Abstract
Both Sys1 and Sygdh, two codon-optimized genes encoding SyS1 and SyGDH, were synthesized based on the carbonyl reductase (S1) and glucose 1-dehydrogenase (GDH) gene sequences, respectively, from Candida magnoliae and Thermoplasma acidophilum, and co-expressed in Escherichia coli BL21(DE3) using two strategies. One strategy involved a recombinant E. coli strain (E. coli/Sygdh-Sys1) constructed by transforming a recombinant plasmid, pETDuet-Sygdh-Sys1, into E. coli BL21. The other strategy involved another recombinant E. coli strain (E. coli/Sys1/Sygdh) obtained by co-transforming the recombinant plasmids pET-22b-Sys1 and pET-28a-Sygdh into E. coli BL21. The enzyme activity assays indicated that the activities of recombinant SyS1 and SyGDH (3.7 and 56.3 U/g wet cells) expressed in E. coli/Sygdh-Sys1 were higher than those (2.8 and 44.1 U/g wet cells) in E. coli/Sys1/Sygdh. Accordingly, E. coli/Sygdh-Sys1 was chosen, and its whole cells were used as catalysts for the asymmetric reduction of m-chlorophenacyl chloride (m-CPC) to the corresponding (R)-2-chloro-1-(3-chlorophenyl)ethanol [(R)-CCE] coupled with the regeneration of NADPH in situ. Under the optimized reaction conditions of 30 mM m-CPC, 50 mg/ml wet cells, 40 mM glucose and 0.2 mM NADP+ at pH 7.0 and 35 °C for 3 h, (R)-CCE was obtained with a molar yield of 99.2 % and an enantiomeric excess (e.e.) value of more than 99 %.
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Acknowledgments
This work was financially supported by the National Nature Science Foundation of China (No. 31271811), Fundamental Research Funds for the Central Universities of China (JUSRP51412B), and Postgraduate Innovation Training Project of Jiangsu, China (No. CXZZ13_0757). We are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.
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Tao Yu and Jian-Fang Li, the two first authors, contributed equally to this work.
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Yu, T., Li, JF., Zhu, LJ. et al. Reduction of m-chlorophenacyl chloride coupled with regeneration of NADPH by recombinant Escherichia coli cells co-expressing both carbonyl reductase and glucose 1-dehydrogenase. Ann Microbiol 66, 343–350 (2016). https://doi.org/10.1007/s13213-015-1114-1
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DOI: https://doi.org/10.1007/s13213-015-1114-1