De novo biosynthesis of medicarpin in metabolically engineered yeast.

Medicarpin exhibits multiple pharmacological activities, including antitumor and antibacterial effects. However, its extraction from natural plant sources is limited, and chemical synthesis is complex and costly. Therefore, developing an economical, green, and sustainable medicarpin production method is of great significance. In this study, we achieved the de novo synthesis of medicarpin in engineered Saccharomyces cerevisiae. Specifically, we overexpressed and mutated endogenous yeast genes involved in the upstream shikimate pathway and aromatic amino acid biosynthesis pathway, which are essential for flavonoid synthesis. Additionally, we constructed the downstream flavonoid biosynthetic pathway in engineered yeast strains by integrating heterologous genes derived from plants and bacteria. Consequently, the synthesis of p-coumaric acid, liquiritigenin, daidzein, and formononetin was successfully achieved in a series of engineered strains, with medicarpin ultimately synthesized in the strain of GlaN22. After 168 h of cultivation, the medicarpin titer of GlaN22 reached 3.13 ± 0.68 μg/L. Further optimization via double-copy integration of some key biosynthetic genes resulted in strains of GlaN25 and GlaN26, which produced 76.23 ± 9.43 μg/L and 157.55 ± 17.05 μg/L of medicarpin, respectively. This work demonstrates the successful de novo production of medicarpin in engineered yeasts, providing a foundation for the green and sustainable biomanufacturing of high-value isoflavonoid compounds derived from traditional Chinese herbs.