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

ABSTRACTEffect of three wood-decaying fungi on decomposition of spruce wood was studied in solid-state cultivation conditions for a period of three months. Two white rot species (Trichaptum abietinum and Phlebia radiata) were challenged by a brown rot species (Fomitopsis pinicola) in varying combinations. Wood decomposition patterns as determined by mass loss, carbon to nitrogen ratio, accumulation of dissolved sugars and release of volatile organic compounds (VOCs) were observed to depend on both fungal combinations and growth time. Similar dependence of fungal species combination, either white or brown rot dominated, was observed for secreted enzyme activities on spruce wood. Fenton chemistry suggesting reduction of Fe3+ to Fe2+ was detected in the presence of F. pinicola, even in co-cultures, together with substantial degradation of wood carbohydrates and accumulation of oxalic acid. Significant correlation was perceived with two enzyme activity patterns (oxidoreductases produced by white rot fungi; hydrolytic enzymes produced by the brown rot fungus) and wood degradation efficiency. Moreover, emission of four signature VOCs clearly grouped the fungal combinations. Our results indicate that fungal decay type, either brown or white rot, determines the loss of wood mass and decomposition of polysaccharides as well as the pattern of VOCs released upon fungal growth on spruce wood.

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.

Chapter 9 - Disaccharides, Oligosaccharides and Polysaccharides

The degrading processes can, under special conditions, slow to a minimum. Environments which are waterlogged, such as lakes, seas, clay soils, and where the water-table is high, often become near-anaerobic and the activity of aggressive wood-decomposing microorganisms decreases dramatically. This is why, under these conditions, we can still find wooden objects which give us new information about the past. If we look 30 years back in time, we see that wood conservators and scientists concentrated for a long period on the hypothesis that the decomposition of waterlogged wooden material was of a chemical nature with hydrolytic reactions. It was in 1988, at the American Chemical Society congress in Los Angeles, that wood biologists first established that the degradation of waterlogged archaeological wood was caused mainly by microorganisms. In 1983, Holt and Nilsson made the first detailed description of wood attack by a bacterium [1]. By using a combination of light microscopy and transmission electron microscopy (TEM) it was possible to investigate and verify a new group of wood-decaying organisms, the tunnelling bacteria [2]. In the years which followed, two other wood-decaying bacteria were found: the cavitation bacteria and the erosion bacteria [3]. The use of new techniques like TEM and scanning electron microscopy (SEM), where the magnification is extremely high, was essential for these new discoveries. Since 1988 a number of archaeological wooden objects have been investigated under the microscope, and microbial attack has been observed in most cases. In dry wood, for example from the Pyramids, attack has mainly been due to fungi [4]. In waterlogged material, soft rot, tunnelling and erosion bacteria dominate [4-8]. The environmental history of wooden objects can be inferred from the results of microscopic observations. Basidiomycete fungi, like white rot and brown rot, require relatively high levels of oxygen for their wood-decaying activities, while soft rot, belonging to the group of ascomycetes and fungi imperfecti, can be active at lower concentrations. Some bacteria require only minimal amounts of oxygen. 'Bulverket' was a peculiar construction in the lake of Tingstdide Trask, Gotland, Sweden, from the early mediaeval period. Houses of wood were built on a construction of poles. Finds of dark-coloured poles led archaeologists to believe that the whole construction had been burned. However, microscopic examination showed that the dark colour was due to attack by soft rot fungi. It was also demonstrated that the house building material showed terrestrial decay forms associated with white and brown rot. Therefore the likely cause of destruction was rot and not fire. The foremast from the ship Vasa was broken, presumably during the wreck, and microscopic examination of wood samples taken next to the fracture revealed a special type of white rot. This white rot fungus grows in living trees and does not occur in aquatic environments. Therefore the wood used for the mast must have come from a tree attacked by white rot when being felled. The use of inferior wood indicates either that the shipwrights were poorly qualified, or that the building project was running out of time or money. In the current project, a great number of wood samples will be studied by light and electron microscopy and the microbial results will be related to environment, age and wood species to see if there is some relationship between the rate and type of decay. A large number of stratified wood fragments were obtained from different archaeological sites in Sweden through the cooperation of field archaeologists. The wood samples were from terrestrial and aquatic sites, including bogs, lake, sea, clay, sand and 'mediaeval deposits'. The age of the samples varies from 7000 BC to AD 1676 and different wood species are represented (mostly pine and oak). The wood samples were sectioned by hand with a razor blade, and stained with safranin or methylene blue in lactic acid. The sections were studied using polarized light. The use of light microscopy made it possible to identify different forms of attack (bacterial or fungal) and to assess the severity of attack. Earlier results [5-8] and preliminary results from this study show that erosion bacteria are responsible for most of the decay in waterlogged archaeological wood. A knowledge of the role of microbial decay in archaeological wood and the different forms of decay must be fundamental for the choice of conservation method.

