Ribosomal Incorporation of Thioxanthone as a Noncanonical Amino Acid Facilitates the Engineering of Photoenzymes

Photocatalysis in biocatalytic systems provides a promising approach for achieving selective and efficient chemical transformations under mild conditions. Naturally occurring photoactive cofactors are rare. To overcome this limitation, genetic code engineering can be applied to equip proteins with additional functionalities beyond those known in the 20 canonical amino acids. Here, we report the engineering of an aminoacyl‐tRNA synthetase (thioXRS) that allows the incorporation of a thioxanthone‐bearing noncanonical amino acid (thioX). As proof‐of‐concept, we utilized the versatile biocatalyst LmrR as a protein scaffold. We identified an active variant able to catalyze the E / Z ‐photoisomerization of a cinnamate ester derivative into coumarin. The reaction design allows direct monitoring through fluorescence measurements, as the fluorescent substrate is converted into a non‐fluorescent product. This work demonstrates that thioXRS is a versatile tool for the future development of new‐to‐nature photoenzymes, expanding the synthetic capabilities of biocatalysis towards light‐driven transformations.