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Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase is one of the extensively studied sugar-oxidizing enzymes used as a biocatalyst for biosensors and biofuel cells. A novel pyranose dehydrogenase (CcPDH) derived from the basidiomycete Coprinopsis cinerea is the first discovered eukaryotic PQQ-dependent enzyme. This enzyme carries a b-type cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase (CDH); thus, CcPDH is a quinohemoprotein. CcPDH catalyzes the oxidation of various aldose sugars and shows significant activity toward the reverse-chair conformation of pyranoses. Interdomain electron transfer occurs in CcPDH similar to CDH, from the PQQ cofactor in the catalytic domain to the heme b in the cytochrome domain. This enzyme is able to direct electrical communication with electrodes, without artificial electron mediators, thus allowing direct electron transfer (DET)-type bioelectrocatalysis. In this review, we briefly describe recent progress in research on the biochemical discovery of CcPDH and the development of (bio)electrochemical applications (an amperometric biosensor) based on DET reactions.

The glycoside hydrolase (GH) 16 family of Coprinopsis cinerea includes 15 members distributed in four subgroups (A1, A2, B and D) by phylogenetic analysis. The expression patterns match well with the requirement of wall-softening in the germination of basidiospores, hyphal growth and branching, primordium formation, stipe elongation, pileus expansion and autolysis. Remarkably, expression levels of different GH16 members varied with different morphogenetic events. Like orthologs of Aspergillus fumigatus GH16 glucanases (ENG2–5), which were expressed in the dormant conidia and conidiogenesis, and essential for segregation of conidia, some members such as ENG in the subgroup A1 in C. cinerea were also predominantly expressed in dormant basidiospores, primordia and maturing pilei during basidiosporogenesis. In contrast, other members in subgroup A2, subgroup B or D were dominantly expressed in the germinating basidiospores, the growing mycelia, and the elongating stipes. We did not find the members of the GH81 or GH55 family in C. cinerea genome, which was different from A. fumigatus. However, C. cinerea contains an extra three subgroups (A2, B and D) compared with A. fumigatus. These extra subgroups of GH16 family members may function as those endo-β-1,3-glucanases belonging to other GH families in the development and growth of C. cinerea.

Laccase Lcc9 from Coprinopsis cinerea heterologously expressed in Pichia pastoris (rLcc9) displayed different molecular weight and specific activity from the native laccase (nLcc9). Glycosylation may play a role in regulating the Lcc9 specific activity. To elucidate this hypothesis, in this study, firstly we demonstrated that rLcc9 and nLcc9 were glycoproteins, and then enzymatically deglycosylated them. The obtained drLcc9 and dnLcc9 showed an apparent decrease in their specific activities. Three putative N-glycosylation sites (N293, N313, and N454) were then predicted in Lcc9 and substituted to evaluate their roles in its specific activity. Molecular weight analysis on those mutants suggested that glycosylation should have occurred on N313 and N454 whereas not on N293 in rLcc9. Comparison of catalytic properties of those mutants revealed that glycosylation at N313 and N454 in rLcc9 could affect the binding affinity to substrates and the catalytic rate, respectively. In addition, the glycosylation could also affect the thermal stability of rLcc9 and nLcc9 since deglycosylation of those Lcc9s resulted in decreases in their thermal stability to some extent. These results will help us to understand the effect of glycosylation on biochemical characteristics of fungal laccases, and provide us support for the improvement of fungal laccase activity based on N-linked glycosylation modification.

Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus Coprinopsis cinerea (CcPDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome b domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database. Here, we present the crystal structures of the AA12 domain in its apo- and holo-forms and the AA8 domain of this enzyme. The crystal structures of the holo-AA12 domain bound to PQQ provide direct evidence that eukaryotes have PQQ-dependent enzymes. The AA12 domain exhibits a six-blade β-propeller fold that is also present in other known PQQ-dependent glucose dehydrogenases in bacteria. A loop structure around the active site and a calcium ion binding site are unique among the known structures of bacterial quinoproteins. The AA8 cytochrome domain has a positively charged area on its molecular surface, which is partly due to the propionate group of the heme interacting with Arg181; this feature differs from the characteristics of cytochrome b in the AA8 domain of the fungal cellobiose dehydrogenase and suggests that this difference may affect the pH dependence of electron transfer.IMPORTANCE Pyrroloquinoline quinone (PQQ) is known as the "third coenzyme" following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQ-dependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected.

Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus Coprinopsis cinerea (CcPDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome b domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database. Here, we present the crystal structures of the AA12 domain in its apo-and holo-forms and the AA8 domain of this enzyme. The crystal structures of the holo-AA12 domain bound to PQQ provide direct evidence that eukaryotes have PQQ-dependent enzymes. The AA12 domain exhibits a six-blade-propeller fold that is also present in other known PQQ-dependent glucose dehydrogenases in bacteria. A loop structure around the active site and a calcium ion binding site are unique among the known structures of bacterial quinoproteins. The AA8 cytochrome domain has a positively charged area on its molecular surface, which is partly due to the propionate group of the heme interacting with Arg181; this feature differs from the characteristics of cytochrome b in the AA8 domain of the fungal cellobiose dehydrogenase and suggests that this difference may affect the pH dependence of electron transfer. Pyrroloquinoline quinone (PQQ) is known as the "third coenzyme" following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQdependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected.

