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Brown rot (BR) decay mechanisms employ carbohydrate-active enzymes (CAZymes) as well as a unique non-enzymatic chelator-mediated Fenton (CMF) chemistry to deconstruct lignocellulosic materials. Unlike white rot fungi, BR fungi lack peroxidases for lignin deconstruction, and also lack some endoglucanase/cellobiohydrolase activities. The role that the CMF mechanism plays in "opening up" the wood cell wall structure in advance of enzymatic action, and any interaction between CMF constituents and the selective CAZyme suite that BRs possess, is still unclear. Expression patterns for CMF redox metabolites and lytic polysaccharide monooxygenase (LPMO–AA9 family) genes showed that some LPMO isozymes were upregulated with genes associated with CMF at early stages of brown rot by Gloeophyllum trabeum. In the structural studies, wood decayed by the G. trabeum was compared to CMF-treated wood, or CMF-treated wood followed by treatment with either the early-upregulated LPMO or a commercial CAZyme cocktail. Structural modification of decayed/treated wood was characterized using small angle neutron scattering. CMF treatment produced neutron scattering patterns similar to that of the BR decay indicating that both systems enlarged the nanopore structure of wood cell walls to permit enzyme access. Enzymatic deconstruction of cellulose or lignin in raw wood samples was not achieved via CAZyme cocktail or LPMO enzyme action alone. CMF treatment resulted in depolymerization of crystalline cellulose as attack progressed from the outer regions of individual crystallites. Multiple pulses of CMF treatment on raw wood showed a progressive increase in the spacing between the cellulose elementary fibrils (EFs), indicating the CMF eroded the matrix outside the EF bundles, leading to less tightly packed EFs. Peracetic acid delignification treatment enhanced subsequent CMF treatment effects, and allowed both enzyme systems to further increase spacing of the EFs. Moreover, even after a single pulse of CMF treatment, both enzymes were apparently able to penetrate the cell wall to further increase EF spacing. The data suggest the potential for the early-upregulated LPMO enzyme to work in association with CMF chemistry, suggesting that G. trabeum may have adopted mechanisms to integrate non-enzymatic and enzymatic chemistries together during early stages of brown rot decay. [ABSTRACT FROM AUTHOR]

Wood decayed by brown rot fungi and wood treated with the chelator-mediated Fenton (CMF) reaction, either alone or together with a cellulose enzyme cocktail, was analyzed by small angle neutron scattering (SANS), sum frequency generation (SFG) spectroscopy, Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Results showed that the CMF mechanism mimicked brown rot fungal attack for both holocellulose and lignin components of the wood. Crystalline cellulose and lignin were both depolymerized by the CMF reaction. Porosity of the softwood cell wall did not increase during CMF treatment, enzymes secreted by the fungi did not penetrate the decayed wood. The enzymes in the cellulose cocktail also did not appear to alter the effects of the CMF-treated wood relative to enhancing cell wall deconstruction. This suggests a rethinking of current brown rot decay models and supports a model where monomeric sugars and oligosaccharides diffuse from the softwood cell walls during non-enzymatic action. In this regard, the CMF mechanism should not be thought of as a “pretreatment” used to permit enzymatic penetration into softwood cell walls, but instead it enhances polysaccharide components diffusing to fungal enzymes located in wood cell lumen environments during decay. SANS and other data are consistent with a model for repolymerization and aggregation of at least some portion of the lignin within the cell wall, and this is supported by AFM and TEM data. The data suggest that new approaches for conversion of wood substrates to platform chemicals in biorefineries could be achieved using the CMF mechanism with >75% solubilization of lignocellulose, but that a more selective suite of enzymes and other downstream treatments may be required to work when using CMF deconstruction technology. Strategies to enhance polysaccharide release from lignocellulose substrates for enhanced enzymatic action and fermentation of the released fraction would also aid in the efficient recovery of the more uniform modified lignin fraction that the CMF reaction generates to enhance biorefinery profitability. [ABSTRACT FROM AUTHOR]

Dry rot fungi are a unique group of brown rot fungi, capable of decaying wood at a distance from soil and moisture sources. When degrading wood in the built environment, these fungi are typically observed in contact with calcium (Ca)-containing materials, but it is unclear how or if this association benefits the fungus. In this work, several wood-degrading fungi, including two dry rot species, were cultured for 10 weeks in agar-block microcosms containing spruce heartwood and a Ca treatment. Calcium treatments were gypsum blocks (>99% pure CaSO4) or 2mM CaCl2 in agar. Controls contained no Ca addition. Scanning electron microscopy and microanalysis confirmed hyphal contact with gyp-board and verified Ca oxalate crystal formation. Cation analysis revealed net Ca enrichment in wood degraded in the presence of gyp-board, including wood degraded by a white rot fungus. Despite wood Ca enrichment in gypsum treatments, wood weight loss and pH were unaffected, while acid-extractable oxalate was often higher. This suggests that Ca extracted from materials by wood decay fungi affects oxalate solubility, but does not necessarily facilitate wood decay. This dynamic should, however, be explored in relation to other building material elements and during incipient decay. [Copyright &y& Elsevier]

