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

## TL;DR

Scientists improved the production of health-benefiting isoflavones in engineered bacteria by linking specific genes together.

## Contribution

Translational coupling of the ifcA and dzr genes enhanced the production of equol and its derivatives in multiple lactic acid bacteria strains.

## Key findings

- Translational coupling improved equol production to 111.15–410.56 μM in soy beverages.
- 5-hydroxy-equol and 5-hydroxy-dehydroequol were produced at 71.00–148.22 μM and 111.15–201.09 μM, respectively.
- Multiple LAB genera, including L. fermentum, L. plantarum, and L. paracasei, achieved high compound production.

## Abstract

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
(dzr), dihydrodaidzein racemase (ifcA), dihydrodaidzein reductase (ddr) and tetrahydrodaidzein
reductase (tdr). Since the production of these isoflavones
is of interest due to their great-health benefits for humans, the
heterologous expression of dzr, ddr, tdr and ifcA from Slackia isoflavoniconvertenes 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 Limosilactobacillus
fermentum 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 ifcA gene. Translational
coupling of ifcA and dzr 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 ifcA and dzr 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 L. fermentum INIA 584L, Lactilactobacillus plantarum WCFS1, and Lactocaseibacillus paracasei BL23. Translational coupling has allowed engineered Laboratories
strains belonging to different genera, such as L. fermentum, L. plantarum, and L. paracasei, 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.