Applied Microbiology and Biotechnology

, Volume 103, Issue 6, pp 2665–2674 | Cite as

Enhanced activity and substrate tolerance of 7α-hydroxysteroid dehydrogenase by directed evolution for 7-ketolithocholic acid production

  • Bin HuangEmail author
  • Qiang Zhao
  • Jing-hui Zhou
  • Gang Xu
Biotechnologically relevant enzymes and proteins


7-Ketolithocholic acid (7-KLCA) is an important intermediate for the synthesis of ursodeoxycholic acid (UDCA). UDCA is the main effective component of bear bile powder that is used in traditional Chinese medicine for the treatment of human cholesterol gallstones. 7α-Hydroxysteroid dehydrogenase (7α-HSDH) is the key enzyme used in the industrial production of 7-KLCA. Unfortunately, the natural 7α-HSDHs reported have difficulty meeting the requirements of industrial application, due to their poor activities and strong substrate inhibition. In this study, a directed evolution strategy combined with high-throughput screening was applied to improve the catalytic efficiency and tolerance of high substrate concentrations of NADP+-dependent 7α-HSDH from Clostridium absonum. Compared with the wild type, the best mutant (7α-3) showed 5.5-fold higher specific activity and exhibited 10-fold higher and 14-fold higher catalytic efficiency toward chenodeoxycholic acid (CDCA) and NADP+, respectively. Moreover, 7α-3 also displayed significantly enhanced tolerance in the presence of high concentrations of substrate compared to the wild type. Owing to its improved catalytic efficiency and enhanced substrate tolerance, 7α-3 could efficiently biosynthesize 7-KLCA with a substrate loading of 100 mM, resulting in 99% yield of 7-KLCA at 2 h, in contrast to only 85% yield of 7-KLCA achieved for the wild type at 16 h.


7-Ketolithocholic acid 7α-Hydroxysteroid dehydrogenase Directed evolution Substrate tolerance NADP+ regeneration Biosynthesis 



This work was supported by the National Science and Technology Major Special Independent Project of China (No. 2017ZX07402003). We are grateful to Prof. Bochu Wang from Chongqing University, for providing us with the wild-type CA 7α-HSDH gene.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9668_MOESM1_ESM.pdf (538 kb)
ESM 1 (PDF 537 kb)


