<p> </p><p>In a time when the amount of biological data present in the public domain is becoming increasingly vast, the need for good classification systems has never been greater. In the field of glycoscience the necessity of a good classification for the enzymes involved in the biosynthesis, modification and degradation of polysaccharides is even more pronounced than in other fields. This is due to the complexity of the substrates, the polysaccharides, as the theoretical number of possible hexa-oligosaccharides from only hexoses exceeds 10¹² isomers! </p><p>An initiative to classify enzymes acting on carbohydrates began around 1990 by the French scientist Bernard Henrissat. The resulting database, the Carbohydrate Active enzymes database (CAZy), classifies enzymes by sequence similarity into families allowing the inference of structure and catalytic mechanism. What CAZy <em>does not </em>provide however, are means to understand how members of a family are related, and in what way they differ from each other. The top-down approach used in this thesis, combining phylogenetic analysis of whole CAZy families, or sub-families, with structural determinations and detailed kinetic analysis allows for exactly that.  </p><p>Finding determinants for transglycosylation <em>versus </em>hydrolysis within the <em>xth </em>gene product family of GH16 as well as restricting the hydrolytic enzymes to a well defined clade are integral parts of paper I. In paper II a new bacterial sub-clade within CE8 was discovered. The structural determination of the<em>Escherichia coli </em>outer membrane lipoprotein YbhC from from the new sub-clade explained the difference in specificity. The information provided in the two papers of this thesis gives a better understanding of the development of different specificities of diverse CAZY families as well as it aids in future gene product annotations. Furthermore this work has begun to fill the white spots uncovered in the phylogenetic trees.</p><p> </p><p> </p>

In a time when the amount of biological data present in the public domain is becoming increasingly vast, the need for good classification systems has never been greater. In the field of glycoscience the necessity of a good classification for the enzymes involved in the biosynthesis, modification and degradation of polysaccharides is even more pronounced than in other fields. This is due to the complexity of the substrates, the polysaccharides, as the theoretical number of possible hexa-oligosaccharides from only hexoses exceeds 1012 isomers!  An initiative to classify enzymes acting on carbohydrates began around 1990 by the French scientist Bernard Henrissat. The resulting database, the Carbohydrate Active enzymes database (CAZy), classifies enzymes by sequence similarity into families allowing the inference of structure and catalytic mechanism. What CAZy does not provide however, are means to understand how members of a family are related, and in what way they differ from each other. The top-down approach used in this thesis, combining phylogenetic analysis of whole CAZy families, or sub-families, with structural determinations and detailed kinetic analysis allows for exactly that.   Finding determinants for transglycosylation versus hydrolysis within the xth gene product family of GH16 as well as restricting the hydrolytic enzymes to a well defined clade are integral parts of paper I. In paper II a new bacterial sub-clade within CE8 was discovered. The structural determination of theEscherichia coli outer membrane lipoprotein YbhC from from the new sub-clade explained the difference in specificity. The information provided in the two papers of this thesis gives a better understanding of the development of different specificities of diverse CAZY families as well as it aids in future gene product annotations. Furthermore this work has begun to fill the white spots uncovered in the phylogenetic trees.

La dégradation des fibres alimentaires est une fonction essentielle des écosystèmes digestifs microbiens. Chez le ruminant, elle est assurée par des bactéries, champignons et protozoaires capables de produire de nombreuses enzymes nécessaires à l’hydrolyse des polysaccharides de paroi végétale. Parmi les facteurs susceptibles d’influencer l’efficacité de dégradation des fibres, qui est une composante importante de la productivité et de la santé animales, des additifs tels que des levures probiotiques apparaissent comme un levier intéressant. Afin d’approfondir les connaissances sur les facteurs de modulation de l’activité fibrolytique, une biopuce ADN fonctionnelle, outil moléculaire haut-débit, ciblant les gènes codant les enzymes clés de la dégradation de la cellulose et des xylanes dans les écosystèmes digestifs a été développée. Aussi, une méthode efficace dédiée à des échantillons ruminaux pour la soustraction des ARNr à partir des ARN totaux a été mise au point afin d’accroitre la sensibilité de l’outil. La biopuce fonctionnelle a été validée sur échantillons de complexité croissante et démontre d’excellents caractères de spécificité et de sensibilité tout en étant exploratoire et quantitative. Des régulations différentielles de l’arsenal des gènes de la fibrolyse de la bactérie du rumen Fibrobacter succinogenes ont pu être montrées. De même, les résultats sur échantillons de rumen suggèrent un rôle des microorganismes eucaryotes dans la fibrolyse pouvant être plus important qu’initialement envisagé. Cette approche métatranscriptomique dirigée pourra in fine continuer d’être appliquée dans l’étude de l’impact de facteurs biotiques et abiotiques sur la fonction fibrolytique microbienne chez les animaux d’élevage. / Dietary fibre degradation is an essential function of microbial digestive ecosystems. In ruminants, this function is ensured by bacteria, fungi and protozoa, producing a large array of enzymes able to degrade plant cell wall polysaccharides. Among factors likely to influence the efficiency of fibre degradation, which is an important component in animal productivity and health, dietary additives such as probiotic yeasts appear as an interesting tool. To provide more insight on factors modulating fibrolytic activity, we designed a functional DNA microarray targeting genes coding for key enzymes involved in cellulose and xylan degradation by digestive microbiota. Also, an efficient method dedicated to rumen samples for removing microorganisms’ rRNA from total RNA samples was developed to increase the sensitivity of the tool. The DNA microarray was validated using targets of increasing complexity and demonstrated sensitivity and specificity as well as explorative and quantitative potential. Differential expression of genes involved in fibrolysis was evidenced in the rumen bacterium Fibrobacter succinogenes. Moreover, results on rumen samples suggest a more important role of eucaryotes in fibre degradation than previously thought. This targeted metatranscriptomic approach will be further applied to the study of the impact of biotic and abiotic factors on the microbial mechanisms of fibre degradation in livestock.