The review "Evolution of Pyrrolysyl-tRNA Synthetase: From Methanogenesis to Genetic Code Expansion" by Nikolaj G. Koch and Nediljko Budisa provides an overview of the discovery and evolution of the pyrrolysine (Pyl) encoding translation system in specific archaea. The focus is on the pyrrolysyl-tRNA synthetase (PyIRS) tRNA pair, which was initially responsible for translating enzymes crucial in methanogenic metabolic pathways. The review discusses the engineering of PyIRS to recognize new substrates and enhance in vivo activity, highlighting the successful incorporation of over 340 noncanonical amino acids (ncAAs) using the PyIRS system. It also summarizes recent applications of the PyIRS system in creating innovative therapeutic solutions, such as new antibody-drug conjugates, advancements in vaccine modalities, and potential production of new antimicrobials. The review covers the history, function, and different classes of PyIRS, strategies for engineering new ncAA recognition, and methods to enhance in vivo efficiency. It also explores the coupling of metabolic engineering with PyIRS-based orthogonal translation systems (OTS) to create synthetic cells and discusses various applications of ncAAs in protein engineering, including probing and engineering protein structure and function, and therapeutic applications. The review concludes with insights and suggestions for advancing the field of genetic code expansion.The review "Evolution of Pyrrolysyl-tRNA Synthetase: From Methanogenesis to Genetic Code Expansion" by Nikolaj G. Koch and Nediljko Budisa provides an overview of the discovery and evolution of the pyrrolysine (Pyl) encoding translation system in specific archaea. The focus is on the pyrrolysyl-tRNA synthetase (PyIRS) tRNA pair, which was initially responsible for translating enzymes crucial in methanogenic metabolic pathways. The review discusses the engineering of PyIRS to recognize new substrates and enhance in vivo activity, highlighting the successful incorporation of over 340 noncanonical amino acids (ncAAs) using the PyIRS system. It also summarizes recent applications of the PyIRS system in creating innovative therapeutic solutions, such as new antibody-drug conjugates, advancements in vaccine modalities, and potential production of new antimicrobials. The review covers the history, function, and different classes of PyIRS, strategies for engineering new ncAA recognition, and methods to enhance in vivo efficiency. It also explores the coupling of metabolic engineering with PyIRS-based orthogonal translation systems (OTS) to create synthetic cells and discusses various applications of ncAAs in protein engineering, including probing and engineering protein structure and function, and therapeutic applications. The review concludes with insights and suggestions for advancing the field of genetic code expansion.