Expanding the Structural Diversity of Protein Building Blocks with Noncanonical Amino Acids Biosynthesized from Aromatic Thiols

Incorporation of structurally novel noncanonical amino acids (ncAAs) into proteins is valuable for both scientific and biomedical applications. To expand the structural diversity of available ncAAs and to reduce the burden of chemically synthesizing them, we have developed a general and simple biosynthetic method for genetically encoding novel ncAAs into recombinant proteins by feeding cells with economical commercially available or synthetically accessible aromatic thiols. We demonstrate that nearly 50 ncAAs with a diverse array of structures can be biosynthesized from these simple small-molecule precursors by hijacking the cysteine biosynthetic enzymes, and the resulting ncAAs can subsequently be incorporated into proteins via an expanded genetic code. Moreover, we demonstrate that bioorthogonal reactive groups such as aromatic azides and aromatic ketones can be incorporated into green fluorescent protein or a therapeutic antibody with high yields, allowing for subsequent chemical conjugation.

Automated summary

Incorporation of structurally novel noncanonical amino acids (ncAAs) into proteins was achieved through biosynthesis, with nearly 50 ncAAs synthesized from simple small-molecule precursors. Furthermore, bioorthogonal reactive groups could be incorporated into proteins with high yields for subsequent chemical conjugation.