Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation

Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods. Incorporation of noncanonical amino acids into proteins holds great promise for altering structure and function of these proteins. Here the authors generate metabolically modified prokaryotic and eukaryotic cells that can biosynthesize sTyr and incorporate it into proteins in a site-specific manner.

Automated summary

This study reports the creation of autonomous cells that can biosynthesize and genetically encode sulfotyrosine (sTyr), which greatly extends the utility of genetic code expansion technology by enhancing the incorporation efficiency of noncanonical amino acids (ncAAs) into proteins.