Production of Recombinant Laccase From Coprinopsis cinerea and Its Effect in Mediator Promoted Lignin Oxidation at Neutral pH.

Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70°C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.

Depolymerization of biorefinery lignin by improved laccases of the white-rot fungus Obba rivulosa.

Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white-rot fungus Obba rivulosa, we mutated their substrate-binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2-D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic α-oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2-D206N is a promising candidate for lignin-related applications.

Fungal Treatment Modifies Kraft Lignin for Lignin- and Cellulose-Based Carbon Fiber Precursors.

The kraft lignin's low molecular weight and too high hydroxyl content hinder its application in bio-based carbon fibers. In this study, we were able to polymerize kraft lignin and reduce the amount of hydroxyl groups by incubating it with the white-rot fungus Obba rivulosa. Enzymatic radical oxidation reactions were hypothesized to induce condensation of lignin, which increased the amount of aromatic rings connected by carbon-carbon bonds. This modification is assumed to be beneficial when aiming for graphite materials such as carbon fibers. Furthermore, the ratio of remaining aliphatic hydroxyls to phenolic hydroxyls was increased, making the structure more favorable for carbon fiber production. When the modified lignin was mixed together with cellulose, the mixture could be spun into intact precursor fibers by using dry-jet wet spinning. The modified lignin leaked less to the spin bath compared with the unmodified lignin starting material, making the recycling of spin-bath solvents easier. The stronger incorporation of modified lignin in the precursor fibers was confirmed by composition analysis, thermogravimetry, and mechanical testing. This work shows how white-rot fungal treatment can be used to modify the structure of lignin to be more favorable for the production of bio-based fiber materials.

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.

Characterization of a feruloyl esterase from Aspergillus terreus facilitates the division of fungal enzymes from Carbohydrate Esterase family 1 of the carbohydrate-active enzymes (CAZy) database.

Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell-wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate-active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A. terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes.

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.

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.

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.

Lignocellulose-converting enzyme activity profiles correlate with molecular systematics and phylogeny grouping in the incoherent genus Phlebia (Polyporales, Basidiomycota).

BACKGROUND: The fungal genus Phlebia consists of a number of species that are significant in wood decay. Biotechnological potential of a few species for enzyme production and degradation of lignin and pollutants has been previously studied, when most of the species of this genus are unknown. Therefore, we carried out a wider study on biochemistry and systematics of Phlebia species. METHODS: Isolates belonging to the genus Phlebia were subjected to four-gene sequence analysis in order to clarify their phylogenetic placement at species level and evolutionary relationships of the genus among phlebioid Polyporales. rRNA-encoding (5.8S, partial LSU) and two protein-encoding gene (gapdh, rpb2) sequences were adopted for the evolutionary analysis, and ITS sequences (ITS1+5.8S+ITS2) were aligned for in-depth species-level phylogeny. The 49 fungal isolates were cultivated on semi-solid milled spruce wood medium for 21 days in order to follow their production of extracellular lignocellulose-converting oxidoreductases and carbohydrate active enzymes. RESULTS: Four-gene phylogenetic analysis confirmed the polyphyletic nature of the genus Phlebia. Ten species-level subgroups were formed, and their lignocellulose-converting enzyme activity profiles coincided with the phylogenetic grouping. The highest enzyme activities for lignin modification (manganese peroxidase activity) were obtained for Phlebia radiata group, which supports our previous studies on the enzymology and gene expression of this species on lignocellulosic substrates. CONCLUSIONS: Our study implies that there is a species-level connection of molecular systematics (genotype) to the efficiency in production of both lignocellulose-converting carbohydrate active enzymes and oxidoreductases (enzyme phenotype) on spruce wood. Thus, we may propose a similar phylogrouping approach for prediction of lignocellulose-converting enzyme phenotypes in new fungal species or genetically and biochemically less-studied isolates of the wood-decay Polyporales.

Mitochondrial genome of Phlebia radiata is the second largest (156 kbp) among fungi and features signs of genome flexibility and recent recombination events.

Mitochondria are eukaryotic organelles supporting individual life-style via generation of proton motive force and cellular energy, and indispensable metabolic pathways. As part of genome sequencing of the white rot Basidiomycota species Phlebia radiata, we first assembled its mitochondrial genome (mtDNA). So far, the 156 348 bp mtDNA is the second largest described for fungi, and of considerable size among eukaryotes. The P. radiata mtDNA assembled as single circular dsDNA molecule containing genes for the large and small ribosomal RNAs, 28 transfer RNAs, and over 100 open reading frames encoding the 14 fungal conserved protein subunits of the mitochondrial complexes I, III, IV, and V. Two genes (atp6 and tRNA-IleGAU) were duplicated within 6.1 kbp inverted region, which is a unique feature of the genome. The large mtDNA size, however, is explained by the dominance of intronic and intergenic regions (sum 80% of mtDNA sequence). The intergenic DNA stretches harness short (≤ 200 nt) repetitive, dispersed and overlapping sequence elements in abundance. Long self-splicing introns of types I and II interrupt eleven of the conserved genes (cox1,2,3; cob; nad1,2,4,4L,5; rnl; rns). The introns embrace a total of 57 homing endonucleases with LAGLIDADGD and GYI-YIG core motifs, which makes P. radiata mtDNA to one of the largest known reservoirs of intron-homing endonucleases. The inverted duplication, intergenic stretches, and intronic features are indications of dynamics and genetic flexibility of the mtDNA, not fully recognized to this extent in fungal mitochondrial genomes previously, thus giving new insights for the evolution of organelle genomes in eukaryotes.

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus.

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 µkat/µg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.