Letter | Published:

Recoded organisms engineered to depend on synthetic amino acids

Nature volume 518, pages 8993 (05 February 2015) | Download Citation

  • A Corrigendum to this article was published on 23 September 2015

Abstract

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 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 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 1011 cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches2,3,6,7,8,9,10. 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.

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Accessions

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

Genome sequences have been deposited in GenBank under the accession numbers CP010455 (rEc.γ.dC.46) and CP010456 (rEc.β.dC.12).

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Acknowledgements

We are grateful to D. Söll and Y. S. Wang for discussion and for providing pTech-supU. We thank N. Carriero and R. Bjornson at the Yale Biomedical High Performance Computing Cluster for assistance with SIFT. Funding received was from the Defense Advanced Research Projects Agency (N66001-12-C-4020, N66001-12-C-4211); NIH-MSTP-TG-T32GM07205 (A.D.H.); DuPont Inc. and the Arnold and Mabel Beckman Foundation (F.J.I.).

Author information

Author notes

    • Adrian D. Haimovich
    •  & Spencer R. Katz

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA

    • Alexis J. Rovner
    • , Adrian D. Haimovich
    • , Spencer R. Katz
    • , Zhe Li
    • , Michael W. Grome
    • , Miriam Amiram
    • , Jaymin R. Patel
    • , Ryan R. Gallagher
    •  & Farren J. Isaacs
  2. Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA

    • Alexis J. Rovner
    • , Adrian D. Haimovich
    • , Spencer R. Katz
    • , Zhe Li
    • , Michael W. Grome
    • , Brandon M. Gassaway
    • , Miriam Amiram
    • , Jaymin R. Patel
    • , Ryan R. Gallagher
    • , Jesse Rinehart
    •  & Farren J. Isaacs
  3. Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA

    • Brandon M. Gassaway
    •  & Jesse Rinehart

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Contributions

A.J.R. and F.J.I. conceived the study, designed experiments and wrote the paper with assistance from A.D.H. and S.R.K.; A.J.R. conducted experiments with assistance from S.R.K., A.D.H., Z.L., M.W.G., M.A., J.R.P. and R.R.G.; B.M.G. and J.R. conducted mass spectrometry. All authors commented on the paper and F.J.I. supervised all aspects of the study.

Competing interests

A.J.R. and F.J.I. have filed a provisional application with the US Patent and Trademark Office on this work. F.J.I. is a founder of enEvolv, Inc.

Corresponding author

Correspondence to Farren J. Isaacs.

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DOI

https://doi.org/10.1038/nature14095

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