Enzyme engineering through genetic code expansion

While the scope of enzyme engineering with the 20 canonical amino acids is powerful, it is limited in terms of the available functional groups and the ability to fine-tune the chemical properties at enzyme active sites. The incorporation of non-canonical amino acids into enzymes can help to overcome this limitation. In this work, we extend the scope and efficiency of non-canonical amino acid incorporation to enable exotic tuning of protein hydrophobicity, acid-base chemistry, and metal coordination. Furthermore, we demonstrate that enzymatic activity can be both studied and improved by applying fundamental chemical principles to guide non-canonical amino acid mutagenesis.