White-rot Fungus Dichomitus Squalens Research Articles

Transcriptional response of the white-rot fungus Dichomitus squalens to polysaccharides reveals a co-expression network of plant biomass conversion related genes

Wood-degrading white-rot fungi can efficiently degrade all plant biomass components, but the molecular mechanisms behind the degradation of plant polysaccharides remain poorly understood. For example, the gene sets and expression levels induced by the plant polysaccharide-derived monosaccharides in white-rot fungi do not reflect those induced by crude plant biomass substrates. To explore the molecular response of the white-rot fungus Dichomitus squalens to plant-derived oligo- and polysaccharides, we investigated the transcriptomes from mono- and dikaryotic strains of the fungus on 10 substrates and compared the expression of carbohydrate-active enzyme-encoding genes to that previously reported for different monosaccharides and cellobiose. Our results revealed that in D. squalens, a robust response to cellulose leads to its effective depolymerization, with an orthologue of the ascomycete Trichoderma reesei ACE3 likely acting as a central transcriptional regulator. The conserved response between cellulose and cellobiose further confirms cellobiose as the main cellulase inducer in D. squalens. Surprisingly, despite low abundance of pectin in the natural wood substrate of D. squalens, we identified polygalacturonic acid as a major inducer of a broad-targeted pectinolytic response including pectinase, pectin-related sugar transporter and catabolism genes, and four fungal specific transcription factors. This indicates that D. squalens has not only maintained its ability to degrade minor polysaccharide components in its biotope, but also a regulatory system spanning from extracellular degradation to metabolic conversion. Our study contributes to a deeper understanding of the molecular mechanisms behind white-rot fungal plant polysaccharide degradation and provides leads for functional studies of potential transcriptional regulators in basidiomycetes.

Glucose-Mediated Repression of Plant Biomass Utilization in the White-Rot Fungus Dichomitus squalens

The extent of carbon catabolite repression (CCR) at a global level is unknown in wood-rotting fungi, which are critical to the carbon cycle and are a source of biotechnological enzymes. CCR occurs in the presence of sufficient concentrations of easily metabolizable carbon sources (e.g., glucose) and involves downregulation of the expression of genes encoding enzymes involved in the breakdown of complex carbon sources. We investigated this phenomenon in the white-rot fungus Dichomitus squalens using transcriptomics and exoproteomics. In D. squalens cultures, approximately 7% of genes were repressed in the presence of glucose compared to Avicel or xylan alone. The glucose-repressed genes included the essential components for utilization of plant biomass-carbohydrate-active enzyme (CAZyme) and carbon catabolic genes. The majority of polysaccharide-degrading CAZyme genes were repressed and included activities toward all major carbohydrate polymers present in plant cell walls, while repression of ligninolytic genes also occurred. The transcriptome-level repression of the CAZyme genes observed on the Avicel cultures was strongly supported by exoproteomics. Protease-encoding genes were generally not glucose repressed, indicating their likely dominant role in scavenging for nitrogen rather than carbon. The extent of CCR is surprising, given that D. squalens rarely experiences high free sugar concentrations in its woody environment, and it indicates that biotechnological use of D. squalens for modification of plant biomass would benefit from derepressed or constitutively CAZyme-expressing strains.IMPORTANCE White-rot fungi are critical to the carbon cycle because they can mineralize all wood components using enzymes that also have biotechnological potential. The occurrence of carbon catabolite repression (CCR) in white-rot fungi is poorly understood. Previously, CCR in wood-rotting fungi has only been demonstrated for a small number of genes. We demonstrated widespread glucose-mediated CCR of plant biomass utilization in the white-rot fungus Dichomitus squalens This indicates that the CCR mechanism has been largely retained even though wood-rotting fungi rarely experience commonly considered CCR conditions in their woody environment. The general lack of repression of genes encoding proteases along with the reduction in secreted CAZymes during CCR suggested that the retention of CCR may be connected with the need to conserve nitrogen use during growth on nitrogen-scarce wood. The widespread repression indicates that derepressed strains could be beneficial for enzyme production.

Transcriptome analysis of differential gene expression in Dichomitus squalens during interspecific mycelial interactions and the potential link with laccase induction.

