Philip J. Kersten

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.

Glyoxal Oxidase from Phanerochaete chrysosporium Is a New Radical-Copper Oxidase

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies. The native (inactive) enzyme is activated by oxidants leading to the elimination of the cupric EPR signal consistent with formation of an antiferromagnetically coupled radical-copper complex. Oxidation also leads to the appearance of a substoichiometric free radical EPR signal with an average g value (gav = 2.0055) characteristic of phenoxyl π-radicals arising from a minority apoenzyme fraction. Optical absorption, CD, and spectroelectrochemical measurements on the active enzyme reveal complex spectra extending into the near IR and define the redox potential for radical formation (E1/2 = 0.64 V versus NHE, pH 7.0). Resonance Raman spectra have identified the signature of a modified (cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active complex. This radical-copper motif has previously been found only in galactose oxidase, with which glyoxal oxidase shares many properties despite lacking obvious sequence identity, and catalyzing a distinct reaction. The enzymes thus represent members of a growing class of free radical metalloenzymes based on the radical-copper catalytic motif and appear to represent functional variants that have evolved to distinct catalytic roles.

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.

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.

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.

Effects of Diterpene Acids on Components of a Conifer Bark Beetle–Fungal Interaction: Tolerance by Ips pini and Sensitivity by Its Associate Ophiostoma ips

Abstract Conifer resin and phloem tissue contain several phytochemical groups, composed primarily of monoterpenes, diterpene acids, and stilbene phenolics. The effects of monoterpenes and phenolics on stem-colonizing bark beetles and their associated microorganisms have been studied to some extent, but the roles of diterpene acids are largely unknown. Diterpene acids are known to have substantial feeding deterrent and growth inhibiting effects on a variety of insect groups and are known to inhibit a variety of fungi. We tested three diterpene acids present in red pine, Pinus resinosa, at various concentrations, on several life history components of the bark beetle Ips pini and the fungus Ophiostoma ips. No diterpene acid affected the host acceptance behavior or larval survival of Ips pini. In contrast, abietic acid and isopimaric acid strongly inhibited spore germination of O. ips, and abietic acid strongly inhibited mycelial growth. The levels of inhibition observed were higher than with any previous assays of monoterpenes or phenolics in this system. These results support the view that conifer defenses against bark beetle–fungal complexes are multifaceted, with all three phytochemical groups being important to P. resinosa, but each with varying relative activity against the beetles and fungi.

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.

Physiological regulation of glyoxal oxidase from Phanerochaete chrysosporium by peroxidase systems

Glyoxal oxidase (GLOX) is an H2O2-producing enzyme secreted by ligninolytic cultures of Phanerochaete chrysosporium. The oxidase is reversibly inactivated during purification, but can be reactived when coupled to lignin peroxidase (LiP) with veratryl alcohol as the peroxidase substrate. To characterize the modulation of this extracellular oxidase activity, we studied effects of pH, peroxide concentration, peroxidase source (fungal vs plant), and peroxidase substrate with recombinant GLOX (rGLOX). Our results show that a peroxidase system is not required for rGLOX activity. However, the activity is transient and the enzyme is partly and reversibly inactivated by the produced peroxide. rGLOX activity is more sustained at pH 6 than pH 4.5, and therefore the activation at pH 4.5 by a coupled peroxidase system is more clearly demonstrable. Results with peroxidase substrates of widely varying redox potentials strongly suggest that oxidized intermediates produced by coupled peroxidases are the GLOX activators. Both LiP and horseradish peroxidase (HRP) may be used to fully activate rGLOX using methoxybenzenes as peroxidase substrates. Notably, rGLOX is activated when lignin itself is used in coupled reactions with LiP. In contrast, guaiacol and catechols are both inactivating and lignin degradation products are expected to have similar effects. Taken together, our results suggest that ligninolysis by peroxidase could be regulated by GLOX activity and influenced by the presence of veratryl alcohol, lignin, and lignin degradation products. Such coordinated metabolism would influence the kinetics of free radical generation by the LiPs and, therefore, the overall efficiency of lignin depolymerization.

Influence of Populus Genotype on Gene Expression by the Wood Decay Fungus Phanerochaete chrysosporium

ABSTRACT 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. chrysosporium gene expression patterns are substantially influenced by lignin composition.

Rapid Analysis of Abietanes in Conifers

Diterpene resin acids are major constituents of conifer oleoresin and play important roles in tree defense against insects and microbial pathogens. The tricyclic C-20 carboxylic acids are generally classified into two groups, the abietanes and the pimaranes. The abietanes have conjugated double bonds and exhibit characteristic UV spectra. Here, we report the analysis of abietanes by reversed-phase high-performance liquid chromatography using multiwavelength detection to optimize quantification of underivatized abietic, neoabietic, palustric, levopimaric, and dehydroabietic acids. The utility of the method is demonstrated with methanol extracts of white spruce (Picea glauca) phloem, and representative concentrations are reported.