The production of <i>S</i>-equol from daidzein is associated with a cluster of three genes in <i>Eggerthella</i> sp. YY7918

Daidzein (DZN) is converted to equol (EQL) by intestinal bacteria. We previously reported that Eggerthella sp. YY7918, which is found in human feces, is an EQL-producing bacterium and analyzed its whole genomic sequence. We found three coding sequences (CDSs) in this bacterium that showed 99% similarity to the EQL-producing enzymes of Lactococcus sp. 20-92. These identified CDSs were designated eqlA, eqlB, and eqlC and thought to encode daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR), respectively. These genes were cloned into pColdII. Recombinant plasmids were then introduced into Escherichia coli BL21 (DE3) and DZNR, DHDR, and THDR were expressed and purified by 6×His-Tag chromatography. We confirmed that these three enzymes were involved in the conversion of DZN to EQL. Purified DZNR converted DZN to dihydrodaizein (DHD) in the presence of NADPH. DHDR converted DHD to tetrahydrodaizein (THD) in the presence of NADPH. Neither enzyme showed activities with NADH. THDR converted THD in the absence of cofactors, NAD(P)H, and also produced DHD as a by-product. Thus, we propose that THDR is not a reductase but a new type of dismutase. The GC content of these clusters was 64%, similar to the overall genomic GC content for Eggerthella and Coriobacteriaceae (56-60%), and higher than that for Lactococcus garvieae (39%), even though the gene cluster showed 99% similarity to that in Lactococcus sp. 20-92. Taken together, our results indicate that the gene cluster associated with EQL production evolved in high-GC bacteria including Coriobacteriaceae and was then laterally transferred to Lactococcus sp. 20-92.