Resumo A resistência à degradação de chapas de madeira-plástico aos fungos de podridão-branca, Trametes versicolor (Linnaeus ex Fries) Pilát, e de podridão-parda, Gloeophyllum trabeum (Persoon ex ...Fries) Murrill, foi avaliada conforme a norma ASTM D 2017-81 (1994). As chapas foram confeccionadas com cavacos de Eucalyptus grandis e pellets de polietileno de baixa densidade (PEBD) pós-consumo nas proporções de plástico/madeira de 40/60, 50/50, 60/40 e 100/0. Utilizou-se como testemunha a madeira de E. grandis. Houve maior ataque do fungo de podridão-parda em relação ao fungo de podridão-branca. Foi observada correlação negativa entre o teor de plástico e o grau de ataque de ambos os fungos. De modo geral, as chapas de madeira-plástico foram classificadas como “altamente resistente” com exceção da proporção com 40% de plástico, que foi classificada como “resistente” ao ataque do fungo de podridão-parda. O índice de susceptibilidade de degradação (DSI) calculado, que leva em conta a densidade do material e a perda de massa real, mostrou que os compósitos foram mais resistentes que a madeira utilizada como referência.
Genes in prokaryotic genomes are often arranged into clusters and co-transcribed into polycistronic RNAs. Isolated examples of polycistronic RNAs were also reported in some higher eukaryotes but ...their presence was generally considered rare. Here we developed a long-read sequencing strategy to identify polycistronic transcripts in several mushroom forming fungal species including Plicaturopsis crispa, Phanerochaete chrysosporium, Trametes versicolor, and Gloeophyllum trabeum. We found genome-wide prevalence of polycistronic transcription in these Agaricomycetes, involving up to 8% of the transcribed genes. Unlike polycistronic mRNAs in prokaryotes, these co-transcribed genes are also independently transcribed. We show that polycistronic transcription may interfere with expression of the downstream tandem gene. Further comparative genomic analysis indicates that polycistronic transcription is conserved among a wide range of mushroom forming fungi. In summary, our study revealed, for the first time, the genome prevalence of polycistronic transcription in a phylogenetic range of higher fungi. Furthermore, we systematically show that our long-read sequencing approach and combined bioinformatics pipeline is a generic powerful tool for precise characterization of complex transcriptomes that enables identification of mRNA isoforms not recovered via short-read assembly.
•MB degradation by G. trabeum was enhanced with the addition of Ralstonia pickettii.•Concentrations of R. pickettii added: 2, 4, 6, 8, and 10 mL (1 mL ≈ 1.39 × 108 CFU).•The highest decolorization ...(85%) was obtained with the addition of 10 mL R. pickettii.•Metabolite products were C12H13N3O6, C14H14N3S, C12H11N3SO6, C12H11N3SO7, and C22H15N3SO5.
Methylene Blue (MB) is a thiazine group dye frequently used in the textile industry but the difficulty in degrading its molecule poses a significant risk of toxicity to humans. Gloeophyllum trabeum, a brown-rot fungus, has been previously shown to degrade MB. However, the decolorization capacity achieved was relatively poor due to the extended incubation time. This study aimed to improve the MB degradation process by G. trabeum with the addition of Ralstonia pickettii bacteria. The concentrations of R. pickettii added included 2, 4, 6, 8, and 10 mL (1 mL ≈ 1.39 × 108 CFU), while the degradation process was conducted at 30 °C within a 7-day incubation period. The results showed that the highest decolorization percentage was obtained with the addition of 10 mL R. pickettii. The mixed cultures decolorized MB by approximately 85%, while G. trabeum achieved 11% decolorization. The metabolite product produced from the process included C12H13N3O6, C14H14N3S, C12H11N3SO6, C12H11N3SO7, and C22H15N3SO5. Therefore, it was concluded that R. pickettii could enhance the capability of G. trabeum to decolorize MB.
•Konjac flying powder extract and its active compounds affect biochemistry of wood decay fungi.•Extract inhibit enzyme activity, respiratory and energy metabolism of wood decay fungi.•Salicylic acid, ...vanillin or cinnamaldehyde inhibits enzyme activity and damage cell membrane of wood decay fungi.•2,4,6-trichlorophenol inhibits enzyme activity and respiratory metabolism of wood decay fungi.