Relationship between basidiospore size, shape and life history characteristics: a comparison of polypores

Esca Complex of Diseases (ECD) is widespread in viticultural areas worldwide, posing a serious risk to modern viticulture. The role of basidiomycetes in the onset of foliar symptoms has recently been reconsidered, highlighting a link between the reduction in these symptoms and the presence of Fomitiporia mediterranea in vines. As several basidiomycetes have been associated with wood decay in grapevine, understanding their degradation mechanisms could help unravel the role of wood degradation in ECD. Thus, a collection of ECD-associated basidiomycetes, including F. capensis, F. langloisii, F. polymorpha, F. australiensis, Tropicoporus texanus, Inonotus vitis, and Stereum hirsutum, was examined in addition to F. mediterranea. Our study investigates the production and activity of lignocellulosic enzymes (particularly class-II peroxidases and laccases), along with the low molecular weight compounds (LMWC) in the secretome of these fungi, to assess their ability to perform the Chelator-Mediated-Fenton (CMF) pathway. Results highlight the production of ligninolytic enzymes with low production of active manganese peroxidases and a virtually total absence of lignin peroxidase activity. Additionally, the study confirms that the CMF mechanism is widespread among these fungi, with all LMW secretomes fulfilling the required steps of the CMF mechanism. This study provides critical insights into the degradation strategies of ECD-associated white rot agents, offering potential new perspectives on studying ECD symptomatology and developing targeted control strategies. The findings emphasize the need to consider the CMF mechanism in the context of fungal wood degradation and its potential role in ECD development.

The aim of fungal treatment of organic matter for ruminants is the improvement of its degradability. So far, such treatment appears to be time-consuming and improvement has been modest. In previous work, we observed within three white rot species that there is modest (Ceriporiopsis subvermispora) or low (Lentinula edodes and Pleurotus eryngii) variation in fiber degradation in wheat straw during seven weeks of incubation. By extending and re-examining the data from all three species, we see that strains of C. subvermispora show the largest variation and improvement in the degradability of treated wheat straw. In addition, C. subvermispora also generated the highest absolute amount of degradable organic matter, a parameter not calculated before, but is very relevant for the economic feasibility of fungal treatment. In estimating fungal growth, we found no good correlation between an increase in ergosterol and a decrease in plant biomass, indicating a variation within fungal species of the ergosterol/fungal biomass ratio and/or a variation in carbon use efficiency, which has also not been analyzed before. This work contributes to the knowledge of how fungi degrade lignocellulose and further specifies what can be targeted for breeding to make fungal pretreatment economically feasible for upgrading organic waste streams into ruminal feed.

