Characterization and Identification of a New Daidzein Reductase Involved in (S)-Equol Biosynthesis in Clostridium sp. ZJ6

(S)-equol (EQ) is an isoflavone with high estrogen-like activity in the human body, and is only produced by some gut bacteria in vivo. It plays an important role in maintaining individual health, however, the dearth of resources associated with (S)-EQ-producing bacteria has seriously restricted the production and application of (S)-EQ. We report here a new functional gene KEC48-07020 (K-07020) that was identified from a chick (S)-EQ-producing bacterium (Clostridium sp. ZJ6, ZJ6). We found that recombinant protein of K-07020 possessed similar function to daidzein reductase (DZNR), which can convert daidzein (DZN) into R/S-dihydrodaidzein (R/S-DHD). Interestingly, K-07020 can reversely convert (R/S)-DHD (DHD oxidase) into DZN even without cofactors under aerobic conditions. Additionally, high concentrations of (S)-EQ can directly promote DHD oxidase but inhibit DZNR activity. Molecular docking and site-directed mutagenesis revealed that the amino acid > Arg75 was the active site of DHD oxidase. Subsequently, an engineered E. coli strain based on K-07020 was constructed and showed higher yield of (S)-EQ than the engineered bacteria from our previous work. Metagenomics analysis and PCR detection surprisingly revealed that K-07020 and related bacteria may be prevalent in the gut of humans and animals. Overall, a new DZNR from ZJ6 was found and identified in this study, and its bidirectional enzyme activities and wide distribution in the gut of humans and animals provide alternative strategies for revealing the individual regulatory mechanisms of (S)-EQ-producing bacteria.