Response of Trametes hirsuta to hexavalent chromium promotes laccase-mediated decolorization of reactive black 5.

The recalcitrant azo dyes combined with heavy metals constitute a major challenge for the bioremediation of industrial effluents. This study aimed to investigate the effect and mechanism of action of a white-rot fungus Trametes hirsuta TH315 on the simultaneous removal of hexavalent chromium [Cr(VI)] and azo dye (Reactive Black 5, RB5). Here, this study discovered that toxic Cr(VI) (1 mM) greatly promoted RB5 decolorization (from 57.15% to 83.65%) by white-rot fungus Trametes hirsuta with high Cr(VI)-reducing ability (>96%), resulting in the simultaneous removal of co-contaminants. On the basis of transcriptomic and biochemical analysis, our study revealed that the oxidative stress in co-contaminants mainly caused by Cr(VI), and a number of dehydrogenases and oxidases showed up-regulation in response to Cr(VI) stress. It was noteworthy that the oxidative stress caused by Cr(VI) in co-contaminants can both significantly induce glutathione S-transferase and laccase expression. Glutathione S-transferase potentially involved in antioxidation against Cr(VI) stress. Laccase was found to play a key role in RB5 decolorization by T. hirsuta. These results suggested that the simultaneous removal of co-contaminants by T. hirsuta could be achieved with Cr(VI) exposure. Overall, the elucidation of the molecular basis in details will help to advance the general knowledge about the fungus by facing harsh environments, and put forward a further possible application of fungi on environmental remediation.

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.

Elimination of fungicides in biopurification systems: Effect of fungal bioaugmentation on removal performance and microbial community structure.

Bioaugmentation with ligninolytic fungi represents a potential way to improve the performance of biomixtures used in biopurification systems for the treatment of pesticide-containing agricultural wastewater. The fungus Trametes versicolor was employed in the bioaugmentation of a biomixture to be used in the simultaneous removal of seven fungicides. Liquid cultures of the fungus were able to remove tebuconazole, while no evidence of carbendazim, metalaxyl and triadimenol depletion was found. When applied in the biomixture, the bioaugmented matrix failed to remove all the triazole fungicides (including tebuconazole) under the assayed conditions, but was efficient to eliminate carbendazim, edifenphos and metalaxyl (the latter only after a second pesticide application). The re-addition of pesticides markedly increased the elimination of carbendazim and metalaxyl; nonetheless, no clear enhancement of the biomixture performance could be ascribed to fungal bioaugmentation, not even after the re-inoculation of fungal biomass. Detoxification efficiently took place in the biomixture (9 d after pesticide applications) according to acute tests on Daphnia magna. DGGE-analysis revealed only moderate time-divergence in bacterial and fungal communities, and a weak establishment of T. versicolor in the matrix. Data suggest that the non-bioaugmented biomixture is useful for the treatment of fungicides other than triazoles.

Induction of wheat straw delignification by Trametes species.

Wheat straw is the major crop residue in European countries which makes it the most promising material for bioconversion into biofuels. However, cellulose and hemicellulose are protected with lignin, so delignification is an inevitable phase in lignocellulose processing. The organisms predominantly responsible for its degradation are white-rot fungi and among them Trametes species represent promising degraders due to a well-developed ligninolytic enzyme system. Although numerous studies have confirmed that low molecular weight compounds can induce the production and activity of ligninolytic enzymes it is not clear how this reflects on the extent of delignification. The aim of the study was to assess the capacity of p-anisidine and veratryl alcohol to induce the production and activity of Mn-oxidizing peroxidases and laccases, and wheat straw delignification by six Trametes species. Significant inter- and intraspecific variations in activity and features of these enzymes were found, as well as differences in the potential of lignocellulose degradation in the presence or absence of inducers. Differences in the catalytic properties of synthesized enzyme isoforms strongly affected lignin degradation. Apart from enhanced lignin degradation, the addition of p-anisidine could significantly improve the selectivity of wheat straw ligninolysis, which was especially evident for T. hirsuta strains.

The GSTome Reflects the Chemical Environment of White-Rot Fungi.

