Blog List

Tuesday, 14 February 2017

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

Author
  1. Dan Cullenm,1
Author Affiliations
  1. Edited by Richard A. Dixon, The Samuel Roberts Noble Foundation, Ardmore, OK, and approved February 22, 2012 (received for review December 6, 2011)
    1. Abstract
    2. Full Text
    3. Authors & Info
    4. Figures
      • SI
      • Metrics
      Next Section

      Abstract

      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.
      The most abundant source of photosynthetically fixed carbon in land ecosystems is plant biomass, composed primarily of cellulose, hemicellulose, and lignin. Many microorganisms are capable of using cellulose and hemicellulose as carbon and energy sources, but a much smaller group of filamentous fungi in the phylum Basidiomycota has also evolved with the unique ability to efficiently depolymerize and mineralize lignin, the most recalcitrant component of plant cell walls. Collectively known as white rot fungi, they remove lignin to gain access to cell wall carbohydrates for carbon and energy sources. These wood-decay fungi are common inhabitants of fallen trees and forest litter. As such, white rot fungi play a pivotal role in the carbon cycle. Their unique metabolic capabilities are of considerable recent interest in bioenergy-related processes (1).
      White rot basidiomycetes differ in their gross morphological patterns of decay (ref. 2 and refs. therein). Phanerochaete chrysosporium simultaneously degrades cellulose, hemicellulose, and lignin, whereas a few others such as the closely related polypore species, Ceriporiopsis subvermispora, have the ability to remove lignin in advance of cellulose. The mechanistic basis of this selectivity is unknown.
      The roles of P. chrysosporium lignin peroxidase [LiP; Enzyme Commission (EC) 1.11.1.14] and manganese peroxidase (EC 1.11.1.13) have been intensively studied (3). Reactions catalyzed by LiP include Cα-Cβcleavage of propyl side chains in lignin and lignin models, hydroxylation of benzylic methylene groups, oxidation of benzyl alcohols to the corresponding aldehydes or ketones, phenol oxidation, and aromatic ring cleavage in nonphenolic lignin model compounds. In addition to P. chrysosporium, multiple ligninolytic peroxidase isozymes and their corresponding genes have been identified in several efficient lignin-degrading fungi (4). In some white rot fungi, such as the oyster mushroom Pleurotus ostreatus and related species, LiP is absent, but a third ligninolytic peroxidase type that combines LiP and MnP catalytic properties, versatile peroxidase (VP; EC 1.11.1.16), has been characterized (4, 5) and identified by genome analysis (6). Repeated and systematic attempts have failed to identify LiP (or VP) activity in C. subvermispora cultures, but substantial evidence implicates MnP in ligninolysis (e.g., refs 7, 8). First discovered in P. chrysosporiumcultures, this enzyme oxidizes Mn2+ to Mn3+, using H2O2 as an oxidant (9, 10). MnP cannot directly cleave the dominant nonphenolic structures within lignin, but it has been suggested that oxidation may be mediated by lipid peroxidation mechanisms that are promoted by Mn3+ (3).
      In addition to peroxidases, laccases (EC 1.10.3.2) have been implicated in lignin degradation. Several have been characterized from C. subvermispora cultures (11), whereas no genes encoding laccase, in the strict sense, are present in the P. chrysosporium genome (12). The mechanism by which laccases might degrade lignin remains unclear, as the enzyme lacks sufficient oxidation potential to cleave nonphenolic linkages within the polymer. However, various mediators have been proposed (13).
      Other components commonly ascribed to ligninolytic systems include extracellular enzymes capable of generating hydrogen peroxide. Glucose–methanol–choline oxidoreductases such as aryl-alcohol oxidase, methanol oxidase and pyranose oxidase, together with copper radical oxidases such as glyoxal oxidase, have been characterized in P. chrysosporium (14), but none of these activities have been reported in C. subvermispora cultures.
      Conceivably, selective lignin degradation patterns may involve modulation of the hydrolytic enzymes commonly associated with cellulose and hemicellulose degradation. These systems are well characterized in P. chrysosporium, whereas little is known about C. subvermispora glycoside hydrolases (GHs) (15).
      To further our understanding of selective ligninolysis, we report here initial analysis of the C. subvermisporagenome. Comparison with the genome, transcriptome, and secretome of P. chrysosporium reveal substantial differences among the genes that are likely to be involved in lignocellulose degradation, providing insight into diversification of the white rot mechanism.

