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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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Comprehensive map of synthetic auxotrophsCircos plot summarizing synthetic auxotrophs generated in this study. Red and green genes reflect knockouts and insertions, respectively. Outermost ticks indicate genomic location, inner blue ticks indicate locations where TAG codons were converted to TAA in the GRO, and green ticks reflect locations of 303 E. coli essential genes. The shaded grey inner circle contains essential TAG loci in synthetic auxotrophs, where yellow ticks represent amino-terminal insertions, blue ticks represent tolerant substitutions, and red ticks represent functional site substitutions. Innermost links represent unique combinations of TAGs in higher-order synthetic auxotrophs. Links of a single color correspond to a single strain.
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Figure 1: Comprehensive map of synthetic auxotrophsCircos plot summarizing synthetic auxotrophs generated in this study. Red and green genes reflect knockouts and insertions, respectively. Outermost ticks indicate genomic location, inner blue ticks indicate locations where TAG codons were converted to TAA in the GRO, and green ticks reflect locations of 303 E. coli essential genes. The shaded grey inner circle contains essential TAG loci in synthetic auxotrophs, where yellow ticks represent amino-terminal insertions, blue ticks represent tolerant substitutions, and red ticks represent functional site substitutions. Innermost links represent unique combinations of TAGs in higher-order synthetic auxotrophs. Links of a single color correspond to a single strain.

Mentions: We pursued three strategies to engineer dependence on nontoxic, membrane permeable, and well-characterized sAAs through the introduction of TAG codons into essential genes: (1) insertion at the amino-terminus, (2) substitution of residues with computationally predicted tolerances19, and (3) substitution of conserved13 residues at functional sites (Fig. 1a). We initially pursued the first two strategies in a GRO containing an OTS optimized for the sAA p-acetyl-l-phenylalanine (pAcF, α). Using MAGE12, we targeted 155 codons for TAG incorporation via four pools of oligonucleotides (Supplementary Table 1, Supplementary Table 2) in permissive media containing pAcF and l-arabinose (aaRS induction) (Fig. 1b). After replica plating on nonpermissive media lacking pAcF and l-arabinose, we isolated eight pAcF auxotrophs with one strain containing two TAGs in essential genes (Fig. 1c and Supplementary Table 3). To determine whether our strategy was capable of creating synthetic auxotrophs dependent on other sAAs, MAGE was used to mutagenize annotated residues in the sAA binding pocket of the pAcF aaRS (Supplementary Table 4) to accommodate p-iodo-l-phenylalanine (pIF, β) or p-azido-l-phenyalanine (pAzF, γ) in two strains. After MAGE-based incorporation of TAGs and selections on permissive and nonpermissive solid media, we obtained eight pIF and 23 pAzF auxotrophs harboring one to four TAGs at 30 distinct loci across 20 essential genes (Supplementary Table 3, Supplementary Table 5). Together, these data demonstrate the modularity of our approach and that synthetic auxotrophs can be engineered across many essential genes using multiple sAAs (Extended Data Fig. 1).


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)

Comprehensive map of synthetic auxotrophsCircos plot summarizing synthetic auxotrophs generated in this study. Red and green genes reflect knockouts and insertions, respectively. Outermost ticks indicate genomic location, inner blue ticks indicate locations where TAG codons were converted to TAA in the GRO, and green ticks reflect locations of 303 E. coli essential genes. The shaded grey inner circle contains essential TAG loci in synthetic auxotrophs, where yellow ticks represent amino-terminal insertions, blue ticks represent tolerant substitutions, and red ticks represent functional site substitutions. Innermost links represent unique combinations of TAGs in higher-order synthetic auxotrophs. Links of a single color correspond to a single strain.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 1: Comprehensive map of synthetic auxotrophsCircos plot summarizing synthetic auxotrophs generated in this study. Red and green genes reflect knockouts and insertions, respectively. Outermost ticks indicate genomic location, inner blue ticks indicate locations where TAG codons were converted to TAA in the GRO, and green ticks reflect locations of 303 E. coli essential genes. The shaded grey inner circle contains essential TAG loci in synthetic auxotrophs, where yellow ticks represent amino-terminal insertions, blue ticks represent tolerant substitutions, and red ticks represent functional site substitutions. Innermost links represent unique combinations of TAGs in higher-order synthetic auxotrophs. Links of a single color correspond to a single strain.
Mentions: We pursued three strategies to engineer dependence on nontoxic, membrane permeable, and well-characterized sAAs through the introduction of TAG codons into essential genes: (1) insertion at the amino-terminus, (2) substitution of residues with computationally predicted tolerances19, and (3) substitution of conserved13 residues at functional sites (Fig. 1a). We initially pursued the first two strategies in a GRO containing an OTS optimized for the sAA p-acetyl-l-phenylalanine (pAcF, α). Using MAGE12, we targeted 155 codons for TAG incorporation via four pools of oligonucleotides (Supplementary Table 1, Supplementary Table 2) in permissive media containing pAcF and l-arabinose (aaRS induction) (Fig. 1b). After replica plating on nonpermissive media lacking pAcF and l-arabinose, we isolated eight pAcF auxotrophs with one strain containing two TAGs in essential genes (Fig. 1c and Supplementary Table 3). To determine whether our strategy was capable of creating synthetic auxotrophs dependent on other sAAs, MAGE was used to mutagenize annotated residues in the sAA binding pocket of the pAcF aaRS (Supplementary Table 4) to accommodate p-iodo-l-phenylalanine (pIF, β) or p-azido-l-phenyalanine (pAzF, γ) in two strains. After MAGE-based incorporation of TAGs and selections on permissive and nonpermissive solid media, we obtained eight pIF and 23 pAzF auxotrophs harboring one to four TAGs at 30 distinct loci across 20 essential genes (Supplementary Table 3, Supplementary Table 5). Together, these data demonstrate the modularity of our approach and that synthetic auxotrophs can be engineered across many essential genes using multiple sAAs (Extended Data Fig. 1).

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