Engineering of enzymes using non-natural amino acids

In enzyme engineering, the main targets for enhancing properties are enzyme activity, stereoselective specificity, stability, substrate range, and the development of unique functions. With the advent of genetic code extension technology, non-natural amino acids (nnAAs) are able to be incorporated into proteins in a site-specific or residue-specific manner, which breaks the limit of 20 natural amino acids for protein engineering. Benefitting from this approach, numerous enzymes have been engineered with nnAAs for improved properties or extended functionality. In the present review, we focus on applications and strategies for using nnAAs in enzyme engineering. Notably, approaches to computational modelling of enzymes with nnAAs are also addressed. Finally, we discuss the bottlenecks that currently need to be addressed in order to realise the broader prospects of this genetic code extension technique.