      Previous SectionNext Section

      Results


      General Features of C. subvermispora Genome. 

      The 39-Mb haploid genome of C. subvermisporamonokaryotic strain B (16) (SI Appendix, Fig. S1) is predicted to encode 12,125 proteins (SI Appendixprovides detailed assembly and annotation information). For comparison, the latest release of the related polypore white rot fungus P. chrysosporium features 35.1 Mb of nonredundant sequence and 10,048 gene models (12, 17). The overall relatedness of these polypore fungi was clearly evident from the syntenic regions between their largest scaffolds and large number of similar (BLAST E-values <10−5) protein sequences, i.e., 74% (n = 9,007) of C. subvermispora models aligned with P. chrysosporium and 82% (n = 8,258) of P. chrysosporium models aligned with C. subvermispora. Most (n = 5,443) of these pairs were also reciprocal “best hits” and are thus likely to represent orthologues. Significant expansions compared with P. chrysosporium and/or other sequenced Agaricomycetes were observed in transporters, various oxidoreductases including peroxidases, cytochrome p450s, and other gene families discussed here.

      Peroxidases.


      Twenty-six C. subvermispora gene models are predicted to encode heme peroxidases. Fifteen were classified as probable ligninolytic peroxidases, which included 13 MnPs, a VP, and an LiP. These classifications were based on homology modeling (18) with particular attention to conserved Mn2+ oxidation and catalytic tryptophan sites (19, 20). Those classified as MnPs include seven typical “long” MnPs specific for Mn2+, and a “short” MnP also able to oxidize phenols and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) in the absence of Mn2+, as previously reported in the P. ostreatus genome (6). The remaining five could be classified as “extra long” MnPs in view of their long C-termini, as reported for the first time in Dichomitus squalens MnPs (21). Only four full-length MnP-encoding genes were previously identified in C. subvermispora(GenBank accession nos. AAB03480, AAB92247, AAO61784, and AF161585). Additional class II peroxidases have long been suspected (22, 23), but no LiP/VP-like transcripts or activities have been identified. Thus, the repertoire of C. subvermispora peroxidases differs from P. chrysosporium, which features 10 LiP and five MnP genes (Fig. 1). Extending comparative analysis to 90 basidiomycete peroxidases (SI Appendix, Fig. S3) suggested that the C. subvermispora VP and LiP represent divergent proteins, an observation consistent with their catalytic properties (as detailed later).
      Fig. 1.
      Fig. 1.
      Phylogenetic analysis of selected peroxidases from C. subvermispora and P. chrysosporium. The analysis was performed in RAxML Blackbox under the model GTRGAMMA, using the substitution matrix WAG with 100 rapid bootstrap replicates. The ascomycete sequences of class II peroxidases were used to root the tree (http://phylobench.vital-it.ch/raxml-bb/) (32). Ball-milled aspen versus glucose transcript ratios (BMA/Glu) are indicated, and complete data are available under Gene Expression Omnibus accession nos. GSE1473 and GSE34636 for P. chrysosporium and C. subvermispora, respectively.
      By using a previously developed Escherichia coli expression system including in vitro activation (24, 25), the C. subvermispora putative LiP (Cesubv118677) and VP (Cesubv99382) were evaluated for their oxidation of three representative substrates, namely Mn2+, the high redox-potential veratryl alcohol (VA), and Reactive Black 5 (RB5) (Table 1). The corresponding steady-state kinetic constants were compared with those of Pleurotus eryngii VP (isozyme VPL; AF007244), a P. chrysosporium LiP (isozyme H8; GenBank accession no. Y00262), and a conventional C. subvermispora MnP (Cesubv117436; Fig. 1) also produced in E. coli. The putative C. subvermispora LiP (protein model Cesubv118677) was unable to oxidize Mn2+, as expected given the absence of a typical manganese oxidation site in its theoretical molecular structure (SI Appendix, Fig. S2). A conventional C. subvermispora MnP protein (Cesubv117436), also predicted based on structure, and the VP from P. eryngii showed Mn2+ oxidation. Surprisingly, the C. subvermispora protein designated Cesubv99382, which we tentatively classified as a VP, was not able to oxidize Mn2+, irrespective of the presence of a putative manganese oxidation site in its structural model (SI Appendix, Fig. S2). The catalytic behaviors of Cesubv99382 and Cesubv118677 are very similar. Both enzymes oxidize VA, the typical LiP (and VP) substrate, and also RB5, a characteristic substrate of VP (that LiP is unable to oxidize in the absence of mediators), with similar Km, kcat, and kcat/Km values (Table 1).
      Table 1.
      Steady-state kinetic constants of three peroxidases from C. subvermispora genome vs. P. chrysosporium LiP and P. eryngii VP
      Peroxidase expression patterns differed significantly between C. subvermispora and P. chrysosporium. In medium containing ball-milled Populus grandidentata (aspen) as sole carbon source, transcript levels of two C. subvermispora MnPs were significantly up-regulated relative to glucose medium. Liquid chromatography/tandem MS (LC-MS/MS) analysis of culture filtrates identified peptides corresponding to three C. subvermispora MnP genes (Fig. 1). In identical media, none of the P. chrysosporium MnP genes were up-regulated, but significant accumulation of two LiP gene transcripts was observed relative to glucose (Fig. 1). No peroxidases were identified by LC-MS/MS analysis of P. chrysosporium culture filtrates.

