Laboratory of Biocatalysis and Bioprocessing, College of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou 213164, PR China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
Laboratory of Biocatalysis and Bioprocessing, College of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou 213164, PR China.
Bioresour Technol. 2014 Jun;161:461-4. doi: 10.1016/j.biortech.2014.03.133. Epub 2014 Apr 2.
An NADH-dependent reductase (CmCR) from Candida magnoliae was discovered by genome mining for carbonyl reductases. After CmCR was overexpressed in Escherichia coli BL21, a robust reductase-producing strain, recombinant E. coli CCZU-K14, was employed for the efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) from the reduction of ethyl 4-chloro-3-oxobutanoate (COBE). After the optimization, the optimum reaction conditions were obtained. Notably, E. coli CCZU-K14 had broad substrate specificity in reducing both aliphatic and aromatic substrates, and excellent enantioselectivity of CCZU-K14 was observed for most of the tested substrates, resulting in chiral alcohols of over 99.9% ee. Moreover, COBE at a high concentration of (3000mM) could be asymmetrically reduced to (S)-CHBE in the high yield (>99.0%) and high enantiometric excess value (>99.9% ee) after 14h. Significantly, E. coli CCZU-K14 shows high potential in the industrial production of (S)-CHBE and its derivatives (>99.9% ee).
通过对羰基还原酶的基因组挖掘，发现了一种来自假丝酵母的 NADH 依赖性还原酶（CmCR）。在大肠杆菌 BL21 中过表达 CmCR 后，构建了一株高效表达还原酶的重组大肠杆菌 CCZU-K14，用于从乙基 4-氯-3-氧代丁酸酯（COBE）还原高效合成（S）-4-氯-3-羟基丁酸乙酯（（S）-CHBE）。经过优化，得到了最佳反应条件。值得注意的是，大肠杆菌 CCZU-K14 在还原脂肪族和芳香族底物时具有广泛的底物特异性，并且对大多数测试的底物都表现出出色的对映选择性，导致手性醇的对映体过量值超过 99.9%ee。此外，在 14 小时后，高浓度（3000mM）的 COBE 可以不对称还原为（S）-CHBE，产率大于 99.0%，对映体过量值大于 99.9%ee。重要的是，大肠杆菌 CCZU-K14 在（S）-CHBE 及其衍生物的工业生产中具有很大的应用潜力（>99.9%ee）。
