Expression and characterization of a family 45 glycosyl hydrolase from Fomitopsis pinicola and comparison to Phanerochaete chrysosporium Cel45A.

To efficiently decompose biomass, fungi have developed various enzymatic and non-enzymatic strategies and are a source of versatile biocatalysts. The endoglucanases in glycosyl hydrolase CAZy family 45 (GH45) are known for their small size, a high thermostability and a broad substrate specificity that has been employed in textile and detergent industries. Here we report the heterologous expression and characterisation of an GH45 endoglucanase from the brown rot Fomitopsis pinicola and its direct comparison to an already characterised GH45 from the white rot Phanerochaete chrysosporium. Both enzymes were recombinantly expressed in Pichia pastoris and purified by two chromatographic steps. The biochemical characterisation highlighted the acidophilic character, with an optimal pH of 4, and a preference for amorphous substrates as carboxymethyl cellulose (CMC) and substrates containing ß-1,4-glucans rather than the previously reported ß-1,3/1,4-glucans lichenan and ß-glucan. The dominating products from ß-1,4-glucans were C3-C6 oligosaccharides, whereas from ß-1,3/1,4-glucans glucose was the main reaction product. From the characterisation no differences between the brown rot and the white rot GH45 was evident.

Functional expression and characterization of two laccases from the brown rot Fomitopsis pinicola.

Laccase is predominantly found in lignin degrading filamentous white rot fungi, where it is involved in the oxidative degradation of this recalcitrant heteropolymer. In brown rot fungi it is much less prevalent: laccases from only a few brown rots have been detected and only two have been characterized. This study tries to understand the role of this ligninolytic enzyme in brown rots by investigating the catalytic properties of laccases secreted by Fomitopsis pinicola FP58527 SS1. When grown on either poplar or spruce wood blocks, several laccases were detected in the secretome. Two of them (FpLcc1 and FpLcc2) were heterologously produced using Trichoderma reesei QM9414 Δxyr1 as expression host and purified to homogeneity by consecutive steps of hydrophobic interaction, anion exchange and size exclusion chromatography. With the substrates 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol both laccases showed similar, low pH-optima below 3 for ABTS and 2,6-DMP and at pH 3.5 for guaiacol which is at the acidic end of laccases isolated from white rot fungi. The determined KM values were low while kcat values measured at acidic conditions were comparable to those reported for other laccases from white rot fungi. While both enzymes showed a moderate decrease in activity in the presence of oxalic and citric acid FpLcc2 was activated by acetic acid up to 3.7 times. This activation effect is much more pronounced at pH 5.0 compared to pH 3.0 and could already be observed at a concentration of 1 mM acetic acid.

Fungal mycelial mats used as textile by indigenous people of North America.

The indigenous people of the United States and Canada long have used forest fungi for food, tinder, medicine, paint, and many other cultural uses. New information about historical uses of fungi continues to be discovered from museums as accessions of fungi and objects made from fungi collected over the last 150+ years are examined and identified. Two textiles thought to be made from fungal mats are located in the Hood Museum of Art, Dartmouth College, and the Oakland Museum of California. Scanning electron microscopy and DNA sequencing were used to attempt to identify the fungus that produced the mats. Although DNA sequencing failed to yield a taxonomic identification, microscopy and characteristics of the mycelial mats suggest that the mats were produced by Laricifomes officinalis. This first report of fungal mats used for textile by indigenous people of North America will help to alert museum curators and conservators as well as mycological researchers to their existence and hopefully lead to more items being discovered that have been made from fungal fabric.

Decolorization and biodegradation of melanoidin contained in beet molasses by an anamorphic strain of Bjerkandera adusta CCBAS930 and its mutants.

We used a ligninolytic strain of the white-rot fungus B. adusta CCBAS 930 and its mutants with modified ligninolytic activity to assess their potential to remove of molasses. The analyzed strains have been shown to be able to decolorize 1% or 2% molasses solutions containing brown-colored toxic melanoidins. It was found that the decolorization process was determined by the transition to the stage of production of sporulating aerial mycelium (liquid and agar cultures) coupled with an increase in peroxidase activity, which was accompanied by a decrease in the level of melanoidin, free radicals, and phenolic compounds. Four different peroxidase activities were detected in post-culture liquids, i.e. horseradish-like (HRP-like), manganese-dependent (MnP), lignin (LiP), and versatile (VP) peroxidase activities. The HRP-like peroxidase was characterized by the highest activity. The efficiency of removal of melanoidins from a 1% molasses solution by the parental strain and the mutants was dependent on the culture method. The highest efficiency was noted in immobilized cultures (threefold higher than in the mycelium-free cultures), which was accompanied by stimulation of HRP-like peroxidase activity. Mutant 930-5 was found to be the most effective in the decolorization and decomposition of melanoidin. The HRP-like activity in the immobilized cultures of B. adusta 930-5 was 640-fold higher than in the mycelium-free cultures of the fungus. Moreover, decolorization and biodegradation of melanoidin by B. adusta CCBAS 930 and 930-5 was coupled with detoxification.

