Author
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Weimin Sun, Jiachao Zhang, Ming Chen, Hongli Huang, Chang Zhang, Jie Liang, Man Yu, Yaoning Chen, Binbin Huang, Guangming Zeng, and Yi Zhu
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Environmental Engineering ,biology ,Inoculation ,Agriculture ,Bioengineering ,Geotrichum ,Biodegradation ,Phanerochaete ,biology.organism_classification ,Galactomyces ,Pollution ,Microbiology ,Coprinopsis cinerea ,Horticulture ,Biodegradation, Environmental ,Environmental Chemistry ,Microbial inoculant ,Soil Microbiology ,Chrysosporium - Abstract
Inoculation with exogenous white-rot fungi has been proven to be an efficient method to promote lignocellulose biodegradation during agricultural waste composting. Indigenous fungal communities, the most important organisms responsible for mineralization and decomposition of lignocellulosic materials in composts, can be affected by sample properties and other biotic factors. This research was conducted to determine the effects of the Phanerochaete chrysosporium inoculation on the indigenous fungal communities during agricultural waste composting. Fungal communities in samples with different inoculation regimes were investigated by sequencing and quantitative PCR. Results showed that P. chrysosporium inoculants produced significant negative effects on the indigenous fungal community abundance during the thermophilic stage. Samples inoculated during Phase II contained higher proportion of Acremonium chrysogenum and Galactomyces geotrichum, while those non-inoculated samples were dominated by Coprinopsis cinerea and Scytalidium thermophilum. Moreover, the indigenous fungal community abundance was significantly correlated with the C/N ratio, water soluble carbon and moisture content (P < 0.05). Redundancy analysis indicated that the most variation in distribution of indigenous fungal community structure was statistically explained by nitrate, C/N ratio, and moisture content, factors which solely explained 29.6 % (F = 30.316, P = 0.002), 25.6 % (F = 26.191, P = 0.002) and 10.0 % (F = 10.249, P = 0.002) of the variation in the indigenous fungal community structure, respectively.
- Published
- 2014
207. Heterologous expression and characterization of laccase 2 fromCoprinopsis cinereacapable of decolourizing different recalcitrant dyes
- Author
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Wang Rongtan, Rihe Peng, Quan-Hong Yao, Yongsheng Tian, and Hu Xu
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Laccase ,ABTS ,Chromatography ,biology ,biology.organism_classification ,Microbiology ,Pichia pastoris ,chemistry.chemical_compound ,Coprinopsis cinerea ,chemistry ,Methyl orange ,Guaiacol ,Crystal violet ,Malachite green ,Biotechnology - Abstract
The gene (CcLcc2) encoding laccase from the basidiomycete Coprinopsis cinerea Okayama-7 #130 was synthesized by polymerase chain reaction-based two-step DNA synthesis, and heterologously expressed in Pichia pastoris. The recombinant protein was purified by ammonium sulphate precipitation and nickel nitrilotriacetic acid chromatography. The molecular mass of CcLcc2 was estimated to be 54 kDa by denaturing polyacrylamide gel electrophoresis. The optimum pH and temperature for laccase catalysis for the oxidation of 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) were 2.6 and 45 °C, respectively. The Km values of the enzyme towards the substrates ABTS, 2,6-dimethoxyphenol (2,6-DMP) and guaiacol were 0.93, 1.02 and 28.07 mmol·L−1, respectively. The decolourization of methyl orange, crystal violet and malachite green, commonly used in the textile industry, was assessed. The decolourization percentage of crystal violet and malachite green was 80% after 4 h of reaction, and that of methyl orange was 50...
- Published
- 2014
208. Dimerization of the fungal defense lectin CCL2 is essential for its toxicity against nematodes
- Author
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Annabelle Varrot, Mario Schubert, Ramon Sieber, Jean-Maurice Mallet, Michael O. Hengartner, Markus Künzler, Katrin Stutz, Mayeul Collot, Silvia Bleuler-Martinez, University of Zurich, Künzler, Markus, Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Zürich University of Applied Sciences (ZHAW), Institute for Molecular Biology and Biophysics (IMBB), Centre de Recherches sur les Macromolécules Végétales (CERMAV ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institute of Medical Microbiology [Zurich], and Universität Zürich [Zürich] = University of Zurich (UZH)
- Subjects
0301 basic medicine ,1303 Biochemistry ,Nematodes ,[SDV]Life Sciences [q-bio] ,Protein dimer ,610 Medicine & health ,Toxin ,Dimer ,Glycan binding ,Carbohydrate recognition ,Fucose ,N-glycan core ,Insects ,142-005 142-005 ,Biochemistry ,Fungal Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Polysaccharides ,Animals ,Lectins, C-Type ,Binding site ,Caenorhabditis elegans ,ComputingMilieux_MISCELLANEOUS ,Binding Sites ,biology ,Effector ,Lectin ,biology.organism_classification ,Coprinopsis cinerea ,030104 developmental biology ,Amino Acid Substitution ,chemistry ,Toxicity ,biology.protein ,Drosophila melanogaster ,Agaricales ,Dimerization ,Trisaccharides - Abstract
Glycobiology, 27 (5), ISSN:0959-6658
- Published
- 2016
209. Extracellular Carbohydrate Esterase from the Basidiomycete Coprinopsis cinerea Released Ferulic and Acetic Acids from Xylan.
- Author
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Hashimoto, Kohsuke, Kaneko, Satoshi, and Yoshida, Makoto
- Subjects
- *
EXTRACELLULAR enzymes , *BASIDIOMYCETES , *RECOMBINANT proteins , *ACETIC acid , *PICHIA pastoris , *XYLANS - Abstract
The article presents a study on extracellular carbohydrate esterase (CcEst1), which is cloned from the basidiomycete Coprinopsis cinerea. It says that the recombinant CcEst1 showed in Pichia pastoris advanced on p-nitrophenyl acetate, α-naphthyl acetate, and methyl hydroxycinnamic acids. It mentions that the enzyme released from the basidiomycete Coprinopsis cinerea which are ferulic and acetic acids are coming from the wheat arabinixylan and acetylated xylan.
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- 2010
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210. Characterization of Glycoside Hydrolase Family 6 Enzymes from Coprinopsis cinerea.
- Author
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Liu, Yuan, Igarashi, Kiyohiko, Kaneko, Satoshi, Tonozuka, Takashi, Samejima, Masahiro, Fukuda, Kiyoharu, and Yoshida, Makoto
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- *
GLYCOSIDES , *CELLULOSE spectra , *BACTERIAL antigens , *ENZYME analysis ,CELLULOSE microbiology - Abstract
The article discusses a study which examines the glycoside hydrolase family 6 (GH6) composed of bacterial and fungal cellulases, enzymes, and cellobiohyrolase. It states that the enzymes have undegone study for hydrolysis of microcrystalline cellulose Avicel. Moreover, significant amount of celloboise is detected in Ce16 from Coprinopsis cinerea (CcCe16) after 72 hours of incubation.
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- 2009
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211. Modified recipe to inhibit fruiting body formation for living fungal biomaterial manufacture
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Man Kit Cheung, Hoi Shan Kwan, Po Lam Chan, Yichun Xie, Jinhui Chang, and Ka Lee Ma
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Fungal Structure ,Organogenesis ,Pleurotus djamor ,Gene Expression ,Biocompatible Materials ,02 engineering and technology ,Biochemistry ,Glycogen Synthase Kinase 3 ,Cell Signaling ,Agaricales ,Enzyme Inhibitors ,Post-Translational Modification ,Phosphorylation ,Mycelium ,0303 health sciences ,Mushroom ,Multidisciplinary ,Protein Kinase Signaling Cascade ,biology ,Chemistry ,Eukaryota ,food and beverages ,Biomaterial ,021001 nanoscience & nanotechnology ,Signaling Cascades ,Enzymes ,Coprinopsis cinerea ,Medicine ,0210 nano-technology ,Research Article ,Signal Transduction ,Science ,Mycology ,03 medical and health sciences ,Genetics ,Fruiting Bodies, Fungal ,030304 developmental biology ,Spore-Bearing Organ Development ,fungi ,Fungi ,Organisms ,Biology and Life Sciences ,Proteins ,Cell Biology ,Fruiting body formation ,biology.organism_classification ,Enzymology ,Lower cost ,Lithium Chloride ,Organism Development ,Protein Kinases ,Biomarkers ,Developmental Biology - Abstract
Living fungal mycelium with abolished ability to form fruiting bodies is a self-healing substance, which is particularly valuable for further engineering and development as materials sensing environmental changes and secreting signals. Suppression of fruiting body formation is also a useful tool for maintaining the stability of a mycelium-based material with ease and lower cost. The objective of this study was to provide a biochemical solution to regulate the fruiting body formation, which may replace heat killing of mycelium in practice. The concentrations of glycogen synthase kinase-3 (GSK-3) inhibitors, such as lithium chloride or CHIR99021 trihydrochloride, were found to directly correlate with the development of fruiting bodies in the mushroom forming fungi such as Coprinopsis cinerea and Pleurotus djamor. Sensitive windows to these inhibitors throughout the fungal life cycle were also identified. We suggest the inclusion of GSK-3 inhibitors in the cultivation recipes for inhibiting fruiting body formation and regulating mycelium growth. This is the first report of using a GSK-3 inhibitor to suppress fruiting body formation in living fungal mycelium-based materials. It provides an innovative strategy for easy, reliable, and low cost maintenance of materials containing living fungal mycelium.
- Published
- 2019
212. Molecular Mechanism by Which the GATA Transcription Factor CcNsdD2 Regulates the Developmental Fate of Coprinopsis cinerea under Dark or Light Conditions.
