White Rot Fungus Phanerochaete Chrysosporium Research Articles

Natural resistance of five woods to Phanerochaete chrysosporium degradation

This research evaluated the natural resistance of five woods to the white-rot wood-destroying fungus Phanerochaete chrysosporium under laboratory conditions and in nature. The studied species were Hymenaea stigonocarpa, Anadenanthera colubrina, Caesalpinia ferrea, Manilkara huberi and Delonix regia. The natural resistance to decay is one of the most important properties of wood, mainly assigned to lignin and extractives of wood. A. colubrina has the highest content of extractives and M. huberi the highest content of lignin; both are known as resistant to xylophagous organisms and were also most resistant to the tested fungus. C. ferrea has the lowest content of extractives and D. regia of lignin; both species did not inhibit the fungus Phanerochaete chrysosporium. H. stigonocarpa occupies an intermediate position in content of extractives and lignin as well in resistance to P. chrysosporium.

Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO 2) the recalcitrant fused-ring high molecular weight (⩾4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30 years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes ( Pc-pah1– Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3–5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493–547 aa) are structurally diverse and novel considering their low overall homology (12–23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.

Effects of different wavelengths of light on lignin peroxidase production by the white-rot fungi Phanerochaete chrysosporium grown in submerged cultures

In this study, the effects of different wavelengths of light (UV, blue, green, yellow, red) and white light on lignin peroxidase (LiP), protein, biomass and exo-polysaccharide production and glucose uptake by Phanerochaete chrysosporium BKM-F-1767 were determined. The experiments were conducted under aerated (CS) and oxygenated (RS) culture conditions. The results showed that only green light significantly increased maximum LiP production (by 20% and 27% in CS and RS cultures respectively). Green light also increased biomass production in oxygenated cultures (RS). Blue and UV light both significantly reduced maximum LiP activity. Yellow, red and white lights had mixed effects on culture properties. This is the first time that the effects of different wavelengths of light on lignin peroxidase production and other culture properties have been investigated. The novel findings may be important in improving the yield of lignin modifying enzymes for biomass conversion processes and understanding their regulation.

Involvement of protein kinase C in lignin peroxidase expression in oxygenated cultures of the white rot fungus Phanerochaete chrysosporium

Hydroxyl radicals (OH ) were previously hypothesized as the principal ROS triggering lip-H2 expression in liquid cultures of Phanerochaete chrysosporium, through signal transduction. We therefore focused on determining the relationship between protein kinase C (PKC), ROS and LIP-H2. Significantly lower PKC activity was measured in high-LIP-producing (oxygenated) vs. low-LIP-producing (aerated—grown with free exchange of atmospheric air) cultures. In oxygenated cultures, inactivation of PKC activity by calphostin C, staurosporin or H7 caused significant elevation in lip-H2 and also in MnSOD1 expression. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) caused the reverse effect. Significantly low levels of lip-H2 expression were detected when O 2 −, H 2O 2 or OH were scavenged by 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), pyruvate or dimethylsulfoxide (DMSO), whether the level of PKC was normal, stimulated or inactivated. In situ generation of OH , via addition of Fenton reagents to aerated cultures, reduced pkc expression. In contrast, OH scavenging stimulated pkc expression. This work suggests that due to high OH levels, fungal cells activate a complex defensive system which regulates the levels of Fenton components by repressing PKC and stimulating LIP, MnSOD1 and catalase.

Ligninase-mediated removal of 17β-estradiol from water in the presence of natural organic matter: Efficiency and pathways

Lignin peroxidase (LiP) is excreted by certain lignin-degrading fungi, such as white rot fungus Phanerochaete chrysosporium, in natural environments and is thus widely present in the natural environment. We have found in our earlier studies that LiP mediates effective reactions of a few natural and synthetic estrogens to form oligomeric products via radical coupling. We in particular examined the identity and property of the products resulting from 17β-estradiol (E2) in LiP-mediated oxidative coupling reactions, and the results suggest that such reactions hold great potential in water/wastewater treatment to remove E2 and estrogenicity. Herein, we report a further investigation to postulate possible reaction pathways of E2 with the assistance of ab initio molecular modeling and to more systematically examine the reaction behavior of E2 under sequenced reaction conditions and in systems containing natural organic matter (NOM) at different levels. Our molecular modeling suggested the coupling of E2 likely proceeded via covalent bonding between two E2 radicals at their unsubstituted carbons in phenolic rings. Results obtained from sequenced reagent feed experiments revealed that the coupling products tended to be consumed with increment enzyme treatments, suggesting that most E2 coupling products may still be LiP substrates that can undergo further coupling reactions under catalysis. Higher concentration of NOM present in the reaction system tended to reduce E2 transformation. NOM moieties seemed to couple to each other upon reaction with LiP, which was evidenced by the development of a characteristic absorbance band.

