Skip to main content
SpringerLink
Log in

Engineering a hydroxysteroid dehydrogenase to improve its soluble expression for the asymmetric reduction of cortisone to 11β-hydrocortisone

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

11β-Hydrocortisone (11β-HC) is an important anti-inflammatory drug and intermediate for the synthesis of other steroids. One of the methods for the synthesis of 11β-HC is the asymmetric reduction of cortisone catalyzed by a highly regioselective and stereoselective 11β-hydroxysteroid dehydrogenase (11β-HSDH). However, this process has been prohibited by the poor soluble expression of the membrane-anchoring protein 11β-HSDH in prokaryotes. To overcome this obstacle, a mutant III-1G1 (Phe80Leu/Thr105Ser/Ala260Thr/Tyr274Stop) truncated at position 274 with improved yield of soluble protein was stepwise obtained from the 11β-HSDH from guinea pig by random mutagenesis combining with structural complementation assay and C-terminal truncating library screening. The improved 11β-HSDH mutant and glucose dehydrogenase (GDH) from Bacillus subtilis were coexpressed in Escherichia coli. The resulting whole-cell biocatalyst catalyzed the reduction of cortisone to 11β-HC with 98 % conversion in 20 h, laying foundation for the development of an asymmetric reduction process for the production of 11β-HC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Chen KC, Wey HC (1990) Dissolution enzyme kinetics of 11 beta-hydroxylation of cortexolone by Curvularia lunata. Enzyme Microb Technol 12(8):616–621

    Article  CAS  Google Scholar 

  • Dlugonski J, Paraszkiewicz K, Sedlaczek L (1997) Maintenance of steroid 11-hydroxylation activity in immobilized Cunninghamella elegans protoplasts. World J Microb Biotechnol 13(4):469–473

    Article  CAS  Google Scholar 

  • Dragan CA, Zearo S, Hannemann F, Bernhardt R, Bureik M (2005) Efficient conversion of 11-deoxycortisol to cortisol (hydrocortisone) by recombinant fission yeast Schizosaccharomyces pombe. Fems Yeast Res 5(6–7):621–625

    Article  PubMed  CAS  Google Scholar 

  • Elleby B, Svensson S, Wu XQ, Stefansson K, Nilsson J, Hallen D, Oppermann U, Abrahmsen L (2004) High-level production and optimization of monodispersity of 11 beta-hydroxysteroid dehydrogenase type Ι. Biochim Biophys Acta-Proteins Proteom 1700(2):199–207

    Article  CAS  Google Scholar 

  • Fline JD, van der Waard FW (1970) Process for preparation of trihydroxy steroids. US Patent 3530038

  • Hakki T, Zearo S, Dragan CA, Bureik M, Bernhardt R (2008) Coexpression of redox partners increases the hydrocortisone (cortisol) production efficiency in CYP11B1 expressing fission yeast Schizosaccharomyces pombe. J Biotechnol 133(3):351–359

    Article  PubMed  CAS  Google Scholar 

  • Hosfield DJ, Wu YQ, Skene RJ, Hilgers M, Jennings A, Snell GP, Aertgeerts K (2005) Conformational flexibility in crystal structures of human 11 beta-hydroxysteroid dehydrogenase type I provide insights into glucocorticoid interconversion and enzyme regulation. J Biol Chem 280(6):4639–4648

    Article  PubMed  CAS  Google Scholar 

  • Jiang SM, Li CH, Zhang WW, Cai YH, Yang YL, Yang S, Jiang WH (2007) Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli. Biochem J 402(3):429–437

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Lawson AJ, Walker EA, White SA, Dafforn TR, Stewart PM, Ride JP (2009) Mutations of key hydrophobic surface residues of11beta-hydroxysteroid dehydrogenase type I increase solubility and monodispersity in a bacterial expression system. Protein Sci 18(7):1552–63

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Liu L, Aigner A, Schmid RD (2011) Identification, cloning, heterologous expression, and characterization of a NADPH-dependent 7 beta-hydroxysteroid dehydrogenase from Collinsella aerofaciens. Appl Microbiol Biotechnol 90(1):127–135

    Article  PubMed  CAS  Google Scholar 

  • Lu WY, Du LX, Wang M, Wen HP, Sun B, Guo YW (2006) Effect of two-steps substrate addition on steroids 11 beta-hydroxylation by Curvularia lunata CL-114. Biochem Eng J 32(3):233–238

    Article  CAS  Google Scholar 

  • Manosroi J, Abe M, Manosroi A (1999) Biotransformation of steroidal drugs using microorganisms screened from various sites in Chiang Mai, Thailand. Bioresource Technol 69(1):67–73

