<p>Lignocellulose is an ubiquitous source of fixed carbon that is presently underexploited for renewable energy technologies. Currently, producing enzyme cocktails that robustly degrade these feedstocks is a significant economic bottleneck. Anaerobic gut fungi native to the digestive tracts of ruminants and hindgut fermenters are widely understudied despite their inherent ability to degrade a significant portion (~50%) of the lignocellulose in herbivorous animals. Challenges in cultivation due to their strict oxygen sensitivity, and the lack of a central repository to maintain axenic stocks substantially impede the progress with anaerobic fungi. Yet, these microbes have evolved elegant strategies and may harbor novel biomass degrading enzymes that could be used to more efficiently hydrolyze lignocellulose. Developing these organisms through characterization and genome engineering will yield significant contributions to the bioenergy community by improving hydrolysis technologies.</p> <p>In this work, we report the isolation of four novel species of anaerobic gut fungi. A more complete characterization of one of our four fungal isolates is investigated, whereby the effects of substrate composition and the corresponding fungal growth rates are compared. I also explore the growth of one of our fungal isolates on transgenic poplar to understand how fungal growth and enzyme secretion adapt to variable lignin composition. Notably, no significant reductions in growth were observed highlighting the ability of anaerobic fungi to degrade diverse feedstocks regardless of lignin composition. I have additionally included preliminary work intended to identify what epigenetic regulational strategies exist for anaerobic fungi, and how they relate to carbohydrate active enzyme expression. We hope to leverage this knowledge to engineer base enzyme cocktails that release significant portions of the fermentable sugars in untreated or mildly treated plant biomass as a means to make bioenergy technologies more efficient.</p>

A parede das plantas é formada por uma matriz composta principalmente por celulose, hemicelulose e lignina. Celulose é o principal polissacarídeo das paredes das plantas, apresentando alta cristalinidade e recalcitrância. Xiloglucano (XyG) é um polissacarídeo complexo envolvido no controle da expansão celular e na biossíntese de componentes da parede celular vegetal. Uma complexa rede entre XyG e celulose é mediada por ligações de hidrogênio. A eficiente hidrólise de XyG e celulose é uma estratégia promissora na desconstrução da biomassa lignocelulósica pela ação orquestrada de CAZymes. Aqui são descritas as caracterizações funcional e estrutural de três novas enzimas das hidrolase de glicosídeos das famílias 9 (BlCel9), 48 (BlCel48) e 74 (XcGH74). XcGH74 é uma xiloglucanase de Xanthomonas campestris altamente específica para XyG. Durante a hidrólise do XyG por XcGH74 XX e XG são os produtos finais. A estrutura cristalográfica dessa enzima foi resolvida e as razões moleculares para sua alta permissibilidade no reconhecimento de XyG analisadas. Os resultados sugerem que XcGH74 cliva XyG preferencialmente entre motivos X&ndash;X; no entanto, não hidrolisa entre os motivos L&ndash;L, onde uma substituição da cadeia lateral é um pré-requisito para o reconhecimento do substrato. BlCel9 e BlCel48 são celulases de Bacillus licheniformis. BlCel48 é cataliticamente estável em uma ampla gama de temperaturas e pHs exibindo atividade em celulose inchada com ácido fosfórico (PASC) e celulose bacteriana (BC). BlCel48 libera predominantemente celobiose, e também pequenas quantidades de celotriose, celotetraose como produtos do PASC e tem processividade aparente 4,6 vezes maior em BC do que em PASC. Análises de espalhamento de raios X a baixo ângulo (SAXS) mostraram que essa enzima é globular e monomérica em solução. A estrutura cristalográfica de BlCel48 foi resolvida na presença de ligantes nas posições -5 a -2 e +1 a +2 no sítio catalítico. A especificidade no reconhecimento de celo-oligossacarídeo foi investigada por cromatografia de troca aniônica de alto desempenho (HPLC-PAD), cristalografia de proteínas e análise de acoplamento estatístico, mostrando que esta enzima possui endo iniciação durante a hidrólise de PASC. BlCel9 é uma endoglucanase que exibe eficiência catalítica máxima em pH 7,0 e 60 &deg;C. Tem maior atividade em PASC, seguida por BC e, em menor grau, carboximetilcelulose (CMC). A análise por HPAEC-PAD dos produtos hidrolíticos demonstrou que o produto final da hidrólise é principalmente celobiose. Análises de dados cristalografia de raios X e SAXS mostraram que essa enzima é monomérica em solução, conforme estimado a partir dos dados do SAXS. Tem uma forma alongada composta por um módulo de ligação à carboidratos (CBM3c) N-terminal ligado ao domínio catalítico (GH9) C-terminal por um linker de 20 aminoácidos. Os domínios estão intimamente justapostos em uma conformação estendida e formam uma estrutura relativamente rígida em solução, indicando que as interações entre os domínios catalíticos e CBM3c desta enzima têm um papel cooperativo no reconhecimento da celulose. Juntos, esses resultados lançam alguma luz sobre a relação entre estrutura e função das hidrolases glicosídicas das famílias 9, 48 e 74. / Plant cell walls form a matrix composed mainly of cellulose, hemicellulose, and lignin. Cellulose is the main polysaccharide of the plant cell walls with high crystallinity and recalcitrance. Xyloglucan (XyG) is a complex polysaccharide involved in the control of cell expansion and biosynthesis of cell walls components. A complex crosslink between XyG and cellulose is mediated by H-bonds. An efficient hydrolysis of XyG and cellulose is a promising strategy to achieve an effective lignocellulosic biomass deconstruction by orchestrated action of CAZymes. Here are described the functional and structural characterization of three novel enzymes belonging to glycoside hydrolase families 9 (BlCel9), 48 (BlCel48) and 74 (XcGH74). XcGH74 is a highly specific xyloglucanase from Xanthomonas campestris . During the XyG hydrolysis, XX and XG are its end products. We also solved the structure of this enzyme and analyzed molecular reasons for its high permissibility in XyG recognition. These results suggest that the XcGH74 is able to cleave XyG preferentially between X-X motifs; however, it is unable to hydrolyze the polysaccharide between L-L motifs where a side-chain substitution is a prerequisite to improved substrate recognition. The BlCel9 and BlCel48 are cellulases from Bacillus licheniformis. BlCel48 is catalytically stable in a broad range of temperatures and pH conditions, exhibiting hydrolytic activity against phosphoric acid swollen cellulose (PASC) and bacterial cellulose (BC). BlCel48 releases predominantly cellobiose, and also small amounts of cellotriose and cello-tetraose as products from PASC with apparent processivity 4.6-times greater performance on BC than on PASC. Small-angle X-ray scattering (SAXS) data analysis revealed a globular molecular shape and monomeric state in solution. The crystal structure of BlCel48 was solved and used in presence of ligands spanning -5 to -2 and +1 to +2 subsites into the catalytic site. The specificity of the recognition of the cello-oligosaccharide was investigated by high-performance anion exchange chromatography (HPLC-PAD), protein crystallography, and statistical coupling analysis, which showed that this enzyme has an endo-like initiation on PASC. BlCel9 is a processive endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 &deg;C, exhibiting highest hydrolytic activity against PASC, followed by BC, and to a lesser extent carboxymethyl-cellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose. SAXS and X-ray crystallographic data analyses revealed that this enzyme adopts a monomeric state in solution, as estimated from SAXS data; has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose recognition. Together, these results shed some light on the structure-function relationship of glycoside hydrolases from families 9, 48 and 74.