- Author
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Liu C, Kang L, Lin M, Bi J, Liu Z, and Yuan S
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- Hyphae, Mycelium, Fungal Proteins genetics, Gene Expression Regulation, Fungal, GATA Transcription Factors genetics, GATA Transcription Factors metabolism, Agaricales metabolism
- Abstract
Coprinopsis cinerea has seven homologs of the Aspergillus nidulans transcription factor NsdD. Of these, CcNsdD1 and CcNsdD2 from C. cinerea show the best identities of 62 and 50% to A. nidulans NsdD, respectively. After 4 days of constant darkness cultivation, CcnsdD2 , but not CcnsdD1 , was upregulated on the first day of light/dark cultivation to induce fruiting bodies, and overexpression of CcnsdD2 , but not CcnsdD1 , produced more fruiting bodies under a light/dark rhythm. Although single knockdown of CcnsdD2 did not affect fruiting body production due to upregulation of its homolog CcnsdD1 , the double-knockdown CcNsdD1/NsdD2-RNAi transformant showed defects in fruiting body formation under a light/dark rhythm. Knockdown of CcnsdD1 / nsdD2 led to the differentiation of primary hyphal knots into sclerotia rather than secondary hyphal knots under a light/dark rhythm, similar to the differentiation of primary hyphal knots into sclerotia of the wild-type strain under darkness. The CcNsdD2-overexpressing transformant produced more primary hyphal knots, secondary hyphal knots, and fruiting bodies under a light/dark rhythm but only more primary hyphal knots and sclerotia under darkness. RNA-seq revealed that some genes reported previously to be involved in formation of hyphal knots and primordia, cyclopropane-fatty-acyl-phospholipid synthases cfs1-3 , galectins cgl1-3 , and hydrophobins hyd1-3 were downregulated in the CcNsdD1/NsdD2-RNAi transformant compared to the mock transformant. ChIP-seq and electrophoretic mobility shift assay demonstrated that CcNsdD2 bound to promoter regulatory sequences containing a GATC motif in cfs1 , cfs2 , cgl1 , and hyd1 . A molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions is proposed. IMPORTANCE The model mushroom Coprinopsis cinerea exhibits remarkable photomorphogenesis during fruiting body development. This study reports that the C. cinerea transcription factor CcNsdD2 promotes primary hyphal knot formation by upregulating cfs1 , cfs2 , cgl1 , and hyd1 . Although the induction of CcnsdD2 is not under direct control of light and photoreceptors, the CcNsdD2-mediated developmental fates of the primary hyphal knots depend on the following light/dark rhythm cultivation or dark cultivation after full growth of mycelia in the constant dark cultivation. This study provides new insight into the molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions. In addition, the result that overexpression of CcnsdD2 induced more secondary hyphal knots, primordia, and fruiting bodies under light/dark rhythm cultivation conditions has potential applied value in the edible mushroom industry.
- Published
- 2021
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213. The Coprinopsis cinerea septin Cc.Cdc3 is involved in stipe cell elongation
- Author
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Noriyoshi Ishii, Noriaki Ozaki, Takashi Kamada, Hajime Muraguchi, Keiju Okano, Naoki Takahashi, Masayuki Kobayashi, Tatsuhiro Shioya, Hiroe Nakamura, and Yuichi Sakamoto
- Subjects
biology ,Hypha ,fungi ,Mutant ,Hyphae ,Wild type ,biology.organism_classification ,Septin ,Microbiology ,Cell biology ,Coprinus ,Fungal Proteins ,Protein Transport ,Coprinopsis cinerea ,Transcription (biology) ,Gene Expression Regulation, Fungal ,Cell cortex ,Botany ,Genetics ,Septins ,Actin - Abstract
We have identified and characterized a Coprinopsis cinerea mutant defective in stipe elongation during fruiting body development. In the wild-type, stipe cells elongate at the maturation stage of fruiting, resulting in very slender cells. In the mutant, the stipe cells fail to elongate, but become rather globular at the maturation stage. We found that the mutant phenotype is rescued by a gene encoding a homolog of Saccharomyces cerevisiae CDC3 septin, Cc.Cdc3. The C. cinerea genome includes 6 septin genes, 5 of which, including Cc.cdc3, are highly transcribed during stipe elongation in the wild type. In the mutant, the level of Cc.cdc3 transcription in the stipe cells remains the same as that in the mycelium, and the level of Cc.cdc10 transcription is approximately 100 times lower than that in the wild-type stipe cells. No increase in transcription of Cc.cdc3 in the mutant may be due to the fact that the Cc.cdc3 gene has a 4-base pair insertion in its promoter and/or that the promoter region is methylated in the mutant. Overexpressed EGFP-Cc.Cdc3 fusion protein rescues the stipe elongation in the transformants, localizes to the cell cortex and assembles into abundant thin filaments in the elongating stipe cells. In contrast, in vegetative hyphae, EGFP-Cc.Cdc3 is localized to the hyphal tips of the apical cells of hyphae. Cellular defects in the mutant, combined with the localization of EGFP-Cc.Cdc3, suggest that septin filaments in the cell cortex provide the localized rigidity to the plasma membrane and allow cells to elongate cylindrically.
- Published
- 2013
214. A comparative genomic analysis of the oxidative enzymes potentially involved in lignin degradation by Agaricus bisporus
- Author
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Venkataramanan Subramanian, Bolei Fu, Dan Cullen, and Harshavardhan Doddapaneni
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Laccase ,Oxidase test ,biology ,Agaricus ,Computational Biology ,Genetic Variation ,Cytochrome P450 ,biology.organism_classification ,Lignin ,Microbiology ,Fungal Proteins ,Coprinopsis cinerea ,Biochemistry ,Oxidative enzyme ,Genetics ,biology.protein ,Phanerochaete ,Gene family ,Genome, Fungal ,Oxidoreductases ,Biotransformation ,Metabolic Networks and Pathways ,Peroxidase - Abstract
The oxidative enzymatic machinery for degradation of organic substrates in Agaricus bisporus (Ab) is at the core of the carbon recycling mechanisms in this fungus. To date, 156 genes have been tentatively identified as part of this oxidative enzymatic machinery, which includes 26 peroxidase encoding genes, nine copper radical oxidase [including three putative glyoxal oxidase-encoding genes (GLXs)], 12 laccases sensu stricto and 109 cytochrome P450 monooxygenases. Comparative analyses of these enzymes in Ab with those of the white-rot fungus, Phanerochaete chrysosporium, the brown-rot fungus, Postia placenta, the coprophilic litter fungus, Coprinopsis cinerea and the ectomychorizal fungus, Laccaria bicolor, revealed enzyme diversity consistent with adaptation to substrates rich in humic substances and partially degraded plant material. For instance, relative to wood decay fungi, Ab cytochrome P450 genes were less numerous (109 gene models), distributed among distinctive families, and lacked extensive duplication and clustering. Viewed together with P450 transcript accumulation patterns in three tested growth conditions, these observations were consistent with the unique Ab lifestyle. Based on tandem gene arrangements, a certain degree of gene duplication seems to have occurred in this fungus in the copper radical oxidase (CRO) and the laccase gene families. In Ab, high transcript levels and regulation of the heme-thiolate peroxidases, two manganese peroxidases and the three GLX-like genes are likely in response to complex natural substrates, including lignocellulose and its derivatives, thereby suggesting an important role in lignin degradation. On the other hand, the expression patterns of the related CROs suggest a developmental role in this fungus. Based on these observations, a brief comparative genomic overview of the Ab oxidative enzyme machinery is presented.
- Published
- 2013
215. Crystal structure of the N-terminal domain of a glycoside hydrolase family 131 protein from Coprinopsis cinerea
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Makoto Yoshida, Kiwamu Umezawa, Atsushi Nishikawa, Takatsugu Miyazaki, Yutaro Tanaka, Mizuki Tamura, and Takashi Tonozuka
- Subjects
Models, Molecular ,Glycoside Hydrolases ,Stereochemistry ,Molecular Sequence Data ,β-Glucanase ,Biophysics ,Crystal structure ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,Glycoside hydrolase family 131 ,Fungal Proteins ,Protein structure ,Structural Biology ,Catalytic Domain ,Coprinopsis cinerea ,Hydrolase ,Genetics ,Glycoside hydrolase ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,biology ,Chemistry ,Hydrogen bond ,Active site ,Hydrogen Bonding ,Cell Biology ,biology.organism_classification ,biology.protein ,Basidiomycete ,Agaricales - Abstract
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.
- Published
- 2013
216. Detection of potential suberinase-encoding genes in Streptomyces scabiei strains and other actinobacteria
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Doaa A. Komeil, Carole Beaulieu, and Anne-Marie Simao-Beaunoir
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Immunology ,Gene Expression ,Cutin ,Applied Microbiology and Biotechnology ,Microbiology ,Streptomyces ,Esterase ,Actinobacteria ,Suberin ,Genetics ,Molecular Biology ,Solanum tuberosum ,integumentary system ,biology ,Esterases ,food and beverages ,General Medicine ,Lipid Metabolism ,biology.organism_classification ,Streptomyces scabies ,Lipids ,Coprinopsis cinerea ,Open reading frame ,Biochemistry - Abstract
Streptomyces scabiei causes common scab, an economically important disease of potato tubers. Some authors have previously suggested that S. scabiei penetration into host plant tissue is facilitated by secretion of esterase enzymes degrading suberin, a lipidic biopolymer of the potato periderm. In the present study, S. scabiei EF-35 showed high esterase activity in suberin-containing media. This strain also exhibited esterase activity in the presence of other biopolymers, such as lignin, cutin, or xylan, but at a much lower level. In an attempt to identify the esterases involved in suberin degradation, translated open reading frames of S. scabiei 87-22 were examined for the presence of protein sequences corresponding to extracellular esterases of S. scabiei FL1 and of the fungus Coprinopsis cinerea VTT D-041011, which have previously been shown to be produced in the presence of suberin. Two putative extracellular suberinase genes, estA and sub1, were identified. The presence of these genes in several actinobacteria was investigated by Southern blot hybridization, and both genes were found in most common-scab-inducing strains. Moreover, reverse transcription – polymerase chain reaction performed with S. scabiei EF-35 showed that estA was expressed in the presence of various biopolymers, including suberin, whereas the sub1 gene appeared to be specifically expressed in the presence of suberin and cutin.
