Engineering Saccharomyces cerevisiae for De Novo Biosynthesis of 3′-Hydroxygenistein

The polyhydroxy isoflavone 3′-hydroxygenistein (3′-OHG) has a wide range of pharmaceutical and nutraceutical benefits. Therefore, it is important to develop an efficient and sustainable method for 3′-OHG production. Here, we engineered the metabolic pathways of Saccharomyces cerevisiae to achieve de novo biosynthesis of 3′-OHG. First, we screened 2-hydroxyisoflavanone synthase (IFS), cytochrome P450 reductase, and 2-hydroxyisoflavanone dehydratase from different sources and optimized the best combination via promoter engineering. Next, we demonstrated that amplification of the rate-limiting enzyme PlIFS from Pueraria lobata improved genistein production. Increasing the availability of the cofactor heme further increased genistein titer to 44.55 ± 1.82 mg/L. Subsequently, screening and multicopy integration of isoflavone-3′-hydroxylase achieved 13.23 ± 0.27 mg/L 3′-OHG from 100 mg/L naringenin. Finally, 1.40 ± 0.02 mg/L 3′-OHG could be achieved via the de novo biosynthesis pathway. The final strain generated in this study will facilitate the production of polyhydroxy isoflavones via the 3′-OHG biosynthetic pathway.