Soy isoflavones are biologically active flavonoid compounds produced during soybean growth. (S)-Equol is the most bioactive metabolic product of soy isoflavones transformed by intestinal microflora[1]. (S)-Equol has a higher antioxidant capacity compared to its precursors, and further research indicates it has preventive and therapeutic effects on cardiovascular diseases, wrinkles, hair loss, obesity, hot flashes, and some hormone-related conditions[2]. However, only 30-50% of people can produce (S)-equol in vivo, meaning only a subset of the population benefits from (S)-equol[3]. Equol has one chiral carbon, producing two isomers, (R)/(S)-equol, with (S)-equol being more bioactive than (R)-equol. Currently, chemically synthesized equol contains both isomers, while biosynthetic pathways can specifically produce (S)-equol[4].

Engineering E. coli to heterologously express (S)-equol biosynthetic enzymes is currently an effective approach to producing (S)-equol. To achieve high-level expression of (S)-equol, this study used mCherry as a model protein and the anaerobically induced nar promoter to control target protein expression, which avoids the use of expensive inducer IPTG during fermentation and provides a microaerobic environment favorable for (S)-equol biosynthesis. Furthermore, to finely control protein expression levels, we optimized the ribosomal binding site (RBS) sequence downstream of the nar promoter, obtaining a nar promoter library with different translational levels. Through fluorescence intensity testing, we characterized the protein expression regulatory abilities of different RBS sequences and obtained RBS sequences with the highest translational efficiency. These optimized RBS sequences will be applied to high-level expression of (S)-equol pathway enzymes.

This study provides a feasible green process for (S)-equol production. Engineering highly efficient bacterial strains to transform soy isoflavone glycosides into (S)-equol in a cost-effective and sustainable manner. By anaerobic induction of pathway enzyme expression, expensive inducer IPTG is avoided, thus reducing fermentation costs and purification difficulties. (S)-Equol in the fermentation broth supernatant is readily extracted for applications in healthcare to improve women’s health and exert (S)-equol’s preventive and therapeutic effects on certain diseases. This method can be applied to produce oral healthcare products or nutritional supplements added to special-purpose foods, with broad market prospects.