- Published
- 2013
217. Protein mapping of Chaetomium globosum, a potential biological control agent through proteomics approach
- Author
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Sapna Sharma, Anita Yadav, Rashmi Aggarwal, and Sangeeta Gupta
- Subjects
Two-dimensional gel electrophoresis ,biology ,Chaetomium globosum ,G protein ,Isoelectric focusing ,Plant Science ,biology.organism_classification ,Proteomics ,Neurospora crassa ,Coprinopsis cinerea ,Biochemistry ,Magnaporthe grisea ,Agronomy and Crop Science ,Biotechnology - Abstract
Chaetomium globosum is a ubiquitous filamentous fungus having biological control properties. The potential isolates mycoparasitize the pathogen and produce antifungal metabolites which suppress the growth of pathogenic fungi. A proteomics approach was undertaken to separate and identify proteins from a mycoparasitic strain Cg1 of C. globosum under normal and heat shock conditions in order to identify differentially expressed proteins. We developed and standardized the procedure for extraction of total proteins and 2D gel electrophoresis, which resulted in profiling of more than 100 protein spots. 48 proteins were identified by a combination of matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF) and liquid chromatography mass spectrometry (LCMS/MS). Out of total proteins identified, 79 % were hypothetical proteins and 21 % proteins were functionally characterized. Out of total 79 % hypothetical proteins 24 % proteins matched with C. globosum while 18 % proteins matched with Aspergillus spp., 13 % with Coprinopsis cinerea, 10 % with Giberrella zaea, 8 % with Magnaporthe grisea and 5 % with Neurospora crassa and Lodderomyces elonisporus. Some of the functionally characterized proteins included MAP kinase, maltose permease, GTP binding protein, dyenin heavy chain, HET- C2, vacuolar Dig A protein, polyketide synthase, peptide prolyl cis trans isomerase and translation elongation factor. This study has generated a protein reference map for Chaetomium globosum, and being the first report on proteomics studies would greatly help to unravel biocontrol mechanism and its survival under heat stress conditions.
- Published
- 2013
218. Oxyfunctionalization of aliphatic compounds by a recombinant peroxygenase fromCoprinopsis cinerea
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Ana Gutiérrez, Ángel T. Martínez, José C. del Río, Esteban D. Babot, and Lisbeth Kalum
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Stereochemistry ,Bioengineering ,010402 general chemistry ,Hydroxylation ,01 natural sciences ,Applied Microbiology and Biotechnology ,Gas Chromatography-Mass Spectrometry ,Mixed Function Oxygenases ,03 medical and health sciences ,chemistry.chemical_compound ,Unspecific peroxygenase ,Alkanes ,Organic chemistry ,Fatty acids ,030304 developmental biology ,Alkane ,chemistry.chemical_classification ,Fatty alcohols ,0303 health sciences ,biology ,Agrocybe ,Fatty Acids ,Peroxygenase ,biology.organism_classification ,Recombinant Proteins ,3. Good health ,0104 chemical sciences ,Coprinopsis cinerea ,chemistry ,Biocatalysis ,Myristoleic acid ,Heterologous expression ,Fatty Alcohols ,Agaricales ,Biotechnology - Abstract
The goal of this study is the selective oxyfunctionalization of aliphatic compounds under mild and environmentally friendly conditions using a low-cost enzymatic biocatalyst. This could be possible taking advantage from a new peroxidase type that catalyzes monooxygenase reactions with H2O2 as the only cosubstrate (peroxygenase). With this purpose, recombinant peroxygenase, from gene mining in the sequenced genome of Coprinopsis cinerea and heterologous expression using an industrial fungal host, is tested for the first time on aliphatic substrates. The reaction on free and esterified fatty acids and alcohols, and long-chain alkanes was followed by gas chromatography, and the different reaction products were identified by mass spectrometry. Regioselective hydroxylation of saturated/unsaturated fatty acids was observed at the w-1 and w-2 positions (only at the w-2 position in myristoleic acid). Alkyl esters of fatty acids and monoglycerides were also w-1 or w-2 hydroxylated, but di- and tri-glycerides were not modified. Fatty alcohols yielded hydroxy derivatives at the w-1 or w-2 positions (diols) but also fatty acids and their hydroxy derivatives. Interestingly, the peroxygenase was able to oxyfunctionalize alkanes giving, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule. The predominance of mono- or di-hydroxylated derivatives seems related to the higher or lower proportion of acetone, respectively, in the reaction medium. The recombinant C. cinerea peroxygenase appears as a promising biocatalyst for alkane activation and production of aliphatic oxygenated derivatives, with better properties than the previously reported peroxygenase from Agrocybe aegerita, and advantages related to its recombinant nature for enzyme engineering and industrial production., This study was funded by the PEROXICATS (KBBE-2010-4-265397) EU project. E.D. Babot thanks the Spanish CSIC for a JAE fellowship co-financed by FSE. R. Ullrich and M. Hofrichter are acknowledged for providing wild-type A. aegerita peroxygenase preparation.
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- 2013
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219. Cc.snf5, a gene encoding a putative component of the SWI/SNF chromatin remodeling complex, is essential for sexual development in the agaricomycete Coprinopsis cinerea
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Kunihiko Oka, Kiyoshi Nakahori, Yuki Ando, Takashi Kamada, and Takehito Nakazawa
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Genetics ,biology ,Chromosomal Proteins, Non-Histone ,Genetic Complementation Test ,Molecular Sequence Data ,Fungal genetics ,Mutagenesis (molecular biology technique) ,Gene targeting ,Sequence Analysis, DNA ,Chromatin Assembly and Disassembly ,biology.organism_classification ,Microbiology ,Chromatin remodeling ,SWI/SNF ,DNA-Binding Proteins ,Fungal Proteins ,Complementation ,Coprinopsis cinerea ,Plasmid ,Agaricales ,DNA, Fungal ,Gene Deletion ,Transcription Factors - Abstract
We characterized a Coprinopsis cinerea mutant strain, Spe20, defective in fruiting initiation, which was isolated after restriction enzyme-mediated integration (REMI) mutagenesis of a homokaryotic fruiting strain, 326. A plasmid rescue followed by complementation experiments, RACE, and cDNA analyses revealed that the gene, a mutation of which is responsible for the phenotype, is predicted to encode a protein that exhibits a high similarity to yeast Snf5p, a key component of the chromatin remodeling complex SWI/SNF, and named Cc.snf5. Cc.Snf5 is, however, different from Snf5p in that the former has, in addition to an Snf5 domain comprising N-terminal repeat1 (rp1) and C-terminal repeat2 (rp2) subdomains in a middle region, a GATA Zn-finger domain in a C-terminal region. In strain Spe20, plasmid pPHT1 used for REMI is inserted in the ORF encoding rp2. This raised the possibility that in strain Spe20, the disrupted Cc.Snf5 is functionally active albeit incompletely because it retains rp1. Thus, we disrupted the whole SNF5 domain and its downstream peptide and found that the disruption results in inhibition of not only fruiting initiation but also dikaryon development, a prerequisite for fruiting. We also found that specific disruption of the Zn-finger domain results in inhibition of fruiting initiation. These results indicate that Cc.Snf5 plays an essential role in sexual development of C. cinerea.
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- 2013
220. Heterologous expression and biochemical characterization of acetyl xylan esterase from Coprinopsis cinerea
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Jin Chuan Wu, Veeresh Juturu, and Christina Aust
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PNGase F ,Glycosylation ,Physiology ,medicine.medical_treatment ,Gene Expression ,Applied Microbiology and Biotechnology ,Esterase ,Pichia ,Pichia pastoris ,Nitrophenols ,Enzyme Stability ,medicine ,Cloning, Molecular ,Peptide sequence ,Protease ,biology ,Molecular mass ,Temperature ,General Medicine ,Hydrogen-Ion Concentration ,biology.organism_classification ,Molecular Weight ,Butyrates ,Kinetics ,Coprinopsis cinerea ,Biochemistry ,Acetylesterase ,Electrophoresis, Polyacrylamide Gel ,Heterologous expression ,Agaricales ,Protein Processing, Post-Translational ,Biotechnology - Abstract
Acetyl xylan esterase (AXE) from basidiomycete Coprinopsis cinerea Okayama 7 (#130) was functionally expressed in Pichia pastoris with a C-terminal tag under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 1.5 mg l(-1). Its molecular mass was estimated to be 65.5 kDa based on the SDS-PAGE analysis, which is higher than the calculated molecular mass of 40 kDa based on amino acid composition. In-silico analysis of the amino acid sequence predicted two potential N-glycosylation sites. Results from PNGase F deglycosylation and mass spectrum confirmed the presence of N-glycosylation on the recombinant AXE with predominant N-glycans HexNAc2Hex9-16. The recombinant AXE showed best activity at 40 °C and pH 8. It showed not only acetyl esterase activity with a Km of 4.3 mM and a Vmax of 2.15 U mg(-1) for hydrolysis of 4-nitrophenyl acetate but also a butyl esterase activity for hydrolysis of 4-nitrophenyl butyrate with a Km of 0.11 mM and Vmax of 0.78 U mg(-1). The presence of two additional amino acid residues at its native N-terminus was found to help stabilize the enzyme against the protease cleavages without affecting its activity.
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- 2012
221. The Modes of Action of ChiIII, a Chitinase from Mushroom Coprinopsis cinerea, Shift with Changes in the Length of GlcNAc Oligomers
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Xin Niu, Zhonghua Liu, Sheng Yuan, Mingmei Yang, Cuicui Liu, Fei Ma, and Yuanjing Xiong
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0301 basic medicine ,030106 microbiology ,Molecular Sequence Data ,Oligosaccharides ,Chitin ,Acetylglucosamine ,Substrate Specificity ,Fungal Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Hydrolysis ,Fungal cell walls ,Amino Acid Sequence ,chemistry.chemical_classification ,Mushroom ,biology ,Chitinases ,Substrate (chemistry) ,General Chemistry ,biology.organism_classification ,carbohydrates (lipids) ,Coprinopsis cinerea ,030104 developmental biology ,Enzyme ,chemistry ,Biochemistry ,Chitinase ,biology.protein ,General Agricultural and Biological Sciences ,Agaricales - Abstract
A putative class III endochitinase (ChiIII) was reported previously to be expressed dominantly in fruiting bodies of Coprinopsis cinerea, and its expression levels increased with the maturation of the fruiting bodies. This paper further reports that ChiIII is a novel chitinase with exo- and endoactivities. When the substrate was (GlcNAc)3-5, ChiIII exhibited exoactivity, releasing GlcNAc processively from the reducing end of (GlcNAc)3-5; when the substrate was (GlcNAc)6-7, the activity of ChiIII shifted to an endoacting enzyme, randomly splitting chitin oligosaccharides to various shorter oligosaccharides. This shift in the mode of action of ChiIII may be related to its stronger hydrolytic capacity to degrade chitin in fungal cell walls. The predicted structure of ChiIII shows that it lacks the α+β domain insertion; however, its substrate binding cleft seems to be deeper than that of common endochitinases but shallower and more open than that of common exochitinases, which may be related to its exo- and endohydrolytic activities.
