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A nonradioactive method to detect Phanerochaete chrysosporium grown in a soil matrix was developed. This method involved DNA extraction, PCR amplification, and restriction enzyme analysis. Amplification of ligninase H8 DNA from pure cultures of P. chrysosporium was not as sensitive as amplification of the internal transcribed spacer (ITS) of the highly repetitive nuclear ribosomal DNA. Amplified ITS DNA was digested with restriction enzymes for analysis. The restriction enzyme pattern of PCR-amplified ITS DNA of P. chrysosporium was unique compared with those of unrelated fungi. Two strains of Phanerochaete chrysosporium and two strains of Phanerochaete sordida were indistinguishable by restriction enzyme analysis, while a third strain of P. chrysosporium had an unique pattern. These results were confirmed by sequence information and indicate that species designations of Phanerochaete spp. should be reexamined. The restriction enzyme pattern of DNA extracted and PCR amplified from P. chrysosporium grown in soil was identical to that from P. chrysosporium grown in pure culture. The ITS sequence was detected in 14 ng of the 100 micrograms of total DNA extracted from 1 g of soil.
Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin-salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8 pH at 30 degrees C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin-salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracellular keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones.
Lignin is a major constituent of lignocellulose and the most abundant aromatic biopolymer on earth. It provides plants with rigidity and protection, but its recalcitrant nature also presents a significant barrier to lignocellulose valorization. The white-rot fungus Phanerochaete chrysosporium is among nature's most efficient lignin degraders, and its ligninolytic capabilities have been subjected to intensive investigations. Genome editing with precision is crucial for elucidating the in vivo mechanisms of its ligninolytic actions, but genetic manipulations of P. chrysosporium are often plagued by imprecision. This technical nuisance is driven primarily by canonical non-homologous end joining (c-NHEJ), a DNA repair system that requires little homology and depends on the binding of the Ku70/Ku80 heterodimer to double-strand break (DSB) ends. Loss of Ku70 or Ku80 abolishes c-NHEJ and significantly improves genome editing precision in many filamentous fungi, but it has yet to be examined and exploited in P. chrysosporium. Here, we constructed a homozygous ku70Δ mutant in a meiotic homokaryon of clear genetic background. Loss of Ku70 minimally impacts growth but significantly increases homologous recombination frequency from ~2% to ~66%, with ~32% of the latter being homozygous. Unexpectedly, loss of Ku70 also promotes mononucleate conidiation, which may facilitate isolation of homozygous mutants. Taken together, our work provides a valuable genetic tool to understand and exploit P. chrysosporium's remarkable ligninolytic capabilities.IMPORTANCEGenome editing with precision is essential to unraveling the intricacies of P. chrysosporium's exceptional ligninolytic capabilities, but the available tools are generally imprecise due to the dominance of non-homologous recombination, a problem that is further exacerbated by the discontinuation of Novozyme 234. We tackle these challenges by reestablishing protoplast-based transformation with Lywallzyme as an alternative. Importantly, we demonstrate that inactivation of c-NHEJ by deleting ku70 significantly increases gene knockout efficiency and report the unexpected involvement of c-NHEJ in regulating the number of nuclei during conidiation. Our work paves the way for future ventures into understanding ligninolysis in P. chrysosporium and building superior chassis for industrial applications.
