AA9 Lytic Polysaccharide Monooxygenases Research Articles

Engineering chitinolytic activity into a cellulose-active lytic polysaccharide monooxygenase provides insights into substrate specificity

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.

Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class Agaricomycetes, associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, LsAA9B, a naturally occurring protein from Lentinus similis The LsAA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of LsAA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass.

Formation of a Copper(II)-Tyrosyl Complex at the Active Site of Lytic Polysaccharide Monooxygenases Following Oxidation by H2O2.

Hydrogen peroxide is a cosubstrate for the oxidative cleavage of saccharidic substrates by copper-containing lytic polysaccharide monooxygenases (LPMOs). The rate of reaction of LPMOs with hydrogen peroxide is high, but it is accompanied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we use UV–vis, CD, XAS, EPR, VT/VH-MCD, and resonance Raman spectroscopies, augmented with mass spectrometry and DFT calculations, to show that the product of reaction of an AA9 LPMO with H2O2 at higher pHs is a singlet Cu(II)–tyrosyl radical species, which is inactive for the oxidation of saccharidic substrates. The Cu(II)–tyrosyl radical center entails the formation of significant Cu(II)–(●OTyr) overlap, which in turn requires that the plane of the d(x2–y2) SOMO of the Cu(II) is orientated toward the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a “hole-hopping” charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.

Comparison of three seemingly similar lytic polysaccharide monooxygenases from Neurospora crassa suggests different roles in plant biomass degradation

Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM. All three LPMOs were active on tamarind xyloglucan and konjac glucomannan, generating similar products but clearly differing in activity levels. Importantly, in some cases, the addition of phosphoric acid-swollen cellulose (PASC) had a major effect on activity: NcLPMO9A was active on xyloglucan only in the presence of PASC, and PASC enhanced NcLPMO9D activity on glucomannan. Interestingly, the three enzymes also exhibited large differences in their interactions with enzymatic electron donors, which could reflect that they are optimized to act with different reducing partners. All three enzymes efficiently used H2O2 as a cosubstrate, yielding product profiles identical to those obtained in O2-driven reactions with PASC, xyloglucan, or glucomannan. Our results indicate that seemingly similar LPMOs act preferentially on different types of copolymeric substructures in the plant cell wall, possibly because these LPMOs are functionally adapted to distinct niches differing in the types of available reductants.

Discovery, activity and characterisation of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae

BackgroundThe quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O2 or H2O2 as a co-substrate.ResultsAlthough classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm Lyrodus pedicellatus represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, Teredinibacter turnerae, the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that T. turnerae encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a “histidine-brace” catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, TtAA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases.ConclusionShipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont T. turnerae produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.

Sequence and Structural Analysis of AA9 and AA10 LPMOs: An Insight into the Basis of Substrate Specificity and Regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes in both the natural carbon cycle and the biorefinery industry. Understanding the molecular basis of LPMOs acting on polysaccharide substrates is helpful for improving industrial cellulase cocktails. Here we analyzed the sequences, structures, and substrate binding modes of LPMOs to uncover the factors that influence substrate specificity and regioselectivity. Our results showed that the different compositions of a motif located on L2 affect the electrostatic potentials of substrate binding surfaces, which in turn affect substrate specificities of AA10 LPMOs. A conserved Asn at a distance of 7 Å from the active center Cu might, together with the conserved Ser immediately before the second catalytic His, determine the localization of LPMOs on substrate, and thus contribute to C4-oxidizing regioselectivity. The findings in this work provide an insight into the molecular basis of substrate specificity and regioselectivity of LPMOs.

