Applied Microbiology and Biotechnology

, Volume 97, Issue 2, pp 633–639 | Cite as

One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multienzymatic system

  • Luo Liu
  • Michael Braun
  • Gabi Gebhardt
  • Dirk Weuster-Botz
  • Ralf Gross
  • Rolf D. SchmidEmail author
Biotechnological Products and Process Engineering


12-ketoursodeoxycholic acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of dehydrocholic acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.


Cofactor regeneration Dehydrocholic acid Formate dehydrogenase-7β-hydroxysteroid dehydrogenase 12-keto ursodeoxycholic acid Ursodeoxycholic acid 



We thank the BMBF (German Federal Ministry of Education and Research), Grant-No. FKZ 0315269, for financial support.


  1. Bovara R, Carrea G, Riva S, Secundo F (1996) A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid. Biotechnol Lett 18(3):305–308CrossRefGoogle Scholar
  2. Braun M, Lünsdorf H, Bückmann AF (1991) 12α-hydroxysteroid dehydrogenase from Clostridium group P, strain C 48-50. Production, purification and characterization. Eur J Biochem 196(2):439–450CrossRefGoogle Scholar
  3. Braun M, Link H, Liu L, Schmid RD, Weuster-Botz D (2011) Biocatalytic process optimization based on mechanistic modeling of cholic acid oxidation with cofactor regeneration. Biotechnol Bioeng 108(6):1307–1317CrossRefGoogle Scholar
  4. Carrea G, Bovara R, Longhi R, Barani R (1984) Enzymatic reduction of dehydrocholic acid to 12-ketochenodeoxycholic acid with NADH regeneration. Enzym Microb Tech 6(7):307–311CrossRefGoogle Scholar
  5. Colombo C, Setchell KD, Podda M, Crosignani A, Roda A, Curcio L, Ronchi M, Giunta A (1990) Effects of ursodeoxycholic acid therapy for liver disease associated with cystic fibrosis. J Pediatr 117(3):482–489CrossRefGoogle Scholar
  6. Combes B, Carithers RLJ, Maddrey WC, Lin D, McDonald MF, Wheeler DE, Eigenbrodt EH, Muñoz SJ, Rubin R, Garcia-Tsao G, Santiago J, Bonner GF, West AB, Boyer JL, Luketic VA, Shiffman ML, Mills AS, Peters MG, White HM, Zetterman RK, Rossi SS, Hofmann AF, Markin RS (1995) A randomized, double-blind, placebo-controlled trial of ursodeoxycholic acid in primary biliary cirrhosis. Hepatol 22(3):759–766Google Scholar
  7. Cravotto G, Binello A, Boffa L, Rosati O, Boccalini M, Chimichi S (2006) Regio- and stereoselective reductions of dehydrocholic acid. Steroids 71(6):469–475CrossRefGoogle Scholar
  8. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci 97(12):6640–6645CrossRefGoogle Scholar
  9. Fossati E, Polentini F, Carrea G, Riva S (2006) Exploitation of the alcohol dehydrogenase-acetone NADP-regeneration system for the enzymatic preparative-scale production of 12-ketochenodeoxycholic acid. Biotechnol Bioeng 93(6):1216–1220CrossRefGoogle Scholar
  10. Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci 100(4):1541–1546CrossRefGoogle Scholar
  11. Hinkley DF, Plainfield, Singleton B, Elizabeth and NJ (1960) Oxidation of cholic acid with chlorine, In US Patent No. 2,966,499 USAGoogle Scholar
  12. Hölsch K, Weuster-Botz D (2010) Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins. Biotechnol Appl Biochem 56(4):131–140CrossRefGoogle Scholar
  13. Huang M (1949) Reduction of steroid ketones and other carbonyl compounds by modified Wolff–Kishner method. J Am Chem Soc 71(10):3301–3303CrossRefGoogle Scholar
  14. Im E, Martinez JD (2004) Ursodeoxycholic acid (UDCA) can inhibit deoxycholic acid (DCA)-induced apoptosis via modulation of EGFR/Raf-1/ERK signaling in human colon cancer cells. J Nutr 134(2):483–486Google Scholar
  15. Jörnvall H, Persson B, Krook M, Atrian S, Gonzalez-Duarte R, Jeffery J, Ghosh D (1995) Short-chain dehydrogenases/reductases (SDR). Biochem 34(18):6003–6013CrossRefGoogle Scholar
