Publications by authors named "Mireille Haon"

31 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

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

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

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

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

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

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

Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6.

Biotechnol Biofuels 2016 20;9:108. Epub 2016 May 20.

INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Aix-Marseille Université, Polytech Marseille, 163 Avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France ; UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy-Polytech Marseille, Aix-Marseille Université, 163 Avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France.

Background: Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date, most LPMOs have been characterized from terrestrial fungi, but novel fungal LPMOs isolated from more extreme environments such as an estuary mangrove ecosystem could offer enzymes with unique properties in terms of salt tolerance and higher stability under harsh condition.

Results: Two LPMOs secreted by the mangrove-associated fungus Pestalotiopsis sp. NCi6 (PsLPMOA and PsLPMOB) were expressed in the yeast Pichia pastoris and produced in a bioreactor with >85 mg L(-1) for PsLPMOA and >260 mg L(-1) for PsLPMOB. Structure-guided homology modeling of the PsLPMOs showed a high abundance of negative surface charges, enabling enhanced protein stability and activity in the presence of sea salt. Both PsLPMOs were activated by a cellobiose dehydrogenase (CDH) from Neurospora crassa, with an apparent optimum of interaction at pH 5.5. Investigation into their regioselective mode of action revealed that PsLPMOA released C1- and C4-oxidized cello-oligosaccharide products, while PsLPMOB released only C4-oxidized products. PsLPMOA was found to cleave polymeric cellulose in the presence of up to 6 % sea salt, which emphasizes the use of sea water in the industrial saccharification process with improved ecological footprints.

Conclusions: Two new LPMOs from the mangrove fungus Pestalotiopsis sp. NCi6 were found to be fully reactive against cellulose. The combined hydrolytic activities of these salt-responsive LPMOs could therefore facilitate the saccharification process using sea water as a reaction medium for large-scale biorefineries.
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http://dx.doi.org/10.1186/s13068-016-0520-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4875668PMC
May 2016

Characterization of a new aryl-alcohol oxidase secreted by the phytopathogenic fungus Ustilago maydis.

Appl Microbiol Biotechnol 2016 Jan 9;100(2):697-706. Epub 2015 Oct 9.

INRA, UMR1163 BBF, Polytech'Marseille, F-13288, Marseille, France.

The discovery of novel fungal lignocellulolytic enzymes is essential to improve the breakdown of plant biomass for the production of second-generation biofuels or biobased materials in green biorefineries. We previously reported that Ustilago maydis grown on maize secreted a diverse set of lignocellulose-acting enzymes including hemicellulases and putative oxidoreductases. One of the most abundant proteins of the secretome was a putative glucose-methanol-choline (GMC) oxidoreductase. The phylogenetic prediction of its function was hampered by the few characterized members within its clade. Therefore, we cloned the gene and produced the recombinant protein to high yield in Pichia pastoris. Functional screening using a library of substrates revealed that this enzyme was able to oxidize several aromatic alcohols. Of the tested aryl-alcohols, the highest oxidation rate was obtained with 4-anisyl alcohol. Oxygen, 1,4-benzoquinone, and 2,6-dichloroindophenol can serve as electron acceptors. This GMC oxidoreductase displays the characteristics of an aryl-alcohol oxidase (E.C.1.1.3.7), which is suggested to act on the lignin fraction in biomass.
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http://dx.doi.org/10.1007/s00253-015-7021-3DOI Listing
January 2016

Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris.

Front Microbiol 2015 23;6:1002. Epub 2015 Sep 23.

Architecture et Fonction des Macromolècules Biologiques, CNRS-Aix-Marseille University UMR 7257 Marseille, France ; INRA, USC 1408 AFMB Marseille, France.

Filamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to set-up the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community.
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http://dx.doi.org/10.3389/fmicb.2015.01002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585289PMC
October 2015

A unique CE16 acetyl esterase from Podospora anserina active on polymeric xylan.

Appl Microbiol Biotechnol 2015 Dec 2;99(24):10515-26. Epub 2015 Sep 2.

Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38, Bratislava, Slovak Republic.

