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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.

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Dose-dependent growth of rEc.γ.dC.46′.ΔtY in pAzF and l-arabinose compared to the non-contained ancestorGrowth in LB media supplemented with different concentrations of pAzF and l-arabinose. Growth profiles for rEc.γacross a gradient of pAzF concentrations in the presence of a, 0%, b, 0.002%, c, 0.02%, and d, 0.2% l-arabinose. Growth profiles for rEc.γ.dC.46′.ΔtY across a gradient of pAzF concentrations in the presence of f, 0%, g, 0.002%, h, 0.02%, and i, 0.2% l-arabinose. e and j, Growth profiles illustrated in parts a-d and f-i are depicted as heat maps in parts e and j, respectively, where the maximum OD600 was obtained from the average of three replicates and plotted in MATLAB. Reported growth profiles and heat map values are averages, where n=3 technical replicates, and error bars are ±s.d.
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Figure 7: Dose-dependent growth of rEc.γ.dC.46′.ΔtY in pAzF and l-arabinose compared to the non-contained ancestorGrowth in LB media supplemented with different concentrations of pAzF and l-arabinose. Growth profiles for rEc.γacross a gradient of pAzF concentrations in the presence of a, 0%, b, 0.002%, c, 0.02%, and d, 0.2% l-arabinose. Growth profiles for rEc.γ.dC.46′.ΔtY across a gradient of pAzF concentrations in the presence of f, 0%, g, 0.002%, h, 0.02%, and i, 0.2% l-arabinose. e and j, Growth profiles illustrated in parts a-d and f-i are depicted as heat maps in parts e and j, respectively, where the maximum OD600 was obtained from the average of three replicates and plotted in MATLAB. Reported growth profiles and heat map values are averages, where n=3 technical replicates, and error bars are ±s.d.

Mentions: To further investigate the dependency to sAAs, liquid growth profiles were collected for synthetic auxotrophs across sAA and l-arabinose concentration gradients. Growth of rEc.γ.dC.46′.ΔtY was not observed below 0.002% l-arabinose and 0.5 mM pAzF (Fig. 3g, Extended Data Fig. 7). Growth increased in a dose-dependent manner with increasing concentrations of pAzF and l-arabinose, where 5 mM pAzF and 0.2% l-arabinose was optimal for fitness (i.e., maximum OD600 and minimum DT). In an equivalent experiment with rEc.β.dC.12′.ΔtY, 1 mMpIF and 0.2% l-arabinose was optimal for fitness (Extended Data Fig. 8). Since growth was not observed in media lacking either l-arabinose or the sAA, these data further support the dependency of synthetic auxotrophs on sAAs.


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)

Dose-dependent growth of rEc.γ.dC.46′.ΔtY in pAzF and l-arabinose compared to the non-contained ancestorGrowth in LB media supplemented with different concentrations of pAzF and l-arabinose. Growth profiles for rEc.γacross a gradient of pAzF concentrations in the presence of a, 0%, b, 0.002%, c, 0.02%, and d, 0.2% l-arabinose. Growth profiles for rEc.γ.dC.46′.ΔtY across a gradient of pAzF concentrations in the presence of f, 0%, g, 0.002%, h, 0.02%, and i, 0.2% l-arabinose. e and j, Growth profiles illustrated in parts a-d and f-i are depicted as heat maps in parts e and j, respectively, where the maximum OD600 was obtained from the average of three replicates and plotted in MATLAB. Reported growth profiles and heat map values are averages, where n=3 technical replicates, and error bars are ±s.d.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 7: Dose-dependent growth of rEc.γ.dC.46′.ΔtY in pAzF and l-arabinose compared to the non-contained ancestorGrowth in LB media supplemented with different concentrations of pAzF and l-arabinose. Growth profiles for rEc.γacross a gradient of pAzF concentrations in the presence of a, 0%, b, 0.002%, c, 0.02%, and d, 0.2% l-arabinose. Growth profiles for rEc.γ.dC.46′.ΔtY across a gradient of pAzF concentrations in the presence of f, 0%, g, 0.002%, h, 0.02%, and i, 0.2% l-arabinose. e and j, Growth profiles illustrated in parts a-d and f-i are depicted as heat maps in parts e and j, respectively, where the maximum OD600 was obtained from the average of three replicates and plotted in MATLAB. Reported growth profiles and heat map values are averages, where n=3 technical replicates, and error bars are ±s.d.
Mentions: To further investigate the dependency to sAAs, liquid growth profiles were collected for synthetic auxotrophs across sAA and l-arabinose concentration gradients. Growth of rEc.γ.dC.46′.ΔtY was not observed below 0.002% l-arabinose and 0.5 mM pAzF (Fig. 3g, Extended Data Fig. 7). Growth increased in a dose-dependent manner with increasing concentrations of pAzF and l-arabinose, where 5 mM pAzF and 0.2% l-arabinose was optimal for fitness (i.e., maximum OD600 and minimum DT). In an equivalent experiment with rEc.β.dC.12′.ΔtY, 1 mMpIF and 0.2% l-arabinose was optimal for fitness (Extended Data Fig. 8). Since growth was not observed in media lacking either l-arabinose or the sAA, these data further support the dependency of synthetic auxotrophs on sAAs.

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