Overexpression of cytochrome p450 125 in Mycobacterium: a rational strategy in the promotion of phytosterol biotransformation

  • Liqiu Su
  • Yanbing ShenEmail author
  • Menglei Xia
  • Zhihua Shang
  • Shuangping Xu
  • Xingjuan An
  • Min WangEmail author
Biocatalysis - Original Paper


Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD+/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD+ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD+ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD+/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.


Mycobacterium Phytosterol biotransformation Proteomic analysis Cyp125 NAD+/NADH 



The authors would like to thank the members of the Industrial Fermentation Microbiology Laboratory in the Tianjin University of Science and Technology for their valuable comments and helpful discussions. This work was supported by the Key Project of Chinese Ministry of Education (213004A); the National Natural Science Foundation of China (21276196 and 21406167); Tianjin Municipal Science and Technology Commission (17PTGCCX00190); and Tianjin Programs for Science and Technology Development (15ZCZDSY00510).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    Alfred W, Ansgar S (2006) Ethercarboxylic acid ester of sterol or stanol. US Patent No.US20060183723A1Google Scholar
  2. 2.
    Capyk JK, Kalscheuer R, Stewart GR, Liu J, Kwon H, Zhao R, Okamoto S, Jacobs WR, Eltis LD, Mohn WW (2009) Mycobacterial cytochrome p450 125 (Cyp125) catalyzes the terminal hydroxylation of C27 steroids. J Biol Chem 284(51):35534–35542CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Chen YR, Huan HH, Cheng TF, Tang TY, Liu WH (2006) Expression of a cholesterol oxidase gene from Arthrobacter simplex in Escherichia coli and Pichia pastoris. Enzyme Microb Technol 39:258–262Google Scholar
  4. 4.
    Ding MZ, Cheng JS, Xiao WH, Qiao B, Yuan YJ (2009) Comparative metabolomic analysis on industrial continuous and batch ethanol fermentation processes by GC-TOF-MS. Metabolomics 5(2):229–238CrossRefGoogle Scholar
  5. 5.
    García JL, Uhía I, Galán B (2012) Catabolism and biotechnological applications of cholesterol degrading bacteria. Microb Biotechnol 5(6):679–699CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Garcia-Villalba R, Leon C, Dinelli G, Segura-Carretero A, Fernandez-Gutierrez A, Garcia-Canas V, Cifuentes A (2008) Comparative metabolomic study of transgenic versus conventional soybean using capillary electrophoresis-time-of-flight mass spectrometry. J Chromatogr A 1195(1–2):164–173CrossRefPubMedGoogle Scholar
  7. 7.
    Geize RVD, Dijkhuizen L (2004) Harnessing the catabolic diversity of rhodococci for environmental and biotechnological applications. Curr Opin Microbiol 7:255–261CrossRefPubMedGoogle Scholar
  8. 8.
    Geize RVD, Yam K, Heuser T, Wilbrink MH, Hara H, Anderton MC, Sim E, Dijkhuizen L, Davies JE, Mohn WW, Eltis LD (2007) A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci USA 104:1947–1952CrossRefPubMedGoogle Scholar
  9. 9.
    Ivashina TV, Nikolayeva VM, Dovbnya DV, Donova MV (2012) Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D. J Steroid Biochem 129:47–53CrossRefGoogle Scholar
  10. 10.
    Li B, Wang W, Wang FQ, Wei DZ (2010) Cholesterol oxidase ChoL is a critical enzyme that catalyzes the conversion of diosgenin to 4-ene-3-keto steroids in Streptomyces virginiae IBL-14. Appl Microbiol Biotechnol 85:1831–1838CrossRefPubMedGoogle Scholar
  11. 11.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔCT) method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  12. 12.
    Malaviya A, Gomes J (2008) Androstenedione production by biotransformation of phytosterols. Bioresour Technol 99:6725–6737CrossRefPubMedGoogle Scholar
  13. 13.
    McLean KJ, Lafite P, Levy C, Cheesman MR, Mast N, Pikuleva IA, Leys D, Munro AW (2009) The structure of Mycobacterium tuberculosis CYP125: molecular basis for cholesterol binding in a P450 needed for host infection. J Biol Chem 284(51):35524–35533CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Meyers PR, Bourn WR, Steyn LM, Helden PDV, Beyers AD, Brown GD (1998) Novel method for rapid measurement of growth of Mycobacteria in detergent-free media. J Clin Microbiol 36(9):2752–2754PubMedPubMedCentralGoogle Scholar