Novel evidence of cytochrome P450-catalyzed oxidation of phenanthrene in Phanerochaete chrysosporium under ligninolytic conditions

The presence of cytochrome P450 and P450-mediated phenanthrene oxidation in the white rot fungus Phanerochaete chrysosporium under ligninolytic condition was first demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (130pmolmg(-1) in the microsomal fraction) by phenanthrene. The microsomal P450 degraded phenanthrene with a NADPH-dependent activity of 0.44±0.02min(-1). One of major detectable metabolites of phenanthrene in the ligninolytic cultures and microsomal fractions was identified as phenanthrene trans-9,10-dihydrodiol. Piperonyl butoxide, a P450 inhibitor which had no effect on manganese peroxidase activity, significantly inhibited phenanthrene degradation and the trans-9,10-dihydrodiol formation in both intact cultures and microsomal fractions. Furthermore, phenanthrene was also efficiently degraded by the extracellular fraction with high manganese peroxidase activity. These results indicate important roles of both manganese peroxidase and cytochrome P450 in phenanthrene metabolism by ligninolytic P. chrysosporium.

P450 Redox Enzymes in the White Rot Fungus Phanerochaete chrysosporium: Gene Transcription, Heterologous Expression, and Activity Analysis on the Purified Proteins

With an aim to understand the cytochrome P450 enzyme system in the white rot fungus Phanerochaete chrysosporium, here we report molecular characterization of its P450 redox proteins including the primary P450 oxidoreductase (POR) and two alternate P450 redox proteins cytochrome b5 (cyt b5) and cytochrome b5 reductase (cyt b5r) in terms of transcriptional regulation and heterologous expression. The transcript abundance followed the order POR>cyt b5r>cyt b5. Interestingly, the three genes showed an overall higher expression in the defined carbon-limited cultures with low nitrogen (LN) or high nitrogen (HN) versus the carbon-rich malt extract (ME) cultures. cDNA cloning and analysis revealed the following deduced protein characteristics: cyt b5 (238 amino acids, 25.38kDa) and cyt b5r (321 amino acids, 35.52kDa). Phylogenetic analysis revealed that the cloned cyt b5 belongs to a novel class of fungal cyt b5-like proteins. The two proteins cyt b5 and cyt b5r were heterologously expressed in E. coli and purified using affinity-based purification in an active form. The POR was heterologously expressed in Saccharomyces cerevisiae and was also purified in active form as evidenced by its cytochrome c reduction activity. This is the first report on cloning, heterologous expression, and purification of the alternate redox proteins cyt b5 and cyt b5r in E. coli and on yeast expression of POR from this model white rot fungus.

Ultrahigh (0.93 Å) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: Implications for the catalytic mechanism

Manganese peroxidase (MnP) is an extracellular heme enzyme produced by the lignin-degrading white-rot fungus Phanerochaete chrysosporium. MnP catalyzes the peroxide-dependent oxidation of Mn II to Mn III. The Mn III is released from the enzyme in complex with oxalate, enabling the oxalate–Mn III complex to serve as a diffusible redox mediator capable of oxidizing lignin, especially under the mediation of unsaturated fatty acids. One heme propionate and the side chains of Glu35, Glu39 and Asp179 have been identified as Mn II ligands in our previous crystal structures of native MnP. In our current work, new 0.93 Å and 1.05 Å crystal structures of MnP with and without bound Mn II, respectively, have been solved. This represents only the sixth structure of a protein of this size at 0.93 Å resolution. In addition, this is the first structure of a heme peroxidase from a eukaryotic organism at sub-Ångstrom resolution. These new structures reveal an ordering/disordering of the C-terminal loop, which is likely required for Mn binding and release. In addition, the catalytic Arg42 residue at the active site, normally thought to function only in the peroxide activation process, also undergoes ordering/disordering that is coupled to a transient H-bond with the Mn ligand, Glu39. Finally, these high-resolution structures also reveal the exact H atoms in several parts of the structure that are relevant to the catalytic mechanism.

