A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids

The use of noncanonical amino acids (ncAAs) to control the viability of an organism provides a strategy for the development of conditional “kill switches” for live vaccines or engineered human cells. We report an approach inspired by the posttranslational acetylation/deacetylation of lysine residues, in which a protein encoded by a gene with an in‐frame nonsense codon at an essential lysine can be expressed in its native state only upon genetic incorporation of N ‐ ϵ ‐acetyl‐ l ‐Lys (AcK), and subsequent enzymatic deacetylation in the host cell. We applied this strategy to two essential E. coli enzymes: the branched‐chain aminotransferase BCAT and the DNA replication initiator protein DnaA. We also devised a barnase‐based conditional suicide switch to further lower the escape frequency of the host cells. This strategy offers a number of attractive features for controlling host viability, including a single small‐molecule‐based kill switch, low escape frequency, and unaffected protein function.