Efficient, complete biosynthesis of isoflavonoid calycosin-7-glucoside by metabolically engineered yeast cell factories

Plant isoflavonoids constitute a class of secondary metabolites that hold great potential in nutraceutical and pharmaceutical industries, considering their prominent human health-promoting properties. As a result, there is increasing interest in establishing alternative sources to these bioactive molecules to cope with the uneconomic and non-sustainable nature of traditional crop-based production. Here, we present the construction of a yeast platform that can de novo produce isoflavonoid calycosin-7-glucoside (CG), one of the quality markers for important medicinal Astragalus herbs. Through identifying active plant catalytic enzymes, optimizing carbon sources, utilizing and providing key precursor daidzein, and engineering biosynthesis of key metabolic cofactors, production of 47.60 mg/L CG from simple carbon sources was achieved in shake flask fermentations. In addition, we showcased the feasibility of modifying the substrate preference of selected plant glycosyltransferase by rational protein engineering. Our work provides an important step toward developing industrial-scale biomanufacturing of CG and may be extended to engineer complete biosynthesis of other value-added isoflavonoids in microbes.