The secretome of two representative lignocellulose-decay basidiomycetes growing on sugarcane bagasse solid-state cultures

Secretome evaluations of lignocellulose-decay basidiomycetes can reveal new enzymes in selected fungal species that degrade specific substrates. Proteins discovered in such studies can support biorefinery development. Brown-rot (Gloeophyllum trabeum) and white-rot (Pleurotus ostreatus) fungi growing in sugarcane bagasse solid-state cultures produced 119 and 63 different extracellular proteins, respectively. Several of the identified enzymes are suitable for in vitro biomass conversion, including a range of cellulases (endoglucanases, cellobiohydrolases and β-glucosidases), hemicellulases (endoxylanases, α-arabinofuranosidases, α-glucuronidases and acetylxylan esterases) and carbohydrate-active auxiliary proteins, such as AA9 lytic polysaccharide monooxygenase, AA1 laccase and AA2 versatile peroxidase. Extracellular oxalate decarboxylase was also detected in both fungal species, exclusively in media containing sugarcane bagasse. Interestingly, intracellular AA6 quinone oxidoreductases were also exclusively produced under sugarcane bagasse induction in both fungi. These enzymes promote quinone redox cycling, which is used to produce Fenton’s reagents by lignocellulose-decay fungi. Hitherto undiscovered hypothetical proteins that are predicted in lignocellulose-decay fungi genomes appeared in high relative abundance in the cultures containing sugarcane bagasse, which suggests undisclosed, new biochemical mechanisms that are used by lignocellulose-decay fungi to degrade sugarcane biomass. In general, lignocellulose-decay fungi produce a number of canonical hydrolases, as well as some newly observed enzymes, that are suitable for in vitro biomass digestion in a biorefinery context.

An AA9-LPMO containing a CBM1 domain in Aspergillus nidulans is active on cellulose and cleaves cello-oligosaccharides

Lytic polysaccharide monooxygenases (LPMOs) are copper dependent enzymes that carry out oxidative cleavage of cellulose and other polysaccharides. Aspergillus nidulans, an ascomycete fungus that contains multiple AA9 LPMOs in the genome, offers an excellent model system to study their activity during the oxidative degradation of biomass. AN1602, a dual domain AA9-LPMO in A. nidulans appended with a carbohydrate-binding module, CBM1, was expressed in Pichia pastoris for analyzing oxidative cleavage on cellulosic substrates. The mass spectral and HPAEC analyses showed that the enzyme cleaves phosphoric acid swollen cellulose (PASC) in the presence of a reducing agent, yielding a range of cello-oligosaccharides. In addition to the polymeric substrate cellulose, AN1602 is also active on soluble cellohexaose, a property that is restricted to only a few characterized LPMOs. Product analysis of AN1602 cleaved cellohexaose revealed that C4 was the sole site of oxidation. The sequence and predicted structure of the catalytic domain of AN1602 matched very closely to known C4 cellohexaose active enzymes.

Methylation of the N-terminal histidine protects a lytic polysaccharide monooxygenase from auto-oxidative inactivation.

The catalytically crucial N-terminal histidine (His1) of fungal lytic polysaccharide monooxygenases (LPMOs) is post-translationally modified to carry a methylation. The functional role of this methylation remains unknown. We have carried out an in-depth functional comparison of two variants of a family AA9 LPMO from Thermoascus aurantiacus (TaLPMO9A), one with, and one without the methylation on His1. Various activity assays showed that the two enzyme variants are identical in terms of substrate preferences, cleavage specificities and the ability to activate molecular oxygen. During the course of this work, new functional features of TaLPMO9A were discovered, in particular the ability to cleave xyloglucan, and these features were identical for both variants. Using a variety of techniques, we further found that methylation has minimal effects on the pKa of His1, the affinity for copper and the redox potential of bound copper. The two LPMOs did, however, show clear differences in their resistance against oxidative damage. Studies with added hydrogen peroxide confirmed recent claims that low concentrations of H2 O2 boost LPMO activity, whereas excess H2 O2 leads to LPMO inactivation. The methylated variant of TaLPMO9A, produced in Aspergillus oryzae, was more resistant to excess H2 O2 and showed better process performance when using conditions that promote generation of reactive-oxygen species. LPMOs need to protect themselves from reactive oxygen species generated in their active sites and this study shows that methylation of the fully conserved N-terminal histidine provides such protection.

Multipoint Precision Binding of Substrate Protects Lytic Polysaccharide Monooxygenases from Self-Destructive Off-Pathway Processes.

