Expression Of AdhR In Escherichia Coli For The Production Of Intermediate For Imipenem Drug

(2S,3R)-methyl-2-benzamidomethyl-3-hydroxybutyrate ((2S,3R)-BHBM) is an important chiral intermediate, which can be further used to synthetize carbapenem and penem antibiotics, such as imipenem, faropenem, meropenem, etc. Asymmetric hydrogenation of racemic methyl-2-benzamidomethyl-3-oxobutyrate (BOBM) to the optically active 2-benzamidomethyl-3-hydroxybutyrate catalyzed by biological method has been shown to be an effective way.In this study, the NADPH-dependent carbonyl reductase AdhR from Lactobacill-us kefiri was discovered by screening, which can convert BOBM to (2S,3R)-BHBM, with the optical purity of >99% (enantiometric efficiency). Then AdhR was cloned and expressed in different expression systems, and the protein expression and catalytic activity of each recombination strain were studied to pick out the best strain E.coli BL21(DE3)/pET30a(+)-AdhR, with the enzyme activity of 12.23U/mL. Then the medium and induction conditions were optimized and the OD600 of 7.18, the enzyme activity of 38.65U/mL were obtained. After that, the batch and the fed-batch fermentation were performed in a 10L fermentor. Finally the cell density(dry weight) of 41.5 g/L and enzymatic activity of 221 U/mL was achieved after fed-batch fermentation, which was 18 times higher than that of LB medium.Asymmetric reduction of BOBM to synthetize (2S,3R)-BHBM need to add NADPH as co-enzyme. In order to solve co-enzyme NADPH regeneration during reaction, glucose dehydrogenase was over expressed and was used to regenerate NADPH. After the comparision of several strategies of enzymatic regeneration of co-enzyme, the two-strain coupled system of E.coli BL21(DE3)/pET30a(+)-AdhR and E.coli BL2(DE3)/pET30a(+)-GDHBM was used in the asymmetric biotransformation of BOBM to (2S,3R)-BHBM.Finally, the catalytic processes by the two-strain coupled system was optimized, the optically active (2S,3R)-BHBM can be efficiently prepared by this coupling system. Under the optimal conditions,450g/L BOBM was completely reducted to (2S,3R)-BHBM, when the cell concentration of E.coli BL21(DE3)/pET30a(+)-AdhR was 7.78gDCW/L, the cell ratio was 1:1 and the NADP+concentration was 0.2mmol/L After reaction in 30℃ for 48h, the yield of >95%, the space-time yield of 9.375g/L·h, the optical purity of >99% was obtained, and the total turnover number(TTN) was 8671, which was better than the literature reports. These results indicate the potential of this two-strain coupled system for industrial production of (2S,3R)-BHBM.