  1. Bakonyi D, Hummel W (2017) Cloning, expression, and biochemical characterization of a novel NADP(+)-dependent 7alpha-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids. Enzym Microb Technol 99:16–24. CrossRefGoogle Scholar
  2. Baron S, Franklund C, Hylemon P (1991) Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708. J Bacteriol 173(15):4558–4569CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bennett MJ, McKnight SL, Coleman JP (2003) Cloning and characterization of the NAD-dependent 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis. Curr Microbiol 47(6).
  4. Carrea G, Pilotti A, Riva S, Canzi E, Ferrari A (1992) Enzymatic synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid in a membrane reactor. Biotechnol Lett 14(12):1131–1134CrossRefGoogle Scholar
  5. Carter JL, Bekhouche M, Noiriel A, Blum LJ, Doumeche B (2014) Directed evolution of a formate dehydrogenase for increased tolerance to ionic liquids reveals a new site for increasing the stability. Chembiochem 15(18):2710–2718. CrossRefPubMedGoogle Scholar
  6. Eggert T, Bakonyi D, Hummel W (2014) Enzymatic routes for the synthesis of ursodeoxycholic acid. J Biotechnol 191:11–21. CrossRefPubMedGoogle Scholar
  7. Ferrandi EE, Bertolesi GM, Polentini F, Negri A, Riva S, Monti D (2012) In search of sustainable chemical processes cloning, recombinant expression, and functional characterization of the 7α- and 7β-hydroxysteroid dehydrogenases from Clostridium absonum. Appl Micrbiol Biotechnol 95(5):1221–1233. CrossRefGoogle Scholar
  8. 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):1216–1220. CrossRefPubMedGoogle Scholar
  9. Giacomo C, Roberto B, Renato L, Sergio R (1985) Preparation of 12-ketochenodeoxycholic acid from cholic acid using coimmobilized 12α-hydroxysteroid dehydrogenase and glutamate dehydrogenase with NADP+ cycling at high efficiency. Enzym Microb Technol 7(12):4Google Scholar
  10. Giovannini PP, Grandini A, Perrone D, Pedrini P, Fantin G, Fogagnolo M (2008) 7α- and 12α-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid. Steroids 73(14):1385–1390. CrossRefPubMedGoogle Scholar
  11. Ji W, Chen Y, Zhang H, Zhang X, Li Z, Yu Y (2014) Cloning, expression and characterization of a putative 7alpha-hydroxysteroid dehydrogenase in Comamonas testosteroni. Microbiol Res 169(2–3):148–154. CrossRefPubMedGoogle Scholar
  12. Johannes TW, Woodyer RD, Zhao H (2005) Directed evolution of a thermostable phosphite dehydrogenase for NAD(P)H regeneration. Appl Environ Microbiol 71(10):5728–5734. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kim JH, Choi GS, Kim SB, Kim WH, Lee JY, Ryu YW, Kim GJ (2004) Enhanced thermostability and tolerance of high substrate concentration of an esterase by directed evolution. J Mol Catal B Enzym 27(4–6):169–175. CrossRefGoogle Scholar
  14. Li W, Xu S, Zhang B, Zhu Y, Hua Y, Kong X, Sun L, Hong J (2017) Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis. PLoS One 12(5):e0177877. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Lou D, Wang B, Tan J, Zhu L (2014) Carboxyl-terminal and Arg38 are essential for activity of the 7α-hydroxysteroid dehydrogenase from Clostridium absonum. Protein Peptide Lett 21(9):894–900CrossRefGoogle Scholar
  16. Lou D, Wang B, Tan J, Zhu L, Cen X, Ji Q, Wang Y (2016) The three-dimensional structure of Clostridium absonum 7α-hydroxysteroid dehydrogenase: new insights into the conserved arginines for NADP(H) recognition. Sci Rep 6:22885. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Medici A, Pedrini P, Bianchini E, Fantina G, Guerrinia A, Natalinib B, Pellicciarib R (2002) 7α-OH epimerisation of bile acids via oxido-reduction with Xanthomonas maltophilia. Steroids 67(1):51–56CrossRefPubMedGoogle Scholar
  18. Miyazaki K, Takenouchi M (2002) Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. BioTechniques 33(5):1033–1036CrossRefPubMedGoogle Scholar
  19. Pedrini P, Andreotti E, Guerrini A, Dean M, Fantin G, Giovannini PP (2006) Xanthomonas maltophilia CBS 897.97 as a source of new 7β-and 7α-hydroxysteroid dehydrogenases and cholylglycine hydrolase: improved biotransformations of bile acids. Steroids 71(3):189–198. CrossRefPubMedGoogle Scholar
  20. Richter N, Zienert A, Hummel W (2011) A single-point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor. Eng Life Sci 11(1):26–36. CrossRefGoogle Scholar
  21. Shi J, Wang J, Yu L, Yang L, Zhao S, Wang Z (2017) Rapidly directional biotransformation of tauroursodeoxycholic acid through engineered Escherichia coli. J Ind Microbiol Biotechnol 44(7):1073–1082. CrossRefPubMedGoogle Scholar
  22. Song C, Wang B, Tan J, Zhu L, Lou D (2017) Discovery of tauroursodeoxycholic acid biotransformation enzymes from the gut microbiome of black bears using metagenomics. Sci Rep 7:45495. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Tanabe T, Tanaka N, Uchikawa K, Kabashima T, Ito K, Nonaka T, Mitsui Y, Tsuru M, Yoshimoto T (1998) Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7α-hydroxysteroid dehydrogenase from Escherichia coli. J Biochem 124(3):634–641CrossRefPubMedGoogle Scholar
  24. Taylor JL, Price JE, Toney MD (2015) Directed evolution of the substrate specificity of dialkylglycine decarboxylase. Biochim Biophys Acta 1854(2):146–155. CrossRefPubMedGoogle Scholar
  25. Tian K, Tai K, Chua BJW, Li Z (2017) Directed evolution of Thermomyces lanuginosus lipase to enhance methanol tolerance for efficient production of biodiesel from waste grease. Bioresour Technol 245(Pt B):1491–1497. CrossRefPubMedGoogle Scholar
  26. Ueda S, Oda M, Imamura S, Ohnishi M (2004) Molecular and enzymatic properties of 7α-hydroxysteroid dehydrogenase from Pseudomonas sp. B0831. J Biol Macromol 4(1):33–38Google Scholar
  27. 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):2173–2179CrossRefPubMedPubMedCentralGoogle Scholar
  28. Zheng MM, Wang RF, Li CX, Xu JH (2015) Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques. Process Biochem 50(4):598–604. CrossRefGoogle Scholar
  29. Zheng MM, Chen KC, Wang RF, Li H, Li CX, Xu JH (2017) Engineering 7β-hydroxysteroid dehydrogenase for enhanced ursodeoxycholic acid production by multiobjective directed evolution. J Agric Food Chem 65(6):1178–1185. CrossRefPubMedGoogle Scholar
  30. Zheng MM, Chen FF, Li H, Li CX, Xu JH (2018) Continuous production of ursodeoxycholic acid by using two cascade reactors with co-immobilized enzymes. Chembiochem 19(4):347–353. CrossRefPubMedGoogle Scholar
  31. Zhu L, Wu Z, Jin JM, Tang SY (2016) Directed evolution of leucine dehydrogenase for improved efficiency of l-tert-leucine synthesis. Appl Micrbiol Biotechnol 100(13):5805–5813. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Hunan Flag Bio-tech Co., Ltd.ChangshaChina
  2. 2.College of Life Science and TechnologyCentral South University of Forestry and TechnologyChangshaChina

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