Identification of Trifolium repens as a New Source of Glycyrrhetinic Acid: Pathway Elucidation and Heterologous Reconstruction in Yeast.

Glycyrrhetinic acid (GA) is a pharmacologically important oleanane-type triterpenoid best known from licorice, but its sustainable production is limited by inefficient enzymes. Here, we report the complete GA biosynthetic pathway in Trifolium repens, a legume not previously known to produce GA. Targeted metabolomics confirmed GA accumulation in multiple tissues, highest in roots. Transcriptomic analyses identified key biosynthetic genes, and heterologous reconstruction in yeast verified their catalytic functions. The pathway comprises three β-amyrin synthases, two β-amyrin 11-oxidases (TrCYP88D37/35b), and an 11-oxo-β-amyrin 30-oxidase (TrCYP72A1633). Phylogenetic analyses revealed these CYP450s are conserved in supertribe Fabodae, hinting at the potential for GA or related triterpenoid production in other legumes. Screening seven cytochrome P450 reductases (CPRs), including two from T. repens, identified AiCPR from Azadirachta indica as optimal for TrCYP72A1633, increasing GA titers in yeast to 3.07 mg/L. This study establishes GA biosynthesis in T. repens and provides a basis for sustainable microbial production.