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

Abstract

(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.

Keywords: Bi-enzyme coupled; Carbonyl reductase; Fusion-expression; High-density fermentation; Substrate feeding strategy.

Fig. 1

Structural diagram of the CpCR recombinant expression plasmids. (a), (b) and (c) stand for pACYCDuet-1-CpCR, pET28a-CpCR, pETDuet-1-CpCR

Fig. 2

Plasmid map of the CpCR/GDH co-expressing recombinant strains. (a), (b) and (c) stand for pETDuet-1-CpCR-GDH, pETDuet-1-GDH-CpCR and pETDuet-1-CpCR/pACYCDuet-1-GDH

Fig. 3

Plasmid map of the CpCR/GDH fusion expression recombinant strain. (a) and (b) stand for pETDuet-1-CpCR-L-GDH and pETDuet-1-GDH-L-CpCR

Fig. 4

SDS-PAGE analysis of CpCR protein. (a) The SDS-PAGE analysis of CpCR expressed by E. coli BL21-pETDuet-1-CpCR, E. coli BL21-pET28a-CpCR and E. coli BL21-pACYCDuet-1-CpCR. (b) The purification of CpCR expressed by E. coli BL21-pETDuet-1-CpCR. ((a): Lane 1: Supernatant of E. coli BL21-pETDuet-1-CpCR; Lane 2: Precipitation of E. coli BL21-pETDuet-1-CpCR; Lane 3: Supernatant of E. coli BL21-pET28a-CpCR; Lane 4: Precipitation of E. coli BL21-pET28a-CpCR; Lane 5: Supernatant of E. coli BL21-pACYCDuet-1-CpCR; Lane 6: Precipitation of E. coli BL21-pACYCDuet-1-CpCR. (b): Lane 1: supernatant of E. coli BL21-pETDuet-1-CpCR breakage solution; Lane 2: precipitate of E. coli BL21-pETDuet-1-CpCR breakage solution; Lane 3: supernatant of concentrated breakage solution; Lane 4-Lane 6: eluent of protein CpCR purification)

Fig. 5

Enzyme activity of CpCR in different expression plasmids

Fig. 6

SDS-PAGE analysis of CpCR/GDH co-expression protein and fusion proteins CpCR-L-GDH and GDH-L-CpCR. (a) SDS-PAGE of CpCR/GDH co-expression (Lane 1: Supernatant of E. coli BL21-pETDuet-1-CpCR/pACYCDuet-1-GDH; Lane 2: Precipitation of E. coli BL21-pETDuet-1-CpCR/pACYCDuet-1-GDH; Lane 3: Supernatant of E. coli BL21-pETDuet-1-CpCR-GDH; Lane 4: Precipitation of E. coli BL21-pETDuet-1-CpCR-GDH; Lane 5: Supernatant of E. coli BL21-pETDuet-1-GDH-CpCR ; Lane 6: Precipitation of E. coli BL21-pETDuet-1-GDH-CpCR.); (b) DS-PAGE analysis of the fusion proteins CpCR-L-GDH and GDH-L-CpCR (Lane M: Protein Marker; lane 1: supernatant of pETDuet-1-CpCR-L-GDH; lane 2: precipitation of pETDuet-1-CpCR-L-GDH; lane 3: supernatant of pETDuet-1-GDH-L-CpCR; lane 4: precipitation of pETDuet-1-GDH-L-CpCR)

Fig. 7

Conversion rates and enantiomeric excess (ee%) of (R)-HPBE by different recombinant strains. (Control: reaction without cells; 1: E. coli BL21-pETDuet-1-CpCR; 2: E. coli BL21-pACYCDuet-1-CpCR; 3: E. coli BL21-pET28a-CpCR; 4:combination of E. coli BL21-pETDuet-1-CpCR and E. coli pACYCDuet-1-GDH; 5: E. coli BL21-pETDuet-1-CpCR/pACYCDuet-1-GDH; 6: E. coli BL21-pETDuet-1-CpCR-GDH; 7: E. coli BL21-pETDuet-1-GDH-CpCR; 8: E. coli BL21-pETDuet-1-CpCR-L-GDH; 9: E. coli BL21-pETDuet-1-GDH-L-CpCR)

Fig. 8

Residual enzyme activity of purified enzymes CpCR, CpCR/GDH, CpCR-L-GDH, and GDH-L-CpCR after incubation under different conditions. a, b, and c stand for the pH stability, thermal stability and time-dependent stability

Fig. 9

SDS-PAGE of GDH-L-CpCR in E. coli BL21-pETDuet-1-GDH-L-CpCR at different induction conditions. (a) IPTG concentrations. Lane 1, 3, 5, 7, and 9 show supernatant from cell extracts with 0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, and 0.9 mM IPTG. Lanes 2, 4, 6, 8, and 10 show the corresponding precipitation; (b) Temperatures. Lane 1, 3, 5, 7, and 9 represent the supernatant of cell-free extracts induced at 18, 23, 28, 30, and 37 °C, respectively. Lane 2, 4, 6, 8, and 10 show the corresponding precipitation at the same induction temperatures; (c) Times. Lane 1, 3, 5, 7, and 9 show the supernatant of cell-free extracts after induction times of 8, 12, 16, 20, and 24 h, respectively. Lane 2, 4, 6, 8, and 10 depict the corresponding pellets for the same induction times

Fig. 10

Optimization of enzyme induction conditions. (a), (b), and (c) represent the effects of IPTG concentration, induction temperature, and induction time, respectively, on the enzyme activity of the fusion protein GDH-L-CpCR

Fig. 11

Optimization of Whole-Cell Catalytic Conditions for Strain E. coli BL21-pETDuet-1-GDH-L-CpCR. (a) pH; (b) Substrate concentration; (c) Metal ions; (d) Glucose concentration; (e) Temperature; (f) Time; (g) Cosolvent; (h) Cosolvent concentration

Fig. 12

Time course of batch and fed-batch bioconversion of OPBE to (R)-HPBE by the strain E. coli BL21-pETDuet-1-GDH-L-CpCR. (a) Batch bioconversion; (b) Fed-batch bioconversion