Encouraged by this promising initial activity and high enantioselectivity, we chose Parent F2 as a starting point for directed evolution to improve enzyme performance using iterative site-saturation mutagenesis (SSM) of residues located in the heme domain (Entries 2-11), screening for improved azetidine yield by gas chromatography. Sites were selected for mutagenesis based on success in previous directed evolution campaigns of P450BM3 as well as prior knowledge of residues responsible for substrate binding and catalysis in the heme domain of this protein scaffold. 17a Ten beneficial mutations were identified during this campaign, resulting in a more efficient 'azetidine synthase' (P411-AzetS) with a net improvement of 16-fold in TTN and improved enantioselection (99:1 er). With P411-AzetS in hand, we next examined the impact of varying the reaction conditions on the product yield (Entries 12-17). Notably, increasing the scale from 4 μmol to 100 μmol resulted in an increase in the reaction yield. When the concentrations of 1 and EDA were doubled to 20 mM and 30 mM, respectively, a decrease in reaction yield was observed (although TTN increased). The ring expansion reaction also proceeded in clarified cell lysate, albeit with decreased yields when compared to analogous reactions performed with whole-cell suspensions. Lastly, decreasing the reaction temperature from 22 to 4 °C did not have a meaningful impact on the reaction yields when run in whole-cell suspensions.