Sustainable asymmetric synthesis of diltiazem precursor enabled by recombinant Escherichia coli whole cells co‐expressing an engineered ketoreductase and glucose dehydrogenase

Author:

Yue Xiaoping123,Li Yitong123,Yang Lin4,Sang Di12,Huang Zedu12,Chen Fener123ORCID

Affiliation:

1. Engineering Center of Catalysis and Synthesis for Chiral Molecules Department of Chemistry Fudan University Shanghai P. R. China

2. Shanghai Engineering Research Center of Industrial Asymmetric Catalysis of Chiral Drugs Shanghai P. R. China

3. Sichuan Research Center for Drug Precision Industrial Technology West China School of Pharmacy Sichuan University Chengdu P. R. China

4. School of Health Jiangxi Normal University Nanchang P. R. China

Abstract

AbstractAs a key synthetic intermediate of the cardiovascular drug diltiazem, methyl (2R,3S)‐3‐(4‐methoxyphenyl) glycidate ((2R,3S)‐MPGM) (1) is accessible via the ring closure of chlorohydrin (3S)‐methyl 2‐chloro‐3‐hydroxy‐3‐(4‐methoxyphenyl)propanoate ((3S)‐2). We report the efficient reduction of methyl 2‐chloro‐3‐(4‐methoxyphenyl)‐3‐oxo‐propanoate (3) to (3S)‐2 using an engineered enzyme SSCRM2 possessing 4.5‐fold improved specific activity, which was obtained through the structure‐guided site‐saturation mutagenesis of the ketoreductase SSCR by reliving steric hindrance and undesired interactions. With the combined use of the co‐expression fine‐tuning strategy, a recombinant E. coli (pET28a‐RBS‐SSCRM2/pACYCDuet‐GDH), co‐expressing SSCRM2 and glucose dehydrogenase, was constructed and optimized for protein expression. After optimizing the reaction conditions, whole‐cell‐catalyzed complete reduction of industrially relevant 300 g L−1 of 3 was realized, affording (3S)‐2 with 99% ee and a space‐time yield of 519.1 g∙L−1∙d−1, representing the highest record for the biocatalytic synthesis of (3S)‐2 reported to date. The E‐factor of this biocatalytic synthesis was 24.5 (including water). Chiral alcohol (3S)‐2 generated in this atom‐economic synthesis was transformed to (2R,3S)‐MPGM in 95% yield with 99% ee.

Funder

National Natural Science Foundation of China

National Key Research and Development Program of China

Publisher

Wiley

Subject

Molecular Medicine,Applied Microbiology and Biotechnology,General Medicine

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