Highly efficient synthesis of the chiral ACE inhibitor intermediate (R)-2-hydroxy-4-phenylbutyrate ethyl ester via engineered bi-enzyme coupled systems.

(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester ((R)-HPBE) is an essential chiral intermediate in the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Its production involves the highly selective asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE), catalyzed by carbonyl reductase (CpCR), with efficient cofactor regeneration playing a crucial role. In this study, an in-situ coenzyme regeneration system was developed by coupling carbonyl reductase (CpCR) with glucose dehydrogenase (GDH), resulting in the construction of five recombinant strains capable of NADPH regeneration. Among these, the recombinant strain E. coli BL21-pETDuet-1-GDH-L-CpCR, where CpCR is fused to the C-terminus of GDH, demonstrated the highest catalytic activity. This strain exhibited an enzyme activity of 69.78 U/mg and achieved a conversion rate of 98.3%, with an enantiomeric excess (ee) of 99.9% during the conversion of 30 mM OPBE to (R)-HPBE. High-density fermentation further enhanced enzyme yield, achieving an enzyme activity of 1960 U/mL in the fermentation broth, which is 16.2 times higher than the volumetric activity obtained from shake flask fermentation. Additionally, the implementation of a substrate feeding strategy enabled continuous processing, allowing the strain to efficiently convert a final OPBE concentration of 920 mM, producing 912 mM of (R)-HPBE. These findings highlight the system's improved catalytic efficiency, stability, and scalability, making it highly suitable for industrial-scale biocatalytic production.