Genome description of Phlebia radiata 79 with comparative genomics analysis on lignocellulose decomposition machinery of phlebioid fungi.

BACKGROUND: The white rot fungus Phlebia radiata, a type species of the genus Phlebia, is an efficient decomposer of plant cell wall polysaccharides, modifier of softwood and hardwood lignin, and is able to produce ethanol from various waste lignocellulose substrates. Thus, P. radiata is a promising organism for biotechnological applications aiming at sustainable utilization of plant biomass. Here we report the genome sequence of P. radiata isolate 79 originally isolated from decayed alder wood in South Finland. To better understand the evolution of wood decay mechanisms in this fungus and the Polyporales phlebioid clade, gene content and clustering of genes encoding specific carbohydrate-active enzymes (CAZymes) in seven closely related fungal species was investigated. In addition, other genes encoding proteins reflecting the fungal lifestyle including peptidases, transporters, small secreted proteins and genes involved in secondary metabolism were identified in the genome assembly of P. radiata. RESULTS: The PACBio sequenced nuclear genome of P. radiata was assembled to 93 contigs with 72X sequencing coverage and annotated, revealing a dense genome of 40.4 Mbp with approximately 14 082 predicted protein-coding genes. According to functional annotation, the genome harbors 209 glycoside hydrolase, 27 carbohydrate esterase, 8 polysaccharide lyase, and over 70 auxiliary redox enzyme-encoding genes. Comparisons with the genomes of other phlebioid fungi revealed shared and specific properties among the species with seemingly similar saprobic wood-decay lifestyles. Clustering of especially GH10 and AA9 enzyme-encoding genes according to genomic localization was discovered to be conserved among the phlebioid species. In P. radiata genome, a rich repertoire of genes involved in the production of secondary metabolites was recognized. In addition, 49 genes encoding predicted ABC proteins were identified in P. radiata genome together with 336 genes encoding peptidases, and 430 genes encoding small secreted proteins. CONCLUSIONS: The genome assembly of P. radiata contains wide array of carbohydrate polymer attacking CAZyme and oxidoreductase genes in a composition identifiable for phlebioid white rot lifestyle in wood decomposition, and may thus serve as reference for further studies. Comparative genomics also contributed to enlightening fungal decay mechanisms in conversion and cycling of recalcitrant organic carbon in the forest ecosystems.

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.

Decomposition of spruce wood and release of volatile organic compounds depend on decay type, fungal interactions and enzyme production patterns.

Effect of three wood-decaying fungi on decomposition of spruce wood was studied in solid-state cultivation conditions for a period of three months. Two white rot species (Trichaptum abietinum and Phlebia radiata) were challenged by a brown rot species (Fomitopsis pinicola) in varying combinations. Wood decomposition patterns as determined by mass loss, carbon to nitrogen ratio, accumulation of dissolved sugars and release of volatile organic compounds (VOCs) were observed to depend on both fungal combinations and growth time. Similar dependence of fungal species combination, either white or brown rot dominated, was observed for secreted enzyme activities on spruce wood. Fenton chemistry suggesting reduction of Fe3+ to Fe2+ was detected in the presence of F. pinicola, even in co-cultures, together with substantial degradation of wood carbohydrates and accumulation of oxalic acid. Significant correlation was perceived with two enzyme activity patterns (oxidoreductases produced by white rot fungi; hydrolytic enzymes produced by the brown rot fungus) and wood degradation efficiency. Moreover, emission of four signature VOCs clearly grouped the fungal combinations. Our results indicate that fungal decay type, either brown or white rot, determines the loss of wood mass and decomposition of polysaccharides as well as the pattern of VOCs released upon fungal growth on spruce wood.

Assessment of the efficiency of U-tube continuous bioreactor and immobilized enzyme beads for dye decolourization.

Two bioreactors (column and U-tube) were compared for continuous dye decolourization efficiency using a laccase-producing white rot fungus, Phlebia radiata. Column bioreactor containing immobilized crude enzyme beads and U-tube continuous bioreactor containing actively growing fungal biomass were established. Synthetic dye (coracryl blue C5G) solution treated with immobilized crude enzyme on alginate beads showed a maximum net decolourization up to 55% (flow rate 1 ml/min). The U-tube bioreactor was more efficient in decolorizing the dye, which showed a net decolourization up to 64% at faster flow rate (2.5 ml/min). The decolorization efficiency in both the systems was positively influenced by the slower flow rate. Thus, the study presents designing and operations of two continuous small-scale bioreactors one with immobilized enzyme while the another one with direct fungal contact.

Single-step, single-organism bioethanol production and bioconversion of lignocellulose waste materials by phlebioid fungal species.

Ethanol production from non-pretreated lignocellulose was carried out in a consolidated bioprocess with wood-decay fungi of phlebioid Polyporales. Ethanol production was attempted on glucose, spruce wood sawdust and waste core board. Substantial quantities of ethanol were achieved, and isolate Phlebia radiata 0043 produced 5.9g/L of ethanol reaching the yield of 10.4% ethanol from core board lignocellulose substrate. Acidic initial culture conditions (pH 3) induced ethanol fermentation compared to the more neutral environment. Together with bioethanol, the fungi were able to produce organic acids such as oxalate and fumarate, thus broadening their capacity and applicability as efficient organisms to be utilized for bioconversion of various lignocelluloses. In conclusion, fungi of Phlebia grow on, convert and saccharify solid lignocellulose waste materials without pre-treatments resulting in accumulation of ethanol and organic acids. These findings will aid in applying fungal biotechnology for production of biofuels and biocompounds.

Comparison of traditional field retting and Phlebia radiata Cel 26 retting of hemp fibres for fibre-reinforced composites.

Classical field retting and controlled fungal retting of hemp using Phlebia radiata Cel 26 (a mutant with low cellulose degrading ability) were compared with pure pectinase treatment with regard to mechanical properties of the produced fibre/epoxy composites. For field retting a classification of the microbial evolution (by gene sequencing) and enzyme profiles were conducted. By phylogenetic frequency mapping, different types of fungi, many belonging to the Ascomycota phylum were found on the fibres during the first 2 weeks of field retting, and thereafter, different types of bacteria, notably Proteobacteria, also proliferated on the field retted fibres. Extracts from field retted fibres exhibited high glucanase activities, while extracts from P. radiata Cel 26 retted fibres showed high polygalacturonase and laccase activities. As a result, fungal retting gave a significantly higher glucan content in the fibres than field retting (77 vs. 67%) and caused a higher removal of pectin as indicated by lower galacturonan content of fibres (1.6%) after fibres were retted for 20 days with P. radiata Cel 26 compared to a galacturonan content of 3.6% for field retted fibres. Effective fibre stiffness increased slightly after retting with P. radiata Cel 26 from 65 to 67 GPa, while it decreased after field retting to 52 GPa. Effective fibre strength could not be determined similarly due to variations in fibre fracture strain and fibre-matrix adhesion. A maximum composite strength with 50 vol% fibres of 307 MPa was obtained using P. radiata Cel 26 compared to 248 MPa with field retting.

Removal of metal and organic pollutants from wastewater by a sequential selective technique.

In this study the application of a sequential selective system that combined biosorption with biodegradation was evaluated as a feasible process for the removal of Cr(VI) and m-cresol from effluents. Cr(VI) biosorption on pretreated chestnut shells showed 100% metal removal and modelling efforts demonstrated that the pseudo-second order kinetic model and Langmuir isotherm fit well the process behaviour. Thus, the treated stream was an appropriate environment for the biodegradation of m-cresol using a laccase-producer fungus, Phlebia radiata. Two bioreactor configurations, rotating drum and modified-airlift, were studied using the fungus grown on chestnut shells, which act as support-substrate as well as oxidative enzyme inductor increasing the laccase activity up to 1000UL(-1). The best bioreactor, rotating drum, reached 100% removal in 7days. Finally, the best configuration for the sequential selective system was modelled operating in continuous mode by the breakthrough curves generated using FASTv2.0 and the design bioreactor flow model.

Time-scale dynamics of proteome and transcriptome of the white-rot fungus Phlebia radiata: growth on spruce wood and decay effect on lignocellulose.

BACKGROUND: The white-rot Agaricomycetes species Phlebia radiata is an efficient wood-decaying fungus degrading all wood components, including cellulose, hemicellulose, and lignin. We cultivated P. radiata in solid state cultures on spruce wood, and extended the experiment to 6 weeks to gain more knowledge on the time-scale dynamics of protein expression upon growth and wood decay. Total proteome and transcriptome of P. radiata were analyzed by peptide LC-MS/MS and RNA sequencing at specific time points to study the enzymatic machinery on the fungus' natural growth substrate. RESULTS: According to proteomics analyses, several CAZy oxidoreductase class-II peroxidases with glyoxal and alcohol oxidases were the most abundant proteins produced on wood together with enzymes important for cellulose utilization, such as GH7 and GH6 cellobiohydrolases. Transcriptome additionally displayed expression of multiple AA9 lytic polysaccharide monooxygenases indicative of oxidative cleavage of wood carbohydrate polymers. Large differences were observed for individual protein quantities at specific time points, with a tendency of enhanced production of specific peroxidases on the first 2 weeks of growth on wood. Among the 10 class-II peroxidases, new MnP1-long, characterized MnP2-long and LiP3 were produced in high protein abundances, while LiP2 and LiP1 were upregulated at highest level as transcripts on wood together with the oxidases and one acetyl xylan esterase, implying their necessity as primary enzymes to function against coniferous wood lignin to gain carbohydrate accessibility and fungal growth. Majority of the CAZy encoding transcripts upregulated on spruce wood represented activities against plant cell wall and were identified in the proteome, comprising main activities of white-rot decay. CONCLUSIONS: Our data indicate significant changes in carbohydrate-active enzyme expression during the six-week surveillance of P. radiata growing on wood. Response to wood substrate is seen already during the first weeks. The immediate oxidative enzyme action on lignin and wood cell walls is supported by detected lignin substructure sidechain cleavages, release of phenolic units, and visual changes in xylem cell wall ultrastructure. This study contributes to increasing knowledge on fungal genetics and lignocellulose bioconversion pathways, allowing us to head for systems biology, development of biofuel production, and industrial applications on plant biomass utilizing wood-decay fungi.