O material lignocelulósico, presente na biomassa vegetal, representa uma importante fonte de energia, entretanto necessita da ação das enzimas lignocelulolíticas para sua degradação. A busca por novas enzimas que atuam na quebra da parede celular da planta em comunidades microbianas evoluídas naturalmente em um ambiente de degradação de biomassa como o rúmen oferece uma estratégia promissora para a prospecção de genes. Com isso, o projeto teve como objetivo a identificação de genes degradarores de biomassa vegetal em microrganismos do rúmen de ovinos usando a abordagem metagenômica. Para tanto, foram coletadas amostras da fase sólida do rúmen de 6 animais fistulados (Ovis aries) divididos em dois grupos e submetidos a duas dietas por 60 dias: tratamento controle e tratamento com dieta contendo bagaço de cana-de-açúcar. O DNA metagenômico total das amostras foi extraído e sequenciado na plataforma MiSeq Personal Sequencer (Illumina&reg;). A análise dos dados para a anotação taxônomica e funcional foi realizada no software MG-RAST. A caracterização dos genes degradadores de biomassa vegetal foi feita na plataforma CLC Genomic Workbench v.5.5.1(CLC Bio, Denmark) e a anotação de 4,68 gigabases de dados foi feita no banco de dados CAZy. A análise taxonômica mostrou uma predominância do domínio Bacteria compondo mais de 96% de todas as amostras, sendo os filos mais abundantes Bacteroidetes, Firmicutes, seguido de Proteobacteria. Entre todos os filos anotados, cinco tiveram a abundância aumentada no tratamento com adição de bagaço de cana-de-açúcar na dieta, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes e Verrucomicrobia, e dois filos foram mais abundantes no tratamento controle, Bacteroidetes e Synergistetes. De modo geral, a análise de ordenação não mostrou correlação entre a composição do microbioma e o tipo de dieta, porém, na análise funcional, essa correlação foi observada uma vez que houve separação entre os tratamentos. A abundância relativa das famílias de enzimas relacionadas à degradação de carboidratos segue um padrão similar em todas as amostras metagenômicas. O módulo catalítico da família de Glycoside Hydrolases (GH), o qual foi anotado em 129 subfamílias diferentes, foi o mais abundante em todas as amostras (45,5%), seguido da família GT (Glicosyl Tranferase), anotada em 97 subfamílias diferentes e CBM (Carbohydrete-Bining Module), em 78 subfamílias. A montagem do metagenoma resultou em aproximadamente 110.000 contigs e possibilitou a identificação de 15 diferentes genes completos codificados nas subfamílias GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 e GH127. A análise comparativa dos diferentes tratamentos mostrou uma maior abundância dessas enzimas no rúmen dos animais alimentados com a dieta enriquecida com bagaço de cana-de-açúcar. Em conclusão, a manipulação da dieta de ovinos por meio da substituição de parte da fração fibrosa da dieta por bagaço de cana-de-açúcar promove o enriquecimento de enzimas que degradam a biomassa vegetal no rúmem, favorecendo a prospecção e identificação de genes ativos em carboidratos. / The lignocellulose present in the plant biomass is a promising source of energy generation. However, the breakdown of plant biomass into simple sugars for bioethanol production is still inefficient and costly due to the recalcitrant nature of the plant fiber. The sheep rumen microbiome is specialized in degradation of plant material, but most members of this complex community are uncultured in the laboratory. Therefore, the search for new lignocellulolytic enzymes in microbial communities naturally evolved in biomass degradation environments, such as the rumen, using the exploration of the metagenome, is a promising strategy for identifying new genes. In this context, this study aimed to prospect plant biomass-degrading genes, selected from the sheep rumen microorganisms. The rumen samples were collected from 6 fistulated animals (Ovis aries), divided into two groups and subjected to two diets: control treatment and a treatment with a diet amended with sugarcane bagasse. The animals were fed for 60 days before sampling. To characterize the composition and functions of the rumen microbiome followed by the search of biomass-degrading genes, the metagenomic DNA was extracted from the solid contents of rumen and sequenced in MiSeq Personal Sequencer platform (Illumina&reg;). The taxonomic and functional data were performed using MG-RAST software. For the characterization of the plant biomass degrading genes, they were analyzed on the CLC platform Genomic Workbench v.5.5.1 (CLC Bio, Denmark) and 4.68 gigabases of data was annotated against the CAZy database. The taxonomic analysis showed a predominance of the Bacteria domain composing more than 96% of all the samples, being the most abundant phyla Bacterioidetes, Firmiutes, followed by Proteobacteria. Five bacterial phyla were significantly more abundant in the treatment were sugarcane bagasse was added, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes and Verrucomicrobia, and two phyla were more abundant in the control treatment, Bacteroidetes and Synergistetes. In general, the ordination analysis did not show correlation between diet type and rumen microbiota, but in the functional analysis, this correlation was observed since there was separation between the treatments. The relative abundance of enzyme families related to carbohydrate degradation follows a similar pattern of abundance across all metagenomic samples. The catalytic module of the GH (Glycoside Hydrolases) family, which was annotated in 129 different subfamilies, was the most abundant in all samples (45.5%), followed by the GT family (Glycosyltransferase), annotated in 97 different subfamilies and CBM (Carbohydre-Bining Module) in 78 sub-families. Metagenome assembly resulted in ~110,000 contigs enabled the retrieval of 15 complete different genes encoded in the subfamilies GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 and GH127. A comparative analysis between the groups of animals in the different treatments showed a greater abundance of enzymes, with no metagenome of the fiber proven from the group of animals fed a diet enriched with sugarcane bagasse. These results show the sheep rumen microbiome as an untapped source of potential new fibrolytic enzymes. Using a diet amended with sugarcane bagasse increases the abundance of CAE and provide a substantially expanded catalog of genes participating in the deconstruction of plant biomass.

To understand and modify the secondary cell walls of plants the project group Enzyme Discovery in Hybrid Aspen for Fiber Engineering (EDEN) was founded composed of nine laboratories with funding from the European Commission. The main target of EDEN´s research is to genetically engineer fiber structure in order to produce transgenic trees with modified properties for the pulp and paper industries. In this target framework, the Populus tremula x tremuloides xyloglucan endotransglycosylase (PttXET16A) was selected for in-depth study of its transglycosylase activity catalyzing cleavage and reconnection of xyloglucan molecules, which is proposed to be involved in secondary cell wall morphogenesis. The creation of a family 16 carbohydrate active enzyme &#61538;-glucanase/XET hybrids were attempted in order to design a chimeric enzyme with one or more of the following altered properties: specificity, activity, and or stability. The two enzymes, Bacillus licheniformis 1,3-1,4-&#61538;-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase, are members of the same enzymatic family and have highly homologous 3-dimensional structures. However, the enzymes exhibit different activities, one a hydrolase the other a transferase; different specificities, one accepts only linear glcosydic substrates while the other branched substrates; and different stabilities. Hybrid enzyme construction represented an investigational challenge in order to understand what physical characteristics of both enzymes attribute to the specific pattern of activity and specificity observed.Removal of the 1,3-1,4-&#61538;-glucanase major loop resulted in a folded protein which still maintained some &#946;-glucan hydrolase activity. However, no xyloglucan endotransglycosylase-like activity or specificity was observed. Next, point mutations of the &#946;-sheets forming the enzymatic binding site cleft were mutated to resemble PttXET16A residues. The final chimeric protein neither exhibited XET nor &#946;-glucanase activities. Structural analysis by X-ray crystallography revealed a major unexpected structural rearrangement providing a clear insight for further enzyme engineering. / Amb la finalitat d'entendre i modificar la paret cel·lular secundària de les plantes, es va fundar el grup Enzyme Discovery in Hibrid Aspen for Fibern Engineering (EDEN) composat per nou laboratoris amb la finançament de la Comissió Europea. El principal objectiu de la recerca del grup EDEN és enginyar genèticament l'estructura de fibres per tal de produir arbres transgènics amb propietats modificades per les indústries de la polpa i el paper.En el marc d'aquest projecte, es va