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Woody biomass represents an important source of carbon on earth, and its global recycling is highly dependent on Agaricomycetes fungi. White-rot Basidiomycetes are a very important group in this regard, as they possess a large and diverse enzymatic repertoire for biomass decomposition. Among these enzymes, the recently discovered lytic polysaccharide monooxygenases (LPMOs) have revolutionized biomass processing with their novel oxidative mechanism of action. The strikingly high representation of LPMOs in fungal genomes raises the question of their functional versatility. In this work, we studied an AA9 LPMO from the white-rot basidiomycete Pycnoporus sanguineus, PsAA9A. Successfully produced as a recombinant secreted protein in Pichia pastoris, PsAA9A was found to be a C1-specific LPMO active on cellulosic substrates, generating native and oxidized cello-oligosaccharides in the presence of an external electron donor. PsAA9A boosted cellulolytic activity of glysoside hydrolases from families GH1, GH5, and GH6.This study serves as a starting point towards understanding the functional versatility and biotechnological potential of this enzymatic family, highly represented in wood decay fungi, in Pycnoporus genus. KEY POINTS: • PsAA9A is the first AA9 from P. sanguineus to be characterized. • PsAA9A has activity on cellulose, producing C1-oxidized cello-oligosaccharides. • Boosting activity with GH1, GH5, and GH6 was proven.

Some plant species colonize mining-contaminated soils. They are adapted to harsh growth conditions and show high native phytoremediation abilities. For environmental cleanup, these species can be improved by genetic manipulation. Laccases are ligninolytic enzymes that oxidise a broad range of substrates by a radical-catalysed reaction mechanism using oxygen as the electron acceptor. Fungi of the genus Pycnoporus produce laccases with important biotechnological, industrial and environmental applications. Here we describe a successful attempt of Agrobacterium rhizogenes mediated stable transformation of Nicotiana glauca, a species naturally resistant to metal-contaminated soils. The coding region of a Pycnoporus sanguineus laccase gene was fused with the Nicotiana plumbaginifolia calreticulin apoplast targeting signal and driven by the constitutive cauliflower mosaic virus (CaMV) 35S promoter (2X35S). The obtained transgenic N. glauca callus cells secreted a recombinant fungal laccase into the growth medium and were capable to degrade an anthraquinone dye, Remazol Brilliant Blue R. Under phenol and cadmium stresses, the transgenic calli not only maintained their growth capacity but their relative growth rate was greater compared to callus transformed with empty vector. This work shows that stable expression of a fungal laccase in a plant species resistant to heavy metals presents a successful strategy that potentially can be used to combat combined organic and inorganic contamination. A fungal laccase was heterologously expressed in a heavy metal-tolerant plant species. The transgenic callus was capable of growth in medium supplemented with organic and inorganic contaminants.

Biorecovery is emerging as a promising approach to retrieve gold from various sources, while its efficiency is usually restricted by the limited functional groups on natural microbial biomass surface. This study aims to intensify Pycnoporus sanguineus boosted sorption-reduction coupled gold biorecovery process via microbial surface modification. Results showed that grafting polyallylamine hydrochloride onto P. sanguineus biomass surface increased amino group content on microbial biomass surface from 1.29 to 2.81 mmol/g. When applying modified biomass to gold biorecovery with initial gold concentrations of 1.0, 2.0 and 3.0 mM, biosorption equilibrium time shortened to the 12.5%, 37.5% and 41.7% of those obtained with pristine biomass, and sorption rate constants correspondingly increased to 11.2, 3.1 and 3.7 folds as well. Maximum sorption capacity increased 30% and the affinity between biomass and gold enhanced heavily after microbial surface modification. Meanwhile, microbial surface modification favored gold reduction and gold nanoparticles (AuNPs) formation. The change of microbial biomass morphology from smooth surface with some branched structure to layered stacking structure with many pores and the increase of amino group content on microbial biomass surface were the main impetus for the gold bioreocovery process intensification.

