Microenvironment accessibility enables rare oxidation type of triterpenoids by plant P450

Triterpenoids are valuable medicinal scaffolds, characterized by excellent pharmacological properties and the presence of hydroxyl and carboxyl groups that allow for further structural modifications. Expanding the scope of oxidative modifications on these molecules is crucial for increasing their synthetic structural diversity and unlocking new potential pharmacological activities. However, the progress has been limited by the scarcity of suitable tailoring enzymes. Here, we reported a break-through in achieving targeted and remote dual-site oxidation of licorice triterpenoids using a single P450 mutant. This approach successfully enabled the selective synthesis of the rare triterpenoid, liquiritic acid and 24-OH-liquiritic acid. Our findings demonstrate that microenvironmental accessibility engineering of triterpenoid substrates within the P450 enzyme is essential for continuous and regioselective oxidation. This study not only sheds light on the mechanistic aspects of P450 catalysis but also expands the enzymatic toolkit for selective oxidative modifications in triterpenoid biosynthesis.