The Translational Coupling of Daidzein Reductase and Dihydrodaidzein Racemase Genes Improves the Production of Equol and Its Analogous Derivatives in Engineered Lactic Acid Bacteria

Equol (EQ) and its analogous derivatives 5-hydroxy-equol (5-OH-EQ) and 5-hydroxy-dehydroequol (5-OH-D-EQ) are isoflavones which benefit human health. They are produced from daidzein and genistein, respectively, in the gut by microorganisms harboring the genes daidzein reductase (<i>dzr</i>), dihydrodaidzein racemase (<i>ifc</i>A), dihydrodaidzein reductase (<i>ddr</i>) and tetrahydrodaidzein reductase (<i>tdr</i>). Since the production of these isoflavones is of interest due to their great-health benefits for humans, the heterologous expression of <i>dzr</i>, <i>ddr</i>, <i>tdr</i> and <i>ifc</i>A from <i>Slackia isoflavoniconvertenes</i> DSM 22006T in lactic acid bacteria (LAB) was used as a strategy to produce EQ, 5-OH-EQ and 5-OH-D-EQ in soy beverages. However, efficient production of these compounds was only demonstrated in two engineered <i>Limosilactobacillus fermentum</i> strains, and it is dependent on dihydrodaidzein racemase (DDRC). In order to increase the production of EQ and its analogous derivatives in different LAB species and genera, different strategies were performed with the <i>ifc</i>A gene. Translational coupling of <i>ifc</i>A and <i>dzr</i> genes (pNZ:TuR.dzr.ifcA) under the influence of a constitutive promoter improved the efficiency of production of EQ, 5-OH-EQ and 5-OH-D-EQ in the engineered LAB strains. The translational coupling of <i>ifc</i>A and <i>dzr</i> genes allowed the production of high concentrations of eq (111.15 ± 9.20-410.56 ± 24.15 μM), 5-OH-eq (71.00 ± 4.25 μM-148.22 ± 9.15 μM) and 5-OH-D-eq (111.15 ± 9.20-201.09 ± 7.65 μM) in soy beverages by different engineered LAB genera, such as <i>L. fermentum</i> INIA 584L, <i>Lactilactobacillus plantarum</i> WCFS1, and <i>Lactocaseibacillus paracasei</i> BL23. Translational coupling has allowed engineered Laboratories strains belonging to different genera, such as <i>L. fermentum</i>, <i>L. plantarum</i>, and <i>L. paracasei</i>, to produce high concentrations of EQ, 5-OH-EQ and 5-OH-D-EQ. Translational coupling could be exploited as a strategy for the efficient production of bioactive compounds.