Polysachharide utilization loci in rumen bacteria. Droplet-based functional metagenomic screening for the discovery of polysaccharide utilization loci using unlabelled oligosaccharides

In this study we overcome the limitation to model substrates in a new functional metagenomic screening approach based on microfluidics. The growth of a fluorescent host strain is used to detect the turnover of complex carbohydrates into metabolizable monosaccharides by a library member and serves as the criterion to identify hits. Screening of a 2x104-member fosmid library from bovine rumen in one hour led to the identification of five new PULs. Subsequent kinetic validations link hits to the unlabelled screening oligosaccharides and also suggest possible natural substrates (polysaccharides isolated from plant cell wall). Liberated from the requirement to use model substrates, droplet microfluidics is shown to efficiently explore metagenomic sequences for activities on real world substrates that contribute to a more comprehensive annotation of the enzyme thesaurus lodged in databases.