Characterization of lignin-degrading enzymes (LDEs) from a dimorphic novel fungus and identification of products of enzymatic breakdown of lignin.
Ontology highlight
ABSTRACT:
SUBMITTER: Sahadevan LDM
PROVIDER: S-EPMC4752945 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Inonotus obliquus is a pathogenic fungus found in living trees and has been widely used as a traditional medicine for cancer therapy. Although lignocellulose-degrading enzymes are involved in the early stages of host infection, the parasitic life cycle of this fungus has not been fully understood. In this study, we aimed to investigate the activities of laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) from I. obliquus cultivated in Kirk's medium. The fungus was subjected to genome sequencing, and genes related to wood degradation were identified. The draft genome sequence of this fungus comprised 21,203 predicted protein-coding genes, of which 134 were estimated to be related to wood degradation. Among these, 47 genes associated with lignin degradation were found to have the highest number of mnp genes. Furthermore, we cloned the cDNA encoding a putative MnP, referred to as IoMnP1, and characterized its molecular structure. The results show that IoMnP1 has catalytic properties analogous to MnP. Phylogenetic analysis also confirmed that IoMnP1 was closely related to the MnPs from Pyrrhoderma noxium, Fomitiporia mediterranea, and Sanghuangporus baumii, which belong to the same family of Hymenochaetaceae. From the above results, we suggest that IoMnP1 is a member of MnPs.
| S-EPMC10258189 | biostudies-literature
Project description:The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degradation, an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period associated with the appearance of these enzymes. These first peroxidases were not able to degrade lignin directly and used diffusible metal cations to attack its phenolic moiety. The phylogenetic analysis of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are included, suggests that later in evolution these enzymes would have acquired the ability to degrade nonphenolic lignin using a tryptophanyl radical interacting with the bulky polymer at the surface of the enzyme. Here, we track this powerful strategy for lignin degradation as a phenotypic trait in fungi and show that it is not an isolated event in the evolution of Polyporales. Using ancestral enzyme resurrection, we study the molecular changes that led to the appearance of the same surface oxidation site in two distant peroxidase lineages. By characterization of the resurrected enzymes, we demonstrate convergent evolution at the amino acid level during the evolution of these fungi and track the different changes leading to phylogenetically distant ligninolytic peroxidases from ancestors lacking the ability to degrade nonphenolic lignin.
| S-EPMC6016776 | biostudies-literature
Project description:Microarrays have become established tools for describing microbial systems, however the assessment of expression profiles for environmental microbial communities still presents unique challenges. Notably, the concentration of particular transcripts are likely very dilute relative to the pool of total RNA, and PCR-based amplification strategies are vulnerable to amplification biases and the appropriate primer selection. Thus, we apply a signal amplification approach, rather than template amplification, to analyze the expression of selected lignin-degrading enzymes in soil. Controls in the form of known amplicons and cDNA from Phanerochaete chrysosporium were included and mixed with the soil cDNA both before and after the signal amplification in order to assess the dynamic range of the microarray. We demonstrate that restored prairie soil expresses a diverse range of lignin-degrading enzymes following incubation with lignin substrate, while farmed agricultural soil does not. The mixed additions of control cDNA with soil cDNA indicate that the mixed biomass in the soil does interfere with low abundance transcript changes, nevertheless our microarray approach consistently reports the most robust signals. Keywords: comparative analysis, microbial ecology, soil microbial communities We used lignin degradation as a model process to demonstrate the use of an oligonucleotide microarray for directly detecting gene expression in soil communities using signal amplification instead of template amplification to avoid the introduction of PCR bias. In the current study, we analyzed mRNA isolated from two distinct soil microbial communities and demonstrate our ability to detect the expression of a small subset of lignin degrading genes following exposure to a lignitic substrate. We also included purified control amplicons and mixed target experiments with pure P. chrysosporium genomic cDNA to determine the level of interference from soil biomass on target hybridization.
