A Single Plasmid System to Addict Bacteria to Non-Canonical Amino Acids

A single plasmid system to addict bacteria to non-canonical amino acids.
Genetic incorporation of non-canonical amino acids (NCAAs) allows the addition of new and novel chemistries to the genetic code. This can be accomplished by using an orthogonal aminoacyl tRNA synthetase:tRNA pair derived from an evolutionarily distant organism to reassign the amber (UAG) stop codon. Unfortunately, global UAG suppression imposes a significant fitness cost on the host cell, leading to deletion or inactivation of the orthogonal translation machinery during serial culture experiments. To solve this problem, and ensure maintenance of functional orthogonal translation machinery, we engineered an essential protein, β-lactamase, to require incorporation of NCAAs. Following library selections with various 3-substituted tyrosine analogues, clones conferring β-lactam resistance were counter-screened to eliminate those active in the presence of common E. coli suppressor tRNAs. We show that by iteratively decreasing the number of canonical amino acid solutions, enzymes can be evolved with a strong dependence on NCAA incorporation. An evolved β-lactamase has been shown to maintain orthogonal translation machinery in E. coli for more than 250 generations, without detectable escape from NCAA dependence. Unlike other approaches that rely on extensive manipulation of the host genome, this system is compact and broadly compatible. To demonstrate the utility of this approach the sub-circuit containing the addicted lactamase and the orthogonal synthetase and tRNA pair were transferred on a broad host range vector to different bacterial species, including Shigella and Salmonella, which immediately resulted in the non-revertible addiction of these strains to the NCAA without the alteration of their underlying codes via other synthetic biology approaches. The use of our or similar addiction modules should quickly allow the introduction of NCAAs into a much wider variety of bacterial species, including industrially-relevant fermentation and production strains.