Firewalled synthetic commensal blocks horizontal gene transfer in the gut

Synthetic biology enables the rational reprogramming of microorganisms into living therapeutics and agents for bioremediation. However, such genetically modified organisms (GMOs) disseminate their synthetic genetic information into natural microbial communities through horizontal gene transfer (HGT), posing biosafety risks that limit clinical and environmental deployment. Reassigning sense codons to an alternative amino acid identity establishes a genetic firewall that simultaneously prevents incoming and outgoing gene flow, but reported implementations compromise fitness, precluding clinical and industrial use. Here, we overcome this limitation using genome design and laboratory evolution to create a high-fitness genetically firewalled Escherichia coli commensal. By directly altering the amino acid identity of TCA and TCG serine codons in the genetic code without an unassigned intermediate, we establish a robust genetic firewall that remains stable for thousands of generations. This firewalled commensal stably colonizes the mouse gastrointestinal tract for more than 100 days and blocks viral infections and HGT. As the long-term within-gut evolution of this firewalled organism identified adaptive mutations in genes responsible for carbon source utilization, we rationally redesigned the strain's genome to increase fitness. Together, this work establishes a genetically firewalled commensal for safer living therapeutics development and provides a strategy for designing high-fitness, virus- and gene-transfer-resistant organisms for clinical and environmental use.