Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis.

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn(2+). Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion.

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative beta-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H(2)O(2). These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H(2)O(2) react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.

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

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.

Structure and transcriptional impact of divergent repetitive elements inserted within Phanerochaete chrysosporium strain RP-78 genes.

We describe the structure, organization, and transcriptional impact of repetitive elements within the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Searches of the P. chrysosporium genome revealed five copies of pce1, a ~1,750-nt non-autonomous, class II element. Alleles encoding a putative glucosyltransferase and a cytochrome P450 harbor pce insertions and produce incomplete transcripts. Class I elements included pcret1, an intact 8.14-kb gypsy-like retrotransposon inserted within a member of the multicopper oxidase gene family. Additionally, we describe a complex insertion of nested transposons within another putative cytochrome P450 gene. The disrupted allele lies within a cluster of >14 genes, all of which encode family 64 cytochrome P450s. Components of the insertion include a disjoint copia-like element, pcret3, the pol domain of a second retroelement, pcret2, and a duplication of an extended ORF of unknown function. As in the case of the pce elements, pcret1 and pcret2/3 insertions are confined to single alleles, transcripts of which are truncated. The corresponding wild-type alleles are apparently unaffected. In aggregate, P. chrysosporium harbors a complex array of repetitive elements, at least five of which directly influence expression of genes within families of structurally related sequences.

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

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.