Discover the Maze-like Pathway for Glabridin Biosynthesis

Flavonoid diversity arises from modifications of a few precursors by tailored enzymes, yet the complexity of their evolved metabolic networks complicates pathway elucidation and design. Here, we identify and reconstruct the glabridin biosynthetic pathway in yeast, enabling de novo production of this antioxidant flavonoid from the rare plant Glycyrrhiza glabra L.. By analyzing 183 licorice transcriptomes, we discover previously unknown enzymes that modify isoflavan scaffolds. Among 13 theoretical routes, we validate six functional pathways, revealing a ladder-like, multi-route tailoring network that challenges the classical linear view of secondary metabolism. This network features a multi-step “protection–deprotection” mechanism driven by a dynamic methylation–demethylation cycle that regulate intermediate hydrophilicity and enzyme affinity, thereby enabling species-specific glabridin synthesis. Reconstruction of this network in yeast shows that metabolic redundancy and interconnectivity enhance robustness and yield compared to single-route designs. These results establish a biosynthetic paradigm with broad implications for natural product discovery and sustainable biomanufacturing.