(S)‐equol, the most active metabolite of the soybean isoflavones in vivo, has exhibited various biological activities and clinical benefits. Existing studies on the heterologous biosynthesis of (S)‐equol via the engineered E. coli constructed have been significantly progressed. In the present study, the engineered E. coli was further improved to be more suitable for (S)‐equol production. The four enzymes involved in the biosynthesis of (S)‐equol and another GDH for NADPH regeneration were combined to construct the recombinant E. coli BL21(DE3). The optimal conditions for (S)‐equol production were explored, respectively. The yield of equol reached 98.05% with 1 mM substrate daidzein and 4% (wt/vol) glucose. Even when the substrate concentration increased to 1.5 mM, (S)‐equol could maintain a high yield of 90.25%. Based on the 100 ml one‐pot reaction system, (S)‐equol was produced with 223.6 mg/L in 1.5 h. The study presented a more suitable engineered E. coli for the production of (S)‐equol.
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