Recoded organisms engineered to depend on synthetic amino acids
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 aminoacids (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 ~10^sup 11^ cells on solid media for 7 days or in liquidmedia for 20 days.This is a significant improvement over existing biocontainment approaches2,3,6-10. 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..
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2015 |
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| Erschienen: |
2015 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:518 |
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| Enthalten in: |
Nature |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Alexis J Rovner [VerfasserIn] |
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| Links: |
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| doi: |
10.1038/nature14095 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 245 | 1 | 0 | |a Recoded organisms engineered to depend on synthetic amino acids |
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| 520 | |a 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 aminoacids (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 ~10^sup 11^ cells on solid media for 7 days or in liquidmedia for 20 days.This is a significant improvement over existing biocontainment approaches2,3,6-10. 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. | ||
| 650 | 4 | |a Genomes | |
| 650 | 4 | |a Organisms | |
| 650 | 4 | |a Amino acids | |
| 650 | 4 | |a Genetic engineering | |
| 650 | 4 | |a Proteins | |
| 650 | 4 | |a Metabolites | |
| 650 | 4 | |a Mutation | |
| 650 | 4 | |a Metabolism | |
| 650 | 4 | |a Escherichia coli Proteins - metabolism | |
| 650 | 4 | |a Synthetic Biology - methods | |
| 650 | 4 | |a Amino Acids - pharmacology | |
| 650 | 4 | |a Microbial Viability - drug effects | |
| 650 | 4 | |a RNA, Transfer - genetics | |
| 650 | 4 | |a Escherichia coli - genetics | |
| 650 | 4 | |a Genetic Engineering - methods | |
| 650 | 4 | |a Escherichia coli - metabolism | |
| 650 | 4 | |a Codon - genetics | |
| 650 | 4 | |a Escherichia coli - cytology | |
| 650 | 4 | |a Amino Acids - chemical synthesis | |
| 650 | 4 | |a Phenylalanine - metabolism | |
| 650 | 4 | |a Escherichia coli - drug effects | |
| 650 | 4 | |a Culture Media - chemistry | |
| 650 | 4 | |a Amino Acids - metabolism | |
| 650 | 4 | |a Gene Transfer, Horizontal - genetics | |
| 650 | 4 | |a Escherichia coli Proteins - biosynthesis | |
| 650 | 4 | |a Catalytic Domain - genetics | |
| 650 | 4 | |a Organisms, Genetically Modified - growth & development | |
| 650 | 4 | |a Culture Media - pharmacology | |
| 650 | 4 | |a Phenylalanine - chemistry | |
| 650 | 4 | |a Escherichia coli Proteins - chemistry | |
| 650 | 4 | |a Amino Acids - chemistry | |
| 650 | 4 | |a Peptide Termination Factors - genetics | |
| 650 | 4 | |a Organisms, Genetically Modified - metabolism | |
| 650 | 4 | |a Amino Acyl-tRNA Synthetases - metabolism | |
| 650 | 4 | |a Containment of Biohazards - methods | |
| 650 | 4 | |a Escherichia coli Proteins - genetics | |
| 650 | 4 | |a Protein Multimerization - genetics | |
| 650 | 4 | |a Organisms, Genetically Modified - genetics | |
| 650 | 4 | |a Genome, Bacterial - genetics | |
| 650 | 4 | |a Microbial Viability - genetics | |
| 650 | 4 | |a Genetic Code - genetics | |
| 650 | 4 | |a Amino Acyl-tRNA Synthetases - genetics | |
| 650 | 4 | |a Genes, Essential - genetics | |
| 700 | 0 | |a Adrian D Haimovich |4 oth | |
| 700 | 0 | |a Spencer R Katz |4 oth | |
| 700 | 0 | |a Zhe Li |4 oth | |
| 700 | 0 | |a Michael W Grome |4 oth | |
| 700 | 0 | |a Brandon M Gassaway |4 oth | |
| 700 | 0 | |a Miriam Amiram |4 oth | |
| 700 | 0 | |a Jaymin R Patel |4 oth | |
| 700 | 0 | |a Ryan R Gallagher |4 oth | |
| 700 | 0 | |a Jesse Rinehart |4 oth | |
| 700 | 0 | |a Farren J Isaacs |4 oth | |
| 773 | 0 | 8 | |i Enthalten in |t Nature <London> |d London : Macmillan, 1869 |g 518(2015), 7537, Seite 89-93 |w (DE-627)129292834 |w (DE-600)120714-3 |w (DE-576)014473941 |x 0028-0836 |7 nnns |
| 773 | 1 | 8 | |g volume:518 |g year:2015 |g number:7537 |g pages:89-93 |
| 856 | 4 | 1 | |u http://dx.doi.org/10.1038/nature14095 |3 Volltext |
| 856 | 4 | 2 | |u http://www.ncbi.nlm.nih.gov/pubmed/25607356 |
| 856 | 4 | 2 | |u http://search.proquest.com/docview/1654998807 |
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| 952 | |d 518 |j 2015 |e 7537 |h 89-93 | ||