Publications by authors named "Jean-Guy Berrin"

79 Publications

Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs).

J Biol Chem 2020 Nov 24;296:100086. Epub 2020 Nov 24.

INRAE, Aix-Marseille University, Polytech Marseille, UMR1163 BBF, Marseille, France. Electronic address:

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides, thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose, but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides, and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharide degradation suggest that the subtle differences in amino-acid sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO subclusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.
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http://dx.doi.org/10.1074/jbc.RA120.015545DOI Listing
November 2020

Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs).

J Biol Chem 2020 Nov 16. Epub 2020 Nov 16.

BBF, INRAE, France.

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharides degradation suggests that the subtle differences in amino acids sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO sub-clusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.
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http://dx.doi.org/10.1074/jbc.RA120.015545DOI Listing
November 2020

Evaluation of the Enzymatic Arsenal Secreted by During Growth on Sugarcane Bagasse With a Focus on LPMOs.

Front Bioeng Biotechnol 2020 25;8:1028. Epub 2020 Aug 25.

INRAE, Faculté des Sciences de Luminy, Aix Marseille Université, UMR 1163 Biodiversité et Biotechnologie Fongiques, Polytech Marseille, Marseille, France.

The high demand for energy and the increase of the greenhouse effect propel the necessity to develop new technologies to efficiently deconstruct the lignocellulosic materials into sugars monomers. Sugarcane bagasse is a rich polysaccharide residue from sugar and alcohol industries. The thermophilic fungus (syn. ) is an interesting model to study the enzymatic degradation of biomass. The genome of encodes an extensive repertoire of cellulolytic enzymes including 23 lytic polysaccharide monooxygenases (LPMOs) from the Auxiliary Activity family 9 (AA9), which are known to oxidatively cleave the β-1,4 bonds and boost the cellulose conversion in a biorefinery context. To achieve a deeper understanding of the enzymatic capabilities of on sugarcane bagasse, we pretreated this lignocellulosic residue with different methods leading to solids with various cellulose/hemicellulose/lignin proportions and grew on these substrates. The secreted proteins were analyzed using proteomics taking advantage of two mass spectrometry methodologies. This approach unraveled the secretion of many CAZymes belonging to the Glycosyl Hydrolase (GH) and AA classes including several LPMOs that may contribute to the biomass degradation observed during fungal growth. Two AA9 LPMOs, called LPMO9B and LPMO9H, were selected from secretomic data and enzymatically characterized. Although LPMO9B and LPMO9H were both active on cellulose, they differed in terms of optimum temperatures and regioselectivity releasing either C1 or C1-C4 oxidized oligosaccharides, respectively. LPMO activities were also measured on sugarcane bagasse substrates with different levels of complexity. The boosting effect of these LPMOs on bagasse sugarcane saccharification by a commercial cocktail was also observed. The partially delignified bagasse was the best substrate considering the oxidized oligosaccharides released and the acid treated bagasse was the best one in terms of saccharification boost.
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http://dx.doi.org/10.3389/fbioe.2020.01028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477043PMC
August 2020

A new synergistic relationship between xylan-active LPMO and xylobiohydrolase to tackle recalcitrant xylan.

Biotechnol Biofuels 2020 10;13:142. Epub 2020 Aug 10.

Industrial Biotechnology and Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780 Athens, Greece.

Background: Hemicellulose accounts for a significant part of plant biomass, and still poses a barrier to the efficient saccharification of lignocellulose. The recalcitrant part of hemicellulose is a serious impediment to the action of cellulases, despite the use of xylanases in the cellulolytic cocktail mixtures. However, the complexity and variety of hemicelluloses in different plant materials require the use of highly specific enzymes for a complete breakdown. Over the last few years, new fungal enzymes with novel activities on hemicelluloses have emerged. In the present study, we explored the synergistic relationships of the xylan-active AA14 lytic polysaccharide monooxygenase (LPMO), AA14B, with the recently discovered glucuronoxylan-specific xylanase Xyn30A, of the (sub)family GH30_7, displaying xylobiohydrolase activity, and with commercial cellobiohydrolases, on pretreated natural lignocellulosic substrates.

Results: AA14B and Xyn30A showed a strong synergistic interaction on the degradation of the recalcitrant part of xylan. AA14B was able to increase the release of xylobiose from Xyn30A, showing a degree of synergism (DS) of 3.8 on birchwood cellulosic fibers, and up to 5.7 on pretreated beechwood substrates. The increase in activity was dose- and time- dependent. A screening study on beechwood materials pretreated with different methods showed that the effect of the AA14B-Xyn30A synergism was more prominent on substrates with low hemicellulose content, indicating that AA14B is mainly active on the recalcitrant part of xylan, which is in close proximity to the underlying cellulose fibers. Simultaneous addition of both enzymes resulted in higher DS than sequential addition. Moreover, AA14B was found to enhance cellobiose release from cellobiohydrolases during hydrolysis of pretreated lignocellulosic substrates, as well as microcrystalline cellulose.

Conclusions: The results of the present study revealed a new synergistic relationship not only among two recently discovered xylan-active enzymes, the LPMO AA14B, and the GH30_7 glucuronoxylan-active xylobiohydrolase Xyn30A, but also among AA14B and cellobiohydrolases. We hypothesize that AA14B creates free ends in the xylan polymer, which can be used as targets for the action of Xyn30A. The results are of special importance for the design of next-generation enzymatic cocktails, able to efficiently remove hemicelluloses, allowing complete saccharification of cellulose in plant biomass.
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http://dx.doi.org/10.1186/s13068-020-01777-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419196PMC
August 2020

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus.

DNA Res 2020 Apr;27(2)

INRAE, UMR1163, Biodiversity and Biotechnology of Fungi, Aix Marseille University, 13009 Marseille, France.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.
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http://dx.doi.org/10.1093/dnares/dsaa011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406137PMC
April 2020

Enzymes to unravel bioproducts architecture.

Biotechnol Adv 2020 Jul - Aug;41:107546. Epub 2020 Apr 8.

INRAE, Université de Reims Champagne Ardenne, FARE, F-51100 Reims, France. Electronic address:

Enzymes are essential and ubiquitous biocatalysts involved in various metabolic pathways and used in many industrial processes. Here, we reframe enzymes not just as biocatalysts transforming bioproducts but also as sensitive probes for exploring the structure and composition of complex bioproducts, like meat tissue, dairy products and plant materials, in both food and non-food bioprocesses. This review details the global strategy and presents the most recent investigations to prepare and use enzymes as relevant probes, with a focus on glycoside-hydrolases involved in plant deconstruction and proteases and lipases involved in food digestion. First, to expand the enzyme repertoire to fit bioproduct complexity, novel enzymes are mined from biodiversity and can be artificially engineered. Enzymes are further characterized by exploring sequence/structure/dynamics/function relationships together with the environmental factors influencing enzyme interactions with their substrates. Then, the most advanced experimental and theoretical approaches developed for exploring bioproducts at various scales (from nanometer to millimeter) using active and inactive enzymes as probes are illustrated. Overall, combining multimodal and multiscale approaches brings a better understanding of native-form or transformed bioproduct architecture and composition, and paves the way to mainstream the use of enzymes as probes.
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http://dx.doi.org/10.1016/j.biotechadv.2020.107546DOI Listing
August 2020

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases.

J Am Chem Soc 2020 03 26;142(10):4648-4662. Epub 2020 Feb 26.

York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York YO10 5DD, U.K.

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.
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http://dx.doi.org/10.1021/jacs.9b11351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068720PMC
March 2020

A fungal family of lytic polysaccharide monooxygenase-like copper proteins.

Nat Chem Biol 2020 03 13;16(3):345-350. Epub 2020 Jan 13.

INRA, Biodiversité et Biotechnologie Fongiques (BBF), UMR1163, Aix Marseille Université, Marseille, France.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation.
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http://dx.doi.org/10.1038/s41589-019-0438-8DOI Listing
March 2020

Influence of the carbohydrate-binding module on the activity of a fungal AA9 lytic polysaccharide monooxygenase on cellulosic substrates.

Biotechnol Biofuels 2019 3;12:206. Epub 2019 Sep 3.

2Biodiversité et Biotechnologie Fongiques, UMR1163, INRA, Aix Marseille Université, Marseille, France.

Background: Cellulose-active lytic polysaccharide monooxygenases (LPMOs) secreted by filamentous fungi play a key role in the degradation of recalcitrant lignocellulosic biomass. They can occur as multidomain proteins fused to a carbohydrate-binding module (CBM). From a biotech perspective, LPMOs are promising innovative tools for producing nanocelluloses and biofuels, but their direct action on cellulosic substrates is not fully understood.

Results: In this study, we probed the role of the CBM from family 1 (CBM1) appended to the LPMO9H from (LPMO9H) using model cellulosic substrates. Deletion of the CBM1 weakened the binding to cellulose nanofibrils, amorphous and crystalline cellulose. Although the release of soluble sugars from cellulose was drastically reduced under standard conditions, the truncated LPMO retained some activity on soluble oligosaccharides. The cellulolytic action of the truncated LPMO was demonstrated using synergy experiments with a cellobiohydrolase (CBH). The truncated LPMO was still able to improve the efficiency of the CBH on cellulose nanofibrils in the same range as the full-length LPMO. Increasing the substrate concentration enhanced the performance of LPMO9H without CBM in terms of product release. Interestingly, removing the CBM also altered the regioselectivity of LPMO9H, significantly increasing cleavage at the C1 position. Analysis of the insoluble fraction of cellulosic substrates evaluated by optical and atomic force microscopy confirmed that the CBM1 module was not strictly required to promote disruption of the cellulose network.

Conclusions: Absence of the CBM1 does not preclude the activity of the LPMO on cellulose but its presence has an important role in driving the enzyme to the substrate and releasing more soluble sugars (both oxidized and non-oxidized), thus facilitating the detection of LPMO activity at low substrate concentration. These results provide insights into the mechanism of action of fungal LPMOs on cellulose to produce nanocelluloses and biofuels.
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http://dx.doi.org/10.1186/s13068-019-1548-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721207PMC
September 2019

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

J Biol Chem 2019 11 30;294(45):17117-17130. Epub 2019 Aug 30.

Department of Chemistry, University of Copenhagen, 2100 Copenhagen, Denmark

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 , 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, AA9B, a naturally occurring protein from The AA9B 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 AA9B 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.
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http://dx.doi.org/10.1074/jbc.RA119.009223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851306PMC
November 2019

Dynamic and Functional Profiling of Xylan-Degrading Enzymes in Secretomes Using Activity-Based Probes.

ACS Cent Sci 2019 Jun 24;5(6):1067-1078. Epub 2019 May 24.

Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands.

Plant polysaccharides represent a virtually unlimited feedstock for the generation of biofuels and other commodities. However, the extraordinary recalcitrance of plant polysaccharides toward breakdown necessitates a continued search for enzymes that degrade these materials efficiently under defined conditions. Activity-based protein profiling provides a route for the functional discovery of such enzymes in complex mixtures and under industrially relevant conditions. Here, we show the detection and identification of β-xylosidases and -β-1,4-xylanases in the secretomes of , by the use of chemical probes inspired by the β-glucosidase inhibitor cyclophellitol. Furthermore, we demonstrate the use of these activity-based probes (ABPs) to assess enzyme-substrate specificities, thermal stabilities, and other biotechnologically relevant parameters. Our experiments highlight the utility of ABPs as promising tools for the discovery of relevant enzymes useful for biomass breakdown.
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http://dx.doi.org/10.1021/acscentsci.9b00221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598175PMC
June 2019

Lytic polysaccharide monooxygenases (LPMOs) facilitate cellulose nanofibrils production.

Biotechnol Biofuels 2019 24;12:156. Epub 2019 Jun 24.

1UR1268 Biopolymères Interactions Assemblages, INRA, 44316 Nantes, France.

Background: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharides through an oxidative mechanism. These enzymes are major contributors to the recycling of carbon in nature and are currently used in the biorefinery industry. LPMOs are commonly used in synergy with cellulases to enhance biomass deconstruction. However, there are few examples of the use of monocomponent LPMOs as a tool for cellulose fibrillation. In this work, we took advantage of the LPMO action to facilitate disruption of wood cellulose fibers as a strategy to produce nanofibrillated cellulose (NFC).

Results: The fungal LPMO from AA9 family (LPMO9E) was used in this study as it displays high specificity toward cellulose and its recombinant production in bioreactor is easily upscalable. The treatment of birchwood fibers with LPMO9E resulted in the release of a mixture of C1-oxidized oligosaccharides without any apparent modification in fiber morphology and dimensions. The subsequent mechanical shearing disintegrated the LPMO-pretreated samples yielding nanoscale cellulose elements. Their gel-like aspect and nanometric dimensions demonstrated that LPMOs disrupt the cellulose structure and facilitate the production of NFC.

