Improvement of ligninolytic properties by recombinant expression of glyoxal oxidase gene in hyper lignin-degrading fungus Phanerochaete sordida YK-624

Glyoxal oxidase (GLOX) is a source of the extracellular H₂O₂ required for the oxidation reactions catalyzed by the ligninolytic peroxidases. In the present study, the GLOX-encoding gene (glx) of Phanerochaete chrysosporium was cloned, and bee2 promoter of P. sordida YK-624 was used to drive the expression of glx. The expression plasmid was transformed into a P. sordida YK-624 uracil auxotrophic mutant (strain UV-64), and 16 clones were obtained as GLOX-introducing transformants. These transformants showed higher GLOX activities than wild-type P. sordida YK-624 and control transformants harboring marker plasmid. RT-PCR analysis indicated that the increased GLOX activity was associated with elevated recombinant glx expression. Moreover, these transformants showed higher ligninolytic activity than control transformants. These results suggest that the ligninolytic properties of white-rot fungi can be improved by recombinant expression of glx.     

Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624

Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 × 10⁷ protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.     

Bleaching of Hardwood Kraft Pulp with Manganese Peroxidase Secreted from Phanerochaete sordida YK-624

In vitro bleaching of an unbleached hardwood kraft pulp was performed with manganese peroxidase (MnP) from the fungus Phanerochaete sordida YK-624. When the kraft pulp was treated with partially purified MnP in the presence of MnSO₄, Tween 80, and sodium malonate with continuous addition of H₂O₂ at 37°C for 24 h, the pulp brightness increased by about 10 points and the kappa number decreased by about 6 points compared with untreated pulp. The pulp brightness was also increased by 43 points to 75.5% by multiple (six) treatments with MnP combined with alkaline extraction. Our results indicate that in vitro degradation of residual lignin in hardwood kraft pulp with MnP is possible.     

Dimerization of Bisphenol A by Hyper Lignin-Degrading Fungus Phanerochaete sordida YK-624 Under Ligninolytic Condition

Bisphenol A (BPA) was treated with hyper lignin-degrading fungus Phanerochaete sordida YK-624 under ligninolytic condition. After preculturing P. sordida YK-624 for 4 days, BPA (final concentration, 1 and 0.1 mM) was added to cultures. Both 1- and 0.1-mM BPA were effectively decreased within a 24-h treatment and two metabolites were detected. Two metabolites (5,5′-bis-[1-(4-hydroxy-phenyl)1-methyl-ethyl]-biphenyl-2,2′-diol and 4-(2-(4-hydroxy-phenyl) propan-2-yl)-2-(4-(2-(4-hydroxyphenyl) propan-2-yl) phenoxy)phenol) were identified by ESI–MS and NMR analysis. These results indicated that BPA was oxidized to BPA phenoxy radicals by ligninolytic enzymes and then dimerized at extracellular region.     

Trapping of 2,7-Dichlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-Dioxin in Aqueous Solution by Enzymatic Reaction of Fungal Manganese Peroxidase in the Presence of Polyunsaturated Fatty Acids

Abstract:In the presence of polyunsaturated fatty acids, including cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), 2,7-dichlorodibenzo-p-dioxin (DCDD) was treated with manganese peroxidase (MnP) from white rot basidiomycete Phanerochaete sordida YK-624. After incubation with MnP, DCDD could not be extracted from the reaction mixture with n-hexane and was trapped in the water layer. DCDD was released by alkalification of the water layer. DCDD was also trapped after treatment with lipoxidase, which produces hydroperoxides from unsaturated lipids. The amounts of thiobarbituric acid-reactive substances produced in the MnP reactions with three highly unsaturated fatty acids were higher than the amounts produced with three fatty acids with a lower degree of unsaturation. These results suggest that a DCDD-trapping compound may be produced by peroxidation of the polyunsaturated fatty acids.Received: 22 October 2002 / Accepted: 6 December 2002     