seleccionar el Populus tremula x tremuloides xiloglucà endotransglicosilasa (PttXET16A) per estudiar en profunditat la seva activitat transglicosilasa catalitzant el trencament i la reconnexió de molècules de xiloglucà, el qual sembla estar involucrat en la morfogènesi de la paret cel·lular secundària. D'aquesta manera, s'intentà crear una família 16 d'híbrids de l'enzim actiu amb carbohidrats &#61538;-glucanasa/XET per tal de dissenyar un enzim quimèric amb una o més de les propietats següents alterades: especificitat, activitat i/o estabilitat.Els dos enzims, Bacillus licheniformis 1,3-1,4-&#61538;-glucanasa i Populus tremula x tremuloides xiloglucà endotransglicosilasa, són membres de la mateixa família enzimàtica i tenen una gran homologia en les seves estructures en 3-dimensions. Tot i així, aquests enzims presenten diferents activitats, un presenta activitat hidrolasa i l'altre, transferasa; diferents especificitats, un accepta només substrats glicosílics lineals mentre l'altre, substrats ramificats; i diferents estabilitats. La construcció d'un enzim híbrid representa un repte en la investigació amb la finalitat d'entendre quines característiques físiques dels dos enzims s'atribueixen al model específic de l'activitat i especificitat observada.L'extracció del llaç més gran de l'1,3-1,4-&#61538;-glucanasa va resultar en l'obtenció d'una proteïna plegada que encara manté certa activitat hidrolasa del &#61538;-glucà. Tot i això, no s'observà activitat o especificitat similar a la xiloglucà endotransglicosilasa. A partir d'aquí, es realitzaren mutacions puntuals a diferents punts de les fulles &#61538; que formen l'escletxa del lloc d'unió de l'enzim per assemblar-se als residus del PttXET16A. La proteïna quimèrica final tampoc presentava activitat XET ni &#61538;-glucanasa. L'anàlisi de l'estructura per cristal·lografia de raigs X revelà una major reorganització estructural de l'esperada proveint el nou enzim d'un clar espai intern que obra moltes més portes a l'enginyeria de l'enzim. / Con la finalidad de entender y modificar la pared celular secundaria de las plantas, se fundó el grupo Enzyme Discovery in Hibrid Aspen for Fibern Engineering (EDEN) compuesto por nueve laboratorios con la financiación de la Comisión Europea. El principal objetivo de la búsqueda del grupo EDEN es ingeniar genéticamente la estructura de fibras para producir árboles transgénicos con propiedades modificadas para las industrias de la pulpa y el papel. En el marco de este proyecto, se seleccionó el Populus tremula x tremuloides xiloglucán endotransglicosilasa (PttXET16A) para estudiar en profundidad su actividad transglicosilasa catalizando la rotura y la reconnexión de moléculas de xiloglucán, el cual parece estar involucrado en la morfogénesis de la pared celular secundaria. De esta forma, se intentó crear una familia 16 de híbridos de la enzima activa con carbohidratos &#61538;-glucanasa/XET con la finalidad de diseñar una enzima quimérica con una o más de las propiedades siguientes alteradas: especificidad, actividad y/o estabilidad. Las dos enzimas, Bacillus licheniformis 1,3-1,4-&#61538;-glucanasa y Populus tremula x tremuloides xiloglucà endotransglicosilasa, son miembros de la misma familia enzimática y tienen una gran homología en sus estructuras en 3-dimensiones. Aún así, estas enzimas presentan diferentes actividades, una tiene actividad hidrolasa y la otra, transferasa; diferentes especificidades, una acepta sólo sustratos glicosílicos lineales mientras la otra, sustratos ramificados; y diferentes estabilidades. La construcción de una enzima híbrida representa un reto dentro de la investigación con la finalidad de entender qué características físicas de las dos enzimas se atribuyen al modelo específico de la actividad y especificidad observada. La extracción del lazo más grande de la 1,3-1,4-&#61538;-glucanasa resultó en la obtención de una proteína plegada que todavía mantiene cierta actividad hidrolasa del &#61538;-glucán. Aún así, no se observó actividad o especificidad similar a la xiloglucán endotransglicosilasa. A partir de este punto, se realizaron mutaciones puntuales a diferentes puntos de las hojas &#61538; que forman la brecha del lugar de unión de la enzima por asemejarse a los residuos del PttXET16A. La proteína quimérica final tampoco presentaba actividad XET ni &#61538;-glucanasa. El análisis de la estructura por cristalografía de rayos X reveló una mayor reorganización estructural de la esperada proveyendo la nueva enzima de un claro espacio interno que obre muchas más puertas a la ingeniería de la enzima.

