Under cellulolytic conditions, the white-rot fungus Trametes versicolor produces cellobiose dehydrogenase (CDH), an enzyme with a number of biochemical properties that are potentially relevant to the degradation of lignin and cellulose. To clarify its biochemical properties, CDH was purified from cultures of T. versicolor. Two isoforms of CDH were found: a 97 kDa isoform with both heme and flavin cofactors, and an 81 kDa isoform with a flavin cofactor. Both isoforms of CDH were found to be quite non-specific in their reductive half reactions. The flavin enzyme catalyzed many of the same reactions as the heme/flavin enzyme, but less efficiently. The flavin isoform reduced Fe(III) and Cu(II) only at concentrations well above those found physiologically. Thus the heme/flavin enzyme, but not the flavin enzyme, could be involved in promoting and sustaining the generation of hydroxyl radicals (·OH) by Fenton's chemistry. / To characterize further the structural features of CDH, a genomic clone was isolated and sequenced. CDH was found to consist of 748 amino acids, without its predicted 19 amino acid signal peptide. Consistent with the domain structure of other CDHs, T. versicolor CDH appeared to be divided into an amino terminal heme domain and a carboxy terminal flavin domain, connected by a hydroxyamino acid-rich linker. Within the flavin domain, a putative cellulose-binding domain (CBD) was found by alignment to the hypothesized CBD of P. chrysosporium CDH. The CBD of CDH appeared to be structurally unrelated to other CBDs which have been reported. / A cDNA clone encoding T. versicolor CDH was isolated by RT-PCR. Using this clone, three vectors for the heterologous expression in Aspergillus oryzae of CDH were prepared. These vectors were built by performing in-frame fusions of the cDNA to control sequences from the highly expressed A. oryzae amylase gene. These vectors were transformed into A. oryzae and one strain was isolated which contained the expression construct DNA. / A rapid method for cloning cdh-like genes was developed. Using short stretches of amino acids completely conserved within T. versicolor and P. chrysosporium CDH, PCR primers were designed to amplify a homologous gene from other fungi. The primers were tested using genomic DNA of Pycnoporus cinnabarinus. A 1.8-kb fragment of P. cinnabarinus cdh was thereby amplified and cloned, and its sequence was determined. The three CDHs displayed very high homology at the amino acid level. / Finally, to probe the role of CDH in lignocellulose degradation by T. versicolor, a "knockout" vector was constructed consisting of a phleomycin-resistance cassette inserted into the protein coding sequence of cloned T. versicolor cdh. T. versicolor was transformed with the knockout vector and the transformants were analyzed for their CDH-producing phenotype. Three isolates were found that produced no detectable CDH. Biobleaching and delignification by the CDH(-) strains appeared to be unaffected, suggesting that CDH does not play an important role in these processes.

No description available.

En réponse aux préoccupations environnementales, les procédés industriels comme la production de bioéthanol de deuxième génération sont apparus. Basés sur la conversion enzymatique de la cellulose, ces processus font face à un problème majeur, la réticence de la biomasse lignocellulosique à l'hydrolyse. Afin de résoudre ce problème et celui lié aux coûts d'utilisation de cocktails de cellulases, les recherches se sont axées sur diverses méthodes permettant d'augmenter l'hydrolyse de la cellulose. Les champignons filamenteux sont connus pour être des dégradeurs naturels du bois et, par conséquent, sont utilisés dans de nombreuses applications biotechnologiques. Récemment, quelques études ont révélé l'importance d'enzymes fongiques telles que la CDH et les GH61 dans la dégradation oxydative de la lignocellulose. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose. L'utilisation de ces enzymes oxydatives offre de réelles voies d'amélioration de la production de bioéthanol et de compréhension de la dégradation in vivo des lignocelluloses par les champignons. / In response to environmental concerns, industrial processes such as second generation bioethanol production have emerged. Based on enzymatic cellulose conversion, these processes are confronted with a major problem, the recalcitrance of lignocellulosic biomass. To solve the problem caused by substrate recalcitrance and high cost of cellulase cocktails, research has focused on various methods to enhance cellulose hydrolysis. Fungi are known to be natural degraders of wood and consequently are used in derived biotechnological applications. Recently, several studies have revealed the importance of fungal enzymes such as GH61 and CDH in the oxidative degradation of lignocellulose. During the work done on this thesis, we demonstrated implication of these oxidative enzymes in lignocellulose deconstruction to enhance hydrolysis performed by more classical cellulases. Utilization of oxidative enzymes offers a suitable way for bioethanol processing enhancement and comprehension of the in vivo lignocellulosic degradation by fungi.