    Article  CAS  Google Scholar 

  • Naim N, Adham NZ, Abd El-Rehim H, Abd El-Hady A. (2003) Prednisolone production using Pseudomonas fluorescens cells immobilized with polymer carrier produced by radiation polymerisation.Process Biochem 38(7):1083–1089

  • Ogg D, Elleby B, Norstrom C, Stefansson K, Abrahmsen L, Oppermann U, Svensson S (2005) The crystal structure of guinea pig 11beta-hydroxysteroid dehydrogenase type I provides a model for enzyme-lipid bilayer interactions. J Biol Chem 280(5):3789–94

    Article  PubMed  CAS  Google Scholar 

  • Pu X, Yang K (2000) Guinea pig 11beta-hydroxysteroid dehydrogenase type I: primary structure and catalytic properties. Steroids 65(3):148–56

    Article  PubMed  CAS  Google Scholar 

  • Sivashanmugam A, Murray V, Cui CX, Zhang YH, Wang JJ, Li QQ (2009) Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 8(5):936–948

    Article  Google Scholar 

  • Sonomoto K, Hoq MM, Tanaka A, Sukui S (1983) 11-Beta-hydroxylation of cortexolone (Reichstein Compound S) to hydrocortisone by Curvularialunata entrapped in photo-pross-pinked resin gels. Appl Environ Microb 45(2):436–443

    CAS  Google Scholar 

  • Sun BQ, Kantzow C, Bresch S, Castiglione K, Weuster-Botz D (2013) Multi-enzymatic one-pot reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid with whole-cell biocatalysts. Biotechnol Bioeng 110(1):68–77

    Article  PubMed  CAS  Google Scholar 

  • Suzuki K, Sanga K, Chikaoka Y, Itagaki E (1993) Purification and properties of cytochromeP-450 (P-450lun) catalyzing steroid 11-beta-hydroxylation in Curvularia lunata. Biochim Biophys Acta 1203(2):215–223

    Article  PubMed  CAS  Google Scholar 

  • Szczebara FM, Chandelier C, Villeret C, Masurel A, Bourot S, Duport C, Blanchard S, Groisillier A, Testet E, Costaglioli P, Cauet G, Degryse E, Balbuena D, Winter J, Achstetter T, Spagnoli R, Pompon D, Dumas B (2003) Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nat Biotechnol 21(2):143–149

    Article  PubMed  CAS  Google Scholar 

  • Wang J, Chen CZ, Li BA, Zhang JH, Yu YT (1998) Production of hydrocortisone from cortexolone-21-acetate by immobilized Absidia orchidis in cosolventcontaining media. Enzyme Microb Technol 22(5):368–373

    Article  CAS  Google Scholar 

  • Wigley WC, Stidham RD, Smith NM, Hunt JF, Thomas PJ (2001) Protein solubility and folding monitored in vivo by structural complementation of a genetic marker protein. Nat Biotechnol 19(2):131–136

    Article  PubMed  CAS  Google Scholar 

  • Wilmanska D, Milczarek K, Rumijowska A, Bartnicka K, Sedlaczek L (1992) Elimination of by-products in 11beta-hydroxylation of substance S using Curvularia lunata clones regenerated from NTG-treated protoplasts. Appl Microbiol Biotechnol 37(5):626–630

    Article  PubMed  CAS  Google Scholar 

  • Zhang BQ, Zhu HM, Liu XF (2004) Effect of supercritical fluids on C11 beta-hydroxylation activity of Absidia coerulea. Biotechnol Progr 20(6):1885–1887

    Article  CAS  Google Scholar 

Download references

Conflict of interest

The authors have no conflict of interest to declare.

Author information

Authors and Affiliations

  1. National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

    Dalong Zhang, Rui Zhang, Wenyue Dong, Qing Zhao, Qiaqing Wu & Dunming Zhu

  2. Tianjin Pharmaceuticals Research Institute Co., Ltd., Tianjin, 300457, China

    Jie Zhang, Liying Chen & Chunxia Zhao

Authors
  1. Dalong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rui Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Liying Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chunxia Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wenyue Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Qing Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Qiaqing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dunming Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dunming Zhu.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 341 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, D., Zhang, R., Zhang, J. et al. Engineering a hydroxysteroid dehydrogenase to improve its soluble expression for the asymmetric reduction of cortisone to 11β-hydrocortisone. Appl Microbiol Biotechnol 98, 8879–8886 (2014). https://doi.org/10.1007/s00253-014-5967-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-5967-1

Keywords

Search

Navigation