Reconstitution of metabolic reactions within self-assembled, multi-compartment protein vesicles

Artificial cells that reproduce the spatial organization of metabolism offer a powerful platform for understanding and controlling complex biochemical pathways. In this work, we engineer globular protein vesicles (GPVs) by exhibiting octopine dehydrogenase (ODH), a model monomeric enzyme, on vesicle membranes through the self-assembly of recombinant fusion proteins. The enzymatic GPVs are constructed from two complementary fusion protein building blocks: ODH fused with a glutamic acid-rich leucine zipper (ODH-Z_E) and an elastin-like polypeptide fused with an arginine-rich leucine zipper (Z_R-ELP). The oppositely charged leucine zipper pairs (Z_E and Z_R) form a heterodimer via electrostatic interactions, driving vesicle assembly. Because these interactions are sensitive to ionic strength and stoichiometry, we investigate the effects of salt concentration and molar ratio on self-assembly behavior and vesicle morphology. Enzymatic assays show that ODH displayed on GPVs exhibits enhanced stability and sustained catalytic activity compared to the free enzyme. To recapitulate a two-step enzyme cascade representing the terminal steps of glycolysis and anaerobic fermentation observed in marine invertebrates, we created a hierarchical multicompartment architecture consisting of nanoscale ODH-displaying vesicles (∼500 nm in diameter) encapsulated within giant GPVs (tens of micrometers). We further engineered co-encapsulation and nested configurations to control pyruvate generation and transport across compartments. Fluorescence-based monitoring of NADH consumption reveals that these architectures produce distinct reaction kinetics, underscoring the role of spatial organization in modulating enzymatic behavior. Together, these results highlight the potential of GPVs as customizable platforms for rebuilding metabolic processes within artificial cell-like compartments.