Hypoxia is regulating enzymatic wood decomposition and intracellular carbohydrate metabolism in filamentous white rot fungus

Background Fungal decomposition of wood is considered as a strictly aerobic process. However, recent findings on wood-decaying fungi to produce ethanol from various lignocelluloses under oxygen-depleted conditions lead us to question this. We designed gene expression study of the white rot fungus Phlebia radiata (isolate FBCC0043) by adopting comparative transcriptomics and functional genomics on solid lignocellulose substrates under varying cultivation atmospheric conditions. Results Switch to fermentative conditions was a major regulator for intracellular metabolism and extracellular enzymatic degradation of wood polysaccharides. Changes in the expression profiles of CAZy (carbohydrate-active enzyme) encoding genes upon oxygen depletion, lead into an alternative wood decomposition strategy. Surprisingly, we noticed higher cellulolytic activity under fermentative conditions in comparison to aerobic cultivation. In addition, our results manifest how oxygen depletion affects over 200 genes of fungal primary metabolism including several transcription factors. We present new functions for acetate generating phosphoketolase pathway and its potential regulator, Adr1 transcription factor, in carbon catabolism under oxygen depletion. Conclusions Physiologically resilient wood-decomposing Basidiomycota species P. radiata is capable of thriving under respirative and fermentative conditions utilizing only untreated lignocellulose as carbon source. Hypoxia-response mechanism in the fungus is, however, divergent from the regulation described for Ascomycota fermenting yeasts or animal-pathogenic species of Basidiomycota.Peer reviewe

Basidiomycota fungi and ROS: genomic perspective on key enzymes involved in generation and mitigation of reactive oxygen species

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Interactions affect hyphal growth and enzyme profiles in combinations of coniferous wood-decaying fungi of Agaricomycetes

Fomitopsis pinicola is a species of Polyporales frequently encountered in Nordic temperate and boreal forests. In nature, the fungus causes destructive brown rot in wood, colonizing tree trunks often occupied by other Basidiomycota species. We mimicked these species-species interactions by introducing F. pinicola to five white rot species, all common saprotrophs of Norway spruce. Hyphal interactions and mycelial growth in various combinations were recorded, while activities of lignocellulose-acting CAZymes and oxidoreductases were followed in co-cultures on two different carbon-source media. Of the species, Phlebia radiata and Trichaptum abietinum were the strongest producers of lignin-modifying oxidoreductases (laccase, manganese peroxidase) when evaluated alone, as well as in co-cultures, on the two different growth media (low-nitrogen liquid medium containing ground coniferous wood, and malt extract broth). F. pinicola was an outstanding producer of oxalic acid (up to 61 mM), whereas presence of P. radiata prevented acidification of the growth environment in the liquid malt-extract cultures. When enzyme profiles of the species combinations were clustered, time-dependent changes were observed on wood-supplemented medium during the eight weeks of growth. End-point acidity and production of mycelium, oxalic acid and oxidoreductase activities, in turn clustered the fungal combinations into three distinct functional groups, determined by the presence of F. pinicola and P. radiata, by principal component analysis. Our findings indicate that combinations of wood-decay fungi have dramatic dynamic effects on the production of lignocellulose-active enzymes, which may lead to divergent degradative processes of dead wood and forest litter.Peer reviewe

Metabolic activities and ultrastructure imaging at late-stage of wood decomposition in interactive brown rot - white rot fungal combinations

Basidiomycota brown rot fungus (Fomitopsis pinicola) and two white rot fungi (Phlebia radiata, Trichaptum abietinum) were cultivated on thin slices of spruce wood individually and in interspecies combinations. Within 12 months, F. pinicola substantially decomposed spruce wood observed as mass loss, also in three-species combinations. However, white rot fungi through hyphal interactions negatively affected the brown-rot indicative iron reduction capacity of F. pinicola. Decay-signature gene expression in mycelial interaction zones indicated suppression of brown rot mechanism but stimulation of enzymatic white-rot lignin attack by P. radiata. Wood ultrastructure imaging showed white-rot dominance in the fungal combinations, whereas destructive brown-rot was evident with F. pinicola alone. Our results confirm the dynamic pattern of enzyme production in fungal combinations, and transition from brown to white rot decomposition metabolism during the late stage of wood decay after one year of interspecific interactions.Peer reviewe

Enzyme Activity Profiles Produced on Wood and Straw by Four Fungi of Different Decay Strategies

Four well-studied saprotrophic Basidiomycota Agaricomycetes species with different decay strategies were cultivated on solid lignocellulose substrates to compare their extracellular decomposing carbohydrate-active and lignin-attacking enzyme production profiles. Two Polyporales species, the white rot fungus Phlebia radiata and brown rot fungus Fomitopsis pinicola, as well as one Agaricales species, the intermediate “grey” rot fungus Schizophyllum commune, were cultivated on birch wood pieces for 12 weeks, whereas the second Agaricales species, the litter-decomposing fungus Coprinopsis cinerea was cultivated on barley straw for 6 weeks under laboratory conditions. During 3 months of growth on birch wood, only the white rot fungus P. radiata produced high laccase and MnP activities. The brown rot fungus F. pinicola demonstrated notable production of xylanase activity up to 43 nkat/mL on birch wood, together with moderate β-glucosidase and endoglucanase cellulolytic activities. The intermediate rot fungus S. commune was the strongest producer of β-glucosidase with activities up to 54 nkat/mL, and a notable producer of xylanase activity, even up to 620 nkat/mL, on birch wood. Low lignin-attacking but moderate activities against cellulose and hemicellulose were observed with the litter-decomposer C. cinerea on barley straw. Overall, our results imply that plant cell wall decomposition ability of taxonomically and ecologically divergent fungi is in line with their enzymatic decay strategy, which is fundamental in understanding their physiology and potential for biotechnological applications

