Abstract
Over the past 16 years, genetic code expansion and reprogramming in living organisms has been transformed by advances that leverage the unique properties of pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl) pairs. Here we summarize the discovery of the pyrrolysine system and describe the unique properties of PylRS/tRNA(Pyl) pairs that provide a foundation for their transformational role in genetic code expansion and reprogramming. We describe the development of genetic code expansion, from E. coli to all domains of life, using PylRS/tRNA(Pyl) pairs, and the development of systems that biosynthesize and incorporate ncAAs using pyl systems. We review applications that have been uniquely enabled by the development of PylRS/tRNA(Pyl) pairs for incorporating new noncanonical amino acids (ncAAs), and strategies for engineering PylRS/tRNA(Pyl) pairs to add noncanonical monomers, beyond α-L-amino acids, to the genetic code of living organisms. We review rapid progress in the discovery and scalable generation of mutually orthogonal PylRS/tRNA(Pyl) pairs that can be directed to incorporate diverse ncAAs in response to diverse codons, and we review strategies for incorporating multiple distinct ncAAs into proteins using mutually orthogonal PylRS/tRNA(Pyl) pairs. Finally, we review recent advances in the encoded cellular synthesis of noncanonical polymers and macrocycles and discuss future developments for PylRS/tRNA(Pyl) pairs.