The genome of the coprophilous fungus Podospora anserina displays an impressive array of genes encoding hemicellulolytic enzymes. In this study, we focused on a putative carbohydrate esterase (CE) from family 16 (CE16) that bears a carbohydrate-binding module from family CBM1. The protein was heterologously expressed in Pichia pastoris and purified to electrophoretic homogeneity. The P. anserina CE16 enzyme (PaCE16A) exhibited different catalytic properties than so far known CE16 esterases represented by the Trichoderma reesei CE16 acetyl esterase (TrCE16). A common property of both CE16 esterases is their exodeacetylase activity, i.e., deesterification at positions 3 and 4 of monomeric xylosides and the nonreducing end xylopyranosyl (Xylp) residue of oligomeric homologues. However, the PaCE16A showed lower positional specificity than TrCE16 and efficiently deacetylated also position 2. The major difference observed between PaCE16A and TrCE16 was found on polymeric substrate, acetylglucuronoxylan. While TrCE16 does not attack internal acetyl groups, PaCE16A deacetylated singly and doubly acetylated Xylp residues in the polymer to such an extent that it resulted in the polymer precipitation. Similarly as typical acetylxylan esterases belonging to CE1, CE4, CE5, and CE6 families, PaCE16A did not attack 3-O-acetyl group of xylopyranosyl residues carrying 4-O-methyl-D-glucuronic acid at position 2. PaCE16A thus represents a CE16 member displaying unique catalytic properties, which are intermediate between the TrCE16 exodeacetylase and acetylxylan esterases designed to deacetylate polymeric substrate. The catalytic versatility of PaCE16A makes the enzyme an important candidate for biotechnological applications.
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http://dx.doi.org/10.1007/s00253-015-6934-1DOI Listing
December 2015

Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

Biotechnol Biofuels 2015 20;8:90. Epub 2015 Jun 20.

INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, ESIL Polytech, F-13288 Marseille, France.

Background: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.

Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).

Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.
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http://dx.doi.org/10.1186/s13068-015-0274-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487207PMC
July 2015

Fast solubilization of recalcitrant cellulosic biomass by the basidiomycete fungus Laetisaria arvalis involves successive secretion of oxidative and hydrolytic enzymes.

Biotechnol Biofuels 2014 8;7(1):143. Epub 2014 Oct 8.

INRA, UMR1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France.

Background: Enzymatic breakdown of lignocellulosic biomass is a known bottleneck for the production of high-value molecules and biofuels from renewable sources. Filamentous fungi are the predominant natural source of enzymes acting on lignocellulose. We describe the extraordinary cellulose-deconstructing capacity of the basidiomycete Laetisaria arvalis, a soil-inhabiting fungus.

Results: The L. arvalis strain displayed the capacity to grow on wheat straw as the sole carbon source and to fully digest cellulose filter paper. The cellulolytic activity exhibited in the secretomes of L. arvalis was up to 7.5 times higher than that of a reference Trichoderma reesei industrial strain, resulting in a significant improvement of the glucose release from steam-exploded wheat straw. Global transcriptome and secretome analyses revealed that L. arvalis produces a unique repertoire of carbohydrate-active enzymes in the fungal taxa, including a complete set of enzymes acting on cellulose. Temporal analyses of secretomes indicated that the unusual degradation efficiency of L. arvalis relies on its early response to the carbon source, and on the finely tuned sequential secretion of several lytic polysaccharide monooxygenases and hydrolytic enzymes targeting cellulose.

Conclusions: The present study illustrates the adaptation of a litter-rot fungus to the rapid breakdown of recalcitrant plant biomass. The cellulolytic capabilities of this basidiomycete fungus result from the rapid, selective and successive secretion of oxidative and hydrolytic enzymes. These enzymes expressed at critical times during biomass degradation may inspire the design of improved enzyme cocktails for the conversion of plant cell wall resources into fermentable sugars.
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http://dx.doi.org/10.1186/s13068-014-0143-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197297PMC
October 2014

The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown.

BMC Genomics 2014 Jun 18;15:486. Epub 2014 Jun 18.

INRA, UMR1163 Biotechnologie des Champignons Filamenteux, Aix-Marseille Université, Polytech Marseille, 163 avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France.

Background: Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology.

Results: The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases.

Conclusions: With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.
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http://dx.doi.org/10.1186/1471-2164-15-486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4101180PMC
June 2014

First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies.

J Biol Chem 2014 Feb 6;289(8):5261-73. Epub 2014 Jan 6.

From the Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse.

α-L-arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-L-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-L-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-L-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed β-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-L-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.
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http://dx.doi.org/10.1074/jbc.M113.528133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931082PMC
February 2014

Characterization of a broad-specificity β-glucanase acting on β-(1,3)-, β-(1,4)-, and β-(1,6)-glucans that defines a new glycoside hydrolase family.

Appl Environ Microbiol 2012 Dec 28;78(24):8540-6. Epub 2012 Sep 28.