O material lignocelulósico, presente na biomassa vegetal, representa uma importante fonte de energia, entretanto necessita da ação das enzimas lignocelulolíticas para sua degradação. A busca por novas enzimas que atuam na quebra da parede celular da planta em comunidades microbianas evoluídas naturalmente em um ambiente de degradação de biomassa como o rúmen oferece uma estratégia promissora para a prospecção de genes. Com isso, o projeto teve como objetivo a identificação de genes degradarores de biomassa vegetal em microrganismos do rúmen de ovinos usando a abordagem metagenômica. Para tanto, foram coletadas amostras da fase sólida do rúmen de 6 animais fistulados (Ovis aries) divididos em dois grupos e submetidos a duas dietas por 60 dias: tratamento controle e tratamento com dieta contendo bagaço de cana-de-açúcar. O DNA metagenômico total das amostras foi extraído e sequenciado na plataforma MiSeq Personal Sequencer (Illumina&reg;). A análise dos dados para a anotação taxônomica e funcional foi realizada no software MG-RAST. A caracterização dos genes degradadores de biomassa vegetal foi feita na plataforma CLC Genomic Workbench v.5.5.1(CLC Bio, Denmark) e a anotação de 4,68 gigabases de dados foi feita no banco de dados CAZy. A análise taxonômica mostrou uma predominância do domínio Bacteria compondo mais de 96% de todas as amostras, sendo os filos mais abundantes Bacteroidetes, Firmicutes, seguido de Proteobacteria. Entre todos os filos anotados, cinco tiveram a abundância aumentada no tratamento com adição de bagaço de cana-de-açúcar na dieta, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes e Verrucomicrobia, e dois filos foram mais abundantes no tratamento controle, Bacteroidetes e Synergistetes. De modo geral, a análise de ordenação não mostrou correlação entre a composição do microbioma e o tipo de dieta, porém, na análise funcional, essa correlação foi observada uma vez que houve separação entre os tratamentos. A abundância relativa das famílias de enzimas relacionadas à degradação de carboidratos segue um padrão similar em todas as amostras metagenômicas. O módulo catalítico da família de Glycoside Hydrolases (GH), o qual foi anotado em 129 subfamílias diferentes, foi o mais abundante em todas as amostras (45,5%), seguido da família GT (Glicosyl Tranferase), anotada em 97 subfamílias diferentes e CBM (Carbohydrete-Bining Module), em 78 subfamílias. A montagem do metagenoma resultou em aproximadamente 110.000 contigs e possibilitou a identificação de 15 diferentes genes completos codificados nas subfamílias GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 e GH127. A análise comparativa dos diferentes tratamentos mostrou uma maior abundância dessas enzimas no rúmen dos animais alimentados com a dieta enriquecida com bagaço de cana-de-açúcar. Em conclusão, a manipulação da dieta de ovinos por meio da substituição de parte da fração fibrosa da dieta por bagaço de cana-de-açúcar promove o enriquecimento de enzimas que degradam a biomassa vegetal no rúmem, favorecendo a prospecção e identificação de genes ativos em carboidratos. / The lignocellulose present in the plant biomass is a promising source of energy generation. However, the breakdown of plant biomass into simple sugars for