Wood products are vulnerable to fungal degradation that reduces service life. Traditional wood preservatives can provide excellent service life but efforts are underway to develop more natural methods for wood protection. Konjac flying powder is a residual waste produced during the processing of corms of Konjac (Amorphophallus konjac K. Koch) to produce Konjac flour. This waste material contains a number of compounds including salicylic acid (SA), 2,4,6-trichlorophenol (TCP), vanillin (VL), and cinnamaldehyde (CMA) has been found to be active against decay fungi; however, the antifungal mode of action is uncertain. The effects of konjac flying powder extract (KFPE) and its active compounds on cellulase, hemicellulose and ligninase activity, respiratory metabolism, cell membrane permeability, protein, and energy metabolism was studied on the white-rot fungus, Trametes versicolor (L. ex Fr.) Quél. (T. versicolor) and brown-rot fungus, Gloeophyllum trabeum (Pers.: Fr.) Murr. (G. trabeum). Konjac flying powder inhibited cellulase and hemicellulase activity of both fungi and ligninase activity of T. versicolor. Respiratory metabolism and energy metabolism were also inhibited. The four active compounds had different effects on activities suggesting that konjac flying powder functioned against multiple metabolic activities of the test fungi, potentially increasing its ability to provide broad spectrum wood protection.
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that perform oxidative cleavage of recalcitrant polysaccharides. We have purified and characterized a recombinant family AA9 ...LPMO, LPMO9B, from
(
LPMO9B) which is active on both cellulose and xyloglucan. Activity of the enzyme was tested in the presence of three different reductants: ascorbic acid, gallic acid, and 2,3-dihydroxybenzoic acid (2,3-DHBA). Under standard aerobic conditions typically used in LPMO experiments, the first two reductants could drive LPMO catalysis whereas 2,3-DHBA could not. In agreement with the recent discovery that H
O
can drive LPMO catalysis, we show that gradual addition of H
O
allowed LPMO activity at very low, substoichiometric (relative to products formed) reductant concentrations. Most importantly, we found that while 2,3-DHBA is not capable of driving the LPMO reaction under standard aerobic conditions, it can do so in the presence of externally added H
O
At alkaline pH, 2,3-DHBA is able to drive the LPMO reaction without externally added H
O
, and this ability overlaps entirely the endogenous generation of H
O
by
LPMO9B-catalyzed oxidation of 2,3-DHBA. These findings support the notion that H
O
is a cosubstrate of LPMOs and provide insight into how LPMO reactions depend on, and may be controlled by, the choice of pH and reductant.
Lytic polysaccharide monooxygenases promote enzymatic depolymerization of lignocellulosic materials by microorganisms due to their ability to oxidatively cleave recalcitrant polysaccharides. The properties of these copper-dependent enzymes are currently of high scientific and industrial interest. We describe a previously uncharacterized fungal LPMO and show how reductants, which are needed to prime the LPMO by reducing Cu(II) to Cu(I) and to supply electrons during catalysis, affect enzyme efficiency and stability. The results support claims that H
O
is a natural cosubstrate for LPMOs by demonstrating that when certain reductants are used, catalysis can be driven only by H
O
and not by O
Furthermore, we show how auto-inactivation resulting from endogenous generation of H
O
in the LPMO-reductant system may be prevented. Finally, we identified a reductant that leads to enzyme activation without any endogenous H
O
generation, allowing for improved control of LPMO reactivity and providing a valuable tool for future LPMO research.
Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, ...and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.
Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.
Many various industries use synthetic dyes as their raw materials. These dyes have triggered environmental problems because of the occurring effluents, and one of the environmentally safe solutions ...for this problem is biodegradation through microorganisms. Reactive Black 5 (RB5) dye degradation was performed by utilizing a metal-organic framework Universitetet i Oslo-66 (UiO-66) and Gloeophyllum trabeum (GT) fungus biocomposite. The UiO-66@GT composite was fabricated by inoculating the fungal culture in flasks with the PDB medium that contained UiO-66. This biocomposite was applied to decolorize and degrade RB5 dye, while pure GT culture can decolorize about 36.47% in five days. The percentage of RB5 decolorization was shown to be increased with the addition of UiO-66; the composite could decolorize RB5 up to 72.55% after five days incubation period. Moreover, the optimum conditions for the 100% targeted rate of RB5 decolorization found by the Response Surface Methodology (RSM) are: initial RB5 concentration (72.54 mg L-1), pH (6.53), and temperature (38.06 °C). Two novel metabolites from RB5 decolorization by the composite were detected based on LCMS-QTOF analysis and were used to propose a degradation pathway: 6-((1-amino-7,8-dihydroxy-6-sulfonaphthalen-2-yl) diazinyl) cyclohexa-2,4-dien-1-ide (m/z = 360) and 3,4-diamino-5,6-dihydroxy-1,2,7,8-tetrahydronaphthalene-2,7-disulfonic acid (m/z = 354).
