In the first 2.5 years of this project we have taken major strides toward achieving our ambitious research vision. We have established engineered cellular translation components to selectively install non-canonical amino acids containing new functional side chains, and are making progress in developing mutually orthogonal translation systems to allow selective introduction of two distinct non-standard amino acids into the same active site (unpublished data). Genetically encoding the non-canonical functionality greatly facilitates the production of well-defined, homogeneous proteins; it allows the non-canonical amino acid to be introduced at any site, in any protein scaffold; and, perhaps most significantly, we have shown that this approach allows rapid optimization of enzyme properties using directed evolution. We have exploited these advanced protein engineering methods to create several metalloenzymes with non-canonical coordination environments (e.g. J. Am. Chem. Soc. 2018, 140, 1535. Chem. Eur. J. 2018, 24, 11821.). A non-canonical ligand has also allowed us to unravel the active site features that control the reactivity of iconic ferryl (metal-oxo) intermediates in haem enzymes (ACS Catalysis, 2020, 10, 2735). In parallel, we have been able to capture elusive reactive intermediates in copper dependent lytic polysaccharide monooxygenases, a recently discovered family of enzymes that show great potential as auxiliary biocatalysts for commercially viable cellulose deconstruction (manuscript under review). We have also recently demonstrated that our protein engineering strategies can be extended beyond metalloenzyme design. Taking inspiration from the field of small molecule organocatalysis, our lab have used a combination of genetic code expansion, computational enzyme design and laboratory evolution to create enzymes that exploit non-canonical amino acids as catalytic nucleophiles (Nature, 2019, 570, 219. Curr. Opin. Chem. Biol. 2020, 55, 136).