Michael D. Mozuch

Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium

Abstract Phanerochaete chrysosporium BKM-1767 secretes multiple lignin peroxidase isoenzymes when grown under nitrogen-limited conditions. Here we report the purification of these heme-containing peroxidases, and their physical and catalytic characterization. Ten hemeproteins, designated H1–H10, were separated by anion exchange HPLC. Six of them, H1, H2, H6, H7, H8, and H10, were lignin peroxidases, oxidizing veratryl alcohol in the presence of H 2 O 2 . The other four (three peaks were resolved) exhibited manganese-dependent peroxidase activity, oxidizing vanillylacetone in the presence of H 2 O 2 and Mn +2 . The lignin peroxidases have different isoelectric points, between p14.7 and 3.3, and molecular weights between 38 and 43 kDa, determined by SDS-PAGE. All are N - and probably O -glycosylated. Three organic substrates and H 2 O 2 were used to compare their kinetic properties: the organic substrates were veratryl alcohol, 1,4-dimethoxybenzene, and the lignin model compound 1-(3,4-dimethoxyphenyl)-2-( o -methoxyphenoxy)-propane-1,3-diol. K M and TN values for each of these substrates varied significantly; e.g. for veratryl alcohol K M values were from 86 to 480 μ m and TN values were from 1.3 to 8.3 s -1 . The ranking of the isoenzyme activities differed with the different substrates, suggesting differences in affinities or in active site accessibilities. The K M for H 2 O 2 varied between 13 and 77 μ m . Immunological blot analysis and partial proteolytic digestion patterns showed that the isoenzymes have a high degree of homology. The isoenzyme concentrations in extracellular culture fluid were found to vary relatively and absolutely with culture time. A nomenclature scheme for these 10 hemeproteins has been proposed. This scheme should simplify identification of these proteins in the literature as well as be adaptable to others found in Phanerochaete chrysosporium .

Comparative transcriptome and secretome analysis of wood decay fungi Postia placenta and Phanerochaete chrysosporium.

ABSTRACT Cellulose degradation by brown rot fungi, such as Postia placenta, is poorly understood relative to the phylogenetically related white rot basidiomycete, Phanerochaete chrysosporium. To elucidate the number, structure, and regulation of genes involved in lignocellulosic cell wall attack, secretome and transcriptome analyses were performed on both wood decay fungi cultured for 5 days in media containing ball-milled aspen or glucose as the sole carbon source. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a total of 67 and 79 proteins were identified in the extracellular fluids of P. placenta and P. chrysosporium cultures, respectively. Viewed together with transcript profiles, P. chrysosporium employs an array of extracellular glycosyl hydrolases to simultaneously attack cellulose and hemicelluloses. In contrast, under these same conditions, P. placenta secretes an array of hemicellulases but few potential cellulases. The two species display distinct expression patterns for oxidoreductase-encoding genes. In P. placenta, these patterns are consistent with an extracellular Fenton system and include the upregulation of genes involved in iron acquisition, in the synthesis of low-molecular-weight quinones, and possibly in redox cycling reactions.

Transcriptome and Secretome Analyses of Phanerochaete chrysosporium Reveal Complex Patterns of Gene Expression

ABSTRACT The wood decay basidiomycete Phanerochaete chrysosporium was grown under standard ligninolytic or cellulolytic conditions and subjected to whole-genome expression microarray analysis and liquid chromatography-tandem mass spectrometry of extracellular proteins. A total of 545 genes were flagged on the basis of significant changes in transcript accumulation and/or peptide sequences of the secreted proteins. Under nitrogen or carbon limitation, lignin and manganese peroxidase expression increased relative to nutrient replete medium. Various extracellular oxidases were also secreted in these media, supporting a physiological connection based on peroxide generation. Numerous genes presumed to be involved in mobilizing and recycling nitrogen were expressed under nitrogen limitation, and among these were several secreted glutamic acid proteases not previously observed. In medium containing microcrystalline cellulose as the sole carbon source, numerous genes encoding carbohydrate-active enzymes were upregulated. Among these were six members of the glycoside hydrolase family 61, as well as several polysaccharide lyases and carbohydrate esterases. Presenting a daunting challenge for future research, more than 190 upregulated genes are predicted to encode proteins of unknown function. Of these hypothetical proteins, approximately one-third featured predicted secretion signals, and 54 encoded proteins detected in extracellular filtrates. Our results affirm the importance of certain oxidative enzymes and, underscoring the complexity of lignocellulose degradation, also support an important role for many new proteins of unknown function.

Degradation of gymnosperm (Guaiacyl) vs. angiosperm (syringyl/guaiacyl) lignins by Phanerochaete chrysosporium

This study examined the relative degradabilities of guaiacyl and syringyl/guaiacyl lignins by the white-rot fungus Phanerochaete chrysosporium Burds. Synthetic syringyl/guaiacyl lignin (Cpin syringyl units) was depolymerized much more rapidly than synthetic guaiacyl lignin (Cp), although the two were oxidized to CO2 at the same rate. Milled wood lignin or birch, labeled with H at Ce, was also depolymerized more rapidly than similarly labeled spruce milled wood lignin. During degradation two to three times äs much of both the synthetic and natural guaiacyl lignins became mycelium-bound äs the syringyl/guaiacyl lignins. This result is interpreted to reflect a greater resistance of the guaiacyl lignins. Collectively, our results point to a more facile initial degradation of the syringyl/guaiacyl type of lignins. This finding helps explain the more rapid degradation of angiosperm wood than gymnosperm wood by white-rot fungi.

