Engineered ketoreductase-catalyzed stereoselective reduction of ethyl 2′-ketopantothenate and its analogues: chemoenzymatic synthesis of d-pantothenic acid

Abstract

D-Pantothenic acid (D-PaA) is an essential vitamin for human beings, and the global market value of calcium pantothenate, the commercial form of D-PaA, has been estimated to be over one billion US dollars. Therefore, development of a stereoselective, green, and cost-effective synthesis of D-PaA (calcium pantothenate) is highly desirable. In the present work, a chemoenzymatic synthesis route to D-PaA was devised and executed, in which ketoreductase (KRED)-overexpressing recombinant E. coli strain-catalyzed reduction of ethyl 2′-ketopantothenate (K-PaOEt) to ethyl (R)-pantothenate ((R)-PaOEt) served as the key transformation and has been accomplished for the first time, to the best of our knowledge. Enzyme screening, followed by structure-guided semi-rational protein engineering, including alanine-scanning and site-saturation mutagenesis, resulted in an efficient mutant enzyme designated as M3, possessing three point mutations of F97L, S173I, and P243L, and displaying a 2.3-fold enhanced specific activity (249.1 U mg⁻¹) and a 5.2-fold improved catalytic efficiency (27.08 s⁻¹ mM⁻¹) compared with the wild-type ketoreductase SSCR. After constructing and evaluating seven recombinant E. coli strains co-expressing M3 and glucose dehydrogenase (GDH), and further optimizing the reaction conditions, complete reduction of 100 g L⁻¹ of K-PaOEt at a gram-scale was realized, without the cumbersome operation of (semi)continuous feeding, by using the wet whole-cells of E. coli (pET28a-M3/pACYCDuet-1-GDH) as biocatalysts, delivering the desired (R)-PaOEt in 86% isolated yield with >99% ee. The current biocatalytic synthesis is characteristic of an excellent space–time yield of 520 g L⁻¹ d⁻¹ and a decent E-factor of 58 (including water). The subsequent conversion of (R)-PaOEt to D-PaA succeeded with 97% yield. Finally, the synthesis potential of E. coli (pET28a-M3/pACYCDuet-1-GDH) was showcased by the successful preparation of ten structural analogues of (R)-PaOEt with (potential) bioactivity with 50–82% isolated yields and perfect stereoselectivities (all >99% ee or >99% de). The current study not only demonstrates an efficient and green synthesis of (R)-PaOEt with an excellent level of stereocontrol, but also sets a solid basis for establishing a sustainable, cost-effective, and industrially applicable production avenue for D-pantothenic acid.