Franz J. St. John

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.

Characterization of XynC from Bacillus subtilis subsp. subtilis Strain 168 and Analysis of Its Role in Depolymerization of Glucuronoxylan

Secretion of xylanase activities by Bacillus subtilis 168 supports the development of this well-defined genetic system for conversion of methylglucuronoxylan (MeGAXn [where n represents the number of xylose residues]) in the hemicellulose component of lignocellulosics to biobased products. In addition to the characterized glycosyl hydrolase family 11 (GH 11) endoxylanase designated XynA, B. subtilis 168 secretes a second endoxylanase as the translated product of the ynfF gene. This sequence shows remarkable homology to the GH 5 endoxylanase secreted by strains of Erwinia chrysanthemi. To determine its properties and potential role in the depolymerization of MeGAXn, the ynfF gene was cloned and overexpressed to provide an endoxylanase, designated XynC, which was characterized with respect to substrate preference, kinetic properties, and product formation. With different sources of MeGAXn as the substrate, the specific activity increased with increasing methylglucuronosyl substitutions on the beta-1,4-xylan chain. With MeGAXn from sweetgum as a preferred substrate, XynC exhibited a Vmax of 59.9 units/mg XynC, a Km of 1.63 mg MeGAXn/ml, and a k(cat) of 2,635/minute at pH 6.0 and 37 degrees C. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and 1H nuclear magnetic resonance data revealed that each hydrolysis product has a single glucuronosyl substitution penultimate to the reducing terminal xylose. This detailed analysis of XynC from B. subtilis 168 defines the unique depolymerization process catalyzed by the GH 5 endoxylanases. Based upon product analysis, B. subtilis 168 secretes both XynA and XynC. Expression of xynA was subject to MeGAXn induction; xynC expression was constitutive with growth on different substrates. Translation and secretion of both GH 11 and GH 5 endoxylanases by the fully sequenced and genetically malleable B. subtilis 168 recommends this bacterium for the introduction of genes required for the complete utilization of products of the enzyme-catalyzed depolymerization of MeGAXn. B. subtilis may serve as a model platform for development of gram-positive biocatalysts for conversion of lignocellulosic materials to renewable fuels and chemicals.

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.

Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase.

Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGX(n)) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGX(n) by these enzymes results in singly substituted aldouronates having a 4-O-methylglucuronate moiety linked to a xylose penultimate from the reducing terminal xylose and some number of xylose residues toward the nonreducing terminus. This novel mode of action distinguishes GH30 xylanases from the more common xylanase families that cleave MeGX(n) in accessible regions. To help understand this unique biochemical function, we have determined the structure of XynC in its native and ligand-bound forms. XynC structure models derived from diffraction data of XynC crystal soaks with the simple sugar glucuronate (GA) and the tetrameric sugar 4-O-methyl-aldotetrauronate resulted in models containing GA and 4-O-methyl-aldotriuronate, respectively. Each is observed in two locations within XynC surface openings. Ligand coordination occurs within the XynC catalytic substrate binding cleft and on the structurally fused side β-domain, demonstrating a substrate targeting role for this putative carbohydrate binding module. Structural data reveal that GA acts as a primary functional appendage for recognition and hydrolysis of the MeGX(n) polymer by the protein. This work compares the structure of XynC with a previously reported homologous enzyme, XynA, from Erwinia chrysanthemi and analyzes the ligand binding sites. Our results identify the molecular interactions that define the unique function of XynC and homologous GH30 enzymes.

Proteomic and Functional Analysis of the Cellulase System Expressed by Postia placenta during Brown Rot of Solid Wood

ABSTRACT Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these—two in glycoside hydrolase family 5, one in family 10, and one in family 12—have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.

Holo-Ni(II)HpNikR is an asymmetric tetramer containing two different nickel binding sites

The metalloregulatory protein NikR from Helicobacter pylori (HpNikR) is a master regulator of gene expression which both activates and represses specific genes in response to nickel availability. Here, we report the first crystal structure (at 2.37 Å resolution) of Ni(II)HpNikR prepared directly from the holo protein. The protein contains four nickel ions located in two distinct coordination environments. Two nickel ions are bound to sites in a four-coordinate square-planar geometry as predicted on the basis of the structures of NikR from Escherichia coli and Pyrococcus horikoshii . The remaining two nickel ions are bound to sites with unexpected 5- or 6-coordination geometries which were previously thought to be involved in nickel incorporation into the protein. The nickel with 5-/6-coordination geometry utilizes three histidines from two separate monomeric HpNikR units along with two or three water molecules as ligands. The spatial location of the nickel in the 5-/6-coordinate site is within approximately 5 Å of the expected site if a 4-coordinate square-planar geometry occurred. Two of the histidines that participate as ligands in the 5-/6-coordinate site would also participate as ligands if the 4-coordinate site was occupied, making it impossible for both sites to be occupied simultaneously. DFT calculations show that the 5-/6-coordinate geometries are energetically favorable when the local protein environment is included in the calculations. The presence of two distinct coordination environments in HpNikR is suggested to be related to the specificity and binding affinity of this transcription factor for DNA.