Enzyme activities and decolourization of single and mixed azo dyes by the white-rot fungus Phanerochaete chrysosporium

Decolourization of two azo dyes, namely Direct Red–80 (DR–80) and Mordant Blue–9 (MB–9), by Phanerochaete chrysosporium was investigated both individually and in mixtures in batch shake flasks. Mixture experiments containing these dyes were performed at five different initial concentrations ranging from 10 to 200 mg l −1 each. While the results of dye decolourization in mixture indicated decolourization efficiencies of more than 92%, the efficiencies were more than 94% in the single dye system. During the experiments, enzyme activities of lignin peroxidase (LiP) and manganese peroxidase (MnP) were also analyzed. Maximum LiP and MnP activities of 136 U l −1 and 16 U l −1, respectively, were achieved after 24 h of the fungal culture for respective initial DR–80 concentrations of 10 mg l −1 and 150 mg l −1 in the single dye system. In case of MB–9, maximum enzyme activities were 118 U l −1 (LiP) and 34 U l −1 (MnP) at initial concentrations of 50 mg l −1 and 100 mg l −1, respectively. When the two dyes were added together to the culture, maximum LiP and MnP activities were found to be 139 U l −1 and 132 U l −1 respectively for an initial concentration combination of 50 mg l −1 DR–80 and 10 mg l −1 MB–9. These profiles of enzyme activities and dye decolourization by the fungus, in both single and mixed dye systems, suggest the fungal peroxidase enzymes play a strong role in dye decolourization.

Metabolism of mono- and dichloro-dibenzo-p-dioxins by Phanerochaete chrysosporium cytochromes P450

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min(-1)) and 2-MCDD (13 min(-1)) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichlorodibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzop-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.

Enhanced decolourization of Direct Red-80 dye by the white rot fungus Phanerochaete chrysosporium employing sequential design of experiments

Decolourization of Direct Red 80 (DR-80) by the white rot fungus Phanerochaete chrysosporium MTCC 787 was investigated employing sequential design of experiments. Media components for growing the white rot fungus were first screened using Plackett-Burman design and then optimized using response surface methodology (RSM), which resulted in enhancement in the efficiency of dye removal by the fungus. For determining the effect of media constituents on the dye removal, both percent dye decolourization and specific dye removal due to maximum enzyme activity were chosen as the responses from the experiments, and the media constituents glucose, veratryl alcohol, KH(2)PO(4), CaCl(2) and MgSO(4) were screened to be the most effective with P values less than 0.05. Central composite design (CCD) followed by RSM in the optimization study revealed the following optimum combinations of the screened media constituents: glucose, 11.9 g l(-1); veratryl alcohol, 12.03 mM; KH(2)PO(4), 23.08 g l(-1); CaCl(2), 2.4 g l(-1); MgSO(4), 10.47 g l(-1). At the optimum settings of the media constituents, complete dye decolourization (100% removal efficiency) and a maximum specific dye removal due to lignin peroxidase enzyme of 0.24 mg U(-1) by the white rot fungus were observed.

In vitro degradation of carbamazepine and diclofenac by crude lignin peroxidase

Carbamazepine and diclofenac were frequently detected in water bodies. In this study, crude lignin peroxidase, produced from a white rot fungus Phanerochaete chrysosporium, was studied on its in vitro degradation of both drugs. The influencing parameters were studied, including pH, the hydrogen peroxide concentration, veratryl alcohol and the temperature. It was found that LiP completely degraded diclofenac at pH 3.0–4.5 and 3–24 ppm H 2O 2 while the degradation efficiency of carbamazepine was mostly below 10%. The addition of veratryl alcohol and the increased temperature did not enhance the degradation of carbamazepine.

Atypical kinetics of cytochromes P450 catalysing 3'-hydroxylation of flavone from the white-rot fungus Phanerochaete chrysosporium

We cloned full-length cDNAs of 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium and successfully expressed 70 isoforms in Saccharomyces cerevisiae. To elucidate substrate specificity of P. chrysosporium P450s, we examined various substrates including steroid hormones, several drugs, flavonoids and polycyclic aromatic hydrocarbons using the recombinant S. cerevisiae cells. Of these P450s, two CYPs designated as PcCYP50c and PcCYP142c with 14% identity in their amino acid sequences catalyse 3'-hydroxylation of flavone and O-deethylation of 7-ethoxycoumarin. Kinetic data of both enzymes on both reactions fitted not to the Michaelis-Menten equation but to Hill's equation with a coefficient of 2, suggesting that two substrates bind to the active site. Molecular modelling of PcCYP50c and a docking study of flavone to its active site supported this hypothesis. The enzymatic properties of PcCYP50c and PcCYP142c resemble mammalian drug-metabolizing P450s, suggesting that their physiological roles are metabolism of xenobiotics. It is noted that these unique P. chrysosporium P450s have a potential for the production of useful flavonoids.

