Positional assembly of enzymes on bacterial outer membrane vesicles for cascade reactions

The systematic organization of enzymes is a key feature for the efficient operation of cascade reactions in nature. Here, we demonstrate a facile method to create nanoscale enzyme cascades by using engineered bacterial outer membrane vesicles (OMVs) that are spheroid nanoparticles (roughly 50 nm in diameter) produced by Gram-negative bacteria during all phases of growth. By taking advantage of the fact that OMVs naturally contain proteins found in the outer cell membrane, we displayed a trivalent protein scaffold containing three divergent cohesin domains for the position-specific presentation of a three-enzyme cascade on OMVs through a truncated ice nucleation protein anchoring motif (INP). The positional assembly of three enzymes for cellulose hydrolysis was demonstrated. The enzyme-decorated OMVs provided synergistic cellulose hydrolysis resulting in 23-fold enhancement in glucose production than free enzymes.