Conclusions: This study demonstrates the potential use of LPMOs as a pretreatment in the NFC production process. LPMOs weaken fiber cohesion and facilitate fiber disruption while maintaining the crystallinity of cellulose.
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http://dx.doi.org/10.1186/s13068-019-1501-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589874PMC
June 2019

Tracking of enzymatic biomass deconstruction by fungal secretomes highlights markers of lignocellulose recalcitrance.

Biotechnol Biofuels 2019 1;12:76. Epub 2019 Apr 1.

2INRA, Aix Marseille Univ., UMR1163, BBF, Biodiversité et Biotechnologie Fongiques, Marseille, France.

Background: Lignocellulose biomass is known as a recalcitrant material towards enzymatic hydrolysis, increasing the process cost in biorefinery. In nature, filamentous fungi naturally degrade lignocellulose, using an arsenal of hydrolytic and oxidative enzymes. Assessment of enzyme hydrolysis efficiency generally relies on the yield of glucose for a given biomass. To better understand the markers governing recalcitrance to enzymatic degradation, there is a need to enlarge the set of parameters followed during deconstruction.

Results: Industrially-pretreated biomass feedstocks from wheat straw, miscanthus and poplar were sequentially hydrolysed following two steps. First, standard secretome from was used to maximize cellulose hydrolysis, producing three recalcitrant lignin-enriched solid substrates. Then fungal secretomes from three basidiomycete saprotrophs ( and ) displaying various hydrolytic and oxidative enzymatic profiles were applied to these recalcitrant substrates, and compared to the secretome. As a result, most of the glucose was released after the first hydrolysis step. After the second hydrolysis step, half of the remaining glucose amount was released. Overall, glucose yield after the two sequential hydrolyses was more dependent on the biomass source than on the fungal secretomes enzymatic profile. Solid residues obtained after the two hydrolysis steps were characterized using complementary methodologies. Correlation analysis of several physico-chemical parameters showed that released glucose yield was negatively correlated with lignin content and cellulose crystallinity while positively correlated with xylose content and water sorption. Water sorption appears as a pivotal marker of the recalcitrance as it reflects chemical and structural properties of lignocellulosic biomass.

Conclusions: Fungal secretomes applied to highly recalcitrant biomass samples can further extend the release of the remaining glucose. The glucose yield can be correlated to chemical and physical markers, which appear to be independent from the biomass type and secretome. Overall, correlations between these markers reveal how nano-scale properties (polymer content and organization) influence macro-scale properties (particle size and water sorption). Further systematic assessment of these markers during enzymatic degradation will foster the development of novel cocktails to unlock the degradation of lignocellulose biomass.
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http://dx.doi.org/10.1186/s13068-019-1417-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442405PMC
April 2019

AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes.

Biotechnol Biofuels 2019 16;12:55. Epub 2019 Mar 16.

1Biodiversité et Biotechnologie Fongiques, UMR1163, INRA, Aix Marseille Université, Marseille, France.

Background: Lignocellulosic biomass is considered as a promising alternative to fossil resources for the production of fuels, materials and chemicals. Efficient enzymatic systems are needed to degrade the plant cell wall and overcome its recalcitrance. A widely used producer of cellulolytic cocktails is the ascomycete , but this organism secretes a limited set of enzymes. To improve the saccharification yields, one strategy is to upgrade the enzyme cocktail with enzymes produced by other biomass-degrading filamentous fungi isolated from biodiversity.

Results: In this study, the enzymatic cocktails secreted by five strains from the genus ( strains BRFM 405, 1487, 1489, 1490 and strain BRFM 430) were tested for their ability to boost a reference cocktail for the saccharification of pretreated biomass. Proteomic analysis of fungal secretomes that significantly improved biomass degradation showed that the presence of proteins belonging to a putative LPMO family previously identified by genome analysis and awaiting experimental demonstration of activity. Members of this novel LPMO family, named AA16, are encountered in fungi and oomycetes with life styles oriented toward interactions with plant biomass. One AA16 protein from (AaAA16) was produced to high level in LPMO-type enzyme activity was demonstrated on cellulose with oxidative cleavage at the C1 position of the glucose unit. AaAA16 LPMO was found to significantly improve the activity of CBHI on cellulosic substrates.

