High-throughput functional metagenomics for the discovery of glycan metabolizing pathways

Glycans are widely distributed in nature. Produced by almost all organisms, they are involved in numerous cellular processes, such as energy supply and storage, cell structuration, protein maturation and signalling, and cell recognition. Glycans are thus key elements mediating the interactions between mammals, plants, bacteria, fungi and even viruses. They also represent a reliable source of carbon for microbes, which have developed complex strategies to face their structural diversity and to harvest them. However between 70 and 99% of these microorganisms are still uncultured, while they represent a goldmine for the discovery of new enzymes. In order to boost their identification and characterization, a functional metagenomic approach was developed, based on the design of various high-throughput, robust and sensitive screening strategies. The functional potential of Gbp of metagenomic DNA from various origins was explored, revealing dozens of novel enzyme families and functions. Integration of biochemical, structural, meta-omic and omic data allowed us to decipher, from the molecular to the ecosystemic scale, novel mechanisms of plant, microbial and mammal glycan metabolization. These new metabolic pathways involve batteries of glycoside-hydrolases, glycoside-phosphorylases and sugar transporters. These fascinating proteins appear as new targets to control host-microbe interactions. They also constitute very efficient biotechnological tools for biorefineries and synthetic biology

Investigating Host Microbiota Relationships Through Functional Metagenomics

The human Intestinal mucus is formed by glycoproteins, the O- and N-linked glycans which constitute a crucial source of carbon for commensal gut bacteria, especially when deprived of dietary glycans of plant origin. In recent years, a dozen carbohydrate-active enzymes from cultivated mucin degraders have been characterized. But yet, considering the fact that uncultured species predominate in the human gut microbiota, these biochemical data are far from exhaustive. In this study, we used functional metagenomics to identify new metabolic pathways in uncultured bacteria involved in harvesting mucin glycans. First, we performed a high-throughput screening of a fosmid metagenomic library constructed from the ileum mucosa microbiota using chromogenic substrates. The screening resulted in the isolation of 124 clones producing activities crucial in the degradation of human O- and N-glycans, namely sialidases, beta-D-N-acetyl-glucosaminidase, beta-D-N-acetyl-galactosaminidase, and/or beta-D-mannosidase. Thirteen of these clones were selected based on their diversified functional profiles and were further analyzed on a secondary screening. This step consisted of lectin binding assays to demonstrate the ability of the clones to degrade human intestinal mucus. In total, the structural modification of several mucin motifs, sialylated mucin ones in particular, was evidenced for nine clones. Sequencing their metagenomic loci highlighted complex catabolic pathways involving the complementary functions of glycan sensing, transport, hydrolysis, deacetylation, and deamination, which were sometimes associated with amino acid metabolism machinery. These loci are assigned to several Bacteroides and Feacalibacterium species highly prevalent and abundant in the gut microbiome and explain the metabolic flexibility of gut bacteria feeding both on dietary and human glycans