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.

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.

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.

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.

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.

Oxalate decarboxylase of the white-rot fungus Dichomitus squalens demonstrates a novel enzyme primary structure and non-induced expression on wood and in liquid cultures.

Oxalate decarboxylase (ODC) catalyses the conversion of oxalic acid to formic acid and CO(2) in bacteria and fungi. In wood-decaying fungi the enzyme has been linked to the regulation of intra- and extracellular quantities of oxalic acid, which is one of the key components in biological decomposition of wood. ODC enzymes are biotechnologically interesting for their potential in diagnostics, agriculture and environmental applications, e.g. removal of oxalic acid from industrial wastewaters. We identified a novel ODC in mycelial extracts of two wild-type isolates of Dichomitus squalens, and cloned the corresponding Ds-odc gene. The primary structure of the Ds-ODC protein contains two conserved Mn-binding cupin motifs, but at the N-terminus, a unique, approximately 60 aa alanine-serine-rich region is found. Real-time quantitative RT-PCR analysis confirmed gene expression when the fungus was cultivated on wood and in liquid medium. However, addition of oxalic acid in liquid cultures caused no increase in transcript amounts, thereby indicating a constitutive rather than inducible expression of Ds-odc. The detected stimulation of ODC activity by oxalic acid is more likely due to enzyme activation than to transcriptional upregulation of the Ds-odc gene. Our results support involvement of ODC in primary rather than secondary metabolism in fungi.

Molecular characterization of the basidiomycete isolate Nematoloma frowardii b19 and its manganese peroxidase places the fungus in the corticioid genus Phlebia.

The basidiomycete isolate b19, originally identified by morphological characteristics of the fruiting body as Nematoloma frowardii, efficiently produces manganese peroxidase (MNP) and is used for degradation of natural, persistent aromatic polymers (lignin, humic acids and brown coal components). The N. frowardii MNP has shown good activity in conversion of xenobiotic compounds such as polycyclic hydrocarbons and trinitrotoluene. However, this biotechnologically promising fungus has not previously been studied at the molecular biology level. We show here that according to the molecular characterization of its main MNP isozyme, Nf b19 MNP2, and partial sequencing of its MNP3-, three lignin peroxidase- and two laccase-encoding genes, and the gene encoding the ribosomal SSU 18S RNA, that the fungus has a close phylogenetic relationship to the white-rot basidiomycete Phlebia radiata (Fr.). Ribosomal internal transcribed spacer (ITS) sequence (ITS1+5.8S+ITS2) phylogeny reclassifies Nf b19 as a possible representative of a new species of the genus Phlebia, nearest to the Phlebia acerina clade. The genus Phlebia belongs to a completely different family (Corticiaceae) and order (Aphyllophorales) within the phylum Basidiomycota than the genus Nematoloma, which is classified in the order Agaricales, family Strophariaceae. Our results thus indicate a need for systematic re-identification of the previously named N. frowardii isolate b19.

Novel thermotolerant laccases produced by the white-rot fungus Physisporinus rivulosus.

The white-rot basidiomycete Physisporinus rivulosus strain T241i is highly selective for degradation of softwood lignin, which makes this fungus suitable for biopulping. In order to promote laccase production, P. rivulosus was cultivated in nutrient-nitrogen sufficient liquid media containing either charcoal or spruce sawdust as supplements. Two laccases with distinct pI values, Lac-3.5 and Lac-4.8, were purified from peptone-spruce sawdust-charcoal cultures of P. rivulosus. Both laccases showed thermal stability at up to 60 degrees C. Lac-4.8 was thermally activated at 50 degrees C. Surprisingly, both laccases displayed atypically low pH optima (pH 3.0-3.5) in oxidation of the commonly used laccase substrates syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine), 2,6-dimethoxyphenol and guaiacol (2-methoxyphenol). Steady-state kinetic measurements pointed to unusually low affinity to guaiacol at low pH, whereas the kinetic constants for the methoxyphenols and ABTS were within the ranges reported for other fungal laccases. The combination of thermotolerance with low pH optima for methoxylated phenol substrates suggests that the two P. rivulosus T241i laccases possess potential for use in biotechnological applications.

Expression and molecular properties of a new laccase of the white rot fungus Phlebia radiata grown on wood.

Laccases are phenol-oxidizing, multicopper enzymes produced by fungi, plants, insects and bacteria. Fungal laccases are involved in ecologically important processes such as decomposition of lignocellulose (wood and plant material). In this work, in order to find out the role of fungal laccases upon wood colonisation and lignin decay, we describe expression of laccase-encoding genes in the white rot basidiomycete Phlebia radiata 79, when the fungus grows on its natural substrates, that is on softwood (Alnus incana) and hardwood (Picea abies). Clones for two laccase-encoding genes, the previously described Pr-lac1 and a new gene Pr-lac2 were characterized. Pr-lac2 coding region is interrupted by 12 introns and the deduced Lac2 protein displays a higher pI value (5.8) than Lac1 (pI 3.2-3.5). Phylogenetic analysis indicates differential evolution for the two laccases, and Lac2 demonstrates the highest sequence identity with Trametes laccases (66%). Transcripts of Pr-lac1 were the most abundant both in solid-state softwood and semi-solid hardwood cultures, as analyzed by competitive RT-PCR and Northern hybridization. On spruce wood chips, Pr-lac1 and Pr-lac2 were expressed within 2-3 weeks of growth together with manganese and lignin peroxidase-encoding genes. Our results indicate wood-promoted but time-dependent regulation of expression for the two, at protein and gene level distinct P. radiata laccases.

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem.

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce ( Picea abies (L.) H. Karsten) and silver birch ( Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-microm cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.

Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy-immunogold labeling.

The lignification process in mature Norway spruce [ Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)-immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.