A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth

摘要

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.