title: Recoded organisms engineered to depend on synthetic amino acids.
abstract: Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems, which include bioremediation and probiotics. Although safeguards have been designed to control cell growth by essential gene regulation, inducible toxin switches and engineered auxotrophies, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations. Here we describe the construction of a series of genomically recoded organisms (GROs) 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 engineering, 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 residues of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼10(11) cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches. 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 transfer and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Alexis J
authors.initials: AJ
authors.lastname: Rovner
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Adrian D
authors.initials: AD
authors.lastname: Haimovich
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Spencer R
authors.initials: SR
authors.lastname: Katz
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Li
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Grome
authors.affiliation: 1] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA [2] Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Gassaway
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Miriam
authors.initials: M
authors.lastname: Amiram
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Jaymin R
authors.initials: JR
authors.lastname: Patel
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Ryan R
authors.initials: RR
authors.lastname: Gallagher
authors.affiliation: 1] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA [2] Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
authors.affiliation: 1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA [2] Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
authors.forename: Farren J
authors.initials: FJ
authors.lastname: Isaacs
mesh_terms: D000596:Amino Acids / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D003062:Codon / Q000235:genetics; D003264:Containment of Biohazards / Q000379:methods*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004777:Environment; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D022761:Gene Transfer, Horizontal / Q000235:genetics; D020043:Genes, Essential / Q000235:genetics; D005815:Genetic Code / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D016680:Genome, Bacterial / Q000235:genetics; D050296:Microbial Viability / Q000187:drug effects* / Q000235:genetics; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D010454:Peptide Termination Factors / Q000235:genetics; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D000596:Amino Acids; D003062:Codon; D003470:Culture Media; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C489005:prfA protein, E coli; D010649:Phenylalanine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases