Whole-Genome De Novo Sequencing of the Lignin-Degrading Wood Rot Fungus Phanerochaete chrysosporium (ATCC 20696).
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ABSTRACT:
SUBMITTER: Hong CY
PROVIDER: S-EPMC5552983 | biostudies-other | 2017 Aug
REPOSITORIES: biostudies-other
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Project description:Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.
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Project description:The Target Of Rapamycin (TOR) signaling pathway is known to regulate growth in response to nutrient availability and stress in eukaryotic cells. In the present study, we have investigated the TOR pathway in the white-rot fungus Phanerochaete chrysosporium. Inhibition of TOR activity by rapamycin affects conidia germination and hyphal growth highlighting the conserved mechanism of susceptibility to rapamycin. Interestingly, the secreted protein content is also affected by the rapamycin treatment. Finally, homologs of the components of TOR pathway can be identified in P. chrysosporium. Altogether, those results indicate that the TOR pathway of P. chrysosporium plays a central role in this fungus.
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Project description:Phanerochaete chrysosporium, the model white rot fungus, has been the focus of research for the past about four decades for understanding the mechanisms and processes of biodegradation of the natural aromatic polymer lignin and a broad range of environmental toxic chemicals. The ability to degrade this vast array of xenobiotic compounds was originally attributed to its lignin-degrading enzyme system, mainly the extracellular peroxidases. However, subsequent physiological, biochemical, and/or genetic studies by us and others identified the involvement of a peroxidase-independent oxidoreductase system, the cytochrome P450 monooxygenase system. The whole genome sequence revealed an extraordinarily large P450 contingent (P450ome) with an estimated 149 P450s in this organism. This review focuses on the current status of understanding on the P450 monooxygenase system of P. chrysosproium in terms of pre-genomic and post-genomic identification, structural and evolutionary analysis, transcriptional regulation, redox partners, and functional characterization for its biodegradative potential. Future research on this catalytically diverse oxidoreductase enzyme system and its major role as a newly emerged player in xenobiotic metabolism/degradation is discussed.
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Project description:The white rot basidiomycete Phanerochaete chrysosporium completely degrades lignin and a variety of aromatic pollutants during the secondary metabolic phase of growth. Two families of secreted heme enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP), are major components of the extracellular lignin degradative system of this organism. MnP and LiP both are encoded by families of genes, and the lip genes appear to be clustered. The lip genes contain eight or nine short introns; the mnp genes contain six or seven short introns. The sequences surrounding active-site residues are conserved among LiP, MnP, cytochrome c peroxidase, and plant peroxidases. The eight LiP cysteine residues align with 8 of the 10 cysteines in MnP. LiPs are synthesized as preproenzymes with a 21-amino-acid signal sequence followed by a 6- or 7-amino-acid propeptide. MnPs have a 21- or 24-amino-acid signal sequence but apparently lack a propeptide. Both LiP and MnP are regulated at the mRNA level by nitrogen, and the various isozymes may be differentially regulated by carbon and nitrogen. MnP also is regulated at the level of gene transcription by Mn(II), the substrate for the enzyme, and by heat shock. The promoter regions of mnp genes contain multiple heat shock elements as well as sequences that are identical to the consensus metal regulatory elements found in mammalian metallothionein genes. DNA transformation systems have been developed for P. chrysosporium and are being used for studies on gene regulation and for gene replacement experiments.
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Project description:The effectiveness of RNA interference (RNAi) is demonstrated in the lignin-degrading fungus Phanerochaete chrysosporium. The manganese-containing superoxide dismutase gene (MnSOD1) was used as the target for RNAi. The plasmid constructed for gene silencing contained a transcriptional unit for hairpin RNA expression. Significantly lower MnSOD expression at both the mRNA and protein activity levels was detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies of the MnSOD gene, this RNAi construct was sufficient to decrease the enzymatic activity by as much as 70% relative to control levels. Implementation of the RNAi technique in P. chrysosporium provides an alternative genetic tool for studies of gene function, particularly of essential genes or gene families.
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Project description:A glutathione S-transferase (GST) was purified to homogeneity from the white-rot fungus, Phanerochaete chrysosporium, by affinity chromatography on glutathione-agarose followed by Mono-Q ion-exchange FPLC. This protein immunoblotted with antisera to rat Theta class GST 5-5 and also showed N-terminal sequence similarity to the Theta class, including the presence of a conserved serine residue that has been specifically implicated in catalysis in this class [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133-2143] and other residues conserved in plant sequences. Catalytic activity was found to be highly labile in the purified protein, although preliminary evidence for activity (approx. 120 m-units/mg) with 1,2-epoxy-3-(p-nitrophenoxy)propane was obtained in some preparations. The enzyme seems to be a dimer with a subunit molecular mass of 25 kDa by SDS/PAGE. The native molecular masses estimated by non-denaturing electrophoresis and by Superose-12 gel filtration were 58 and 45 kDa respectively. A second protein purified in this study also gave low level of activity with 1,2-epoxy-3-(p-nitrophenoxy)propane and had a subunit molecular mass of 28 kDa (native size 62-63 kDa), but did not immunoblot with any GST class and seemed to be N-terminally blocked.
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Project description:Fullerenes are widely produced and applied carbon nanomaterials that require a thorough investigation into their environmental hazards and risks. In this study, we compared the toxicity of pristine fullerene (C60) and carboxylated fullerene (C60-COOH) to white rot fungus Phanerochaete chrysosporium. The influence of fullerene on the weight increase, fibrous structure, ultrastructure, enzyme activity, and decomposition capability of P. chrysosporium was investigated to reflect the potential toxicity of fullerene. C60 did not change the fresh and dry weights of P. chrysosporium but C60-COOH inhibited the weight gain at high concentrations. Both C60 and C60-COOH destroyed the fibrous structure of the mycelia. The ultrastructure of P. chrysosporium was changed by C60-COOH. Pristine C60 did not affect the enzyme activity of the P. chrysosporium culture system while C60-COOH completely blocked the enzyme activity. Consequently, in the liquid culture, P. chrysosporium lost the decomposition activity at high C60-COOH concentrations. The decreased capability in degrading wood was observed for P. chrysosporium exposed to C60-COOH. Our results collectively indicate that chemical functionalization enhanced the toxicity of fullerene to white rot fungi and induced the loss of decomposition activity. The environmental risks of fullerene and its disturbance to the carbon cycle are discussed.
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Project description:cDNA encoding a hemoprotein similar to the cytochrome domain of extracellular flavocytochrome cellobiose dehydrogenase (CDH) was cloned from the white-rot fungus Phanerochaete chrysosporium. The deduced amino acid sequence implies that there is a two-domain structure consisting of an N-terminal cytochrome domain and a C-terminal family 1 carbohydrate-binding module (CBM1) but that the flavin-containing domain of CDH is not present. The gene transcripts were observed in cultures in cellulose medium but not in cultures in glucose medium, suggesting that there is regulation by carbon catabolite repression. The gene was successfully overexpressed in Pichia pastoris, and the recombinant protein was designated carbohydrate-binding cytochrome b562 (CBCyt. b562). The resonance Raman spectrum suggested that the heme of CBCyt. b562 is 6-coordinated in both the ferric and ferrous states. Moreover, the redox potential measured by cyclic voltammetry was similar to that of the cytochrome domain of CDH. These results suggest that the redox characteristics may be similar to those of the cytochrome domain of CDH, and so CBCyt. b562 may have an electron transfer function. In a binding study with various carbohydrates, CBCyt. b562 was adsorbed with high affinity on both cellulose and chitin. As far as we know, this is the first example of a CBM1 connected to a domain without apparent catalytic activity for carbohydrate; this CBM1 may play a role in localization of the redox protein on the surface of cellulose or on the fungal sheath in vivo.
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