Living fungal mycelium with suppressed or abolished fruit-forming ability is a self-healing substance particularly valuable biomaterial for further engineering and development in applications such as monitoring/sensing environmental changes and secreting signals. The ability to suppress fungal fruiting is also a useful tool for maintaining stability (e.g., shape, form) of a mycelium-based biomaterial with ease and lower cost. The objective of this present study is to provide a biochemical solution to regulate the fruiting body formation to replace heat killing of mycelium during production. We discovered that GSK-3 activity directly correlates with the development of fruiting bodies in fungi, especially mushroom forming fungi such as Coprinopsis cinerea. By regulating GSK-3 expression and activity, one can control the fungal fruiting body development. We successfully demonstrated that treatment of an inhibitor of GSK-3 kinase activity resulted in acceleration in mycelium growth rate, absence of fruiting body and general decrease in GSK-3 gene expression. Therefore, GSK-3 inhibitor is suggested to be included in the mycelium cultivation recipes for regulating the growth of fungal mycelium and for inhibiting the development of fruiting bodies. This is the first report of using a GSK-3 inhibitor, such as lithium or any other GSK-3 inhibitor, to suppress or abolish fruiting body formation in living fungal mycelium-based biomaterial. It also provides an innovative strategy for easy, reliable, and low cost maintenance of biomaterial containing live fungal mycelium.

The functions of glycogen synthase kinase 3 (GSK3) have been well-studied in animal, plant and yeast. However, information on its roles in basidiomycetous fungi is still limited. In this study, we used the model mushroom Coprinopsis cinerea to study the characteristics of GSK3 in fruiting body development. Application of a GSK3 inhibitor Lithium chloride (LiCl) induced enhanced mycelial growth and inhibited fruiting body formation in C. cinerea. RNA-Seq of LiCl-treated C. cinerea resulted in a total of 14128 unigenes. There were 1210 differentially expressed genes (DEGs) between the LiCl-treated samples and control samples in the mycelium stage (first time point), whereas 1402 DEGs were detected at the stage when the control samples formed hyphal knots and the treatment samples were still in mycelium (second time point). Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis of the DEGs revealed significant associations between the enhanced mycelium growth in LiCl treated C. cinerea and metabolism pathways such as “biosynthesis of secondary metabolite” and “biosynthesis of antibiotics”. In addition, DEGs involved in cellular process pathways, including “cell cycle-yeast” and “meiosis-yeast”, were identified in C. cinerea fruiting body formation suppressed by LiCl under favorable environmental conditions. Our findings suggest that GSK3 activity is essential for fruiting body formation as it affects the expression of fruiting body induction genes and genes in cellular processes. Further functional studies of GSK3 in basidiomycetous fungi may help understand the relationships between environmental signals and fruiting body development.

Active movement of mRNAs by sophisticated transport machineries determines precise spatiotemporal expression of encoded proteins. A prominent example discovered in fungi is microtubule-dependent transport via endosomes. This mode of transport was thought to be only operational in the basidiomycete Ustilago maydis. Here, we report that distinct core components are evolutionarily conserved in fungal species of distantly related phyla like Mucoromycota. Interestingly, orthologues of the key RNA-binding protein Rrm4 from the higher basidiomycete Coprinopsis cinerea and the mucoromycete Rhizophagus irregularis shuttle on endosomes in hyphae of U. maydis. Thus, endosomal mRNA transport appears to be more wide-spread than initially anticipated.Highlights‐Core transport components Upa1 and Rrm4 are conserved in different fungal phyla‐Components of the Rrm4 machinery were most likely secondarily lost in ascomycetes‐Upa1 from Microbotryum lychnidis-dioicae is functional in U. maydis‐Rrm4 orthologues from Basidio- and Mucoromycota shuttle in hyphae of U. maydis

Fruiting bodies that exhibit strong autolysis of Coprinopsis cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications.

Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1,1-6 which can be complemented by introduction of the trp1 + wild-type gene. Regularly in C. cinerea, single-trp1 +-vector transformations yield about half the numbers of clones than cotransformations with a non-trp1 +-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB + gene shown here to function as a selection marker in C. cinerea. Parts of single-trp1 + - or single-trpB + -vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single-trp1 +-vector transformants and also sets off loss of clones in cotransformation with a non-trp1 +-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1 + copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Δku70 mutant abolished the effect of doubling clone numbers in cotransformations due to preferred single trp1 + integration by homologous recombination at its native genomic site.

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

Agaricomycetes exhibit a remarkable morphological differentiation from vegetative mycelia to huge fruiting bodies. To investigate the molecular mechanism underlying the fruiting body development, we have isolated and characterized many Coprinopsis cinerea mutant strains defective in fruiting initiation to date. Dikaryon formation in agaricomycetes, which is followed by fruiting development, is governed by the mating type loci, A and B. Recently, mutations in the Cc.snf5 gene, which encodes a putative component of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF), were shown to cause defects in A-regulated clamp cell morphogenesis, as well as in fruiting initiation. Here, we demonstrate that Cc.arp9, which encodes a putative actin-related protein associated with two chromatin remodeling complexes, SWI/SNF and remodels the structure of chromatin (RSC), is also essential for fruiting initiation. In contrast to Cc.snf5 mutants, Cc.arp9 mutants were not defective in clamp cell formation. The effects of mutations in Cc.arp9 and Cc.snf5 on oidia production and the transcriptional expression levels of clp1 and pcc1, which are under the control of the A gene, were also examined. These indicated that Cc.Snf5 is involved in A-regulated pathways, whereas Cc.Arp9 is not apparently. Cc.arp9/Cc.snf5 double-gene disruptants were generated and their phenotypes were analyzed, which suggested a complicated developmental regulation mechanism mediated by chromatin remodeling.