Insights into the mechanism of cyanobacteria removal by the algicidal fungi Bjerkandera adusta and Trametes versicolor.

Fungal mycelia can eliminate almost all cocultured cyanobacterial cells within a short time. However, molecular mechanisms of algicidal fungi are poorly understood. In this study, a time-course transcriptomic analysis of algicidal fungus Bjerkandera adusta T1 was applied to investigate gene expression and regulation. A total of 132, 300, 422, and 823 differentially expressed genes (DEGs) were identified at 6, 12, 24, and 48 hr, respectively. Most DEGs exhibited high endopeptidase activity, cellulose catabolic process, and transmembrane transporter activity by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Many decomposition genes encoding endopeptidases were induced a little later in B. adusta T1 when compared with previously investigated algicidal fungus Trametes versicolor F21a. Besides, the accumulated expression of Polysaccharide lyases8 (PL8) gene with peptidoglycan and alginate decomposition abilities was greatly delayed in B. adusta T1 relative to T. versicolor F21a. It was implied that endopeptidases and enzymes of PL8 might be responsible for the strong algicidal ability of B. adusta T1 as well as T. versicolor F21a.

A Wild Fomes fomentarius for Biomediation of One Pot Synthesis of Titanium Oxide and Silver Nanoparticles for Antibacterial and Anticancer Application.

The present study offers an alternative method for green synthesis of the formation of two types of nanoparticles (NPs). These NPs, titanium oxide and silver NPs (TiO2 and Ag NPs, respectively), were obtained from the amalgamation of intracellular extract of a wild mushroom, Fomes fomentarius, with aqueous solutions of titanium isopropoxide and silver nitrate, respectively. F. fomentarius was identified phenotypically and by 18S ribosomal RNA gene sequencing (Gene accession no: MK635351). The biosynthesis of TiO2 and Ag NPs was studied and characterized by X-ray diffraction (XRD), diffuse reflectance UV-Visible spectroscopy (DR-UV), fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and transmission electron microscope (TEM). Success was achieved in obtaining NPs of differing sizes and shapes. The antibacterial and anticancer activity of the NPs was significant with morphological damage being caused by both, although Ag NPs (10-20 nm) were found to have profound effects on bacterial and cancer cells in comparison to TiO2 NPs (100-120 nm). These metal NPs, synthesized using wild mushrooms, hold a great potential in biomedicinedue to an effective enzyme combination, which permits them to modify different chemical compounds to less toxic forms, which is required for ecofriendly and safe biomaterials.

Starch-degrading enzymes from the brown-rot fungus Fomitopsis palustris.

Brown-rot fungi preferentially degrade softwood and cause severe breakdown of wooden structures. At the initial stage of the brown-rot decay, penetrating hyphae of the fungi are observed in ray parenchyma. Since starch grains are known to be present in the ray parenchyma of sapwood, investigation of the functions and roles of the starch-degrading enzymes is important to understand the initial stage of brown-rot decay. We purified and characterized two starch-degrading enzymes, an α-amylase (FpAmy13A) and a glucoamylase (FpGLA15A), from the brown-rot fungus, Fomitopsis palustris, and cloned the corresponding genes. The optimal temperature for both enzymes was 60 °C. FpAmy13A showed higher activity at a broad range of pH from 2.0 to 5.0, whereas FpGLA15A was most active at pH 5.0-6.0. Notable thermal stability was found for FpGLA15A. Approximately 25% of the activity remained even after treatment at 100 °C for 30 min in sodium phosphate buffer at pH 7.0. These different characteristics imply the different roles of these enzymes in the starch degradation of wood.

Fomitopsis mounceae and F. schrenkii-two new species from North America in the F. pinicola complex.

