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.

Polyporales Brown Rot Species Fomitopsis pinicola: Enzyme Activity Profiles, Oxalic Acid Production, and Fe3+-Reducing Metabolite Secretion.

Basidiomycota fungi in the order Polyporales are specified to decomposition of dead wood and woody debris and thereby are crucial players in the degradation of organic matter and cycling of carbon in the forest ecosystems. Polyporales wood-decaying species comprise both white rot and brown rot fungi, based on their mode of wood decay. While the white rot fungi are able to attack and decompose all the lignocellulose biopolymers, the brown rot species mainly cause the destruction of wood polysaccharides, with minor modification of the lignin units. The biochemical mechanism of brown rot decay of wood is still unclear and has been proposed to include a combination of nonenzymatic oxidation reactions and carbohydrate-active enzymes. Therefore, a linking approach is needed to dissect the fungal brown rot processes. We studied the brown rot Polyporales species Fomitopsis pinicola by following mycelial growth and enzyme activity patterns and generating metabolites together with Fenton-promoting Fe3+-reducing activity for 3 months in submerged cultures supplemented with spruce wood. Enzyme activities to degrade hemicellulose, cellulose, proteins, and chitin were produced by three Finnish isolates of F. pinicola Substantial secretion of oxalic acid and a decrease in pH were notable. Aromatic compounds and metabolites were observed to accumulate in the fungal cultures, with some metabolites having Fe3+-reducing activity. Thus, F. pinicola demonstrates a pattern of strong mycelial growth leading to the active production of carbohydrate- and protein-active enzymes, together with the promotion of Fenton biochemistry. Our findings point to fungal species-level "fine-tuning" and variations in the biochemical reactions leading to the brown rot type of wood decay.IMPORTANCEFomitopsis pinicola is a common fungal species in boreal and temperate forests in the Northern Hemisphere encountered as a wood-colonizing saprotroph and tree pathogen, causing a severe brown rot type of wood degradation. However, its lignocellulose-decomposing mechanisms have remained undiscovered. Our approach was to explore both the enzymatic activities and nonenzymatic Fenton reaction-promoting activities (Fe3+ reduction and metabolite production) by cultivating three isolates of F. pinicola in wood-supplemented cultures. Our findings on the simultaneous production of versatile enzyme activities, including those of endoglucanase, xylanase, ß-glucosidase, chitinase, and acid peptidase, together with generation of low pH, accumulation of oxalic acid, and Fe3+-reducing metabolites, increase the variations of fungal brown rot decay mechanisms. Furthermore, these findings will aid us in revealing the wood decay proteomic, transcriptomic, and metabolic activities of this ecologically important forest fungal species.

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.

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.

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and ß-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Biochemical and molecular characterization of an atypical manganese peroxidase of the litter-decomposing fungus Agrocybe praecox.

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer.

Toxicity Screening of Fungal Extracts and Metabolites, Xenobiotic Chemicals, and Indoor Dusts with In Vitro and Ex Vivo Bioassay Methods.

It is controversial how useful bioassays are for identifying the in vivo toxicity of hazardous environmental exposures. In this study, fruiting bodies of forest mushrooms (n = 46), indoor mold colonies (n = 412), fungal secondary metabolites (n = 18), xenobiotic chemicals such as biocides and detergents (n = 6), and methanol extracts of indoor dusts from urban buildings (n = 26) were screened with two different bioactivity assays: boar sperm motility inhibition (BSMI) and inhibition of cell proliferation (ICP) tests. For the forest mushrooms, the toxicity testing result was positive for 100% of poisonous-classified species, 69% of non-edible-classified species, and 18% of edible-classified species. Colonies of 21 isolates of Ascomycota mold fungal species previously isolated from water-damaged buildings proved to be toxic in the tests. Out of the fungal metabolites and xenobiotic chemicals, 94% and 100% were toxic, respectively. Out of the indoor dusts from moldy-classified houses (n = 12) and from dry, mold-free houses (n = 14), 50% and 57% were toxic, respectively. The bioassay tests, however, could not differentiate the samples from indoor dusts of moldy-classified buildings from those from the mold-free buildings. Xenobiotic chemicals and indoor dusts were more toxic in the BSMI assay than in the ICP assay, whereas the opposite results were obtained with the Ascomycota mold colonies and fungal secondary metabolites. The tests recognized unknown methanol-soluble thermoresistant substances in indoor settled dusts. Toxic indoor dusts may indicate a harmful exposure, regardless of whether the toxicity is due to xenobiotic chemicals or microbial metabolites.

