t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is a key chiral diol precursor of atorvastatin calcium (Lipitor (R)). 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(p) > 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 (1)-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 (1)-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 onefifth 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 3 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. (C) 2017 American Institute of Chemical Engineers