Conclusions: Although spp. has been investigated for decades for their CAZymes diversity, we identified members of a new fungal LPMO family using secretomics and functional assays. Properties of the founding member of the AA16 family characterized herein could be of interest for use in biorefineries.
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http://dx.doi.org/10.1186/s13068-019-1394-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420742PMC
March 2019

Broad-specificity GH131 β-glucanases are a hallmark of fungi and oomycetes that colonize plants.

Environ Microbiol 2019 08 21;21(8):2724-2739. Epub 2019 Apr 21.

INRA, Aix-Marseille Univ, UMR1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with β-1,4, β-1,3 and mixed β-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.
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http://dx.doi.org/10.1111/1462-2920.14596DOI Listing
August 2019

Recent insights into lytic polysaccharide monooxygenases (LPMOs).

Biochem Soc Trans 2018 12 31;46(6):1431-1447. Epub 2018 Oct 31.

Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100-DK, Copenhagen, Denmark

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.
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http://dx.doi.org/10.1042/BST20170549DOI Listing
December 2018

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis.

Appl Microbiol Biotechnol 2018 Dec 28;102(23):10091-10102. Epub 2018 Sep 28.

Department of Biotechnology, Ghent University, Ghent, Belgium.

Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
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http://dx.doi.org/10.1007/s00253-018-9389-3DOI Listing
December 2018

Lavender- and lavandin-distilled straws: an untapped feedstock with great potential for the production of high-added value compounds and fungal enzymes.

Biotechnol Biofuels 2018 2;11:217. Epub 2018 Aug 2.

1UMR1163 BBF Biodiversité et Biotechnologie Fongiques, INRA, Aix Marseille Univ, 13288 Marseille Cedex 09, France.

Background: Lavender () and lavandin (a sterile hybrid of  × ) essential oils are among those most commonly used in the world for various industrial purposes, including perfumes, pharmaceuticals and cosmetics. The solid residues from aromatic plant distillation such as lavender- and lavandin-distilled straws are generally considered as wastes, and consequently either left in the fields or burnt. However, lavender- and lavandin-distilled straws are a potentially renewable plant biomass as they are cheap, non-food materials that can be used as raw feedstocks for green chemistry industry. The objective of this work was to assess different pathways of valorization of these straws as bio-based platform chemicals and fungal enzymes of interest in biorefinery.

Results: Sugar and lignin composition analyses and saccharification potential of the straw fractions revealed that these industrial by-products could be suitable for second-generation bioethanol prospective. The solvent extraction processes, developed specifically for these straws, released terpene derivatives (e.g. τ-cadinol, β-caryophyllene), lactones (e.g. coumarin, herniarin) and phenolic compounds of industrial interest, including rosmarinic acid which contributed to the high antioxidant activity of the straw extracts. Lavender and lavandin straws were also suitable inducers for the secretion of a wide panel of lignocellulose-acting enzymes (cellulases, hemicellulases and oxido-reductases) from the white-rot model fungus Interestingly, high amounts of laccase and several lytic polysaccharide monooxygenases were identified in the lavender and lavandin straw secretomes using proteomics.

Conclusions: The present study demonstrated that the distilled straws of lavender and lavandin are lignocellulosic-rich materials that can be used as raw feedstocks for producing high-added value compounds (antioxidants, aroma) and fungal oxidative enzymes, which represent opportunities to improve the decomposition of recalcitrant lignocellulose into biofuel. Hence, the structure and the physico-chemical properties of these straws clearly open new perspectives for use in biotechnological processes involving especially filamentous fungi. These approaches represent sustainable strategies to foster the development of a local circular bioeconomy.
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http://dx.doi.org/10.1186/s13068-018-1218-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071384PMC
August 2018

The ectomycorrhizal basidiomycete Laccaria bicolor releases a secreted β-1,4 endoglucanase that plays a key role in symbiosis development.

New Phytol 2018 12 6;220(4):1309-1321. Epub 2018 Apr 6.

UMR 1136 INRA-Université de Lorraine 'Interactions Arbres/Microorganismes', Laboratoire d'Excellence ARBRE, Centre INRA-Lorraine, 54280, Champenoux, France.

