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Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity

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Abstract

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40°C and 7.5–8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0–7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.

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Acknowledgments

This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science and Technology, Republic of Korea and the 2009 Research Fund of the Catholic University of Korea.

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Authors and Affiliations

  1. Division of Biotechnology, The Catholic University of Korea, Bucheon, 420-743, Republic of Korea

    Yun Hee Choi, Hye Jeong Choi & Hyung-Kwoun Kim

  2. Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 305-806, Republic of Korea

    Dooil Kim

  3. Equispharm Ltd., Daejeon, 305-811, Republic of Korea

    Ki-Nam Uhm

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  1. Yun Hee Choi
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  2. Hye Jeong Choi
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  3. Dooil Kim
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Correspondence to Hyung-Kwoun Kim.

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Supplementary data 1

SDS–PAGE of expressed microbial reductases. Recombinant enzymes were expressed in E. coli BL21 (DE3) cells, and each of the soluble (S) and insoluble (P) fractions was analyzed. Arrows indicate the expected protein bands (PPT 434 kb)

Supplementary data 2

Amino acid sequence and secondary structure of YOL151W aligned with those of SSCR. Blue arrows and orange bars indicate β-strands and α-helices, respectively (PPT 246 kb)

Supplementary data 3

3D model of YOL151W (green) superimposed on the X-ray crystal structure of SSCR (magenta) (PPT 226 kb)

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Choi, Y.H., Choi, H.J., Kim, D. et al. Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity. Appl Microbiol Biotechnol 87, 185–193 (2010). https://doi.org/10.1007/s00253-010-2442-5

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  • DOI: https://doi.org/10.1007/s00253-010-2442-5

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