Functional exploration of naturally and artificially enriched rumen microbiomes reveals novel enzymes involved in polysaccharide breakdown
Plant cell walls form the main carbon sources for bovine ruminal bacteria, which have evolved sophisticated multi-functional enzyme cocktails in response to the structural diversity of lignocelulloses. In order to isolate them from the vast world of uncultured bacteria, a high-throughput activity-based metagenomic approach was developed.   A multi-step screening methodology was implemented to identify metagenomic clones acting on polysaccharides and polyaromatic compounds. This approach was used to explore the functional potential of two different bovine ruminal microbiomes, totalling 1.5 Gb, derived from in vivo and in vitro enrichments on wheat straw. One hundred and sixty-eight fosmid metagenomic clones were isolated, producing various original glycoside-hydrolases, esterases and oxidoreductases mostly issued from unknown bacterial genera. Functional analysis evidenced that most of the identified xylanases come from Firmicutes that were not selected during enrichment in the fermenter, while most cellulases and mannanases originate from Bacteroidota.  These novel enzymes, that, for most of them, had not been previously identified by in depth-metagenome sequencing, present a high potential for numerous biotechnological processes, as they could be used alone or in cocktails to break down crude plant cell walls. The relationships established between enzyme function and taxonomy highlight the various roles played by ruminal Firmicutes and Bacteroidoda in plant cell wall degradation.