Recoded organisms engineered to depend on synthetic amino acids

Construction of a series of genomically recoded organisms whose growth is restricted by the expression of essential genes dependent on exogenously supplied synthetic amino acids introduces novel orthogonal barriers between these engineered organisms and the environment, thereby creating safer genetically modified organisms. Two manuscripts published in this issue of Nature describe independent approaches towards generating an organism dependent on unnatural amino acids, a development which could find applications for biocontainment and exploration of previously unsampled fitness landscapes. George Church and colleagues redesigned essential enzymes in an organism (Escherichia coli) with an altered genetic code to make it metabolically dependent on non-standard amino acids for survival. The resulting genetically modified organisms (GMOs) cannot metabolically circumvent their biocontainment mechanisms and show unprecedented resistance to evolutionary escape. The few escapees are rapidly outcompeted by unmodified organisms. Using multiplex automated genome engineering, Farren Isaacs and colleagues construct a series of genomically recoded organisms whose growth is restricted by the expression of essential genes that depend on exogenously supplied synthetic amino acids. They constructed synthetic auxotrophs with advanced orthogonal barriers between engineered organisms and the environment, thereby creating safer GMOs. Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals1. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems2,3, which include bioremediation4 and probiotics5. Although safeguards have been designed to control cell growth by essential gene regulation6, inducible toxin switches7 and engineered auxotrophies8, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations9,10. Here we describe the construction of a series of genomically recoded organisms (GROs)11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering12, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼1011 cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches2,3,6,7,8,9,10. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.