A new poroid wood-inhabiting fungal genus, Leifiporia, is proposed, based on morphological and molecular evidence, which is typified by L. rhizomorpha sp. nov. The genus is characterized by an annual growth habit, resupinate basidiocarps with white to cream pore surface, a dimitic hyphal system with generative hyphae bearing clamp connections and branching mostly at right angles, skeletal hyphae present in the subiculum only and distinctly thinner than generative hyphae, IKI–, CB–, and ellipsoid, hyaline, thin-walled, smooth, IKI–, CB– basidiospores. Sequences of ITS and LSU nrRNA gene regions of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and Bayesian inference methods. The phylogenetic analysis based on molecular data of ITS + nLSU sequences showed that Leifiporia belonged to the core polyporoid clade and was closely related to Diplomitoporus overholtsii and Lopharia cinerascens, and then grouped with Pycnoporus and Trametes. Further investigation was obtained for more representative taxa in the Polyporaceae based on nLSU sequences, in which the results demonstrated that the genus Leifiporia formed a monophyletic lineage with a strong support (100 % BS, 100 % BP, 1.00 BPP). Both morphological and molecular evidence confirmed the placement of the new genus in the core polyporoid clade. In addition, a new combination, Leifiporia eucalypti, is proposed based on examination of its type material and phylogeny.

The effects of Cu(2+) on the activity and expression of laccase were investigated in seven different strains of Pycnoporus coccineus collected from different regions in Korea. Cu(2+) was toxic to mycelial growth at concentrations greater than 0.5 mM CuSO4 and showed complete growth inhibition at 1 mM in the liquid culture. However, Cu(2+) significantly upregulated the extracellular laccase activity at 0.2 mM in five strains of P. coccineus, IUM4209, IUM0032, IUM0450, IUM0470, and IUM4093, whereas two strains, IUM0253 and IUM0049, did not respond to Cu(2+), despite being closely related to the other five strains. Subsequent RT-PCR analysis also showed that the laccase mRNA was highly expressed only in the former five strains in the presence of Cu(2+). Taken together, these results indicate that Cu(2+) regulates expression of the laccase gene in a strain-dependent manner. The five strains commonly produced a single predominant laccase protein with a molecular weight of 68 kDa. Peptide sequencing revealed that the laccase was a homolog of Lcc1 of P. coccineus, which was isolated in China. The Cu(2+)-induced culture supernatants exhibited high degradation of polycyclic aromatic hydrocarbons, indicating that the 68-kDa laccase is the primary extracellular degradative enzyme in P. coccineus.

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L(-1 )days(-1), or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L(-1).

We conducted experiments to culture Pycnoporus sp. SYBC-L3 in a medium comprising an industrial waste (dye-containing textile effluent) and a lignocellulosic waste (Phragmites australis) that achieved laccase production while having the color removed from the wastewater. Our experimental results showed that the fungus grew well in liquid submerged cultivation with the diluted textile effluent as the sole culture medium, but relatively low extracellular laccase activity (1.8 U/mL) was produced. Addition of the lignocellulosic biomass enhanced laccase production and color removal. The highest laccase activity was found to be 6.5 U/mL in the presence of Phragmites australis stem. Under this condition, 70 % color removal occurred in the culture medium. This study provided an alternative novel scheme to remove color in textile wastewater while having an economic value added by producing laccase.

This study presents the biodecolorization potential of basidiomycete fungi Trametes hirsuta, Pycnoporus sp., and Irpex sp. for different reactive dyes viz. Reactive Red 120, Remazol Brilliant Blue R (RBBR), Reactive Orange G, and Reactive Orange 16 under static and shaking conditions. The screening trials revealed that T. hirsuta exhibited maximum potential (83.75 %) for biodecolorization of RBBR dye under static conditions after the fifth day of incubation. However, the rate of biodecolorization of RBBR dye by Pycnoporus sp. was much slow and reached maximum (81.25 %) after 15 days of incubation under shaking conditions. By process optimization, enhanced decolorization (91.2 %) of RBBR by T. hirsuta was achieved at pH 5.5 within 24 h using a defined salt medium amended with p-coumaric acid under static conditions. pH was found to be an important parameter for the enzymatic system involved in RBBR dye decolorization by T. hirsuta and Pycnoporus sp. Biodecolorization of RBBR dye was determined by a reduction in optical density at the wavelength of maximum absorbance (λ, 578 nm) by UV-vis spectrophotometer. The shift in maximum wavelength toward shorter/longer wavelength in UV-vis scanning spectrum revealed the degradation of RBBR dye into different transformation products.