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the degradation of polysaccharides in biomass by catalyzing powerful oxidative chemistry using only a single copper ion as a cofactor. Despite the natural abundance and importance of these powerful monocopper enzymes, the structural determinants of their functionality have remained largely unknown. We have used site-directed mutagenesis to probe the roles of 13 conserved amino acids located on the flat substrate-binding surface of CBP21, a chitin-active family AA10 LPMO from Serratia marcescens, also known as SmLPMO10A. Single mutations of residues that do not interact with the catalytic copper site, but rather are involved in substrate binding had remarkably strong effects on overall enzyme performance. Analysis of product formation over time showed that these mutations primarily affected enzyme stability. Investigation of protein integrity using proteomics technologies showed that loss of activity was caused by oxidation of essential residues in the enzyme active site. For most enzyme variants, reduced enzyme stability correlated with a reduced level of binding to chitin, suggesting that adhesion to the substrate prevents oxidative off-pathway processes that lead to enzyme inactivation. Thus, the extended and highly evolvable surfaces of LPMOs are tailored for precise multipoint substrate binding, which provides the confinement that is needed to harness and control the remarkable oxidative power of these enzymes. These findings are important for the optimized industrial use of LPMOs as well as the design of LPMO-inspired catalysts.

Catalytic Mechanism of Fungal Lytic Polysaccharide Monooxygenases Investigated by First-Principles Calculations.

Lytic polysaccharide monooxygenases (LPMOs) are Cu-containing enzymes that facilitate the degradation of recalcitrant polysaccharides by the oxidative cleavage of glycosidic bonds. They are gaining rapidly increasing attention as key players in biomass conversion, especially for the production of second-generation biofuels. Elucidation of the detailed mechanism of the LPMO reaction is a major step toward the assessment and optimization of LPMO efficacy in industrial biotechnology, paving the way to utilization of sustainable fuel sources. Here, we used density functional theory calculations to study the reaction pathways suggested to date, exploiting a very large active-site model for a fungal AA9 LPMO and using a celloheptaose unit as a substrate mimic. We identify a copper oxyl intermediate as being responsible for H-atom abstraction from the substrate, followed by a rapid, water-assisted hydroxyl rebound, leading to substrate hydroxylation.

Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type

Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery.

The effect of a lytic polysaccharide monooxygenase and a xylanase from Gloeophyllum trabeum on the enzymatic hydrolysis of lignocellulosic residues using a commercial cellulase.

Hydrolysis of lignocellulosic biomass depends on the concerted actions of cellulases and accessory proteins. In this work we examined the combined action of two auxiliary proteins from the brown rot fungus Gloeophyllum trabeum: a family AA9 lytic polysaccharide monooxygenase (GtLPMO) and a GH10 xylanase (GtXyn10A). The enzymes were produced in the heterologous host Pichia pastoris. In the presence of an electron source, GtLPMO increased the activity of a commercial cellulase on filter paper, and the xylanase activity of GtXyn10A on beechwood xylan. Mixtures of GtLPMO, GtXyn10A and Celluclast 1.5L were used for hydrolysis of pretreated wheat straw. Results showed that a mixture of 60% Celluclast 1.5L, 20% GtXyn10A and 20% GtLPMO increased total reducing sugar production by 54%, while the conversions of glucan to glucose and xylan to xylose were increased by 40 and 57%, respectively. This suggests that GtLPMO can contribute to lignocellulose hydrolysis, not only by oxidative activity on glycosidic bonds, but also to hemicellulose through the oxidation of xylosyl bonds in xylan. The concerted action of these auxiliary enzymes may significantly improve large-scale recovery of sugars from lignocellulose.

Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates

Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides. Here we present a functional and structural characterization of two closely related AA9-family LPMOs from Lentinus similis (LsAA9A) and Collariella virescens (CvAA9A). LsAA9A and CvAA9A cleave a range of polysaccharides, including cellulose, xyloglucan, mixed-linkage glucan and glucomannan. LsAA9A additionally cleaves isolated xylan substrates. The structures of CvAA9A and of LsAA9A bound to cellulosic and non-cellulosic oligosaccharides provide insight into the molecular determinants of their specificity. Spectroscopic measurements reveal differences in copper co-ordination upon the binding of xylan and glucans. LsAA9A activity is less sensitive to the reducing agent potential when cleaving xylan, suggesting that distinct catalytic mechanisms exist for xylan and glucan cleavage. Overall, these data show that AA9 LPMOs can display different apparent substrate specificities dependent upon both productive protein–carbohydrate interactions across a binding surface and also electronic considerations at the copper active site.

