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Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst

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Abstract

A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.

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Acknowledgements

This work was supported by the Major Basic Research Program of China (Grant No. 2009CB724700), National Key Technology RD Program (Grant No. 2008BAI63B07). Qi YE was supported by the Innovation Fund for Doctoral Dissertation of Nanjing University of Technology (BSCX200911) and the Program (KJ103736).

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Author notes

  1. Hanjie Ying

    Present address: College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Xin mofan Road 5, Nanjing, 210009, People’s Republic of China

Authors and Affiliations

  1. State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, 210009, People’s Republic of China

    Qi Ye, Hou Cao, Pingkai Ouyang & Hanjie Ying

  2. College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 210009, People’s Republic of China

    Qi Ye, Hou Cao, Lan Mi, Ming Yan, Yan Wang, Ximu Li, Jian Li, Lin Xu, Pingkai Ouyang & Hanjie Ying

  3. National Engineering Technique Research Center for biotechnology, Nanjing, 211816, People’s Republic of China

    Qi Ye, Yueyuan Zhang, Jian Xiong, Pingkai Ouyang & Hanjie Ying

  4. National Center for Quality Supervision and Test of Chinese Liquor, Suqian, 223800, People’s Republic of China

    Qi Ye & Guanglou Zang

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  1. Qi Ye
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  2. Hou Cao
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  3. Guanglou Zang
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Corresponding author

Correspondence to Hanjie Ying.

Additional information

Qi Ye, Hou Cao, and Lan Mi have equally contributed to this study.

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Ye, Q., Cao, H., Zang, G. et al. Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst. Appl Microbiol Biotechnol 88, 1277–1285 (2010). https://doi.org/10.1007/s00253-010-2836-4

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