Induction of functional cytochrome P450 and its involvement in degradation of benzoic acid by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium has the largest cytochrome P450 contingent known to date in fungi, but the study on the function of these P450s is limited. In this study, induction of functional P450 in P. chrysosporium was first shown and P450-mediate degradation of benzoic acid was demonstrated in this fungus. Carbon monoxide difference spectra indicated significant induction of P450 by benzoic acid, m-chlorobenzoic acid, p-chlorobenzoic acid and n-hexane, and showed the effect of inducer concentration and nutrient condition on the induction of P450. The high contents of P450 in the microsomal fractions facilitated the study on the function of P450. While the n-hexane-induced P450 could not interact with benzoic acid, the microsomal P450 induced by benzoic acid produced type I substrate binding spectra upon the addition of benzoic acid. The benzoic acid degradation by the microsomal P450 was NADPH-dependent at a specific rate of 194 +/- 14 min(-1), and significantly inhibited by piperonyl butoxide (a P450 inhibitor). However, inhibition of benzoic acid degradation by piperonyl butoxide was slight or not detectable in the cultures of this fungus, suggesting presumable involvement of other enzyme in benzoic acid degradation. The extracellular ligninolytic enzymes, lignin peroxidase and manganese-dependent peroxidase, were not involved in initial metabolism of benzoic acid under the test conditions.

Enzyme activities during degradation of polycyclic aromatic hydrocarbons by white rot fungus Phanerochaete chrysosporium in soils

The degradation of three polycyclic aromatic hydrocarbons (PAHs), phenanthrene, pyrene and benzo[a]pyrene in soils by Phanerochaete chrysosporium, and the enzyme activities of lignin peroxidase (LiP) and manganese peroxidase (MnP) produced during degradation, were analyzed. The results showed that the 19-d percentage degradation ranged from 72.77 ± 1.39% to 25.50 ± 3.41% for the three compounds, and the maximum LiP and MnP activities ranged from 0.16 ± 0.005 to 0.05 ± 0.002 U g −1 and from 1.92 ± 0.03 to 0.54 ± 0.03 U g −1, respectively. Degradation percentage and enzyme activities both exhibited inverse relationships with the octanol/water partition coefficient ( K ow) of the compounds, indicating that LiP and MnP from P. chrysosporium may be the primary enzymes responsible for PAH degradation in soil. As the soil organic matter (SOM) content increased from 0.3% for Soil 1 to 19% for Soil 4, the 19-d degradation percentage of pyrene decreased from 66.20 ± 2.72% to 32.42 ± 1.05%, and correspondingly, the maximum of LiP and MnP activities increased from 0.05 ± 0.002 to 1.78 ± 0.15 U g −1 and from 0.34 ± 0.03 to 1.78 ± 0.15 U g −1, respectively. Hence, it is plausible to conclude that the P. chrysosporium appeared to degrade not only the PAHs with small molecular size but also the macromolecular SOM. When SOM differences are large, as in this study, SOM has greater influence on enzyme activity than low-level exotic pollutants.

Polychlorinated biphenyls fractioning in aqueous bioremediation assay with Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium is able to degrade PCBs in water and in soil. This study aims at estimating the fractioning of truly degraded PCBs, adsorbed PCBs and residual PCBs in an aqueous bioremediation assay with Phanerochaete chrysosporium. Di-, tri- tetra-, penta-, hexa-, hepta-chlorinated biphenyls (IUPAC numbers: PCB28, PCB52, PCB101, PCB118, PCB138, PCB153, PCB180) were extracted from 500 mL aqueous bioremediation assays using a liquid–liquid extraction with n-hexane. Analyses were performed by gas chromatography coupled to mass spectrometry. The study reveals that the adsorbed PCBs fraction ranges from 42% to 54%, whereas the degraded one ranges from 39% to 49%. No PCBs were detected in the residual water (limit of detection: 13 ng L−1).

