Assembly of cellulases with synthetic protein scaffolds in vitro

Background: Enzymatic cascades in metabolic pathways are spatially organized in such a way as to facilitate the flow of substrates. The construction of artificial cellulase complexes that mimic natural multienzyme assemblies can potentially enhance the capacity for cellulose hydrolysis. In this study, an artificial cellulase complex was constructed by tethering three cellulases to a synthetic protein scaffold. Results: Three pairs of interacting proteins were selected and characterized. The artificial protein scaffolds were constructed by fusing three interacting proteins. Cellulases were tethered to these synthetic scaffolds in different orders. The optimal assembly resulted in a 1.5-fold higher hydrolysis of cellulose than that achieved by unassembled cellulases. Conclusions: A novel artificial protein scaffold was constructed and used to assemble three cellulases. The resultant increase in enzymatic activity suggests that this can be used as a strategy for enhancing the biocatalytic capacity of enzyme cascades.