Cloning, Expression Of Carbonyl Reductase And Its Application In Synthesis Of Statin-type Drugs

Carbonyl reductase or alcohol dehydrogenases can catalyze the inter-conversion of aldehydes and alcohols. The t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate is a key chiral intermediate in the synthesis of statin-type drugs such as Crestor (rosuvastatin calcium) and Lipitor (atorvastatin). Herein, a novel carbonyl reductase (named as KleADH) discovered from Klebsiella oxytoca by a genome mining method was cloned and characterized. The KleADH was functionally overexpressed in E. coli Rosetta (DE3) and the recombinant KleADH showed a single band of approximately 30 kDa on the SDS-PAGE gel.The whole cell biocatalyst was able to convert t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate to t-butyl 6-chloro-(3R, 5S)-dihydroxyhexanoate with more than 99% diastereomeric excess (de) and 99% yield in 24h without adding any expensive cofactors.Homology model of KleADH was constructed using Discovery Studio 2.5,then modeled KleADH docked with coenzyme and t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate. We get the 3D structure of KleADH and to validate the catalytic tetrad, we performed an alanine scan for the four residues Asp44, Tyr49, Lys74 and His 107. The mutants were transformed and expressed in E. coli Transetta. As we expected, the reductase activities of all the mutants were totally lost. These results confirmed that Asp44, Tyr49, Lys74, and His 107 are indeed involved in the catalytic tetrad.A systematic study of all factors influencing the whole cell catalyst activity such as temperature, pH, the effects of metal ions and organic solvent were determined. The optimum enzyme activity was achieved at 30? and pH 7.0 and it was shown that 0.1 mM Mg2+ can increase the enzyme activity by 1.45 times. N-hexane/water and n-heptane/water biphasic systems can also increase the activity of KleADH. Besides t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate, substrate specificity studies showed that KleADH also exhibited notable activity towards several aryl ketones with high stereoselectivity. Our investigations on this novel carbonyl reductase KleADH offers a promising biocatalyst for producing chiral alcohols for the preparation of valuable pharmaceuticals chemicals.