<p>The enzymatic degradation of cellulose is an important process in nature. This thesis has focused on the degradation of cellulose by enzymes from two cellulose-degrading fungi, <i>Hypocrea jecorina</i> and <i>Phanerochaete chrysosporium</i>, including both the action of the individual enzymes and their synergistic interplay. </p><p>The end-preference of cellobiohydrolases on crystalline cellulose was studied. Cellobiohydrolases belonging to glycosyl hydrolase (GH) family 7 were found to hydrolyse cellulose processively, starting from the reducing end of the cellulose chain. End-labelled cellulose can serve as a tool for functional classification of cellulases.</p><p>The synergy mechanism between endoglucanases and cellobiohydrolases was studied using substrates with different physical properties derived from bacterial cellulose. A new mechanism for synergism between endo- and exoacting enzymes was proposed whereby endoglucanases, in addition to creating nicks in amorphous parts of cellulose, thereby making new starting-points for processively acting cellobiohydrolases, also “polish” the cellulose surface by removing shorter chains from cellulose surface.</p><p>A new small endoglucanase belonging to the GH12 family was isolated and characterised. The proposed role of this enzyme is to make the cellulose in wood more accessible to other cellulases.</p><p>Oxygen conversion by cellobiose dehydrogenase was studied. Hydrogen peroxide produced by cellobiose dehydrogenase can be decomposed even by traces of certain metal ions into a hydroxyl radical and a hydroxyl ion. As an example, reduced metal ions will be continuously regenerated by cellobiose dehydrogenase, which thus stimulates the degradation.</p><p>Interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were studied by fluorescence spectroscopy and kinetic tests. o-nitrophenyl cellobioside was used as indicator ligand to determine the dissociation constants for cellobiose binding to catalytically inactive Cel7A mutants by displacement binding experiments.</p>

The enzymatic degradation of cellulose is an important process in nature. This thesis has focused on the degradation of cellulose by enzymes from two cellulose-degrading fungi, Hypocrea jecorina and Phanerochaete chrysosporium, including both the action of the individual enzymes and their synergistic interplay. The end-preference of cellobiohydrolases on crystalline cellulose was studied. Cellobiohydrolases belonging to glycosyl hydrolase (GH) family 7 were found to hydrolyse cellulose processively, starting from the reducing end of the cellulose chain. End-labelled cellulose can serve as a tool for functional classification of cellulases. The synergy mechanism between endoglucanases and cellobiohydrolases was studied using substrates with different physical properties derived from bacterial cellulose. A new mechanism for synergism between endo- and exoacting enzymes was proposed whereby endoglucanases, in addition to creating nicks in amorphous parts of cellulose, thereby making new starting-points for processively acting cellobiohydrolases, also “polish” the cellulose surface by removing shorter chains from cellulose surface. A new small endoglucanase belonging to the GH12 family was isolated and characterised. The proposed role of this enzyme is to make the cellulose in wood more accessible to other cellulases. Oxygen conversion by cellobiose dehydrogenase was studied. Hydrogen peroxide produced by cellobiose dehydrogenase can be decomposed even by traces of certain metal ions into a hydroxyl radical and a hydroxyl ion. As an example, reduced metal ions will be continuously regenerated by cellobiose dehydrogenase, which thus stimulates the degradation. Interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were studied by fluorescence spectroscopy and kinetic tests. o-nitrophenyl cellobioside was used as indicator ligand to determine the dissociation constants for cellobiose binding to catalytically inactive Cel7A mutants by displacement binding experiments.

