Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate.

tert -Butyl (3 R ,5 S ) - 6-chloro-3,5-dihydroxyhexanoate ((3 R ,5 S )-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3 R ,5 S )-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies ( k cat/ K m) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward ( S ) - 6-chloro-5-hydroxy-3-oxohexanoate (( S )-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L −1 of ( S )-CHOH to produce (3 R ,5 S )-CDHH with >99% yield and >99% e.e. , and the highest space-time yield achieved at 752.76 mmol L −1 h −1  g −1 wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3 R ,5 S )-CDHH by carbonyl reductase. [ABSTRACT FROM AUTHOR]