Characterization and identification of a novel daidzein reductase involved in (S)-equol biosynthesis in Clostridium C1

Abstract

(S)-equol is an isoflavone with high estrogen-like activity and no toxic effects 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)-equol-producing bacteria has seriously restricted the production and application of (S)-equol. We report here a novel functional gene C1-07020 that was identified from a chick (S)-equol-producing bacterium (Clostridium C1). We found that recombinant protein of C1-07020 possessed similar function to daidzein reductase (DZNR), which can convert daidzein (DZN) into R/S-dihydrodaidzein (R/S-DHD). Interestingly, C1-07020 can reverse convert (R/S)-DHD (DHD oxidases) into DZN even without cofactors or anaerobic conditions. Additionally, high concentrations of (S)-equol can directly promote DHD oxidase but inhibit DZNR activity. Molecular docking and site-directed mutagenesis revealed that the amino acid Arg 75 was the active site of DHD oxidases. Subsequently, an engineered E. coli strain based on C1-07020 was constructed and showed higher yield of (S)-equol than the engineered bacteria from our previous work. Metagenomics analysis and PCR detection surprisingly revealed that C1-07020 and related bacteria may be prevalent in the gut of humans and animals and their (S)-equol production state may cause differed between (S)-equol producer and non-producer. Overall, a novel DZNR from Clostridium C1 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)-equol-producing bacteria. Importance(S)-equol is a final product of DZN that metabolized by some enteric bacteria. Although (S)-equol played very important roles in maintaining human health, larger differences in equol production varied between different populations. Here, a novel DZNR gene C1-07020, which related to (S)-equol production, was reported. The bidirectional enzyme functions and wide distribution of C1-07020 in human and animal gut provided additional insights into the metabolic regulation of (S)-equol. Additionally, C1-07020 can be used for improving the production of (S)-equol in vitro.