Discovery and Engineering of Prenyltransferases in Glycyrrhiza inflata Enable Diversified Biosynthesis of Prenylated Flavonoids

Abstract

Prenylated flavonoids represent a significant class of bioactive natural products widely distributed in plants, and prenyltransferases (PTs) play a key role in their biosynthesis. Although a number of flavonoid prenyltransferases have been reported, they generally exhibit narrow substrate promiscuity, low catalytic efficiency, and limited modification sites (primarily at C6 or C8 of the A-ring). Herein, six flavonoid prenyltransferases were identified from Glycyrrhiza inflata, which can be classified into two groups based on their regioselectivity. GinPT1, GinPT2, and GinPT5 catalyze C6 prenylation of flavonoid substrates, whereas GinPT3, GinPT4, and GinPT6 catalyze C3′ prenylation. Through rational design, a single-site mutation M171I was found to enhance both the substrate promiscuity and catalytic efficiency of GinPT5. This mutant is capable of prenylating over 30 phenolic compounds, with the highest conversion rate reaching 98%. GinPT5-M171I represents the plant PT with the broadest substrate promiscuity reported to date. Furthermore, residue #171 may be a general key amino acid that determines the substrate selectivity of plant PTs. Using the above native and engineered enzymes, a biosynthesis platform was established that can produce a variety of prenylated phenolic compounds with promising pharmacological activities.