      Multicopper Oxidases.


      Nine multicopper (MCO)-encoding C. subvermispora genes may be relevant to lignin degradation. Multiple alignments emphasizing signature regions (26, 27) revealed the presence of seven laccases, in the strictest sense, one of which was previously known (28). This observation is in distinct contrast to the P. chrysosporium genome, which contains no laccases (12) (Fig. 2). Consistent with a role in lignocellulose modification, transcript levels corresponding to C. subvermispora laccase was significantly up-regulated (more than threefold; P < 0.01) in media containing ball-milled P. grandidentata wood (aspen) relative to glucose medium (Fig. 2).
      Fig. 2.
      Fig. 2.
      Phylogenetic analysis of all MCO oxidases from C. subvermispora, P. chrysosporium, and the related polypore P. placenta. Analysis was performed by using RAxML with the WAG substitution matrix, γ-distributed rates among sites, a proportion of invariant sites and empirical amino acid frequencies (i.e., m = PROTGAMMAIWAGF). Shown is the maximum-likelihood tree found by using 1,000 heuristic searches, with bootstrap support shown for nodes with values greater than 50%. As in Fig. 1, transcript level ratios are adjacent to protein identification numbers. Complete P. placenta microarray data are available under Gene Expression Omnibus accession no. GSE12540 (33).
      In addition to the laccases, C. subvermispora MCO-encoding genes included a canonical ferroxidase (Fet3). Involved in high-affinity iron uptake, the Fet3 genes of C. subvermispora (Cesubv67172) and Postia placenta(Pospl129808) show significant up-regulation on aspen-containing medium, whereas the P. chrysosporiumorthologue (Phchr26890) is sharply down-regulated under identical conditions (Fig. 2). This strongly suggests that iron homeostasis is achieved by different mechanisms in these fungi.

      Other Enzymes Potentially Involved in Extracellular Redox Processes.


      Peroxide and free radical generation are considered key components of ligninolysis, and analysis of the C. subvermispora genome, transcriptome, and secretome revealed a diverse array of relevant proteins. These included four copper radical oxidases, cellobiose dehydrogenase, various other glucose–methanol–choline oxidoreductases, and several putative transporters. Possibly related to selectivity of ligninolysis, expression patterns exhibited by certain genes, e.g., methanol oxidase, differed significantly between P. chrysosporium and C. subvermispora. (SI Appendix and SI Appendix, Table S1, include detailed listings of all annotated genes, transcript levels, and LC-MS/MS identification of extracellular proteins.)
      Of particular relevance to lignin degradation by MnP, we observed a significant expansion of the genes putatively involved in fatty acid metabolism (Table 2). Relative to the single gene in P. chrysosporium(encoding Phchr125220) the Δ-12 fatty acid desaturase gene family was particularly expanded (five paralogues) in C. subvermispora. The P. chrysosporium and C. subvermispora genes were previously designated Pcfad2 and Csfad2 (29, 30), respectively. Transcript levels of P. chrysosporium Pcfad2 were significantly reduced (0.25-fold; P < 0.01) in media with aspen relative to glucose, whereas a C. subvermispora Δ-12 fatty acid desaturase (Cesubv124119) was up-regulated (2.9-fold; P < 0.01). With regard to Δ-9 fatty acid desaturases, only two P. chrysosporium genes were detected and, as in the case of Δ-12 fatty acid synthetases, both were down-regulated more than twofold (P < 0.01). Modest transcript accumulation (1.48-fold; P = 0.03) was observed for one of the four C. subvermispora Δ-9 fatty acid desaturases (Cesubv117066) in aspen wood media relative to glucose media. Increased numbers of MnP and lipid metabolism genes, viewed together with their expression patterns, are consistent with an important role for peroxyl radical attack on nonphenolic substructures of lignin.
      Table 2.
      Number, overall relatedness, and transcript levels of genes putatively involved in lipid metabolism

      Carbohydrate Active Enzymes.


