Production of a lignocellulolytic enzyme system for simultaneous bio-delignification and saccharification of corn stover employing co-culture of fungi.

Aiming at improving the efficiency of transferring corn stover into sugars, an efficient lignocellulolytic enzyme system was developed and investigated by co-cultivation of the Coprinus comatus with Trichoderma reesei in a single bioreactor. The results showed that the lignocellulolytic enzyme activities of the co-culture exceeded that of the monoculture, suggesting synergistic interaction between two fungi. The highest laccase activity from the co-culture was 2.6-fold increase over that of the C. comatus monoculture and reached a peak 3days earlier. The maximum delignification obtained was 66.5% and about 82% of the original polysaccharides were converted into fermentable sugars by simultaneous bio-delignification and saccharification process. Correlation analysis showed that sugar yields were directly proportional to the lignin degradation. Our results suggested that co-fungi cultivation was a valuable technique for corn stover bioconversion, which could produce high efficiency of lignocellulolytic enzyme system as a cheaper alternative to commercial enzymes for industrial utilization.

The Coprinopsis cinerea septin Cc.Cdc3 is involved in stipe cell elongation.

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.

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized Coprinus cinereus in fluidized bed bioreactor.

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g(-1) h(-1) with a 50% conversion time (t1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus).

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.

Optimization of the functional expression of Coprinus cinereus peroxidase in Pichia pastoris by varying the host and promoter.

Peroxidase from Coprinus cinereus (CiP) has attracted attention for its high specific activity and broad substrate spectrum compared with other peroxidases. In this study, the functional expression of this peroxidase was successfully achieved in the methylotrophic yeast Pichia pastoris. The expression level of CiP was increased by varying the microbial hosts and the expression promoters. Since a signal sequence, such as the alpha mating factor of Saccharomyces cerevisiae, was placed preceding the cDNA of the CiP coding gene, expressed recombinant CiP (rCiP) was secreted into the culture broth. The Mut+ Pichia pastoris host showed a 3-fold higher peroxidase activity, as well as 2-fold higher growth rate, compared with the Muts Pichia pastoris host. Furthermore, the AOX1 promoter facilitated a 5-fold higher expression of rCiP than did the GAP promoter.

Meiotic cytogenetics in Coprinus cinereus.

The basidiomycete fungus Coprinus cinereus has naturally synchronous meiosis and is amenable to analysis using an array of well-developed genetic and molecular tools. In this chapter, we explain in detail the two methods most commonly employed for C. cinereus, staining of intact gill segments and chromosome spreads, with an example of the application of each. We describe iron-hematoxylin staining of intact gill segments for the brightfield examination of meiotic progression, and the use of surface spreads and fluorescence in situ hybridization (FISH) to investigate meiotic chromosome pairing. Gill segments can alternatively be stained with DAPI for the determination of meiotic stage, or propidium iodide for the quantitation of nuclear DNA content, and the chromosome fixation and spreading techniques used for FISH are also suitable for immunolocalization studies of chromosomal proteins.

Identification and characterisation of structural maintenance of chromosome 1 (smc1) mutants of Coprinopsis cinerea.

A Coprinopsis cinerea homokaryotic fruiting strain was mutagenised, identifying a mutant that exhibited a hyphal growth temperature sensitive defect and hyphal knot development defect at an early fruiting stage, even at the hyphal growth permissive temperature. Microscopic observation suggested that the mutant nuclei exhibited defects in the metaphase to anaphase transition at the restrictive temperature. The gene in which the mutation occurred was cloned, sequenced and determined to be homologous to smc1. Sequence analyses of the mutant revealed deletion of 28 base pairs in the 19th intron of the Cc.smc1 gene, resulting in complete failure of splicing of that intron and in insertion of 14 amino acids in the C-terminal region of the Cc.Smc1 protein. We isolated eight hyphal growth revertants and identified four intragenic suppressors. All were the result of amino acid substitutions in the C-terminal region. Three of the suppressors caused reversion of the arrest in an early fruiting stage. One of the suppressors exhibited cold sensitivity and failed to suppress the fruiting defect, suggesting that flexibility of a lobe in the C-terminal region is important for proper function of Cc.Smc1.

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity.

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

DNA ligase IV from a basidiomycete, Coprinus cinereus, and its expression during meiosis.