Biological control agents are a promising niche to replace chemical pesticides for treating plant pathogens in agriculture. A potential biocontrol agent is the microparasitic fungi Clonostachys rosea which has the ability to attack various plant pathogens such as other fungi and nematodes. One key feature in the interaction between mycoparasite and prey is degradation of the fungal cell wall where cell wall degrading enzymes are important. One cell wall degrading enzyme is cellobiose dehydrogenase of which it has been found a high number of genes for in C. rosea compared to other mycoparasites. The reason for these many cellobiose dehydrogenase genes being present in C. rosea is what this study aimed to find out. To do so, the different cellobiose dehydrogenase proteins 001, 002, 003 and 004 were successfully expressed in Pichia pastoris. The 003 protein had significantly higher expression levels and were further purified with size exclusion chromatography where some of the resulting purified protein was used to set up a crystallization screen. Unfortunately, no crystals have been formed so far. The enzymatic activity against lactose, cellobiose and laminaribiose of all produced cellobiose dehydrogenase proteins were also analyzed using a 2,6-dichloroindophenol activity assay. The proteins 001 and 002 showed a low activity against lactose and cellobiose whereas the other protein showed no activity for the tested conditions. That these proteins have developed variations in their activities may be one reason for why they are all still existing.

A produção de biocombustíveis e a obtenção de alguns compostos químicos a partir de materiais renováveis, como a biomassa lignocelulósica, ainda não são processos triviais, principalmente devido a recalcitrância destes materiais. Estudos recentes reconheceram as enzimas acessórias, como xilanases e enzimas com Atividade Auxiliar, como potencializadores da atividade de celulases no processo de despolimerização da lignocelulose. A prospecção de enzimas com características termoestáveis é vantajosa para este tipo de aplicação e além disso, estudos sobre o secretoma de diversos fungos cultivados em biomassa como fonte de carbono, tem encontrado enzimas com mecanismo oxidativo, dentre eles, o fungo termofílico Myceliophthora thermophila M77. Porém, estas enzimas tem sido pouco estudadas quanto a sua aplicação na sacarificação da biomassa. Sendo assim, este trabalho visou a expressão heteróloga, a caracterização bioquímica e a ação da enzima oxidativa celobiose desidrogenase do fungo M. thermophila (M77CDH) em conjunto com outras celulases no processo de sacarificação da biomassa. Pela análise filogenética a M77CDH prospectada foi classificada como pertencente a Classe IIB das CDHs. O gene que codifica esta enzima foi clonado no vetor pEXPYR e heterólogamente expresso em A. nidulans. A proteína recombinante M77CDH foi purificada e teve sua identidade confirmada por espectrometria de massas. Nas análises bioquímicas, apresentou atividade ótima a 65 °C e reteve mais de 80% da sua atividade a 50°C por 2 horas e pela análise de dicroísmo circular apresentou um desenovelamento da sua estrutura na temperatura de transição de 62,8 °C. Apresentou mais de 80% de atividade em uma faixa ampla de pH (4,5 - 9), em que o domínio citocromo mostrou maior afinidade em pHs alcalinos, característica incomum entre as CDHs descritas na literatura. A atividade da M77CDH foi ligeiramente aumentada pela adição de MgCl2 e Na2MoO4 e altamente afetada por CuSO4 e FeCl3. A eficiência catalítica (kcat/km=266 mM-1s-1) utilizando celobiose foi bastante similar aos valores indicados por CDHs da Classe IIA. O envelope da M77CDH gerado por SAXS foi satisfatório e conveniente com a literatura. Na sacarificação de bagaço de cana pré-tratado hidrotermicamente, utilizando coquetel de A. niveus suplementado com M77CDH, foi possível observar que a adição de M77CDH modificou o perfil de produtos liberados na desconstrução da biomassa. Por fim, na sacarificação do PASC observou-se a sacarificação e produção de ácido celobiônico. / The production of biofuels and chemicals from renewable materials such as lignocellulosic biomass are non-trivial processes mainly due to the recalcitrance of the material. Recent studies have recognized accessory enzymes such as xylanases and Auxiliary Activity enzymes as potentiators in cellulase activity during the depolymerization of lignocellulose. The prospection of thermostable enzymes can be an advantage the improve the depolymerization of these materials. In addition, several enzymes showing oxidative mode of action were found in the secretoma of the thermophilic fungus Myceliophthora thermophila strain M77. However, these enzymes are poor studied regarding their application in biomass saccharification. Therefore, this project aimed the heterologous expression and biochemical characterization of the oxidative enzyme cellobiose dehydrogenase of the fungus M. thermophila (M77CDH). By phylogenetic analysis the M77CDH was classified as belonging to Class IIB of CDHs. The gene encoding this enzyme was cloned and heterologously expressed in A. nidulans, the M77CDH was purified and had its identity confirmed by mass spectrometry. In the biochemical analyzes the M77CDH showed an optimum activity at 65 °C and retained more than 80% of its activity at 50 °C for 2 hours. The circular dichroism analysis showed a denaturation of its structure at the transition temperature of 62.8 ° C. M77CDH also kept more than 80% of its activity in a wide pH range (4.5 - 9), in which the cytochrome domain showed higher affinity at alkaline pH, an unusual behavior compared with other CDHs described in the literature. The activity of M77CDH was increased slightly in the presence of MgCl2 and Na2MoO4 and was highly affected by CuSO4 and FeCl3. The catalytic efficiency (kcat/km = 266 mM-1s-1) in cellobiose was quite similar to the values indicated by CDHs from Class IIA. The envelope of M77CDH generated by SAXS was satisfactory and convenient with the literature. In saccharification of sugarcane bagasse hydrothermally pretreated using A. niveus cocktail supplemented with M77CDH was possible to observe the addition of M77CDH modified the profile of released products in the deconstruction of the biomass. Finally, in the action on PASC was observed the saccharification and production of cellobionic acid.

