Chiaki Hori

The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes

Dating Wood Rot Specific lineages within the basidiomycete fungi, white rot species, have evolved the ability to break up a major structural component of woody plants, lignin, relative to their non–lignin-decaying brown rot relatives. Through the deep phylogenetic sampling of fungal genomes, Floudas et al. (p. 1715; see the Perspective by Hittinger) mapped the detailed evolution of wood-degrading enzymes. A key peroxidase and other enzymes involved in lignin decay were present in the common ancestor of the Agaricomycetes. These genes then expanded through gene duplications in parallel, giving rise to white rot lineages. The enzyme family that enables fungi to digest lignin expanded around the end of the coal-forming Carboniferous period. Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non–lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.

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 Mn2+. 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.

Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay

To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (LPMO, formerly GH61), also have been implicated in cellulose degradation. To examine polysaccharide-degrading potential between white- and brown-rot fungi, we performed genomewide analysis of CAZys and these oxidative enzymes in 11 Polyporales, including recently sequenced monokaryotic strains of Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora. Furthermore, we conducted comparative secretome analysis of seven Polyporales grown on wood culture. As a result, it was found that genes encoding cellulases belonging to families GH6, GH7, GH9 and carbohydrate-binding module family CBM1 are lacking in genomes of brown-rot polyporales. In addition, the presence of CDH and the expansion of LPMO were observed only in white-rot genomes. Indeed, GH6, GH7, CDH and LPMO peptides were identified only in white-rot polypores. Genes encoding aldose 1-epimerase (ALE), previously detected with CDH and cellulases in the culture filtrates, also were identified in white-rot genomes, suggesting a physiological connection between ALE, CDH, cellulase and possibly LPMO. For hemicellulose degradation, genes and peptides corresponding to GH74 xyloglucanase, GH10 endo-xylanase, GH79 β-glucuronidase, CE1 acetyl xylan esterase and CE15 glucuronoyl methylesterase were significantly increased in white-rot genomes compared to brown-rot genomes. Overall, relative to brown-rot Polyporales, white-rot Polyporales maintain greater enzymatic diversity supporting lignocellulose attack.

Comparative genomics of the white-rot fungi, Phanerochaete carnosa and P. chrysosporium, to elucidate the genetic basis of the distinct wood types they colonize

BackgroundSoftwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome.ResultsP. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood.ConclusionsThe P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.

Characterization of an Endoglucanase Belonging to a New Subfamily of Glycoside Hydrolase Family 45 of the Basidiomycete Phanerochaete chrysosporium

ABSTRACT The wood decay fungus Phanerochaete chrysosporium has served as a model system for the study of lignocellulose conversions, but aspects of its cellulolytic system remain uncertain. Here, we report identifying the gene that encodes the glycoside hydrolase (GH) family 45 endoglucanase (EG) from the fungus, cloning the cDNA, determining its heterologous expression in the methylotrophic yeast Pichia pastoris, and characterizing the recombinant protein. The cDNA consisted of 718 bp, including an open reading frame encoding a 19-amino-acid signal peptide, a 7-amino-acid presequence at the N-terminal region, and a 180-amino-acid mature protein, which has no cellulose binding domain. Analysis of the amino acid sequence revealed that the protein has a low similarity (<22%) to known fungal EGs belonging to the GH family 45 (EGVs). No conserved domain of this family was found by a BLAST search, suggesting that the protein should be classified into a new subdivision of this GH family. The recombinant protein has hydrolytic activity toward amorphous cellulose, carboxylmethyl cellulose, lichenan, barley β-glucan, and glucomannan but not xylan. Moreover, a synergistic effect was observed with the recombinant GH family 6 cellobiohydrolase from the same fungus toward amorphous cellulose as a substrate, indicating that the enzyme may act in concert with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.

Effects of xylan and starch on secretome of the basidiomycete Phanerochaete chrysosporium grown on cellulose

Lignocellulosic biomass contains cellulose and xylan as major structural components, and starch as a storage polysaccharide. In the present study, we have used comparative secretomic analysis to examine the effects of xylan and starch on the expression level of proteins secreted by the basidiomycete Phanerochaete chrysosporium grown on cellulose,. Forty-seven spots of extracellular proteins expressed by P. chrysosporium separated by two-dimensional electrophoresis were identified by liquid chromatography-tandem mass spectrometry analysis. Addition of starch to the cellulolytic culture did not affect fungal growth significantly, but did decrease the production of total extracellular enzymes, including cellulases and xylanases. In contrast, addition of xylan increased mycelial volume and the production of extracellular proteins. Xylan increased synthesis of several glycoside hydrolase (GH) family 10 putative endoxylanases and a putative glucuronoyl esterase belonging to carbohydrate esterase family 15, for which plant cell wall xylan may be a substrate. Moreover, cellobiose dehydrogenase and GH family 61 proteins, which are known to promote cellulose degradation, were also increased in the presence of xylan. These enzymes may contribute to degradation by the fungus of not only cellulose but also complex carbohydrate components of the plant cell wall.

Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. giganteas extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

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.

Transcriptional response of the cellobiose dehydrogenase gene to cello- and xylooligosaccharides in the basidiomycete Phanerochaete chrysosporium.

ABSTRACT Cellobiose dehydrogenase (CDH) gene transcripts were quantified by reverse transcription-PCR (RT-PCR) in cultures of Phanerochaete chrysosporium supplemented with various cello- and xylooligosaccharides in order to elucidate the mechanism of enhanced CDH production in xylan/cellulose culture. Cellotriose and cellotetraose induced cdh expression, while xylobiose and xylotriose induced expression of cellobiohydrolase genes, especially cel7C.

First aid for flood-damaged paper using saltwater: The inhibiting effect of saltwater on mold growth

Abstract With the aim of developing a new easy-to-use method for rescuing flood-damaged paper, the effect of saltwater on the inhibition of fungal growth on paper was investigated. This procedure could be used instead of, or assisted by freeze drying. Cellulose-digesting Trichoderma reesei, three types of fungi representative of fungi growing on paper (T. reesei, Aspergillus terreus, and Aureobasidium pullulans) and various naturally airborne fungi were cultured on cellulosic materials in liquid media containing artificial seawater with different salt concentrations. The addition of salts successfully inhibited the growth of T. reesei on microcrystalline cellulose at the concentration of 3.2% (m/m) or higher. The critical salt concentration, 3.2%, is within the general range of salt content in seawater. Other solutions of salts similar to sodium chloride also inhibited fungal growth. Although the observed growth-inhibiting effect was attributed to the high osmotic pressure of the salt solution, physiological effects depending on ion species used were also considered to be possible. The growth of all three types of fungi on copy paper was inhibited effectively when the salt concentration was increased. The growth of various fungi on pure cellulose, with enough oxygen supplied to pores, was completely inhibited (as assessed by visual examination) for 24 days at salt concentrations of 3.5% (m/m) or greater. The fact that the effect of saltwater on cellulosic materials was observed even under optimum medium conditions implies that fungi would be considerably inhibited on flood-damaged paper immersed in saltwater. This method is a promising first aid measure when circumstances do not allow for flood-damaged paper to be dried immediately.