BackgroundFungi are organisms with the highest natural capacity to degrade lignocellulose substrates, which is enabled by complex systems of extracellular enzymes, whose expression and secretion depend on the characteristics of substrates and the environment.ResultsThis study reports a secretome analysis for white-rot basidiomycete Trametes hirsuta cultivated on a synthetic media and a lignocellulose substrate. We demonstrate that T. hirsuta st. 072 produces multiple extracellular ligninolytic, cellulolytic, hemicellulolytic, peroxide generating, and proteolytic enzymes, as well as cerato-platanins. In contrast to other white rot species described earlier, which mostly secreted glucanases and mannosidases in response to the presence of the lignocellulose substrate, T. hirsuta expressed a spectrum of extracellular cellulolytic enzymes containing predominantly cellobiases and xylanases. As proteomic analysis could not detect lignin peroxidase (LiP) among the secreted lignin degrading enzymes, we attributed the observed extracellular LiP - like activity to the expressed versatile peroxidase (VP). An accessory enzyme, glyoxal oxidase, was found among the proteins secreted in the media during submerged cultivation of T. hirsuta both in the presence and in the absence of copper. However, aryl-alcohol oxidase (AAO) was not identified, despite the presence of AAO enzymatic activity secreted by the fungus.The spectra of the expressed enzymes dramatically changed depending on the growth conditions. Transfer from submerged cultivation to surface cultivation with the lignocellulose substrate switched off expression of exo-β-1,3-glucanase and α-amylase and turned on secretion of endo-β-1,3-glucanase and a range of glycosidases. In addition, an aspartic peptidase started being expressed instead of family S53 protease. For the first time, we report production of cerato-platanin proteins by Trametes species. The secretion of cerato-platanins was observed only in response to contact with lignocellulose, thus indicating a specific role of these proteins in degradation of the lignocellulose substrates.ConclusionsOur results suggest a sequential mechanism of natural substrate degradation by T. hirsuta, in which the fungus produces different sets of enzymes to digest all main components of the substrate during cultivation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0729-0) contains supplementary material, which is available to authorized users.

BackgroundPhanerochaete chrysosporium, the model white rot basidiomycetous fungus, has the extraordinary ability to mineralize (to CO2) lignin and detoxify a variety of chemical pollutants. Its cytochrome P450 monooxygenases have recently been implied in several of these biotransformations. Our initial P450 cloning efforts in P. chrysosporium and its subsequent whole genome sequencing have revealed an extraordinary P450 repertoire ("P450ome") containing at least 150 P450 genes with yet unknown function. In order to understand the functional diversity and the evolutionary mechanisms and significance of these hemeproteins, here we report a genome-wide structural and evolutionary analysis of the P450ome of this fungus.ResultsOur analysis showed that P. chrysosporium P450ome could be classified into 12 families and 23 sub-families and is characterized by the presence of multigene families. A genome-level structural analysis revealed 16 organizationally homogeneous and heterogeneous clusters of tandem P450 genes. Analysis of our cloned cDNAs revealed structurally conserved characteristics (intron numbers and locations, and functional domains) among members of the two representative multigene P450 families CYP63 and CYP505 (P450foxy). Considering the unusually complex structural features of the P450 genes in this genome, including microexons (2–10 aa) and frequent small introns (45–55 bp), alternative splicing, as experimentally observed for CYP63, may be a more widespread event in the P450ome of this fungus. Clan-level phylogenetic comparison revealed that P. chrysosporium P450 families fall under 11 fungal clans and the majority of these multigene families appear to have evolved locally in this genome from their respective progenitor genes, as a result of extensive gene duplications and rearrangements.ConclusionP. chrysosporium P450ome, the largest known todate among fungi, is characterized by tandem gene clusters and multigene families. This enormous P450 gene diversity has evolved by extensive gene duplications and intragenomic recombinations of the progenitor genes presumably to meet the exceptionally high metabolic demand of this biodegradative group of basidiomycetous fungi in ecological niches. In this context, alternative splicing appears to further contribute to the evolution of functional diversity of the P450ome in this fungus. The evolved P450 diversity is consistent with the known vast biotransformation potential of P. chrysosporium. The presented analysis will help design future P450 functional studies to understand the underlying mechanisms of secondary metabolism and oxidative biotransformation pathways in this model white rot fungus.

Retroviruses evolved from long terminal repeat (LTR) retrotransposons by acquisition of envelope functions, and subsequently reinvaded host genomes. Together, endogenous retroviruses and LTR retrotransposons represent major components of animal, plant, and fungal genomes. Sequences from these elements have been exapted to perform essential host functions, including placental development, synaptic communication, and transcriptional regulation. They encode a Gag polypeptide, the capsid domains of which can oligomerize to form a virus-like particle. The structures of retroviral capsids have been extensively described. They assemble an immature viral particle through oligomerization of full-length Gag. Proteolytic cleavage of Gag results in a mature, infectious particle. In contrast, the absence of structural data on LTR retrotransposon capsids hinders our understanding of their function and evolutionary relationships. Here, we report the capsid morphology and structure of the archetypal Gypsy retrotransposon Ty3. We performed electron tomography (ET) of immature and mature Ty3 particles within cells. We found that, in contrast to retroviruses, these do not change size or shape upon maturation. Cryo-ET and cryo-electron microscopy of purified, immature Ty3 particles revealed an irregular fullerene geometry previously described for mature retrovirus core particles and a tertiary and quaternary arrangement of the capsid (CA) C-terminal domain within the assembled capsid that is conserved with mature HIV-1. These findings provide a structural basis for studying retrotransposon capsids, including those domesticated in higher organisms. They suggest that assembly via a structurally distinct immature capsid is a later retroviral adaptation, while the structure of mature assembled capsids is conserved between LTR retrotransposons and retroviruses.