Brown rot fungi are wood-degrading fungi that employ both oxidative and hydrolytic mechanisms to degrade wood. Hydroxyl radicals that facilitate the oxidative component are powerful nonselective ...oxidants and are incompatible with hydrolytic enzymes unless they are spatially segregated in wood. Differential gene expression has been implicated in the segregation of these reactions in
, but it is unclear if this two-step mechanism varies in other brown rot fungi with different traits and life history strategies that occupy different niches in nature. We employed proteomics to analyze a progression of wood decay on thin wafers, using brown rot fungi with significant taxonomic and niche distances:
(Boletales; "dry rot" lumber decay) and
(order Gloeophyllales; slash, downed wood). Both fungi produced greater oxidoreductase diversity upon wood colonization and greater glycoside hydrolase activity later, consistent with a two-step mechanism. The two fungi invested very differently, however, in terms of growth (infrastructure) versus protein secretion (resource capture), with the ergosterol/extracted protein ratio being 7-fold higher with
than with
In line with the native substrate associations of these fungi, hemicellulase-specific activities were dominated by mannanase in
and by xylanase in
Consistent with previous observations,
did not produce glycoside hydrolase 6 (GH6) cellobiohydrolases (CBHs) in this study, despite taxonomically belonging to the order Boletales, which is distinguished among brown rot fungi by having CBH genes. This work suggests that distantly related brown rot fungi employ staggered mechanisms to degrade wood, but the underlying strategies vary among taxa.
Wood-degrading fungi are important in forest nutrient cycling and offer promise in biotechnological applications. Brown rot fungi are unique among these fungi in that they use a nonenzymatic oxidative pretreatment before enzymatic carbohydrate hydrolysis, enabling selective removal of carbohydrates from lignin. This capacity has independently evolved multiple times, but it is unclear if different mechanisms underpin similar outcomes. Here, we grew fungi directionally on wood wafers and we found similar two-step mechanisms in taxonomically divergent brown rot fungi. The results, however, revealed strikingly different growth strategies, with
investing more in biomass production than secretion of proteins and
showing the opposite pattern, with a high diversity of uncharacterized proteins. The "simplified"
secretomic system could help narrow gene targets central to oxidative brown rot pretreatments, and a comparison of its distinctions with
and other brown rot fungi (e.g.,
) might offer similar traction in noncatabolic genes.
Woody biomass is anticipated to be a resource for a decarbonized society, but the difficulty of isolating woody components is a significant challenge. Brown-rot fungi, a type of wood rotting fungi, ...decompose hemicellulose particularly efficiently. However, there are few reports on the hemicellulases from brown-rot fungi. An α-L-arabinofuranosidase belonging to glycoside hydrolase family 51 (GH51) from the brown-rot fungus Gloeophyllum trabeum (GtAbf51A) was cloned and characterized in the present study. Analyses of the phylogeny of GH51 enzymes in wood rotting fungi revealed the existence of two groups, intercellular and extracellular enzymes. After deglycosylation, the recombinant GtAbf51A produced by Pichia pastoris appeared on SDS-PAGE as approximately 71,777 daltons, which is the expected molecular weight based on the amino acid sequence of GtAbf51A. Maximum enzyme activity occurred between pH 2.2 and 4.0 and at 50°C, while it was stable between pH 2.2 and 10.0 and up to 40°C. Due to the presence of a signal peptide, GtAbf51A was thought to hydrolyze polysaccharide containing arabinose. However, the hydrolysis rate of arabinosyl linkages in polysaccharides was only 3%–4% for arabinoxylan and 17% for arabinan. GtAbf51A, in contrast, efficiently hydrolyzed arabinoxylooligosaccharides, particularly O-α-L-arabinofuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-β-Dxylopyranose, which is the principal product of GH10 β-xylanase. These data suggest that GtAbf51A cooperates with other xylan-degrading enzymes, such as β-xylanase, to degrade xylan in nature.
Brown rot fungi have great potential in biorefinery wood conversion systems because they are the primary wood decomposers in coniferous forests and have an efficient lignocellulose degrading system. ...Their initial wood degradation mechanism is thought to consist of an oxidative radical-based system that acts sequentially with an enzymatic saccharification system, but the complete molecular mechanism of this system has not yet been elucidated. Some studies have shown that wood degradation mechanisms of brown rot fungi have diversity in their substrate selectivity. Gloeophyllum trabeum, one of the most studied brown rot species, has broad substrate selectivity and even can degrade some grasses. However, the basis for this broad substrate specificity is poorly understood. In this study, we performed RNA-seq analyses on G. trabeum grown on media containing glucose, cellulose, or Japanese cedar (Cryptomeria japonica) as the sole carbon source. Comparison to the gene expression on glucose, 1,129 genes were upregulated on cellulose and 1,516 genes were upregulated on cedar. Carbohydrate Active enZyme (CAZyme) genes upregulated on cellulose and cedar media by G. trabeum included glycoside hyrolase family 12 (GH12), GH131, carbohydrate esterase family 1 (CE1), auxiliary activities family 3 subfamily 1 (AA3_1), AA3_2, AA3_4 and AA9, which is a newly reported expression pattern for brown rot fungi. The upregulation of both terpene synthase and cytochrome P450 genes on cedar media suggests the potential importance of these gene products in the production of secondary metabolites associated with the chelator-mediated Fenton reaction. These results provide new insights into the inherent wood degradation mechanism of G. trabeum and the diversity of brown rot mechanisms.
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