The dikaryon is the persistent vegetative phase of most basidiomycetes and arises by cell fusion of two haploids carrying different mating type alleles. The diplophase is restricted to a single cell (the basidium) where meiosis takes place. Cell fusion and karyogamy are thus temporally and spatially separated. The dikaryon is characterized by the presence in each cell of two genetically distinct nuclei in a 1:1 ratio and by clamp connections, structures involved in partitioning of the nuclei during their synchronous division. In Coprinopsis cinerea and Schizophyllum commune, with two mating type loci (A and B), heterokaryons with identical A alleles exhibit nuclear migration but no clamps, and those with identical B alleles have unfused clamps (pseudoclamps) and no nuclear migration. Armillaria species are exceptional among basidiomycetes because of their persistent vegetative diploid phase with no clamp connections. Cell fusion is followed immediately by karyogamy. Haploidization of the diploid nucleus may occur in the fruiting body, resulting in a new dikaryophase–diplophase. Diploids in Ustilago maydis were the first to be synthesized in any basidiomycete. They played a critical role in pioneering studies on DNA recombination and repair. Subsequently diploids were generated in C. cinerea, S. commune, Microbotryum violaceum, and Cryptococcus neoformans. Dikaryons (or heterokaryons) and diploids of the same genotype exhibit phenotypic differences. The reason is not known but evidence indicates that gene expression differs when the same genetic information resides in two nuclei versus one nucleus in the same cell. Diploids in basidiomycete fungi are very stable by themselves but become unstable in 2N + N or 2N + 2N dikaryons. Instability results in haploidization (gradual loss of chromosomes) of the diploid nucleus. The mechanism that triggers this instability is not known.

Fungi do not have different sexes characterized by specific morphological organs but control sexual development by physiological differences defined by mating types. While other lineages in the fungal kingdom have only two distinct mating types, multiple mating types are common in the Basidiomycetes, presumably for outcomes related to enhanced outbreeding. Mating types in Basidiomycetes are determined by genes that encode two types of homeodomain transcription factors (HD genes) and by genes that encode lipopeptide pheromones and pheromone receptors (PR genes). In the tetrapolar mushroom Coprinopsis cinerea, these genes reside at two distinct loci on different chromosomes and regulate distinct steps in dikaryon formation and maintenance and fruiting body formation for sexual reproduction by controlling the expression of large sets of genes. The matA (HD) locus in C. cinerea has an estimated 160 alleles in nature, and the matB (PR) locus has 79. The respective products of both loci interact with each other in a lock-and-key manner. Proteins encoded in allelic sets of mating type genes recognize each other and interact in order to either form a functional transcription factor complex that is transported into the nucleus (matA proteins) or to initiate a pheromone response signaling cascade (matB proteins). Products encoded by the same matA or matB allele do not interact, which is crucial to the functionality of the two systems controlling sexual development. Multiple alleles of mating type genes arose in evolution through the evolution of distinct DNA sequences and the resulting encoded proteins perform the tasks of recognition and discrimination. In a second modus of multiplication, paralogous sets of HD and PR genes evolved by gene duplications and subsequent sequence diversification in order to mediate functional independency and respective allele variation. In C. cinerea, there are thus three paralogous groups of functional mating type genes at both the matA and the matB locus. Because groups of paralogous genes are freely interchangeable, only a few alleles of each of the actual gene groups (between 2 and 9 are known per group) are required to generate the large number of distinct matA and matB mating types found in nature. As deduced from the ever increasing number of genome projects, the basic principles of evolution of multiple mating types as unraveled in C. cinerea by Lorna A. Casselton and her coworkers are to different degrees also operative in other mushroom species.

In the filamentous basidiomycetes Coprinopsis cinerea and Schizophyllum commune, mating is regulated by the tetrapolar mating-type system consisting of two unlinked genetic complexes, named A and B. In the nineties, the molecular structure of A and B mating type loci and genes was revealed side by side in C. cinerea and S. commune, first the A complex and quite soon thereafter the B complex genes. The clear molecular structure of C. cinerea mating type genes has led to their use as models for genomic approaches to investigate several other filamentous basidiomycetes. In filamentous fungi, hyphal fusions are important for the distribution of available nutrients in a fungal colony. In ascomycetes and basidiomycetes they are also important for sexual reproduction. These aspects have been approached, especially in filamentous ascomycetes, but in filamentous basidiomycetes the role of fusions in the fungal life cycle has received less attention. Several proteins encoded by the genes required for hyphal fusion in filamentous ascomycetes show homology with proteins forming the striatin-interacting phosphatase and kinase (STRIPAK) complex in eukaryotic cells. Homologs to the genes encoding STRIPAK complex proteins can be identified in C. cinerea and S. commune genomes suggesting that a STRIPAK-like complex could also regulate hyphal fusions of filamentous basidiomycetes. The STRIPAK complex is a conserved signaling complex also homologous to the Saccharomyces cerevisiae Far complex involved in cell cycle arrest at yeast mating, while the fission yeast Schizosaccharomyces pombe SIP complex is involved in transition signaling from mitosis to cytokinesis. In filamentous basidiomycetes the signaling pathway regulated by the B mating type genes, the pheromone response pathway, is assumed to follow the same pattern as in the yeast S. cerevisiae. This poses an interesting question as to the relationship between the STRIPAK complex proteins and the proteins in cellular processes such as cell cycle, septal dissolution, nuclear migration, clamp cell development and fusion, known to take place after hyphal fusions. All these processes are also dependent on the activation of the B and A mating type pathways in C. cinerea and S. commune.