<p>Microorganisms that degrade lignocellulose play an important role in maintaining the global carbon cycle. Under cellulolytic conditions, the fungus <i>Phanerochaete chrysosporium</i> produces an extracellular flavocytochrome, cellobiose dehydrogenase (CDH), with a proposed role in lignocellulose degradation. CDH consists of 755 amino acids including a C-terminal flavodehydrogenase linked by a peptide hinge to an N-terminal <i>b</i>-type cytochrome. The enzyme catalyses the oxidation of cellobiose to cellobiono-1,5-lactone, followed by transfer of electrons to an electron acceptor, either directly by the flavodehydrogenase domain, or via the cytochrome domain. This thesis presents a structural study on the individual domains of <i>P. chrysosporium</i> cellobiose dehydrogenase.</p><p>The crystal structure of the cytochrome was determined at 1.9 Å resolution. It folds as a β-sandwich with the topology of the antibody Fab V(H) domain, and the haem iron is ligated by Met65 and His163. This is only the second example of a <i>b</i>-type cytochrome with this ligation. The haem propionates are surface exposed to facilitate interdomain electron transfer.</p><p>The structure of a cytochrome Met65His mutant was determined at 1.9 Å resolution. In the mutant, the iron is ligated by the histidyl δ and ε nitrogens, rather than the usual N-ε/N-εligation. This is the first example of a <i>bis</i>-His N-ε/N-δ coordinated protoporphyrin IX iron. The structure of the flavoprotein domain was determined at 1.5 Å resolution. It is partitioned into an FAD-binding subdomain of α/β-type and a substrate-binding subdomain consisting of a seven-stranded β-sheet and six α-helices. Furthermore, the structure of the flavoprotein with the inhibitor cellobiono-1,5-lactam at 1.8 Å resolution lends support to a hydride-transfer mechanism for the reductive-half reaction of CDH although a radical mechanism cannot be excluded.</p>

