| Resumo: | The plant cell wall is, in its majority, constituted by complex and structurally diverse polysaccharides that are valuable resources for industrial and biotechnological applications. Anaerobic microbial organisms are highly efficient for plant cell wall polysaccharide biodegradation and have evolved a multi-enzyme complex system, the Cellulosome, where catalytic enzymes have non-catalytic Carbohydrate Binding Modules (CBMs) appended that highly potentiate the enzymes’ catalytic efficiency. Deciphering at molecular level the mechanisms underlying plant cell wall carbohydrate recognition and deconstruction by different cellulolytic bacteria is crucial to elucidate these complex biological systems, as well as to further promote novel potential applications. The work developed in this Thesis focused on the unique approach of combining carbohydrate microarrays with X-ray crystallography, to uncover carbohydrate ligands for CBMs and to structurally characterize novel CBM-carbohydrate interactions of two anaerobic bacteria that reside in different ecological niches: Clostridium thermocellum, found in soils, and Ruminococcus flavefaciens FD-1, present in the rumen of herbivorous. To this end, microarrays featuring carbohydrate probes with polysaccharide and oligosaccharide sequences representative of the structural diversity found on plant cell walls, but also in fungal and bacterial cell walls, were developed and then used to screen the carbohydrate-binding and ligand-specificity of 150 CBMs of C. thermocellum and R. flavefaciens CBMomes. The groups of polysaccharides that are differentially recognised were revealed for 59 CBMs and novel CBM-ligand specificities were identified for 23 modules from C. thermocellum and 21 from R. flavefaciens. Overall, the two bacteria differentially expressed CBM families with different carbohydrate-binding specificities, which may reflect adaptation to substrate availability in their specific ecological niche or the complexity of their Cellulosome. Using the information derived from the high-throughput microarray analysis, and according to their biotechnological relevance or novelty, CBMs and the respective ligands were selected for further structural studies. The novel CBM structures solved, complemented with biochemical and biophysical data, enabled the characterization of the molecular determinants for the recognition of mixed-linked β1,3-1,4-glucans by C. thermocellum family 11 CBM, chitin and peptidoglycan-derived sequences by a novel LysM domain from C. thermocellum family 50 CBMs, and pectic arabinans by R. flavefaciens family 13 CBM. The results reported here allow to assign a functional role for these CBMs and CBM families and contribute to the classification of the novel CBMs identified in the genome of the two bacteria, particularly those from R. flavefaciens FD1. Furthermore, the information derived from this integrative study, can promote a better understanding of cellulolytic capabilities of these bacteria, as well as to potentiate biotechnological applications of CBMs.
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