A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes was evaluated in combination with endoglucanase and beta-glucosidase. Two enzymes (TrCel61A and Aspte6) showed the ability to release more than 36% of the pretreated soy spent flake glucose – a greater than 75% increase over the same treatment without LPMO addition.

Boosting LPMO-driven lignocellulose degradation by polyphenol oxidase-activated lignin building blocks

BackgroundMany fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). The application of LPMOs is expected to contribute to ecologically friendly conversion of biomass into fuels and chemicals. Moreover, applications of LPMO-modified cellulose-based products may be envisaged within the food or material industry.ResultsHere, we show an up to 75-fold improvement in LPMO-driven cellulose degradation using polyphenol oxidase-activated lignin building blocks. This concerted enzymatic process involves the initial conversion of monophenols into diphenols by the polyphenol oxidase MtPPO7 from Myceliophthora thermophila C1 and the subsequent oxidation of cellulose by MtLPMO9B. Interestingly, MtPPO7 shows preference towards lignin-derived methoxylated monophenols. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota reveals a high correlation between MtPPO7 and AA9 LPMO genes.ConclusionsThe activity towards methoxylated phenolic compounds distinguishes MtPPO7 from well-known PPOs, such as tyrosinases, and ensures that MtPPO7 is an excellent redox partner of LPMOs. The correlation between MtPPO7 and AA9 LPMO genes is indicative for the importance of the coupled action of different monooxygenases in the concerted degradation of lignocellulosic biomass. These results will contribute to a better understanding in both lignin deconstruction and enzymatic lignocellulose oxidation and potentially improve the exploration of eco-friendly routes for biomass utilization in a circular economy.

The Podospora anserina lytic polysaccharide monooxygenase PaLPMO9H catalyzes oxidative cleavage of diverse plant cell wall matrix glycans

BackgroundThe enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMO) that catalyze oxidative cleavage of polysaccharides. These powerful enzymes are secreted by a large number of fungal saprotrophs and are important components of commercial enzyme cocktails used for industrial biomass conversion. Among the 33 AA9 LPMOs encoded by the genome of Podospora anserina, the PaLPMO9H enzyme catalyzes mixed C1/C4 oxidative cleavage of cellulose and cello-oligosaccharides. Activity of PaLPMO9H on several hemicelluloses has been suggested, but the regioselectivity of the cleavage remained to be determined.ResultsIn this study, we investigated the activity of PaLPMO9H on mixed-linkage glucans, xyloglucan and glucomannan using tandem mass spectrometry and ion mobility–mass spectrometry. Structural analysis of the released products revealed that PaLPMO9H catalyzes C4 oxidative cleavage of mixed-linkage glucans and mixed C1/C4 oxidative cleavage of glucomannan and xyloglucan. Gem-diols and ketones were produced at the non-reducing end, while aldonic acids were produced at the reducing extremity of the products.ConclusionThe ability of PaLPMO9H to target polysaccharides, differing from cellulose by their linkages, glycosidic composition and/or presence of sidechains, could be advantageous for this coprophilous fungus when catabolizing highly variable polysaccharides and for the development of optimized enzyme cocktails in biorefineries.

Cultivation of Podospora anserina on soybean hulls results in an efficient enzyme cocktail for plant biomass hydrolysis

The coprophilic ascomycete fungus Podospora anserina was cultivated on three different plant biomasses, i.e. cotton seed hulls (CSH), soybean hulls (SBH) and acid-pretreated wheat straw (WS) for four days, and the potential of the produced enzyme mixtures was compared in the enzymatic saccharification of the corresponding lignocellulose feedstocks. The enzyme cocktail P. anserina produced after three days of growth on SBH showed superior capacity to release reducing sugars from all tested plant biomass feedstocks compared to the enzyme mixtures from CSH and WS cultures. Detailed proteomics analysis of the culture supernatants revealed that SBH contained the most diverse set of enzymes targeted on plant cell wall polymers and was particularly abundant in xylan, mannan and pectin acting enzymes. The importance of lytic polysaccharide monooxygenases (LPMOs) in plant biomass deconstruction was supported by identification of 20 out of 33 AA9 LPMOs in the SBH cultures. The results highlight the suitability of P. anserina as a source of plant cell wall degrading enzymes for biotechnological applications and the importance of selecting the most optimal substrate for the production of enzyme mixtures.

