A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth

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.

Directed-evolution of translation system for efficient unnatural amino acids incorporation and generalizable synthetic auxotroph construction

“…In this context, several strategies have been reported to construct UAA-dependent synthetic auxotrophs with low escape frequencies 19 , 20 , 39 – 43 . Despite exciting progress, simple and general strategies to efficiently construct UAA-dependent common bacterial strains with very low escape frequency remain to be an outstanding challenge 42 , 44 , 45 . And the utility of UAA-dependent synthetic auxotrophs in living animals is almost unknown.…”

“…To construct an AzF-dependent synthetic auxotroph, we planned to insert an in-frame amber codon (from two to four amber codons) into the essential gene, dnaN , encoding a subunit of the DNA polymerase III holoenzyme 42 , 47 . The chromosomal dnaN gene was knocked out using the lambda Red recombinase system encoded in the pTKRed vector (Fig.…”

Noncanonical Amino Acids in Synthetic Biosafety and Post‐translational Modification Studies

“…When a biphenylalanine‐specific orthogonal AARS‐tRNA pair and the corresponding ncAA were used, the introduction of a TAG stop codon into multiple proteins could dramatically reduce the escape frequency to 2.2×10 −12 [53] . Recently, researchers established an analogous system using benzoylphenylalanine incorporation [54] . As a reporter, they used a sliding clamp protein that is essential for DNA replication.…”

Addicting Escherichia coli to New-to-Nature Reactions

“…10,15 We anticipate this gap to widen once other catalyst/transformation combinations are evaluated for their ability to rescue synthetic auxotrophs dependent on ncAAs and unnatural nucleotides as well as designer organisms that have undergone full-genome recoding. [16][17][18][19][20]23 The resulting modularity should allow for flexibility and customization, which are attractive features when considering the variety of environments to which GMOs could potentially be applied.…”

“…Both noncanonical amino acids (ncAAs) and unnatural nucleobases have been successfully employed for restricting bacterial growth to artificial environments in which these xenobiotics are supplied (= synthetic auxotrophy, Figure B). − For example, the suppression of in-frame stop codons by orthogonal translation systems (OTS, Figure C) enables the site-specific incorporation of ncAAs into essential proteins. , In the absence of the non-natural building block, premature termination of translation is triggered, resulting in a truncated and inactive protein fragment. As a result, this functional connection renders ncAAs essential metabolites and also can prevent metabolic cross-feeding, if the ncAA is not readily available in natural habitats.…”