DNA ligase IV is thought to be involved in DNA double-strand break repair and DNA non-homologous end-joining pathways, but these mechanisms are still unclear. To investigate the roles of DNA ligase IV from a biologically functional viewpoint, the authors studied its relationship to meiosis in a basidiomycete, Coprinus cinereus, which shows a highly synchronous meiotic cell cycle. The C. cinereus cDNA homologue of DNA ligase IV (CcLIG4) was successfully cloned. The 3.2 kb clone including the ORF encoded a predicted product of 1025 amino acid residues with a molecular mass of 117 kDa. A specific inserted sequence composed of 95 amino acids rich in aspartic acid and glutamic acid could be detected between tandem BRCT domains. The inserted sequence had no sequence identity with other eukaryotic counterparts of DNA ligase IV or with another aspartic acid and glutamic acid rich sequence inserted in C. cinereus proliferating cell nuclear antigen (CcPCNA), although the length and the percentages of aspartic and glutamic acids were similar. In addition, the recombinant CcLIG4 protein not only showed ATP-dependent ligase activity, but also used (dT)(16)/poly(dA) and (dT)(16)/poly(rA) as substrates, and had double-strand ligation activity, like human DNA ligase IV. Northern hybridization analysis and in situ hybridization indicated that CcLIG4 was expressed not only at the pre-meiotic S phase but also at meiotic prophase I. Intense signals were observed in leptotene and zygotene. Based on these observations, the possible role(s) of C. cinereus DNA ligase IV during meiosis are discussed.

The Coprinus cinereus adherin Rad9 functions in Mre11-dependent DNA repair, meiotic sister-chromatid cohesion, and meiotic homolog pairing.

Mitotic sister-chromatid cohesion (SCC) is known to depend in part on conserved proteins called adherins, which although necessary for SCC are not themselves localized between sister chromatids. We have examined mitotic DNA-repair and meiotic chromosome behavior in the Coprinus cinereus adherin mutant rad9-1. Genetic pathway analysis established that Rad9 functions in an Mre11-dependent pathway of DNA repair. Using fluorescence in situ hybridization, we found that the rad9-1 mutant is defective in the establishment of meiotic homolog pairing at both interstitial and subtelomeric sites but in the maintenance of pairing at only interstitial loci. To determine the role of Rad9 in meiotic SCC, we hybridized nuclear spreads simultaneously with a homolog-specific probe and a probe that recognizes both members of a homologous pair. We found that Rad9 is required for wild-type levels of meiotic SCC, and that nuclei showing loss of cohesion were twice as likely also to fail at homolog pairing. To ask whether the contribution of Rad9 to homolog pairing is solely in the establishment of SCC, we examined a rad9-1;msh5-22 double mutant, in which premeiotic DNA replication is inhibited. The msh5-22 mutation partially suppressed the deleterious effects of the rad9-1 mutation on homolog pairing; however, pairing in the double mutant still was significantly lower than in the msh5-22 single mutant control. Because the role of Rad9 in homolog pairing is not obviated by the absence of a sister chromatid, we conclude that adherins have one or more early meiotic functions distinct from the establishment of cohesion.

Replication-dependent early meiotic requirement for Spo11 and Rad50.

Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S. cerevisiae and the basidiomycete Coprinus cinereus, spo11 and rad50 mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in C. cinereus spo11 and rad50 mutants if premeiotic DNA replication is prevented. Double mutants were constructed with spo11-1 or rad50-4 and another mutant, spo22-1, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.

Multiple roles of Spo11 in meiotic chromosome behavior.

Spo11, a type II topoisomerase, is likely to be required universally for initiation of meiotic recombination. However, a dichotomy exists between budding yeast and the animals Caenorhabditis elegans and Drosophila melanogaster with respect to additional roles of Spo11 in meiosis. In Saccharomyces cerevisiae, Spo11 is required for homolog pairing, as well as axial element (AE) and synaptonemal complex (SC) formation. All of these functions are Spo11 independent in C.elegans and D.melanogaster. We examined Spo11 function in a multicellular fungus, Coprinus cinereus. The C.cinereus spo11-1 mutant shows high levels of homolog pairing and occasionally forms full-length AEs, but no SC. In C.cinereus, Spo11 is also required for maintenance of meiotic chromosome condensation and proper spindle formation. Meiotic progression in spo11-1 is aberrant; late in meiosis basidia undergo programmed cell death (PCD). To our knowledge, this is the first example of meiotic PCD outside the animal kingdom. Ionizing radiation can partially rescue spo11-1 for both AE and SC formation and viable spore production, suggesting that the double-strand break function of Spo11 is conserved and is required for these functions.

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus.

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12 h light/12 h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.

A new meiotic endonuclease from Coprinus meiocytes.

Two different types of Coprinus meiotic nuclease have been previously reported by the authors which are believed to be involved in meiotic chromosome recombination [1,2]. A third meiotic endonuclease was purified from the cap tissues of the basidiocarp of Coprinus cinereus. The enzyme is a 60 kDa molecule composed of a monopolypeptide as revealed by SDS-PAGE and FPLC-Sephacryl S-300 gel filtration. The enzyme belongs to a type of endonuclease which can preferentially digest single-stranded DNA and requires divalent cations as a co-factor, most commonly Mg2+ ions. In the presence of this co-factor, the enzyme converts the supercoiled plasmid DNA (form I) to both the relaxed form (form II) and the linear form (form III). Ca2+ ions can also function as a co-factor, though, in this case, not only is form I plasmid converted to form II, but a few ladder bands between form I and form II are also produced. The Ca2+ ion effect as a cofactor can be prevented with ATP. Immunohistochemical observation shows that the enzyme is distributed in the surface of the gills, which contain the meiotic tissues. These characteristics clearly differ from those of the meiotic nucleases reported previously.