Two new species, Fomitopsis mounceae and F. schrenkii (Polyporales, Basidiomycota) in the F. pinicola species complex in North America, are described and illustrated. Previous molecular phylogenetic analyses identified three well-delimited lineages that represent F. mounceae and F. ochracea from Canada, the Appalachian Mountains, and the northern United States and F. schrenkii from western and southwestern regions of the United States. Fomitopsis pinicola sensu stricto is restricted to Eurasia and does not occur in North America. Morphological descriptions of basidiocarps and cultures for F. mounceae, F. schrenkii, and F. ochracea are presented. The three species are readily differentiated by nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) sequence, geographic distribution, and basidiospore size. Polyporus ponderosus H. Schrenk is an earlier illegitimate synonym of F. schrenkii. Both F. mounceae and F. schrenkii have a heterothallic multiallelic incompatibility system.

Real-Time PCR Detection of the Basidiomycetous Fungus Bjerkandera adusta: A Tool to Identify Itraconazole Responder Patients with Unexplained Chronic Cough.

BACKGROUND: Filamentous basidiomycetes (f-BMs) are involved in some unexplained chronic cough (UCC) cases that can be improved by the administration of antifungal agents. The disease concept was termed fungus-associated chronic cough (FACC). The current diagnostic criteria warrant environmental fungi isolation from respiratory specimens, which is hardly conceivable for such fungi. OBJECTIVES: This study aimed to detect the f-BMs Bjerkandera adusta, the most common pathogen in FACC, from respiratory specimens of patients with UCC using real-time polymerase chain reaction (PCR). It also evaluated the applicability of the PCR system to detect antifungal agent responders among patients with unexplained cough. METHODS: The PCR system specific to B. adusta was developed and its utility was evaluated using sputum samples from 23 patients with chronic cough. RESULTS: B. adusta was detected in 10 out of 14 patients with UCC (71.4%), in contrast to only 2 out of 9 patients with non-UCC (22.2%; p < 0.05 with the Fisher's exact test). The copy number of the samples correlated with the therapeutic impact score for cough symptoms following the oral administration of itraconazole. CONCLUSION: Development of the real-time PCR system enabled us to demonstrate that many patients with UCC might be influenced by B. adusta, a fact evidenced by the improvement of symptoms with itraconazole administration in most PCR-positive patients. This method would help in detecting itraconazole responders among patients with UCC when the isolation of f-BMs is not achievable.

Chemical composition and biological activity of extracts from fruiting bodies and mycelial cultures of Fomitopsis betulina.

Fomitopsis betulina (Bull.) B.K. Cui, M.L. Han & Y.C. Dai has been used for medicinal purposes for over 5000 years. Numerous studies have confirmed the biological activity of compounds found in this species. The purpose of this study was a comparative analysis of selected groups of metabolites in the extracts from fruiting bodies and mycelial cultures. Phenolic acids (syringic, gallic, p-hydroxybenzoic, 3,4-dihydrophenylacetic), indole compounds (L-tryptophan, 5-hydroxy-L-tryptophan, 5-methyltryptamine), sterols (ergosterol, ergosterol peroxide, hexestrol, cholecalciferol), and triterpenes (betulinic acid, betulin) were determined quantitatively by high performance liquid chromatography with UV-Vis/DAD detection, while fatty acids were assessed with the gas chromatography method. Cytotoxic activity against selected human cancer cell lines was determined using the lactate dehydrogenase test. Anti-inflammatory activity was evaluated on lipopolysaccharide activated A549 cells. Those extracts with anti-inflammatory activity were evaluated for their inhibition of pro-inflammatory enzymes. The mycelium extract exhibited significant cytotoxic activity against prostate cancer cells, while the fruiting body extract indicated a moderate effect on the viability of melanoma and prostate cancer. Incubation of lung epithelial cells with biomass extract significantly decreased cyclooxygenase-2 levels compared to LPS activated A549 cells. This paper is the first report of a comparative quantitative analysis of the metabolites in mycelial cultures and fruiting bodies. In addition, a novel element of this study is its comparison of the cytotoxic and anti-inflammatory activity of the obtained extracts. The results of comparing the composition and activity of mycelium and fruiting bodies shows that the cultures could be proposed as a potential biotechnological source for selected biologically active compounds.

Classification of fungal glucuronoyl esterases (FGEs) and characterization of two new FGEs from Ceriporiopsis subvermispora and Pleurotus eryngii.

