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

Area of Science:

• Biotechnology
• Enzyme Engineering
• Metabolic Engineering

Background:

• The isoflavone metabolite (S)-equol possesses diverse physiological activities, making its efficient and sustainable manufacturing crucial.
• Daidzein reductase (DZNR) is a key enzyme in the (S)-equol biotransformation pathway, but its catalytic efficiency requires improvement for industrial applications.

Purpose of the Study:

• To enhance the catalytic performance of daidzein reductase (DZNR) through semi-rational design.
• To develop an efficient biocatalytic system for the industrial-scale biomanufacturing of (S)-equol.

Main Methods:

• Semi-rational design of DZNR based on multiple sequence alignment and 3D structural analysis.
• Construction and screening of single and combinatorial enzyme variants.
• Whole-cell biocatalysis using recombinant *Escherichia coli* (*E. coli*) expressing the engineered DZNR.

Main Results:

• Identification of critical residues Gly30 and Ala105 in DZNR.
• Development of a double mutant, DZNR30S+105S, with substantially enhanced catalytic performance.
• Achieved (S)-equol yields of 238.3 mg/L at 1 mM daidzein and 384.6 mg/L at 2 mM daidzein using the engineered *E. coli* strain.

Conclusions:

• The engineered DZNR variant offers significantly improved catalytic efficiency for (S)-equol production.
• The developed whole-cell biocatalytic system demonstrates potential for high-substrate-concentration applications in (S)-equol biomanufacturing.
• This study advances green biosynthesis technologies for the sustainable production of (S)-equol.