Improved 11α-hydroxycanrenone production by modification of cytochrome P450 monooxygenase gene in Aspergillus ochraceus

Qianqian Li 1 , Li Shi 1 , Yingying Liu 1 , Shimin Guan 1 , Shuo Zhang 1 , Baoguo Cai 1 ,  and Shaofeng Rong 1
  • 1 Department of Bioengineering Shanghai Institute of Technology, , Fengxian, Shanghai, 201418, China


Eplerenone is a drug that protects the cardiovascular system. 11α-Hydroxycanrenone is a key intermediate in eplerenone synthesis. We found that although the cytochrome P450 (CYP) enzyme system in Aspergillus ochraceus strain MF018 could catalyse the conversion of canrenone to 11α-hydroxycanrenone, its biocatalytic efficiency is low. To improve the efficiency of 11α-hydroxycanrenone production, the CYP monooxygenase-coding gene of MF018 was predicted and cloned based on whole-genome sequencing results. A recombinant A. ochraceus strain MF010 with the high expression of CYP monooxygenase was then obtained through homologous recombination. The biocatalytic rate of this recombinant strain reached 93 % at 60 h without the addition of organic solvents or surfactants and was 17–18 % higher than that of the MF018 strain. Moreover, the biocatalytic time of the MF010 strain was reduced by more than 30 h compared with that of the MF018 strain. These results show that the recombinant A. ochraceus strain MF010 can overcome the limitation of substrate biocatalytic efficiency and thus holds a high poten tial for application in the industrial production of eplerenone.

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  • 1. L. O. L. Ríos, J. M. Luengo and J. M. Fernández-Cañón, Steroid 11-alpha-hydroxylation by the fungi Aspergillus nidulans and Aspergillus ochraceus, Methods Mol. Biol. 1645 (2017) 271–287;

  • 2. L. Jia, J. Dong, R. Wang, S. Mao, F. Lu, S. Singh, Z. Wang and X. Liu, Identification and characterization of the steroid 15α-hydroxylase gene from Penicillium raistrickii, Appl. Microbiol. Biotechnol. 101 (2017) 6409–6418;

  • 3. S. Huang, J. Xie, J. Cui, L. Liu, Y. Liang, Y. Liu and Q. Xiao, Comparative investigation of binding interactions between three steroidal compounds and human serum albumin: Multispectroscopic and molecular modeling techniques, Steroids 128 (2017) 136–146;

  • 4. X. Wang, J. Feng, D. Zhang, Q. Wu, D. Zhu and Y. Ma, Characterization of new recombinant 3-ketosteroid-Δ1-dehydrogenases for the biotransformation of steroids, Appl. Microbiol. Biotechnol. 101 (2017) 6049–6060;

  • 5. S. Mao, X. Wang, Z. Ge, A. Su, L. Zhang, Y. Li, X. Liu and F. Lu, Microbial hydroxylation of steroids by Penicillium decumbens, J. Mol. Catal. B-Enzym. 133 (2017) S346–S351;

  • 6. E. Kozłowska, N. Hoc, J. Sycz, M. Urbaniak, M. Dymarska, J. Grzeszczuk, E. Kostrzewa-Susłow, L. Stępień, E. Pląskowska and T. Janeczko, Biotransformation of steroids by entomopathogenic strains of Isaria farinose, Microb. Cell Fact. 17 (2018) 71;

  • 7. A. Świzdor, A. Panek, P. Ś. Ostrowska, Metabolic fate of pregnene-based steroids in the lactonization pathway of multifunctional strain Penicillium lanosocoeruleum, Microb. Cell Fact. 17 (2018) 100;

  • 8. C. Yarnold, J. M. Bainbridge, H. S. Boehm, B. Mark, C. Stephen, D. Hervé, D. L. Raffaella, G. Paul, H. Estelle, J. Jörn, L. Amedeo, M. Mirco, R. Carlo, S. Andreas, S. Suganthan, S. Sakthi, S. Giorgio, T. Paolo and W. David, Steroidal mineralocorticoid receptor antagonists: synthesis and biology, ChemistrySelect. 2 (2017) 175–189;

  • 9. P. Rossignol, N. Girerd, G. Bakris, O. Vardeny, B. Claggett, J. J. V. McMurray, K. Swedberg, H. Krum, D. J. van Veldhuisen, H. Shi, S. Spanyers, J. Vincent, R. Fay, Z. Lamiral, S. D. Solomon, F. Zannad and B. Pitt, Impact of eplerenone on cardiovascular outcomes in heart failure patients with hypokalaemia, Eur. J. Heart Fail. 19 (2017) 792–799;