Interspecific mycelial interactions between white rot fungi are always accompanied by an increased production of laccase. In this study, the potential of the white rot fungus Dichomitus squalens to enhance laccase production during interactions with two other white rot fungi, Trametes versicolor or Pleurotus ostreatus, was assessed. To probe the mechanism of laccase induction and the role that laccase plays during combative interaction, we analyzed the differential gene expression profile of the laccase induction response to stressful conditions during fungal interaction. We further confirmed the expression patterns of 16 selected genes by qRT-PCR analysis. We noted that many differentially expressed genes (DEGs) encoded proteins that were involved in xenobiotic detoxification and reactive oxygen species (ROS) generation or reduction, including aldo/keto reductase, glutathione S-transferases, cytochrome P450 enzymes, alcohol oxidases and dehydrogenase, manganese peroxidase and laccase. Furthermore, many DEG-encoded proteins were involved in antagonistic mechanisms of nutrient acquisition and antifungal properties, including glycoside hydrolase, glucanase, chitinase and terpenoid synthases. DEG analyses effectively revealed that laccase induction was likely caused by protective responses to oxidative stress and nutrient competition during interspecific fungal interactions.

Delamination of plastic-coated waste paper by enzymes of the white rot fungus Dichomitus squalens

Many paper products are coated with plastic to improve their quality and stability. However, this limits recycling and recovery options and the plastic-coated waste paper is mostly disposed in landfills. Such practices are uneconomical and contrary to sustainable waste management. In this work enzymes of the white rot fungus Dichomitus squalens were investigated for possible delamination of plastic-coated waste paper. Enzymes were found capable to release the polyethylene foil from plastic-coated paper which resulted in 88.6–91.5% mass loss. The delamination rate, however, was depended on the ratio between plastic-coated paper and volume of enzyme filtrate. Results of a consequent experiment showed that enzymes are also efficient when plastic-coated paper is treated in a sequencing batch reactor resulting in 88.2–90.6% mass loss. The system was fully functional up to the 5th cycle; afterwards, the delamination rate reduced due to high thickness of the waste paper sludge. The enzyme activity, however, was still very high; with the laccase activity at the end of the experiment above 900 U/L and manganese peroxidase above 250 U/L. Our results demonstrated, that plastic-coated waste paper has the potential to be efficiently recovered instead of being disposed in landfills.

Selective Cleavage of Lignin β-O-4 Aryl Ether Bond by β-Etherase of the White-Rot Fungus Dichomitus squalens.

Production of value-added compounds from a renewable aromatic polymer, lignin, has proven to be challenging. Chemical procedures, involving harsh reaction conditions, are costly and often result in nonselective degradation of lignin linkages. Therefore, enzymatic catalysis with selective cleavage of lignin bonds provides a sustainable option for lignin valorization. In this study, we describe the first functionally characterized fungal intracellular β-etherase from the wood-degrading white-rot basidiomycete Dichomitus squalens. This enzyme, Ds-GST1, from the glutathione-S-transferase superfamily selectively cleaved the β-O-4 aryl ether bond of a dimeric lignin model compound in a glutathione-dependent reaction. Ds-GST1 also demonstrated activity on polymeric synthetic lignin fractions, shown by a decrease in molecular weight distribution of the laccase-oxidized guaiacyl dehydrogenation polymer. In addition to a possible role of Ds-GST1 in intracellular catabolism of lignin-derived aromatic compounds, the cleavage of the most abundant linkages in lignin under mild reaction conditions makes this biocatalyst an attractive green alternative in biotechnological applications.

The molecular response of the white-rot fungus Dichomitus squalens to wood and non-woody biomass as examined by transcriptome and exoproteome analyses.

The ability to obtain carbon and energy is a major requirement to exist in any environment. For several ascomycete fungi, (post-)genomic analyses have shown that species that occupy a large variety of habitats possess a diverse enzymatic machinery, while species with a specific habitat have a more focused enzyme repertoire that is well-adapted to the prevailing substrate. White-rot basidiomycete fungi also live in a specific habitat, as they are found exclusively in wood. In this study, we evaluated how well the enzymatic machinery of the white-rot fungus Dichomitus squalens is tailored to degrade its natural wood substrate. The transcriptome and exoproteome of D. squalens were analyzed after cultivation on two natural substrates, aspen and spruce wood, and two non-woody substrates, wheat bran and cotton seed hulls. D. squalens produced ligninolytic enzymes mainly at the early time point of the wood cultures, indicating the need to degrade lignin to get access to wood polysaccharides. Surprisingly, the response of the fungus to the non-woody polysaccharides was nearly as good a match to the substrate composition as observed for the wood polysaccharides. This indicates that D. squalens has preserved its ability to efficiently degrade plant biomass types not present in its natural habitat.

Saccharification of Lignocelluloses by Carbohydrate Active Enzymes of the White Rot Fungus Dichomitus squalens.

