Applied Microbiology and Biotechnology

, Volume 87, Issue 1, pp 185–193 | Cite as

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

  • Yun Hee Choi
  • Hye Jeong Choi
  • Dooil Kim
  • Ki-Nam Uhm
  • Hyung-Kwoun Kim
Biotechnologically Relevant Enzymes and Proteins


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.


Antidepressant drugs Chiral intermediate Docking model Enantioselectivity Reductase 

Supplementary material

253_2010_2442_MOESM1_ESM.ppt (434 kb)
Supplementary data 1SDS–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)
253_2010_2442_MOESM2_ESM.ppt (246 kb)
Supplementary data 2Amino 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)
253_2010_2442_MOESM3_ESM.ppt (226 kb)
Supplementary data 33D model of YOL151W (green) superimposed on the X-ray crystal structure of SSCR (magenta) (PPT 226 kb)


  1. Andrews M, Brown A, Chiva JY, Fradet D, Gordon D, Lansdell M, MacKenny M (2009) Design and optimization of selective serotonin re-uptake inhibitors with high synthetic accessibility. Part 1. Bioorg Med Chem Lett 19:2329–2332CrossRefGoogle Scholar
  2. Brautigam S, Bringer-Meyer S, Weuster-Botz D (2007) Asymmetric whole cell biotransformations in biphasic ionic liquid/water-systems by use of recombinant Escherichia coli with intracellular cofactor regeneration. Tetrahedron Asymmetr 18:1883–1887CrossRefGoogle Scholar
  3. Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM, Onufriev A, Simmerling C, Wang B, Woods RJ (2005) The Amber biomolecular simulation programs. J Comput Chem 26:1668–1688CrossRefGoogle Scholar
  4. Ema T, Yagasaki H, Okita N, Takeda M, Sakai T (2006) Asymmetric reduction of ketones using recombinant E. coli cells that produce a versatile carbonyl reductase with high enantioselectivity and broad substrate specificity. Tetrahedron 62:6143–6149CrossRefGoogle Scholar
  5. Ernst M, Kaup B, Muller M, Bringer-Meyer S, Sahm H (2005) Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase. Appl Microbiol Biotechnol 66:629–634CrossRefGoogle Scholar
  6. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GERMA, Cheeseman JR, Montgomery JA, Vreven T, Kudin KN, Burant JC, Millam JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi M, Scalmani G, Rega N, Petersson GA, Nakatsuji H, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Klene M, Li X, Knox JE, Hratchian HP, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Ayala PY, Morokuma K, Voth GA, Salvador P, Dannenberg JJ, Zakrzewski VG, Dapprich S, Daniels AD, Strain MC, Farkas O, Malick DK, Rabuck AD, Raghavachari K, Foresman JB, Ortiz JV, Cui Q, Baboul AG, Clifford S, Cioslowski J, Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Martin RL, Fox DJ, Keith T, Al-Laham MA, Peng CY, Nanayakkara A, Challacombe M, Gill PMW, Johnson B, Chen W, Wong MW, Gonzalez C, Pople JA (2004) Gaussian 03. Gaussian, Inc., WallingfordGoogle Scholar
  7. Goldberg K, Schroer K, Lütz S, Liese A (2007) Biocatalytic ketone reduction-a powerful tool for the production of chiral alcohols-part I: processes with isolated enzymes. Appl Microbiol Biotechnol 76:237–248CrossRefGoogle Scholar
  8. Huang B, Schroeder M (2008) Using protein binding site prediction to improve protein docking. Gene 422:14–21CrossRefGoogle Scholar
  9. Huey R, Morris GM, Olson AJ, Goodsell DS (2007) A semiempirical free energy force field with charge-based desolvation. J Comput Chem 28:1145–1152CrossRefGoogle Scholar
  10. Inoue K, Makino Y, Itoh N (2005) Production of (R)-chiral alcohols by a hydrogen-transfer bioreduction with NADH-dependent Leifsonia alcohol dehydrogenase (LSADH). Tetrahedron: Assymmetry 16:2539–2549CrossRefGoogle Scholar
  11. Kaluzna IA, Feske BD, Wittayanan W, Ghiviriga I, Stewart JD (2005) Stereoselective, biocatalytic reductions of α-chloro-β-keto esters. J Org Chem 70:342–345CrossRefGoogle Scholar
  12. Kamitori S, Iguchi A, Ohtaki A, Yamada M, Kita K (2005) X-ray structures of NADPH-dependent carbonyl reductase from Sporobolomyces salmonicolor provide insights into stereoselective reductions of carbonyl compounds. J Mol Biol 352:551–558CrossRefGoogle Scholar
  13. Kayser MM, Drolet M, Stewart JD (2005) Application of newly available bio-reducing agents to the synthesis of chiral hydroxy-β-lactams: model for aldose reductase selectivity. Tetrahedron Asymmetr 16:4004–4009CrossRefGoogle Scholar
  14. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26:283–291CrossRefGoogle Scholar
  15. Luthy R, Bowie UJ, Eisenberg D (1992) Assessment of protein models with three dimensional profiles. Nature 356:83–85CrossRefGoogle Scholar
  16. Makino Y, Inoue K, Dairi T, Itoh N (2005) Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol. Appl Environ Microbiol 71:4713–4720CrossRefGoogle Scholar
  17. Moore JC, Pollard DJ, Kosjek B, Devine PN (2007) Advances in the enzymatic reduction of ketones. Acc Chem Res 40:1412–1419CrossRefGoogle Scholar
  18. Pfruender H, Jones R, Weuster-Botz D (2006) Water immiscible ionic liquids as solvents for whole cell biocatalysis. J Biotechnol 124:182–190CrossRefGoogle Scholar
  19. Sali A, Blundell TL (1993) Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 234:779–815CrossRefGoogle Scholar
  20. Schroer K, Mackfeld U, Tan IAW, Wandrey C, Heuser F, Bringer-Mayer S, Weckbecker A, Hummel W, Daußmann T, Pfaller R, Liese A, Lütz S (2007) Continuous asymmetric ketone reduction processes with recombinant Escherichia coli. J Biotechnol 132:438–444CrossRefGoogle Scholar
  21. Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L (2005) The FoldX web server: an online force field. Nucleic Acids Res 33:W382–W388CrossRefGoogle Scholar
  22. Söding J, Biegert A, Lupas AN (2005) The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33:W244–W248CrossRefGoogle Scholar
  23. Vaswani M, Linda FK, Ramesh S (2003) Role of selective serotonin reuptake inhibitors in psychiatric disorders: a comprehensive review. Prog Neuropsychopharmacol Biol Psychiatry 27:85–102CrossRefGoogle Scholar
  24. Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput Chem 25:1157–1174CrossRefGoogle Scholar
  25. Xu Z, Liu Y, Fang L, Jiang X, Jing K, Cen P (2006) Construction of a two-strain system for asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydroxybutanoate ethyl ester. Appl Micobiol Biotechnol 70:40–46CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Yun Hee Choi
    • 1
  • Hye Jeong Choi
    • 1
  • Dooil Kim
    • 2
  • Ki-Nam Uhm
    • 3
  • Hyung-Kwoun Kim
    • 1
  1. 1.Division of BiotechnologyThe Catholic University of KoreaBucheonRepublic of Korea
  2. 2.Systems Microbiology Research CenterKorea Research Institute of Bioscience and BiotechnologyDaejeonRepublic of Korea
  3. 3.Equispharm Ltd.DaejeonRepublic of Korea

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