Agaricus bisporus and related Agaricus species on lignocellulose: production of manganese peroxidase and multicopper oxidases.

Biotechnological, microbiological, and genetic studies of Agaricus species other than A. bisporus, the white button mushroom, have been limited so far. To expand the knowledge in the genus Agaricus, six novel wild-type isolates of Agaricus spp. were studied on their nutritional demands for enzyme production and mycelial growth. All the selected Agaricus species produced extracellular manganese peroxidase (MnP) and laccase activities in semi-solid rye bran cultures. Moderate MnP activities were measured for A. bisporus, A. bernardii and A. campestris. The highest laccase activities were obtained for A. bisporus and A. campestris. On soy medium, the highest mycelial tyrosinase activity was determined for A. bernardii. For A. bisporus, addition of copper caused no increase in laccase or tyrosinase activities on soy or malt extract media. Hyphal growth rate of the isolates was studied on lignocellulose amended agar plates. Fastest growth was obtained for A. bisporus on wheat bran and birch leaf litter agar. Except for A. bernardii, hyphal growth rates correlated well with MnP and laccase production levels between Agaricus species. Molecular taxonomy of the novel Agaricus spp. positioned them to distinct phylogenetic clusters with species-level identity. In conclusion, our data point to the importance of both MnP and multicopper enzymes in Agaricus spp. while growing on lignocelluloses.

Genes associated with lignin degradation in the polyphagous white-rot pathogen Heterobasidion irregulare show substrate-specific regulation.

The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicellulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. We identified and followed the regulation of expression of genes belonging to families encoding ligninolytic enzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool of class II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Furthermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in lignin oxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcohol oxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on defined lignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiological roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels upon fungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compounds unique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, other oxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than on heartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differs between spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities of laccases are related to the detoxification of phenolic compounds involved in host-defense. Expression of the several short-MnP enzymes indicated an important role for these enzymes in effective delignification of wood by H. irregulare.

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.

Lignin-degrading peroxidases in Polyporales: an evolutionary survey based on 10 sequenced genomes.

The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) were sequenced to expand our knowledge on the diversity of ligninolytic and related peroxidase genes in this Basidiomycota order that includes most wood-rotting fungi. The survey was completed by analyzing the heme-peroxidase genes in the already available genomes of seven more Polyporales species representing the antrodia, gelatoporia, core polyporoid and phlebioid clades. The study confirms the absence of ligninolytic peroxidase genes from the manganese peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP) families, in the brown-rot fungal genomes (all of them from the antrodia clade), which include only a limited number of predicted low redox-potential generic peroxidase (GP) genes. When members of the heme-thiolate peroxidase (HTP) and dye-decolorizing peroxidase (DyP) superfamilies (up to a total of 64 genes) also are considered, the newly sequenced B. adusta appears as the Polyporales species with the highest number of peroxidase genes due to the high expansion of both the ligninolytic peroxidase and DyP (super)families. The evolutionary relationships of the 111 genes for class-II peroxidases (from the GP, MnP, VP, LiP families) in the 10 Polyporales genomes is discussed including the existence of different MnP subfamilies and of a large and homogeneous LiP cluster, while different VPs mainly cluster with short MnPs. Finally, ancestral state reconstructions showed that a putative MnP gene, derived from a primitive GP that incorporated the Mn(II)-oxidation site, is the precursor of all the class-II ligninolytic peroxidases. Incorporation of an exposed tryptophan residue involved in oxidative degradation of lignin in a short MnP apparently resulted in evolution of the first VP. One of these ancient VPs might have lost the Mn(II)-oxidation site being at the origin of all the LiP enzymes, which are found only in species of the order Polyporales.