  16. Kanazawa T, Shimazaki A, Sato T, Hoshino T (1955) Study on the ursodeoxycholic acid synthesis. Nippon Kagaku Zasshi 76:297–301CrossRefGoogle Scholar
  17. Khare S, Cerda S, Wali RK, von Lintig FC, Tretiakova M, Joseph L, Stoiber D, Cohen G, Nimmagadda K, Hart J, Sitrin MD, Boss GR, Bissonnette M (2003) Ursodeoxycholic acid inhibits Ras mutations, wild-type Ras activation, and cyclooxygenase-2 expression in colon cancer. Cancer Res 63(13):3517–3523Google Scholar
  18. Liu L, Aigner A, Schmid RD (2011) Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7beta-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Appl Microbiol Biotechnol 90(1):127–135CrossRefGoogle Scholar
  19. MacDonald IA, Williams CN, Mahony DE (1973) 7α-hydroxysteroid dehydrogenase from Escherichia coli B: preliminary studies. Biochim Biophys Acta 309(2):243–253CrossRefGoogle Scholar
  20. Makino I, Shinozaki K, Yoshino K, Nakagawa S (1975) Dissolution of cholesterol gallstones by long-term administration of ursodeoxycholic acid. Nippon Shokakibyo Gakkai Zasshi 72(6):690–702Google Scholar
  21. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60(3):512–538Google Scholar
  22. Mobus E, Maser E (1998) Molecular cloning, overexpression, and characterization of steroid-inducible 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. A novel member of the short-chain dehydrogenase/reductase superfamily. J Biol Chem 273(47):30888–30896CrossRefGoogle Scholar
  23. Monti D, Ferrandi E, Zanellato I, Hua L, Polentini F, Carrea G, Riva S (2009) One-pot multienzymatic synthesis of 12-ketoursodeoxycholic acid: subtle cofactor specificities rule the reaction equilibria of five biocatalysts working in a row. Adv Synth Catal 351(9):1303–1311CrossRefGoogle Scholar
  24. Salen G, Colalillo A, Verga D, Bagan E, Tint GS, Shefer S (1980) Effect of high and low doses of ursodeoxycholic acid on gallstone dissolution in humans. Gastroenterol 78(6):1412–1418Google Scholar
  25. Sawada H, Kulprecha S, Nilubol N, Yoshida T, Kinoshita S, Taguchi H (1982) Microbial production of ursodeoxycholic acid from lithocholic acid by Fusarium equiseti M41. Appl Environ Microbiol 44(6):1249–1252Google Scholar
  26. Shoda M (1927) Über die Ursodeoxycholsäure aus Bärengallen und ihre physiologische Wirkung. J Biochem 7:505–517Google Scholar
  27. Stiehl A, Czygan P, Kommerell B, Weis HJ, Holtermuller KH (1978) Ursodeoxycholic acid versus chenodeoxycholic acid. Comparison of their effects on bile acid and bile lipid composition in patients with cholesterol gallstones. Gastroenterol 75(6):1016–1020Google Scholar
  28. Sutherland JD, MacDonald IA, Forrest TP (1982) The enzymic and chemical synthesis of ursodeoxycholic and chenodeoxycholic acid from cholic acid. Prep Biochem 12(4):307–321CrossRefGoogle Scholar
  29. Tishkov VI, Popov VO (2006) Protein engineering of formate dehydrogenase. Biomol Eng 23(2–3):89–110CrossRefGoogle Scholar
  30. Yoshimoto T, Higashi H, Kanatani A, Lin XS, Nagai H, Oyama H, Kurazono K, Tsuru D (1991) Cloning and sequencing of the 7α-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme. J Bacteriol 173(7):2173–2179Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Luo Liu
    • 1
    • 4
  • Michael Braun
    • 2
  • Gabi Gebhardt
    • 2
  • Dirk Weuster-Botz
    • 2
  • Ralf Gross
    • 3
  • Rolf D. Schmid
    • 1
    • 5
    Email author
  1. 1.Institute of Technical BiochemistryUniversity of StuttgartStuttgartGermany
  2. 2.Institute of Biochemical EngineeringTechnische Universität MünchenGarchingGermany
  3. 3.PharmaZell GmbHRaublingGermany
  4. 4.Beijing Key Laboratory of Bioprocess, College of Life Science and TechnologyBeijing University of Chemical TechnologyBeijingPeople’s Republic of China
  5. 5.StuttgartGermany

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