INRA, Aix Marseille Université, UMR1163 BCF, Polytech Marseille, Marseille, France.

Here we report the cloning of the Pa_3_10940 gene from the coprophilic fungus Podospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeast Pichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of β-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this β-glucanase is an exo-acting enzyme on β-(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan substrates. Hydrolysis of short β-(1,3), β-(1,4), and β-(1,3)/β-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-β-(1,3)/(1,6) and endo-β-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.
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http://dx.doi.org/10.1128/AEM.02572-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3502917PMC
December 2012

Exploring the natural fungal biodiversity of tropical and temperate forests toward improvement of biomass conversion.

Appl Environ Microbiol 2012 Sep 6;78(18):6483-90. Epub 2012 Jul 6.

INRA, UMR1163 BCF, Marseille, France.

In this study, natural fungal diversity in wood-decaying species was explored for biomass deconstruction. In 2007 and 2008, fungal isolates were collected in temperate forests mainly from metropolitan France and in tropical forests mainly from French Guiana. We recovered and identified 74 monomorph cultures using morphological and molecular identification tools. Following production of fungal secretomes under inductive conditions, we evaluated the capacity of these fungal strains to potentiate a commercial Trichoderma reesei cellulase cocktail for the release of soluble sugars from biomass. The secretome of 19 isolates led to an improvement in biomass conversion of at least 23%. Of the isolates, the Trametes gibbosa BRFM 952 (Banque de Ressources Fongiques de Marseille) secretome performed best, with 60% improved conversion, a feature that was not universal to the Trametes and related genera. Enzymatic characterization of the T. gibbosa BRFM 952 secretome revealed an unexpected high activity on crystalline cellulose, higher than that of the T. reesei cellulase cocktail. This report highlights the interest in a systematic high-throughput assessment of collected fungal biodiversity to improve the enzymatic conversion of lignocellulosic biomass. It enabled the unbiased identification of new fungal strains issued from biodiversity with high biotechnological potential.
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http://dx.doi.org/10.1128/AEM.01651-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426683PMC
September 2012

FunGene-DB: a web-based tool for Polyporales strains authentication.

J Biotechnol 2012 Oct 2;161(3):383-6. Epub 2012 Jul 2.

INRA, UMR 1163 Biotechnologie des Champignons Filamenteux ESIL, 163 avenue de Luminy, CP 925, 13288 Marseille Cedex 09, France.

Polyporales are extensively studied wood-decaying fungi with applications in white and green biotechnologies and in medicinal chemistry. We developed an open-access, user-friendly, bioinformatics tool named FunGene-DB (http://www.fungene-db.org). The goal was to facilitate the molecular authentication of Polyporales strains and fruit-bodies, otherwise subjected to morphological studies. This tool includes a curated database that contains ITS1-5.8S-ITS2 rDNA genes screened through a semi-automated pipeline from the International Nucleotide Sequence Database (INSD), and the similarity search BLASTn program. Today, the web-accessible database compiles 2379 accepted sequences, among which 386 were selected as reference sequences (most often fully identified ITS sequences for which a voucher, strain or specimen, has been deposited in a public-access collection). The restriction of the database to one reference sequence per species (or per clade for species complex) allowed most often unequivocal analysis. We conclude that FunGene-DB is a promising tool for molecular authentication of Polyporales. It should be especially useful for scientists who are not expert mycologists but who need to check the identity of strains (e.g. for culture collections, for applied microbiology).
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http://dx.doi.org/10.1016/j.jbiotec.2012.06.023DOI Listing
October 2012

Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis.

BMC Genomics 2012 Feb 2;13:57. Epub 2012 Feb 2.

INRA, UMR BCF, Marseille, France.

Background: Filamentous fungi are potent biomass degraders due to their ability to thrive in ligno(hemi)cellulose-rich environments. During the last decade, fungal genome sequencing initiatives have yielded abundant information on the genes that are putatively involved in lignocellulose degradation. At present, additional experimental studies are essential to provide insights into the fungal secreted enzymatic pools involved in lignocellulose degradation.

Results: In this study, we performed a wide analysis of 20 filamentous fungi for which genomic data are available to investigate their biomass-hydrolysis potential. A comparison of fungal genomes and secretomes using enzyme activity profiling revealed discrepancies in carbohydrate active enzymes (CAZymes) sets dedicated to plant cell wall. Investigation of the contribution made by each secretome to the saccharification of wheat straw demonstrated that most of them individually supplemented the industrial Trichoderma reesei CL847 enzymatic cocktail. Unexpectedly, the most striking effect was obtained with the phytopathogen Ustilago maydis that improved the release of total sugars by 57% and of glucose by 22%. Proteomic analyses of the best-performing secretomes indicated a specific enzymatic mechanism of U. maydis that is likely to involve oxido-reductases and hemicellulases.