      Overall, the number of GHs encoded by the C. subvermispora genome is slightly lower than that of other plant cell wall degrading basidiomycetes whose genomes have been sequenced (Dataset S1 and SI Appendix, Table S1). The number of GHs in C. subvermispora (n = 171) is close to that in P. chrysosporium (n = 177), and noticeably different in total number and in family distribution compared with the phylogenetically related brown rot fungus P. placenta (n = 145; Fig. 3). Differences between C. subvermispora and P. chrysosporium are limited to a few families, but these distinctions might have consequences for degradation of plant cell wall polysaccharides. For example, C. subvermisporacontained only three predicted proteins belonging to family GH7, an important group typically featuring “exo” cellobiohydrolases. In contrast, at least six GH7 protein models were identified in the P. chrysosporiumgenome. Family GH3, containing β-glucosidases involved in the hydrolysis of cellobiose, was represented by only six gene models in the C. subvermispora genome, unlike the 11 GH3 models found in P. chrysosporium. In addition, the C. subvermispora genome revealed only 16 cellulose binding modules (CBM1s), compared with 31 CBM1-containing protein models found in the P. chrysosporium genome.
      Fig. 3.
      Fig. 3.
      Distribution of GHs in P. placenta (inner ring), C. subvermispora (middle ring), and P. chrysosporium(outer ring). Families absent from at least one species are underlined. Detailed listings of gene numbers within these and other species appear in Dataset S1, and expression patterns (transcript and protein) are presented in SI Appendix, Table S1.
      In contrast to the oxidative systems, transcriptome and secretome analysis of GHs generally showed lower expression in C. subvermispora relative to P. chrysosporium (Table 3 and SI Appendix, Table S1). Transcripts corresponding to 30 C. subvermispora GH-encoding genes accumulated more than twofold (P < 0.05) in aspen wood- vs. glucose-containing media. In contrast, 52 P. chrysosporium GH-encoding genes were up-regulated (more than twofold; P < 0.05). MS unambiguously identified 60 and 121 proteins in filtrates from aspen wood media of P. chrysosporium and C. subvermispora cultures, respectively, among which 18 and three, respectively, corresponded to GHs.
      Table 3.
      Expression of C. subvermispora and P. chrysosporiumcellulases
      Genes encoding likely cellulases showed only modest transcript levels in C. subvermispora (Table 3). C. subvermispora transcripts corresponding to single copies of a CBM1-containing cellobiohydrolase (GH7), a CBM1-containing endo-β-1,4-glucanase (GH5), and a GH12 endoglucanase, all canonical cellulases, were significantly up-regulated (more than twofold; P < 0.01) in aspen wood relative to glucose media. Under identical conditions, accumulating P. chrysosporium transcripts included four GH7 cellobiohydrolases, two GH5 endo-β-1,4-glucanases, and two GH12 endoglucanases (Table 3).
      The foregoing analysis is limited to expression patterns of genes with putative function inferred from sequence comparisons. However, many of the predicted proteins that show no significant sequence similarity to known proteins could be important in selective ligninolysis. Specifically, we identified 139 “hypothetical” C. subvermispora proteins whose sequences show no significant similarity to P. chrysosporium models but were otherwise highly expressed, i.e., transcript levels more than two SDs above the genome-wide mean (n = 12084, X = 10.56) or more than twofold transcript accumulation in aspen wood media vs. glucose or unambiguously identified via MS (at least two unique peptide sequences).