Engineering the Xylan Utilization System in Bacillus subtilis for Production of Acidic Xylooligosaccharides

ABSTRACT Xylans are the predominant polysaccharides in hemicelluloses and an important potential source of biofuels and chemicals. The ability of Bacillus subtilis subsp. subtilis strain 168 to utilize xylans has been ascribed to secreted glycoside hydrolase family 11 (GH11) and GH30 endoxylanases, encoded by the xynA and xynC genes, respectively. Both of these enzymes have been defined with respect to structure and function. In this study, the effects of deletion of the xynA and xynC genes, individually and in combination, were evaluated for xylan utilization and formation of acidic xylooligosaccharides. Parent strain 168 depolymerizes methylglucuronoxylans (MeGX n ), releasing the xylobiose and xylotriose utilized for growth and accumulating the aldouronate methylglucuronoxylotriose (MeGX3) with some methylglucuronoxylotetraose (MeGX4). The combined GH11 and GH30 activities process the products generated by their respective actions on MeGX n to release a maximal amount of neutral xylooligosaccharides for assimilation and growth, at the same time forming MeGX3 in which the internal xylose is substituted with methylglucuronate (MeG). Deletion of xynA results in the accumulation of β-1,4-xylooligosaccharides with degrees of polymerization ranging from 4 to 18 and an average degree of substitution of 1 in 7.2, each with a single MeG linked α-1,2 to the xylose penultimate to the xylose at the reducing terminus. Deletion of the xynC gene results in the accumulation of aldouronates comprised of 4 or more xylose residues in which the MeG may be linked α-1,2 to the xylose penultimate to the nonreducing xylose. These B. subtilis lines may be used for the production of acidic xylooligosaccharides with applications in human and veterinary medicine.

GH115 α-glucuronidase and GH11 xylanase from Paenibacillus sp. JDR-2: potential roles in processing glucuronoxylans

Paenibacillus sp. JDR-2 (Pjdr2) has been studied as a model for development of bacterial biocatalysts for efficient processing of xylans, methylglucuronoxylan, and methylglucuronoarabinoxylan, the predominant hemicellulosic polysaccharides found in dicots and monocots, respectively. Pjdr2 produces a cell-associated GH10 endoxylanase (Xyn10A1) that catalyzes depolymerization of xylans to xylobiose, xylotriose, and methylglucuronoxylotriose with methylglucuronate-linked α-1,2 to the nonreducing terminal xylose. A GH10/GH67 xylan utilization regulon includes genes encoding an extracellular cell-associated Xyn10A1 endoxylanase and an intracellular GH67 α-glucuronidase active on methylglucuronoxylotriose generated by Xyn10A1 but without activity on methylglucuronoxylotetraose generated by a GH11 endoxylanase. The sequenced genome of Pjdr2 contains three paralogous genes potentially encoding GH115 α-glucuronidases found in certain bacteria and fungi. One of these, Pjdr2_5977, shows enhanced expression during growth on xylans along with Pjdr2_4664 encoding a GH11 endoxylanase. Here, we show that Pjdr2_5977 encodes a GH115 α-glucuronidase, Agu115A, with maximal activity on the aldouronate methylglucuronoxylotetraose selectively generated by a GH11 endoxylanase Xyn11 encoded by Pjdr2_4664. Growth of Pjdr2 on this methylglucuronoxylotetraose supports a process for Xyn11-mediated extracellular depolymerization of methylglucuronoxylan and Agu115A-mediated intracellular deglycosylation as an alternative to the GH10/GH67 system previously defined in this bacterium. A recombinantly expressed enzyme encoded by the Pjdr2 agu115A gene catalyzes removal of 4-O-methylglucuronate residues α-1,2 linked to internal xylose residues in oligoxylosides generated by GH11 and GH30 xylanases and releases methylglucuronate from polymeric methylglucuronoxylan. The GH115 α-glucuronidase from Pjdr2 extends the discovery of this activity to members of the phylum Firmicutes and contributes to a novel system for bioprocessing hemicelluloses.