t-Butyl 6 cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-1b) is a valuable chiral synthon of atorvastatin calcium. A novel NADPH-specific aldo-keto reductase (AKR) was identified from a thermotolerant yeast Kluyveromyces Marxianus ZJB14056 by genome database mining, displaying t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-la) reducing activity and moderate diastereoselectivity (de(p)-similar to 80.5%). Molecular homology modeling and docking studies demonstrated that the side chain of Trp297 blocks binding of (5R)-la to KmAKR. The mutation of Trp297 to His led to dramatic conformational changes and significant improvement in both diastereoselectivity and activity. In comparison with KmAKR, KmAKR-W297H displayed strict diastereoselectivity, and 2.8-fold, 3.9-fold improvement in kcat and kat/Km toward (5R)-la, which were 10.36s-1 and 6.56 s-l-mM-1 respectively. Coupling KmAKR-W297H with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for coenzyme regeneration, 100 mM (5R)-la was completely reduced to (3R,5R)-lb within 12 h, in a de(p) > 99.5%.