Identification of pterocarpan reductases and prenyltransferases reveals bifurcated biosynthetic pathways for glabridin in Glycyrrhiza glabra

Glabridin is a key chemical and biological marker of Glycyrrhiza glabra L., with significant market demand due to its anti-inflammatory, antioxidant and skin-brightening properties. Currently, glabridin is primarily extracted from the roots of G. glabra, but as a prenylated isoflavonoid, its purification process is complex and time-consuming. Therefore, elucidating the biosynthetic pathways mediated by pterocarpan reductases (PTRs) and prenyltransferases (PTs) is therefore essential for enabling glabridin production through synthetic biology approaches. In this study, we identified three functional GgPTRs and two GgPTs that collectively generated 4′-O-methylpreglabridin, a precursor for glabridin, through metabolic grids. In vitro enzymatic assays indicated that GgPTR1, GgPTR6, and GgPTR9 catalyzed the cleavage of C-O bond in the furan ring of pterocarpan, converting (-)-medicarpin to (-)-vestitol. Two prenyltransferases, GgPT1 and GgPT4, which share 77% sequence identity, exhibited distinct substrate preferences while catalyzing C-4′ prenylation: GgPT1 acted on furan-ring-containing (-)-medicarpin, whereas GgPT4 specifically prenylated ring-opened (-)-vestitol. Through site-directed mutagenesis, 5 mutants with enhanced activity were obtained for GgPTR1. Furthermore, N-terminal truncation markedly enhanced PT activity, yielding GgPT1∆1–85 and GgPT4∆1–58 with 3.26-fold and 2.09-fold increase in catalytic efficiency, respectively.