Engineering Tetrahydrodaidzein Reductase to Eliminate Reverse Catalysis for Efficient Biosynthesis of (S)-Equol

(S)-Equol, an isoflavone metabolite, is used as a raw ingredient in health food products, phytoestrogen supplements, and functional cosmetics. However, tetrahydrodaidzein reductase (THDR), a flavoenzyme involved in the reversible catalytic activity of converting daidzein to (S)-equol, substantially limits substrate conversion efficiency. In this study, the quantum chemistry cluster model calculation was used to elucidate the reversible catalytic reaction mechanism involving E50 and N5-H of FADH2 docked in THDR. Subsequently, the substrate channel, catalytic pocket, and water channel of THDR were redesigned to obtain the M3-THDR variant (E50D-L136A-D263F-L293V-G294V), which retains high forward dehydroxylation efficiency and suppresses the oxygen-dependent reverse hydroxylation reaction. Finally, the M3-THDR variant significantly enhanced (S)-equol production to 4700 mg/L in a 5 L bioreactor, which is the highest reported (S)-equol yield obtained through microbial fermentation to date. Overall, this study provides a blueprint for translating a mechanism-guided pathway optimization into an industrial-scale production process for functional foods and cosmetic ingredients.