Fungal glucuronoyl esterases (FGEs) catalyze cleavage of the ester bond connecting a lignin alcohol to the xylan-bound 4-O-methyl-D-glucuronic acid of glucuronoxylans. Thus, FGEs are capable of degrading lignin-carbohydrate complexes and have potential for biotechnological applications toward woody biomass utilization. Therefore, identification and characterization of new FGEs are of critical importance. Firstly, in this study, we built a phylogenetic tree from almost 400 putative FGEs obtained on BLAST analysis and defined six main clades. In the phylogenetic tree, all the putative FGEs of ascomycetes cluster in clades I to IV, and most of the putative FGEs of basidiomycetes (B-FGEs) cluster in clades V to VI. Interestingly, several B-FGEs were found to cluster in clade II; most FGEs of clade II were found to have higher theoretical isoelectric points than those in the other five clades. To gain an insight into the putative FGEs in the clades that have not been characterized yet, we chose the FGEs of Ceriporiopsis subvermispora (CsGE) and Pleurotus eryngii (PeGE), which belong to clades V and II, respectively. The catalytic domains of both CsGE and PeGE were successfully expressed using Pichia pastoris, and then purified. Benzyl glucuronic acid was used as a substrate to confirm the activities of the CsGE and PeGE, and the hydrolyzed product, glucuronic acid, was quantified spectrophotometrically. Both CsGE and PeGE clearly exhibited the esterase activity. Additionally, we demonstrated that PeGE exhibits high tolerance toward several denaturing agents, which may make it a potentially more applicable enzyme.

Genetic Variability of the Medicinal Tinder Bracket Polypore, Fomes fomentarius (Agaricomycetes), from the Asian Part of Russia.

We analyzed intraspecies genetic variability of the medicinal tinder bracket polypore, Fomes fomentarius, from the Asian part of Russia, including the Ural, Altai, Western Sayan, and Baikal regions. We used nuclear ribosomal DNA internal transcribed spacer (ITS) sequence data as a standard marker for fungal DNA barcoding. In the Asian part of Russia, lineage A occurs as sublineage A2, which differs from sublineage A1 by a single nucleotide insertion at ITS2.3. Sublineage A2 is distributed up to Lake Baikal in the Ural, Altai, and Western Sayan regions. It can be characterized as a Eurasian sublineage of F. fomentarius. Lineage B is also represented by 2 sublineages (B1 and B2), which differ from each other by nucleotide sequences at ITS2.1. Sublineage B1 is represented by a small group of isolates from Asia (Iran, China, Nepal, South Korea), whereas sublineage B2 mainly includes isolates from Europe (Great Britain, Italy, Latvia, Slovakia, Slovenia) and 2 separate samples from Asia (Iran, China); these locales compose the distribution area of F. fomentarius. In the Asian part of Russia, lineage B is represented by sublineage B2 found in the Southern Urals (at the border between Europe and Asia), which is the only area where sublineages A2 and B2 are present. These sublineages are characterized by different substrate spectra: sublineage A2 is predominantly associated with Betula spp. and rarely with Alnus and Larix trees, whereas sublineage B2 does not have a pronounced substrate preference and is found in basidiomes collected from Acer, Duschekia, Prunus, and Salix trees, but not Betula trees. In general, the spectrum of substrates for F. fomentarius lineages A and B in the Asian part of Russia corresponds to that in other parts of this polypore's distribution area. Data are needed on genetic intraspecies variability (polymorphism) in relation to pharmacological properties for further biotechnological cultivation and use of the medicinal fungus F. fomentarius.

Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola.

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed the gene expression levels and RNA editing profiles of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression and RNA editing were observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression and RNA editing encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. There was no overlap between differentially expressed and differentially edited genes, suggesting that these may provide F. pinicola with independent mechanisms for responding to different conditions. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. In contrast, the suites of genes subject to RNA editing were much less affected by culture conditions. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi.IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that enable fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species, aspen, pine, and spruce, under various culture conditions. We examined both gene expression (transcription levels) and RNA editing (posttranscriptional modification of RNA, which can potentially yield different proteins from the same gene). We found that F. pinicola is able to modify both gene expression and RNA editing profiles across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This work provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

BadGluc, a ß-glucosidase from Bjerkandera adusta with anthocyanase properties.

