Elucidation and de novo Reconstitution of Glyceollins Biosynthesis

Abstract 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 the physiological functions of glyceollins and achieving sustainable production through synthetic biology. In this study, we identified previously uncharacterized genes encoding two reductases for 7,2’,4’-trihydroxyisoflavanol (THI) biosynthesis and five P450 enzymes responsible for the final oxidative cyclization in glyceollin I, II, and III biosynthesis, thereby completing the entire glyceollin 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 the understanding of glyceollin biosynthesis in soybean, facilitates sustainable production in microbial hosts and offers new opportunities for their application in agriculture and biology. Competing Interest Statement The authors have declared no competing interest. Footnotes The titer of glyceollins I,II and III was inproved through metabolic engineering, and Figure 6 has been revised.