Enhanced activity and substrate tolerance of 7α-hydroxysteroid dehydrogenase by directed evolution for 7-ketolithocholic acid production
- 241 Downloads
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.
Keywords7-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.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 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. https://doi.org/10.1016/j.enzmictec.2016.12.006 CrossRefGoogle Scholar
- Bennett MJ, McKnight SL, Coleman JP (2003) Cloning and characterization of the NAD-dependent 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis. Curr Microbiol 47(6). https://doi.org/10.1007/s00284-003-4079-4
- 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. https://doi.org/10.1007/s00253-011-3798-x CrossRefGoogle Scholar
- 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. https://doi.org/10.1002/bit.20753 CrossRefPubMedGoogle Scholar
- 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
- 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. https://doi.org/10.1016/j.steroids.2008.06.013 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1038/srep22885 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.steroids.2005.10.002 CrossRefPubMedGoogle Scholar
- 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