Complete mitochondrial genome sequence of the Agaricomycetes brown rot fungus Fomitopsis pinicola isolate FBCC1181.

The mitochondrial genome of the brown rot fungus Fomitopsis pinicola isolate FBCC 1181 is a 66.5 kbp circular chromosome. It contains 64 predicted genes, including a set typical for Basidiomycota Agaricomycetes mitogenomes. Introns of cox and cob genes contain several homing endonucleases of both LAGLIDADG and GIY-YIG types.

Basidiomycota Fungi and ROS: Genomic Perspective on Key Enzymes Involved in Generation and Mitigation of Reactive Oxygen Species.

Our review includes a genomic survey of a multitude of reactive oxygen species (ROS) related intra- and extracellular enzymes and proteins among fungi of Basidiomycota, following their taxonomic classification within the systematic classes and orders, and focusing on different fungal lifestyles (saprobic, symbiotic, pathogenic). Intra- and extracellular ROS metabolism-involved enzymes (49 different protein families, summing 4170 protein models) were searched as protein encoding genes among 63 genomes selected according to current taxonomy. Extracellular and intracellular ROS metabolism and mechanisms in Basidiomycota are illustrated in detail. In brief, it may be concluded that differences between the set of extracellular enzymes activated by ROS, especially by H2O2, and involved in generation of H2O2, follow the differences in fungal lifestyles. The wood and plant biomass degrading white-rot fungi and the litter-decomposing species of Agaricomycetes contain the highest counts for genes encoding various extracellular peroxidases, mono- and peroxygenases, and oxidases. These findings further confirm the necessity of the multigene families of various extracellular oxidoreductases for efficient and complete degradation of wood lignocelluloses by fungi. High variations in the sizes of the extracellular ROS-involved gene families were found, however, among species with mycorrhizal symbiotic lifestyle. In addition, there are some differences among the sets of intracellular thiol-mediation involving proteins, and existence of enzyme mechanisms for quenching of intracellular H2O2 and ROS. In animal- and plant-pathogenic species, extracellular ROS enzymes are absent or rare. In these fungi, intracellular peroxidases are seemingly in minor role than in the independent saprobic, filamentous species of Basidiomycota. Noteworthy is that our genomic survey and review of the literature point to that there are differences both in generation of extracellular ROS as well as in mechanisms of response to oxidative stress and mitigation of ROS between fungi of Basidiomycota and Ascomycota.

Microbial Communities of Cladonia Lichens and Their Biosynthetic Gene Clusters Potentially Encoding Natural Products.

Lichens have been widely used in traditional medicine, especially by indigenous communities worldwide. However, their slow growth and difficulties in the isolation of lichen symbionts and associated microbes have hindered the pharmaceutical utilisation of lichen-produced compounds. Advances in high-throughput sequencing techniques now permit detailed investigations of the complex microbial communities formed by fungi, green algae, cyanobacteria, and other bacteria within the lichen thalli. Here, we used amplicon sequencing, shotgun metagenomics, and in silico metabolomics together with compound extractions to study reindeer lichens collected from Southern Finland. Our aim was to evaluate the potential of Cladonia species as sources of novel natural products. We compared the predicted biosynthetic pathways of lichen compounds from isolated genome-sequenced lichen fungi and our environmental samples. Potential biosynthetic genes could then be further used to produce secondary metabolites in more tractable hosts. Furthermore, we detected multiple compounds by metabolite analyses, which revealed connections between the identified biosynthetic gene clusters and their products. Taken together, our results contribute to metagenomic data studies from complex lichen-symbiotic communities and provide valuable new information for use in further biochemical and pharmacological studies.

Laccase as a Tool in Building Advanced Lignin-Based Materials.