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- 2016
222. Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline
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Shin-ichi Nakakita, Yasunari Eshima, Yujiro Higuchi, Kaoru Takegawa, Yibo Huang, Wataru Sumiyoshi, and Takashi Kinoshita
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0301 basic medicine ,Glycosylation ,Stereochemistry ,Oligosaccharides ,Bioengineering ,Oxazoline ,Applied Microbiology and Biotechnology ,Endoglycosidase ,03 medical and health sciences ,chemistry.chemical_compound ,Sugar ,chemistry.chemical_classification ,biology ,General Medicine ,Coprinopsis ,Complex type ,Oligosaccharide ,biology.organism_classification ,Coprinopsis cinerea ,030104 developmental biology ,Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase ,Biochemistry ,chemistry ,biology.protein ,Glycoprotein ,Biotechnology - Abstract
To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities.Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase (Endo-CCThis transglycosylation method using SG-oxazoline and Endo-CC
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- 2016
223. Purification, characterization and function analysis of an extracellular β-glucosidase from elongating stipe cell walls in Coprinopsis cinerea
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Sheng Yuan, Liqin Kang, Zhonghua Liu, Mingmei Yang, Jun Wang, Wenming Zhang, and Yajun Zhou
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0301 basic medicine ,beta-Glucans ,030106 microbiology ,Oligosaccharides ,Trimer ,Microbiology ,Cell wall ,03 medical and health sciences ,Hydrolysis ,Cell Wall ,Hydrolase ,Extracellular ,Genetics ,Molecular Biology ,chemistry.chemical_classification ,biology ,beta-Glucosidase ,Glycosyltransferases ,biology.organism_classification ,Coprinopsis cinerea ,Stipe (mycology) ,Enzyme ,Biochemistry ,chemistry ,Biophysics ,Electrophoresis, Polyacrylamide Gel ,Agaricales - Abstract
A β-glycoside hydrolase was isolated from cell walls material in Coprinopsis cinerea elongating stipes. By analysis of SDS-PAGE, MALDI-TOF/TOF MS and substrate specificity, this enzyme was characterized as an extracellular β-glucosidase which is a trimer consisting of three homosubunits. β-Glucosidase did not degrade β-glucans with modified ends, whereas it hydrolyzed various β-glucans with free ends and related oligosaccharides with β-1,3-, β-1,4- or β-1,6-linkages. Although this β-glucosidase possesses glycosyltransferase activity on laminarioligosaccharides, it did not transfer glucose residues from laminaritriose to β-glucan in stipe cell walls to produce larger β-glucan molecules; instead, it caused a decrease in the molecular size of stipe wall β-glucan by removing glucose. Relatively, the molecular size of wall β-glucans in the elongating apical stipe was less than that found in the non-elongating basal stipes, and this β-glucosidase was more highly expressed in the elongating apical stipe than in non-elongating basal regions. Therefore, we propose that β-glucosidase functions by trimming or cutting the β-glucan side chains on the β-1,3-glucan backbone to prevent them from forming longer branches, keeping the wall plastic to promote diffuse wall growth.
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- 2016
224. An aryl-alcohol oxidase of Pleurotus sapidus: heterologous expression, characterization, and application in a 2-enzyme system
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Hanno Luig, Aysha Javeed, Ilya Galperin, Martin Rühl, and Günter Lochnit
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0301 basic medicine ,030106 microbiology ,Gene Expression ,Alcohol oxidoreductase ,Dehydrogenase ,Anthraquinones ,Biology ,Pleurotus ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,Aryl-alcohol oxidase ,Cloning, Molecular ,Peroxidase ,fungi ,food and beverages ,General Medicine ,biology.organism_classification ,Recombinant Proteins ,Coprinopsis cinerea ,Alcohol Oxidoreductases ,030104 developmental biology ,Biochemistry ,Pleurotus ostreatus ,Heterologous expression ,Agaricales ,Agaricus bisporus ,Biotechnology - Abstract
Aryl-alcohol oxidases (AAOs) are enzymes supporting the degradation of lignin by fungal derived class II peroxidases produced by white-rot fungi. AAOs are able to generate H2O2 as a by-product via oxidation of an aryl-alcohol into its correspondent aldehyde. In this study, an AAO was heterologously expressed in a basidiomycete host for the first time. The gene for an AAO of the white-rot fungus Pleurotus sapidus, a close relative to the oyster mushroom Pleurotus ostreatus, was cloned into an expression vector and put under control of the promotor of the glyceraldehyde-3-phosphate dehydrogenase gene 2 (gpdII) of the button mushroom Agaricus bisporus. The expression vector was transformed into the model basidiomycete Coprinopsis cinerea, and several positive transformants were obtained. The best producing transformants were grown in shake-flasks and in a stirred tank reactor reaching enzymatic activities of up to 125 U L(-1) using veratryl alcohol as a substrate. The purified AAO was biochemically characterized and compared to the previously described native and recombinant AAOs from other Pleurotus species. In addition, a two-enzyme system comprising a dye-decolorizing peroxidase (DyP) from Mycetinis scorodonius and the P. sapidus AAO was successfully employed to bleach the anthraquinone dye Reactive Blue 5.
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- 2016
225. Molecular determinants for selective C25-hydroxylation of vitamins D2 and D3 by fungal peroxygenases
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Lisbeth Kalum, René Ullrich, José C. del Río, Esteban D. Babot, Victor Guallar, Marina Cañellas, Ángel T. Martínez, Fátima Lucas, Ana Gutiérrez, Martin Hofrichter, Ministerio de Economía y Competitividad (España), and Barcelona Supercomputing Center
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0301 basic medicine ,Vitamines ,Stereochemistry ,A. aegerita ,Vitamins Computational studies ,Hydroxylation ,Molecular determinants ,Catalysis ,Experimental conversion ,03 medical and health sciences ,chemistry.chemical_compound ,Coprinopsis cinerea ,medicine ,Regioselective conversion ,Reactivity (chemistry) ,Molècules ,Relative distances ,Site selectivity ,chemistry.chemical_classification ,biology ,Agrocybe ,Enginyeria mecànica::Impacte ambiental [Àrees temàtiques de la UPC] ,Oxygenations of aliphatic ,Vitamins ,Molecules ,biology.organism_classification ,3. Good health ,Ergocalciferol ,Molecular oxygen ,030104 developmental biology ,Enzyme ,chemistry ,Molecular simulations ,Cholecalciferol ,Molecular structure ,Methyl group ,medicine.drug - Abstract
8 páginas.-- 7 figuras.-- 45 referencias.-- Electronic supplementary information (ESI) available: Molecular model for Ccinerea peroxygenase obtained using A. aegerita's crystal structure as the template. See DOI: 10.1039/c5cy00427f, Hydroxylation of vitamin D by Agrocybe aegerita and Coprinopsis cinerea peroxygenases was investigated in a combined experimental and computational study. 25-Monohydroxylated vitamins D3 (cholecalciferol) and D2 (ergocalciferol), compounds of high interest in human health and animal feeding, can be obtained through a reaction with both fungal enzymes. Differences in conversion rates, and especially in site selectivity, were observed. To rationalize the results, diffusion of D2 and D3 on the molecular structure of the two enzymes was performed using the PELE software. In good agreement with experimental conversion yields, simulations indicate more favorable energy profiles for the substrates' entrance in C. cinerea than for A. aegerita enzyme. On the other hand, GC-MS analyses show that while a full regioselective conversion of D2 and D3 into the active C25 form is catalyzed by C. cinerea peroxygenase, A. aegerita yielded a mixture of the hydroxylated D3 products. From the molecular simulations, relative distance distributions between the haem compound I oxygen atom and H24/H25 atoms (hydrogens on C24 and C25, respectively) were plotted. Results show large populations for O–H25 distances below 3 Å for D2 and D3 in C. cinerea in accordance with the high reactivity observed for this enzyme. In A. aegerita, however, cholecalciferol has similar populations (below 3 Å) for O–H25 and O–H24, which can justify the hydroxylation observed in C24. In the case of ergocalciferol, due to the bulky methyl group in position C24, very few structures are found with O–H24 distances below 3 Å and thus, as expected, the reaction was only observed at the C25 position., This work was supported by the INDOX (KBBE-2013-7-613549) and PELE (ERC-2009-Adg 25027) EU projects, and by the BIO2011-26694 and CTQ2013-48287 projects of the Spanish Ministry of Economy and Competitiveness.
