Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in <Emphasis Type="Italic">Phlebia tremellosa</Emphasis>

A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.     

Analyses of the Temporal Dynamics of Fungal Communities Colonizing the Healthy Wood Tissues of Esca Leaf-Symptomatic and Asymptomatic Vines

Esca, a Grapevine Trunk Disease (GTD), is of major concern for viticulture worldwide. Our study compares the fungal communities that inhabit the wood tissues of vines that expressed or not foliar esca-symptoms. The trunk and rootstock tissues were apparently healthy, whether the 10 year-old plants were symptomatic or not. The only difference was in the cordon, which contained white rot, a typical form of esca, in 79% of symptomatic plants. Observations over a period of one year using a fingerprint method, Single Strand Conformation Polymorphism (SSCP), and the ITS-DNA sequencing of cultivable fungi, showed that shifts occurred in the fungal communities colonizing the healthy wood tissues. However, whatever the sampling time, spring, summer, autumn or winter, the fungi colonizing the healthy tissues of asymptomatic or symptomatic plants were not significantly different. Forty-eight genera were isolated, with species of Hypocreaceae and Botryosphaeriaceae being the most abundant species. Diverse fungal assemblages, made up of potentially plant-pathogenic and -protective fungi, colonized these non-necrotic tissues. Some fungi, possibly involved in GTD, inhabited the non-necrotic wood of young plants, but no increase in necrosis areas was observed over the one-year period.     

Manganese Peroxidase, Produced by Trametes versicolor during Pulp Bleaching, Demethylates and Delignifies Kraft Pulp

Previous work has shown that Trametes (Coriolus) versicolor bleaches kraft pulp brownstock with the concomitant release of methanol. In this work, the fungus is shown to produce both laccase and manganese peroxidase (MnP) but not lignin peroxidase during pulp bleaching. MnP production was enhanced by the presence of pulp and/or Mn(II) ions. The maximum level of secreted MnP was coincident with the maximum rate of fungal bleaching. Culture filtrates isolated from bleaching cultures produced Mn(II)- and hydrogen peroxide-dependent pulp demethylation and delignification. Laccase and MnP were separated by ion-exchange chromatography. Purified MnP alone produced most of the demethylation and delignification exhibited by the culture filtrates. On the basis of the methanol released and the total and phenolic methoxyl contents of the pulp, it appears that MnP shows a preference for the oxidation of phenolic lignin substructures. The extensive increase in brightness observed in the fungus-treated pulp was not found with MnP alone. Therefore, either the MnP effect must be optimized or other enzymes or compounds from the fungus are also required for brightening.     

Major changes in grapevine wood microbiota are associated with the onset of esca,a devastating trunk disease

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%–90% and P. chlamydospora: 5%–15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.     

The selective visualization of lignin peroxidase,manganese peroxidase and laccase,produced by white rot fungi on solid media

A visual method for the selective screening of lignin degrading enzymes, produced by white rot fungi (WRF), was investigated by the addition of coloring additives to solid media. Of the additives used in the enzyme production media, guaiacol and RBBR could be used for the detection of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. Syringaldazine and Acid Red 264 were able for the detection of both the MnP and laccase, and the LiP and laccase, respectively, and a combination of these two additives was able to detect each of the ligninases produced by the WRF on solid media.     

Effect of phenolic acids on manganese peroxidase-dependent peroxidation of linoleic acid and degradation of a non-phenolic lignin model compound

The influence of aromatic phenolic and non-phenolic acids on manganese peroxidase (MnP)-dependent peroxidation of linoleic acid, and oxidation of a non-phenolic lignin model compound (LMC) was studied. Phenolic compounds inhibited both the MnP-dependent lipid peroxidation (LPO) and non-phenolic LMC degradation in the system. The antioxidant activity of the aromatic compounds in the enzymatic system with MnP-dependent LPO depends on the presence of the phenolic hydroxyl groups attached to the aromatic ring structure, the methoxylation of the hydroxyl group in the ortho position in diphenolics, and number of carbon atoms in the side chain. Natural phenolic compounds inhibit the oxidation of non-phenolic lignin in the system based on MnP-mediated LPO, but do not prevent it. This result indicates that MnP-mediated LPO may play an important role in lignin degradation even in the presence of the phenolic antioxidant compounds, and supports the possibility of the involvement of LPO in the degradation of lignin in wood.     

Molecular Evolution and Diversity of Lignin Degrading Heme Peroxidases in the Agaricomycetes

The plant and microbial peroxidase superfamily encompasses three classes of related protein families. Class I includes intracellular peroxidases of prokaryotic origin, class II includes secretory fungal peroxidases, including the lignin degrading enzymes manganese peroxidase (MnP), lignin peroxidase (LiP), and versatile peroxidase (VP), and class III includes the secretory plant peroxidases. Here, we present phylogenetic analyses using maximum parsimony and Bayesian methods that address the origin and diversification of class II peroxidases. Higher-level analyses used published full-length sequences from all members of the plant and microbial peroxidase superfamily, while lower-level analyses used class II sequences only, including 43 new sequences generated from Agaricomycetes (mushroom-forming fungi and relatives). The distribution of confirmed and proposed catalytic sites for manganese and aromatic compounds in class II peroxidases, including residues supposedly involved in three different long range electron transfer pathways, was interpreted in the context of phylogenies from the lower-level analyses. The higher-level analyses suggest that class II sequences constitute a monophyletic gene family within the plant and microbial peroxidase superfamily, and that they have diversified extensively in the basidiomycetes. Peroxidases of unknown function from the ascomycete Magnaporthe grisea were found to be the closest relatives of class II sequences and were selected to root class II sequences in the lower-level analyses. LiPs evidently arose only once in the Polyporales, which harbors many white-rot taxa, whereas MnPs and VPs are more widespread and may have multiple origins. Our study includes the first reports of partial sequences for MnPs in the Hymenochaetales and Corticiales.     

Mycoremediation (bioremediation with fungi) – growing mushrooms to clean the earth

Abstract

Some of the prospects of using fungi, principally white-rot fungi, for cleaning contaminated land are surveyed. That white-rot fungi are so effective in degrading a wide range of organic molecules is due to their release of extra-cellular lignin-modifying enzymes, with a low substrate-specificity, so they can act upon various molecules that are broadly similar to lignin. The enzymes present in the system employed for degrading lignin include lignin-peroxidase (LiP), manganese peroxidase (MnP), various H₂O₂ producing enzymes and laccase. The degradation can be augmented by adding carbon sources such as sawdust, straw and corn cob at polluted sites.     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.     

高效木质素降解菌的筛选及其对玉米秸秆的降解效果

本研究利用愈创木酚和苯胺蓝固体培养基对菌株进行初筛,利用形态学和分子生物学对筛选出的菌株进行鉴定,以黄孢原毛平革菌Phanerochaete chrysosporium CGMCC 5.0776为对照,利用其对玉米秸秆进行预处理并测定木质素和纤维素的降解率,测定筛选菌株在预处理玉米秸秆过程中漆酶、锰过氧化物酶（manganese peroxidase,MnP）和木质素过氧化物酶（lignin peroxidase,LiP）活性。结果表明:利用愈创木酚和苯胺蓝固体培养基,从16株白腐真菌菌株中筛选出2株具有较高漆酶或MnP活性的菌株,鉴定其为桦栓孔菌Trametes betulina (L.) Pilát（ZT-153）和亚黑管孔菌Bjerkandera fumosa (Pers.) P. Karst.（ZT-307）,测定T. betulina ZT-153和B. fumosa ZT-307对玉米秸秆酸不溶木质素降解效率分别为13.60%和21.87%,较对照P. chrysosporium CGMCC 5.0776高1.58%和9.85%,对纤维素的降解率较低,分别为4.10%和4.50%。2株菌株在预处理玉米秸秆过程中,T. betulina ZT-153表现出漆酶和MnP活性,B. fumosa ZT-307只表现出LiP活性。其中B. fumosa ZT-307对玉米秸秆酸不溶木质素的降解效率最高,在秸秆资源的综合利用方面具有较好的潜力和应用前景。     

