Update of the Pyrrolysyl-tRNA Synthetase/tRNA Pyl Pair and Derivatives for Genetic Code Expansion.

The cotranslational incorporation of pyrrolysine (Pyl), the 22nd proteinogenic amino acid, into proteins in response to the UAG stop codon represents an outstanding example of natural genetic code expansion. Genetic encoding of Pyl is conducted by the pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA, tRNA Pyl . Owing to the high tolerance of PylRS toward diverse amino acid substrates and great orthogonality in various model organisms, the PylRS/tRNA Pyl -derived pairs are ideal for genetic code expansion to insert noncanonical amino acids (ncAAs) into proteins of interest. Since the discovery of cellular components involved in the biosynthesis and genetic encoding of Pyl, synthetic biologists have been enthusiastic about engineering PylRS/tRNA Pyl -derived pairs to rewrite the genetic code of living cells. Recently, considerable progress has been made in understanding the molecular phylogeny, biochemical properties, and structural features of the PylRS/tRNA Pyl pair, guiding its further engineering and optimization. In this review, we cover the basic and updated knowledge of the PylRS/tRNA Pyl pair's unique characteristics that make it an outstanding tool for reprogramming the genetic code. In addition, we summarize the recent efforts to create efficient and (mutually) orthogonal PylRS/tRNA Pyl -derived pairs for incorporation of diverse ncAAs by genome mining, rational design, and advanced directed evolution methods.