Engineering Artificial Mitochondria with Self-Amplifying Proton Generation for Autonomous Energy Supply and Metabolic...

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 H₂O₂ to generate O₂, 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.