The mitochondrion is a vital organelle in eukaryotic cells. Unlike the inheritance of nuclear genes, that of mitochondrial DNA (mtDNA) does not follow Mendelian Laws. In the great majority of sexual plants and animals, mtDNA is uniparentally inherited from the maternal parent. However, there is a diversity of mtDNA inheritance patterns in basidiomycete fungi. In this commemorative review of mtDNA inheritance in basidiomycetes, we first summarize Dr. Lorna Casselton's pioneering contributions to the subject using Coprinopsis cinerea as a model organism. Using her findings as a benchmark, we then describe other mtDNA inheritance patterns found in basidiomycete fungi, both in laboratory crosses and in natural populations. We review recent research on the genes, molecular mechanisms, and environmental factors that impact mtDNA inheritance in basidiomycete fungi. The patterns and mechanisms of mitochondrial inheritance are compared between unicellular and filamentous basidiomycetes, and among basidiomycetes, ascomycetes and representatives from other groups of eukaryotes. Our analyses suggest that basidiomycetes can be excellent models for understanding the molecular, cellular, and evolutionary mechanisms governing mtDNA inheritance in eukaryotes.

The balance and interplay between sexual and asexual reproduction is one of the most intriguing mysteries in the study of fungi. The choice of developmental strategy reflects the ability of fungi to adapt to the changing environment. However, the evolution of developmental paths and the metabolic regulation during differentiation and morphogenesis are poorly understood. Here, an analysis was performed of carbohydrate metabolism and gene expression regulation during the early differentiation process from the vegetative mycelium, to the differentiated structures, fruiting body, oidia and sclerotia, of a homokaryotic fruiting Coprinopsis cinerea strain A43mutB43mut pab1-1 #326. Changes during morphogenesis and the evolution of developmental strategies were followed. Conversion between glucose and glycogen and between glucose and beta-glucan were the main carbon flows in the differentiation processes. Genes related to carbohydrate transport and metabolism were significantly differentially expressed among paths. Sclerotia displayed a set of specifically up-regulated genes that were enriched in the carbon metabolism and energy production and conversion processes. Evolutionary transcriptomic analysis of four developmental paths showed that all transcriptomes were under the purifying selection, and the more stressful the environment, the younger the transcriptome age. Oidiation has the lowest value of transcriptome age index (TAI) and transcriptome divergence index (TDI), while the fruiting process has the highest of both indexes. These findings provide new insights into the regulations of carbon metabolism and gene expressions during the early stages of fungal developmental paths differentiation, and improve our understanding of the evolutionary process of life history and reproductive strategy in fungi.

Coprinopsis cinerea is a model mushroom-forming basidiomycete which produces basidiospores during sexual reproduction. This fungus is widely used to study fruiting body formation and development. Molecular mechanisms controlling its growth from vegetative mycelium to multicellular mature fruiting body have been studied extensively. However, little is known about the underlying biological processes during germ tube outgrowth or the transition from basidiospores to multinucleate hyphae. To better understand sexual spore germination in fungi, here we examined the time-dependent cellular events at resting, germinating and fully germinated basidiospores of C. cinerea by genome-wide transcriptional and post-transcriptional analyses and by carbohydrate composition analysis. Our results revealed a high demand of protein degradation, and biosynthesis of various compounds at the early stage of basidiospore gemination and dynamic changes of carbohydrate metabolism throughout the germination process. Seven microRNA-like RNAs (milRNAs) were identified in the resting basidiospores of C. cinerea, six of which were basidiospore-specific. Glycogen and trehalose were shown to be the carbon sources supporting the initiation of germ tube outgrowth. One basidiospore-specific milRNA, cci-milR-37, was found to be a potential regulator of glycogen metabolic pathways related to vegetative hyphal growth. Our results demonstrated the mRNA and miRNA-mediated regulation on energy production, protein and carbohydrate metabolisms at the early developmental stages of germ tube to form totipotent hyphae. To our knowledge, this is the first study to show the roles of miRNAs in mushroom basidiospore germination and out-growth.

