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

Hailiang Li,Xin Wang,Liying Zhu,Huahai Chen,Yeshi Yin,Wei Liu,Shaoming Mao

doi:10.3389/fmicb.2018.01182

Abstract

(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.

Highlights

Soy isoflavones have multiple health benefits due to their anti-carcinogenic, anti-oxidant, and anti-atherosclerotic properties (Xiao et al, 2017)
In order to evaluate the equol-producing activity of the engineered E. coli, 50 μg/mL (∼200 μM) daidzein was added to Lennox broth (LB) culturing media and (S)-equol production was detected under both anaerobic and aerobic conditions at different time points after IPTG induction
Previous studies have reported that the equol produced by gut bacteria is (S)-equol (Setchell et al, 2005), and that the metabolite produced by Lactococcus strain 20–92 is (S)-equol (Shimada et al, 2011, 2012), the metabolite produced by the engineered E. coli was likely to be (S)-equol (Supplementary Figure 3)

Summary

Introduction

Soy isoflavones have multiple health benefits due to their anti-carcinogenic, anti-oxidant, and anti-atherosclerotic properties (Xiao et al, 2017). These chemicals interact with the estrogen receptor, enabling them to act as weak to moderate phytoestrogens (Nielsen and Williamson, 2007). Only 30–50% of the human population can produce equol (Low et al, 2005; Ozasa et al, 2005; Setchell and Cole, 2006; Hall et al, 2007) This suggests that health effects of functional foods supplemented with (S)-equol could be more beneficial than daidzein. Several studies have demonstrated that a diet supplemented with natural (S)-equol alleviates menopausal symptoms, such as hot flushes and crow’s feet wrinkles (Aso et al, 2012; Oyama et al, 2012)

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