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.