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Abstract

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 CO2 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. All life must transform metabolites through enzyme-catalyzed reactions and transmit the information necessary to produce those enzymes for future generations. Synthetic biology systems are often limited to either a metabolic or genetic focus. Giaveri et al. combined two existing artificial systems, a carbon dioxide–fixing metabolic cycle and an in vitro transcription and translation platform, to create a complex hybrid system that can incorporate carbon dioxide–derived glycine into DNA-encoded protein products. When provided with the appropriate starting conditions, a droplet-confined system can self-regenerate by producing missing enzymes. This integrated metabolic and genetic biosynthetic system may be a useful system in which to study metabolic networks and could serve as a platform for additional metabolic modules. —Michael A. Funk