Discover the maze-like network 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, by combining chromosome-scale genome assembly with large-scale transcriptome analysis, we identify and reconstruct the glabridin biosynthetic pathway in yeast, enabling de novo production of this antioxidant flavonoid from Glycyrrhiza glabra L. By analyzing 183 licorice transcriptomes, we discover four classes of enzymes capable of modify isoflavan scaffolds. Among 13 theoretical routes, we validate six functional pathways, revealing a ladder-like, multi-route tailoring network. 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 biomanufacturing. The complexity of the evolved metabolic networks of flavonoids complicates pathway elucidation and design. Here, the authors identify enzymes that modify isoflavan scaffolds from the genome and transcriptome data of Glycyrrhiza glabra, and reconstruct de novo glabridin biosynthesis in yeast.