Conclusion: This study provides insight into the lignocellulose-degradation mechanisms by filamentous fungi and allows for the identification of a number of enzymes that are potentially useful to further improve the industrial lignocellulose bioconversion process.
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http://dx.doi.org/10.1186/1471-2164-13-57DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298532PMC
February 2012

A thermostable GH45 endoglucanase from yeast: impact of its atypical multimodularity on activity.

Microb Cell Fact 2011 Dec 6;10:103. Epub 2011 Dec 6.

INRA, UMR 1163 BCF, 13288 Marseille, France.

Background: The gene encoding an atypical multi-modular glycoside hydrolase family 45 endoglucanase bearing five different family 1 carbohydrate binding modules (CBM1), designated PpCel45A, was identified in the Pichia pastoris GS115 genome.

Results: PpCel45A (full-length open reading frame), and three derived constructs comprising (i) the catalytic module with its proximal CBM1, (ii) the catalytic module only, and (iii) the five CBM1 modules without catalytic module, were successfully expressed to high yields (up to 2 grams per litre of culture) in P. pastoris X33. Although the constructs containing the catalytic module displayed similar activities towards a range of glucans, comparison of their biochemical characteristics revealed striking differences. We observed a high thermostability of PpCel45A (Half life time of 6 h at 80°C), which decreased with the removal of CBMs and glycosylated linkers. However, both binding to crystalline cellulose and hydrolysis of crystalline cellulose and cellohexaose were substantially boosted by the presence of one CBM rather than five.

Conclusions: The present study has revealed the specific features of the first characterized endo β-1,4 glucanase from yeast, whose thermostability is promising for biotechnological applications related to the saccharification of lignocellulosic biomass such as consolidated bioprocessing.
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http://dx.doi.org/10.1186/1475-2859-10-103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247070PMC
December 2011

Phylogeographic relationships in the polypore fungus Pycnoporus inferred from molecular data.

FEMS Microbiol Lett 2011 Dec 3;325(1):37-48. Epub 2011 Oct 3.

INRA, UMR 1163 de Biotechnologie des Champignons Filamenteux, ESIL, Marseille, France.

The genus Pycnoporus forms a group of four species known especially for producing high redox potential laccases suitable for white biotechnology. A sample of 36 Pycnoporus strains originating from different geographical areas was studied to seek informative molecular markers for the typing of new strains in laboratory culture conditions and to analyse the phylogeographic relationships in this cosmopolitan group. ITS1-5.8S-ITS2 ribosomal DNA and partial regions of β-tubulin and laccase lac3-1 gene were sequenced. Phylogenetic trees inferred from these sequences clearly differentiated the group of Pycnoporus cinnabarinus strains from the group of Pycnoporus puniceus strains into strongly supported clades (100% bootstrap value). Molecular clustering based on lac 3-1 sequences enabled the distribution of Pycnoporus sanguineus and Pycnoporus coccineus through four distinct, well supported clades and sub-clades. A neotropical sub-clade, grouping the P. sanguineus strains from French Guiana and Venezuela, corresponded to P. sanguineus sensu stricto. A paleotropical sub-clade, clustering the strains from Madagascar, Vietnam and New Caledonia, was defined as Pycnoporus cf. sanguineus. The Australian clade corresponded to P. coccineus sensu stricto. The Eastern Asian region clade, clustering the strains from China and Japan, formed a P. coccineus-like group. Laccase gene (lac 3-1) analysis within the Pycnoporus species can highlight enzyme functional diversity associated with biogeographical origin.
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http://dx.doi.org/10.1111/j.1574-6968.2011.02412.xDOI Listing
December 2011

Podospora anserina hemicellulases potentiate the Trichoderma reesei secretome for saccharification of lignocellulosic biomass.

Appl Environ Microbiol 2011 Jan 29;77(1):237-46. Epub 2010 Oct 29.

INRA, UMR 1163, Universités de Provence et de la Méditerranée, Laboratoire de Biotechnologie des Champignons Filamenteux, Marseille, France.