White-rot fungi possess the unique ability to degrade and mineralize all the different components of wood. In other respects, wood durability, among other factors, is due to the presence of extractives that are potential antimicrobial molecules. To cope with these molecules, wood decay fungi have developed a complex detoxification network including glutathione transferases (GST). The interactions between GSTs from two white-rot fungi, Trametes versicolor and Phanerochaete chrysosporium, and an environmental library of wood extracts have been studied. The results demonstrate that the specificity of these interactions is closely related to the chemical composition of the extracts in accordance with the tree species and their localization inside the wood (sapwood vs heartwood vs knotwood). These data suggest that the fungal GSTome could reflect the chemical environment encountered by these fungi during wood degradation and could be a way to study their adaptation to their way of life.

Use of new endophytic fungi as pretreatment to enhance enzymatic saccharification of Eucalyptus globulus.

New endophytic fungi are assessed for the first time as pretreatment to enhance saccharification of Eucalyptus globulus wood. The fungi are all laccase-producing ascomycetes and were isolated from eucalyptus trees in Spain. After five endophytes had been assayed alone or in combination with white-rot fungus Trametes sp. I-62, three were pre-selected. To improve sugar production, an autohydrolysis pretreatment was performed before or after fungal treatment. Pretreatment increased sugar production 2.7 times compared to non-pretreated wood. When fungal and autohydrolysis pretreatments were combined, a synergistic increase in saccharification was observed in all cases. Endophytic fungi Ulocladium sp. and Hormonema sp. produced greater enhancements in saccharification than Trametes sp. I-62 (increase in sugar yields of 8.5, 8.0 and 6.0 times, respectively), demonstrating the high potential of these new endophytic fungi for saccharification enhancement.

Potential of extracellular enzymes from Trametes versicolor F21a in Microcystis spp. degradation.

Studies have shown that microorganisms may be used to eliminate cyanobacteria in aquatic environments. The present study showed that the white-rot fungus Trametes versicolor F21a could degrade Microcystis aeruginosa. After T. versicolor F21a and Microcystis spp. were co-incubated for 60h, >96% of Microcystis spp. cells were degraded by T. versicolor F21a. The activities of extracellular enzymes showed that cellulase, ß-glucosidase, protease, and laccase were vital to Microcystis spp. degradation in the early stage (0h to 24h), while ß-glucosidase, protease, laccase, and manganese peroxidase in the late stage (24h to 60h). The positive and significant correlation of the degradation rate with these enzyme activities indicated that these enzymes were involved in the degradation rate of Microcystis spp. cells at different phases. It suggested that the extracellular enzymes released by T. versicolor F21a might be vital to Microcystis spp. degradation. The results of this study may be used to develop alternative microbial control agents for cyanobacterial control.

Degradation of pharmaceuticals from membrane biological reactor sludge with Trametes versicolor.

Emerging contaminants are a wide group of chemical products that are found at low concentrations in the environment. These contaminants can be either natural, e.g., estrogens, or synthetics, such as pesticides and pharmaceuticals, which can enter the environment through the water and sludge from wastewater treatment plants (WWTP). The growth of Trametes versicolor on membrane biological reactor (MBR) sludge in bioslurry systems at the Erlenmeyer scale was assessed and its capacity for removing pharmaceutical and personal care products (PPCPs) was evaluated. The ability of the fungus to remove hydrochlorothiazide (HZT) from liquid media cultures was initially assessed. Consequently, different bioslurry media (complete nutrient, glucose and no-nutrient addition) and conditions (sterile and non-sterile) were tested, and the removal of spiked HZT was monitored under each condition. The highest spiked HZT removal was assessed under non-sterile conditions without nutrient addition (93.2%). Finally, the removal assessment of a broad set of pharmaceuticals was performed in non-spiked bioslurry. Under non-sterile conditions, the fungus was able to completely degrade 12 out of the 28 drugs initially detected in the MBR sludge, achieving an overall degradation of 66.9%. Subsequent microbial analysis showed that the microbial diversity increased after 15 days of treatment, but there was still some T. versicolor in the bioslurry. Results showed that T. versicolor can be used to remove PPCPs in bioslurry systems under non-sterile conditions, without extra nutrients in the media, and in matrices as complex as an MBR sludge.