  15. 15.
    Ouellet H, Guan S, Johnston JB, Chow ED, Kells PM, Burlingame AL, Cox JS, Podust LM, Ortiz de Montellano PR (2010) Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest-4-en-3-one. Mol Microbiol 77(3):730–742CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ouellet H, Johnston JB, de Montellano PR (2011) Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis. Trends Microbiol 19(11):530–539CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Perez C, Falero A, Duc HL, Balcinde Y, Hung BR (2006) A very efficient bioconversion of soybean phytosterols mixtures to androstanes by mycobacteria. J Ind Microbiol Biotechnol 33(8):719–723CrossRefPubMedGoogle Scholar
  18. 18.
    Rosłoniec KZ, Wilbrink M, Moccia JK, Mohn WW, Geize RVD, Dijkhuizen L, Eltis LD (2009) Cytochrome P450 125 (CYP125) catalyses C26-hydroxylation to initiate sterol side-chain degradation in Rhodococcus jostii RHA1. Mol Microbiol 74(5):1031–1043CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Shen YB, Wang M, Li HN, Wang YB, Luo JM (2012) Infuence of hydroxypropyl-β cyclodextrin on phytosterol biotransformation by different strains of Mycobacterium neoaurum. J Ind Microbiol Biotechnol 39(9):1253–1259CrossRefPubMedGoogle Scholar
  20. 20.
    Su LQ, Shen YB, Gao T, Luo JM, Wang M (2017) Improvement of AD biosynthesis response to enhanced oxygen transfer by oxygen vectors in Mycobacterium neoaurum TCCC 11979. Appl Biochem Biotechnol 182(4):1564–1574CrossRefPubMedGoogle Scholar
  21. 21.
    Su LQ, Shen YB, Zhang WK, Gao T, Shang ZH, Wang M (2017) Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation. Microb Cell Fact 16(1):182–192CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Szentirmai A (1990) Microbial physiology of side chain degradation of sterols. J Ind Microbiol Biotechnol 6(2):101–115Google Scholar
  23. 23.
    Uhía I, Galán B, Morales V, García JL (2011) Initial step in the catabolism of cholesterol by Mycobacterium smegmatis mc2 155. Environ Microbiol 13(4):943–959CrossRefPubMedGoogle Scholar
  24. 24.
    Wang J, Liu HH, Huang D, Jin LN, Wang C, Wen JP (2017) Comparative proteomic and metabolomic analysis of Streptomyces tsukubaensis reveals the metabolic mechanism of FK506 overproduction by feeding soybean oil. Appl Microbiol Biotechnol 101:2447–2465CrossRefPubMedGoogle Scholar
  25. 25.
    Wei W, Fan SY, Wang FQ, Wei DZ (2014) Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01. World J Microbiol Biotechnol 30:1947–1954CrossRefPubMedGoogle Scholar
  26. 26.
    Wei W, Wang FQ, Fan SY, Wei DZ (2010) Inactivation and augmentation of the primary 3-ketosteroid-delta-1-dehydrogenase in Mycobacterium neoaurum NwIB-01: biotransformation of soybean phytosterols to 4-androstene-3,17-dione or 1,4-androstadiene-3,17-dione. Appl Environ Microb 76(13):4578–4582CrossRefGoogle Scholar
  27. 27.
    Yao K, Wang FQ, Zhang HC, Wei DZ (2013) Identifcation and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum. Metab Eng 15:75–87CrossRefPubMedGoogle Scholar
  28. 28.
    Yao K, Xu LQ, Wang FQ, Wei DZ (2014) Characterization and engineering of 3-ketosteroid-delta-1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4- androstene-3, 17-dione through the catabolism of sterols. Metab Eng 24:181–191CrossRefPubMedGoogle Scholar
  29. 29.
    Yang X, Dubnau E, Smith I, Sampson NS (2007) Rv1106c from Mycobacterium tuberculosis is a 3β-hydroxysteroid dehydrogenase. Biochemistry 46(31):9058–9067CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Zhang YP, Huang ZH, Du CY, Li Y, Cao ZA (2009) Introduction of an NADH regeneration system into Klebsiella oxytoca leads to an enhanced oxidative and reductive metabolism of glycerol. Metab Eng 11:101–106CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  1. 1.Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science and Technology), Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process Control, College of BiotechnologyTianjin University of Science and TechnologyTianjinPeople’s Republic of China

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