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PubMed1992

Enhanced biodegradation of phenanthrene in oil tar-contaminated soils supplemented with Phanerochaete chrysosporium.

In recent years, the white rot fungus Phanerochaete chrysosporium has shown promise as an organism suitable for the breakdown of a broad spectrum of environmental pollutants, including polynuclear aromatic hydrocarbons (PAHs). The focus of this study was to determine whether P. chrysosporium could effectively operate in an actual field sample of oil tar-contaminated soil. The soil was loaded with [14C]phenanthrene to serve as a model compound representative of the PAHs. Soil with the native flora present under static, aerobic conditions with buffering (pH 5.0 to 5.5) displayed full mineralization on the order of 20% in 21 days. The addition of P. chrysosporium was synergistic, with full mineralization on the order of 38% in 21 days. In addition to full mineralization, there was an increase in the proportion of radiolabelled polar extractives, both soluble and bound, in the presence of P. chrysosporium. From this study, it is apparent that the native soil microflora can be prompted into full mineralization of PAHs in some contaminated soils and that this mineralization can be enhanced when supplemented with the white rot fungus P. chrysosporium. With further refinement, this system may prove an effective bioremediation technology for soils contaminated with PAHs.

Adsorption↗
PubMed1998

Cloning of Phanerochaete chrysosporium leu2 by complementation of bacterial auxotrophs and transformation of fungal auxotrophs.

A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in both Escherichia coli and Saccharomyces cerevisiae. This expression vector, lambda YES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the beta-isopropylmalate dehydrogenase from P. chrysosporium was characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.

3-Isopropylmalate Dehydrogenase↗
PubMed2001

The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium.

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3' and pUGiGM3' contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3' and pUMiGM3' contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3' untranslated region. In pUGGM3' and pUMGM3', the promoters were fused directly with egfp, whereas in pUGiGM3' and pUMiGM3', following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5' end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5' of the egfp gene (pUGiGM3' and pUMiGM3') exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5' intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5' intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3' paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.

Base Sequence↗
PubMed2005

[Agrobacterium tumefaciens-mediated transformation of the white-rot basidiomycete, phanerochaete chrysosporium].

Agrobacterium tumefaciens has been used to transfer its T-DNA to filamentous fungus Phanerochaete chrysosporium. A DNA fragment containing recombinant hygromycin B phosphotransferase gene (rhyg(r)) which is controlled by a promoter from P. chrysosporium and the lignin peroxidase gene terminator was cut out from pCH6, and inserted into a plant binary plasmid pCAMB1300, resulted in the recombinant plasmid pCH6-1300. The plasmid pCH6-1300 was then transformed into Agrobacterium tumefaciens strain A208, which was used to infect conidia or protoplasts of P. chrysosporium. Sixteen hygromycin B-resistant P. chrysosporium colonies were obtained. The transformation frequency is about 8 - 9 transformants per 106 conidia or protoplasts, which had no significant difference with the values obtained from conidia or protoplasts. One of the transformants named AT18, whose colony morphology was different from the wild-type strain. The results from both PCR and Southern blot analysis of transformants demonstrated that the rHyg(r) gene was integrated into the host genome with single copy. This approach may not require any instrument and digestion of fungal cell walls. A . tumefaciens-facilitated transformation should be very useful for further studies of the molecular genetics of P. chrysosporium.

Agrobacterium tumefaciens↗
PubMed1982

The involvement of hydroxyl radical derived from hydrogen peroxide in lignin degradation by the white rot fungus Phanerochaete chrysosporium.

The possible involvement of hydrogen peroxide (H2O2)-derived hydroxyl radical (.OH) in lignin degradation ([14C]lignin leads to 14CO2) by Phanerochaete chrysosporium was investigated. When P. chrysosporium was grown in low nitrogen medium (2.4 mM N), an increase in the specific activity for H2O2 production in cell extracts was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered stationary phase. The production of .OH in ligninolytic cultures of P. chrysosporium was demonstrated by alpha-keto-gamma-methiolbutyric acid-dependent formation of ethylene. Hydrogen peroxide-dependent .OH formation was also shown in cell extracts of ligninolytic cultures. The radical species was demonstrated to be .OH by the .OH-dependent hydroxylation of p-hydroxybenzoic acid to form protocatechuic acid and by using 5,5-dimethyl-1-pyrroline-N-oxide and detecting the production of the nitroxide radical of 5,5-dimethyl-1-pyrroline-N-oxide by EPR. These reactions were inhibited by .OH-scavenging agents and were stimulated when azide was added to inhibit endogenous catalase. Lignin degradation by P. chrysosporium was markedly suppressed in the presence of the .OH-scavenging agents mannitol, benzoate, and the nonspecific radical scavenging agent butylated hydroxytoluene. The above results indicate that .OH derived from H2O2 is involved in lignin biodegradation by P. chrysosporium.