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- 2016
226. Identification of a Novel Nematotoxic Protein by Challenging the Model Mushroom Coprinopsis cinerea with a Fungivorous Nematode
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Erika Lindquist, Anna Lipzen, Stefanie S. Schmieder, David F. Plaza, and Markus Künzler
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0301 basic medicine ,Nematoda ,Physiological ,fungal defense ,030106 microbiology ,Basidiomycete ,Fungal defense ,RNA sequencing ,CCTX2 ,Transcriptomics ,Bacillus subtilis ,QH426-470 ,Investigations ,Stress ,Microbiology ,Fungal Proteins ,03 medical and health sciences ,transcriptomics ,Stress, Physiological ,Antibiosis ,Genetics ,Aphelenchus avenae ,Animals ,Molecular Biology ,Genetics (clinical) ,Mycelium ,Mushroom ,Fungal protein ,biology ,Gene Expression Profiling ,fungi ,Computational Biology ,High-Throughput Nucleotide Sequencing ,food and beverages ,biology.organism_classification ,Coprinopsis cinerea ,Nematode ,Infectious Diseases ,Agaricales ,Transcriptome ,Biotechnology ,basidiomycete - Abstract
The dung of herbivores, the natural habitat of the model mushroom Coprinopsis cinerea, is a nutrient-rich but also very competitive environment for a saprophytic fungus. We showed previously that C. cinerea expresses constitutive, tissue-specific armories against antagonists such as animal predators and bacterial competitors. In order to dissect the inducible armories against such antagonists, we sequenced the poly(A)-positive transcriptome of C. cinerea vegetative mycelium upon challenge with fungivorous and bacterivorous nematodes, Gram-negative and Gram-positive bacteria and mechanical damage. As a response to the fungivorous nematode Aphelenchus avenae, C. cinerea was found to specifically induce the transcription of several genes encoding previously characterized nematotoxic lectins. In addition, a previously not characterized gene encoding a cytoplasmic protein with several predicted Ricin B-fold domains, was found to be strongly upregulated under this condition. Functional analysis of the recombinant protein revealed a high toxicity toward the bacterivorous nematode Caenorhabditis elegans. Challenge of the mycelium with A. avenae also lead to the induction of several genes encoding putative antibacterial proteins. Some of these genes were also induced upon challenge of the mycelium with the bacteria Escherichia coli and Bacillus subtilis. These results suggest that fungi have the ability to induce specific innate defense responses similar to plants and animals., G3: Genes, Genomes, Genetics, 6 (1), ISSN:2160-1836
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- 2016
227. Global Gene Expression in Coprinopsis cinerea Meiotic Mutants Reflects Checkpoint Arrest
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Claire Burns, Erika Anderson, and Miriam E. Zolan
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DNA Replication ,Premeiotic DNA replication ,checkpoint arrest ,Investigations ,Coprinus ,Fungal Proteins ,03 medical and health sciences ,Prophase ,Meiosis ,Gene Expression Regulation, Fungal ,Gene expression ,Genetics ,Meiotic S phase ,Molecular Biology ,Gene ,MRN complex ,Genetics (clinical) ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,Gene Expression Profiling ,fungi ,Wild type ,Cell Cycle Checkpoints ,biology.organism_classification ,meiotic S phase ,DNA-Binding Proteins ,Coprinopsis cinerea ,mushroom development ,Mutation ,microarray analysis - Abstract
The basidiomycete Coprinopsis cinerea is well-suited to studies of meiosis because meiosis progresses synchronously in 10 million cells within each mushroom cap. Approximately 20% of C. cinerea genes exhibit changing expression during meiosis, but meiosis and mushroom development happen concurrently and therefore differentially expressed genes might not be directly involved in meiotic processes. By using microarrays, we examined global gene expression across a meiotic time course in two mutants in which meiosis arrests but mushrooms develop normally. Genes differentially expressed in the mutants compared with the wild type are likely to be involved in meiosis and sporulation as opposed to mushroom development. In rad50-1, which arrests in late prophase, RNA abundance for a group of early meiotic genes remains high, whereas the expression of a group of late meiotic genes is never induced. In contrast, in msh5-22 (which fails to undergo premeiotic DNA replication), both early and late meiotic genes are underexpressed relative to wild type at late meiotic time points as the cells die. Genes that are differentially expressed relative to wild type in both mutants are particularly strong candidates for playing roles in meiosis and sporulation.
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- 2012
228. The activity of phosphoinositide-specific phospholipase C is required for vegetative growth and cell wall regeneration in Coprinopsis cinerea
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Chang-Won Lee, Young Taek Oh, Chun-Seob Ahn, Jae-Won Kim, Kyung-Jin Lee, Hyeon-Su Ro, and Jeong Geun Kim
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Hyphal growth ,Hypha ,Hyphae ,Biology ,Phosphatidylinositols ,Applied Microbiology and Biotechnology ,Microbiology ,Cell wall ,Cell Wall ,Regeneration ,Enzyme Inhibitors ,Estrenes ,Structural analog ,Phospholipase C ,Regeneration (biology) ,fungi ,Computational Biology ,General Medicine ,Spores, Fungal ,biology.organism_classification ,Pyrrolidinones ,Coprinopsis cinerea ,Biochemistry ,Germination ,Type C Phospholipases ,Genome, Fungal ,Agaricales - Abstract
Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.
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- 2012
229. Comparison of the structural changes in two cellobiohydrolases, CcCel6A and CcCel6C, from Coprinopsis cinerea - a tweezer-like motion in the structure of CcCel6C
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Makoto Yoshida, Yutaro Tanaka, Takashi Tonozuka, Takatsugu Miyazaki, Mizuki Tamura, and Atsushi Nishikawa
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biology ,Stereochemistry ,Active site ,Cell Biology ,Cellobiose ,Cellulase ,Ligand (biochemistry) ,biology.organism_classification ,Biochemistry ,chemistry.chemical_compound ,Coprinopsis cinerea ,chemistry ,Hydrolase ,biology.protein ,Glycoside hydrolase ,Cellulose ,Molecular Biology - Abstract
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.
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- 2012
230. Structural Basis of Trypsin Inhibition and Entomotoxicity of Cospin, Serine Protease Inhibitor Involved in Defense of Coprinopsis cinerea Fruiting Bodies
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Petra Avanzo Caglič, Miha Renko, Sandra Kallert, Markus Künzler, Dušan Turk, Borut Štrukelj, Markus Aebi, Janko Kos, Silvia Bleuler-Martinez, and Jerica Sabotič
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Proteases ,animal structures ,medicine.medical_treatment ,Molecular Sequence Data ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,Microbiology ,Fungal Proteins ,Serine ,03 medical and health sciences ,medicine ,Amino Acid Sequence ,Fruiting Bodies, Fungal ,Molecular Biology ,030304 developmental biology ,Serine protease ,0303 health sciences ,Fungal protein ,Protease ,biology ,030306 microbiology ,fungi ,food and beverages ,Cell Biology ,biology.organism_classification ,Trypsin ,Protein Structure, Tertiary ,Coprinopsis cinerea ,nervous system ,Protein Structure and Folding ,biology.protein ,Agaricales ,Trypsin Inhibitors ,psychological phenomena and processes ,Clitocybe nebularis ,medicine.drug - Abstract
Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with K(i) in the picomolar range. Determination of the crystal structure revealed that the protein has a β-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the β2 and β3 strands, distinguishing cospin from other β-trefoil-fold serine protease inhibitors in which β4-β5 or β5-β6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut.
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- 2012
231. Substrate oxidation by dye-decolorizing peroxidases (DyPs) from wood- and litter-degrading agaricomycetes compared to other fungal and plant heme-peroxidases
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Holger Zorn, Martin Hofrichter, Christiane Liers, Anja Worrich, Kari Steffen, René Ullrich, Harald Kellner, and Marek J. Pecyna
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Models, Molecular ,Protein Conformation ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,chemistry.chemical_compound ,Bjerkandera adusta ,Enzyme Stability ,Lignin ,Organic chemistry ,Versatile peroxidase ,Coloring Agents ,030304 developmental biology ,Dye decolorizing peroxidase ,0303 health sciences ,biology ,Sequence Homology, Amino Acid ,030306 microbiology ,Chemistry ,Basidiomycota ,General Medicine ,Lignin peroxidase ,Hydrogen-Ion Concentration ,biology.organism_classification ,Molecular Weight ,Coprinopsis cinerea ,Biochemistry ,Peroxidases ,biology.protein ,Phanerochaete ,Oxidation-Reduction ,Biotechnology ,Peroxidase ,Chromatography, Liquid - Abstract
Catalytic and physicochemical properties of representative fungal dye-decolorizing peroxidases (DyPs) of wood- (WRF) and litter-decomposing white-rot fungi (LDF) are summarized and compared, including one recombinant Mycetinis scorodonius DyP (rMscDyP; LDF), the wild-type Auricularia auricula-judae DyP (AauDyP; WRF), and two new DyPs secreted by the jelly fungi Exidia glandulosa (EglDyP; WRF) and Mycena epipterygia (MepDyP; LDF). Homogeneous preparations of these DyPs were obtained after different steps of fast protein liquid chromatography, and they increase the total number of characterized fungal DyP proteins to eight. The peptide sequences of AauDyP, MepDyP, and EglDyP showed highest homologies (52–56 %) to the DyPs of M. scorodonius. Five out of the eight characterized fungal DyPs were used to evaluate their catalytic properties compared to classic fungal and plant heme peroxidases, namely lignin peroxidase of Phanerochaete chrysosporium (PchLiP; WRF), versatile peroxidase of Bjerkandera adusta (BadVP; WRF), and generic peroxidases of Coprinopsis cinerea (CiP) and Glycine max (soybean peroxidase = SBP). All DyPs tested possess unique properties regarding the stability at low pH values: 50–90 % enzymatic activity remained after 4-h exposition at pH 2.5, and the oxidation of nonphenolic aromatic substrates (lignin model compounds) was optimal below pH 3. Furthermore, all DyPs efficiently oxidized recalcitrant dyes (e.g., Azure B) as well as the phenolic substrate 2,6-dimethoxyphenol. Thus, DyPs combine features of different peroxidases on the functional level and may be part of the biocatalytic system secreted by fungi for the oxidation of lignin and/or toxic aromatic compounds.