Coordinated cell elongation alone drives tropic bending in stems of the mushroom fruit body of Coprinus cinereus.

During tropic bending in the stem of the mushroom fruit body of Coprinus cinereus the majority of extension occurred in the upper 20-30% of the stem. By attaching inert markers to the stem, it was shown that the outer flank of the bend initially has a faster rate of extension, although the inner flank matches this growth rate later in the response. Thus bending results from differential enhancement of growth rate rather than sustained differences. Large voids, up to 85 micrometers in diameter, observed in tropically bent stems showed no significant difference in number between inner and outer flanks but are implicated in bending because of their absence from unbent stems. Such voids may prevent the propagation of cracks through the stem tissue during bending. Creases at the external and lumen surfaces were also peculiar to bent stems and could represent constrictions caused by localized accumulation of stresses. Cell morphometric analysis of transverse sections of both flanks of the bend revealed no significant differences in hyphal diameter, distribution, or populations of cell types, but cells of the outer flank were four to five times longer than those of the inner. Thus, tropic bending requires only an increase in length of pre-existing inflated hyphae in the outer flank tissue.

A DNA polymerase alpha catalytic subunit is purified independently from the tissues at meiotic prometaphase I of a basidiomycete, Coprinus cinereus.

A 135kDa DNA polymerase alpha lacking primase activity has been purified to near homogeneity from Coprinus meiotic tissues. The activity of the DNA polymerase was sensitive to aphidicolin and N-ethylmaleimide, but was insensitive to dideoxythymidine triphosphate. DNA synthesis was proceeded with a low processivity. Neither activity nor processivity were affected by PCNA in the presence or absence of KCI. Monovalent cation inhibited its activity. These biochemical properties are almost identical to those of Coprinus DNA polymerase alpha -primase complex. However, the 135kDa DNA polymerase did not use activated DNA as a template-primer, inconsistent with Coprinus DNA polymerase alpha-primase complex. The 135kDa DNA polymerase was purified from the tissues at meiotic pro-metaphase I, suggesting that the alpha- DNA polymerase-primase complex dissociates as the meiotic cell cycle progresses and only the catalytic subunit remains at this stage.

Gravimorphogenesis in agarics.

The shape changes which occur in agaric fruit bodies in response to change in the direction of gravity, usually referred to as gravitropism are morphogenetic changes. Our interest in what we prefer to call gravimorphogenesis is to use it to examine morphogenesis experimentally. We are examining two agarics, Coprinus cinereus and Flammulina velutipes, and applying the best available technologies, including video analysis, all forms of electron microscopy, computer-aided image analysis and experiments in orbit in Spacelab. Responses to gravity of the two organisms differ in ways which can be related to their ecological and structural adaptations. C. cinereus reacts extremely rapidly; its fruit body can regain the vertical within 3 h of being placed horizontal, whereas F. velutipes requires 12 h to bend through 90 degrees. The fungi also differ in the bulk of tissue involved in the response. In Coprinus, a zone extending several cm down from the apex is normally involved in bending. In Flammulina, gravisensing is limited to a region just a few mm immediately below the cap, although curvature is performed in a zone of up to 2 cm below. Flammulina cultures were flown on the Spacelab D-2 mission in 1993, and fruit body disorientation in orbit provides the first definitive proof that 'gravitropism' really is a response to the unidirectional gravity vector. Experiments with different clinostat rotation rates in Flammulina indicate that the perception threshold is about 10(-4) x g. Analysis of different times of exposure to an altered gravity vector prior to clinorotation in Coprinus reveals that the perception time is 7 minutes and that continued response requires continued exposure. Cell size determinations in Coprinus demonstrate that cells of the stem increase in length, not diameter, to produce the growth differential. In Flammulina a unique population of highly electron-transparent microvacuoles changes in distribution; decreasing in upper cells and increasing in the lower cells in a horizontal fruit body within a few minutes of disorientation. These are thought to contribute to vacuolar expansion which accompanies/drives cell elongation. Application of a variety of metabolic inhibitors indicates that the secondary messenger calcium is also involved in regulating the growth differentials of gravimorphogenesis but that gravity perception is unaffected by inhibitors of calcium signalling. In both Flammulina and Coprinus, gravity perception seems to be dependent on the actin cytoskeleton since cytochalasin treatment suppresses gravitropic curvature in Flammulina and, in Coprinus, significantly delays curvature without affecting stem extension. This, together with altered nuclear motility observed in living hyphae during reorientation suggests that gravity perception involves statoliths (possibly nuclei) acting on the actin cytoskeleton and triggering specific vesicle/microvacuole release from the endomembrane system.