Lignin is an abundant natural feedstock that offers great potential as a renewable substitute for fossil-based resources. Its polyaromatic structure and unique properties have attracted significant research efforts. The advantages of an enzymatic over chemical or thermal approach to construct or deconstruct lignins are that it operates in mild conditions, requires less energy, and usually uses non-toxic chemicals. Laccase is a widely investigated oxidative enzyme that can catalyze the polymerization and depolymerization of lignin. Its dual nature causes a challenge in controlling the overall direction of lignin-laccase catalysis. In this Review, the factors that affect laccase-catalyzed lignin polymerization were summarized, evaluated, and compared to identify key features that favor lignin polymerization. In addition, a critical assessment of the conditions that enable production of novel lignin hybrids via laccase-catalyzed grafting was presented. To assess the industrial relevance of laccase-assisted lignin valorization, patented applications were surveyed and industrial challenges and opportunities were analyzed. Finally, our perspective in realizing the full potential of laccase in building lignin-based materials for advanced applications was deduced from analysis of the limitations governing laccase-assisted lignin polymerization and grafting.

Oxalate decarboxylase: biotechnological update and prevalence of the enzyme in filamentous fungi.

Oxalate decarboxylase (ODC) is a manganese-containing, multimeric enzyme of the cupin protein superfamily. ODC is one of the three enzymes identified to decompose oxalic acid and oxalate, and within ODC catalysis, oxalate is split into formate and CO(2). This primarily intracellular enzyme is found in fungi and bacteria, and currently the best characterized enzyme is the Bacillus subtilis OxdC. Although the physiological role of ODC is yet unidentified, the feasibility of this enzyme in diverse biotechnological applications has been recognized for a long time. ODC could be exploited, e.g., in diagnostics, therapeutics, process industry, and agriculture. So far, the sources of ODC enzyme have been limited including only a few fungal and bacterial species. Thus, there is potential for identification and cloning of new ODC variants with diverse biochemical properties allowing e.g. more enzyme fitness to process applications. This review gives an insight to current knowledge on the biochemical characteristics of ODC, and the relevance of oxalate-converting enzymes in biotechnological applications. Particular emphasis is given to fungal enzymes and the inter-connection of ODC to fungal metabolism of oxalic acid.

New and classic families of secreted fungal heme peroxidases.

Heme-containing peroxidases secreted by fungi are a fascinating group of biocatalysts with various ecological and biotechnological implications. For example, they are involved in the biodegradation of lignocelluloses and lignins and participate in the bioconversion of other diverse recalcitrant compounds as well as in the natural turnover of humic substances and organohalogens. The current review focuses on the most recently discovered and novel types of heme-dependent peroxidases, aromatic peroxygenases (APOs), and dye-decolorizing peroxidases (DyPs), which catalyze remarkable reactions such as peroxide-driven oxygen transfer and cleavage of anthraquinone derivatives, respectively, and represent own separate peroxidase superfamilies. Furthermore, several aspects of the "classic" fungal heme-containing peroxidases, i.e., lignin, manganese, and versatile peroxidases (LiP, MnP, and VP), phenol-oxidizing peroxidases as well as chloroperoxidase (CPO), are discussed against the background of recent scientific developments.

Lignin-modifying enzymes in filamentous basidiomycetes--ecological, functional and phylogenetic review.

Filamentous fungi owe powerful abilities for decomposition of the extensive plant material, lignocellulose, and thereby are indispensable for the Earth's carbon cycle, generation of soil humic matter and formation of soil fine structure. The filamentous wood-decaying fungi belong to the phyla Basidiomycota and Ascomycota, and are unique organisms specified to degradation of the xylem cell wall components (cellulose, hemicelluloses, lignins and extractives). The basidiomycetous wood-decaying fungi form brackets, caps or resupinaceous (corticioid) fruiting bodies when growing on wood for dissemination of their sexual basidiospores. In particular, the ability to decompose the aromatic lignin polymers in wood is mostly restricted to the white rot basidiomycetes. The white-rot decay of wood is possible due to secretion of organic acids, secondary metabolites, and oxidoreductive metalloenzymes, heme peroxidases and laccases, encoded by divergent gene families in these fungi. The brown rot basidiomycetes obviously depend more on a non-enzymatic strategy for decomposition of wood cellulose and modification of lignin. This review gives a current ecological, genomic, and protein functional and phylogenetic perspective of the wood and lignocellulose-decaying basidiomycetous fungi.

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.

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 ß-glucosidase and endoglucanase cellulolytic activities. The intermediate rot fungus S. commune was the strongest producer of ß-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.