Development Of Biocatalytic Process For Tert-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate Employing Recombinant E.Coli Co-expressing Carbonyl Reductase And Glucose Dehydrogenase

Atorvastatin is a kind of hydroxylmethylglutaryl-CoA(HMG-CoA)reductase inhibitor,which inhibits cholesterol synthesis and has been developed to an important drug for cardiovascular diseasetreatment.In 2006,due to its outstanding efficiency and safety,atorvastatin became the top-selling hypolipidemic drug with annual sale of 13.6 billion dollars.t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is an important building block of atorvastatin.Compared with chemical synthesis,biodatalytic synthesis of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate has advantages of mild reaction conditions,environment friendly and excellent stereochemical specificity.In this thesis,we optimized the cultivation conditions for E.coli BL21(DE3)/pCDFDuet-gdh-cr-X,which was preserved in our lab.Results showed that the optimal cultivation conditions are as follows:seed age 8-10 h grown at 37 ℃;inoculum size 5.0%(V/V),culture temperature 37 ℃ and 0.50 mM IPTG supplemented at OD600 of about 0.80,induction at 28 ℃and lasted 13 h.Based on the redults from shake flask culture,we further studied the cultivation conditions for E.coli BL21(DE3)/pCDFDuet-gdh-cr-X on 5 L fermenter.After optimization of cultivation and induction condition,the biomass and volumetric activity of E.coli BL21(DE3)/pCDFDuet-gdh-cr-X were increased up to 55.67 g DCW/L and 8317.15 U/L,which was 13.57 and 7.60 times higher than those of shake flasks,respectively.We investigated the asymmetrically reduction of t-butyl 6-cyano-(5R)-hy droxy 1-3-oxohexanoate into t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate by E.coli BL21(DE3)/pCDFDuet-gdh-cr-X.Result indicated that under the condition of 28 ℃,pH 7.0 K2HPO4-KH2PO4(100.0 mM),mass ratio of glucose to substrate at 1:1(w/w),200.0 g/L of substrate was bio-reduced to t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate within 3h with 99.0%conversion and d.e.value over 99.5%by resting cells.Unexpectedly,300.0 g/L of substrate was converted to t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate in 8 h with 99.0%conversion and d.e.over 99.5%by freezed-cells.In addition,t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate was reacted to structurally stable t-butyl 6-cyano-3,5-O-i sopropylidene-(3R,5R)-dihydroxyhexanoate.Besides,the structure of 6-cyano-3,5-O-isopropylidene-(3R,5R)-dihydroxyhexanoate was elucidated by single crystal diffraction.,further confirming the structure and configuration of t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate.