Identification of Two Novel Reductases Involved in Equol Biosynthesis in Lactococcus Strain 20–92

Lactococcus strain 20–92 is a bacterium that produces equol directly from daidzein under anaerobic conditions. In this study, we reveal that the transcription of the gene encoding daidzein reductase in Lactococcus strain 20–92 (L-DZNR), which is responsible for the first stage of the biosynthesis of equol from daidzein, is regulated by the presence of daidzein. We analyzed the sequence surrounding the L-DZNR gene and found six novel genes, termed orf-US4, orf-US3, orf-US2, orf-US1, orf-DS1 and orf-DS2. These genes were expressed in Escherichia coli, and the resulting gene products were assayed for dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) activity. The results showed that orf-US2 and orf-US3 encoded DHDR and THDR, respectively. DHDR in Lactococcus strain 20–92 (L-DHDR) was similar to the 3-oxoacyl-acyl-carrier-protein reductases of several bacteria and belonged to the short chain dehydrogenase/reductase family. THDR in Lactococcus strain 20–92 (L-THDR) was similar to several putative fumarate reductase/succinate dehydrogenase flavoprotein domain proteins. L-DHDR required NAD(P)H for its activity, whereas L-THDR required neither NADPH nor NADH. Thus, we succeeded in identifying two novel enzymes that are related to the second and third stages of the biosynthetic pathway that converts daidzein to equol.