Surpassing Substrate-Enzyme Competition by Compartmentalization

Enzymecompartmentalization is one of the main strategies exploitedby nature to create physically separated chemical environments thatallow simultaneous enzyme reactions within the cell metabolic networks.However, designing nanostructured architectures that mimic cellularcompartments remains a challenge when two competing enzymes must worksimultaneously over the same substrate. Herein, we develop a methodto fabricate soft hybrids that physically separate two oxidoreductasesthat compete for NADH with greatly different kinetics. The less competitiveenzyme is encapsulated into polymeric capsules capable of recruitingNADH, which are then assembled on porous agarose microbeads wherethe most competitive enzyme is immobilized. As a result, this functionalhybrid enables the simultaneous action of two competing enzymes inthe same reaction media, which would otherwise be impossible in anon-compartmentalized system. We demonstrate that substrate recruitmentis a powerful approach to building up enzymatic reaction networkswith complex dynamics. Moreover, single-particle analysis under operando conditions reveals the impact of enzyme spatialorganization on the overall performance of these soft hybrids, underliningthe importance of understanding the functional variability withincompartmentalized systems. Finally, integrating this compartmentalizedsystem into a model cell-free biosynthetic cascade, we transform vinylacetate into (S)-& beta;-hydroxybutyrate with a 2times higher titer than the non-compartmentalized free system. Theproposed strategy can be generalized to produce compartmentalizedcell-free biosynthetic pathways and multienzyme cascades where enzymecompetition is an issue.