Seizing the key nodes in glabridin biosynthesis network: identification of enzymes for producing licorice hairy roots with high levels of glabridin.

Glabridin holds significant value in the pharmaceutical and cosmetics industry. Due to the challenges associated with chemical synthesis, Glycyrrhiza glabra (licorice) remains the only source of glabridin. However, its naturally low content is insufficient to meet the increasing market demand. In this study, several inducers of glabridin accumulation-alkali stress, salt-alkali stress, hydroxylamine, 5-azacytidine, and methyl jasmonate (MeJA)-were identified. Using integrated multi-omics analyses and in vitro enzyme assays, two interconnected biosynthetic routes were elucidated: an OMT/ODMT-dependent pathway involving methylation-demethylation cycles and an OMT/ODMT-independent route. Five enzymes, GgIFR (isoflavone reductase), GgTHIS1/2 (7,2',4'-trihydroxyisoflavanol synthases), GgPTS (pterocarpan synthase), GgPTR1/4 (pterocarpan reductases), and GgODMT (O-demethyltransferase), were functionally validated. Using an optimized Agrobacterium rhizogenes-mediated transformation system, we generated transgenic hairy root lines overexpressing these enzymes were generated. GgIFR-overexpressing lines achieved a 44-fold increase in glabridin content (0.507 mg/g DW), comparable to levels in 4-year-old wild roots. This study not only elucidates the complex biosynthetic network of glabridin biosynthesis but also establishes a scalable and sustainable hairy root platform for its industrial production via synthetic biology.