Elucidation and de novo reconstitution of glyceollin biosynthesis.

Glyceollins are phytoalexins, soybean-produced compounds that respond to pathogen invasion, injury, and environmental challenges. While these compounds have diverse bioactivities, their limited accessibility hinders further physiological and biochemical studies. Additionally, the incomplete understanding of glyceollin biosynthesis, particularly cyclization steps, remains a major barrier to sustainable production through synthetic biology. In this study, we uncover the complete biosynthetic pathway of glyceollins through a combinatorial approach involving transient expression in Nicotiana benthamiana, in vitro enzyme characterization, and yeast feeding studies. We identified previously uncharacterized genes encoding reductases for 7,2',4'-trihydroxyisoflavanol biosynthesis and five P450 enzymes that mediate the final oxidative cyclization to produce glyceollins I, II, and III. By de novo reconstruction of the pathway through synthetic biology and metabolic engineering, we successfully produced glyceollins from simple carbon sources in baker's yeast. This work advances the understanding of glyceollin biosynthesis in soybeans, enables sustainable production in microbial hosts, and offers new opportunities for their application in agriculture and biology.