Understanding the DNA variation in regulation of carbohydrate-active enzymes (CAZymes) is fundamental to the use of wood-decaying basidiomycetes in lignocellulose conversion into renewable energy. Our goal is to identify the regulators of lignocellulolytic enzymes in Coprinopsis cinerea, of which the genome harbors high number of Auxiliary Activities enzymes.The DNA sequence of C. cinerea family including 46 single spore isolates (SSIs) from crosses of two homozygous strains are used to develop a panel of SNP markers. Then the RNA sequence were used to characterize the gene expression profiles. The RNA were extracted from cultures grown on softwood-enriched sawdust to induce lignocellulolytic enzymes and CCR de-repression genes. To assess the genetic contribution to enzyme expression variations among the 46 SSIs, associations between SNPs and gene expressions were examined genome-widely. 5148 local eQTLs and 7738 distant eQTLs were obtained. By analyzing these eQTLs, the potential regulatory factors of the CAZymes expression and the de-repression of Carbon Catabolism Repression (CCR) were identified,.The eQTL network is characterized in terms of hotspots, evolutionary age and post-translational modifications (PTMs). In the eQTL network of C. cinerea, the non-regulatory genes are younger than the regulatory genes. The proteins regulated by combinational multiple types of PTMs are more likely to function as super regulatory hotspots in protein-protein interactions. The evolutionary age analysis and the PTMome analysis could serve as alternative methods to identify master regulators from genomic data.This work demonstrates a comprehensive bioinformatics approach to identify regulatory factors with next-generation sequencing data. The results provide candidate genes for bioengineering to increase the enzyme production, which will practically benefit the bioethanol production from lignocellulose.SignificanceThis eQTL analysis is designed to study the fungal CAZymes and carbon catabolism repression, especially during the mycelium stage.In Coprinopsis cinerea, only the regions near two ends of the chromosomes have high recombination rate, and suitable for family based eQTL analysis.A sugar transporter is a hotspot controlling many CCR genes.CAZymes are not regulated by a master regulator, but by individual regulators. This indicates that CAZymes are under specific regulatory pathways, so can response to specific conditions.In the eQTL network, the rGenes are evolutionarily older, with more types of PTM sites than eGenes.In the eQTL network, the proteins with more types of PTM sites are more likely associated with Information Storage and Processing, and act as super-hub in the network.

In 2014, the first fungal pyrroloquinoline-quinone (PQQ)-dependent enzyme was discovered as a pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH). This discovery laid the foundation for a new Auxiliary Activities (AA) family, AA12, in the Carbohydrate-Active enZymes (CAZy) database and revealed a novel enzymatic activity potentially involved in biomass conversion. This review summarizes recent progress made in research on this fungal oxidoreductase and related enzymes. CcPDH consists of the catalytic PQQ-binding AA12 domain, an N-terminal cytochrome b AA8 domain, and a C-terminal family 1 carbohydrate-binding module (CBM1). CcPDH oxidizes 2-keto-d-glucose (d-glucosone), l-fucose, and rare sugars such as d-arabinose and l-galactose, and can activate lytic polysaccharide monooxygenases (LPMOs). Bioinformatic studies suggest a widespread occurrence of quinoproteins in eukaryotes as well as prokaryotes.

Local rice is rice that has been cultivated for generations by the community and commonly cultivated without using chemical inputs. Endophytic fungi are fungi that live in the plant tissue and does not cause disease symptoms in the host plants. This study aimed to molecular identifying isolates of MDTA and MDTB endophytic fungi which have been isolated from the local Pulu Mandoti rice plant tissue and growth test on the four types of culture media those were synthetic PDA, natural PDA, MPA, and MEA. The fungi DNA isolation using DNesay Kit. DNA sequencing analysis using the mega BLAST program showed that the MDTB fungus has similarities to Podoscypha bolleana strain 32034 no accession JQ675334 and Podoscypha bolleana strain 32032 no accession JQ675332, whereas the MDTA fungus has similarities to Coprinopsis cinerea A2S3-5 isolate and Coprinopsis cinerea strain CNRMA / F 07-32. The best culture media and sporulation of endophytic fungi is MPA media. This research is the first study to molecular identifying with endophytic fungi from local rice and viability test on the four types of culture media. The results of this study contribute to the diversity of local rice endophytic fungi in Sulawesi.