Enzyme Activity Profiles Produced on Wood and Straw by Four Fungi of Different Decay Strategies

Four well-studied saprotrophic Basidiomycota Agaricomycetes species with different decay strategies were cultivated on solid lignocellulose substrates to compare their extracellular decomposing carbohydrate-active and lignin-attacking enzyme production profiles. Two Polyporales species, the white rot fungus Phlebia radiata and brown rot fungus Fomitopsis pinicola, as well as one Agaricales species, the intermediate “grey” rot fungus Schizophyllum commune, were cultivated on birch wood pieces for 12 weeks, whereas the second Agaricales species, the litter-decomposing fungus Coprinopsis cinerea was cultivated on barley straw for 6 weeks under laboratory conditions. During 3 months of growth on birch wood, only the white rot fungus P. radiata produced high laccase and MnP activities. The brown rot fungus F. pinicola demonstrated notable production of xylanase activity up to 43 nkat/mL on birch wood, together with moderate β-glucosidase and endoglucanase cellulolytic activities. The intermediate rot fungus S. commune was the strongest producer of β-glucosidase with activities up to 54 nkat/mL, and a notable producer of xylanase activity, even up to 620 nkat/mL, on birch wood. Low lignin-attacking but moderate activities against cellulose and hemicellulose were observed with the litter-decomposer C. cinerea on barley straw. Overall, our results imply that plant cell wall decomposition ability of taxonomically and ecologically divergent fungi is in line with their enzymatic decay strategy, which is fundamental in understanding their physiology and potential for biotechnological applications

Conversion of Milled Pine Wood by Manganese Peroxidase from Phlebia radiata

Purified manganese peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tw-een 20, Mn2+, bon-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid redsidues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from P. radiata (MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37 degreesC with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to > 6.5. An aromatic nonphenolic lignin structure (beta -O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols

Transcription of lignocellulose-decomposition associated genes, enzyme activities and production of ethanol upon bioconversion of waste substrate by Phlebia radiata

Previously identified twelve plant cell wall degradation-associated genes of the white rot fungus Phlebia radiata were studied by RT-qPCR in semi-aerobic solid-state cultures on lignocellulose waste material, and on glucose-containing reference medium. Wood-decay-involved enzyme activities and ethanol production were followed to elucidate both the degradative and fermentative processes. On the waste lignocellulose substrate, P. radiata carbohydrate-active enzyme (CAZy) genes encoding cellulolytic and hemicellulolytic activities were significantly upregulated whereas genes involved in lignin modification displayed a more complex response. Two lignin peroxidase genes were differentially expressed on waste lignocellulose compared to glucose medium, whereas three manganese peroxidase-encoding genes were less affected. On the contrary, highly significant difference was noticed for three cellulolytic genes (cbhI_1, eg1, bgl1) with higher expression levels on the lignocellulose substrate than on glucose. This indicates expression of the wood-attacking degradative enzyme system by the fungus also on the recycled, waste core board material. During the second week of cultivation, ethanol production increased on the core board to 0.24 g/L, and extracellular activities against cellulose, xylan, and lignin were detected. Sugar release from the solid lignocellulose resulted with concomitant accumulation of ethanol as fermentation product. Our findings confirm that the fungus activates its white rot decay system also on industrially processed lignocellulose adopted as growth substrate, and under semi-aerobic cultivation conditions. Thus, P. radiata is a good candidate for lignocellulose-based renewable biotechnology to make biofuels and biocompounds from materials with less value for recycling or manufacturing.Peer reviewe

Deadwood substrate and species-species interactions determine the release of volatile organic compounds by wood-decaying fungi

Wood-decaying fungi in the phylum Basidiomycota play a significant role in the global carbon cycle, as they decompose deadwood effectively. Fungi may compete for utilizable substrate and growth space by producing soluble metabolites and by releasing volatile organic compounds (VOCs). We determined the role of wood substrate (Scots pine or Norway spruce) on the generation of hyphal biomass, secreted metabolites and enzyme activities, wood decomposition rate, and fungal species-species interactions on VOC release. We studied one brown-rot species (Fomitopsis pinicola) and two white-rot species (Phlebia radiata and Trichaptum abietinum) cultivated individually or in combinations. Wood substrate quality influences VOC release by the wood-decaying fungi, with signature differences caused by the decomposition trait (brown rot or white rot) and species-species interactions. VOC release was higher in the cultures of Basidiomycota than in uncolonized sawdust. Fungal biomass, decomposition activity, iron reduction, enzyme activities, oxalate anion content, and oxalic acid production explained VOC release from decaying wood.Peer reviewe