2010-05-18 | E-GEOD-13977 | biostudies-arrayexpress
Project description:Microarrays have become established tools for describing microbial systems, however the assessment of expression profiles for environmental microbial communities still presents unique challenges. Notably, the concentration of particular transcripts are likely very dilute relative to the pool of total RNA, and PCR-based amplification strategies are vulnerable to amplification biases and the appropriate primer selection. Thus, we apply a signal amplification approach, rather than template amplification, to analyze the expression of selected lignin-degrading enzymes in soil. Controls in the form of known amplicons and cDNA from Phanerochaete chrysosporium were included and mixed with the soil cDNA both before and after the signal amplification in order to assess the dynamic range of the microarray. We demonstrate that restored prairie soil expresses a diverse range of lignin-degrading enzymes following incubation with lignin substrate, while farmed agricultural soil does not. The mixed additions of control cDNA with soil cDNA indicate that the mixed biomass in the soil does interfere with low abundance transcript changes, nevertheless our microarray approach consistently reports the most robust signals. Keywords: comparative analysis, microbial ecology, soil microbial communities
2009-06-01 | GSE13977 | GEO
Project description:BackgroundValorization of lignin has the potential to significantly improve the economics of lignocellulosic biorefineries. However, its complex structure makes conversion to useful products elusive. One promising approach is depolymerization of lignin and subsequent bioconversion of breakdown products into value-added compounds. Optimizing transport of these depolymerization products into one or more organism(s) for biological conversion is important to maximize carbon utilization and minimize toxicity. Current methods assess internalization of depolymerization products indirectly-for example, growth on, or toxicity of, a substrate. Furthermore, no method has been shown to provide visualization of depolymerization products in individual cells.ResultsWe applied mass spectrometry to provide direct measurements of relative internalized concentrations of several lignin depolymerization compounds and single-cell microscopy methods to visualize cell-to-cell differences in internalized amounts of two lignin depolymerization compounds. We characterized internalization of 4-hydroxybenzoic acid, vanillic acid, p-coumaric acid, syringic acid, and the model dimer guaiacylglycerol-beta-guaiacyl ether (GGE) in the lignolytic organisms Phanerochaete chrysosporium and Enterobacter lignolyticus and in the non-lignolytic but genetically tractable organisms Saccharomyces cerevisiae and Escherichia coli. The results show varying degrees of internalization in all organisms for all the tested compounds, including the model dimer, GGE. Phanerochaete chrysosporium internalizes all compounds in non-lignolytic and lignolytic conditions at comparable levels, indicating that the transporters for these compounds are not specific to the lignolytic secondary metabolic system. Single-cell microscopy shows that internalization of vanillic acid and 4-hydroxybenzoic acid analogs varies greatly among individual fungal and bacterial cells in a given population. Glucose starvation and chemical inhibition of ATP hydrolysis during internalization significantly reduced the internalized amount of vanillic acid in bacteria.ConclusionsMass spectrometry and single-cell microscopy methods were developed to establish a toolset for providing direct measurement and visualization of relative internal concentrations of mono- and di-aryl compounds in microbes. Utilizing these methods, we observed broad variation in intracellular concentration between organisms and within populations and this may have important consequences for the efficiency and productivity of an industrial process for bioconversion. Subsequent application of this toolset will be useful in identifying and characterizing specific transporters for lignin-derived mono- and di-aryl compounds.
| S-EPMC6607601 | biostudies-literature
Project description:The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.
| S-EPMC3351359 | biostudies-literature
Project description:Previous works have demonstrated that ligninolytic enzymes mediated effective degradation of lignin wastes. The degrading ability greatly relied on the interactions of ligninolytic enzymes with lignin. Ligninolytic enzymes mainly contain laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP). In the present study, the binding modes of lignin to Lac, LiP and MnP were systematically determined, respectively. Robustness of these modes was further verified by molecular dynamics (MD) simulations. Residues GLU460, PRO346 and SER113 in Lac, residues ARG43, ALA180 and ASP183 in LiP and residues ARG42, HIS173 and ARG177 in MnP were most crucial in binding of lignin, respectively. Interactional analyses showed hydrophobic contacts were most abundant, playing an important role in the determination of substrate specificity. This information is an important contribution to the details of enzyme-catalyzed reactions in the process of lignin biodegradation, which can be used as references for designing enzyme mutants with a better lignin-degrading activity.