Fungi secrete a set of glycoside hydrolases and oxidoreductases, including lytic polysaccharide monooxygenases (LPMOs), for the degradation of plant polysaccharides. LPMOs catalyze the oxidative cleavage of glycosidic bonds after activation by an external electron donor. So far, only flavin-dependent oxidoreductases (from the auxiliary activity [AA] family AA3) have been shown to activate LPMOs. Here, we present LPMO activation by a pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase (PDH) from Coprinopsis cinerea , Cc PDH, the founding member of the recently discovered auxiliary activity family AA12. Cc PDH contains a C-terminal family 1 carbohydrate binding module (CBM1), an N-terminal family AA8 cytochrome domain, and a central AA12 dehydrogenase domain. We have studied the ability of full-length Cc PDH and its truncated variants to drive catalysis by two Neurospora crassa LPMOs. The results show that Cc PDH indeed can activate the C-1-oxidizing N. crassa LPMO 9F ( Nc LPMO9F) and the C-4-oxidizing Neurospora crassa LPMO 9C ( Nc LPMO9C), that this activation depends on the cytochrome domain, and that the dehydrogenase and the LPMO reactions are strongly coupled. The two tested Cc PDH-LPMO systems showed quite different efficiencies, and this difference disappeared upon the addition of free PQQ acting as a diphenol/quinone redox mediator, showing that LPMOs differ when it comes to their direct interactions with the cytochrome domain. Surprisingly, removal of the CBM domain from Cc PDH had a considerable negative impact on the efficiency of the Cc PDH-LPMO systems, suggesting that electron transfer in the vicinity of the substrate is beneficial. Cc PDH does not oxidize cello-oligosaccharides, which makes this enzyme a useful tool for studying cellulose-oxidizing LPMOs. IMPORTANCE Lytic polysaccharide monooxygenases (LPMOs) are currently receiving increasing attention because of their importance in degrading recalcitrant polysaccharides and their potential roles in biological processes, such as bacterial virulence. LPMO action requires an external electron donor, and fungi growing on biomass secrete various so-called glucose-methanol-choline (GMC) oxidoreductases, including cellobiose dehydrogenase, which can donate electrons to LPMOs. This paper describes how an enzyme not belonging to the GMC oxidoreductase family, Cc PDH, can activate LPMOs, and it provides new insights into the activation process by (i) describing the roles of individual Cc PDH domains (a dehydrogenase, a cytochrome, and a carbohydrate-binding domain), (ii) showing that the PDH and LPMO enzyme reactions are strongly coupled, (iii) demonstrating that LPMOs differ in terms of their efficiencies of activation by the same activator, and (iv) providing indications that electron transferring close to the substrate surface is beneficial for the overall efficiency of the Cc PDH-LPMO system.

Coprinopsis cinerea is a model mushroom particularly suited to study fungal fruiting body development and the evolution of multicellularity in fungi. While microRNAs (miRNAs) are extensively studied in animals and plants for their essential roles in post-transcriptional regulation of gene expression, miRNAs in fungi are less well characterized and their potential roles in controlling mushroom development remain unknown. To identify miRNA-like RNAs (milRNAs) in C. cinerea and explore their expression patterns during the early developmental transition of mushroom development, small RNA libraries of vegetative mycelium and primordium were generated and putative milRNA candidates were identified following the standards of miRNA prediction in animals and plants. Two out of 22 novel predicted milRNAs, cci-milR-12c and cci-milR-13e-5p, were validated by northern blot and stem-loop reverse transcription real-time PCR. Cci-milR-12c was differentially expressed whereas the expression levels of cci-milR-13e-5p were similar in the two developmental stages. Target prediction of the validated milRNAs resulted in genes associated with fruiting body development, including pheromone, hydrophobin, cytochrome P450, and protein kinase. Besides, essential genes for miRNA biogenesis, including three coding for Dicer-like (DCL), two for Argonaute-like (AGO-like) and one for quelling deficient-2 (QDE-2) proteins, were identified in the C. cinerea genome. Phylogenetic analysis showed that the DCL and AGO-like proteins of C. cinerea were more closely related to those in other basidiomycetes and ascomycetes than to animals and plants. Taken together, our findings provided the first evidence of milRNAs in the model mushroom and their potential roles in regulating fruiting body development. Information on the evolutionary relationship of milRNA biogenesis proteins across kingdoms has also provided new insights into further functional and evolutionary studies of miRNAs.

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions.

Inky cap mushroom (Coprinus sp) possesses vital antifungal properties against the major wilt causing pathogens of banana, tomato and chilli. Wilt disease caused by Fusarium oxysporum f. sp. cubense in banana, F. o. f. sp. lycopersici in tomato and F.brachygibbosum in chilli were obtained and the antifungal activities of Coprinus cinerea was assessed. Methanolic extract from the fruiting bodies of inky cap C. cinerea consisted of molecules which could inhibit the growth of Fusarium spp at 2000 ppm. Results showed 54.5, 52.2 and 51.1 per cent of mycelia inhibition of F. oxysporum f. sp. cubense, F. o. f. sp. lycopersici and F. brachygibbosum, respectively when the methanolic extract of C. cinerea was used in agar wells in petri dishes. GC-MS analysis of the methanolic extract of the fruiting bodies of inky cap indicated the presence of 9 different compounds viz., formic acid, 2-propenyl ester; acetic acid; D-alanine; N-propargyloxycarbonyl-; isohexyl ester; 4H-Pyran-4-one; 2,3-dihydro-3,5-dihydroxy-6-methyl-; n-hexadecanoic acid; octasiloxane; 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl-; 2,7-diphenyl-1,6-dioxopyridazino [4,5:2’,3’] pyrrolo[4’,5’-d]pyridazine; squalene and 2-Nonadecanone 2,4-dinitrophenylhydrazine. Among these compounds, formic acid, 2-propenyl ester, acetic acid; n-hexadecanoic acid; 2,7-diphenyl-1,6-dioxopyridazino [4,5:2’,3’] pyrrolo[4’,5’-d]pyridazine; squalene; 2-nonadecanone 2,4-dinitrophenylhydrazine are have been reported to possess antifungal activities.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

8 páginas.-- 4 figuras.-- 4 tablas.-- 34 referencias.-- Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cy00272j, This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H2O2 as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200000) than MroUPO (26000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (kcat/Km) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18O from H218O2 into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydroxylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemical synthesis for the production of DHS, RSV and oxyRSV., This study was funded by the EnzOx2 (H2020-BBI-PPP-2015-2-1-720297) EU-project, and the BIORENZYMERY (AGL2014- 53730-R) project of the Spanish MINECO (co-financed by FEDER). H. Lund (Novozymes) is acknowledged for rCciUPO