Proteomics investigation reveals cell death-associated proteins of basidiomycete fungus Trametes versicolor treated with Ferruginol.

Ferruginol has antifungal activity against wood-rot fungi (basidiomycetes). However, specific research on the antifungal mechanisms of ferruginol is scarce. Two-dimensional gel electrophoresis and fluorescent image analysis were employed to evaluate the differential protein expression of wood-rot fungus Trametes versicolor treated with or without ferruginol. Results from protein identification of tryptic peptides via liquid chromatography­electrospray ionization tandem mass spectrometry (LC­ESI-MS/MS) analyses revealed 17 protein assignments with differential expression. Downregulation of cytoskeleton ß-tubulin 3 indicates that ferruginol has potential to be used as a microtubule-disrupting agent. Downregulation of major facilitator superfamily (MFS)­multiple drug resistance (MDR) transporter and peroxiredoxin TSA1 were observed, suggesting reduction in self-defensive capabilities of T. versicolor. In addition, the proteins involved in polypeptide sorting and DNA repair were also downregulated, while heat shock proteins and autophagy-related protein 7 were upregulated. These observations reveal that such cellular dysfunction and damage caused by ferruginol lead to growth inhibition and autophagic cell death of fungi.

Huaier suppresses proliferation and induces apoptosis in human pulmonary cancer cells via upregulation of miR-26b-5p.

Various studies have reported that Huaier possesses anti-tumor effects. However, the mechanisms are not completely elucidated. Here, we found 66 differentially expressed miRNAs in Huaier-treated pulmonary adenocarcinoma A549 cells, with upregulation of miR-26b-5p. Transfection of A549 cells with miR-26b-5p mimic inhibited proliferation and induced apoptosis, while transfection of Huaier-treated A549 cells with a miR-26b-5p inhibitor reversed the effects of Huaier. EZH2 was verified as the target of miR-26b-5p. Thus, our findings indicate that Huaier might suppress proliferation and induce apoptosis in lung cancer cells via a miR-26b-5p-EZH2-mediated approach, which provides a new perspective for understanding the anti-tumor effects of Huaier.

Bacterial-fungal interactions enhance power generation in microbial fuel cells and drive dye decolourisation by an ex situ and in situ electro-Fenton process.

In this work, the potential for sustainable energy production from wastes has been exploited using a combination fungus-bacterium in microbial fuel cell (MFC) and electro-Fenton technology. The fungus Trametes versicolor was grown with Shewanella oneidensis so that the bacterium would use the networks of the fungus to transport the electrons to the anode. This system generated stable electricity that was enhanced when the electro-Fenton reactions occurred in the cathode chamber. This configuration reached a stable voltage of approximately 1000 mV. Thus, the dual benefits of the in situ-designed MFC electro-Fenton, the simultaneous dye decolourisation and the electricity generation, were demonstrated. Moreover, the generated power was effectively used to drive an ex situ electro-Fenton process in batch and continuous mode. This newly developed MFC fungus-bacterium with an in situ electro-Fenton system can ensure a high power output and a continuous degradation of organic pollutants.

Study of the physiological characteristics of the medicinal mushroom Trametes pubescens (higher Basidiomycetes) during the laccase-producing process.

The study of the physiological characteristics of the medicinal mushroom Trametes pubescens was conducted under submerged cultures, suggesting that the laccase activity was positively correlated with oxidative level and culture conditions. Mycelial biomass and laccase activity in medium I were higher than those in medium II, which indicated that laccase activity was correlated with mycelium growth. The enhancement in mycelial biomass presented the logarithmic increase at days 6-8 and the peak value on the day 12 after inoculation. During liquid cultivation, increases in the amounts of malondialdehyde, hydrogen peroxide, and ascorbic acid were observed. In addition, the higher activities of superoxide dismutase and total antioxidative capacity still could be detected during this period. However, better ability to restrain hydroxyl free radical and catalase had a negative influence on laccase activity. It was evident that the fungal strain T. pubescens was under oxidative stress during the laccase-producing process. When the concentrations of H2O2 and Fe2+ were 3 and 30 mmol/L, respectively, the laccase activity reached to its peak at 37.21 U/L after a 14-day incubation period. It was concluded that a relationship between laccase synthesis and antioxidative capability existed in fungal cells, which could be regulated by reactive oxygen.

