Substrate Versatility and Regiocontrol of Licorice Flavonoid Prenyltransferase GgFPT3 for the Biosynthesis of Diverse Prenylated Flavonoid Natural Products

Abstract Species of Glycyrrhiza (licorice) produce a diverse array of bioactive prenylated flavonoids. Prenyltransferases (PTs) are crucial for this structural diversification, yet their diversity and functional roles in licorice remain largely unexplored. Here, we identified three root-specific, stress-induced flavonoid PT candidates (GgFPT1-3) from G. glabra. While all three mainly localized to the chloroplast, only GgFPT3 exhibited catalytic activity in vitro under the assay conditions tested. GgFPT3 demonstrated remarkable substrate promiscuity, catalyzing the monoprenylation of diverse backbones including dihydroflavones, isoflavans, isoflavanones, isoflavones, chalcones, and pterocarpans, with a regioselective preference for B-ring (e.g., C-3′) prenylation. Intriguingly, despite sharing high amino acid sequence identity (>90%) with GgFPT3, its orthologs in G. inflata and G. uralensis showed distinct expression patterns, enzymatic activities, and substrate preferences. These functional divergences likely underpin the species-specific chemotypic variations of prenylated flavonoids across the Glycyrrhiza genus, expanding our understanding of flavonoid diversification mechanisms.