White rot fungus Dichomitus squalens is an efficient lignocellulose degrading basidiomycete and a promising source for new plant cell wall polysaccharides depolymerizing enzymes. In this work, we focused on cellobiohydrolases (CBHs) of D. squalens. The native CBHI fraction of the fungus, consisting three isoenzymes, was purified and it maintained the activity for 60 min at 50°C, and was stable in acidic pH. Due to the lack of enzyme activity assay for detecting only CBHII activity, CBHII of D. squalens was produced recombinantly in an industrially important ascomycete host, Trichoderma reesei. CBH enzymes of D. squalens showed potential in hydrolysis of complex lignocellulose substrates sugar beet pulp and wheat bran, and microcrystalline cellulose, Avicel. Recombinant CBHII (rCel6A) of D. squalens hydrolysed all the studied plant biomasses. Compared to individual activities, synergistic effect between rCel6A and native CBHI fraction of D. squalens was significant in the hydrolysis of Avicel. Furthermore, the addition of laccase to the mixture of CBHI fraction and rCel6A significantly enhanced the amount of released reducing sugars from sugar beet pulp. Especially, synergy between individual enzymes is a crucial factor in the tailor-made enzyme mixtures needed for hydrolysis of different plant biomass feedstocks. Our data supports the importance of oxidoreductases in improved enzyme cocktails for lignocellulose saccharification.

Oxalate-metabolising genes of the white-rot fungus Dichomitus squalens are differentially induced on wood and at high proton concentration.

Oxalic acid is a prevalent fungal metabolite with versatile roles in growth and nutrition, including degradation of plant biomass. However, the toxicity of oxalic acid makes regulation of its intra- and extracellular concentration crucial. To increase the knowledge of fungal oxalate metabolism, a transcriptional level study on oxalate-catabolising genes was performed with an effective lignin-degrading white-rot fungus Dichomitus squalens, which has demonstrated particular abilities in production and degradation of oxalic acid. The expression of oxalic-acid decomposing oxalate decarboxylase (ODC) and formic-acid decomposing formate dehydrogenase (FDH) encoding genes was followed during the growth of D. squalens on its natural spruce wood substrate. The effect of high proton concentration on the regulation of the oxalate-catabolising genes was determined after addition of organic acid (oxalic acid) and inorganic acid (hydrochloric acid) to the liquid cultures of D. squalens. In order to evaluate the co-expression of oxalate-catabolising and manganese peroxidase (MnP) encoding genes, the expression of one MnP encoding gene, mnp1, of D. squalens was also surveyed in the solid state and liquid cultures. Sequential action of ODC and FDH encoding genes was detected in the studied cultivations. The odc1, fdh2 and fdh3 genes of D. squalens showed constitutive expression, whereas ODC2 and FHD1 most likely are the main responsible enzymes for detoxification of high concentrations of oxalic and formic acids. The results also confirmed the central role of ODC1 when D. squalens grows on coniferous wood. Phylogenetic analysis revealed that fungal ODCs have evolved from at least two gene copies whereas FDHs have a single ancestral gene. As a conclusion, the multiplicity of oxalate-catabolising genes and their differential regulation on wood and in acid-amended cultures of D. squalens point to divergent physiological roles for the corresponding enzymes.

Fungal and enzymatic treatment of mature municipal landfill leachate

The aim of our study was to evaluate biotreatability of mature municipal landfill leachate by using white rot fungus and its extracellular enzymes. Leachates were collected in one active and one closed regional municipal landfill. Both chosen landfills were operating for many years and the leachates generated there were polluted by organic and inorganic compounds. The white rot fungus Dichomitus squalens was able to grow in the mature leachate from the closed landfill and as it utilizes present organic matter as a source of carbon, the results were showing 60% of DOC and COD removal and decreased toxicity to the bacterium Aliivibrio fischeri. On the other hand, growth of the fungus was inhibited in the presence of the leachate from the active landfill. However, when the leachate was introduced to a crude enzyme filtrate containing extracellular ligninolytic enzymes, removal levels of COD and DOC reached 61% and 44%, respectively. Furthermore, the treatment led to detoxification of the leachate to the bacterium Aliivibrio fischeri and to reduction of toxicity (42%) to the plant Sinapis alba. Fungal and enzymatic treatment seems to be a promising biological approach for treatment of mature landfill leachates and their application should be further investigated.