A glycosidase of the basidiomycete Bjerkandera adusta (BadGluc) was found in screenings to possess a strong decolorizing ability towards malvidin-3-galactoside, an anthocyanin abundant in various berry fruits. The BadGluc was purified from the culture supernatant via FPLC, and the corresponding gene was identified which showed low similarity to other characterized glucosidases. Scanning the primary sequence with PROSITE no active site motif was detected. Eventually, a specific 18 aa consensus pattern was identified manually. The active site motif possessed an undescribed sequence which was only found in a few hypothetical proteins. The corresponding gene was cloned and expressed in Pichia pastoris GS115 yielding activities up to 100 U/L using 4-nitrophenyl-ß-d-glucopyranoside (pNPG) as substrate. The enzyme possessed a good temperature (70% after 1 h at 50°C) and pH stability (70% between pH 2 and 7.5), and preferably catalysed the hydrolysis of delphinidin-3-glucoside and cyanidin-3-glucoside, regardless of the position of the terminal Hexa-His tag. This novel glucosidase worked in aqueous solution as well as on pre-stained fabrics making it the first known candidate anthocyanase for applications in the detergent and food industries.

Overview of the Biological Activities of a Methanol Extract from Wild Red Belt Conk, Fomitopsis pinicola (Agaricomycetes), Fruiting Bodies from Central Italy.

Fomitopsis pinicola (Sw.) P. Karst. (Fomitopsidaceae) is a medicinal mushroom with a variety of healthy properties. In this study we tested the radical scavenging activity and antimicrobial and anticancer potential of methanol extracts of F. pinicola from central Italy. Molecular identification confirmed that the samples were F. pinicola; a Basic Local Alignment Search Tool search showed a close match (99% sequence identity) with European isolates of this species. The free radical scavenging capacities, measured by DPPH assay, showed that the extract activity was 3.5% that of Trolox. The MTT test, evaluated after 72 hours of treatment with increasing doses of extract (5-500 µg · mL-1), considerably inhibited proliferation in a dose-dependent manner in 2 human tumor cell lines. This reduction was coupled with a relevant induction of apoptosis in the human leukemia THP-1 cell line after 24 hours of treatment, but a relevant toxic effect occurred in the human colon adenocarcinoma HT29 cell line. The genotoxic potential of the methanol extracts was studied by single-cell gel electrophoresis of normal human leukocytes exposed to 20 µg extract at 37°C for 30 minutes; no DNA damage was observed. The F. pinicola methanol extract was found to have varying degrees of antifungal effects against the pathogenic fungi tested (minimum inhibitory concentration from 23.63 to 66.81 µg · mL-1). The results show that the tested F. pinicola extract has strong antimicrobial and chemo-preventive activities, but is a poor antioxidant.

Purification and characterization of two novel peroxidases from the dye-decolorizing fungus Bjerkandera adusta strain CX-9.

Two extracellular peroxidases from Bjerkandera adusta strain CX-9, namely a lignin peroxidase (called LiP BA45) and manganese peroxidase (called MnP BA30), were purified simultaneously by applying successively, ammonium sulfate precipitation-dialysis, Mono-S Sepharose anion-exchange and Sephacryl S-200 gel filtration and biochemically characterized. The sequence of their NH2-terminal amino acid residues showed high homology with those of fungi peroxidases. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzymes MnP BA30 and LiP BA45 were a monomers with a molecular masses 30125.16 and 45221.10Da, respectively. While MnP BA30 was optimally active at pH 3 and 70°C, LiP BA45 showed optimum activity at pH 4 and 50°C. The two enzymes were inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in their tertiary structures. The Km and Vmax for LiP BA45 toward 2,4-Dichlorolphenol (2,4-DCP) were 0.099mM and 9.12U/mg, respectively and for MnP BA30 toward 2,6-Dimethylphenol (2,6-DMP), they were 0.151mM and 18.60U/mg, respectively. Interestingly, MnP BA30 and LiP BA45 demonstrated higher catalytic efficiency than that of other tested peroxidases (MnP, LiP, HaP4, and LiP-SN) and marked organic solvent-stability and dye-decolorization efficiency. Data suggest that these peroxidases may be considered as potential candidates for future applications in distaining synthetic-dyes.

Role of carbon source in the shift from oxidative to hydrolytic wood decomposition by Postia placenta.

