P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

( S )-equol, a gut bacterial isoflavone derivative, has drawn great attention to its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for daidzein to ( S )-equol conversion. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of ( S )-equol limit its efficient production using only natural gut bacteria. In this study, the low ( S )-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into the E. coli BL21 (DE3) strain. The reaction conditions were optimized for ( S )-equol production using the recombinant strain, and this recombinant system enabled the efficient conversion of 200 μM and 1 mM of daidzein to ( S )-equol under aerobic condition to achieve the yields of 95% and 85%, respectively. Since the biosynthesis of trans -tetrahydrodaidzein was found to be a rate-determining step for ( S )-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the ( S )-equol productivity from 59.0 mg/L/h to 69.8 mg/L/h in the whole-cell reaction. The P212A mutation caused an increase in the ( S )-dihydrodaidzein enantioselectivity by decreasing overall activities of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, so that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3 S ,4 R )-tetrahydrodaidzein with an enantioselectivity above 99%.