Experimental method to quantify progressive stages of decay of wood by basidiomycete fungi

Sequencing the Fungal Tree of Life F. Martin 1 , D. Cullen 2 , D. Hibbett 3 , A. Pisabarro 4 , J. W. Spatafora 5 , S. E. Baker 6,7, I. V. Grigoriev 7 UMR INRA/UHP 1136, Interactions Arbres/Micro-Organismes, INRA-Nancy, 54280 Champenoux, France University of Wisconsin-Madison, Madison, WI, USA Clark University, Worcester, MA, USA Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Spain Dept. Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland, Washington, USA, 99352 US DOE Joint Genome Institute, Walnut Creek, California, USA March 2011 The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02- 05CH11231 DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California.

In the past, only a few incidences of esca diseased grapevines were reported from the Slanghoekand Rawsonville areas of South Africa, with the damage believed to be of little importance so that the diseasehas not been studied in South Africa. In the present study, vines with internal or external symptoms of the esca disease complex were sampled from table, raisin and wine grape cultivars from 37 production areas inthe Western Cape, Northern Cape and Limpopo provinces of that country. Most vines were greater than 10years old, but younger vines (3 and 5 years old) were also found to be infected. External symptoms, includingdieback, tiger striped leaves, berry symptoms (shrivelling, insufficient colouring) and apoplexy, resembledthose found on grapevines in Europe and the USA, although the typical tiger stripe symptom was observedless frequently. The internal stem and trunk symptoms were similar to European symptoms, and includedwhite rot, black and brown wood streaking, brown necrosis within white rot, sectorial brown necrosis andbrown/red/margins next to decay, which often included back lines delimiting white decay. The fungi isolatedmostly from the white rot were basidiomycetes species (30.4%). Black and brown wood streaking was primarily caused by Phaeomoniella chlamydospora (45.4%). Brown necrosis within the white rot was linked to colonization by basidiomycetes (20.4%), Phaeoacremonium aleophilum (15.9%) and Pa. chlamydospora (13.6%). Phaeomoniella chlamydospora (20.8%) and Botryosphaeriaceae species (10.7%) were isolated the most fromthe sectorial brown necrosis and Pa. chlamydospora (29.1%) from the brown/red margins and black lines next to decay. Given the wide distribution of esca complex wood and foliar symptoms in the grape growing regions investigated, this disease should be considered as an important limiting factor in the productive lifespan of vineyards and the quality of produce from grapevine in South Africa.

ABSTRACT: Dyes released by the textile industries pose a threat to environmental quality. Ligninolytic white-rot basidiomycetes can effectively degrade colored effluents and conventional dyes. White-rot fungi produce various isoforms of extracellular oxidases including laccase, Mn peroxidase and lignin peroxidase (LiP), which are involved in the degradation of lignin in their natural lignocellulosic substrates. The textile industry, by far the most avid user of synthetic dyes, is in need of eco-efficient solutions for its colored effluents. White rot basidiomycetous fungi comprise the only group of organisms known to completely degrade lignin. Ligninolytic enzymes have potential applications in a large number of fields, including the chemical, fuel, food, agricultural, paper, textile, cosmetic industrial sectors and more. This ligninolytic system of white-rot fungi is also directly involved in the degradation of various xenobiotic compounds apart from textile dyes. Their capacities to remove xenobiotic substances make them a useful tool for bioremediation purposes. This paper reviews involvement of ligninolytic enzymes of white rot basidiomycetes in the degradation of textiles dyes and xenobiotic compounds for their industrial and biotechnological applications.

Bark beetles have a decisive impact on fungal communities in Norway spruce stem sections