Transcriptional analysis of selected cellulose-acting enzymes encoding genes of the white-rot fungus Dichomitus squalens on spruce wood and microcrystalline cellulose

The recent discovery of oxidative cellulose degradation enhancing enzymes has considerably changed the traditional concept of hydrolytic cellulose degradation. The relative expression levels of ten cellulose-acting enzyme encoding genes of the white-rot fungus Dichomitus squalens were studied on solid-state spruce wood and in microcrystalline Avicel cellulose cultures. From the cellobiohydrolase encoding genes, cel7c was detected at the highest level and showed constitutive expression whereas variable transcript levels were detected for cel7a, cel7b and cel6 in the course of four-week spruce cultivation. The cellulolytic enzyme activities detected in the liquid cultures were consistent with the transcript levels. Interestingly, the selected lytic polysaccharide monooxygenase (LPMO) encoding genes were expressed in both cultures, but showed different transcription patterns on wood compared to those in submerged microcrystalline cellulose cultures. On spruce wood, higher transcript levels were detected for the lpmos carrying cellulose binding module (CBM) than for the lpmos without CBMs. In both cultures, the expression levels of the lpmo genes were generally higher than the levels of cellobiose dehydrogenase (CDH) encoding genes. Based on the results of this work, the oxidative cellulose cleaving enzymes of D. squalens have essential role in cellulose degrading machinery of the fungus.

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.

Decolorization of Orange G and Remazol Brilliant Blue R by the white rot fungus Dichomitus squalens: Toxicological evaluation and morphological study

Dichomitus squalens efficiently decolorized both Orange G and Remazol Brilliant Blue R (RBBR) at concentrations of 0.5 g l −1 and 3 g l −1 in static and shaken culture and also on solid medium within 14 d. The presence of the dyes in the culture medium mostly caused a decrease in biomass production and in growth rate, which was more significant in the case of RBBR. After 14 d of cultivation, electron microscopy showed substantial morphological changes in mycelia of D. squalens growing in media containing dyes. The hyphae deformations were more intensively manifested in solid media than in liquid culture. In all the cases, the morphological changes were more prominent in the presence of RBBR. Higher concentrations of both dyes brought about more intensive changes. The toxicity of synthetic dyes Orange G and RBBR was tested using a bioassay based on the growth inhibition of duckweed Lemna minor. Two endpoints such as the number of fronds and their weight were studied during the bioassay. The results showed higher toxicity of RBBR than that of Orange G. The toxicity of both dyes decreased after the decolorization process.

Synthetic dye decolorization capacity of white rot fungus Dichomitus squalens

The ability to decolorize eight chemically different synthetic dyes (Orange G, Amaranth, Orange I, Remazol Brilliant Blue R (RBBR), Cu-phthalocyanin, Poly R-478, Malachite Green and Crystal Violet) by the white rot fungus Dichomitus squalens was evaluated on agar plates. The fungus showed high decolorization capacity and was able to decolorize all dyes tested, but not to the same extent. Some of the dyes did not limit the decolorization capacity of the strain tested even at a concentration of 2 g/l. The presence of the dyes in solid media reduced the mycelial growth rate of D. squalens; a positive correlation was found between the growth rate and the decolorization ability. Decolorization of Orange G and RBBR was studied also in liquid culture, where both dyes caused an enhancement of ligninolytic enzyme and overall hydrogen peroxide production and a decrease of biomass production. RBBR was removed to a higher extent than Orange G.

Fermentation of wheat straw xylan by the white-rot fungus Dichomitus squalens

The pattern of degradation of purified wheat straw xylan by submerged cultures of the ligninolytic white-rot fungus Dichomitus squalens has been determined. After a 22-h lag period, extracellular xylanase and protease activities developed during the first phase of secretion, the exponential growth phase. A rapid drop in the degree of polymerization of the soluble xylan was observed, corresponding to the release of xylobiose, followed by the release of xylotriose and xylose. After 70 h cultivation, soluble sugars were almost exhausted; laminarinase activities were secreted, followed by endoxylanases, β-xylosidases, CM-cellulases, and an α-L-arabinofuranosidase. All the endoxylanases exhibited β-xylosidase activity. No α-D-glucuronidase could be determined. Accumulation of an undegradable soluble polymer, highly substituted by glucuronic acid residues, was observed. Since no free arabinose or free glucuronic acid could be detected in the culture medium, it is suggested that a progressive increase of unsubstituted xylose units arises from preferential attack on the more highly substituted regions of the xylan backbone by endoxylanases.

