A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth

Minseob Koh; Anzhi Yao; Patrick R. Gleason; Jeremy H. Mills; Peter G. Schultz

doi:10.1021/jacs.9b08491

A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth

Minseob Koh, Anzhi Yao, Patrick R. Gleason, Jeremy H. Mills, Peter G. Schultz

Molecular Sciences, School of (SMS)

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

Synthetic auxotrophy in which bacterial viability depends on the presence of a synthetic amino acid provides a robust strategy for the containment of genetically modified organisms and the development of safe, live vaccines. However, a simple, general strategy to evolve essential proteins to be dependent on synthetic amino acids is lacking. Using a temperature-sensitive selection system, we evolved an Escherichia coli (E. coli) sliding clamp variant with an orthogonal protein-protein interface, which contains a Leu273 to p-benzoylphenyl alanine (pBzF) mutation. The E. coli strain with this variant DNA clamp has a very low escape frequency (<10^-10), and its growth is strictly dependent on the presence of pBzF. This selection strategy can be generally applied to create ncAA dependence of other organisms with DNA clamp homologues.

Original language	English (US)
Pages (from-to)	16213-16216
Number of pages	4
Journal	Journal of the American Chemical Society
Volume	141
Issue number	41
DOIs	https://doi.org/10.1021/jacs.9b08491
State	Published - Oct 16 2019

ASJC Scopus subject areas

Catalysis
Chemistry(all)
Biochemistry
Colloid and Surface Chemistry

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abstract = "Synthetic auxotrophy in which bacterial viability depends on the presence of a synthetic amino acid provides a robust strategy for the containment of genetically modified organisms and the development of safe, live vaccines. However, a simple, general strategy to evolve essential proteins to be dependent on synthetic amino acids is lacking. Using a temperature-sensitive selection system, we evolved an Escherichia coli (E. coli) sliding clamp variant with an orthogonal protein-protein interface, which contains a Leu273 to p-benzoylphenyl alanine (pBzF) mutation. The E. coli strain with this variant DNA clamp has a very low escape frequency (<10-10), and its growth is strictly dependent on the presence of pBzF. This selection strategy can be generally applied to create ncAA dependence of other organisms with DNA clamp homologues.",

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AU - Schultz, Peter G.

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A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth

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