Limits...
Recoded organisms engineered to depend on synthetic amino acids.

Rovner AJ, Haimovich AD, Katz SR, Li Z, Grome MW, Gassaway BM, Amiram M, Patel JR, Gallagher RR, Rinehart J, Isaacs FJ - Nature (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH

Related in: MedlinePlus

Investigating the viability of synthetic auxotrophs on diverse media typesRescue by cross-feeding shown through spotting on diverse media types +/- pAzF/l-arabinose and biotin supplementation; EcNR2, rEc.γ, and rEc.γ.dC.46′ are auxotrophic for biotin (ΔbioA/B) and rEc.γ.dC.46′ is also a pAzF auxotroph.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4590768&req=5

Figure 13: Investigating the viability of synthetic auxotrophs on diverse media typesRescue by cross-feeding shown through spotting on diverse media types +/- pAzF/l-arabinose and biotin supplementation; EcNR2, rEc.γ, and rEc.γ.dC.46′ are auxotrophic for biotin (ΔbioA/B) and rEc.γ.dC.46′ is also a pAzF auxotroph.

Mentions: To determine whether a synthetic auxotroph could be rescued by metabolic cross-feeding, we evaluated the viability of strains on diverse media types. We grew wild-type MG1655 E. coli, a biotin auxotroph (EcNR212), a non-contained GRO (rEc.γ), and the pAzF synthetic auxotroph (rEc.γ.dC.46′) on solid media containing both pAzF/L-arabinose and biotin, either pAzF/L-arabinose or biotin, and on plates lacking small molecules (Fig. 4). Despite biotin auxotrophy, growth of EcNR2 on rich defined media without biotin was rescued in close proximity to wild-type E. coli, suggesting cross-feeding of essential metabolites (Extended Data Fig. 9). Blood agar and soil extracts without biotin or pAzF/l-arabinose supplementation supported growth of all strains except the synthetic auxotroph, which only grew on media supplemented with pAzF and l-arabinose. These data suggest that synthetic auxotrophies could lead to a more viable containment strategy for clinical (e.g., blood) and environmental (e.g., soil) settings, where metabolic auxotrophies can be overcome by proximal, metabolically competent strains.


Recoded organisms engineered to depend on synthetic amino acids.

Rovner AJ, Haimovich AD, Katz SR, Li Z, Grome MW, Gassaway BM, Amiram M, Patel JR, Gallagher RR, Rinehart J, Isaacs FJ - Nature (2015)

Investigating the viability of synthetic auxotrophs on diverse media typesRescue by cross-feeding shown through spotting on diverse media types +/- pAzF/l-arabinose and biotin supplementation; EcNR2, rEc.γ, and rEc.γ.dC.46′ are auxotrophic for biotin (ΔbioA/B) and rEc.γ.dC.46′ is also a pAzF auxotroph.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590768&req=5

Figure 13: Investigating the viability of synthetic auxotrophs on diverse media typesRescue by cross-feeding shown through spotting on diverse media types +/- pAzF/l-arabinose and biotin supplementation; EcNR2, rEc.γ, and rEc.γ.dC.46′ are auxotrophic for biotin (ΔbioA/B) and rEc.γ.dC.46′ is also a pAzF auxotroph.
Mentions: To determine whether a synthetic auxotroph could be rescued by metabolic cross-feeding, we evaluated the viability of strains on diverse media types. We grew wild-type MG1655 E. coli, a biotin auxotroph (EcNR212), a non-contained GRO (rEc.γ), and the pAzF synthetic auxotroph (rEc.γ.dC.46′) on solid media containing both pAzF/L-arabinose and biotin, either pAzF/L-arabinose or biotin, and on plates lacking small molecules (Fig. 4). Despite biotin auxotrophy, growth of EcNR2 on rich defined media without biotin was rescued in close proximity to wild-type E. coli, suggesting cross-feeding of essential metabolites (Extended Data Fig. 9). Blood agar and soil extracts without biotin or pAzF/l-arabinose supplementation supported growth of all strains except the synthetic auxotroph, which only grew on media supplemented with pAzF and l-arabinose. These data suggest that synthetic auxotrophies could lead to a more viable containment strategy for clinical (e.g., blood) and environmental (e.g., soil) settings, where metabolic auxotrophies can be overcome by proximal, metabolically competent strains.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus