Abstract

ent -Kaurane diterpenoids ( ent -KTs) represent a structurally diverse class of natural products renowned for their antitumor and anti-inflammatory bioactivities. The C-14 hydroxyl modification is crucial for enhancing their potency; however, introducing this functional group remains a considerable challenge. Here, we present a computational heme-guided site-specific (CHS) strategy to identify three bacterial P450s (CYP260A1, CYP105N1, and CYP154C5) for C-14 hydroxylation. Further computationally guided enzyme engineering and redox partner screening identify the CYP260A1 L162V variant paired with CamA/CamB, achieving a 52-fold increase in production titer and a yield of 84.2 mg/L of (14 R ,16 R )- ent -kauran-14,16-diol ( 2 ) in Escherichia coli . Substrate scope test reveals functional groups affecting reactivity. Structure-activity relationship studies demonstrate the synergistic effect between the C-14 hydroxyl and C15–C16 Michael acceptor, resulting in a potent derivative ( 27 ) with strong cytotoxicity (IC 50 HCT116  = 1.4 μM). This study demonstrates a framework combining CHS-guided P450 discovery and computational enzyme engineering to advance ent -KT modifications.