Enhanced diastereoselective synthesis of t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate by using aldo‐keto reductase and glucose dehydrogenase co‐producing engineered Escherichia coli

Abstract

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo-keto reductase mutant KlAKR-Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (de > 99.5%) toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co-producer E. coli BL 21(DE3) pETDuet-esgdh (MCS1)/pET-28b (+)-klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet-esgdh (MCS1)/pET-28b (+)-klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5R)-1 at substrate loading size of up to 60.0 g/L (5R)-1 in the absence of exogenous NADH, which was one-fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)-2 with productivity of 243.2 kg/m d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co-expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235-1242, 2017.

Engineering ketoreductases for the enantioselective synthesis of chiral alcohols

“…65 In contrast, co-expression of the KlAKR-Y295W/W296L variant, derived from site-saturation mutagenesis, in E. coli BL21 (DE3) together with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH), enabled the formation of 2 in 243 kg m −3 . 66 Immobilisation of reconstructed E. coli BL21 cells in Celite-polyethyleneimine (PEI)-glutaraldehyde improved the stability of the biocatalyst which could be reused 7 times giving a total of 17.5 g 2 per gram in d e p > 99.5%. 43 Zheng and Wang and coworkers used a variant of the Km AKR from Kluyveromyces marxianus , obtained following multiple rounds of directed evolution, for the efficient asymmetric reduction of 1 .…”