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

The biological delignification of lignocellulosic feedstocks, Prosopis juliflora and Lantana camara was carried out with Pycnoporus cinnabarinus, a white rot fungus, at different scales under solid-state fermentation (SSF) and the fungal treated substrates were evaluated for their acid and enzymatic saccharification. The fungal fermentation at 10.0 g substrate level optimally delignified the P. juliflora by 11.89% and L. camara by 8.36%, and enriched their holocellulose content by 3.32 and 4.87%, respectively, after 15 days. The fungal delignification when scaled up from 10.0 g to 75.0, 200.0 and 500.0 g substrate level, the fungus degraded about 7.69-10.08% lignin in P. juliflora and 6.89-7.31% in L. camara, and eventually enhanced the holocellulose content by 2.90-3.97 and 4.25-4.61%, respectively. Furthermore, when the fungal fermented L. camara and P. juliflora was hydrolysed with dilute sulphuric acid, the sugar release was increased by 21.4-42.4% and the phenolics content in hydrolysate was decreased by 18.46 and 19.88%, as compared to the unfermented substrate acid hydrolysis, respectively. The reduction of phenolics in acid hydrolysates of fungal treated substrates decreased the amount of detoxifying material (activated charcoal) by 25.0-33.0% as compared to the amount required to reduce almost the same level of phenolics from unfermented substrate hydrolysates. Moreover, an increment of 21.1-25.1% sugar release was obtained when fungal treated substrates were enzymatically hydrolysed as compared to the hydrolysis of unfermented substrates. This study clearly shows that fungal delignification holds potential in utilizing plant residues for the production of sugars and biofuels.

A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml(-1), which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0 degrees C but also a high thermostable enzyme. The half-lives at 60, 70 and 80 degrees C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.

Unprotected L-phenylalanine was derivatized by an innovative enzymatic method by means of laccases from Pycnoporus cinnabarinus and Myceliophthora thermophila. During the incubation of L-phenylalanine with para-hydroquinones using laccase as biocatalyst, one or two main products were formed. Dependent on the substitution grade of the hydroquinones mono- and diaminated products were detected. Differences of the used laccases are discussed. The described reactions are of interest for the derivatization of amino acids and a synthesis of pharmacological-active amino acid structures in the field of white biotechnology.

Brazilian tropical fungi were screened for lignin degradation, and the ability to increase or decrease thein vitro digestibility and pH of wheat straw used as a substrate (at 25 and 30 °C after 30 and 60 d of incubation). Out of 72 species and strains ofAgrocybe, Antrodiella, Auricularia, Coriolopsis, Cymatoderma, Fomitopsis, Ganoderma, Gerronema, Gloeophyllum, Gymnopilus, Irpex, Lentinus, Melanoporia, Oligoporus, Oudemansiella, Panaeolus, Peniophora, Phellinus, Pherotus, Psathyrella, Psilocybe, Pycnoporus, Rigidoporus, Schizophyllum, Trametes, Trichaptum andTyromyces, 22 decomposed more than 50% of lignin and 10 increased thein vitro substrate digestibility by more than 30 U. The highest degradation of lignin was observed withLentinus crinitus (80 %, 60 d) and the highest increase inin vitro substrate digestibility was caused byPeniophora utriculosa (36 U, 30 d).

Laccase, a ligninolytic enzyme, was secreted by each ofthe white rot fungi Trametes versicolor and Pycnoporus cinnabarinusduring growth in a nitrogen-rich medium under agitated conditions. Afteraddition of 2-hydroxydibenzofuran to cell-freesupernatants of the cultures, yellow precipitates wereformed. These precipitates were poorly soluble in waterand therefore readily separated from the supernatant. Theproducts formed were more hydrophobic than thesubstrate, as indicated by their longer retention times on areverse phase high-performance liquid chromatographycolumn. Mass spectrometric analysis of the purifiedproducts indicated the formation of oligomers. Analysis ofthe mixture of products by gas chromatography and massspectrometry after derivatization with diazomethanesuggested the formation of at least three dimeric and ninetrimeric products. Carbon-carbon and carbon-oxygenbonds were identified in the dimers and trimers,respectively. The nuclear magnetic resonance spectrum ofthe main dimer suggested coupling of the two monomersat the carbon one position.

Distinctive basidiomes of Pycnoporus sp. were observed on 5–6-year-old living Paulownia trees at two commercial plantations in Western Australia. The basidiomes were erupting through the bark, which readily peeled away to expose the rotting wood beneath. Based on internal transcribed spacer-polymerase chain reaction, the fungus was identified as Pycnoporus cinnabarinus. Under-bark inoculation of 3-year-old Paulownia fortunei trees in the glasshouse, with mycelium isolated from basidiomes on trees at the commercial plantations, resulted in the formation of dark lesions and the pathogen was re-isolated ahead of the lesion front. Benomyl and cyprodinil were the most effective fungicides at inhibiting the growth of mycelium in vitro. This is the first report of Pyc. cinnabarinus acting as a pathogen on Paulownia trees. It is likely that the pathogen enters living trees through pruning wounds or damage from sun scald. Further work is required to confirm methods of control in the field.