Noncanonical Amino Acid Addictions in Biological Systems

Engineered orthogonal translation systems (OTSs) for the incorporation of non-canonical amino acids (NCAAs) allow for the addition of new chemistries to genetic code.  OTSs are generally detrimental to cell fitness, and are quickly lost in natural bacterial systems.  We have demonstrated that a single, essential enzyme can be engineered to require a NCAA, 3-nitrotyrosine, for activity. Furthermore, that enzyme can be used to leverage a cellular addiction to a NCAA for hundreds of generations without escape from NCAA dependence.  A β-lactamase engineered for NCAA addiction is sufficient for organismal addiction to the NCAA, and this addiction operon, consisting of the engineered β-lactamase and an engineered OTS, provided addiction in E. coli for over 500 generations without detectable escape (<10^-9 escape frequency).  At the conclusion of 500 generations, not only do the E. coli become more tolerant to the NCAA, but a portion of the population appears to be significantly hindered in growth in the absence of the NCAA, suggesting that the NCAA is not only required for lactam resistance, but incorporated as a part of the bacterial genome. Additionally, the addiction operon can be quickly transferred to a wide variety of bacterial orders, including Pseudomonadales, Vibrionales, Pasteurellales, and Enterbacteriales, by changing lactamase specificity from penicillins to cephalosporins, demonstrating for the first time that the Methanocaldococcus jannaschii tyrosine OTS functions in a diverse array of bacteria.  Our work paves the way for the use of OTSs, and their many chemistries, in a variety of bacteria, and may lay a foundation for the use of NCAAs as a facile means for biocontainment using single engineered essential enzymes.