Surpassing Substrate–Enzyme Competition by Compartmentalization

Abstract

Enzyme compartmentalization is one of the main strategies exploited by nature to create physically separated chemical environments that allow simultaneous enzyme reactions within the cell metabolic networks. However, designing nanostructured architectures that mimic cellular compartments remains a challenge when two competing enzymes must work simultaneously over the same substrate. Herein, we develop a method to fabricate soft hybrids that physically separate two oxidoreductases that compete for NADH with greatly different kinetics. The less competitive enzyme is encapsulated into polymeric capsules capable of recruiting NADH, which are then assembled on porous agarose microbeads where the most competitive enzyme is immobilized. As a result, this functional hybrid enables the simultaneous action of two competing enzymes in the same reaction media, which would otherwise be impossible in a non-compartmentalized system. We demonstrate that substrate recruitment is a powerful approach to building up enzymatic reaction networks with complex dynamics. Moreover, single-particle analysis under operando conditions reveals the impact of enzyme spatial organization on the overall performance of these soft hybrids, underlining the importance of understanding the functional variability within compartmentalized systems. Finally, integrating this compartmentalized system into a model cell-free biosynthetic cascade, we transform vinyl acetate into (S)-β-hydroxybutyrate with a 2 times higher titer than the non-compartmentalized free system. The proposed strategy can be generalized to produce compartmentalized cell-free biosynthetic pathways and multienzyme cascades where enzyme competition is an issue.

Coenzyme A Thioester Intermediates as Platform Molecules in Cell‐Free Chemical Biomanufacturing

“…Finally, we envision that system compartmentalization can alleviate some of the compatibility issues found in these synthetic pathways that ultimately limit their further implementation. [60]…”

Exploring Emergent Properties in Enzymatic Reaction Networks: Design and Control of Dynamic Functional Systems

“…Please note, the compartmentalization of enzymes or the immobilization of enzymes on different types of support can enhance the reaction rate and therefore the total output of an enzymatic cascade. For more information about compartmentalization, − in DNA nanocages, − immobilization in metal organic frameworks (MOFs), − or immobilization of enzymes on other supports, − we would refer to other reviews.…”

Single‐Particle and Single‐Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts

“…Specifically, the slower enzyme, alcohol dehydrogenase, can be confined within micrometric polymeric capsules composed of poly(allylamine)/ poly(styrene sulfonate). [53] These capsulespartially entrap NADH and effectively segregate the dehydrogenases. By immobilizing these capsules on agarose beads functionalized with aldehyde groups, where NADH oxidase is already anchored, products generated by the two enzymes utilizing NADH could be identified in the bulk.…”

“…Furthermore, SP studies have elucidated the feasibility of circumventing the competition for NADH between an NADH‐dependent alcohol dehydrogenase (slower enzyme) and a NADH oxidase (faster enzyme) through strategic compartmentalization. Specifically, the slower enzyme, alcohol dehydrogenase, can be confined within micrometric polymeric capsules composed of poly(allylamine)/poly(styrene sulfonate) [53] . These capsulespartially entrap NADH and effectively segregate the dehydrogenases.…”