Two cDNA sequences representing putative allelic variants of the Phanerochaete chrysosporium cbhII gene were isolated by hybridization to the Trichoderma reesei cbhII gene. Both of the equivalent genomic sequences were subsequently isolated by the inverse PCR technique. DNA sequencing showed that the cbhII open reading frame of 1,380 bp codes for a putative polypeptide of 460 amino acids which is interrupted by six introns. The domain structure found in T. reesei cbhII is conserved in the equivalent P. chrysosporium protein. The overall similarity between the two gene products is 54%, with the region of highest conservation being found in the cellulose-binding domain (65%). Unlike the cbhI gene of P. chrysosporium, cbhII does not appear to be a member of a class of closely related genes. CBHII is a new member of family B of the beta-1, 4-glucanases. Alignment of the P. chrysosporium and T. reesei CBHII protein sequences showed that all of the residues important for the formation of the extended loops of the catalytic domain and those residues that are involved in the catalytic action of the T. reesei enzyme are also present in the P. chrysosporium equivalent. The profiles of cbh gene expression in P. chrysosporium reveal that while cbhI.1 and cbhI.2 could be coregulated, cbhII can be independently controlled. The latter is so far the only cellulase gene found to be expressed when the fungus is grown on oat spelt arabinoxylan, suggesting that it may play an active role in the xylanolytic as well as the cellulolytic systems.
4-Chlorophenol (4-CP) degradation was investigated by suspended and immobilized Phanerochaete chrysosporium conducted in static and agitated cultures. The best results were achieved when experiment was carried out in a rotating biological contactor instead of an Erlenmeyer flask, for both batch degradation and repeated batch degradation. The relative contribution of lignin peroxidase (LiP) versus manganese peroxidase (MnP) to the 4-CP degradation by P. chrysosporium was investigated. 4-CP degradation slightly increased and a high level of MnP (38 nKat ml(-1)) was produced when P. chrysosporium was grown at high Mnll concentration. High LiP production in the medium had no significant effect on 4-CP degradation. 4-CP degradation occurred when P. chrysosporium was grown in a medium that repressed LiP and MnP production. This result indicates that LiP and MnP are not directly involved in 4-CP degradation by P. chrysosporium.
Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. The pH optimum of the cellobiose-cytochrome c oxidoreductase activity of P. chrysosporium CDH was acidic, whereas that of H. insolens CDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains of P. chrysosporium and H. insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3-9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain from H. insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporium CDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor from H. insolens was found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporium CDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.
The white-rot basidiomycete Phanerochaete chrysosporium produces lignin peroxidases (LiPs), a family of extracellular glycosylated heme proteins, as major components of its lignin-degrading system. Up to 15 LiP isozymes, ranging in M(r) values from 38,000 to 43,000, are produced depending on culture conditions and strains employed. Manganese-dependent peroxidases (MnPs) are a second family of extracellular heme proteins produced by P. chrysosporium that are also believed to be important in lignin degradation by this organism. LiP and MnP production is seen during secondary metabolism and is completely suppressed under conditions of excess nitrogen and carbon. Excess Mn(II) in the medium, on the other hand, suppresses LiP production but enhances MnP production. Nitrogen regulation of LiP and MnP production is independent of carbon and Mn(II) regulation. LiP activity is also affected by idiophasic extracellular proteases. Intracellular cAMP levels appear to be important in regulating the production of LiPs and MnPs, although LiP production is affected more than MnP production. Studies on the sequencing and characterization of lip cDNAs and genes of P. chrysosporium have shown that the major LiP isozymes are each encoded by a separate gene. Each lip gene encodes a mature protein that is 343-344 amino acids long, contains 1 putative N-glycosylation site, a number of putative O-glycosylation sites, and is preceded by a 27-28-amino acid leader peptide ending in a Lys-Arg cleavage site. The coding region of each lip gene is interrupted by 8-9 introns (50-63 bp), and the positions of the last two introns appear to be highly conserved. There are substantial differences in the temporal transcription patterns of the major lip genes. The sequence data suggest the presence of three lip gene subfamilies. The genomic DNA of P. chrysosporium strain BKMF-1767 was resolved into 10 chromosomes (genome size of 29 Mb), and that of strain ME-446 into 11 chromosomes (genome size of 32 Mb). The lip genes have been localized to five chromosomes in BKMF-1767 and to four chromosomes in ME-446. DNA transformation studies have reported both integrative and non-integrative transformation in P. chrysosporium.
The role of lignin peroxidases (LIPs) and manganese peroxidases (MNPs) of Phanerochaete chrysosporium in decolorizing kraft bleach plant effluent (BPE) was investigated. Negligible BPE decolorization was exhibited by a per mutant, which lacks the ability to produce both the LIPs and the MNPs. Also, little decolorization was seen when the wild type was grown in high-nitrogen medium, in which the production of LIPs and MNPs is blocked. A lip mutant of P. chrysosporium, which produces MNPs but not LIPs, showed about 80% of the activity exhibited by the wild type, indicating that the MNPs play an important role in BPE decolorization. When P. chrysosporium was grown in a medium with 100 ppm of Mn(II), high levels of MNPs but no LIPs were produced, and this culture also exhibited high rates of BPE decolorization, lending further support to the idea that MNPs play a key role in BPE decolorization. When P. chrysosporium was grown in a medium with no Mn(II), high levels of LIPs but negligible levels of MNPs were produced and the rate and extent of BPE decolorization by such cultures were quite low, indicating that LIPs play a relatively minor role in BPE decolorization. Furthermore, high rates of BPE decolorization were seen on days 3 and 4 of incubation, when the cultures exhibit high levels of MNP activity but little or no LIP activity. These results indicate that MNPs play a relatively more important role than LIPs in BPE decolorization by P. chrysosporium.
Twenty-two azo dyes were used to study the influence of substituents on azo dye biodegradability and to explore the possibility of enhancing the biodegradabilities of azo dyes without affecting their properties as dyes by changing their chemical structures. Streptomyces spp. and Phanerochaete chrysosporium were used in the study. None of the actinomycetes (Streptomyces rochei A10, Streptomyces chromofuscus A11, Streptomyces diastaticus A12, S. diastaticus A13, and S. rochei A14) degraded the commercially available Acid Yellow 9. Decolorization of monosulfonated mono azo dye derivatives of azobenzene by the Streptomyces spp. was observed with five azo dyes having the common structural pattern of a hydroxy group in the para position relative to the azo linkage and at least one methoxy and/or one alkyl group in an ortho position relative to the hydroxy group. The fungus P. chrysosporium attacked Acid Yellow 9 to some extent and extensively decolorized several azo dyes. A different pattern was seen for three mono azo dye derivatives of naphthol. Streptomyces spp. decolorized Orange I but not Acid Orange 12 or Orange II. P. chrysosporium, though able to transform these three azo dyes, decolorized Acid Orange 12 and Orange II more effectively than Orange I. A correlation was observed between the rate of decolorization of dyes by Streptomyces spp. and the rate of oxidative decolorization of dyes by a commercial preparation of horseradish peroxidase type II, extracellular peroxidase preparations of S. chromofuscus A11, or Mn(II) peroxidase from P. chrysosporium. Ligninase of P. chrysosporium showed a dye specificity different from that of the other oxidative enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Restriction mapping and sequence analysis of cosmid clones revealed a cluster of three cellobiohydrolase genes in Phanerochaete chrysosporium. P. chrysosporium cbh1-1 and cbh1-2 are separated by only 750 bp and are located approximately 14 kb upstream from a cellulase gene previously cloned from P. chrysosporium (P. Sims, C. James, and P. Broda, Gene 74:411-422, 1988). Within a well-conserved region, the deduced amino acid sequences of P. chrysosporium cbh1-1 and cbh1-2 are, respectively, 80 and 69% homologous to that of the Trichoderma reesei cellobiohydrolase I gene. The conserved cellulose-binding domain typical of microbial cellulases is absent from cbh1-1. Transcript levels of the three P. chrysosporium genes varied substantially, depending on culture conditions. cbh1-1 and cbh1-2 were not induced in the presence of cellulose, nor did they appear to be subject to glucose repression. Therefore, aspects of the chromosomal organization, structure, and transcription of these genes are unlike those of any previously described cellulase genes.
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.
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.
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.
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.
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.