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- 2012
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232. Cloning and Expression Analysis of Vvlcc3, a Novel and Functional Laccase Gene Possibly Involved in Stipe Elongation
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Lingdan Lian, Lixian Guo, Bingzhi Chen, Zhiyun Yang, Juan Miao, Baogui Xie, Guangmei Wu, Yuanping Lu, and Wei Wang
- Subjects
Volvariella volvacea ,laccase ,fruiting body formation ,enzyme ,Molecular Sequence Data ,Gene Expression ,Article ,Catalysis ,lcsh:Chemistry ,Inorganic Chemistry ,Gene Expression Regulation, Fungal ,Universal veil ,Complementary DNA ,Gene Order ,Gene expression ,Amino Acid Sequence ,Fruiting Bodies, Fungal ,Cloning, Molecular ,Physical and Theoretical Chemistry ,lcsh:QH301-705.5 ,Molecular Biology ,Gene ,Phylogeny ,Spectroscopy ,Laccase ,biology ,Basidiomycota ,Gene Expression Profiling ,Organic Chemistry ,General Medicine ,biology.organism_classification ,Computer Science Applications ,Coprinopsis cinerea ,lcsh:Biology (General) ,lcsh:QD1-999 ,Biochemistry ,Genetic Loci ,Multigene Family ,Genome, Fungal ,Transcriptome ,Sequence Alignment ,Flammulina - Abstract
Volvariella volvacea, usually harvested in its egg stage, is one of the most popular mushrooms in Asia. The rapid transition from the egg stage to elongation stage, during which the stipe stretches to almost full length leads to the opening of the cap and rupture of the universal veil, and is considered to be one of the main factors that negatively impacts the yield and value of V. volvacea. Stipe elongation is a common phenomenon in mushrooms, however, the mechanisms, genes and regulation involved in stipe elongation are still poorly understood. In order to study the genes related to the stipe elongation, we analyzed the transcription of laccase genes in stipe tissue of V. volvacea, as some laccases have been suggested to be involved in stipe elongation in Flammulina velutipes. Based on transcription patterns, the expression of Vvlcc3 was found to be the highest among the 11 laccase genes. Moreover, phylogenetic analysis showed that VvLCC3 has a high degree of identity with other basidiomycete laccases. Therefore, we selected and cloned a laccase gene, named Vvlcc3, a cDNA from V. volvacea, and expressed the cDNA in Pichia pastoris. The presence of the laccase signature L1-L4 on the deduced protein sequence indicates that the gene encodes a laccase. Phylogenetic analysis showed that VvLCC3 clusters with Coprinopsis cinerea laccases. The ability to catalyze ABTS (2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation proved that the product of the Vvlcc3 gene was a functional laccase. We also found that the expression of the Vvlcc3 gene in V. volvacea increased during button stage to the elongation stage, it reached its peak in the elongation stage, and then decreased in the maturation stage, which was similar to the trend in the expression of Fv-lac3 and Fv-lac5 in F. velutipes stipe tissue. The similar trend in expression level of these laccase genes of F. velutipes suggested that this gene could be involved in stipe elongation in V. volvacea.
- Published
- 2015
- Full Text
- View/download PDF
233. Basidiomycetous wood endophytes from Platanus acerifolia (Platanaceae) of Argentina: notes and culture studies
- Author
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Cecilia Cristina Carmaran, Carolina Analía Robles, and Silvia Edith Lopez
- Subjects
BASIDIOMICETES ,Plant Science ,Biology ,basidiomicetes ,Trichosporon sporotrichoides ,purl.org/becyt/ford/1 [https] ,Ciencias Biológicas ,Árboles urbanos ,lcsh:Botany ,Botany ,Platanus acerifolia ,ENDOFITOS DE MADERA ,Inonotus rickii ,lcsh:Science ,purl.org/becyt/ford/1.6 [https] ,Ecology, Evolution, Behavior and Systematics ,Chrysosporium ,endofitos de madera ,Ecología ,biology.organism_classification ,ÁRBOLES URBANOS ,Granulobasidium vellereum ,lcsh:QK1-989 ,Coprinopsis cinerea ,Platanaceae ,Phanerochaete ,lcsh:Q ,Micología ,CIENCIAS NATURALES Y EXACTAS - Abstract
Conocer las características de cepas fúngicas locales aumenta nuestra capacidad de poder hacer uso de sus propiedades e implementar, en consecuencia, nuevas y mejores formas de aprovechamiento de los recursos biológicos nativos. Con el fin de contribuir al conocimiento de estos organismos, en este trabajo se presentan descripciones detalladas y estudios de cultivo de 5 especies previamente reportadas como basidiomicetes endofíticos: Coprinopsis cinerea, Granulobasidium vellereum, Inonotus rickii, Phanerochaete chrysosporium y Trichosporon sporotrichoides, aisladas como endofitos de madera de plátanos de sombra (Platanus acerifolia) en la Ciudad de Buenos Aires, Argentina. Coprinopsis cinerea es descripta por primera vez en cultivo y se señalan las diferencias que presentan los cultivos de G. vellereum e I. rickii con reportes previos. Los caracteres de cultivo de P. chrysosporium y T. sporotrichoides no presentaron diferencias apreciables con descripciones previas. The knowledge of local fungal strains' features increases our ability to make use of their properties and to implement, in consequence, new and better ways of using the native biological resources. To contribute to the knowledge of these organisms, in this work we present descriptions and cultural studies of 5 species previously reported as endophytic basidiomycetes: Coprinopsis cinerea, Granulobasidium vellereum, Inonotus rickii, Phanerochaete chrysosporium and Trichosporon sporotrichoides, isolated from wood of London plane trees (Platanus acerifolia) in Buenos Aires City, Argentina. Coprinopsis cinerea is described in culture for the first time and differences in cultures of G. vellereum and I. rickii with previous reports are listed. Culture characters of P. chrysosporium and T. sporotrichoides did not show appreciable differences with previous descriptions. Fil: Robles, Carolina Analía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Lopez, Silvia Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Carmaran, Cecilia Cristina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina
- Published
- 2015
234. Molecular determinants for selective C25-hydroxylation of vitamins D2 and D3 by fungal peroxygenases
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Ministerio de Economía y Competitividad (España), Lucas, Fátima, Babot, Esteban Daniel, Cañella, Marina, Río Andrade, José Carlos del, Kalum, Lisbeth, Ullrich, René, Hofrichter, Martin, Guallar, Victor, Martínez, Ángel T., Gutiérrez Suárez, Ana, Ministerio de Economía y Competitividad (España), Lucas, Fátima, Babot, Esteban Daniel, Cañella, Marina, Río Andrade, José Carlos del, Kalum, Lisbeth, Ullrich, René, Hofrichter, Martin, Guallar, Victor, Martínez, Ángel T., and Gutiérrez Suárez, Ana
- Abstract
Hydroxylation of vitamin D by Agrocybe aegerita and Coprinopsis cinerea peroxygenases was investigated in a combined experimental and computational study. 25-Monohydroxylated vitamins D3 (cholecalciferol) and D2 (ergocalciferol), compounds of high interest in human health and animal feeding, can be obtained through a reaction with both fungal enzymes. Differences in conversion rates, and especially in site selectivity, were observed. To rationalize the results, diffusion of D2 and D3 on the molecular structure of the two enzymes was performed using the PELE software. In good agreement with experimental conversion yields, simulations indicate more favorable energy profiles for the substrates' entrance in C. cinerea than for A. aegerita enzyme. On the other hand, GC-MS analyses show that while a full regioselective conversion of D2 and D3 into the active C25 form is catalyzed by C. cinerea peroxygenase, A. aegerita yielded a mixture of the hydroxylated D3 products. From the molecular simulations, relative distance distributions between the haem compound I oxygen atom and H24/H25 atoms (hydrogens on C24 and C25, respectively) were plotted. Results show large populations for O–H25 distances below 3 Å for D2 and D3 in C. cinerea in accordance with the high reactivity observed for this enzyme. In A. aegerita, however, cholecalciferol has similar populations (below 3 Å) for O–H25 and O–H24, which can justify the hydroxylation observed in C24. In the case of ergocalciferol, due to the bulky methyl group in position C24, very few structures are found with O–H24 distances below 3 Å and thus, as expected, the reaction was only observed at the C25 position.
- Published
- 2016
235. Recombinant multi-functional cellulase activity in submerged fermentation of lignocellulosic wastes
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Shujie Cheng, Liqiong Guo, Junfang Lin, Nannan Lou, and Peizhou Yang
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chemistry.chemical_classification ,biology ,Renewable Energy, Sustainability and the Environment ,Substrate (chemistry) ,Dehydrogenase ,Cellulase ,biology.organism_classification ,Coprinopsis cinerea ,Lentinula ,Enzyme ,Biochemistry ,chemistry ,biology.protein ,Agaricus bisporus ,Flammulina - Abstract
The expression of the multi-functional cellulase gene mfc in Coprinopsis cinerea is modulated using glyceraldehyde-3-phosphate dehydrogenase ( gpd ) promoter fragments from Agaricus bisporus , Flammulina velutipes , and Lentinula edodes . In submerged fermentation, with banana peels as the growth substrate, highest activities of endo-β-1,4-glucanase (0.48 U/ml), total cellulase (0.26 U/ml) and endo-β-1,4-xylanase (38.10 U/ml) were noted in the culture liquid Cabm44, which uses a 275 bp gpd fragment from A. bisporus . The lignocellulolytic enzymatic activities were increased nearly twofold in comparison with the wild-type strain.
- Published
- 2011
236. Molecular breeding of a novel Coprinopsis cinerea strain possessing a heterologous laccase gene, lccK, driven by a constitutive promoter
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Hajime Muraguchi, Sonoe O. Yanagi, Yasuhiro Ito, and Manami Kondoh
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Signal peptide ,Laccase ,biology ,fungi ,food and beverages ,biology.organism_classification ,Molecular biology ,Gene expression profiling ,Coprinopsis cinerea ,genomic DNA ,Terminator (genetics) ,Heterologous expression ,Gene ,Ecology, Evolution, Behavior and Systematics - Abstract
Genomic DNA encoding the Pleurotus ostreatus LccK laccase was fused with the Coprinopsis cinerea β-tubulin promoter and terminator, and introduced into a C. cinerea strain. Linkage analysis, native PAGE separations, substrate specificity investigations and expression profiling indicated that C. cinerea transformants secrete P. ostreatus LccK, suggesting that the introns of the lccK gene are correctly spliced and the signal peptide for secretion is functional in C. cinerea. Transformants constitutively expressing laccase may be useful for the degradation of aromatic compounds.
