An engineered bacterium auxotrophic for an unnatural amino acid: a novel biological containment system

Abstract

Biological containment is a genetic technique that programs dangerous organisms to grow only in the laboratory and to die in the natural environment. Auxotrophy for a substance not found in the natural environment is an ideal biological containment. Here, we constructed an Escherichia coli strain that cannot survive in the absence of the unnatural amino acid 3-iodo-L-tyrosine. This synthetic auxotrophy was achieved by conditional production of the antidote protein against the highly toxic enzyme colicin E3. An amber stop codon was inserted in the antidote gene. The translation of the antidote mRNA was controlled by a translational switch using amber-specific 3-iodo-L-tyrosine incorporation. The antidote is synthesized only when 3-iodo-L-tyrosine is present in the culture medium. The viability of this strain rapidly decreased with less than a 1 h half-life after removal of 3-iodo-L-tyrosine, suggesting that the decay of the antidote causes the host killing by activated colicin E3 in the absence of this unnatural amino acid. The contained strain grew 1.5 times more slowly than the parent strains. The escaper frequency was estimated to be 1.4 mutations (95% highest posterior density 1.1–1.8) per 105 cell divisions. This containment system can be constructed by only plasmid introduction without genome editing, suggesting that this system may be applicable to other microbes carrying toxin-antidote systems similar to that of colicin E3.

Long-Term Stable and Tightly Controlled Expression of Recombinant Proteins in Antibiotics-Free Conditions

“…Moreover, the F factor-based system has been used for the stable inheritance of genetic information, stable expression, and reduced metabolic burden on the host cell [25,30]. To overcome the metabolic burden, antibiotics-free systems based on auxotrophy [31,32] and post-segregational killing systems [33,34] have been evaluated.…”

The Use of Non-Canonical Amino Acids as a Novel Biocontainment Strategy

“…In negative selection, the plasmid is transferred to cells that contain an essential gene that contains a premature amber stop codon. In the presence of the ncAA, the novel tRNA/synthetase pair successfully incorporates the ncAA, resulting in an essential, functional gene and the survival of the bacteria (Kato et al 2015). Wang et al (2001) aimed to expand the genetic code of E.coli by incorporating tRNA tyr and tyrosyl-tRNA synthetase (TyrRS) from another organism in an attempt to avoid crossaminoacylation while maintaining and enhancing orthogonality.…”

“…Surviving cells were resistant to ampicillin and contained tRNAs that were efficiently amino-acylated by the M. jannaschii TyrRS (Wang et al 2001). More recently, Kato et al (2015) constructed an E. coli strain that was auxotrophic for the ncAA 3-iodo-L tyrosine (IY). The strain contained two plasmids encoding for a toxinantidote system: one with an amber codon inserted next to a synthetic antidote gene, and the other expressing a novel tRNA-synthetase pair specific for the amber codon.…”

“…3 colony forming units were counted, indicating that IY is required for the survival of the bacteria (Kato et al 2015). If this system is to be used for biocontainment, it is important that the survival of the bacteria can be controlled after it has served its purpose.…”

“…To determine if the bacteria can survive for long periods of time in the absence of the auxotrophic substance, the researchers examined the rate of killing after IY is removed from the media. It was found that the survival rate of the bacteria decreased significantly after two hours, and the half-life was determined to be approximately 50 minutes (Kato et al 2015). In addition to these factors, it is important that the rate of mutation in response to IY is low to prevent the escape of bacteria and unwanted proliferation.…”