Enzymatic properties of cytochrome P450 catalyzing 3′-hydroxylation of naringenin from the white-rot fungus Phanerochaete chrysosporium

We cloned full-length cDNAs of more than 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium, and successfully expressed 70 isoforms using a co-expression system of P. chrysosporium P450 and yeast NADPH-P450 reductase in Saccharomyces cerevisiae. Of these P450s, a microsomal P450 designated as PcCYP65a2 consists of 626 amino acid residues with a molecular mass of 68.3 kDa. Sequence alignment of PcCYP65a2 and human CYP1A2 revealed a unique structure of PcCYP65a2. Functional analysis of PcCYP65a2 using the recombinant S. cerevisiae cells demonstrated that this P450 catalyzes 3′-hydroxylation of naringenin to yield eriodictyol, which has various biological and pharmacological properties. In addition, the recombinant S. cerevisiae cells expressing PcCYP65a2 metabolized such polyaromatic compounds as dibenzo- p-dioxin (DD), 2-monochloroDD, biphenyl, and naphthalene. These results suggest that PcCYP65a2 is practically useful for both bioconversion and bioremediation.

Melanin synthesis by Sclerotinia sclerotiorum

We confirmed that the melanin produced by Sclerotinia sclerotiorum is a dihydroxynaphthalene (DHN). The specific DHN melanogenesis inhibitor test that uses tricyclazole at low levels (typically2–5 ppm) to cause a confirmatory appearance of soluble red-brown inhibition products does not work when analyzing melanin synthesis in the sclerotia of S. sclerotiorum. We demonstrated the presence of scytalone dehydratase, an enzyme specific to DHN melanogenesis, in melanized sclerotia and melanized nonsclerotial mycelia and observed formation of mycelial nonsclerotial melanin when the fungus was grown on the surface of sterilized dialysis membrane or in rich organic media. Nonsclerotial melanized hyphae in wild type and mutant strains showed the typical excretion of pigmented inhibition products of the DHN pathway in the presence of tricyclazole, and one of these products, 2-hydroxyjuglone, was identified by thin layer chromatography and spectroscopy. We report basic conditions for sclerotial melanin degradation by the white rot fungus Phanerochaete chrysosporium.

The first genome-level transcriptome of the wood-degrading fungus Phanerochaete chrysosporium grown on red oak

As part of an effort to determine all the gene products involved in wood degradation, we have performed massively parallel pyrosequencing on an expression library from the white rot fungus Phanerochaete chrysosporium grown in shallow stationary cultures with red oak as the carbon source. Approximately 48,000 high quality sequence tags (246 bp average length) were generated. 53% of the sequence tags aligned to 4,262 P. chrysosporium gene models, and an additional 18.5% of the tags reliably aligned to the P. chrysosporium genome providing evidence for 961 putative novel fragmented gene models. Due to their role in lignocellulose degradation, the secreted proteins were focused upon. Our results show that the four enzymes required for cellulose degradation: endocellulase, exocellulase CBHI, exocellulase CBHII, and beta-glucosidase are all produced. For hemicellulose degradation, not all known enzymes were produced, but endoxylanases, acetyl xylan esterases and mannosidases were detected. For lignin degradation, the role of peroxidases has been questioned; however, our results show that lignin peroxidase is highly expressed along with the H(2)O(2) generating enzyme, alcohol oxidase. The transcriptome snapshot reveals that H(2)O(2) generation and utilization are central in wood degradation. Our results also reveal new transcripts that encode extracellular proteins with no known function.

Biodegradation of the High Explosive Hexanitrohexaazaiso-wurtzitane (CL-20)

The aerobic biodegradability of the high explosive CL-20 by activated sludge and the white rot fungus Phanerochaete chrysosporium has been investigated. Although activated sludge is not effective in degrading CL-20 directly, it can mineralize the alkaline hydrolysis products. Phanerochaete chrysosporium degrades CL-20 in the presence of supplementary carbon and nitrogen sources. Biodegradation studies were conducted using various nutrient media under diverse conditions. Variables included the CL-20 concentration; levels of carbon (as glycerol) and ammonium sulfate and yeast extract as sources of nitrogen. Cultures that received CL-20 at the time of inoculation transformed CL-20 completely under all nutrient conditions studied. When CL-20 was added to pre-grown cultures, degradation was limited. The extent of mineralization was monitored by the 14CO2 time evolution; up to 51% mineralization was achieved when the fungus was incubated with [14C]-CL-20. The kinetics of CL-20 biodegradation by Phanerochaete chrysosporium follows the logistic kinetic growth model.