| S-EPMC3183068 | biostudies-literature
Project description:Lignin is the most abundant aromatic polymer in nature and a promising renewable source for the provision of aromatic platform chemicals and biofuels. β-Etherases are enzymes with a promising potential for application in lignin depolymerization due to their selectivity in the cleavage of β-O-4 aryl ether bonds. However, only a very limited number of these enzymes have been described and characterized so far. Using peptide pattern recognition (PPR) as well as phylogenetic analyses, 96 putatively novel β-etherases have been identified, some even originating from bacteria outside the order Sphingomonadales A set of 13 diverse enzymes was selected for biochemical characterization, and β-etherase activity was confirmed for all of them. Some enzymes displayed up to 3-fold higher activity than previously known β-etherases. Moreover, conserved sequence motifs specific for either LigE- or LigF-type enzymes were deduced from multiple-sequence alignments and the PPR-derived peptides. In combination with structural information available for the β-etherases LigE and LigF, insight into the potential structural and/or functional role of conserved residues within these sequence motifs is provided. Phylogenetic analyses further suggest the presence of additional bacterial enzymes with potential β-etherase activity outside the classical LigE- and LigF-type enzymes as well as the recently described heterodimeric β-etherases.IMPORTANCE The use of biomass as a renewable source and replacement for crude oil for the provision of chemicals and fuels is of major importance for current and future societies. Lignin, the most abundant aromatic polymer in nature, holds promise as a renewable starting material for the generation of required aromatic structures. However, a controlled and selective lignin depolymerization to yield desired aromatic structures is a very challenging task. In this regard, bacterial β-etherases are especially interesting, as they are able to cleave the most abundant bond type in lignin with high selectivity. With this study, we significantly expanded the toolbox of available β-etherases for application in lignin depolymerization and discovered more active as well as diverse enzymes than previously known. Moreover, the identification of further β-etherases by sequence database mining in the future will be facilitated considerably through our deduced etherase-specific sequence motifs.
| S-EPMC6952239 | biostudies-literature
Project description:To investigate how ligninolytic peroxidases acquired the uniquely high redox potential they show today, their ancestors were resurrected and characterized. Unfortunately, the transient Compounds I (CI) and II (CII) from peroxide activation of the enzyme resting state (RS) are unstable. Therefore, the reduction potentials (E°') of the three redox couples (CI/RS, CI/CII and CII/RS) were estimated (for the first time in a ligninolytic peroxidase) from equilibrium concentrations analyzed by stopped-flow UV/Vis spectroscopy. Interestingly, the E°' of rate-limiting CII reduction to RS increased 70 mV from the common peroxidase ancestor to extant lignin peroxidase (LiP), and the same boost was observed for CI/RS and CI/CII, albeit with higher E°' values. A straightforward correlation was found between the E°' value and the progressive displacement of the proximal histidine Hϵ1 chemical shift in the NMR spectra, due to the higher paramagnetic effect of the heme Fe3+ . More interestingly, the E°' and NMR data also correlated with the evolutionary time, revealing that ancestral peroxidases increased their reduction potential in the evolution to LiP thanks to molecular rearrangements in their heme pocket during the last 400 million years.
| S-EPMC6582443 | biostudies-literature
Project description:Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17?-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2?±?6.7?U g?¹ of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T.?versicolor, Irpex lacteus was also able to remove 10?mg l?¹ EE2 within 3 days of cultivation. While an increase to I.?lacteus?MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation.
| S-EPMC3815924 | biostudies-literature