      Previous SectionNext Section

      Discussion

      C. subvermispora and P. chrysosporium are both members of the order Polyporales, but they differ sharply in their ability to selectively degrade lignin. The genetics and physiology of P. chrysosporium have been intensively studied for decades. Largely because of its efficient degradation of plant cell walls, including the recalcitrant lignin, P. chrysosporium was selected as the first sequenced basidiomycete (12). In contrast, C. subvermispora has received less attention, although its selective lignin degradation is well known (2). Overall, our comparisons of C. subvermispora and P. chrysosporium gene repertoires, together with expression patterns on a complex lignocellulose substrate, suggest divergent strategies of plant cell wall degradation and provide clues about mechanisms of selective delignification.
      Generally accepted as important components of lignin degradation systems, class II peroxidases were skewed toward expansion of the number of MnPs and accompanied by a putative LiP (Cesubv118677) and a VP (Cesubv99382). To confirm these predictions, both peroxidases were obtained by E. coli expression, and their steady-state kinetic constants for oxidation of selected peroxidase substrates were compared with those of a typical MnP from the C. subvermispora genome (Cesubv117436), a well characterized VP from P. eryngii(GenBank AF007244), and the well studied P. chrysosporium LiP isozyme H8 (all expressed in E. coli). Cesubv118677 and Cesubv99382 are able to directly oxidize VA and RB5, a unique characteristic of VP, exhibiting similar catalytic efficiency values to those observed for typical VPs. Moreover, both peroxidases are unable to oxidize Mn2+, despite the presence in Cesubv99382 of a putative oxidation site for this cation. Thus, considering their sequences (Fig. 1 and SI Appendix) and catalytic activities (Table 1), these two peroxidases seem to represent an intermediate evolutionary state between LiP and VP.
      In addition to the distinct repertoire of class II peroxidases, selective ligninolysis of C. subvermispora may be related, in part, to the expansion and coexpression of the genes putatively involved in lipid metabolism. Substantial evidence implicates MnP involvement (7, 8) in lignin degradation, but this enzyme cannot directly cleave the dominant nonphenolic structures within lignin. Nevertheless, several studies support mechanisms involving peroxidation of lipids (3). The expansion of C. subvermispora desaturase and MnP gene families, together with their high expression levels relative to P. chrysosporium (Table 2 and Fig. 1), are consistent with a role in lignin degradation.
      Overall numbers and family distributions of GH-encoding genes were similar between C. subvermispora and P. chrysosporium (Fig. 3), but subtle differences in number and expression were noted. Among the cellulases, cellobiohydrolases (cel7s) and endoglucanases (cel5s and cel12s) were particularly notable in their transcript and protein accumulation in P. chrysosporium cultures (Table 3). In contrast, expression of the C. subvermispora cellulolytic system was substantially lower than P. chrysosporium, whereas the converse was observed for enzymes important in extracellular oxidative systems (Figs. 1 and 2, Table 2, and SI Appendix, Table S1).
      These observations provide functional models that may explain the shift toward selective ligninolysis by C. subvermispora. Definitive mechanisms remain uncertain, but our investigations identify a subset of potentially important genes, including those encoding hypothetical proteins. More detailed functional analysis is complicated by the insoluble nature of lignocellulose substrates and by the slow, asynchronous hyphal growth of lignin degrading fungi. Direct and persuasive proof of gene function would be aided by development of experimental tools such as gene disruption/suppression or isozyme-specific immunolocalization of secreted proteins.


      Methods


      Genome Sequencing, Assembly, and Annotation.


      A whole genome shotgun approach was used to sequence C. subvermispora monokaryotic strain B (16) (US Department of Agriculture Forest Mycology Center, Madison, WI). Assembly and annotations are available through interactive visualization and analysis tools from the Joint Genome Institute genome portal (http://www.jgi.doe.gov/Ceriporiopsis) and at DNA Data Base in Japan/European Molecular Biology Laboratory/GenBank under project accession no. AEOV00000000. Details regarding the assembly, repetitive elements (Dataset S2), ESTs annotation, and specific gene sets are provided separately (SI Appendix, Figs. S1–S6).

      MS.


      Soluble extracellular proteins were concentrated from C. subvermispora cultures containing ball-milled aspen as previously described for P. chrysosporium (31) This medium allows rapid growth on a lignocellulose substrate more relevant than glucose- or cellulose-containing media. However, the milling process pulverizes wood cell walls and the culture conditions may not replicate “natural” decay processes. Sample preparation and nano-LC-MS/MS analyses were performed as described in SI Appendix. Peptides were identified by using a Mascot search engine (Matrix Science) against protein sequences of 12,125 predicted gene models described earlier. Complete listings of carbohydrate active enzymes and oxidative enzymes, including peptide sequences and scores, are provided in SI Appendix, Table S1.

      Expression Microarrays.


