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

(<i>S</i>)-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 (<i>S</i>)-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 (<i>S</i>)-equol production to 4700 mg/L in a 5 L bioreactor, which is the highest reported (<i>S</i>)-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.