Brown rot fungi were incubated in agar and agar-wood microcosms containing metallic or hydroxide forms of Al, Cu, and Fe. Metal dissolution was associated with elevated oxalate concentrations in agar, but metals translocated into wood did not affect oxalate accumulation, crystal production, or decay rate, demonstrating a substrate-dependent oxalate dynamic. [ABSTRACT FROM AUTHOR]

The expression of intracellular NADH-dependent 1,4-benzoquinone reductase in the brown-rot fungus Gloeophyllum trabeum was increased in the presence of 2,6-dimethoxy-1,4-benzoquinone (2,6-DMBQ) and, to a lesser extent, vanillic acid. Expression was time-dependent and influenced by nitrogen levels. An intracellular NADH-dependent 1,4-benzoquinone reductase was purified from G. trabeum. The subunit molecular mass was 22 kDa. Flavin mononucleotide was the coenzyme. The pI of the enzyme was 4.2. The quinone reductase catalyzed the reduction of multiple 1,4-quinones and exhibited Ping–Pong kinetics. For enzyme-catalyzed 2,6-DMBQ reduction, the apparent KM was 6.8 μM and the kcat1.0×103 s−1; the pH optimum was between 5.5 and 7; the activation energy of the reaction was estimated at 43.7 kJ mol−1. The one-to-one stoichiometric ratio of NADH oxidation versus 2,6-DMBQ reduction suggested a two-electron transfer mechanism for the enzyme. Dicumarol and Cibacron blue were competitive inhibitors with Ki values of 0.5 and 0.2 μM, respectively. [Copyright &y& Elsevier]

The transplasma membrane redox system of the brown rot fungus Gloeophyllum trabeum was characterized with ferricyanide reduction kinetics. Nitrogen deficiency did not statistically affect the ferricyanide reduction rate, which depended on initial ferricyanide concentration and initial mycelial mass. The reduction rate increased with pH above pH 5.0, and was statistically lower in HEPES buffer (pH 8.0) than in potassium phosphate buffer (pH 8.0). Carbonyl cyanide m-chloromethoxyphenyl hydrazone, 2,4-dinitrophenol and sodium azide were efficient inhibitors of the transplasma membrane redox system. The quinone reducing activity of extracellular, membrane-bound, and intracellular quinone reductases of G. trabeum was tested, extracellular quinone reducing activity was not observed under the examined culture conditions. Quinone reduction by mycelium and the intracellular enzyme showed different kinetic constants. The fungus produced constitutive intracellular benzoquinone reductases, which are NAD(P)H-dependent. [Copyright &y& Elsevier]

Near infrared (NIR) spectroscopy and pyrolysis-molecular beam mass spectrometry (py-MBMS) analysis can be used in conjunction with multivariate regression and principal components analysis to differentiate brown-rot-degraded wood from non-degraded spruce and to follow the temporal changes in wood undergoing brown-rot degradation. Regression of NIR test results vs. percent weight loss for Postia placenta- and Gloeophyllum trabeum-infected spruce wood blocks yielded a correlation coefficient of 0.96. Regression of MBMS test results for the same samples yielded a correlation coefficient of 0.96. Principle components analysis was used to differentiate non-infected wood and P. placenta- and G. trabeum-infected wood. These techniques may be used to detect different types of biodegradation and to develop a better understanding of the chemical changes that the wood undergoes when it is subjected to brown-rot biodegradation. [Copyright &y& Elsevier]

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-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 andMSand that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolyticLPMOactivity 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. [ABSTRACT FROM AUTHOR]

Brown rot fungi utilize iron as part of a chelator-mediated Fenton (CMF) reaction during wood biodegradation. Research suggests these fungi reduce Fe3+ to promote oxygen radical generation resulting in depolymerization of the wood cell wall. High levels of Mn are also found in wood decayed by brown rot fungi. However, little is known about the oxidation states of Fe and Mn during the decay process. X-ray absorption near edge spectroscopy (XANES) can be used to examine metal oxidation states and coordination chemistry. XANES experiments were conducted on wood decayed by Gloeophyllum trabeum over 2–8 weeks with results showing that Mn2+ and Fe3+ predominated for metal oxidation states. However, Fe2+ was present at sites of greater fungal growth In certain cases, the μXANES measurements showed that the fraction of Fe2+ in the wood samples was as high as 50%. Localized areas of reduced iron corresponded with areas of greater fungal hyphal mass which is in agreement with how brown rot fungi decay wood via the CMF reaction. The limited change in oxidation state of Mn observed in wood with active fungal activity suggests that the role of manganese in CMF biodegradation chemistry should be further explored. • We utilize XANES to probe Fe and Mn oxidation states in wood undergoing decay. • Virtually all the Fe in the soil and wood was in the Fe3+ oxidation state. • Mn was predominately in the Mn2+ oxidation state but had as much as 8% Mn4+. [ABSTRACT FROM AUTHOR]