Agaricales↗
PubMed2002

Morphological characterization of in-vitro human hair keratinolysis, produced by identified wild strains of Chrysosporium species.

Chrysosporium species were isolated from soil and keratinized material. Primary isolation was performed following the general method of hair baiting on modified Czapek-agar media with washed, defated and sterilized human hair fragments added. Strains were maintained in test tubes of potato dextrose agar at 29 degrees C and cultivated on phytone yeast extract agar at 28 degrees C for 14 days for identification. Isolates were characterized using Van Oorschot's key. Keratinolytic activity was expressed following a subjective scale representing degree/severity of attack upon hair surface and presence of fungal structures observed in substrate. Culture results and characterization methods were effective for soil Chrysosporium strain isolation. A new hair attack mode is described. Of 71 keratinolytic fungal isolates, eight (12%) Chrysosporium species were identified. One keratinolytic Chrysosporium sp. isolate is yet to be identified.

Chrysosporium↗
PubMed1997

Chrysosporium species, potential spoilage organisms of chocolate.

Standard methods of analysing foods for the presence of moulds are inadequate for the detection of genera such as Chrysosporium which do not grow at the high water activities of most mycological media. The use of malt, yeast, 50% glucose agar (MY50G) in sealed containers as an enrichment medium allowed time for germination and growth of heat-stressed spores. Three Chrysosporium spp., C. xerophilum Pitt, C. inops (Carmichael) and C. farinicola (Burnside) Skou, were isolated from commercial chocolate bars with a water activity (aw) of approximately 0.28. Chrysosporium inops was isolated from commercial milk crumb and a new Chrysosporium sp. was isolated from Ghanaian cocoa beans. In chocolates made by coating MY50G agar (aw = 0.89) with chocolate (aw = 0.27) containing C. inops arthroconidia, two types of deterioration were seen after storage. The first was fat bloom due to recrystallization of the cocoa butter on the outer and inner chocolate surface. The second was growth of C. inops which occurred on the inside chocolate surface adjacent to the MY50G agar filling and on the outside surface after holding at 92% equilibrium relative humidity (erh) for 12 d. There was some evidence that C. inops could grow on the outside of chocolates held at 5.7% erh after 4 months' storage at 25 degrees C. The appearance of the white fungal growth was not unlike fat bloom to the naked eye but was clearly different with the electron microscope.

Cacao↗
PubMed2001

Determination of the toxicity of several aromatic carbonylic compounds and their reduced derivatives on Phanerochaete chrysosporium using a Pseudomonas putida test system.

We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium.

Alcohols↗
PubMed2001

Heterologous expression of athermostable manganese peroxidase from Dichomitus squalens in Phanerochaete chrysosporium.

Dichomitus squalens belongs to a group of white-rot fungi which express manganese peroxidase (MnP) and laccase but do not express lignin peroxidase (LiP). To facilitate structure/function studies of MnP from D. squalens, we heterologously expressed the enzyme in the well-studied basidiomycete, Phanerochaete chrysosporium. The glyceraldehyde-3-phosphate-dehydrogenase (gpd) promoter of P. chrysosporium was fused to the coding region of the mnp2 gene of D. squalens, 5 bp upstream of the translation start site, and placed in a vector containing the ural gene as a selectable marker. Purified recombinant protein (rDsMnP) was similar in kinetic and spectral characteristics to both the wild-type MnPs from D. squalens and P. chrysosporium (PcMnP). The N-terminal amino acid sequence of the rDsMnP was determined and was identical to the predicted sequence. Cleavage of the propeptide followed a conserved amino acid motif (A-A-P-S/T) in both rDsMnP and PcMnP. However, the protein from D. squalens was considerably more thermostable than its P. chrysosporium homolog with half-lives 15- to 40-fold longer at 55 degrees C. As previously demonstrated for PcMnP, addition of exogenous MnII and CdII conferred additional thermal stability to rDsMnP. However, unlike PcMnP, ZnII also confers some additional thermal stability to rDsMnP at 55 degrees C. Some differences in the metal-specific effects on thermal stability of rDsMnP at 65 degrees C were noted.