The fine structures of Fc N-glycan modulate the biological functions and physicochemical properties of antibodies. By remodeling N-glycan to obtain a homogeneous glycoform or chemically modified glycan, antibody characteristics can be controlled or modified. Such remodeling can be achieved by transglycosylation reactions using a mutant of endoglycosidase from Streptococcus pyogenes (Endo-S) and glycan oxazoline. In this study, we generated improved mutants of Endo-S by introducing additional mutations to the D233Q mutant. Notably, Endo-S D233Q/Q303L, D233Q/E350Q, and several other mutations resulted in transglycosylation efficiencies exceeding 90%, with a single-digit donor-to-substrate ratio of five, and D233Q/Y402F/D405A and several other mutations resulted in slightly reduced transglycosylation efficiencies accompanied by no detectable hydrolysis activity for 48 h. We further demonstrated that the combined use of mutants of Endo-S with Endo-M or Endo-CC, endoglycosidases from Mucor hiemalis and Coprinopsis cinerea, enables one-pot transglycosylation from sialoglycopeptide to antibodies. This novel reaction enables glycosylation remodeling of antibodies, without the chemical synthesis of oxazoline in advance or in situ.

A large amount of granular protrusions overlie the outer cell wall surfaces in both elongating and non-elongating stipe regions but overlie the inner cell wall surfaces only in non-elongating stipe regions. Removal of granular protrusions using alkali, amorphous materials overlying on both the inner and outer cell wall surfaces were explored in the non-elongating stipe regions. β-1,3-Glucanase treatment not only removed above those granular protrusions and underlying amorphous materials on the wall surfaces but also removed wall matrices embedding chitin microfibrils on the cell walls of most stipe regions, except for the outer cell wall surfaces of the non-elongating stipe regions where most of the wall matrices remained. The chitin microfibrils were closely and transversely arranged on both the inner and outer cell wall surfaces in the elongating apical stipe region, whereas they were loosely and transversely arranged on the inner cell wall surfaces and further became sparser and even randomly arranged on the outer cell wall surface in the non-elongating stipe regions. We propose that the surface deposition of granular protrusions and amorphous materials and the change of microfibril architecture and wall matrices may cause loss of wall plasticity and cessation of stipe elongation.

The effectiveness of fungal treatment by Coprinopsis cinerea and Polyporus tricholoma on biodegradability and methane yield of paragrass was investigated. After 15 days fungal treatment, reduction in cellulose crystallinity was found in grass treated with C. cinerea and P. tricholoma . The maximum methane production rate occurred at day 10 for the grass treated with C. cinerea , day 13 for the grass treated with P. tricholoma , and day 22 for the untreated grass. However, after 140 day anaerobic digestion, the methane yield of the grass treated by C. cinerea and by P. tricholoma was approximately 15% lower than that of the untreated grass, which was 368 mL STP/g VS added. Using two-stage fungal treatment and anaerobic digestion, the recalcitrant cellulose in the fungal treated grass was significantly lower than that of the untreated grass ( P

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

Fruiting body formation in Agaricomycetes (Agaricomycotina) represents the most complex developmental processes known in the fungal kingdom. Shapes range from simple resupinate forms with open hymenia through to closed puff-balls and false truffles with internally hidden hymenia and include brackets and stiped mushrooms, which may have open caps throughout or which open during development, where the hymenia cover the surfaces of gills or pores. Mushroom shapes and features do not necessarily reflect close or distant phylogenetic relationships. Thus, morphological characteristics have lost some of their former significance in taxonomy. The onset and progress of courses of processes in mushroom formation are determined by the sum of various genetic, physiological and environmental factors. Shapes of mushrooms can be dramatically changed by mutations and by adverse environmental conditions. Events in normal fruiting body formation may run in parallel or behind each other in the form of ‘subroutines’ that have different degrees of independency to each other. Alterations in details or in places and orders of distinct subroutines and omissions can result in abnormal mushrooms. Developmental processes, time courses and tissue structures have been described in more details for a few model species (such as the hemiangiocarpous Coprinopsis cinerea and the gymnocarpous Schizophyllum commune) and some species of commercial interest (e.g. the gymnocarpous Auricularia auricula-judae and the hemiangiocarpous Agaricus bisporus). Morphological descriptions of fruiting body development in these four species are summarized here. Agaricomycetes have relatively large genomes with more than 10,000 different genes, many of which are expressed during the fruiting process in specific pseudoparenchymatous tissues (plectenchyma) or possibly only in individual cells within a tissue and at specific times. To understand the distinct functions of all these genes in space and time will require very fine dissection and analysis of distinct mushroom tissues and cells in future studies.

Two new compounds, 10-dehydroxymelleolide D (1) and 13-hydroxymelleolide K (2), along with seven known compounds, 5'-O-methylmelledonal (3), melleolide D (4), 13-hydroxydihydromelleolide (5), melleolide (6), armillarinin (7), armillaridin (8), and armillarikin (9), were isolated from the culture broth of Armillaria sp. Their structures were determined by spectroscopic data analysis. All the compounds inhibited plant growth of lettuce. Melleolide (6) and armillarikin (9) inhibited mycelial growth of Coprinopsis cinerea and/or Flammulina velutipes.

