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Front Microbiol. 2018 Jun 4;9:1182. doi: 10.3389/fmicb.2018.01182. eCollection 2018.

To Construct an Engineered (S)-Equol Resistant E. coli for in Vitro (S)-Equol Production.

Li H1,2,3, Mao S3, Chen H1,2, Zhu L2, Liu W2, Wang X2, Yin Y1,2.

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Key Laboratory of Comprehensive Utilization of Advantage Plants Resources in Hunan South, College of Chemistry and Bioengineering, Hunan University of Science and Engineering, Yongzhou, China.
State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China.
Hunan Provincial Key Laboratory for Forestry Biotechnology, College of Life Science and Technology, Central South University of Forestry and Technology, Changsha, China.


(S)-equol is one of the major metabolites of daidzein that is produced by human and animal gut bacteria. Most of the physiological functions of soybean isoflavones, such as anti-oxidative activity, anti-cancer activity, and cardiovascular protection have been ascribed to (S)-equol. However, only 30-50% people contain this kind of equol-producing bacteria, and therefore are able to convert daidzein to (S)-equol. Administration of (S)-equol may be more beneficial than soybean isoflavones. The aim of this study was to construct an engineered (S)-equol resistant Escherichia coli to enhance (S)-equol production in vitro. First, transposon mutagenesis libraries were constructed and screened to isolate the (S)-equol resistant mutant E. coli strain BL21 (ydiS) in order to overcome the inhibitory effects of (S)-equol on bacterial growth. Bacterial full genome scan sequencing and in vitro overexpression results revealed that the ydiS gene was responsible for this resistance. Second, the (S)-equol-producing genes L-dznr, L-ddrc, L-dhdr, and L-thdr of Lactococcus strain 20-92 were synthesized and cloned into compatible vectors, pETDuet-1 and pCDFDuet-1. These plasmids were subsequently transformed into BL21 (DE3) and its mutant BL21 (ydiS). Both engineered BL21 (DE3) and BL21 (ydiS) could use daidzein as substrate to produce (S)-equol under both anaerobic and aerobic conditions. As expected, engineered BL21 (ydiS) had faster growth rates than BL21 (DE3) when supplemented with high concentrations of (S)-equol. The yield and the daidzein utilization ratio were higher for engineered BL21 (ydiS). Interestingly, engineered BL21 (ydiS) was able to convert daidzein to (S)-equol efficiently under aerobic conditions, providing a convenient method for (S)-equol production in vitro. In addition, a two-step method was developed to produce (S)-equol using daidzin as substrate.


(S)-equol production; (S)-equol resistance; soybean isoflavone; transposon mutagenesis; ydiS gene

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