In ectomycorrhiza, root ingress and colonization of the apoplast by colonizing hyphae is thought to rely mainly on the mechanical force that results from hyphal tip growth, but this could be enhanced by secretion of cell-wall-degrading enzymes, which have not yet been identified. The sole cellulose-binding module (CBM1) encoded in the genome of the ectomycorrhizal Laccaria bicolor is linked to a glycoside hydrolase family 5 (GH5) endoglucanase, LbGH5-CBM1. Here, we characterize LbGH5-CBM1 gene expression and the biochemical properties of its protein product. We also immunolocalized LbGH5-CBM1 by immunofluorescence confocal microscopy in poplar ectomycorrhiza. We show that LbGH5-CBM1 expression is substantially induced in ectomycorrhiza, and RNAi mutants with a decreased LbGH5-CBM1 expression have a lower ability to form ectomycorrhiza, suggesting a key role in symbiosis. Recombinant LbGH5-CBM1 displays its highest activity towards cellulose and galactomannans, but no activity toward L. bicolor cell walls. In situ localization of LbGH5-CBM1 in ectomycorrhiza reveals that the endoglucanase accumulates at the periphery of hyphae forming the Hartig net and the mantle. Our data suggest that the symbiosis-induced endoglucanase LbGH5-CBM1 is an enzymatic effector involved in cell wall remodeling during formation of the Hartig net and is an important determinant for successful symbiotic colonization.
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http://dx.doi.org/10.1111/nph.15113DOI Listing
December 2018

Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, DyP1 and VP2, Identified from the Secretome of Trametes versicolor.

Appl Environ Microbiol 2018 04 2;84(8). Epub 2018 Apr 2.

INRA, Aix-Marseille Université, UMR1163, Biodiversité et Biotechnologie Fongiques, Marseille, France

is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase DyP1 and the versatile peroxidase VP2. Both peroxidases were heterologously produced in , biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and DyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while VP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and DyP1 was globally found to be more stable than VP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of VP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, DyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and VP2 was shown to accept a wider range of reducing substrates than DyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with VP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and VP2 presented a greater ability to oxidize and decolorize the dye substrates than DyP1. is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the basidiomycete has been extensively studied for its ability to degrade wood, specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of , one dye-decolorizing peroxidase and one versatile peroxidase, were biochemically characterized in depth and compared to other fungal peroxidases.
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http://dx.doi.org/10.1128/AEM.02826-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881066PMC
April 2018

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation.

Nat Chem Biol 2018 03 29;14(3):306-310. Epub 2018 Jan 29.

INRA, Aix Marseille University, Biodiversité et Biotechnologie Fongiques (BBF), Marseille, France.

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans-a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxidative cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications.
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http://dx.doi.org/10.1038/nchembio.2558DOI Listing
March 2018

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

Sci Rep 2017 12 19;7(1):17792. Epub 2017 Dec 19.

FARE Laboratory, INRA, University of Reims Champagne-Ardenne, 51100, Reims, France.

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.
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http://dx.doi.org/10.1038/s41598-017-17938-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736606PMC
December 2017

The yeast encodes functional lytic polysaccharide monooxygenases.

Biotechnol Biofuels 2017 12;10:215. Epub 2017 Sep 12.

INRA, Aix Marseille University BBF, Biodiversité et Biotechnologie Fongiques, 13288 Marseille, France.

Background: Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that have revolutionized our understanding of lignocellulose degradation. Fungal LPMOs of the AA9 family target cellulose and hemicelluloses. AA9 LPMO-coding genes have been identified across a wide range of fungal saprotrophs (Ascomycotina, Basidiomycotina, etc.), but so far they have not been found in more basal lineages. Recent genome analysis of the yeast (Saccharomycotina) revealed the presence of several LPMO genes, which belong to the AA9 family.

Results: In this study, three AA9 LPMOs from were successfully produced and biochemically characterized. The use of native signal peptides was well suited to ensure correct processing and high recombinant production of LPMO9A, LPMO9B, and LPMO9C in . We show that LPMO9A and LPMO9B were both active on cellulose and xyloglucan, releasing a mixture of soluble C1- and C4-oxidized oligosaccharides from cellulose. All three enzymes disrupted cellulose fibers and significantly improved the saccharification of pretreated lignocellulosic biomass upon addition to a commercial cellulase cocktail.

Conclusions: The unique enzymatic arsenal of compared to other yeasts could be beneficial for plant cell wall decomposition in a saprophytic or pathogenic context. From a biotechnological point of view, LPMOs are promising candidates to further enhance enzyme cocktails used in biorefineries such as consolidated bioprocessing.
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http://dx.doi.org/10.1186/s13068-017-0903-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596469PMC
September 2017

GH62 arabinofuranosidases: Structure, function and applications.

Biotechnol Adv 2017 Nov 29;35(6):792-804. Epub 2017 Jun 29.

Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark, Elektrovej, Building 375, DK-2800 Kgs. Lyngby, Denmark. Electronic address:

Motivated by industrial demands and ongoing scientific discoveries continuous efforts are made to identify and create improved biocatalysts dedicated to plant biomass conversion. α-1,2 and α-1,3 arabinofuranosyl specific α-l-arabinofuranosidases (EC 3.2.1.55) are debranching enzymes catalyzing hydrolytic release of α-l-arabinofuranosyl residues, which decorate xylan or arabinan backbones in lignocellulosic and pectin constituents of plant cell walls. The CAZy database classifies α-l-arabinofuranosidases in Glycoside Hydrolase (GH) families GH2, GH3, GH43, GH51, GH54 and GH62. Only GH62 contains exclusively α-l-arabinofuranosidases and these are of fungal and bacterial origin. Twenty-two GH62 enzymes out of 223 entries in the CAZy database have been characterized and very recently new knowledge was acquired with regard to crystal structures, substrate specificities, and phylogenetics, which overall provides novel insights into structure/function relationships of GH62. Overall GH62 α-l-arabinofuranosidases are believed to play important roles in nature by acting in synergy with several cell wall degrading enzymes and members of GH62 represent promising candidates for biotechnological improvements of biofuel production and in various biorefinery applications.
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http://dx.doi.org/10.1016/j.biotechadv.2017.06.005DOI Listing
November 2017

Fungal secretomics to probe the biological functions of lytic polysaccharide monooxygenases.

Carbohydr Res 2017 Aug 17;448:155-160. Epub 2017 May 17.

Protein Glycoscience and Biotechnology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Elektrovej 375, 2800 Kgs Lyngby, Denmark. Electronic address:

Enzymatic degradation of plant biomass is of growing interest for the development of a sustainable bio-based industry. Filamentous fungi, which degrade complex and recalcitrant plant polymers, are proficient secretors of enzymes acting on the lignocellulose composite of plant cell walls in addition to starch, the main carbon storage reservoir. In this review, we focus on the identification of lytic polysaccharide monooxygenases (LPMOs) and their redox partners in fungal secretomes to highlight the biological functions of these remarkable enzyme systems and we discuss future trends related to LPMO-potentiated bioconversion.
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http://dx.doi.org/10.1016/j.carres.2017.05.010DOI Listing
August 2017

The integrative omics of white-rot fungus Pycnoporus coccineus reveals co-regulated CAZymes for orchestrated lignocellulose breakdown.

PLoS One 2017 10;12(4):e0175528. Epub 2017 Apr 10.

Aix-Marseille Université, INRA, UMR 1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

Innovative green technologies are of importance for converting plant wastes into renewable sources for materials, chemicals and energy. However, recycling agricultural and forestry wastes is a challenge. A solution may be found in the forest. Saprotrophic white-rot fungi are able to convert dead plants into consumable carbon sources. Specialized fungal enzymes can be utilized for breaking down hard plant biopolymers. Thus, understanding the enzymatic machineries of such fungi gives us hints for the efficient decomposition of plant materials. Using the saprotrophic white-rot fungus Pycnoporus coccineus as a fungal model, we examined the dynamics of transcriptomic and secretomic responses to different types of lignocellulosic substrates at two time points. Our integrative omics pipeline (SHIN+GO) enabled us to compress layers of biological information into simple heatmaps, allowing for visual inspection of the data. We identified co-regulated genes with corresponding co-secreted enzymes, and the biological roles were extrapolated with the enriched Carbohydrate-Active Enzyme (CAZymes) and functional annotations. We observed the fungal early responses for the degradation of lignocellulosic substrates including; 1) simultaneous expression of CAZy genes and secretion of the enzymes acting on diverse glycosidic bonds in cellulose, hemicelluloses and their side chains or lignin (i.e. hydrolases, esterases and oxido-reductases); 2) the key role of lytic polysaccharide monooxygenases (LPMO); 3) the early transcriptional regulation of lignin active peroxidases; 4) the induction of detoxification processes dealing with biomass-derived compounds; and 5) the frequent attachments of the carbohydrate binding module 1 (CBM1) to enzymes from the lignocellulose-responsive genes. Our omics combining methods and related biological findings may contribute to the knowledge of fungal systems biology and facilitate the optimization of fungal enzyme cocktails for various industrial applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175528PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386290PMC
September 2017

The lytic polysaccharide monooxygenase LPMO9H catalyzes oxidative cleavage of diverse plant cell wall matrix glycans.

