Identification and characterization of key enzymes pterocarpan prenyltransferase and pterocarpan reductase in the biosynthetic pathway of glabridin

Glabridin is a uniquely isopentenyl-modified isoflavonoid component specific to Glycyrrhiza glabra L., which not only possesses multiple functions including antioxidant, anti-inflammatory, anti-atherosclerotic and Anti-aging properties, but also serves as a primary raw material for high-end whitening cosmetics. Currently, glabridin is mainly extracted from G. glabra L. roots, with limited production capacity and restricted sources that cannot meet the growing market demand. In this study, the complete biosynthetic pathway of glabridin was deduced through comparative transcriptome analysis and literature studies. Among these, pterocarpan prenyltransferase (PcM4DT) and pterocarpan reductase (PTR) were identified as two key Enzymes catalyzing the synthesis of 4'-O-methylpreglabridin, the precursor of glabridin from medicarpin. Furthermore, eight potential key PTR Enzymes involved in the conversion of the glabridin precursor licoagrocarpin into 4'-O-methylpreglabridin were screened. Through in vitro enzymatic activity assays, it was confirmed that PcM4DT from Psoralea corylifolia Linn. and PTR8 from G. glabra L. could serve as crucial Enzymes for synthesizing glabridin precursor 4'-O-methylpreglabridin. Concurrently, molecular docking and site-directed mutagenesis of PTR8 revealed that the H270A mutant could enhance the conversion rate of substrate licoagrocarpin. This study not only provides powerful enzymatic tools for biocatalytic synthesis of 4'-O-methylpreglabridin but also demonstrates the potential to advance the heterologous biosynthesis of glabridin.