Brown rot fungi initiate wood decay using oxidative pretreatments to improve access for cellulolytic enzymes. These pretreatments are incompatible with enzymes, and we recently showed that Postia placenta overcomes this issue by delaying glycoside hydrolase (GH) gene upregulation briefly (<48h) until expression of oxidoreductases (ORs) is repressed. This implies an inducible cellulase system rather than a constitutive system, as often reported, and it remains unclear what cues this transition. To address this, we grew P. placenta along wood wafers and spatially mapped expression (via quantitative PCR) of twelve ORs and GHs targeted using functional genomics analyses. By layering expression patterns over solubilized sugar data (via HPLC) from wood, we observed solubilization of wood glucose, cellobiose, mannose, and xylose coincident with the OR-GH transition. We then tested effects of these soluble sugars, plus polymeric carbon sources (spruce powder, cellulose), on P. placenta gene expression in liquid cultures. Expression of ORs was strictly (aox1, cro5) or progressively repressed over time (qrd1, lcc1) by all soluble sugars, including cellobiose, but not by polymeric sources. Simple sugars repressed hemicellulase gene expression over time, but these sugars did not repress cellulases. Cellulase genes were upregulated, however, along with hemicellulases in the presence of soluble cellobiose and in the presence of polymeric carbon sources, relative to starvation (carbon-free). This verifies an inducible cellulase system in P. placenta that lacks carbon catabolite repression (CCR), and it suggests that brown rot fungi use soluble sugars, particularly cellobiose, to cue a critical oxidative-hydrolytic transition.

Recombinant expression of a laccase from Coriolopsis gallica in Pichia pastoris using a modified α-factor preproleader.

In this work we communicate the heterologous expression of a laccase from Coriolopsis gallica in Pichia pastoris. This enzyme has been reported to efficiently degrade a variety of pollutants such as industrial dyes. The expression strategy included using a previously reported modified α-factor preproleader for enhanced secretion and pAOX1, a methanol-responsive promoter. Methanol concentration, copper salts concentration and temperature were varied in order to enhance laccase expression in this heterologous system. A volumetric activity of 250 U/L was achieved after 12-day culture in Fernbach flasks. The protein was recovered from the supernatant and purified, obtaining a preparation with 90% electrophoretic purity. The catalytic constants of the recombinant enzyme are almost identical to the fungal enzyme, thus rendering this system a useful tool for protein engineering of laccase from C. gallica.

Differences between two strains of Ceriporiopsis subvermispora on improving the nutritive value of wheat straw for ruminants.

AIM: This study evaluated differences between two strains of Ceriporiopsis subvermispora on improving the nutritive value and in vitro degradability of wheat straw. METHODS AND RESULTS: Wheat straw was treated with the fungi for 7 weeks. Weekly samples were analysed for ergosterol content, in vitro gas production (IVGP), chemical composition and lignin-degrading enzyme activity. Ergosterol data showed CS1 to have a faster initial growth than CS2 and reaching a stationary phase after 3 weeks. The IVGP of CS1-treated wheat straw exceeded the control earlier than CS2 (4 vs 5 weeks). CS1 showed a significantly higher (P < 0·001) selectivity in lignin degradation compared to CS2. Both strains showed peak activity of laccase and manganese peroxidase (MnP) at week 1. CS1 showed a significantly higher (P < 0·001) laccase activity, but lower (P = 0·008) MnP activity compared to CS2. CONCLUSION: Both CS strains improved the nutritive value of wheat straw. Variation between strains was clearly demonstrated by their growth pattern and enzyme activities. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences among the two strains provide an opportunity for future selection and breeding programs in improving the extent and selectivity of lignin degradation in agricultural biomass.

Whole-genome de novo sequencing of wood rot fungus Fomitopsis palustris (ATCC62978) with both a cellulolytic and ligninolytic enzyme system.

Fomitopsis palustris is a model brown rot fungus causing destructive wood decay based on the cellulase system. Endoglucanase secreted by F. palustris hydrolyzes cellulose in both the crystalline and amorphous form. In this study, whole-genome sequencing was conducted to identify genes related to F. palustris cellulose degradation and their functions. We determined the 43-Mb complete draft genome of F. palustris (ATCC 62978), comprising 14,592 predicted gene models. Gene annotation provided crucial information about the location and function of protein-encoding genes. Three types of endoglucanases were expressed: endo-1,3-beta-glucanase, endo-1,4-beta-d-glucanase, and endoglucanase. In addition, various ligninolytic enzymes such as laccase, aromatic compound dioxygenase, and aryl alcohol dehydrogenase were expressed in F. palustris (ATCC 62978). Colony polymerase chain reaction (PCR) indicated that the endo-1,4-beta-d-glucanase gene comprises 732bp. Optimization of the expression conditions of endoglucanase by real-time PCR revealed that endoglucanase was highly expressed after 7days in all conditions, which was secreted during the secondary metabolism. Studies for large-scale cellulase production from this fungus and investigation of its ligninolytic system will promote its extensive use in various applications. The genomic information determined herein provides a basis for molecular genetics studies to understand the genome functions of F. palustris (ATCC 62978).