To Construct an Engineered ()-Equol Resistant for ()-Equol Production.

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