Functional screening of highly divergent, unclassified or putative polysaccharides degrading enzymes – 1000Cazymes

GH and PL have been classified as a function of the reaction they catalyse (about 160 EC numbers) and by sequence homology (133 GH families and 23 PL families, CAZy classification: www.cazy.org). Despite the exponential deluge of sequence data resulting from genomic programs, the pace of discovery of new GH and PL enzymes remains constant and comparatively modest. The new sequence data essentially increase the number of enzymes classified in existing families, and do not contribute to the number of biochemically-characterized enzymes or of enzymes with a known 3-D structure. In this context, we propose to assign the catalytic function to 1000 recombinant proteins which peptide sequences are highly divergent with GHs and PLs already biochemically characterized, and putative proteins whose genomics context suggests their involvement in carbohydrate breakdown. Based on a medium throughput screening method including 200 oligo- and polysaccharides substrates, we will ascribe the function of selected proteins which will be over-expressed recombinantly in E. coli.

476 genes of CAZymes have been cloned and overexpressed. Functionnal screening have been undertaken. Preliminary results demonstrate the relevance of the strategy.

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Polysaccharides are very complex biomacromolecules and represent the most abundant biomass on land and in oceans. As such they mediate a plethora of biological functions, from energy storage, to structural molecules, or as the mediators of intra- and intercellular recognition within one organism or between organisms. Exploitation of this resource, from the production of original oligosaccharides (e.g. bioactives) toward fermentable monosaccharides (e.g. bioenergy), rely on the use of enzymes: the glycosides hydrolases (GH) and the polysaccharides lyases (PL).
GH and PL have been classified as a function of the reaction they catalyse (about 160 EC numbers) and by sequence homology (133 GH families and 23 PL families, CAZy classification: www.cazy.org). Despite the exponential deluge of sequence data resulting from genomic programs, the pace of discovery of new GH and PL enzymes remains constant and comparatively modest. The new sequence data essentially increase the number of enzymes classified in existing families, and do not contribute to the number of biochemically-characterized enzymes or of enzymes with a known 3-D structure.
In this context, we propose to assign the catalytic function to 1000 recombinant proteins which peptide sequences are highly divergent with GHs and PLs already biochemically characterized, and putative proteins whose genomics context suggests their involvement in carbohydrate breakdown. Based on a medium throughput screening method including 200 oligo- and polysaccharides substrates, we will ascribe the function of selected proteins which will be over-expressed recombinantly in E. coli.
At the end of the project, we will have biochemically characterized novel GH and PL enzymes which will be the first representatives of new families or sub-families of CAZymes. Beside the functional exploration, the project will substantially enrich the catalog of available enzymes and will allow the preparation of new series of oligosaccharides, which present strong potential of biotechnological valorization.