Amino Acid Motifs↗
PubMed1992

Genomic organization of a cellulase gene family in Phanerochaete chrysosporium.

Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were found to hybridize to at least three different chromosomes, one of which is dimorphic. The organization of these genes was similar in another P. chrysosporium strain, ME 446. It is clear that, unlike T. reesei, the most well-studied cellulolytic fungus, P. chrysosporium contains a complex, cbh1-like gene family.

Amino Acid Sequence↗
PubMed2006

Metabolism of 4,4'-dichlorobiphenyl by white-rot fungi Phanerochaete chrysosporium and Phanerochaete sp. MZ142.

Degradation experiment of model polychlorinated biphenyl (PCB) compound 4,4'-dichlorobiphenyl (4,4'-DCB) and its metabolites by the white-rot fungus Phanerochaete chrysosporium and newly isolated 4,4'-DCB-degrading white-rot fungus strain MZ142 was carried out. Although P. chrysosporium showed higher degradation of 4,4'-DCB in low-nitrogen (LN) medium than that in potato dextrose broth (PDB) medium, Phanerochaete sp. MZ142 showed higher degradation of 4,4'-DCB under PDB medium condition than that in LN medium. The metabolic pathway of 4,4'-DCB was elucidated by the identification of metabolites upon addition of 4,4'-DCB and its metabolic intermediates. 4,4'-DCB was initially metabolized to 2-hydroxy-4,4'-DCB and 3-hydroxy-4,4'-DCB by Phanerochaete sp. MZ142. On the other hand, P. chrysosporium transformed 4,4'-DCB to 3-hydroxy-4,4'-DCB and 4-hydroxy-3,4'-DCB produced via a National Institutes of Health shift of 4-chlorine. 3-Hydroxy-4,4'-DCB was transformed to 3-methoxy-4,4'-DCB; 4-chlorobenzoic acid; 4-chlorobenzaldehyde; and 4-chlorobenzyl alcohol in the culture with Phanerochaete sp. MZ142 or P. chrysosporium. LN medium condition was needed to form 4-chlorobenzoic acid, 4-chlorobenzaldehyde, and 4-chlorobenzyl alcohol from 3-hydroxy-4,4'-DCB, indicating the involvement of secondary metabolism. 2-Hydroxy-4,4'-DCB was not methylated. In this paper, we proved for the first time by characterization of intermediate that hydroxylation of PCB was a key step in the PCB degradation process by white-rot fungi.

Biodegradation, Environmental↗
PubMed2003

Homologous expression of Phanerochaete chrysosporium manganese peroxidase, using bialaphos resistance as a dominant selectable marker.

Manganese peroxidase (MnP) is a major extracellular component of the lignin-degrading system of the white-rot fungus, Phanerochaete chrysosporium. Homologous expression of recombinant MnP isozyme 1 (rMnP1) in P. chrysosporium was achieved using a novel transformation system for this fungus, which utilizes the Streptomyces hygroscopicus bialaphos-resistant gene, bar, as the selectable marker. The transformation frequency for this system is approximately 100 bialaphos-resistant transformants per microgram of plasmid DNA. Transformed strains all contain plasmid DNA, ectopically integrated into the fungal genome. Using this transformation system, the promoter region of the P. chrysosporium translation elongation factor gene was used to drive expression of mnp1, encoding MnP1, in primary metabolic cultures of P. chrysosporium, where endogenous MnP was not expressed. Approximately 2-3 mg of active recombinant MnP1 per liter of extracellular medium was produced in agitated cultures of transformants.

Aminobutyrates↗
PubMed1991

An improved transformation vector for the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium.

In this study, a lignin peroxidase-encoding gene (LIP) of Phanerochaete chrysosporium was disrupted by inserting into its coding region the kanamycin-resistance determinant from Tn903. The resulting recombinant plasmid, pUGLG1: kan, was transformed into P. chrysosporium with the expectation that the disrupted gene might replace the homologous LIP gene in the chromosome. However, the results showed that pUGLG1: kan sequences do not integrate into the chromosome; instead, the plasmid is maintained intact in the transformants in an extrachromosomal state. Our data also show that pUGLG1: kan undergoes replication in P. chrysosporium, is maintained as a circular element, is recoverable from meiotic and mitotic progeny, although at a low frequency, and can be recovered intact by Escherichia coli transformation. These results suggest that the GLG1 component of pUGLG1: kan contains as yet unidentified sequences that allow its autonomous replication in P. chrysosporium transformants.

