# A Whole-Cell Catalytic System for Equol Production Based on Daidzein Reductase Engineering

## TL;DR

This study improves the production of (S)-equol by engineering an enzyme called daidzein reductase, achieving higher yields in a sustainable biocatalytic system.

## Contribution

A novel double mutant of daidzein reductase is developed, significantly enhancing catalytic efficiency for (S)-equol production.

## Key findings

- The DZNR30S+105S mutant achieved 238.3 mg/L (S)-equol at 1 mM daidzein.
- At 2 mM daidzein, the mutant produced 384.6 mg/L (S)-equol within 5 hours.
- The mutant shows excellent potential for high-substrate-concentration industrial applications.

## Abstract

As an isoflavone metabolite with diverse physiological activities, the development of efficient and sustainable manufacturing technologies for (S)-equol holds significant importance. This study focuses on the semi-rational design of daidzein reductase (DZNR), the first key enzyme in the (S)-equol biotransformation pathway. Through multiple sequence alignment and three-dimensional structural analysis, two critical residues, Gly30 and Ala105, were identified in DZNR. A library of single and combinatorial mutants was constructed and screened, yielding the double variant DZNR30S+105S with substantially enhanced catalytic performance. In a whole-cell biocatalytic system, the recombinant E. coli (Escherichia coli) strain harboring this combinatorial mutant achieved a yield of 238.3 mg/L (S)-equol at a substrate concentration of 1 mM daidzein, demonstrating markedly improved catalytic efficiency. Upon increasing the daidzein concentration to 2 mM, the reaction reached equilibrium within 5 h, producing 384.6 mg/L (S)-equol, which highlights the mutant’s excellent potential for high-substrate-concentration applications. This study not only provides novel mechanistic insights into DZNR catalysis but also successfully establishes a DZNR variant with enhanced activity, offering an efficient biocatalytic component for the industrial-scale biomanufacturing of (S)-equol and thereby advancing the development of green biosynthesis technologies for this valuable compound.