  • 10. J. A. Delyani, R. Rocha, C. S. Cook, D. S. Tobert, S. Levin, B. Roniker, D. L. Workman, Y. L. Sing and B. Whelihan, Eplerenone: a selective aldosterone receptor antagonist (SARA), Cardiovasc. Drug Rev. 19 (2001) 185–200;

  • 11. M. A. Tantawy, M. S. Nafie, G. A. Elmegeed and I. A. I. Ali, Auspicious role of the steroidal heterocyclic derivatives as a platform for anti-cancer drugs, Bioorg. Chem. 73 (2017) 128–146;

  • 12. T. S. Tam, M. H. Wu, S. C. Masson, M. P. Tsang, S. N. Stabler, A. Kinkade, A. Tung and A. M. Tejani, Eplerenone for hypertension, Cochrane Database Syst. Rev. 2 (2017) CD008996;

  • 13. M. Boehm, N. Arnold, A. Braithwaite, J. Pickworth, C. Lu, T. Novoyatleva, D. G. Kiely, F. Grim-minger, H. A. Ghofrani, N. Weissmann, W. Seeger, A. Lawrie, R. T. Schermuly and B. Kojonazarov, Eplerenone attenuates pathological pulmonary vascular rather than right ventricular remodeling in pulmonary arterial hypertension, BMC Pulm. Med. 18 (2018) 41;

  • 14. J. P. Ferreira, K. Duarte, J. J. V. McMurra, B. Pitt, D. J. van Veldhuisen, J. Vincent, T. Ahmad, J. Tromp, P. Rossignol and F. Zannad, Data-driven approach to identify subgroups of heart failure with reduced ejection fraction patients with different prognoses and aldosterone antagonist response patterns, Circ-Heart. Fail. 11 (2018) e004926;

  • 15. J. S. NG, P. T. Wang, J. A. Baez, C. Liu, D. K. Anderson, J. P. Lawson, D. Erb, J. Wieczorek, G. Mucciariello, F. Vanzanella, S. A. Kunda, L. J. Letendre, M. J. Pozzo, Y. L. Sing and E. E. Yonan, Processes for Preparation of 9,11-epoxy Steroids and Intermediates Useful therein, U. S. Pat. 7,112,669 B2, 26 Aug 2003.

  • 16. I. Dams, A. Białońska, P. Cmoch, M. Krupa, A. Pietraszek, A. Ostaszewska and M. Chodyński, Synthesis and physicochemical characterization of the process-related impurities of eplerenone, an antihypertensive drug, Molecules. 22 (2017) E1354;

  • 17. D. M. Huang, T. Z. Zhang, F. J. Cui, W. J. Sun, L. M. Zhao, M. Y. Yang and Y. J. Wang, Simultaneous identification and quantification of canrenone and 11-α-hydroxy-canrenone by LC-MS and HPLCUVD, J. Biomed. Biotechnol. 2011 (2011) 917232;

  • 18. P. Durairaj, J. S. Hur and H. Yun, Versatile biocatalysis of fungal cytochrome P450 monooxygenases, Microb. Cell Fact. 15 (2016) 125;

  • 19. V. V. Kollerov, A. A. Shutov, A. Kazantsev and M. V. Donova, Biocatalytic modifications of pregnenolone by selected filamentous fungi, Biocatal. Biotransfor. 1 (2019) 1–12;

  • 20. G. D. Saratale, R. P. Humnabadkar and S. P. Govindwar, Study of mixed function oxidase system in Aspergillus ochraceus (NCIM 1146), Indian J. Microbiol. 47 (2007) 304–309;

  • 21. Rédei and P. George, Encyclopedia of Genetics, Genomics, Proteomics and Informatics, Springer, Dordrecht, Berlin 2008, pp. 1087–1087.

