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

Abstract As a key synthetic intermediate of the cardiovascular drug diltiazem, methyl (2 R ,3 S )‐3‐(4‐methoxyphenyl) glycidate ((2 R ,3 S )‐MPGM) ( 1 ) is accessible via the ring closure of chlorohydrin (3 S )‐methyl 2‐chloro‐3‐hydroxy‐3‐(4‐methoxyphenyl)propanoate ((3 S )‐ 2 ). We report the efficient reduction of methyl 2‐chloro‐3‐(4‐methoxyphenyl)‐3‐oxo‐propanoate ( 3 ) to (3 S )‐ 2 using an engineered enzyme SSCR M2 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‐SSCR M2 /pACYCDuet‐GDH), co‐expressing SSCR M2 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 (3 S )‐ 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 (3 S )‐ 2 reported to date. The E‐factor of this biocatalytic synthesis was 24.5 (including water). Chiral alcohol (3 S )‐ 2 generated in this atom‐economic synthesis was transformed to (2 R ,3 S )‐MPGM in 95% yield with 99% ee.