- Published
- 2011
237. Protease inhibitors clitocypin and macrocypin are differentially expressed within basidiomycete fruiting bodies
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Kristina Gruden, Meti Buh Gašparič, Maruška Budič, Gary D. Foster, Andy M. Bailey, Janko Kos, Jerica Sabotič, and Sreedhar Kilaru
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Reporter gene ,Proteases ,Protease ,biology ,Basidiomycota ,medicine.medical_treatment ,Immunoblotting ,Promoter ,General Medicine ,Cysteine Proteinase Inhibitors ,biology.organism_classification ,Polymerase Chain Reaction ,Biochemistry ,Cysteine protease ,Fungal Proteins ,Coprinopsis cinerea ,medicine ,Fruiting Bodies, Fungal ,Gene ,Clitocybe nebularis - Abstract
Clitocypin and macrocypin are cysteine protease inhibitors of the mycocypin family which is unique to basidiomycetes. We have established that Clitocybe nebularis and Macrolepiota procera each contain genes for both macrocypin and clitocypin. Both are expressed in M. procera but only clitocypin in C. nebularis. Further analysis of mycocypin expression at the mRNA and protein levels in mature fruiting bodies of M. procera revealed that clitocypin is expressed evenly throughout the fruiting body, while the level of expression of macrocypins varies, and, at the protein level, is much higher in the veil fragments and the ring. The expression patterns of various mycocypins were determined in Coprinopsis cinerea, using promoters linked to a reporter gene. The expression profile of the clitocypin promoter was similar to that of the constitutive promoter gpdII from Agaricus bisporus, while that of the macrocypin 4 promoter was limited to the outer edges of the fruiting body throughout development. In addition, the activity of the macrocypin 3 promoter was different, indicating different regulation of expression for different macrocypin genes. The complex, tissue specific expression patterns for mycocypin genes suggest different biological roles for the products, either in regulation of endogenous proteases or in defense against pathogens or predators.
- Published
- 2011
238. Purification of a fungal cutinase by adsorptive bubble separation: A statistical approach
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Juliane Merz, Holger Zorn, Bernhard Burghoff, and Gerhard Schembecker
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Cutinase ,Chromatography ,Downstream processing ,biology ,Chemistry ,Bubble ,biology.organism_classification ,Separation process ,Coprinopsis cinerea ,Colloid and Surface Chemistry ,Chemical engineering ,Scientific method ,Foam fractionation ,Column design - Abstract
Nowadays, the use of enzymes for the pharmaceutical, cosmetic or food industry steadily increases. Although the specification and production of enzymes are steadily enlarged, the downstream processing strategies lag behind. Thus, the development or improvement of enzyme purification methods to efficient and competitive techniques is a main issue. Adsorptive bubble separation e.g. foam fractionation represents such a improvable separation process. In this study, foam fractionation was used to separate an extracellular esterase from submerged cultures of the basidiomycete Coprinopsis cinerea. Parameters such as pH-value and column design affect the transport of active enzymes into the foam phase and thus the efficiency of the foam fractionation process. The influence and interactions of various parameters regarding the performance of the foam fractionation process were analyzed using Design of Experiments (DoE). The use of Design of Experiments allowed a fast identification of impact parameters and a direct optimization of the foam fractionation process.
- Published
- 2011
239. Fungal photobiology: a synopsis
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Luis M. Corrochano and Universidad de Sevilla. Departamento de Genética
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Article ,Aspergillus nidulans ,Neurospora crassa ,Coprinopsis cinerea ,Mycology ,Botany ,Mucor circinelloides ,Phycomyces blakesleeanus ,Ecology, Evolution, Behavior and Systematics ,Blue light ,phytochrome ,biology ,fungi ,biology.organism_classification ,photoreceptor ,blue light ,Agricultural and Biological Sciences (miscellaneous) ,red light ,Photobiology ,white collar complex - Abstract
Fungi respond and adapt to many environmental signals including light. The photobiology of fungi has been extensively investigated, but in recent years the identification of the first fungal photoreceptor, WC-1 in the ascomycete Neurospora crassa, and the discovery that similar photoreceptors are required for photoreception in other ascomycete, basidiomycete and zygomycete fungi has allowed the molecular characterization of light reception and the early steps of signal transduction in a number of model fungi. This contribution is based on presentations made at the Special Interest Group Meeting on “Fungal Photobiology” held during IMC9. The contributions summarize the current status of fungal photobiology in Aspergillus nidulans, Neurospora crassa, Mucor circinelloides, and Coprinopsis cinerea.
- Published
- 2011
240. A lectin-mediated resistance of higher fungi against predators and parasites
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Eva Potthoff, M. Garbani, Markus Aebi, Therese Wohlschlager, J. Sabotiĉ, Peter Lüthy, Silvia Bleuler-Martinez, Jure Pohleven, Alex Butschi, Markus Künzler, Martin A. Wälti, and Michael O. Hengartner
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0303 health sciences ,Glycan ,biology ,030306 microbiology ,media_common.quotation_subject ,Lectin ,Insect ,biology.organism_classification ,Microbiology ,03 medical and health sciences ,Coprinopsis cinerea ,Nematode ,Genetics ,biology.protein ,Aphelenchus avenae ,Gene ,Ecology, Evolution, Behavior and Systematics ,Mycelium ,030304 developmental biology ,media_common - Abstract
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.
- Published
- 2011
241. Coprinopsis cinerea from rice husks forming sclerotia in agar culture
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Masahiro Tagawa, Satoshi Hanada, Hideyuki Tamaki, and Tsuneo Watanabe
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food.ingredient ,biology ,fungi ,food and beverages ,Basidiomycota ,Fungus ,biology.organism_classification ,Husk ,Coprinopsis cinerea ,food ,Botany ,Agar ,Clamp connection ,Ecology, Evolution, Behavior and Systematics - Abstract
Sclerotia were formed in agar culture by a fungus with clamp connections isolated from rice husks at Tsukuba, Japan. The sclerotia were brown, globose to ellipsoidal, small, up to 200 μm in diameter, and composed of external rind tissue and internal medulla tissue. Such tiny sclerotia have not been commonly reported among basidiomycetous fungi in the literature. The fungus was identified as Coprinopsis cinerea on the basis of morphological characteristics together with molecular analyses. Three reference strains of C. cinerea formed sclerotia similarly under identical cultural conditions.
- Published
- 2011
242. Correction to 'Structures and Synthesis of Hitoyopodins: Bioactive Aromatic Sesquiterpenoids Produced by the Mushroom Coprinopsis cinerea'
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Junnosuke Otaka, Daisuke Hashizume, Takeshi Shimizu, Hiroyuki Osada, and Yushi Futamura
- Subjects
Mushroom ,Coprinopsis cinerea ,biology ,Chemistry ,Organic Chemistry ,Botany ,Physical and Theoretical Chemistry ,biology.organism_classification ,Biochemistry - Published
- 2018
243. Regulation of fruiting body photomorphogenesis in Coprinopsis cinerea
- Author
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Takehito Nakazawa, Hiroaki Sano, Kiyoshi Nakahori, and Takashi Kamada
- Subjects
Genetics ,Regulation of gene expression ,Light ,Neurospora crassa ,fungi ,Mutant ,Shiitake Mushrooms ,Biology ,biology.organism_classification ,Microbiology ,Agaricomycetes ,Fungal Proteins ,Coprinopsis cinerea ,Gene Expression Regulation, Fungal ,Photomorphogenesis ,Fruiting Bodies, Fungal ,Agaricales ,Gene ,Transcription factor ,Signal Transduction - Abstract
The agaricomycete (homobasidiomycete) Coprinopsis cinerea has been used as a model to study the molecular mechanism for photomorphogenesis. Molecular genetic analyses of mutants defective in fruiting body (mushroom) photomorphogenesis of C. cinerea identified two genes, dst1 and dst2. dst1 encodes a homolog of WC-1, a fungal blue-light photoreceptor first identified in Neurospora crassa, while dst2 encodes a novel protein with a putative flavin adenine dinucleotide (FAD)-binding-4 domain. In addition, reverse genetic analysis revealed that disruption of a C. cinerea gene encoding a WC-2 homolog, the partner of WC-1, causes the same blind phenotype. Searches on the genome data show that both WC-1 and WC-2 homologs are present in some agaricomycetes other than C. cinerea. Furthermore, in an agaricomycete, Lentinula edodes, it has been shown in vitro that the WC-1 and WC-2 homologs interact with each other. These findings suggest that the presumptive mechanism for blue-light sensing in agaricomycetes is fundamentally similar to that in Neurospora crassa, in which the WC-1/WC-2 complex plays a central role. Since the WC-1/WC-2 complex operates as a photoreceptor and a transcription factor, future studies will include identification of the targets of the WC-1/WC-2 complex that regulate photomorphogenesis in agaricomycetes. Another future challenge will be elucidation of the role of the newly identified photomorphogenetic protein, Dst2, in the blue-light-sensing mechanism.
- Published
- 2010
244. Improvement of the Coprinopsis cinerea molecular toolkit using new construct design and additional marker genes
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Sreedhar Kilaru, Gary D. Foster, Andy M. Bailey, Catherine M. Collins, and Mary N. Heneghan
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Genetic Markers ,Genetics, Microbial ,Microbiology (medical) ,Genetics ,biology ,Genetic Vectors ,fungi ,Intron ,Gene Expression ,biology.organism_classification ,Microbiology ,Marker gene ,Coprinopsis cinerea ,Transformation, Genetic ,Gene expression ,Multiple cloning site ,Gene silencing ,Expression cassette ,Cloning, Molecular ,Agaricales ,Genetic Engineering ,Molecular Biology ,Gene ,Gene Deletion - Abstract
This paper describes the optimisation of an existing basidiomycete molecular toolkit through the development of new versatile vectors. These vectors enable the straightforward and rapid construction of gene expression and silencing cassettes by allowing the easy exchange of promoters, coding regions and terminator elements. The constructs contain multiple cloning sites (MCS) allowing any gene to be inserted using a range of restriction sites, with the option of a 5′ integral intron for efficient gene expression. We describe the testing of these vectors through marker gene expression in Coprinopsis cinerea. This work also extends the range of marker genes available for use in C. cinerea with the first report of DsRed and monomeric red fluorescent protein (mRFP) expression in C. cinerea and further demonstrates the requirement for an intron in the expression cassette for some marker genes. However, analysis of transformants containing either β-glucuronidase (GUS) or luciferase (LUC) genes, with and without an intron revealed no detectable marker gene expression. The inclusion of an intron does therefore not guarantee expression and other genetic factors may be involved.
