It has been widely reported that the white rot basidiomycete Phanerochaete chrysosporium, unlike most other white rot fungi, does not produce laccase, an enzyme implicated in lignin biodegradation. Our results showed that P. chrysosporium BKM-F1767 produces extracellular laccase in a defined culture medium containing cellulose (10 g/liter) and either 2.4 or 24 mM ammonium tartrate. Laccase activity was demonstrated in the concentrated extracellular culture fluids of this organism as determined by a laccase plate assay as well as a spectrophotometric assay with ABTS [2,2-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] as the substrate. Laccase activity was observed even after addition of excess catalase to the extracellular culture fluid to destroy the endogenously produced hydrogen peroxide, indicating that the observed activity is not due to a peroxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by activity staining with ABTS revealed the presence of a laccase band with an estimated M r of 46,500.

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the Nterminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases.

Lignin peroxidases (LIPs) and manganesedependent peroxidases (MNPs) are major components of the -ignin-degrading enzyme system of Phanerochaete chrysosporium and typically appear during secondary metabolism. The involvement of cAMP in the regulation of production of LIPs and MNPs was investigated in this study. Production of LIPs and MNPs was preceded by a sharp rise in intracellular cAMP concentration. Addition of atropine, theophylline, or histamine to cultures resulted in a drop in intracellular cAMP concentration and a concomitant inhibition of production of LIPs only or of both LIPs and MNPs, depending on the concentration of the inhibitor added. These results were independently confirmed by fast protein liquid chromatographic proffles of the LIPs and MNPs in the extracellular fluid of the inhibitor-treated and untreated control cultures. LIP production was generally more sensitive to the inhibitors than MNP production. Northern blot analyses showed that the inhibitors affect the production of LIPs and MNPs at the level of transcription. Furthermore, LIP and MNP gene expression appears to be differentially regulated depending on the intracellular concentration of cAMP. These results show that cAMP plays a key role in the regulation of production of LIPs and MNPs in P. chrysosporium.The white-rot basidiomycete Phanerochaete chrysosporium has been the focus of intense worldwide research from the standpoint of lignin biodegradation and bioremediation of environmental pollutants (for review, see refs.

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin -* '4CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H202-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.Phanerochaete chrysosporium, a white-rot basidiomycete, has been extensively studied as a model for fungal lignin degradation (25). Two classes of extracellular heme protein peroxidases, designated lignin peroxidases and manganese peroxidases, and an H202-generating system have been identified to date as the major components of the lignin-degrading enzyme system of this organism (25). Lignin peroxidases are glycosylated heme proteins that catalyze H202-dependent oxidation of a variety of phenolic and nonphenolic lignin model compounds, and that catalyze the oxidative cleavage of P-0-4 linkages (the most abundant linkage in lignin polymers), Cot-Co linkages, and other linkages in lignin and lignin substructure model compounds (25). The number of lignin peroxidase isozymes produced by P. chrysosporium is reported to vary from 2 to 15, based on the strain, culture conditions, and separation efficiency (24,30,41). All the lignin peroxidase isozymes oxidize veratryl alcohol to veratraldehyde but exhibit considerable differences in specific activities (9, 24). Manganese peroxidases constitute a second group of extracellular heme proteins that catalyze the H202-dependent oxidation of Mn(II) to Mn(III), which, in turn, oxidizes various phenolic substrates (25,44). Also, the manganese peroxidases have been reported to show properties of both an oxidase and a peroxidase (32). Both lignin peroxidases and manganese peroxidases require H202 for activity (15,40). Several enzymes, including glucose oxidase and glyoxal oxidase, have been reported to contribute to H202 production in lignin-degrading cultures of P. chrysosporium (8,21,22,23,25). Both lignin peroxidases and manganese peroxidases and H202-generating enzymes are produced during secondary metabolism, in response to nitrogen starvation, whereas cultures grown under nitrogenrich conditions produce no detectable pero...

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (14C-synthetic lignin----14CO2), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the other hand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H2O2 in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

DNA probes specific for the genes encoding major lignin peroxidase (LIP) isozymes H2, H8, and H10 of Phanerochaete chrysosporium were constructed. These probes were used to study the temporal expression of the three lip genes in defined low-nitrogen medium. H2 gene transcripts were produced at high levels on days 4, 5, and 7 and at low levels on day 6, while the H8 gene transcripts peaked on day 4 and were produced in

A stable extrachromosomally maintained transformation vector (pG12-1) for the lignin-degrading ifiamentous fungus Phanerochaete chrysosporium is described. The vector is 6.3 kb and contains a Kanr marker, pBR322 on, and a 2.2-kb fragment (ME-1) derived from an endogenous extrachromosomal DNA element of P. chrysosporium. Vector pG12-1 was able to transform P. chrysosporium to G418 resistance and was readily and consistently recoverable from the total DNA of transformants via Escherichia coli transformation. Southern blot analyses indicated that pG12-1 is maintained at a low copy number in the fungal transformants. The vector is demonstrable in the total DNA of individual G418-resistant basidiospore progeny of the transformants only after amplification by polymerase chain reaction. Exonuclease IH and dam methylation analyses, respectively, indicated that pG12-1 undergoes replication in P. chrysosporium and that it is maintained extrachromosomally in a circular form. The vector is stably maintained in the transformants even after long-term nonselective growth. There is no evidence for integration of the vector into the chromosome at any stage.In recent years, research has intensified worldwide on the ligninolytic fungus Phanerochaete chrysosporium because of the industrial potential of this organism in biopulping, in the conversion of lignin and lignocellulosic materials to feeds, fuels, and chemicals (22, 47), and in detoxifying recalcitrant environmental pollutants such as dioxins, polychlorinated biphenyls, and benzo(a)pyrenes (10, 11). Lignin peroxidases and manganese peroxidases, two families of extracellular, glycosylated, heme proteins involved in lignin degradation by P. chrysosporium, have been isolated and characterized (16, 22,47,48). Genetic studies with this organism have been limited, but auxotrophic mutants (15,26,27) and mutants lacking various secondary metabolic activities (7, 21, 24) have been isolated. Both cDNA (13,49,53,55) and genomic clones (3,5,8,20,41,42,52,54) for lignin peroxidases and cDNA clones for manganese peroxidases (31, 34) have been isolated and sequenced. We previously described vector pRR12, which was shown to transform P. chrysosporium to G418 resistance and was rescued in Escherichia coli from the total DNA of pRR12 transformants (35). Alic et al. (1, 2) have described an integrative transformation system in which a plasmid vector carrying either an heterologous ADE2 or ADE5 gene from Schizophyllum commune was used to complement the appropriate adenine auxotroph of P. chrysosporium. We describe here a novel transformation vector for P. chrysosporium that is maintained stably in the transformants in an extrachromosomal circular form and is readily recoverable from the total DNA of transformants. MATERIALS AND METHODSStrains and media. P. chrysosporium ME446 (ATCC 34541) was maintained as previously described (21). E. coli DHSa [F-endAl hsdR17(rK-MK+) supE44 thi-J deoR recAl gyrA96 relAIA (argF-lacZYA)U169 4i80dlacZAMJSA-], was used for the maintenance of plasmid vectors. Gold...