Activation of bacterial lytic polysaccharide monooxygenases with cellobiose dehydrogenase.

Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate-active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11, and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper-dependent and utilize a redox mechanism to cleave glycosidic bonds that is dependent on molecular oxygen and an external electron donor. The electrons can be provided by various sources, such as chemical compounds (e.g., ascorbate) or by enzymes (e.g., cellobiose dehydrogenases, CDHs, from fungi). Here, we demonstrate that a fungal CDH from Myriococcum thermophilum (MtCDH), can act as an electron donor for bacterial family AA10 LPMOs. We show that employing an enzyme as electron donor is advantageous since this enables a kinetically controlled supply of electrons to the LPMO. The rate of chitin oxidation by CBP21 was equal to that of cosubstrate (lactose) oxidation by MtCDH, verifying the usage of two electrons in the LPMO catalytic mechanism. Furthermore, since lactose oxidation correlates directly with the rate of LPMO catalysis, a method for indirect determination of LPMO activity is implicated. Finally, the one electron reduction of the CBP21 active site copper by MtCDH was determined to be substantially faster than chitin oxidation by the LPMO. Overall, MtCDH seems to be a universal electron donor for both bacterial and fungal LPMOs, indicating that their electron transfer mechanisms are similar.

Time-scale dynamics of proteome and transcriptome of the white-rot fungus Phlebia radiata: growth on spruce wood and decay effect on lignocellulose

BackgroundThe white-rot Agaricomycetes species Phlebia radiata is an efficient wood-decaying fungus degrading all wood components, including cellulose, hemicellulose, and lignin. We cultivated P. radiata in solid state cultures on spruce wood, and extended the experiment to 6 weeks to gain more knowledge on the time-scale dynamics of protein expression upon growth and wood decay. Total proteome and transcriptome of P. radiata were analyzed by peptide LC–MS/MS and RNA sequencing at specific time points to study the enzymatic machinery on the fungus’ natural growth substrate.ResultsAccording to proteomics analyses, several CAZy oxidoreductase class-II peroxidases with glyoxal and alcohol oxidases were the most abundant proteins produced on wood together with enzymes important for cellulose utilization, such as GH7 and GH6 cellobiohydrolases. Transcriptome additionally displayed expression of multiple AA9 lytic polysaccharide monooxygenases indicative of oxidative cleavage of wood carbohydrate polymers. Large differences were observed for individual protein quantities at specific time points, with a tendency of enhanced production of specific peroxidases on the first 2 weeks of growth on wood. Among the 10 class-II peroxidases, new MnP1-long, characterized MnP2-long and LiP3 were produced in high protein abundances, while LiP2 and LiP1 were upregulated at highest level as transcripts on wood together with the oxidases and one acetyl xylan esterase, implying their necessity as primary enzymes to function against coniferous wood lignin to gain carbohydrate accessibility and fungal growth. Majority of the CAZy encoding transcripts upregulated on spruce wood represented activities against plant cell wall and were identified in the proteome, comprising main activities of white-rot decay.ConclusionsOur data indicate significant changes in carbohydrate-active enzyme expression during the six-week surveillance of P. radiata growing on wood. Response to wood substrate is seen already during the first weeks. The immediate oxidative enzyme action on lignin and wood cell walls is supported by detected lignin substructure sidechain cleavages, release of phenolic units, and visual changes in xylem cell wall ultrastructure. This study contributes to increasing knowledge on fungal genetics and lignocellulose bioconversion pathways, allowing us to head for systems biology, development of biofuel production, and industrial applications on plant biomass utilizing wood-decay fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0608-9) contains supplementary material, which is available to authorized users.