Creation of Bacterial Cells with 5-Hydroxytryptophan as a 21st Amino Acid Building Block

While most organisms utilize 20 canonical amino acid building blocks for protein synthesis, adding additional candidates to the amino acid repertoire can greatly facilitate the investigation and manipulation of protein structures and functions. In this study, we report the generation of completely autonomous organisms with a 21st noncanonical amino acid, 5-hydroxytryptophan (5HTP). Like 20 canonical amino acids, 5-hydroxytryptophan can be biosynthesized in vivo from simple carbon sources and is subsequently incorporated into proteins in response to the amber stop codon. Using this unnatural organism, we have prepared a single-chain immunoglobulin variable fragment conjugated with a fluorophore and demonstrated the utility of these autonomous cells to monitor oxidative stress. The creation of this and other cells containing the 21st amino acid will provide an opportunity to generate proteins and organisms with novel activities, as well as to determine the evolutionary consequences of using additional amino acid buildings. [Display omitted] •Construction of autonomous organism with 5-hydroxytryptophan as its 21st amino acid•Use of autonomous organism for preparing proteins with site-specific modifications•Creation of a living oxidative stress indicator Most living organisms use 20 canonical amino acids to encode proteins. Despite this universality, nature appears to require an additional level of complexity in protein structure and function. Thus, there is tremendous excitement surrounding the addition of noncanonical amino acids to the genetic code of organisms. In this study, we create the first autonomous organism with 5-hydroxytryptophan as its 21st amino acid. The resulting cells harbor the endogenous ability to biosynthesize 5-hydroxytryptophan and site-specifically incorporate it into proteins. The utility of this unnatural organism is further demonstrated by our preparation of site-specific protein conjugates and our creation of a living indicator for oxidative stress. The creation of unnatural organisms with expanded genetic codes will allow us to examine the evolutionary consequences of adding new building blocks and to generate proteins and organisms with novel and useful activities. The ability to introduce noncanonical amino acid building blocks into proteins in living cells provides a powerful tool for investigating and manipulating the structure and function of proteins and of the organisms themselves. In this study, we have created a complete