Microorganisms that degrade lignocellulose play an important role in maintaining the global carbon cycle. Under cellulolytic conditions, the fungus Phanerochaete chrysosporium produces an extracellular flavocytochrome, cellobiose dehydrogenase (CDH), with a proposed role in lignocellulose degradation. CDH consists of 755 amino acids including a C-terminal flavodehydrogenase linked by a peptide hinge to an N-terminal b-type cytochrome. The enzyme catalyses the oxidation of cellobiose to cellobiono-1,5-lactone, followed by transfer of electrons to an electron acceptor, either directly by the flavodehydrogenase domain, or via the cytochrome domain. This thesis presents a structural study on the individual domains of P. chrysosporium cellobiose dehydrogenase. The crystal structure of the cytochrome was determined at 1.9 Å resolution. It folds as a β-sandwich with the topology of the antibody Fab V(H) domain, and the haem iron is ligated by Met65 and His163. This is only the second example of a b-type cytochrome with this ligation. The haem propionates are surface exposed to facilitate interdomain electron transfer. The structure of a cytochrome Met65His mutant was determined at 1.9 Å resolution. In the mutant, the iron is ligated by the histidyl δ and ε nitrogens, rather than the usual N-ε/N-εligation. This is the first example of a bis-His N-ε/N-δ coordinated protoporphyrin IX iron. The structure of the flavoprotein domain was determined at 1.5 Å resolution. It is partitioned into an FAD-binding subdomain of α/β-type and a substrate-binding subdomain consisting of a seven-stranded β-sheet and six α-helices. Furthermore, the structure of the flavoprotein with the inhibitor cellobiono-1,5-lactam at 1.8 Å resolution lends support to a hydride-transfer mechanism for the reductive-half reaction of CDH although a radical mechanism cannot be excluded.

Dans le contexte actuel, il devient nécessaire de rendre les alternatives au pétrole, tel que le bioéthanol 2G, disponibles à grande échelle. Cependant, l’étape d’hydrolyse par les enzymes de Trichoderma reesei reste un verrou à un procédé économiquement stable et rentable. Ces travaux de thèse, s'intègrent dans le cadre du projet Futurol et ont pour objectifs d'identifier et de caractériser de nouvelles enzymes fongiques pour améliorer l'hydrolyse de la biomasse lignocellulosique. A partir des données protéomiques disponibles pour Podospora anserina et Fusarium verticillioides, une douzaine d'enzymes candidates ont été identifiées dans leurs sécrétomes. Ce travail de thèse s'est plus particulièrement focalisé sur les AA9s « Lytic Polysaccharide Monooxygenases » (LPMOs) de P. anserina. Parmi les LPMOs étudiées, PaLPMO9A, PaLPMO9E et PaLPMO9H, qui possèdent un CBM1, sont les plus actives sur la cellulose. La détermination de la régiosélectivité d'action a mis en évidence que PaLPMO9A et PaLPMO9H clivent la cellulose en position C1 et C4 alors que la PaLPMO9E génère uniquement des produits oxydés en C1. La PaLPMO9H est la plus versatile puisqu’elle est active sur les cello-oligosaccharides solubles et sur les polysaccharides hémicellulosiques liés en β-(1,4) (i.e., xyloglucane, glucomannane). La supplémentation du cocktail de T. reesei avec PaLPMO9E ou PaLPMO9H a permis de doubler les rendements d'hydrolyse du miscanthus prétraité. Les travaux réalisés au cours de cette thèse ont permis de démontrer l'importance de ces enzymes oxydatives dans les phénomènes de déconstruction de la lignocellulose chez les champignons filamenteux. / In the current context, it becomes essential to make alternative to oil, such as the 2G bioethanol, available at large scale. However, the hydrolysis step by Trichoderma reesei enzymes remains the major bottleneck for an economically sustainable process. The present work is part of the Futurol project, and aims at identifying and characterizing new fungal enzymes to improve the hydrolysis of lignocellulosic biomass. From the proteomic data available for Podospora anserina and Fusarium verticillioides, a dozen of interesting enzymes were identified in their secretomes. This work focuses, mainly, on the AA9s « Lytic Polysaccharide Monooxygenases » (LPMOs) from P. anserina. Among all the LPMOs studied, PaLPMO9A, PaLPMO9E and PaLPMO9H that harbored a CBM1 were the most active on cellulose. 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. 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). The hydrolysis yield of the pretreated miscanthus was significantly improved up to 2 fold, when the PaLPMO9E, or PaLPMO9H were supplemented to the T. reesei cocktail. This work demonstrated the importance of these oxidative enzymes for lignocellulose deconstruction by fungi. These biocatalysts open new prospects to improve the enzymatic conversion of plant biomass for 2G bioethanol production.