Engineering and production of laccase from Trametes versicolor in the yeast Yarrowia lipolytica.

The lcc1 gene coding for the laccase from Trametes versicolor DSM11269 was cloned into the genome of Yarrowia lipolytica using either single or multiple integration sites. The levels of the recombinant laccase activity secreted in the culture media were 0.25 and 1 U ml(-1) for single and multiple integrations, respectively. The strain with a single integration was successfully used to express variant libraries which were screened on ABTS substrate. The strain encoding the double mutant L185P/Q214K (rM4A) showed a sixfold enhancement in secreted enzyme activity. The catalytic efficiency of the purified rM-4A laccase was respectively increased 2.4- and 2.8-fold towards ABTS and 2,6-dimethoxyphenol, compared to the rWT. Culture supernatants containing either rWT or rM-4A catalyzed the almost complete decolorization of an Amaranth solution (70 nMs(-1)). Taken together, our results open new perspectives for the use of Y. lipolytica as a molecular evolution platform to engineer laccases with improved properties.

Heat shock treatment improves Trametes versicolor laccase production.

An efficient heat shock strategy has been developed to improve laccase production in submerged Trametes versicolor cultures. The optimized heat shock strategy consists of subjecting T. versicolor mycelial pellets to three heat shock treatments at 45 °C for 45 min, starting at culture day 0, with a 24-h interval between treatments. Laccase production increased by more than 1.6-fold relative to the control in both flasks and a 5-L bioreactor because the expression of the laccase gene was enhanced by heat shock induction. The present work demonstrates that heat shock induction is a promising method because it both improves fungal laccase production and has a good potential in industrial application.

Improved biomass saccharification by Trichoderma reesei through heterologous expression of lacA gene from Trametes sp. AH28-2.

The Trametes sp. AH28-2 laccase gene lacA fused to cellobiohydrolase I signal peptide coding sequence was heterologously expressed in T. reesei. The lacA cDNA was under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter. Native PAGE analysis indicated that two transformants, L8 and L38, were able to secrete recombinant laccase A, and their laccase activities corresponding to ABTS oxidation reached 3.62 IUml(-1) and 1.50 IUml(-1) respectively. Most of the characteristics of the recombinant laccase were similar to those of the native enzyme. Reducing sugar yields of L8 and L38 obtained from saccharification of corn residue by crude enzyme increased by 31.3% and 71.6% respectively compared to the host strain. These results indicated that the engineering strains developed in this work could be potentially used for laccase production and tailoring cellulase properties with laccase proteins through genetic manipulation would be a feasible strategy to improve saccharification efficiency of biomass by cellulase preparation.

Combination of steam explosion and laccase-mediator treatments prior to Eucalyptus globulus kraft pulping.

The effect of a pretreatment consisting of steam explosion (SE) followed by a laccase mediator system (LMS) stage on Eucalyptus globulus kraft pulping has been evaluated and compared with fungal pretreatments. Pretreatment with SE and LMS was more efficient than pretreatments using Pycnoporus sanguineus and Trametes sp. I-62. Steam explosion not only improved the enzyme penetration into the wood chips and shortened the pulping process by 60%, but also extracted around 50% of the hemicelluloses which could be converted into value-added products. The optimal conditions for the LMS treatment were 3h, 3UA/g and 40°C. Compared to SE, the SE/LMS treatment yielded an increase in delignification of 13.9% without affecting pulp properties, provided a similar screened kraft yield, and reduced consumption of chemical reagents Na(2)S and NaOH by 11.5% and 6.3%, respectively. Therefore, SE/LMS is a promising pretreatment for converting the pulp mill into a forest bio-refinery.

Solid-phase treatment with the fungus Trametes versicolor substantially reduces pharmaceutical concentrations and toxicity from sewage sludge.

