The evolutionary strategy consisted of two rounds of saturation mutagenesis targeting residues in the active site and second coordination sphere, which were individually randomised using NNK degenerate codons. Individual variants arrayed in 96-well microtiter plates were irradiated for 30 mins at 365nm using an LED array in the presence of substrate 1 and reactions analysed by high-throughput ultraperformance liquid chromatography (UPLC, SI Figure ). The most active (~1%) clones, identified based on conversion to 1a, were then evaluated as purified proteins for improved activity and enantioselectivity (SI Figure ). Beneficial mutations identified during each round were subsequently combined by DNA shuffling. Following evaluation of ~3500 library members, an EnT1.3 variant emerged with improved activity, stability, and selectivity. EnT1.3 contains five mutations (EnT1.0 M90A Q149D P196R K225E A229S, Extended Data Figure ) and achieves a substantial 10-fold improvement in reaction conversion of 1 to 1a compared with EnT1.0 following 30 minutes of irradiation (Figure &). The enhanced performance of EnT1.3 arises from a combination of a 4fold increase in initial rate (Extended Data Figure ), improved regioselectivity (1a:1b 2:1 for EnT1.0 vs 9:1 for EnT1.3, Figure ), and reduced susceptibility to photo-deactivation, that likely arises in EnT1.0 from an intramolecular photo-crosslinking process involving the benzophenone side chain (Extended Data Figure ). EnT1.3 also offers high levels of enantio-control, generating (-)-1a with >99% e.e.. Interestingly, EnT1.3 is more active at 4 °C than at room temperature (Extended Data Figure ), which may reflect an increased lifetime of the photosensitiser triplet state at lower temperatures. In contrast to small-molecule photocatalysts, which are highly sensitive to oxygen due to triplet quenching, EnT1.3 is tolerant of aerobic buffers (Figure ) and can achieve >300 turnovers under these conditions (Extended Data Figure ). With only 1.5 mol% EnT1.3, near complete conversion of 1 to optically pure (-)-1a can be achieved within 2 hr (SI Table ), underscoring the efficiency of our engineered photoenzyme. We next explored the photocatalytic activity of EnT1.3 towards [2+2]-cycloadditions of allyloxy-, alken-1-yl-, and allyloxy(methyl)-quinolones to generate optically enriched products (2a-12a, Figure ). High conversions and selectivities were achieved in the majority of cases. With substrates 3, 7 and 11, introduction of a gem-dimethyl moiety led to a reduction in enantioselectivity and in these cases EnT1.2 proved to be a more selective photocatalyst. The ability of EnT1.3 to generate 2a and 6a with high levels of stereocontrol is particularly noteworthy. Analogous reactions with small chiral photosensitisers proceed with poor selectivity, as cyclizations to form 6-membered ring analogues are relatively slow and competing dissociation of the excited substrate from the photosensitiser erodes e.e.. Small molecule chiral photosensitisers also rely on complementary dual hydrogen-bonding contacts between the substrate and catalyst to achieve enantioselective photochemistry. To demonstrate that protein catalysts are not constrained in the same manner, we next investigated cycloaddition of an N-methyl derivative 12. EnT1.3 affords optically enriched 12a, along with its regioisomer 12b, with modest selectivity (2:1 r.r., 36% e.e. for 12a), which could be further enhanced (4:1 r.r., 78% e.e. for 12a) by introducing a rational Y121F mutation (vide infra). This example demonstrates how protein active sites can be readily adapted to augment catalytic function and suggests that our photoenzymes can mediate selective transformations of substrate classes that are beyond the reach of existing small-molecule systems.