Isolation and characterization of a non-cellulosomal cellulase complex from a thermopilic microbial community

Cellulases have traditionally been identified and characterized from fungal and bacterial isolates. In natural environments, microbial consortia are responsible for lignocellulose deconstruction. However, these native communities are often too complex to assign functions to individual enzymes. By adapting complex communities from compost to grow aerobically with biomass substrates, we have obtained predictable consortia that generate highly active cellulases and can be grown at 300 L scale. By a combination of meta-omics methods and biochemical techniques we have established that these community-derived cellulases are localized in a protein complex produced by an uncultivated thermophilic Firmicutes species. This complex was partially purified from culture supernatants of a cellulose-adapted community by a combination of affinity digestion with phosphoric acid-swollen cellulose as resin and anion-exchange chromatography. We identified three glycoside hydrolases associated with the complex: an endoglucanase (GH9), a cellobiohydrolase (GH48), and an exoglucanase (GH6/5). Genes encoding these proteins were organized in a 17 kb putative operon together with two xylanases (GH10 and GH10_2), and a lytic polysaccharide monooxygenase (AA10). All of these proteins were multi-domain cellulases containing from one to three CBM3 binding domains. None of these sequences contained domains characteristic of cellulosomal proteins. We were able to reconstitute the complex in vitro by heterologous expression of the cellulase genes (GH9, GH48 and GH6/5) in E. coli. Currently, we are exploring the interaction between these proteins to maintain the complex. Our results emphasize that additional multi-protein structures beyond the cellulosome can be obtained by targeted discovery in natural samples.