Significant Alteration of Gene Expression in Wood Decay Fungi Postia placenta and Phanerochaete chrysosporium by Plant Species

ABSTRACT Identification of specific genes and enzymes involved in conversion of lignocellulosics from an expanding number of potential feedstocks is of growing interest to bioenergy process development. The basidiomycetous wood decay fungi Phanerochaete chrysosporium and Postia placenta are promising in this regard because they are able to utilize a wide range of simple and complex carbon compounds. However, systematic comparative studies with different woody substrates have not been reported. To address this issue, we examined gene expression of these fungi colonizing aspen (Populus grandidentata) and pine (Pinus strobus). Transcript levels of genes encoding extracellular glycoside hydrolases, thought to be important for hydrolytic cleavage of hemicelluloses and cellulose, showed little difference for P. placenta colonizing pine versus aspen as the sole carbon source. However, 164 genes exhibited significant differences in transcript accumulation for these substrates. Among these, 15 cytochrome P450s were upregulated in pine relative to aspen. Of 72 P. placenta extracellular proteins identified unambiguously by mass spectrometry, 52 were detected while colonizing both substrates and 10 were identified in pine but not aspen cultures. Most of the 178 P. chrysosporium glycoside hydrolase genes showed similar transcript levels on both substrates, but 13 accumulated >2-fold higher levels on aspen than on pine. Of 118 confidently identified proteins, 31 were identified in both substrates and 57 were identified in pine but not aspen cultures. Thus, P. placenta and P. chrysosporium gene expression patterns are influenced substantially by wood species. Such adaptations to the carbon source may also reflect fundamental differences in the mechanisms by which these fungi attack plant cell walls.

Characteristics of Cotton Cellulose Depolymerized by a Brown-Rot Fungus, by Acid, or by Chemical Oxidants

Summary Wood decay fungi of the brown-rot type destroy the strength of wood before significant weight loss occurs.This is due to extension depolymerization of the cellulose. Evidence indicates that enzymes cannot gainaccess to the cellulose in wood and that the depolymerizing agent might be oxidative. Our objectiveshere were to gain information about the nature of the agent by characterizing pure cellulose that hadbeen depolymerized by a brown-rot fungus (BR) and to compare its characteristics with those of cellulosedepolymerizcd by acid (A). by Fenton’s reagent (Fe 2+ + H 2 O 2 ) (F), or by periodic acid/bromine/water(P). All four types of depolymerized cellulose exhibited molecular size characteristics indicating that de-polymerization was due to cleavages within the noncrystalline regions. Carbonyl contents of the sampleswere similar, from 1.4 to 1.9 per cellulose molecule. Carboxyl contents were 0.44, 0.00, 0.24, and 7.38per molecule for samples BR, A, F, and P, respectively; uronic acids were absent except for a trace insample P. On complete acid hydrolysis the samples gave the expected amounts of glucose, except forsample P, which contained nonglucosyl moietics that did not contain carbonyl or carboxyl groups detectedby our analytical procedures. With theglucose in the acid hydrolysates we found glyceric, crythronic,ararbonic, and gluconic acids in samples BR and F, and crythronic acid in sample P. Our results indicatethat the brown-rotted cellulose resembles sample F more than samples A or P.

Structure, organization, and transcriptional regulation of a family of copper radical oxidase genes in the lignin-degrading basidiomycete Phanerochaete chrysosporium.

ABSTRACT The white rot basidiomycete Phanerochaete chrysosporium produces an array of nonspecific extracellular enzymes thought to be involved in lignin degradation, including lignin peroxidases, manganese peroxidases, and the H2O2-generating copper radical oxidase, glyoxal oxidase (GLX). Preliminary analysis of the P. chrysosporium draft genome had identified six sequences with significant similarity to GLX and designated cro1 through cro6. The predicted mature protein sequences diverge substantially from one another, but the residues coordinating copper and constituting the radical redox site are conserved. Transcript profiles, microscopic examination, and lignin analysis of inoculated thin wood sections are consistent with differential regulation as decay advances. The cro2-encoded protein was detected by liquid chromatography-tandem mass spectrometry in defined medium. The cro2 cDNA was successfully expressed in Aspergillus nidulans under the control of the A. niger glucoamylase promoter and secretion signal. The recombinant CRO2 protein had a substantially different substrate preference than GLX. The role of structurally and functionally diverse cro genes in lignocellulose degradation remains to be established.

Ligninase-mediated phenoxy radical formation and polymerization unaffected by cellobiose: quinone oxidoreductase☆

Phanerochete chrysosporium ligninase (+ H2O2) oxidized the lignin substructure-related compound acetosyringone to a phenoxy radical which was identified by ESR spectroscopy. Cellobiose:quinone oxidoreductase (CBQase) + cellobiose, previously suggested to be a phenoxy radical reducing system, was without effect on the radical. Ligninase polymerized guaiacol and it increased the molecular size of a synthetic lignin. These polymerizations, reflecting phenoxy radical coupling reactions, were also unaffected by the CBQase system. We conclude that ligninase catalyzes phenol polymerization via phenoxy radicals, which CBQase does not affect. The CBQase system also did not produce H2O2, and its physiological role remains obscure. Glucose oxidase + glucose did produce H2O2 as expected, but, like CBQase, it did not reduce the phenoxy radical of acetosyringone. Because intact cultures of P. chrysosporium depolymerize lignins, it is likely that phenol polymerization by ligninase is prevented or reversed in vivo by an as yet undescribed system.

Isolation and Purification of Pyranose 2-Oxidase from Phanerochaete chrysosporium and Characterization of Gene Structure and Regulation

ABSTRACT Pyranose 2-oxidase (POX) was recovered from Phanerochaete chrysosporium BKM-F-1767 solid substrate culture using mild extraction conditions and was purified. 13C-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.

Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

ABSTRACT 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.