Engineering Artificial Mitochondria with Self‐Amplifying Proton Generation for Autonomous Energy Supply and Metabolic Coupling in Artificial Cells

Abstract A continuous and autonomous energy supply is essential for sustaining life‐like biochemical processes in artificial cells. Although considerable efforts have been devoted to engineering artificial organelles that emulate mitochondrial energy conversion, the generation of a robust transmembrane proton gradient—essential for driving efficient ATP production—remains a major challenge. Here, we present a mitochondria‐mimicking ATP nano‐generator constructed through quantitative co‐compartmentalization of glucose oxidase and catalase within silica nanocapsules. Enzymes are encapsulated in situ during the formation of core‐shell nanocapsules, enabling precise loading, effective protection, and creation of a confined nanoscale reaction chamber that fosters catalytic synergy. Within this microenvironment, catalase rapidly decomposes H 2 O 2 to generate O 2 , which is in turn utilized by glucose oxidase—thus establishing a self‐reinforcing enzymatic cascade that amplifies proton production. After coating the enzyme‐loaded nanocapsules with an ATPase‐integrated liposome bilayer to construct the artificial mitochondrion, the resulting proton gradient across the membrane efficiently drives ATP synthase rotation, enabling high‐yield ATP production. When integrated into giant unilamellar vesicles (GUVs) as synthetic cell models, this system supports autonomous nicotinamide adenine dinucleotide (NADH) biosynthesis and glucose‐powered oxidative phosphorylation, mimicking key metabolic features of living mitochondria. This work establishes an effective and versatile platform for engineering energy‐autonomous artificial living systems, advancing the state of the art of bottom‐up synthetic biology.