The pyrrolysine (Pyl) coding system in protein translation was first discovered in specific archaea over 20 years ago. This system, involving the pyrrolysyl-tRNA synthetase (PylRS), originally played a role in methanogenic metabolic pathways. It has since become a key tool for genetic code expansion (GCE), enabling the incorporation of noncanonical amino acids (ncAAs) into proteins. This review discusses the engineering of PylRS to recognize new substrates and enhance in vivo activity, as well as the applications of the PylRS system in creating innovative therapeutic solutions, such as antibody-drug conjugates, vaccine modalities, and antimicrobials. The review also covers the expansion of the genetic code by reassigning sense codons and designing codon sizes, and highlights the importance of metabolic engineering in conjunction with PylRS-based orthogonal translation systems (OTSs). The PylRS system has been shown to incorporate over 340 substrates, including various α-hydroxy acids and non-alpha amino acids. The review emphasizes the potential of the PylRS system for future applications in biotechnology and medicine, as well as the challenges and opportunities in expanding the scope of GCE. The PylRS system is particularly valuable due to its natural orthogonality across all three domains of life and its ease of engineering for different substrate recognitions while maintaining its original orthogonality. The review also discusses the importance of in vivo efficiency and the challenges in engineering PylRS for new substrates, as well as the potential of combining metabolic engineering with OTSs to create synthetic cells as robust and programmable production units. The review concludes with a discussion of the future directions for GCE and the potential of the PylRS system in advancing the field.The pyrrolysine (Pyl) coding system in protein translation was first discovered in specific archaea over 20 years ago. This system, involving the pyrrolysyl-tRNA synthetase (PylRS), originally played a role in methanogenic metabolic pathways. It has since become a key tool for genetic code expansion (GCE), enabling the incorporation of noncanonical amino acids (ncAAs) into proteins. This review discusses the engineering of PylRS to recognize new substrates and enhance in vivo activity, as well as the applications of the PylRS system in creating innovative therapeutic solutions, such as antibody-drug conjugates, vaccine modalities, and antimicrobials. The review also covers the expansion of the genetic code by reassigning sense codons and designing codon sizes, and highlights the importance of metabolic engineering in conjunction with PylRS-based orthogonal translation systems (OTSs). The PylRS system has been shown to incorporate over 340 substrates, including various α-hydroxy acids and non-alpha amino acids. The review emphasizes the potential of the PylRS system for future applications in biotechnology and medicine, as well as the challenges and opportunities in expanding the scope of GCE. The PylRS system is particularly valuable due to its natural orthogonality across all three domains of life and its ease of engineering for different substrate recognitions while maintaining its original orthogonality. The review also discusses the importance of in vivo efficiency and the challenges in engineering PylRS for new substrates, as well as the potential of combining metabolic engineering with OTSs to create synthetic cells as robust and programmable production units. The review concludes with a discussion of the future directions for GCE and the potential of the PylRS system in advancing the field.