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/β-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.

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

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

To resolve the structure of laminarin, the recombinant endo-β-1,3-glucanase from Coprinopsis cinerea, which has specific activity on β-1,3 glycosidic bond and could hydrolyze the laminarin with complex structure, was used to hydrolyze laminarin. Then, the structures of enzyme-resistant oligosaccharides were quantitatively and qualitatively analysed by high-performance anion exchange chromatography coupled with mass spectrometry. The laminarin from Laminaria digitata contains 9.51% β-1,6 glycosidic bonds only in the branches (branch degree 7.68%). The laminarin from Eisenia bicyclis contains more β-1,6 glycosidic bonds: 19.42% β-1,6 glycosidic bonds in backbone and more and longer β-1,6 branches (branch degree 25.99%). The differences in the ratio of glycosidic bonds and branch degree influence their bioactivity: the antioxidant activity and the antimicrobial activity against Gram positive bacteria of the laminarin from E. bicyclis is stronger than the laminarin from L. digitata, but the antimicrobial activity on Gram negative bacteria of the laminarin from E. bicyclis is weaker.

Defensins make up a class of cysteine-rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Because of their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin-like peptide, previously identified in the mushroom Coprinopsis cinerea. It exerts its activity against a range of Gram-positive bacteria by binding to the peptidoglycan precursor lipid II and prevention of proper cell wall formation. In this study, we present a new workflow for the generation, production, and activity-driven selection of copsin derivatives, based on their expression in Pichia pastoris. One hundred fifty-two single-amino acid mutants and combinations thereof allowed the identification of k-copsin, a peptide variant exhibiting significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k-copsin, in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47 and 35% of the total contacts for copsin and k-copsin, respectively. Mutated amino acids were located in loop regions of k-copsin and shown to be crucial in the perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed toward antibacterial therapies less prone to resistance issues.

Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of pex1 disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that pex1 is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous C. cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

Biofilm formation on fungal hyphae and production of antifungal molecules are strategies of bacteria in their competition with fungi for nutrients. Since these strategies are often coordinated and under control of quorum sensing by the bacteria, interference with this bacterial communication system can be used as a counter-strategy by the fungi in this competition. Hydrolysis of N-acyl-homoserine lactones (HSL), a quorum sensing molecule used by Gram-negative bacteria, by fungal cultures has been demonstrated. However, the enzymes that are responsible for this activity, have not been identified. In this study, we identified and characterized two paralogous HSL hydrolyzing enzymes from the coprophilous fungus Coprinopsis cinerea. The C. cinerea HSL lactonases belong to the metallo-β-lactamase family and show sequence homology to and a similar biochemical activity as the well characterized lactonase AiiA from Bacillus thuringiensis. We show that the fungal lactonases, similar to the bacterial enzymes, are kept intracellularly and act as a sink for the bacterial quorum sensing signals both in C. cinerea and in Saccharomyces cerevisiae expressing C. cinerea lactonases, due to the ability of these signal molecules to diffuse over the fungal cell wall and plasma membrane. The two isogenes coding for the C. cinerea HSL lactonases are arranged in the genome as a tandem repeat and expressed preferentially in vegetative mycelium. The occurrence of orthologous genes in genomes of other basidiomycetes appears to correlate with a saprotrophic lifestyle.

Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.

The aim of this work was to study the molecular breeding of oleaginous filamentous Mortierella alpina for high production of linoleic (LA) or oleic acid (OA). Heterologous expression of the Δ12-desaturase (DS) gene derived from Coprinopsis cinerea in the Δ6DS activity-defective mutant of M. alpina increased the LA production rate as to total fatty acid to 5 times that in the wild strain. By suppressing the endogenous Δ6I gene expression by RNAi in the Δ12DS activity-defective mutant of M. alpina, the OA accumulation rate as to total fatty acid reached 68.0%. The production of LA and OA in these transformants reached 1.44 and 2.76 g/L, respectively, on the 5th day. The Δ6I transcriptional levels of the RNAi-treated strains were suppressed to 1/10th that in the parent strain. The amount of Δ6II RNA in the Δ6I RNAi-treated strain increased to 8 times that in the wild strain.

The recently identified fungal protease inhibitors cnispin, from Clitocybe nebularis, and cospin, from Coprinopsis cinerea, are both β-trefoil proteins highly specific for trypsin. The reactive site residue of cospin, Arg27, is located on the β2-β3 loop. We show here, that the reactive site residue in cnispin is Lys127, located on the β11-β12 loop. Cnispin is a substrate-like inhibitor and the β11-β12 loop is yet another β-trefoil fold loop recruited for serine protease inhibition. By site-directed mutagenesis of the P1 residues in the β2-β3 and β11-β12 loops in cospin and cnispin, protease inhibitors with different specificities for trypsin and chymotrypsin inhibition have been engineered. Double headed inhibitors of trypsin or trypsin and chymotrypsin were prepared by introducing a second specific site residue into the β2-β3 loop in cnispin and into the β11-β12 loop in cospin. These results show that β-trefoil protease inhibitors from mushrooms exhibit broad plasticity of loop utilization in protease inhibition.