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

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

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01182

DOI=10.3389/fmicb.2018.01182

ISSN=1664-302X

ABSTRACT=<p>(<italic>S</italic>)-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 (<italic>S</italic>)-equol. However, only 30–50% people contain this kind of equol-producing bacteria, and therefore are able to convert daidzein to (<italic>S</italic>)-equol. Administration of (<italic>S</italic>)-equol may be more beneficial than soybean isoflavones. The aim of this study was to construct an engineered (<italic>S</italic>)-equol resistant <italic>Escherichia coli</italic> to enhance (<italic>S</italic>)-equol production <italic>in vitro</italic>. First, transposon mutagenesis libraries were constructed and screened to isolate the (<italic>S</italic>)-equol resistant mutant <italic>E. coli</italic> strain BL21 (<italic>ydiS</italic>) in order to overcome the inhibitory effects of (<italic>S</italic>)-equol on bacterial growth. Bacterial full genome scan sequencing and <italic>in vitro</italic> overexpression results revealed that the <italic>ydiS</italic> gene was responsible for this resistance. Second, the (<italic>S</italic>)-equol-producing genes L-<italic>dznr</italic>, L-<italic>ddrc</italic>, L-<italic>dhdr</italic>, and L-<italic>thdr</italic> of <italic>Lactococcus</italic> 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 (<italic>ydiS</italic>). Both engineered BL21 (DE3) and BL21 (<italic>ydiS</italic>) could use daidzein as substrate to produce (<italic>S</italic>)-equol under both anaerobic and aerobic conditions. As expected, engineered BL21 (<italic>ydiS</italic>) had faster growth rates than BL21 (DE3) when supplemented with high concentrations of (<italic>S</italic>)-equol. The yield and the daidzein utilization ratio were higher for engineered BL21 (<italic>ydiS</italic>). Interestingly, engineered BL21 (<italic>ydiS</italic>) was able to convert daidzein to (<italic>S</italic>)-equol efficiently under aerobic conditions, providing a convenient method for (<italic>S</italic>)-equol production <italic>in vitro</italic>. In addition, a two-step method was developed to produce (<italic>S</italic>)-equol using daidzin as substrate.</p>