bioethanol production is still inefficient and costly due to the recalcitrant nature of the plant fiber. The sheep rumen microbiome is specialized in degradation of plant material, but most members of this complex community are uncultured in the laboratory. Therefore, the search for new lignocellulolytic enzymes in microbial communities naturally evolved in biomass degradation environments, such as the rumen, using the exploration of the metagenome, is a promising strategy for identifying new genes. In this context, this study aimed to prospect plant biomass-degrading genes, selected from the sheep rumen microorganisms. The rumen samples were collected from 6 fistulated animals (Ovis aries), divided into two groups and subjected to two diets: control treatment and a treatment with a diet amended with sugarcane bagasse. The animals were fed for 60 days before sampling. To characterize the composition and functions of the rumen microbiome followed by the search of biomass-degrading genes, the metagenomic DNA was extracted from the solid contents of rumen and sequenced in MiSeq Personal Sequencer platform (Illumina&reg;). The taxonomic and functional data were performed using MG-RAST software. For the characterization of the plant biomass degrading genes, they were analyzed on the CLC platform Genomic Workbench v.5.5.1 (CLC Bio, Denmark) and 4.68 gigabases of data was annotated against the CAZy database. The taxonomic analysis showed a predominance of the Bacteria domain composing more than 96% of all the samples, being the most abundant phyla Bacterioidetes, Firmiutes, followed by Proteobacteria. Five bacterial phyla were significantly more abundant in the treatment were sugarcane bagasse was added, Firmicutes, Proteobacteria, Actinobacteria, Spirochaetes and Verrucomicrobia, and two phyla were more abundant in the control treatment, Bacteroidetes and Synergistetes. In general, the ordination analysis did not show correlation between diet type and rumen microbiota, but in the functional analysis, this correlation was observed since there was separation between the treatments. The relative abundance of enzyme families related to carbohydrate degradation follows a similar pattern of abundance across all metagenomic samples. The catalytic module of the GH (Glycoside Hydrolases) family, which was annotated in 129 different subfamilies, was the most abundant in all samples (45.5%), followed by the GT family (Glycosyltransferase), annotated in 97 different subfamilies and CBM (Carbohydre-Bining Module) in 78 sub-families. Metagenome assembly resulted in ~110,000 contigs enabled the retrieval of 15 complete different genes encoded in the subfamilies GH1, GH2, GH3, GH16, GH20, GH25, GH32, GH97 and GH127. A comparative analysis between the groups of animals in the different treatments showed a greater abundance of enzymes, with no metagenome of the fiber proven from the group of animals fed a diet enriched with sugarcane bagasse. These results show the sheep rumen microbiome as an untapped source of potential new fibrolytic enzymes. Using a diet amended with sugarcane bagasse increases the abundance of CAE and provide a substantially expanded catalog of genes participating in the deconstruction of plant biomass.

