The essential processes of life, such as metabolism and signal transduction, are carried out through intricate mechanisms regulated by living cells within multi-compartmentalized structures. Replicating these systems is a key challenge in synthetic cell research. Especially, artificial organelles in a synthetic cell model are important because they mimic how cells organize their internal processes, helping to better control and optimize biochemical reactions. Globular protein vesicles (GPVs), self-assembled from recombinant fusion proteins, have emerged as a promising platform for synthetic cell development, leveraging the biocompatibility and functional versatility of proteins as building blocks. Functional GPV-based compartments enable controlled enzyme localization, providing spatial regulation of enzymatic reactions. In this study, we produce a recombinant fusion protein of octopine dehydrogenase (ODH), which catalyzes the NADH-dependent reductive condensation of pyruvate and arginine to form octopine, playing a role in anaerobic energy metabolism by regenerating NAD⁺. We investigate its self-assembly behavior under various conditions and demonstrate the formation of multicompartment protein vesicles housing ODH-incorporated vesicles as artificial organelles. By pairing with pyruvate kinase, we perform enzyme cascade reactions to mimic pyruvate metabolism under anaerobic conditions in vitro. This approach offers a versatile platform for recreating cellular metabolic networks in confined environments, providing new opportunities for advancing synthetic cell engineering and biotechnological applications.