Basidiomycota↗
PubMed1992

Oxidative degradation of phenanthrene by the ligninolytic fungus Phanerochaete chrysosporium.

The ligninolytic fungus Phanerochaete chrysosporium oxidized phenanthrene and phenanthrene-9,10-quinone (PQ) at their C-9 and C-10 positions to give a ring-fission product, 2,2'-diphenic acid (DPA), which was identified in chromatographic and isotope dilution experiments. DPA formation from phenanthrene was somewhat greater in low-nitrogen (ligninolytic) cultures than in high-nitrogen (nonligninolytic) cultures and did not occur in uninoculated cultures. The oxidation of PQ to DPA involved both fungal and abiotic mechanisms, was unaffected by the level of nitrogen added, and was significantly faster than the cleavage of phenanthrene to DPA. Phenanthrene-trans-9,10-dihydrodiol, which was previously shown to be the principal phenanthrene metabolite in nonligninolytic P. chrysosporium cultures, was not formed in the ligninolytic cultures employed here. These results suggest that phenanthrene degradation by ligninolytic P. chrysosporium proceeds in order from phenanthrene----PQ----DPA, involves both ligninolytic and nonligninolytic enzymes, and is not initiated by a classical microsomal cytochrome P-450. The extracellular lignin peroxidases of P. chrysosporium were not able to oxidize phenanthrene in vitro and therefore are also unlikely to catalyze the first step of phenanthrene degradation in vivo. Both phenanthrene and PQ were mineralized to similar extents by the fungus, which supports the intermediacy of PQ in phenanthrene degradation, but both compounds were mineralized significantly less than the structurally related lignin peroxidase substrate pyrene was.

Basidiomycota↗
PubMed1995

Degradation of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4'-dichlorobiphenyl [DCB], 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, 14C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the 14C was determined to be biomass bound. Results from a time course study done with 4,4'-[14C]DCB to examine 14C partitioning dynamics indicated that the biomass-bound 14C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4'-[14C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process.

Basidiomycota↗
PubMed1995

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters.

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.

Journal Article↗
PubMed1996

Cloning of a cDNA encoding cellobiose dehydrogenase, a hemoflavoenzyme from Phanerochaete chrysosporium.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by cellulose-degrading cultures of the wood-degrading basidiomycete Phanerochaete chrysosporium. CDH contains one flavin adenine dinucleotide (FAD) and one heme b per molecule, and it oxidizes cellobiose to cellobionolactone. In this report, a 2.4-kb cDNA encoding CDH was isolated by screening an expression library of P. chrysosporium OGC101 with a CDH-specific polyclonal antibody. The cDNA encodes a 755-amino-acid protein with a predicted mass of 80,115 Da. Sequence analysis suggests that the heme domain is located at the N terminus and that the falvin domain is located at the C terminus. The flavin domain shows a beta 1-alpha A-beta 2 motif for FAD binding and has high sequence similarity to several FAD-dependent enzymes. Little sequence similarity to hemoflavoenzymes is found. CDH binds to cellulose similarly to cellulases. However, little sequence similarity is observed with the conserved cellulose-binding sequences of cellulases. This suggests that CDH might possess a specific sequence for cellulose binding which is different from that of cellulases. Northern (RNA) blot analysis of total RNA from cellulose-, glucose-, and cellobiose-grown P. chrysosporium indicated that CDH mRNA is produced only in cellulose-grown cells. This suggests that CDH expression is regulated at the transcriptional level by either cellulose or one of its degradation products. Southern blot analysis suggests the presence of only a single gene for CDH in P. chrysosporium OGC101.

Amino Acid Sequence↗
PubMed1996

Fluorene Oxidation In Vivo by Phanerochaete chrysosporium and In Vitro during Manganese Peroxidase-Dependent Lipid Peroxidation.

The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese(II), unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhibited by the free-radical scavenger butylated hydroxytoluene but not by the lignin peroxidase inhibitor NaVO(inf3). Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.

Journal Article↗