      NimbleGen arrays (Roche) were designed to assess expression of 12,084 genes during growth on ball-milled aspen (P. grandidentata) or on glucose as sole carbon sources. Methods are detailed in SI Appendix, and all data deposited under Gene Expression Omnibus accession no. GSE34636.
      Previous SectionNext Section

      Acknowledgments

      We thank Sally Ralph (Forest Products Laboratory) for preparation of ball-milled wood. The major portions of this work were performed under US Department of Agriculture Cooperative State, Research, Education, and Extension Service Grant 2007-35504-18257 (to D.C. and R.A.B.). The US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract DE-AC02-05CH11231. This work was supported by Spanish Projects BIO2008-01533 and BIO2011-26694, European Project Peroxidases as Biocatalysts KBBE-2010-4-265397 (to F.J.R.-D. and A.T.M.), the Chilean National Fund for Scientific and Technological Development Grant 1090513 (to L.F.L.), and a “Ramon y Cajal” contract (to F.J.R.-D.).


      Previous SectionNext Section

      Footnotes

      • Author contributions: S.S.B., K.W.B., E.A.L., S.M.L., I.V.G., R.M.B., R.A.B., P.K., A.T.M., R.V., and D.C. designed research; E.F.-F., J.G., A.V.W., G.S., B.W.H., K.M.L., A.L., R.R., A.A.S., and A.W. performed research; E.F.-F., F.J.R.-D., P.F., D.F., D.S.H., P.C., L.F.L., T.Y.J., D. Seelenfreund, S.L., R.P., M.T., Y. Honda, Takahito Watanabe, Takashi Watanabe, J.S.R., C.P.K., M. Schmoll, J.G., F.J.S.J., A.V.W., G.S., S.S.B., K.S., J.S.Y., H.D., V.S., J.L.L., J.A.O., G.P., A.G.P., L.R., F.S., E.M., P.M.C., B.H., V.L., J.K.M., U.K., C.H., K.I., M. Samejima, B.W.H., K.W.B., K.M.L., A.L., E.A.L., S.M.L., R.R., A.A.S., D.H., D. Schwenk, Y. Hadar, O.Y., R.P.d.V., A.W., J.S., D.E., I.V.G., R.M.B., R.A.B., P.K., A.T.M., R.V., and D.C. analyzed data; and E.F.-F., F.J.R.-D., P.F., D.F., D.S.H., P.C., L.F.L., T.Y.J., D. Seelenfreund, S.L., R.P., M.T., Y. Honda, Takahito Watanabe, Takashi Watanabe, J.S.R., C.P.K., M. Schmoll, J.G., K.E.H., F.J.S.J., G.S., S.S.B., K.S., J.S.Y., H.D., V.S., J.L.L., J.A.O., G.P., A.G.P., L.R., F.S., E.M., P.M.C., B.H., V.L., J.K.M., U.K., C.H., K.I., M. Samejima, B.W.H., K.W.B., K.M.L., A.L., E.A.L., S.M.L., R.R., A.A.S., D.H., D. Schwenk, Y. Hadar, O.Y., R.P.d.V., A.W., J.S., D.E., I.V.G., R.M.B., R.A.B., P.K., A.T.M., R.V., and D.C. wrote the paper.
      • The authors declare no conflict of interest.
      • This article is a PNAS Direct Submission.
      • Data deposition: The annotated genome is available on an interactive web portal, http://jgi.doe.gov/Ceriporiopsis and at DNA Data Base in Japan/European Molecular Biology Laboratory (DDBJ/EMBL)/GenBank (project accession no. AEOV00000000). The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE34636).
      • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1119912109/-/DCSupplemental.
      Previous Section

      References

      1. ↵

        1. United States Department of Energy
         (2006) Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda. Report from the December 2005 Workshop, DOE/SC-0095 (US Department of Energy Office of Science, Washington, DC).

        Google Scholar
      2. ↵

        1. Blanchette R, 
        2. Krueger E, 
        3. Haight J, 
        4. Akhtar M, 
        5. Akin D
         (1997) Cell wall alterations in loblolly pine wood decayed by the white-rot fungus, Ceriporiopsis subvermispora. J Biotechnol 53:203–213.

        CrossRefWeb of ScienceGoogle Scholar
      3. ↵

        1. Hammel KE, 
        2. Cullen D
         (2008) Role of fungal peroxidases in biological ligninolysis. Curr Opin Plant Biol 11:349–355.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      4. ↵

        1. Martinez AT
         (2002) Molecular biology and structure-function of lignin-degrading heme peroxidases. Enzyme Microb Technol 30:425–444.

