Engineering of the genetic code.

The genetic code is a universally conserved mechanism that translates genetic information into proteins, consisting of 64 codons formed by four nucleotide bases. With a few exceptions, the genetic code universally encodes 20 canonical amino acids (AAs) and three stop signals, with many AAs represented by multiple codons. Genetic engineering has expanded this system through approaches like codon reassignment and synthetic base pair introduction, allowing for the incorporation of noncanonical AAs (ncAAs) into proteins, known as genetic code expansion (GCE). These ncAAs add novel functionalities, such as bio-orthogonal handles, fluorophores, and redox-active ncAAs, enhancing the diversity of proteins. Recent advancements include genome-wide recoding, evolution of orthogonal translation components, and synthetic genetic alphabet, with a focus on improving translational efficiency and reducing off-target effects. This review emphasizes strategies for modifying nucleotides, reassessing codons, and engineering translational enzymes, highlighting innovations that tackle challenges in GCE and promote new protein chemistries and biotechnological applications.