Production of extracellular enzyme by the white-rot fungus Dichomitus squalens in cellulose-containing liquid culture

The white-rot fungus Dichomitus squalens has been found to be a potent endo-1,4-β- d-glucanase producer when cultivated on crystalline cellulose in the presence of 0.2% proteose peptone. Several activities were detected in culture supernatant, including endo-1,4-β- d-glucanase [1,4-(1,3;1,4)-β- d-glucan 4-glucanohydrolase, EC 3.2.1.4], d-xylanase (endo-1,3-β- d-xylanase, 1,3-β- d-xylan xylanohydrolase, EC 3.2.1.32), exopoly- d-galacturonase [poly(1,4-α- d-galacturonide) galacturonohydrolase, EC 3.2.1.67], cellobiose dehydrogenase [cellobiose:quinone oxidoreductase, see d-glucose dehydrogenase (acceptor), d-glucose:(acceptor) 1-oxidoreductase, EC 1.1.99.10], β- d-glucosidase (β- d-glucoside glucohydrolase, EC 3.2.1.21) and exo-cellobiohydrolase (1,4-β- d-glucan cellobiohydrolase, EC 3.2.1.91), using 4-nitrophenyl-β- d-cellobioside as a substrate. A crude enzyme preparation was obtained by ammonium sulphate (80% saturation) precipitation of soluble protein. In spite of the presence of both endo- and exo-β- d-glucanase activities, degradation of crystalline cellulose by the enzymic preparation remained weak.

Purification and properties of two enzymes from Dichomitus squalens which exhibit both cellobiohydrolase and xylanase activity

Two fractions (Ex I and II), exhibiting activity towards p-nitrophenyl β-cellobioside (pNPC) have been isolated by chromatofocusing of the proteins obtained from the supernatant solution of a cellulose-containing culture of the white-rot fungus Dichomitus squalens. They were further purified up to 16.0- and 14.2-fold by chromatography on Phenyl-Sepharose CL-4B and ion-exchange chromatography on DEAE-Trisacryl. Each purified enzyme gave a single peak for protein and activity on chromatography on Ultrogel AcA-54 and a single protein band in disc gel electrophoresis, in the absence and presence of sodium dodecyl sulphate, and on isoelectrofocusing. The mol. wts. of Ex I and II were 39,000 and 36,000, respectively, and their isoelectric points were 4.6 and 4.5, respectively. Maximum activity towards pNPC was shown at pH 5.0 and 60°, and each enzyme was stable over the pH range 4.0–8.0, and up to 70° and 60° for Ex I and II, respectively. The enzymes cleaved pNPC, released mainly cellobiose from cellulose, were especially active towards xylan and o-nitrophenyl β- d-xylopyranoside, and exhibited strong transglycosylating activities.

Production, purification, and properties of an α- l-arabinofuranosidase from Cihomitus squalens

A white-rot fungus Dichomitus squalens, when grown on 1% wheat-straw glucuronoarabinoxylan under aerated submerged conditions, secreted an α- l-arabinofuranosidase (4.3 nkat/mL). The enzyme was purified 70-fold by ammonium sulfate precipitation, chromatofocusing on PBE 94, gel filtration on Ultrogel AcA 54, rechromatofocusing on PBE 94, and lectin affinity chromatography on Concanavalin A-Ultrogel. The enzyme is a glycoprotein having a molecular weight of 60,000 and a pI of 5.1. The enzyme exhibited maximal activity at pH 3.5 and at 60°, and was fully inactivated within 30 min at 70°. The K m value for p-nitrophenyl α- l-arabinofuranoside was 1.64m m. The α- l-arabinofuranosidase liberated arabinose from sugar-beet arabinan, wheat-straw and oat-spelt arabinoxylans, and wheat-bran heteroxylan, and was inactive towards gum arabic.

Purification and partial characterisation of three endo-glucanases from dichomitus squalens

Three endo-glucanases (En I-III) were obtained by chromatofocusing fractionation of a culture supernatant from the white-rot fungus Dichomitus squalens. They were purified further on Phenyl-Sepharose CL-4B, DEAE-Trisacryl, and Ultrogel AcA 54; ∼21-, ∼16-, and ∼9-fold purifications were obtained for En I-III, respectively. The enzymes appeared as homogeneous proteins on disc gel electrophoresis with and without SDS (sodium dodecyl sulphate), and on isoelectric focusing; the respective mol. wts. were 42,000, 56,000, and 47,000, and the isoelectric points 4.8, 4.3, and 4.1. Optimum conditions for the hydrolysis of CM-cellulose were pH 4.8 and 55° for each enzyme, and each was stable over the pH range 4.0–8.5 but inactivated completely within 30 min at 70°. None of the purified enzymes exhibited β- d-glucosidase or cellobiohydrolase activity, but En II was weakly active towards laminaran and xylan. En I and En II acted more randomly on CM-cellulose than did En III. Cellotetraose was degraded by each endo-glucanase, whereas only En III could hydrolyse cellotriose.