Identification of a specific manganese peroxidase among ligninolytic enzymes secreted by Phanerochaete chrysosporium during wood decay

The specific enzymes associated with lignin degradation in solid lignocellulosic substrates have not been identified. Therefore, we examined extracts of cultures of Phanerochaete chrysosporium that were degrading a mechanical pulp of aspen wood. Western blot (immunoblot) analyses of the partially purified protein revealed lignin peroxidase, manganese-dependent peroxidase (MnP), and glyoxal oxidase. The dominant peroxidase, an isoenzyme of MnP (pI 4.9), was isolated, and its N-terminal amino acid sequence and amino acid composition were determined. The results reveal both similarities to and differences from the deduced amino acid sequences from cDNA clones of dominant MnP isoenzymes from liquid cultures. Our results suggest, therefore, that the ligninolytic-enzyme-encoding genes that are expressed during solid substrate degradation differ from those expressed in liquid culture or are allelic variants of their liquid culture counterparts. In addition to lignin peroxidase, MnP, and glyoxal oxidase, xylanase and protease activities were present in the extracts of the degrading pulp.     

Biodelignification of rice straw by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in the presence of dirhamnolipid

Lignin degradation by white-rot fungi has received considerable attention as a means for reducing accumulation of lignocellulosic wastes in the environment. The stimulatory effect of surfactants on fungal lignocellulose bioconversion also has attracted wide interest. In this study the influence of dirhamnolipid biosurfactant on biodegradation of rice straw by Phanerochaete chrysosporium was investigated. It was shown that the biodelignification process of rice straw can be significantly enhanced by the presence of dirhamnolipid biosurfactant. In particular, the dirhamnolipid at the concentration of 0.007% increased the peak activity of lignin peroxidase (LiP) by 86% without affecting the manganese peroxidase (MnP) activity. The water-soluble organic carbon (WSOC) contents in the straw substrates as well as the microbial growth and activity were effectively improved by dirhamnolipid, while the degradation rate of lignin increased by 54% with dirhamnolipid of 0.007%. Observed chemical structural and morphological changes showed that the straw substrates were delignified in the presence of dirhamnolipid with the formation of terrace-like fragments separated from the inner cellular fibers and the release of simple compounds. Variation partitioning analysis revealed that the dirhamnolipid addition induced a significant straw biodelignification which explained 22.1% (P = 0.013) of the variance.     

Identification of a specific manganese peroxidase among ligninolytic enzymes secreted by Phanerochaete chrysosporium during wood decay.

The specific enzymes associated with lignin degradation in solid lignocellulosic substrates have not been identified. Therefore, we examined extracts of cultures of Phanerochaete chrysosporium that were degrading a mechanical pulp of aspen wood. Western blot (immunoblot) analyses of the partially purified protein revealed lignin peroxidase, manganese-dependent peroxidase (MnP), and glyoxal oxidase. The dominant peroxidase, an isoenzyme of MnP (pI 4.9), was isolated, and its N-terminal amino acid sequence and amino acid composition were determined. The results reveal both similarities to and differences from the deduced amino acid sequences from cDNA clones of dominant MnP isoenzymes from liquid cultures. Our results suggest, therefore, that the ligninolytic-enzyme-encoding genes that are expressed during solid substrate degradation differ from those expressed in liquid culture or are allelic variants of their liquid culture counterparts. In addition to lignin peroxidase, MnP, and glyoxal oxidase, xylanase and protease activities were present in the extracts of the degrading pulp.     

Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi

The ligninolytic system of white rot fungi is primarily composed of lignin peroxidase, manganese peroxidase (MnP) and laccase. The present work was carried out to determine the best culture conditions for production of MnP and its activity in the relatively little-explored cultures of Dichomitus squalens, Irpex flavus and Polyporus sanguineus, as compared with conditions for Phanerochaete chrysosporium and Coriolus versicolor. Studies on enzyme production under different nutritional conditions revealed veratryl alcohol, guaiacol, Reax 80 and Polyfon H to be excellent MnP inducers. Electronic Publication     

Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Role of laccase in lignin degradation by white-rot fungi

Abstract Laccase is commonly found in white-rot fungi and catalyses the abstraction of one electron from the phenolic hydroxyl group to polymerize or depolymerize lignin model compounds. Laccase degrades both β-1 and β-O-4 dimers via C _α - C _β cleavage, C _α oxidation and alkyl-aryl cleavage. Also, aromatic ring cleavage may be detected following the action of laccase. Laccase can also oxidize non-phenolic compounds when primary mediators, such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate), are co-present. Laccase produces Mn(III) chelates which allow wood-decaying enzymes to penetrate wood cell walls. Laccase is considered to be capable of degrading lignin together with lignin peroxidase and manganese peroxidase.     

Simple and easy method for the determination of fungal growth and decolourative capacity in solid media

A technique was developed for studying the biodegradative ability of white rot fungi in different solid media. This technique enables the gravimetric determination of fungal growth (increase of biomass) and the spectrometric measurement of fungal decolourization ability (both by the determination of the production of the extracellular enzyme manganese-dependent peroxidase (MnP) and by the rate of decolourization of dyes). Bjerkandera sp., strain BOS55, was grown in different solid media. Its growth rate, decolourization of solophenil blue 2BL (azoic dye), neutral red (eurhodin dye), methyl green and crystal violet (triphenylmethane dyes) and the production of MnP were determined. Application of this technique enabled a spectrometric quantification of enzymatic activity. Assays indicate that greater amounts of MnP were present in agar plate cultures of Bjerkandera sp. than in liquid cultures.     

Fungal Decomposition of Natural Aromatic Structures and Xenobiotics: A Review

The review deals with transformation of natural and synthetic aromatic compounds by fungi (causative agents of white rot, brown rot, and soft rot, as well as soil filamentous fungi). Major enzyme types involved in the transformation of lignin and aromatic xenobiotics are discussed, with emphasis on activity regulation under the conditions of secondary metabolism and oxidative stress. Coupling of systems degrading polysaccharides and lignin and non-phenolic lignin structures (without the involvement of lignin peroxidase) is analyzed, together with nonenzymatic mechanisms involving lipoperoxide free radicals, cation radicals, quinoid mediators, or transition metal ions. Metabolic pathways resulting in the formation of aromatic and haloaromatic compounds in fungi are described. Consideration is given to the mechanisms of fungal adaptation to aromatic xenobiotics.     

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

Generation of a transformant showing higher manganese peroxidase (Mnp) activity by overexpression of Mnp gene in Trametes versicolor

Trametes versicolor has a lignin degrading enzyme system, which is also involved in the degradation of diverse recalcitrant compounds. Manganese-dependent peroxidase (MnP) is one of the lignin degrading enzymes in T. versicolor. In this study, a cDNA clone of a putative MnP-coding gene was cloned and transferred into an expression vector (pBARGPE1) carrying a phosphinothricin resistance gene (bar) as a selectable marker to yield the expression vector, pBARTvMnP2. Transformants were generated through genetic transformation using pBARTvMnP2. The genomic integration of the MnP clone was confirmed by PCR with bar-specific primers. One transformant showed higher enzyme activity than the recipient strain did, and was genetically stable even after 10 consecutive transfers on non-selective medium.