Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrat... more Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified. (13)C-nuclear magnetic resonance confirmed production of d-arabino-hexos-2-ulose (glucosone) from d-glucose with the oxidase. Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of tryptic digests and analysis of the corresponding cDNA revealed a structurally unusual sequence for the P. chrysosporium POX. Relatively high levels of pox transcript were detected under carbon-starved culture conditions but not under nutrient sufficiency. This regulation pattern is similar to that observed for lignin peroxidases, manganese peroxidases, and glyoxal oxidase of P. chrysosporium, supporting evidence that POX has a role in lignocellulose degradation.
Expression of Phanerochaete chrysosporium genes encoding ligninolytic enzymes was assessed in woo... more Expression of Phanerochaete chrysosporium genes encoding ligninolytic enzymes was assessed in wood. Poly(A) RNA was extracted from colonized wood chips by magnetic capture, and specific transcripts were quantified by competitive reverse transcriptase PCR. mRNA levels varied substantially among lignin peroxidase genes, and transcript patterns were dramatically different from those in previous studies with defined media.
The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium-co... more The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium-colonized wood chips were quantified. The transcript patterns obtained were dramatically different from the transcript patterns obtained previously in defined media. Cellobiose dehydrogenase transcripts were also detected, which is consistent with the hypothesis that such transcripts play an important role in cellulose degradation.
We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium w... more We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium when it colonizes hybrid poplar (Populus alba × tremula) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons of P. chrysosporium transcript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold, P < 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate that P. chryso...
Determining linkage is problematic for genes lacking easily identifiable phenotypes and for organ... more Determining linkage is problematic for genes lacking easily identifiable phenotypes and for organisms without well-defined genetic recombination systems. Phanerochaete chrysosporium with its lignin peroxidase (LiP) gene family typifies these difficulties. We describe an experimental approach whereby the segregation of specific alleles is directly monitored during sexual fruiting. The method establishes linkage relationships among genes for which there are no mutations, and it is applicable to a wide range of genes, gene families and organisms. Using this approach, five P. chrysosporium linkage groups were identified. Ten LiP genes were distributed among three of these groups. One co-segregating group contained eight closely linked LiP genes. Another LiP gene was linked to a cellobiohydrolase gene cluster. These genetic linkages were consistent with physical mapping by pulsed field gel electrophoresis. Based on the identification of allelic relationships, a uniform nomenclature for LiP genes is also described.
Three lignin peroxidase (LiP) genes from the basidiomycete Phanerochaete chrysosporium were clone... more Three lignin peroxidase (LiP) genes from the basidiomycete Phanerochaete chrysosporium were cloned on a single 30 kb cosmid insert. One gene, GLG5, is the genomic equivalent of a previously reported cDNA clone, CLG5. The other two LiP genes are transcriptionally convergent and map to a position approximately 15 kb downstream of GLG5. The translational stop codons of these genes are separated by 1.3 kb. Analysis of homokaryons established allelic relationships to previously described LiP clones. Using clamped homogeneous electrical field electrophoresis (CHEF), seven chromosomal bands were resolved from P. chrysosporium genomic DNA. On CHEF gel Southern blots, the LiP gene family was localized to a single, dimorphic chromosome.
The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fu... more The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.
Description: We describe here recent methods for quantitative assessment of specific P. chrysospo... more Description: We describe here recent methods for quantitative assessment of specific P. chrysosporium mRNAs in organopollutant contaminated soils and in Aspen wood chips. Magnetic capture techniques were used to rapidly purify poly(A)-RNA, and quantitative RT-PCR protocols were ...
Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrat... more Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified. (13)C-nuclear magnetic resonance confirmed production of d-arabino-hexos-2-ulose (glucosone) from d-glucose with the oxidase. Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of tryptic digests and analysis of the corresponding cDNA revealed a structurally unusual sequence for the P. chrysosporium POX. Relatively high levels of pox transcript were detected under carbon-starved culture conditions but not under nutrient sufficiency. This regulation pattern is similar to that observed for lignin peroxidases, manganese peroxidases, and glyoxal oxidase of P. chrysosporium, supporting evidence that POX has a role in lignocellulose degradation.
Expression of Phanerochaete chrysosporium genes encoding ligninolytic enzymes was assessed in woo... more Expression of Phanerochaete chrysosporium genes encoding ligninolytic enzymes was assessed in wood. Poly(A) RNA was extracted from colonized wood chips by magnetic capture, and specific transcripts were quantified by competitive reverse transcriptase PCR. mRNA levels varied substantially among lignin peroxidase genes, and transcript patterns were dramatically different from those in previous studies with defined media.
The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium-co... more The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium-colonized wood chips were quantified. The transcript patterns obtained were dramatically different from the transcript patterns obtained previously in defined media. Cellobiose dehydrogenase transcripts were also detected, which is consistent with the hypothesis that such transcripts play an important role in cellulose degradation.
We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium w... more We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium when it colonizes hybrid poplar (Populus alba × tremula) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons of P. chrysosporium transcript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold, P < 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate that P. chryso...
Determining linkage is problematic for genes lacking easily identifiable phenotypes and for organ... more Determining linkage is problematic for genes lacking easily identifiable phenotypes and for organisms without well-defined genetic recombination systems. Phanerochaete chrysosporium with its lignin peroxidase (LiP) gene family typifies these difficulties. We describe an experimental approach whereby the segregation of specific alleles is directly monitored during sexual fruiting. The method establishes linkage relationships among genes for which there are no mutations, and it is applicable to a wide range of genes, gene families and organisms. Using this approach, five P. chrysosporium linkage groups were identified. Ten LiP genes were distributed among three of these groups. One co-segregating group contained eight closely linked LiP genes. Another LiP gene was linked to a cellobiohydrolase gene cluster. These genetic linkages were consistent with physical mapping by pulsed field gel electrophoresis. Based on the identification of allelic relationships, a uniform nomenclature for LiP genes is also described.
Three lignin peroxidase (LiP) genes from the basidiomycete Phanerochaete chrysosporium were clone... more Three lignin peroxidase (LiP) genes from the basidiomycete Phanerochaete chrysosporium were cloned on a single 30 kb cosmid insert. One gene, GLG5, is the genomic equivalent of a previously reported cDNA clone, CLG5. The other two LiP genes are transcriptionally convergent and map to a position approximately 15 kb downstream of GLG5. The translational stop codons of these genes are separated by 1.3 kb. Analysis of homokaryons established allelic relationships to previously described LiP clones. Using clamped homogeneous electrical field electrophoresis (CHEF), seven chromosomal bands were resolved from P. chrysosporium genomic DNA. On CHEF gel Southern blots, the LiP gene family was localized to a single, dimorphic chromosome.
The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fu... more The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.
Description: We describe here recent methods for quantitative assessment of specific P. chrysospo... more Description: We describe here recent methods for quantitative assessment of specific P. chrysosporium mRNAs in organopollutant contaminated soils and in Aspen wood chips. Magnetic capture techniques were used to rapidly purify poly(A)-RNA, and quantitative RT-PCR protocols were ...
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