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

作　　者：Yanmei Dai Jinmei Wang Zijuan Tao Liangli Luo Changshun Huang Bo Liu Hanbing Shi Lan Tang Zhimin Ou 

机构地区：[1]College of Pharmaceutical Science,Zhejiang University of Technology,Hangzhou 310014,China [2]College of Biological&Environmental Sciences,Zhejiang Wanli University,Ningbo 315199,China [3]Department of Respiratory Medicine,The Third Affiliated Hospital of Qiqihar Medical College,Qiqihar,China

出　　处：《Bioresources and Bioprocessing》2024年第1期1291-1310,共20页生物资源与生物加工(英文)

基　　金：supported by the Zhejiang Provincial Science and Technology Plan Project(2024C03014);the National Nature Science Foundation of China(Project Nos.21978267,and 22078300);as well as the Natural Science Foundation of Heilongjiang Province(LH2022H107).

摘　　要：(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.

关 键 词：Carbonyl reductase Bi-enzyme coupled Fusion-expression High-density fermentation Substrate feeding strategy 

分 类 号：O62[理学—有机化学]