- Published
- 2010
245. Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea ( Coprinus cinereus )
- Author
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Patricia J. Pukkila, Claire Burns, Mark L. Farman, Gerard Manning, Björn Canbäck, Ashleigh Huggins, Heather J. Palmerini, Hoi Shan Kwan, Aaron J. Mackey, David C. Fargo, Jixin Deng, Chinnapa Kodira, Vardges Ter-Hovhannisyan, Allen C. Gathman, Chun Hang Au, Sreedhar Kilaru, Dag Ahrén, Jonathan M. Goldberg, Fred S. Dietrich, Donald O. Natvig, Patrick J. Hoegger, Mario Stanke, Miriam E. Zolan, Bruce W. Birren, Alexandre Lomsadze, Weixi Li, Bruce A. Roe, Hajime Muraguchi, Sarah K. Wilke, Doris M. Kupfer, Roderic Guigó, Cathy J. Rehmeyer, Ursula Kües, Francis Martin, Walt W. Lilly, Narmada Shenoy, Chi Keung Cheng, Anders Tunlid, Takashi Kamada, Lorna A. Casselton, Li-Jun Ma, Mark Borodovsky, James B. Hooker, Timothy Y. James, Rajesh Velagapudi, Qiandong Zeng, Jason E. Stajich, and Marilee A. Ramesh
- Subjects
Retroelements ,Molecular Sequence Data ,Coprinus ,Genome ,Evolution, Molecular ,Fungal Proteins ,03 medical and health sciences ,Cytochrome P-450 Enzyme System ,Meiosis ,Gene Duplication ,Commentaries ,Gene family ,Gene ,Phylogeny ,DNA Primers ,030304 developmental biology ,Synteny ,Recombination, Genetic ,Genetics ,0303 health sciences ,Multidisciplinary ,Base Sequence ,biology ,030306 microbiology ,fungi ,Fungal genetics ,Chromosome Mapping ,RNA, Fungal ,biology.organism_classification ,Coprinopsis cinerea ,Multigene Family ,Chromosomes, Fungal ,Genome, Fungal ,Protein Kinases - Abstract
The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 10 8 synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor , a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.
- Published
- 2010
246. Mutations in the Cc.rmt1 gene encoding a putative protein arginine methyltransferase alter developmental programs in the basidiomycete Coprinopsis cinerea
- Author
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Takehito Nakazawa, Kiyoshi Nakahori, Yoshiaki Tatsuta, Takashi Fujita, and Takashi Kamada
- Subjects
Regulation of gene expression ,Protein-Arginine N-Methyltransferases ,biology ,Hypha ,Basidiomycota ,Genes, Fungal ,fungi ,Mutant ,Hyphae ,Wild type ,food and beverages ,Mutagenesis (molecular biology technique) ,General Medicine ,biology.organism_classification ,Carbon ,Culture Media ,Cell biology ,Coprinus ,Coprinopsis cinerea ,Biochemistry ,Mutagenesis ,Mutation ,Genetics ,Clamp connection ,Mycelium - Abstract
We characterized two developmental mutants of Coprinopsis cinerea, Apa56 and Sac29, newly isolated from a homokaryotic fruiting strain, 326 (Amut Bmut pab1-1), after restriction enzyme-mediated integration (REMI) mutagenesis. Both Apa56 and Sac29 exhibited slower mycelial growth than the parental wild-type strain and failed to initiate fruiting when grown on standard malt extract-yeast extract-glucose medium under 12 h light/12 h dark cycle. Both mutants exhibited unusual differentiation in aerial hyphae: differentiated hyphae lacked clamp connections and exhibited irregular shapes. The differentiated hyphae were similar to the component cells of hyphal knots, but did not form hyphal knots: they spread as dense mycelial mats. When the carbon source (glucose) in the medium was substituted with sucrose or galactose, both strains formed as many hyphal knots as the parental wild type. The hyphal knots formed, however, did not develop into fruiting-body initials, but developed into sclerotia. Molecular genetic analysis revealed that the gene, designated Cc.rmt1, is disrupted by REMI mutagenesis and is responsible for the phenotypes in both mutants. Cc.rmt1 is predicted to encode a putative protein arginine methyltransferase, some homologs of which have been shown to be involved in the regulation of gene expression in eukaryotes.
- Published
- 2010
247. Identification and characterization of CcCTR1, a copper uptake transporter-like gene, in Coprinopsis cinerea
- Author
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Akira Yano, Yuko Nakagawa, Yuichi Sakamoto, and Sayaka Kikuchi
- Subjects
chemistry.chemical_classification ,Oxidase test ,Saccharomyces cerevisiae Proteins ,biology ,Base Sequence ,Tyrosinase ,fungi ,Saccharomyces cerevisiae ,Molecular Sequence Data ,Transporter ,biology.organism_classification ,Microbiology ,Fungal Proteins ,Coprinopsis cinerea ,Enzyme ,Biochemistry ,chemistry ,Genes, Bacterial ,Heterologous expression ,SLC31 Proteins ,Agaricales ,Gene ,Cation Transport Proteins ,Copper - Abstract
Copper (Cu) is an essential element for the physiological function of organisms. In basidiomycetes, Cu is necessary for the production of phenol oxidase enzymes such as laccase and tyrosinase. We isolated and characterized two genes, CcCTR1 and -2, from the model basidiomycete Coprinopsis cinerea. CcCTR1 and -2 showed similarity to the Cu transporter CTR1 in Saccharomyces cerevisiae. Both CcCTRs had a MLxxM motif that is conserved in other CTR homologs. The addition of Cu to a liquid culture of C. cinerea decreased the mRNA accumulation of CcCTR1 and -2. Heterologous expression of CcCTR1 in S. cerevisiae increased Cu sensitivity, suggesting that CcCTR1 is a Cu uptake transporter. Together, these results suggest that CcCTR1 plays an important role in Cu accumulation in C. cinerea.
- Published
- 2010
- Full Text
- View/download PDF
248. Crystal structure of a glycoside hydrolase family 6 enzyme, CcCel6C, a cellulase constitutively produced by Coprinopsis cinerea
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Kiyoharu Fukuda, Kiyohiko Igarashi, Takashi Tonozuka, Makoto Yoshida, Masahiro Samejima, Yuan Liu, Yuma Kurakata, Atsushi Nishikawa, and Takatsugu Miyazaki
- Subjects
Conformational change ,biology ,Chemistry ,Stereochemistry ,Substrate (chemistry) ,Cell Biology ,Cellulase ,Cellobiose ,biology.organism_classification ,Biochemistry ,Carboxymethyl cellulose ,Coprinopsis cinerea ,chemistry.chemical_compound ,Hydrolase ,medicine ,biology.protein ,Glycoside hydrolase ,Molecular Biology ,medicine.drug - Abstract
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.
- Published
- 2010
249. The dst2 gene essential for photomorphogenesis of Coprinopsis cinerea encodes a protein with a putative FAD-binding-4 domain
- Author
-
Masaki Kuratani, Kazuhisa Terashima, Kiyoshi Nakahori, Takehito Nakazawa, Takashi Kamada, Kanako Tanaka, and Hajime Muraguchi
- Subjects
Mutant ,Biology ,Microbiology ,Coprinus ,Fungal Proteins ,Open Reading Frames ,Gene Expression Regulation, Fungal ,Basidiospore formation ,Botany ,Genetics ,Primordium ,Fruiting Bodies, Fungal ,Cloning, Molecular ,Models, Genetic ,fungi ,Fungal genetics ,Blotting, Northern ,biology.organism_classification ,Phenotype ,Protein Structure, Tertiary ,Cell biology ,Coprinopsis cinerea ,Mutation ,Flavin-Adenine Dinucleotide ,Photomorphogenesis ,Pileus ,Polymorphism, Restriction Fragment Length ,Protein Binding - Abstract
The fruiting-body primordium of Coprinopsis cinerea exhibits remarkable photomorphogenesis. Under a 12-h light/12-h dark regime, the primordium proceeds to the fruiting-body maturation phase in which the primordium successively undergoes basidiospore formation, stipe elongation and pileus expansion, resulting in the mature fruiting-body. In continuous darkness, however, the primordium never proceeds to the maturation phase: the pileus and stipe tissues at the upper part of the primordium remain rudimentary while the basal part of the primordium elongates, producing the etiolated "dark stipe" phenotype. In our previous studies, blind mutants, which produce dark stipes under light conditions that promote fruiting-body maturation in the wild-type, have been isolated, and two genes, dst1 and dst2, responsible for the mutant phenotype have been identified. In this study we show that the dst2-1 mutant exhibits a blind phenotype during asexual spore production in addition to that in fruiting-body photomorphogenesis. We also reveal that dst2 is predicted to encode a protein with a putative flavin adenine dinucleotide (FAD)-binding-4 domain. The two blind phenotypes, together with the existence of an FAD-binding domain in Dst2, suggest that Dst2 may play a role in perceiving blue light.
- Published
- 2010
250. Molecular Basis for Galactosylation of Core Fucose Residues in Invertebrates
- Author
-
Alex Butschi, Markus Aebi, Ebrahim Razzazi-Fazeli, Markus Künzler, Alexander Titz, Michael O. Hengartner, Bernard Henrissat, Yao-Yun Fan, Iain B. H. Wilson, and Thierry Hennet
- Subjects
Galactosyltransferase ,Glycan ,biology ,Cell Biology ,biology.organism_classification ,Biochemistry ,Fucose ,Coprinopsis cinerea ,chemistry.chemical_compound ,chemistry ,Glycosyltransferase ,biology.protein ,Molecular Biology ,Gene ,Caenorhabditis elegans ,Galectin - Abstract
Galectin CGL2 from the ink cap mushroom Coprinopsis cinerea displays toxicity toward the model nematode Caenorhabditis elegans. A mutation in a putative glycosyltransferase-encoding gene resulted in a CGL2-resistant C. elegans strain characterized by N-glycans lacking the β1,4-galactoside linked to the α1,6-linked core fucose. Expression of the corresponding GALT-1 protein in insect cells was used to demonstrate a manganese-dependent galactosyltransferase activity. In vitro, the GALT-1 enzyme showed strong selectivity for acceptors with α1,6-linked N-glycan core fucosides and required Golgi- dependent modifications on the oligosaccharide antennae for optimal synthesis of the Gal-β1,4-fucose structure. Phylogenetic analysis of the GALT-1 protein sequence identified a novel glycosyltransferase family (GT92) with members widespread among eukarya but absent in mammals.
- Published
- 2009
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