To improve the enzymatic hydrolysis (saccharification) of lignocellulosic biomass by Trichoderma reesei, a set of genes encoding putative polysaccharide-degrading enzymes were selected from the coprophilic fungus Podospora anserina using comparative genomics. Five hemicellulase-encoding genes were successfully cloned and expressed as secreted functional proteins in the yeast Pichia pastoris. These novel fungal CAZymes belonging to different glycoside hydrolase families (PaMan5A and PaMan26A mannanases, PaXyn11A xylanase, and PaAbf51A and PaAbf62A arabinofuranosidases) were able to break down their predicted cognate substrates. Although PaMan5A and PaMan26A displayed similar specificities toward a range of mannan substrates, they differed in their end products, suggesting differences in substrate binding. The N-terminal CBM35 module of PaMan26A displayed dual binding specificity toward xylan and mannan. PaXyn11A harboring a C-terminal CBM1 module efficiently degraded wheat arabinoxylan, releasing mainly xylobiose as end product. PaAbf51A and PaAbf62A arabinose-debranching enzymes exhibited differences in activity toward arabinose-containing substrates. Further investigation of the contribution made by each P. anserina auxiliary enzyme to the saccharification of wheat straw and spruce demonstrated that the endo-acting hemicellulases (PaXyn11A, PaMan5A, and PaMan26A) individually supplemented the secretome of the industrial T. reesei CL847 strain. The most striking effect was obtained with PaMan5A that improved the release of total sugars by 28% and of glucose by 18%, using spruce as lignocellulosic substrate.
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http://dx.doi.org/10.1128/AEM.01761-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019743PMC
January 2011

Phylogenetic analysis of the Aspergillus niger aggregate in relation to feruloyl esterase activity.

Res Microbiol 2007 Jun 7;158(5):413-9. Epub 2007 Apr 7.

Muséum National d'Histoire Naturelle, Département Systématique et Evolution, Unité Taxonomie-Collections, Equipe Mycologie, CP 39, 57 rue Cuvier, 75231 Paris Cedex 05, France.

Species of the Aspergillus niger aggregate are known to produce feruloyl esterases, enzymes involved in the degradation of cell wall polymers. However, species delineation is difficult in these fungi. We combined AFLP analysis with ITS rDNA and beta-tubulin sequencing to characterize the isolates of this aggregate in terms of feruloyl esterase production. A preliminary re-examination of isolates based on comparison of ITS rDNA and beta-tubulin sequences with those of typical taxa deposited in international collections led us to re-identify the isolates as members of the species A. niger, A. foetidus and A. tubingensis. Molecular clustering based on beta-tubulin data and AFLP analysis showed that the strains of A. niger formed a homogenous phylogenetic group distinguished by either zero or type A feruloyl esterase activity, while strains A. foetidus and A. tubingensis exhibited type B feruloyl esterase activity when grown on sugar beet pulp.
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http://dx.doi.org/10.1016/j.resmic.2007.03.004DOI Listing
June 2007

Exploration of members of Aspergillus sections Nigri, Flavi, and Terrei for feruloyl esterase production.

Can J Microbiol 2006 Sep;52(9):886-92

Biotechnologie de champignons filamenteux, INRA/Universités de Provence et de la Méditerranée, IFR86 de Biotechnologie agro-industrielle de Marseille, ESIL, France.

The ability of members of Aspergillus sections Nigri, Flavi, and Terrei to produce feruloyl esterases was studied according to their substrate specificity against synthetic methyl esters of hydroxycinnamic acids. Type A feruloyl esterases (FAEA), induced during growth on cereal-derived products, show a preference for the phenolic moiety of substrates that contain methoxy substitutions, as found in methyl sinapinate, whereas type B feruloyl esterases (FAEB) show a preference for the phenolic moiety of substrates that contain hydroxyl substitutions, as occurs in methyl caffeate. All the strains of Aspergillus section Nigri (e.g., A. niger and A. foetidus) were able to produce feruloyl esterases with activity profiles similar to those reported for FAEA and FAEB of A. niger when grown on oat-spelt xylan and sugar beet pulp, respectively. The two genes encoding these proteins, faeA and faeB, were identified by Southern blot analysis. The strains of Aspergillus sections Flavi (e.g., A. flavus, A. flavo-furcatus, and A. tamarii) and Terrei (e.g., A. terreus) were able to produce type A and type B enzymes. faeA was revealed in genomic DNA of these strains, and FAEA was determined by immunodetection in cultures grown in oat-spelt xylan. In addition, type B enzymes, not related to faeB, were efficiently induced by oat-spelt xylan and exhibited very original activity profiles on sugar beet pulp. This work confirms that the members of the genus Aspergillus are good feruloyl esterase producers.
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http://dx.doi.org/10.1139/w06-046DOI Listing
September 2006