In further directed evolution, F91L was found to be another key mutation providing 6.6-times higher yield and improved enantioselectivity of 2a (entry 5, (29.5 ± 0.5)% yield and 84:16 e.r.). Notably, ACCO I184A K158I F91L was the first variant in this evolutionary lineage favouring the formation of C-H fluorination over the undesired reduction (2a:3a = 82:18), giving rise to substantially enhanced chemoselectivity. Ultimately, after an additional three rounds of SSM and screening (entries 6-8), directed evolution led to ACCO I184A K158I F91L K172Y K93Q T89A (ACCOCHF, CHF = C-H fluorination), furnishing the enantioenriched fluorination product 2a in (97 ± 2)% yield, 95:5 e.r., 98:2 chemoselectivity and (211 ± 5) total turnover number (TTN, entry 8). By further lowering the biocatalyst loading via decreasing the optical density of E. coli cells (OD600 = 10) overexpressing ACCOCHF, a TTN of (601 ± 5) could be achieved without lowering the yield of 2a (entry 9). Further decreasing OD600 to 5 led to a TTN of (820 ± 10). Importantly, among all the nonheme Fe enzymes we investigated, ACCOCHF represents the only biocatalyst capable of delivering enantioenriched fluorinated products in excellent yields without forming other side products, such as the reduction products and C-H hydroxylation products 57 . Furthermore, 550 mg of evolved ACCOCHF could be obtained from 1L E. coli culture in TB, which represented a 1.7-fold improvement in protein expression level relative to wt ACCO and underscored its utility in preparative biotransformations (Table and Fig. ).