Abstract
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.
Highlights
In living organisms, many crucial cellular functions such as biosynthesis and cellular signaling [1] are controlled by multi-step enzymatic reactions that take place simultaneously with unsurpassed efficiency and specificity
Scaf3 have been successfully displayed onto the surface of Saccharomyces cerevisiae [17], it is unclear whether this large scaffold can be functionally displayed on the surface of E. coli
The truncated ice nucleation protein anchor (INP), which has been used as an surface anchor to display proteins up to 119 kDa in E. coli [19], was used to target Scaf3 to the surface of the outer membrane vesicles (OMVs)-hyperproducing strain JC8031 (Fig. 1) [20]
Summary
Many crucial cellular functions such as biosynthesis and cellular signaling [1] are controlled by multi-step enzymatic reactions that take place simultaneously with unsurpassed efficiency and specificity. A key characteristic of these highly efficient enzyme pathways is the cooperative and spatial organization of enzymes to ensure the sequential conversion of substrates [2]. This molecular-level organization of enzymes has a distinct enhancement effect on the overall efficiency by increasing the local enzyme and substrate concentrations, by channeling of intermediates between consecutive enzymes, and to avoid competition with other reactions present in the cell [3,4]. Formation of this highly ordered structure is mediated by the high-affinity proteinprotein interaction (Kd ,10-9 M) between the dockerin and cohesin domain, allowing the assembly of multiple cellulases in a spatially defined manner [6]
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