Controlling Chemo- and Regioselectivity of a Plant P450 in Yeast Cell toward Rare Licorice Triterpenoid Biosynthesis

The hallmark reaction of P450 monooxygenase involves activation of C–H bond and the production of a hydroxyl. P450s tailoring natural product could further oxidize the hydroxyl to carboxylic acid. However, heterogeneously expressed plant P450s display poor chemo- and regioselectivity in microbes, restricting the efficient biosynthesis of related natural products. CYP72A63 is a P450 enzyme previously used for the biosynthesis of glycyrrhetinic acid, and its poor selectivity resulted in oxidation of 11-oxo-β-amyrin to a mixture of rare licorice triterpenoids (glycyrrhetol, glycyrrhetaldehyde, glycyrrhetinic acid, and 29-OH-11-oxo-β-amyrin). In this study, we have identified key residues, which influence the enzyme–substrate hydrophobic interaction, in controlling the chemo- and regioselectivity of the enzyme and engineered the enzyme toward selectivity oxidation to hydroxyl and carboxylic acid. Moreover, tuning the redox partner of the P450 leads to selective production of glycyrrhetaldehyde, a good starting point for further modification. In this study, controlling the catalytic property of plant P450s prove to be of great use in the synthesis of desired licorice triterpenoids, which can be used in biosynthesis of other terpenoid natural products.