PROJECT SUMMARY/ABSTRACT With few exceptions, all organisms encode encodes 20 amino acid building blocks used in the synthesis of proteins. The overall goal of the PI's research program is to develop interdisciplinary tools to reprogram the genetic code to precisely probe and manipulate biological systems. Our ability to incorporate noncanonical amino acids (ncAAs) with diverse physical, chemical, and biological properties into proteins of interest is central to the reprogramming of genetic codes. The overall goal of this proposal is to develop cells able to biosynthesize and utilize ncAAs and explore the utility of these unnatural organisms in the biosynthesis of bioactive drugs and in vivo sensors for posttranslational modification enzymes. To achieve this goal, the first research goal is to identify more ncAA biosynthetic pathways and use them for the generation of prokaryotic and eukaryotic organisms with additional amino acids. The additional amino acid can be biosynthesized without exogenous addition, followed by the genetic incorporation of it into proteins using a bioorthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair in a site-specific manner. The biosynthesis pathway of ncAAs will be obtained via metabolic engineering or using bioinformatic strategies. The resulting organisms with 21st amino acids will be used to prepare bioactive compounds with novel activities as well as the development of in vivo sensors for posttranslational modification enzymes. To biosynthesize bioactive compounds with ncAAs, we will first introduce the bioorthogonal aaRS for aminoacylation of tRNAs with the ncAA, and then engineer synthetases of natural products towards the tRNA charged with the ncAA. The resulting natural product derivatives will exhibit enhanced biological activities or unprecedented biophysiological properties. Next, we will explore the utility of these unnatural organisms with additional protein building blocks for monitoring the activities of posttranslational modification enzymes in vivo. The eukaryotic cells able to biosynthesize and utilize amino acids with posttranslational modification handles will be used to produce caged luciferase or fluorescent proteins. The resulting engineered cells will allow for the evaluation of posttranslational modification enzymes' activities in real-time, thus providing a strategy to guide the treatment of epigenetic inhibitors in vivo. Our efforts in this project will yield a collection of organisms with a 21st amino acid, and will result in new platforms for the evolution of ncAA-based therapeutic molecules or novel living sensors.