Enhancement of biodegradation of 2,7-dichlorodibenzo-p-dioxin by addition of fungal culture filtrate

The hydrophilicity of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), a model dioxin compound, increased when incubated with the culture filtrates of several strains of fungi. The possibility that the addition of these filtrates could enhance the biodegradation of 2,7-DCDD by the white-rot basidiomycetous fungus Phanerochaete sordida YK-624 was examined. The decrease of 2,7-DCDD after 3 weeks incubation in a YK-624 culture containing these filtrates was greater (30%) than that in the culture of YK-624 alone (15%). This is the first report describing the enhancement of dioxin decrease by the addition of a fungal filtrate.     

In Vitro Bleaching of Hardwood Kraft Pulp by Extracellular Enzymes Excreted from White Rot Fungi in a Cultivation System Using a Membrane Filter

To clarify the role of excreted extracellular enzymes during long-term incubation in a pulp biobleaching system with white rot fungi, we developed a cultivation system in which a membrane filter is used; this membrane filter can prevent direct contact between hyphae and kraft pulp, but allows extracellular enzymes to attack the kraft pulp. Phanerochaete sordida YK-624 brightened the pulp 21.4 points to 54.0% brightness after a 5-day in vitro treatment; this value was significantly higher than the values obtained with Phanerochaete chrysosporium and Coriolus versicolor after a 7-day treatment. Our results indicate that cell-free, membrane-filtered components from the in vitro bleaching system are capable of delignifying unbleached kraft pulp. Obvious candidates for filterable reagents capable of delignifying and bleaching kraft pulp are peroxidase and phenoloxidase proteins. The level of secreted manganese peroxidase activity in the filterable components was substantial during strain YK-624 in vitro bleaching. A positive correlation between the level of manganese peroxidase and brightening of the pulp was observed.     

Biotransformation of acetamiprid by the white-rot fungus Phanerochaete sordida YK-624

Acetamiprid (ACE) belongs to the neonicotinoid class of systemic broad-spectrum insecticides, which are the most highly effective and largest-selling insecticides worldwide for crop protection. As neonicotinoid insecticides persist in crops, biotransformation of these insecticides represents a promising approach for improving the safety of foods. Here, the elimination of ACE from a liquid medium by the white-rot fungus Phanerochaete sordida YK-624 was examined. Under ligninolytic and non-ligninolytic conditions, 45% and 30% of ACE were eliminated, respectively, after 15 days of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite identified in the culture supernatant suggested that ACE was N-demethylated to (E)-N ¹-[(6-chloro-3-pyridyl)-methyl]-N ²-cyano-acetamidine, which has a much lower toxicity than ACE. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the elimination of ACE. The elimination rate of ACE by P. sordida YK-624 was markedly reduced by the addition of either 0.01 or 0.1 mM PB to the culture medium. These results suggest that cytochrome P450 plays an important role in the N-demethylation of ACE by P. sordida YK-624.     

Removal of diclofenac and mefenamic acid by the white rot fungus <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> YK-624 and identification of their metabolites after fungal transformation

The non-steroidal anti-inflammatory drugs diclofenac (DCF) and mefenamic acid (MFA) were treated with the white rot fungus Phanerochaete sordida YK-624. DCF completely disappeared and MFA decreased by about 90% after 6 days of treatment. It was also confirmed that the fungus almost completely removed the acute lethal toxicity of DCF and MFA towards the freshwater crustacean Thamnocephalus platyurus after 6 days of treatment. Mass spectrometric and ¹H nuclear magnetic resonance analyses demonstrated that two mono-hydroxylated DCFs (4′-hydroxydiclofenac and 5-hydroxydiclofenac) and one di-hydroxylated DCF (4′,5-dihydroxydiclofenac) were formed via fungal transformation. The four metabolites of MFA were identified as 3′-hydroxymethylmefenamic acid (mono-hydroxylated MFA), 3′-hydroxymethyl-5-hydroxymefenamic acid (di-hydroxylated MFA), 3′-hydroxymethyl-6′-hydroxymefenamic acid (di-hydroxylated MFA) and 3′-carboxymefenamic acid. These results suggest that hydroxylation catalyzed by cytochrome P450 (CYP) in P. sordida YK-624 may be involved in the elimination and detoxification of DCF and MFA. This notion was further supported by the fact that smaller decreases in DCF and MFA were observed in cultures of P. sordida YK-624 incubated with 1-aminobenzotriazole, a known inhibitor of CYP.     

Removal of estrogenic activity of endocrine-disrupting genistein by ligninolytic enzymes from white rot fungi

Endocrine-disrupting genistein was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. Genistein decreased by 93% after 4 days of treatment and the activities of ligninolytic enzymes, manganese peroxidase (MnP) and laccase, were detected during treatment, thus suggesting that the disappearance of genistein is related to ligninolytic enzymes produced extracellularly by white rot fungi. Therefore, genistein was treated with MnP, laccase, and the laccase-mediator system with 1-hydroxybenzotriazole (HBT) as a mediator. HPLC analysis demonstrated that genistein disappeared almost completely in the reaction mixture after 4 h of treatment with either MnP, laccase, or the laccase-HBT system. Using the yeast two-hybrid assay system, it was also confirmed that three enzymatic treatments completely removed the estrogenic activity of genistein after 4h. These results strongly suggest that ligninolytic enzymes are effective in removing the estrogenic activity of genistein.     

Trapping of 2,7-dichlorodibenzo-p-dioxin in aqueous solution by enzymatic reaction of fungal manganese peroxidase in the presence of polyunsaturated fatty acids

In the presence of polyunsaturated fatty acids, including cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), 2,7-dichlorodibenzo-p-dioxin (DCDD) was treated with manganese peroxidase (MnP) from white rot basidiomycete Phanerochaete sordida YK-624. After incubation with MnP, DCDD could not be extracted from the reaction mixture with n-hexane and was trapped in the water layer. DCDD was released by alkalification of the water layer. DCDD was also trapped after treatment with lipoxidase, which produces hydroperoxides from unsaturated lipids. The amounts of thiobarbituric acid-reactive substances produced in the MnP reactions with three highly unsaturated fatty acids were higher than the amounts produced with three fatty acids with a lower degree of unsaturation. These results suggest that a DCDD-trapping compound may be produced by peroxidation of the polyunsaturated fatty acids.     

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.     

Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624

Aflatoxin B(1) (AFB(1) ) is a potent mycotoxin with mutagenic, carcinogenic, teratogenic, hepatotoxic, and immunosuppressive properties. In order to develop a bioremediation system for AFB(1) -contaminated foods by white-rot fungi or ligninolytic enzymes, AFB(1) was treated with manganese peroxidase (MnP) from the white-rot fungus Phanerochaete sordida YK-624. AFB(1) was eliminated by MnP. The maximum elimination (86.0%) of AFB(1) was observed after 48 h in a reaction mixture containing 5 nkat of MnP. The addition of Tween 80 enhanced AFB(1) elimination. The elimination of AFB(1) by MnP considerably reduced its mutagenic activity in an umu test, and the treatment of AFB(1) by 20 nkat MnP reduced the mutagenic activity by 69.2%. (1) H-NMR and HR-ESI-MS analysis suggested that AFB(1) is first oxidized to AFB(1) -8,9-epoxide by MnP and then hydrolyzed to AFB(1) -8,9-dihydrodiol. This is the first report that MnP can effectively remove the mutagenic activity of AFB(1) by converting it into AFB(1) -8,9-dihydrodiol.     

Lignin peroxidase is involved in the biobleaching of manganese-less oxygen-delignified hardwood kraft pulp by white-rot fungi in the solid-fermentation system

Biobleaching of manganese-less oxygen-delignified hardwood kraft pulp (E-OKP) by the white-rot fungi Phanerochaete sordida YK-624 and P. chrysosporium was examined in the solid-state fermentation system. P. sordida YK-624 possessed a higher brightening activity than P. chrysosporium, increasing pulp brightness by 13.4 points after seven days of treatment. In these fermentation systems, lignin peroxidase (LiP) activity was detected as the principle ligninolytic enzyme, and manganese peroxidase and laccase activities were scarcely detected over the course of treatment of E-OKP by either fungus. Moreover, a linear relationship between brightness increase and cumulative LiP activity was observed under all tested culture conditions with P. sordida YK-624 and P. chrysosporium. These results indicated that LiP is involved in the brightening of E-OKP by both white-rot fungi.     

Bleaching of Hardwood Kraft Pulp with Manganese Peroxidase from Phanerochaete sordida YK-624 without Addition of MnSO(inf4)

In vitro bleaching of an unbleached hardwood kraft pulp was performed with partially purified manganese peroxidase (MnP) from the fungus Phanerochaete sordida YK-624 without the addition of MnSO(inf4) in the presence of oxalate, malonate, or gluconate as manganese chelator. When the pulp was treated without the addition of MnSO(inf4), the pulp brightness increased by about 10 points in the presence of 2 mM oxalate, but the brightness did not significantly increase in the presence of 50 mM malonate, a good manganese chelator. Residual MnP activity decreased faster during the bleaching with MnP without MnSO(inf4) in the presence of malonate than in the presence of oxalate. Oxalate reduced MnO(inf2) which already existed in the pulp or was produced from Mn(sup2+) by oxidation with MnP and thus supplied Mn(sup2+) to the MnP system. The presence of gluconate, produced by the H(inf2)O(inf2)-generating enzyme glucose oxidase, also improved the pulp brightness without the addition of MnSO(inf4), although treatment with gluconate was inferior to that with oxalate with regard to increase of brightness. It can be concluded that bleaching of hardwood kraft pulp with MnP, using manganese originally existing in the pulp, is possible in the presence of oxalate, a good manganese chelator and reducing reagent.     

Effective Removal of Endocrine-Disrupting Compounds by Lignin Peroxidase from the White-Rot Fungus <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> YK-624

The removal of endocrine-disrupting compounds (EDCs) by lignin peroxidase from white-rot fungus Phanerochaete sordida YK-624 (YK-LiP1) was investigated. Five endocrine disruptors, p–t-octylphenol (OP), bisphenol A (BPA), estrone (E₁), 17β-estradiol (E₂), and ethinylestradiol (EE₂) were eliminated by YK-LiP1 more effectively than lignin peroxidase from P. chrysosporium (Pc-LiP), and OP and BPA were disappeared almost completely in the reaction mixture containing YK-LiP1 after a 24-h treatment. Particularly, the removal of estrogenic activities of E₂ and EE₂, which show much higher estrogenic activities than other EDCs such as BPA and OP, were removed following 24-h treatment with YK-LiP1. Moreover, 5,5′-bis(1,1,3,3-tetramethylbutyl)-[1,1′-biphenyl]-2,2′-diol and 5,5′-bis-[1-(4-hydroxy-phenyl)-1-methyl-ethyl]-biphenyl-2,2′-diol were identified as the main metabolite from OP or BPA, respectively. These results suggest that YK-LiP1 is highly effective in removing of EDCs by the oxidative polymerization of these compounds.     

Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes

Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.     

Overexpression of the laccase gene,lcc1, in Lentinula edodes using the pChG vector

Lentinula edodes secretes laccase (Lcc: EC 1.10.3.2), an industrially useful enzyme. In this study, we introduced and expressed the L. edodes Lcc gene, lcc1, driven by L. edodes glyceraldehyde-3-phosphate dehydrogenase gene promoter into L. edodes. The resulting transformants showed 2-fold Lcc activity than that of the host strain, and expression of the recombinant lcc1 was confirmed by RT-PCR.     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.