To improve EnT1.0 activity, we initially mutated eight residues that lie in proximity to BpA173 to alanine (Extended Data Figure ). An M90A mutation gave rise to a substantial 3-fold increase in conversion to 1a along with a modest improvement in enantioselectivity to 60% e.e. (EnT1.1, Figure , Extended Data Figure ). This improved variant facilitated detection of enzyme activity in clarified cell lysate, and enabled the development of a directed evolution workflow suitable for high-throughput engineering of triplet energy transfer photoenzymes. This evolutionary workflow relies on uniform irradiation of enzyme variants arrayed in microtiter plates, which was achieved using a commercial LED array (see Experimental Procedures). The observed coefficient of variance across an assay plate using purified EnT1.0 was shown to be less than 5% (Supplementary Figure ), a value that is in line with established high-throughput screening methods.