Elucidation and de novo Reconstitution of Glyceollin Biosynthesis

Glyceollins are phytoalexins produced by soybeans in response to stressors such as pathogen invasion, injury, and environmental challenges. In addition to their antibacterial and antifungal activities, these compounds have attracted significant attention for their potential anticancer, anti-inflammatory, and antioxidant properties. However, their limited accessibility-due to the challenges and high costs associated with organic synthesis or purification from treated soybean seedlings-has hindered further physiological and biochemical studies. Moreover, the incomplete understanding of glyceollin biosynthesis, particularly the final cyclization steps, remains a major barrier to elucidating their physiological functions and achieving sustainable production through synthetic biology. In this study, we identified previously uncharacterized genes encoding two reductases for 7,2’,4’-trihydroxy-isoflavanol (THI) synthesis and five P450 enzymes responsible for the final oxidative cyclization in glyceollins I, II, and III biosynthesis, thereby completing the entire biosynthetic pathway. By reconstructing the pathway de novo through synthetic biology, we achieved the successful production of glyceollins from simple carbon sources in Baker’s yeast. This work advances understanding of glyceollin biosynthesis, facilitates sustainable production in microbial hosts and offers new opportunities for their applications in agriculture and biology.