One of the hallmarks of living organisms is their capacity for self-organization and regeneration, which requires a tight integration of metabolic and genetic networks. We sought to construct a linked metabolic and genetic network in vitro that shows such lifelike behavior outside of a cellular context and generates its own building blocks from nonliving matter. We integrated the metabolism of the crotonyl-CoA/ethyl-malonyl-CoA/hydroxybutyryl-CoA cycle with cell-free protein synthesis using recombinant elements. Our network produces the amino acid glycine from CO ₂ and incorporates it into target proteins following DNA-encoded instructions. By orchestrating ~50 enzymes we established a basic cell-free operating system in which genetically encoded inputs into a metabolic network are programmed to activate feedback loops allowing for self-integration and (partial) self-regeneration of the complete system.