        CrossRefWeb of ScienceGoogle Scholar
      5. ↵

        1. Tsukihara T, 
        2. Honda Y, 
        3. Sakai R, 
        4. Watanabe T, 
        5. Watanabe T
         (2008) Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2. Appl Environ Microbiol74:2873–2881.

        Abstract/FREE Full Text
      6. ↵

        1. Ruiz-Dueñas FJ, 
        2. Fernández E, 
        3. Martínez MJ, 
        4. Martínez AT
         (2011) Pleurotus ostreatus heme peroxidases: An in silico analysis from the genome sequence to the enzyme molecular structure. C R Biol 334:795–805.

        CrossRefMedlineGoogle Scholar
      7. ↵

        1. Urzúa U, 
        2. Fernando Larrondo L, 
        3. Lobos S, 
        4. Larraín J, 
        5. Vicuña R
         (1995) Oxidation reactions catalyzed by manganese peroxidase isoenzymes from Ceriporiopsis subvermispora. FEBS Lett 371:132–136.
        CrossRefMedlineWeb of ScienceGoogle Scholar
      8. ↵

        1. Jensen KA, 
        2. Bao W, 
        3. Kawai S, 
        4. Srebotnik E, 
        5. Hammel KE
         (1996) Manganese-dependent cleavage of nonphenolic lignin structures by Ceriporiopsis subvermispora in the absence of lignin peroxidase. Appl Environ Microbiol 62:3679–3686.

        Abstract/FREE Full Text
      9. ↵

        1. Paszczynski A, 
        2. Huynh V-B, 
        3. Crawford RL
         (1985) Enzymatic activities of an extracellular, manganese-dependent peroxidase from Phanerochaete chrysosporium. FEMS Microbiol Lett 29:37–41.

        CrossRefWeb of ScienceGoogle Scholar
      10. ↵

        1. Gold MH, 
        2. Kuwahara M, 
        3. Chiu AA, 
        4. Glenn JK
         (1984) Purification and characterization of an extracellular H2O2-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium. Arch Biochem Biophys 234:353–362.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      11. ↵

        1. Larrondo LF, 
        2. Avila M, 
        3. Salas L, 
        4. Cullen D, 
        5. Vicuña R
         (2003) Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns. Microbiology 149:1177–1182.

        Abstract/FREE Full Text
      12. ↵

        1. Martinez D, 
        2. et al.
         (2004) Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nat Biotechnol 22:695–700.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      13. ↵

        1. Camarero S, 
        2. Ibarra D, 
        3. Martínez MJ, 
        4. Martínez AT
         (2005) Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl Environ Microbiol71:1775–1784.

        Abstract/FREE Full Text
      14. ↵

        1. Kersten P, 
        2. Cullen D
         (2007) Extracellular oxidative systems of the lignin-degrading basidiomycete Phanerochaete chrysosporium. Fungal Genet Biol 44:77–87.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      15. ↵

        1. Baldrian P, 
        2. Valásková V
         (2008) Degradation of cellulose by basidiomycetous fungi. FEMS Microbiol Rev 32:501–521.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      16. ↵

        1. Tello M, 
        2. et al.
         (2001) Isolation and characterization of homokaryotic strains from the ligninolytic basidiomycete Ceriporiopsis subvermispora. FEMS Microbiol Lett 199:91–96.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      17. ↵

        1. Vanden Wymelenberg A, 
        2. et al.
         (2006) Computational analysis of the Phanerochaete chrysosporium v2.0 genome database and mass spectrometry identification of peptides in ligninolytic cultures reveal complex mixtures of secreted proteins. Fungal Genet Biol 43:343–356.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      18. ↵

        1. Bordoli L, 
        2. et al.
         (2009) Protein structure homology modeling using SWISS-MODEL workspace. Nat Protoc 4:1–13.

        CrossRefMedlineGoogle Scholar
      19. ↵

        1. Ruiz-Dueñas FJ, 
        2. et al.
         (2009) Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases. J Exp Bot 60:441–452.

        Abstract/FREE Full Text
      20. ↵

        1. Ruiz-Duenas FJ, 
        2. Martinez AT
         (2010) in Biocatalysts Based on Heme Peroxidase, Structural and functional features of peroxidases with a potential as industrial biocatalysts, eds Torres  E, Ayala  M(Springer, Berlin), pp 37–59.