La présence de sucres complexes constitue une source nutritive importante pour le microbiote qui assure leur dégradation via des CAZymes. Dans le cadre de cette thèse, nous avons construit in silico un modèle de type minimicrobiome contenant 177 génomes représentatifs des communautés bactériennes dans un microbiote intestinal conventionnel. L’analyse du contenu de ce minimicrobiome nous a permis d’estimer leur abondance et leur diversité. De plus, la comparaison du contenu CAZymes par groupe bactérien de type « phylum » a révélé une variabilité inter-phylum, notamment une diversité de familles CAZymes et une abondance en gènes bien plus élevées chez les Bacteroidetes. Dans un deuxième temps, nous avons développé une puce à ADN sur laquelle nous avons greffé des sondes non redondantes ciblant plus de 6500 gènes codant des CAZymes. Nous avons ensuite testé la "puce CAZyme" par hybridation d’ADN bactérien extrait d’échantillons de selles. Nos résultats suggèrent que cette méthode serait plus sensible dans la détection de CAZymes provenant de bactéries rares par rapport à la métagénomique. Ainsi, il est intéressant de noter qu’en utilisant la puce CAZyme, nous avons pu détecter un gène codant pour une famille GH6, alors que les études métagénomiques n’ont jamais réussi à détecter ce gène dans le microbiome intestinal humain et animal. Enfin, l’examen de huit échantillons de selles a permis l’identification d’un noyau CAZome contenant 46 familles de GHs et PLs, ce qui suggérerait que le microbiote intestinal est caractérisé par une stabilité fonctionnelle en dépit de variations taxonomiques importantes entre les individus testés et indépendamment de leur état de santé. / The bacterial communities that inhabit our gut ensure their growth and survival by extracting their carbon source from the food that transits through the intestines. The complex carbohydrates included in the human diet are almost exclusively degraded by the gut microbiota using CAZymes. We built a minimicrobiome model using 177 genomes associated to gut microbiota. The CAZyme content analysis revealed their huge diversity and abundance in our minimicrobiome model. At the phylum level, the Bacteroidetes genomes showed the greatest CAZyme diversity and abundance. Interestingly, as most of CAZymes found in Bacteroidetes genomes contain a signal peptide allowing their secretion in the intestinal lumen and/or in periplasmic space, members of this phylum are suggested to be the primary degraders of complex carbohydrates. Further, we developed a microarray containing probes to target more than 6,500 CAZyme genes. We then validated the CAZyme microarray by the hybridization of bacterial DNA extracted from the stool samples of individuals. Our results suggest that a microarray-based study can detect genes from low-abundance bacteria better than metagenomic-based studies. A striking example was the detection of gene encoding a GH6-family in all subjects examined, whereas metagenomic studies have consistently failed to detect this gene in both human and animal gut microbiomes. In addition, an examination of eight stool samples allowed the identification of a corresponding core CAZome containing 46 CAZymes families that suggests a functional stability of the gut microbiota despite large taxonomical variations between individuals and independently of health state.

With its unique consortium of microorganisms from all domains of life, termite gut is considered one of the most efficient lignocellulose degrading systems in nature. Recently, host diet and taxonomy as well as gut microenvironmental conditions have emerged as main factors shaping microbial communities in termite guts. The aim of this thesis was to investigate this highly efficient lignocellulolytic system at holobiont level, with a particular focus on gut microbiome function and composition in relation to the host diet. As a starting point, we optimised a complete framework for an accurate termite gut prokaryote-oriented metatranscriptomics, which was at the basis of all subsequent sequencing assay designs and analyses performed in the course of the work. Afterwards, we characterised the compositions and functions of biomass-degrading bacterial communities in guts of plant fibre- and soil-feeding higher termites, proving the existence of functional equivalence across microbial populations from different termite hosts. We also showed that each termite is a reservoir of unique microorganisms and their accompanying genes. We further extended above approach to metagenomics and bacterial genomes reconstruction and we applied it to explore the process of biomass digestion in the different sections of the highly compartmented gut of soil feeding Labiotermes labralis. We showed that primarily cellulolytic activity of the termite host was restricted to foregut and midgut, while bacterial contribution was most pronounced in P1 and P3 hindgut compartments and included activities targeting broad range of lignocellulose components. Finally, we investigated the adaptation of a laboratory-maintained grass-feeding higher termite colony of Cortaritermes spp. to Miscanthus diet at host and symbiont levels. A natural system of a termite gut was shown to progressively change in composition to yield a consortium of microbes specialised in degradation of a specific biomass. Overall, the integrative omics approach proposed here provide a framework for a better understanding of a complex lignocellulose degradation by a higher termite gut system and pave a road towards its future bioprospecting. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished