Unnatural amino acid mutagenesis-based enzyme engineering

•Enzyme engineering using in vivo incorporation of unnatural amino acids (UAA).•Enhancing and altering enzyme activity using residue- and site-specific incorporation of UAA.•Enzyme evolution with UAA by integrating directed evolution and UAA incorporation.

Traditional enzyme engineering relies on substituting one amino acid by one of the other 19 natural amino acids to change the functional properties of an enzyme. However, incorporation of unnatural amino acids (UAAs) has been harnessed to engineer efficient enzymes for biocatalysis. Residue-specific and site-specific in vivo incorporation methods are becoming the preferred approach for producing enzymes with altered or improved functions. We describe the contribution of in vivo UAA incorporation methodologies to enzyme engineering as well as the future prospects for the field, including the integration of UAAs with other new advances in enzyme engineering.