        Google Scholar
      21. ↵

        1. Li D, 
        2. Li N, 
        3. Ma B, 
        4. Mayfield MB, 
        5. Gold MH
         (1999) Characterization of genes encoding two manganese peroxidases from the lignin-degrading fungus Dichomitus squalens(1) Biochim Biophys Acta1434:356–364.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      22. ↵

        1. Rajakumar S, 
        2. et al.
         (1996) Lip-like genes in Phanerochaete sordida and Ceriporiopsis subvermispora, white rot fungi with no detectable lignin peroxidase activity. Appl Environ Microbiol62:2660–2663.

        Abstract/FREE Full Text
      23. ↵

        1. Ruttimann C, 
        2. Schwember E, 
        3. Salas L, 
        4. Cullen D, 
        5. Vicuna R
         (1992) Ligninolytic enzymes of the white rot basidiomycetes Phlebia brevispora and Ceriporiopsis subvermispora. Biotechnol Appl Biochem16:64–76.

        Web of ScienceGoogle Scholar
      24. ↵

        1. Doyle WA, 
        2. Smith AT
         (1996) Expression of lignin peroxidase H8 in Escherichia coli: Folding and activation of the recombinant enzyme with Ca2+ and haem. Biochem J 315:15–19.

        MedlineWeb of ScienceGoogle Scholar
      25. ↵

        1. Perez-Boada JM, 
        2. et al.
         (2002) Expression of Pleurotus eryngii versatile peroxidase in Escherichia coli and optimization of in vitro folding. Enzyme Microb Technol 30:518–524.

        CrossRefWeb of ScienceGoogle Scholar
      26. ↵

        1. Hoegger PJ, 
        2. Kilaru S, 
        3. James TY, 
        4. Thacker JR, 
        5. Kües U
         (2006) Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. FEBS J 273:2308–2326.
        CrossRefMedlineGoogle Scholar
      27. ↵

        1. Kumar SV, 
        2. Phale PS, 
        3. Durani S, 
        4. Wangikar PP
         (2003) Combined sequence and structure analysis of the fungal laccase family. Biotechnol Bioeng 83:386–394.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      28. ↵

        1. Karahanian E, 
        2. Corsini G, 
        3. Lobos S, 
        4. Vicuña R
         (1998) Structure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora. Biochim Biophys Acta 1443:65–74.
        MedlineWeb of ScienceGoogle Scholar
      29. ↵

        1. Watanabe T, 
        2. Tsuda S, 
        3. Nishimura H, 
        4. Honda Y, 
        5. Watanabe T
         (2010) Characterization of a Delta12-fatty acid desaturase gene from Ceriporiopsis subvermispora, a selective lignin-degrading fungus. Appl Microbiol Biotechnol 87:215–224.

        CrossRefMedlineWeb of ScienceGoogle Scholar
      30. ↵

        1. Minto RE, 
        2. Blacklock BJ, 
        3. Younus H, 
        4. Pratt AC
         (2009) Atypical biosynthetic properties of a Delta 12/nu+3 desaturase from the model basidiomycete Phanerochaete chrysosporium. Appl Environ Microbiol 75:1156–1164.

        Abstract/FREE Full Text
      31. ↵

        1. Vanden Wymelenberg A, 
        2. et al.
         (2011) Significant alteration of gene expression in wood decay fungi Postia placenta and Phanerochaete chrysosporium by plant species. Appl Environ Microbiol77:4499–4507.

        Abstract/FREE Full Text
      32. ↵

        1. Stamatakis A, 
        2. Hoover P, 
        3. Rougemont J
         (2008) A rapid bootstrap algorithm for the RAxML Web servers. Syst Biol 57:758–771.

        Abstract/FREE Full Text
      33. ↵

        1. Vanden Wymelenberg A, 
        2. et al.
         (2010) Comparative transcriptome and secretome analysis of wood decay fungi Postia placenta and Phanerochaete chrysosporium. Appl Environ Microbiol 76:3599–3610.
        Abstract

      For further details log on website :
      http://www.pnas.org/content/109/14/5458.long
      at

      No comments:

      Post a Comment

      Subscribe to: Post Comments (Atom)

      Advantages and Disadvantages of Fasting for Runners

      Author BY   ANDREA CESPEDES  Food is fuel, especially for serious runners who need a lot of energy. It may seem counterintuiti...