Integrating the Biosynthesis and Genetic Encoding of Noncanonical Amino Acids for Enzyme Design and Catalysis

Artificial enzymes engineered by site-specific incorporation of catalytically active noncanonical amino acids (ncAAs) into protein scaffolds represent a rapidly advancing class of biocatalysts, particularly for chemical transformations lacking natural enzymatic counterparts. Integrating biosynthesis and genetic incorporation of ncAAs has rapidly expanded the toolkit for enzyme design and catalysis. This review surveys engineered metabolic routes and precursor feeding strategies that supply diverse ncAAs in vivo, and contrasts cell-free and cellular approaches for their production. We examine advances in orthogonal translation systems and site-specific incorporation that enable installation of catalytic, redox, and spectroscopic functionalities, and highlight applications where ncAA-bearing proteins catalyze new-to-nature reactions, and improve selectivity for directed evolution. Recent work on in-cell biosynthesis of ncAAs coupled to on-demand incorporation is discussed for its potential to streamline workflows and enable enzyme design and catalysis in one-pot. Finally, we identify remaining challenges and outline opportunities for coupling metabolic engineering and protein engineering to create next-generation artificial enzymes.