Screening And Application Of Carbonyl Reductase For The Synthesis Of Chiral Intermediate Of Rosuvastatin

Tert-butyl?3R,5S?-6-chloro-3,5-dihydroxyhexanoate??3R,5S?-CDHH?is one of the key chiral intermediates for the synthesis of side chain of the cholesterol-lowering drug rosuvastatin.The biosynthesis of?3R,5S?-CDHH has been drawn extensive attentions,it is very necessary to study the biosynthesis process of?3R,5S?-CDHH and to enhance its productivity.In this paper,we screened stereoselective carbonyl reductases through gene mining and laboratory preserved strains.One recombinant E.coli stain containing a stereoselective carbonyl reductase?pET-28b-SCR1?was isolated with the capability to catalyze the asymmetric reduction of?S?-CHOH,and obtained the product of?3R,5S?-CDHH with d.e.of 99%.Cultivation conditions of recombinant E.coli expressing carbonyl reductase were investigated.The optimized fermentation medium was as follows:peptone 15.0 g/L,yeast extract 12.0 g/L,NaCl 10.0 g/L,?NH₄?₂SO₄ 5 g/L,glycerol 15 g/L,KH₂PO₄ 1.36 g/L,K₂HPO₄·3H₂O 2.28g/L,MgSO₄·7H₂O 0.375 g/L and initial pH 7.0.50?g/mL kanamycin sulfate was added into medium after sterilization.The optimal fermentation conditions were as follows:fermentation temperature was 37?C,inoculum size was 10%?v/v?,10 g/L lactose was added after 2 h fermentation at 150 rpm to induce enzyme expression for 8 h at 28?C.The production of recombinant E.coli by upscale fermentations was carried out in the 50 L?500 L and 5000 L fermentors,resulting the biomass,enzyme activity and specific activity reached 12.70 g dry cell weight?DCW?/L,152104.60 U/L,11976.70 U/g DCW and 9.70 g dry cell weight?DCW?/L,152774.5 U/L,15749.95 U/g DCW and 10.97 g DCW/L,210735.07 U/L,19210.12 U/g DCW,respectively.The trends of batch fermentation for cell growth and enzyme production by different fermentors are similar.In this study,the asymmetric reduction from?S?-CHOH to?3R,5S?-CDHH was further studied,and a co-substrate coenzyme cycle was constructured.The results showed that the recombinant E.coli is efficient and robust to synthesize?3R,5S?-CDHH using?S?-CHOH with the concentration of 400 g/L in the presence of 40%isopropanol and 10g DCW/L whole cells at 30?.Under the optimal conditions,the upscale synthesis reaction was performed in 5000 L bioreactor with the substrate concentration of 400g/L,after 12 h reaction without adding exogenous NADPH,?3R,5S?-CDHH was obtained with yield of 97.5%and d.e.of 99.5%,and the space-time yield per gram of biomass reached 13.7 mM/h/g DCW,which is highest ever reported.After isolation and purification,the amber product was obtained with whole yield of 84.4%.