  • 22. Q. Gao, Y. Qiao, Y. Shen, M. Wang, X. Wang and Y. Liu, Screening for strains with 11α-hydroxylase activity for 17α-hydroxy progesterone biotransformation, Steroids 124 (2017) 67–71;

  • 23. M. K. Refai, N. H. Aziz, F. El-Far and A. A. Hassan, Detection of ochratoxin produced by A. ochraceus in feedstuffs and its control by γ radiation, Appl. Radiat. Isot. 47 (1996) 617–621;

  • 24. D. Sun, L. Wang, X. Mao, M. Fei, Y. Chen, M. Shen and J. Qiu, Chemical transformation mediated CRISPR/Cas9 genome editing in Escherichia coli, Biotechnol. Lett. 41 (2019) 293–303;

  • 25. International Conference on Applied Biotechnology, Engineering of Industrial Aspergillus ochraceus Strains for Improved Steroid 11α-Hydroxylation Efficiency via Overexpression of the 11α-Hydroxylase Gene CYP68J5, November 2016;; last access date October 8, 2017

  • 26. I. Weyda, L. Yang, J. Vang, B. K. Ahring, M. Lübeck and P. S. Lübeck, A comparison of agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius, J. Microbiol. Methods 135 (2017) 26–34;

  • 27. R. Wang, L. Zhang, Z. Zhang and Y. Tian, Comparison of ESI-and APCI-LC-MS/MS methods: a case study of levonorgestrel in human plasma, J. Pharm. Anal. 6 (2016) 356–362;

  • 28. S. Bolten, R. Clayton, A. Easton, L. Engel, D. Messing, J. S. Ng, B. Reitz, M. C. Walker and P. T. Wang, Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase, U.S. Pat. 20,040,900,856, 1 Jun 2006.

  • 29. T. Du Toit, M. A. Stander and A. C. Swart, A high-throughput UPC2-MS/MS method for the separation and quantification of C19 and C21 steroids and their C11-oxy steroid metabolites in the classical, alternative, backdoor and 11OHA4 steroid pathways, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1080 (2018) 71–81;

  • 30. J. Y. Houng, W. P. Chiang, K. C. Chen and C. Tiu, 11α-Hydroxylation of progesterone in biphasic media using alginate-entrapped Aspergillus ochraceus gel beads coated with polyurea, Enzyme Microb. Technol. 16 (1994) 485–491;

  • 31. S. Rong, J. Wang, Q. Li and S. Guan, The enhanced production of 11α-hydroxyandrosta-1,4-diene-3,17-dione based on the application of organic silica hollow spheres in the biotransformation of β-sitosterol, J. Chem. Technol. Biot. 92 (2017) 69–75;

  • 32. R. Wang, P. Sui, X. Hou, T. Cao, L. Jia, F. Lu, S. Singh and Z. Wang, Cloning and identification of a novel steroid 11α-hydroxylase gene from Absidia coerulea, J. Steroid Biochem. Mol. Biol. 171 (2017) 254–161;

  • 33. S. Petrič, T. Hakki, R. Bernhardt, D. Zigon and B. Crešnar, Discovery of a steroid 11α-hydroxylase from Rhizopus oryzae and its biotechnological application, J. Biotechnol. 150 (2010) 428–437;

  • 34. C. M. Hull, A. G. S. Warrilow, N. J. Rolley, C. L. Price, I. S. Donnison, D. E. Kelly and S. L. Kelly, Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases, Biotechnol. Biofuels 10 (2017) 226;

  • 35. A. W. Munro, K. J. Mclean, J. L. Grant and T. M. Makris, Structure and function of the cytochrome P450 peroxygenase enzymes, Biochem. Soc. Trans. 46 (2018) 183–196;

  • 36. P. Córdova, A-M. Gonzalez, D. R. Nelson, M. S. Gutiérrez, M. Baeza, V. Cifuentes and J. Alcaíno, Characterization of the cytochrome P450 monooxygenase genes (P450ome) from the carotenogenic yeast Xanthophyllomyces dendrorhous, BMC Genomics 18 (2017) 540;

  • 37. G. Reguera, Biological electron transport goes the extra mile, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) 5632–5634;

  • 38. X. Han, A. Chakrabortti, J. Zhu, Z. X. Lian and J. Li, Sequencing and functional annotation of the whole genome of the filamentous fungus Aspergillus westerdijkiae, BMC Genomics 17 (2016) 633;

  • 39. K. Tanuja, K. Hemalatha, R. Karuna and B. Sashidhar Rao, Effect of various surfactants (cationic, anionic and non-ionic) on the growth of Aspergillus parasiticus (NRRL 2999) in relation to aflatoxin production, Mycotoxin Res. 26 (2010) 155–170;


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