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

  • Biocatalysis - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

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.

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References

  1. Alfred W, Ansgar S (2006) Ethercarboxylic acid ester of sterol or stanol. US Patent No.US20060183723A1

  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):35534–35542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 39:258–262

    Google Scholar 

  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):229–238

    Article  CAS  Google Scholar 

  5. García JL, Uhía I, Galán B (2012) Catabolism and biotechnological applications of cholesterol degrading bacteria. Microb Biotechnol 5(6):679–699

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):164–173

    Article  CAS  PubMed  Google Scholar 

  7. Geize RVD, Dijkhuizen L (2004) Harnessing the catabolic diversity of rhodococci for environmental and biotechnological applications. Curr Opin Microbiol 7:255–261

    Article  CAS  PubMed  Google Scholar 

  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 104:1947–1952

    Article  CAS  PubMed  Google Scholar 

  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 129:47–53

    Article  CAS  Google Scholar 

  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 85:1831–1838

    Article  CAS  PubMed  Google Scholar 

  11. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔCT) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  12. Malaviya A, Gomes J (2008) Androstenedione production by biotransformation of phytosterols. Bioresour Technol 99:6725–6737

    Article  CAS  PubMed  Google Scholar 

  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):35524–35533

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):2752–2754

    CAS  PubMed  PubMed Central  Google Scholar 

  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):730–742

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ouellet H, Johnston JB, de Montellano PR (2011) Cholesterol catabolism as a therapeutic target in Mycobacterium tuberculosis. Trends Microbiol 19(11):530–539

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):719–723

    Article  CAS  PubMed  Google Scholar 

  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):1031–1043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):1253–1259

    Article  CAS  PubMed  Google Scholar 

  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):1564–1574

    Article  CAS  PubMed  Google Scholar 

  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):182–192

    Article  PubMed  PubMed Central  Google Scholar 

  22. Szentirmai A (1990) Microbial physiology of side chain degradation of sterols. J Ind Microbiol Biotechnol 6(2):101–115

    CAS  Google Scholar 

  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):943–959

    Article  CAS  PubMed  Google Scholar 

  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 101:2447–2465

    Article  CAS  PubMed  Google Scholar 

  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 30:1947–1954

    Article  CAS  PubMed  Google Scholar 

  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):4578–4582

    Article  CAS  Google Scholar 

  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 15:75–87

    Article  CAS  PubMed  Google Scholar 

  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 24:181–191

    Article  CAS  PubMed  Google Scholar 

  29. Yang X, Dubnau E, Smith I, Sampson NS (2007) Rv1106c from Mycobacterium tuberculosis is a 3β-hydroxysteroid dehydrogenase. Biochemistry 46(31):9058–9067

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 11:101–106

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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).

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Authors and Affiliations

  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 Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, People’s Republic of China

    Liqiu Su, Yanbing Shen, Menglei Xia, Zhihua Shang, Shuangping Xu, Xingjuan An & Min Wang

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  1. Liqiu Su
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  2. Yanbing Shen
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  7. Min Wang
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Correspondence to Yanbing Shen or Min Wang.

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Su, L., Shen, Y., Xia, M. et al. Overexpression of cytochrome p450 125 in Mycobacterium: a rational strategy in the promotion of phytosterol biotransformation. J Ind Microbiol Biotechnol 45, 857–867 (2018). https://doi.org/10.1007/s10295-018-2063-z

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  • DOI: https://doi.org/10.1007/s10295-018-2063-z

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