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

Abstract

(R)-2-Hydroxy-4-phenylbutyric acid ethyl ester ((R)-HPBE) represents a crucial chiral intermediate in the synthesis of angiotensin-converting enzyme (ACE) inhibitors. Its preparation entails an asymmetrical reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) with high selectivity by carbonyl reductase, a process that necessitates the regeneration of the cofactor as a pivotal aspect. The carbonyl reductase gene (CpCR) in Candida parapsilosis ATCC 7330 was successfully cloned in our laboratory. A series of recombinant engineering bacteria were constructed based on the cloned CpCR gene. These included E. coli BL21-pETDuet-1-CpCR, E. coli BL21-pET28a-CpCR and E. coli BL21-pACYCDuet-1-CpCR, which expressed the CpCR monoenzyme. To address the issue of cofactor regeneration, glucose dehydrogenase (GDH) was incorporated into the reaction system to construct a bi-enzyme cascade system, which included three co-expression recombinant engineering bacteria (E. coli BL21-pETDuet-1-CpCR/pACYCDuet-1-GDH, The following recombinant engineering bacteria were constructed: E. coli BL21-pETDuet-1-CpCR-GDH and BL21-pETDuet-1-GDH-CpCR, as well as E. coli BL21-pETDuet-1-CpCR-L-GDH and E. coli BL21-pETDuet-1-GDH-L-CpCR, which were created through fusion expression. Subsequent studies demonstrated that the fusion-expressed bi-enzyme cascade recombinant engineering bacteria E. coli BL21-pETDuet-1-GDH-L-CpCR exhibited superior carbonyl reductase activity compared to CpCR single-enzyme recombinant engineering bacteria and bi-enzyme co-expression recombinant engineering bacteria. This was evidenced by a 3-fold increase in substrate processing capacity at a reaction temperature of 30 ℃and a reaction time of 24 h. With a substrate loading of 30 mM OPBE, (R)-HPBE was achieved in 92.1% conversion with an enantiomeric excess value of 99.9%.