Biotechnol Biofuels 2017 11;10:63. Epub 2017 Mar 11.

Polytech Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques, INRA, Aix-Marseille Université, Avenue de Luminy, 13288 Marseille, France.

Background: The 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 , the LPMO9H enzyme catalyzes mixed C1/C4 oxidative cleavage of cellulose and cello-oligosaccharides. Activity of LPMO9H on several hemicelluloses has been suggested, but the regioselectivity of the cleavage remained to be determined.

Results: In this study, we investigated the activity of LPMO9H on mixed-linkage glucans, xyloglucan and glucomannan using tandem mass spectrometry and ion mobility-mass spectrometry. Structural analysis of the released products revealed that LPMO9H 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.

Conclusion: The ability of LPMO9H 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.
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http://dx.doi.org/10.1186/s13068-017-0749-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346257PMC
March 2017

Characterization of a mycobacterial cellulase and its impact on biofilm- and drug-induced cellulose production.

Glycobiology 2017 05;27(5):392-399

Université de Montpellier, CNRS, Centre d'étude d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), FRE3689, 34293 Montpellier, France.

It was recently shown that Mycobacterium tuberculosis produces cellulose which forms an integral part of its extracellular polymeric substances within a biofilm set-up. Using Mycobacterium smegmatis as a proxy model organism, we demonstrate that M. smegmatis biofilms treated with purified MSMEG_6752 releases the main cellulose degradation-product (cellobiose), detected by using ionic chromatography, suggesting that MSMEG_6752 encodes a cellulase. Its overexpression in M. smegmatis prevents spontaneous biofilm formation. Moreover, the method reported here allowed detecting cellobiose when M. smegmatis cultures were exposed to a subinhibitory dose of rifampicin. Overall, this study highlights the role of the MSMEG_6752 in managing cellulose production induced during biofilm formation and antibiotic stress response.
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http://dx.doi.org/10.1093/glycob/cwx014DOI Listing
May 2017

Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure.

Sci Rep 2017 01 10;7:40262. Epub 2017 Jan 10.

BIA, INRA, 44300, Nantes, France.

Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers.
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http://dx.doi.org/10.1038/srep40262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223172PMC
January 2017

Structural insights into a family 39 glycoside hydrolase from the gut symbiont Bacteroides cellulosilyticus WH2.

J Struct Biol 2017 03 24;197(3):227-235. Epub 2016 Nov 24.

CNRS, Aix Marseille Univ, AFMB, Marseille, France; INRA, USC 1408, AFMB, Marseille, France. Electronic address:

Bacteria from the human gut are equipped with an arsenal of carbohydrate-active enzymes that degrade dietary and host-derived glycans. In this study, we present the 2.5Å resolution crystal structure of a member (GH39wh2) from the human gut bacteria Bacteroides cellulosilyticus WH2 representative of a new subgroup within family GH39. Together with 6 other GHs, GH39wh2 belongs to a polysaccharide utilization locus (PUL) that could be involved in detecting, binding and hydrolysing a specific carbohydrate species from the intestinal tract. GH39wh2 shares a similar architecture as other members of family GH39 dominated by a typical (β/α)-barrel fold harboring the catalytic residues and decorated by β-sandwich accessory domains. The GH39wh2 structure unveils an atypical shallow groove rather than a deep pocket due to drastic rearrangements in surface loops surrounding the catalytic interface. These structural adaptations seem to favour recognition of large branched substrates and may explain the lack of activity of GH39wh2 toward small xylose-based and other typical substrates from GH39 members, emphasizing the molecular diversity within the GH39 family. A phylogenetic analysis of the entire GH39 family assigns GH39wh2 as a new subgroup, consistent with the extensive remodelling of the active site region that may confer new substrate specificity toward a complex glycan chain.
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http://dx.doi.org/10.1016/j.jsb.2016.11.004DOI Listing
March 2017