For safe biosolid-land-applying, sludge should be contaminant-free. However, it may contain important amounts of micropollutants, not removed in the wastewater-treatment-processes. An alternative treatment with the fungus Trametes versicolor was applied in sterile solid-phase systems consisting of sludge and a lignocellulosic substrate. Fungal colonization and activity were demonstrated during the process, according to monitoring of ergosterol, laccase activity and the naproxen-degradation test (ND24). Fourteen out of 43 analyzed pharmaceuticals were found in the raw sludge. After treatment, phenazone, bezafibrate, fenofibrate, cimetidine, clarithromycin, sulfamethazine and atenolol were completely removed, while removals between 42% and 80% were obtained for the remaining pharmaceuticals. Toxicological analyses (Daphnia magna, Vibrio fischeri and seed germination) showed an important reduction in sludge toxicity after treatment. Results suggest that a solid-phase treatment with T. versicolor may reduce the ecotoxicological impact of micropollutants present in sewage sludge. This is the first report of a fungal-approach for elimination of emerging pollutants from biosolids.

Microarray analysis of differential gene expression elicited in Trametes versicolor during interspecific mycelial interactions.

Trametes versicolor is an important white rot fungus of both industrial and ecological interest. Saprotrophic basidiomycetes are the major decomposition agents in woodland ecosystems, and rarely form monospecific populations, therefore interspecific mycelial interactions continually occur. Interactions have different outcomes including replacement of one species by the other or deadlock. We have made subtractive cDNA libraries to enrich for genes that are expressed when T. versicolor interacts with another saprotrophic basidiomycete, Stereum gausapatum, an interaction that results in the replacement of the latter. Expressed sequence tags (ESTs) (1920) were used for microarray analysis, and their expression compared during interaction with three different fungi: S. gausapatum (replaced by T. versicolor), Bjerkandera adusta (deadlock) and Hypholoma fasciculare (replaced T. versicolor). Expression of significantly more probes changed in the interaction between T. versicolor and S. gausapatum or B. adusta compared to H. fasciculare, suggesting a relationship between interaction outcome and changes in gene expression.

Changes in oxidative enzyme activity during interspecific mycelial interactions involving the white-rot fungus Trametes versicolor.

Interspecific fungal antagonism leads to biochemical changes in competing mycelia, including up-regulation of oxidative enzymes. Laccase, manganese peroxidase (MnP), manganese-repressed peroxidase (MRP) and lignin peroxidase (LiP) gene expression and enzyme activity were compared during agar interactions between Trametes versicolor and five other wood decay fungi resulting in a range of interaction outcomes from deadlock to replacement of one fungus by another. Increased laccase and Mn-oxidising activities were detected at all interaction zones, but there were few changes in activity in regions away from the interaction zone in T. versicolor mycelia compared to self-pairings. Whilst no LiP activity was detected in any pairing, low level LiP gene expression was detected. MnP activity was detected but not expression of MnP genes; instead, MRP could explain the observed activity. No relationship was found between extent of enzyme activity increase and interaction outcome. Similarities between patterns of gene expression and enzyme activity are discussed.

[Effect of biological pretreatment with Trametes vesicolor on the enzymatic hydrolysis of softwood and hardwood].

We evaluated the effect of biological pretreatment with white rot fungus Trametes vesicolor on the enzymatic hydrolysis of two wood species, Chinese willow (Salix babylonica, hardwood) and China-fir (Cunninghamia lanceolata, softwood). The result indicated that the pretreated woods showed significant increases in the final conversion ratios of enzymatic hydrolysis (4.78-fold for hardwood and 4.02-fold for softwood). In order to understand the role of biological pretreatment we investigated the enzyme-substrate interactions. Biological pretreatment enhanced the substrate accessibility to cellulase but not always correlated with the initial conversion rate. However, the change of the conversion rate decreased dramatically with increased desorption values after biological pretreatment. Thus, the biological pretreatment slowed down the declines in conversion rates during enzymatic hydrolysis by reducing the irreversible adsorption of cellulase and then improved the enzymatic hydrolysis. Moreover, the decreases of the irreversible adsorption may be attributed to the partial lignin degradation and alteration in lignin structure after biological pretreatment.