AJMB  Vol.3 No.4 , October 2013
Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli
Abstract
Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.

Cite this paper
Makeeva, D. , Dovbnya, D. , Donova, M. and Novikova, L. (2013) Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli. American Journal of Molecular Biology, 3, 173-182. doi: 10.4236/ajmb.2013.34023.
References

[1]   Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J., Feyereisen, R., Waxman, D., Waterman, M.R., Gotoh, O., Coon, M.J., Estabrook, R.W., Gunsulus, I.C. and Nebert, D.W. (1996) P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics, 6, 1-42.
http://dx.doi.org/10.1097/00008571-199602000-00002

[2]   Orme-Johnson, N.R. (1990) Distinctive properties of adrenal cortex mitochondria. Biochimica et Biophysica Acta, 1020, 213-231.
http://dx.doi.org/10.1016/0005-2728(90)90151-S

[3]   Miller, W.L. (2008) Steroidogenic enzymes. Endocrine Development, 13, 1-18.

[4]   Szczebara, F.M., 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. and Dumas, B. (2003) Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nature Biotechnology, 21, 143-149.
http://dx.doi.org/10.1038/nbt775

[5]   Nelson, D.R., Kamataki, T., Waxman, D.J., Guengerich, F.P., Estabrook, R.W., Feyereisen, R., Gonzalez, F.J., Coon, M.J., Gunsulus, I.C., Gotoh, O.,Okuda, K. and Nebert, D.W. (1993) The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA and Cell Biology, 12, 1-51.
http://dx.doi.org/10.1089/dna.1993.12.1

[6]   Wada, A., Mathew, P.A., Barnes, H.J., Sanders, D., Estabrook, R.W. and Waterman, M.R. (1991) Expression of functional bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) in E. coli. Archives of Biochemistry and Biophysics, 290, 376-380.
http://dx.doi.org/10.1016/0003-9861(91)90554-V

[7]   Novikova, L.A., Faletrov, Y.V., Kovaleva, I.E., Mauersberger, S., Luzikov, V.N. and Shkumatov, V.M. (2009) From structure and functions of steroidogenic enzymes to new technologies of gene engineering. Biochemistry (Moscow), 74, 1482-1504.
http://dx.doi.org/10.1134/S0006297909130057

[8]   Heinz, A., Hannemann, F., Müller, J.J., Heinemann, U. and Bernhardt, R. (2005) The interaction domain of the redox protein adrenodoxin is mandatory for binding of the electron acceptor CYP11A1, but is not required for binding of the electron donor adrenodoxin reductase. Biochemical and Biophysical Research Communications, 338, 491-498.
http://dx.doi.org/10.1016/j.bbrc.2005.08.077

[9]   Pikuleva, I.A., Mackman, R.L., Kagawa, N., Waterman, M.R. and Ortiz de Montellano, P.R. (1995) Active-site topology of bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) and evidence for interaction of tyrosine 94 with the side chain of cholesterol. Archives of Biochemistry and Biophysics, 322, 189-197.
http://dx.doi.org/10.1006/abbi.1995.1451

[10]   Headlam, M. J., Wilce, M. C. and Tuckey, R. C. (2003) The F-G loop region of cytochrome P450scc (CYP11A1) interacts with the phospholipid membrane. Biochimica et Biophysica Acta, 1617, 96-108.
http://dx.doi.org/10.1016/j.bbamem.2003.09.007

[11]   Strushkevich, N., MacKenzie, F., Cherkesova, T., Grabovec, I., Usanov, S. and Park, H.W. (2011) Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system. Proceedings of the National Academy of Sciences of USA, 108, 10139-10143.
http://dx.doi.org/10.1073/pnas.1019441108

[12]   Shet, M.S., Fisher, C.W., Tremblay, Y., Belanger, A., Conley, A.J., Mason, J.I. and Estabrook, R.W. (2007) Comparison of the 17 alpha-hydroxylase/C17,20-lyase activities of porcine, guinea pig and bovine P450c17 using purified recombinant fusion proteins containing P450c17 linked to NADPH-P450 reductase. Drug Metabolism Reviews, 39, 289-307.
http://dx.doi.org/10.1080/03602530701468391

[13]   Oyama, T., Kagawa, N., Sugio, K., Uramoto, H., Hatano, O., Harada, N., Kaneko, K., Kawamoto, T. and Yasumoto, K. (2009) Expression of aromatase CYP19 and its relationship with parameters in NSCLC. Frontiers in Bioscience, 14, 2285-2292.
http://dx.doi.org/10.2741/3379

[14]   Mast, N., Linger, M. and Pikuleva, I.A. (2012) Inhibition and stimulation of activity of purified recombinant CYP11A1 by therapeutic agents. Molecular and Cellular Endocrinology, 371, 100-106.
http://dx.doi.org/10.1016/j.mce.2012.10.013

[15]   Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Headlam, M.J. and Tuckey, R.C. (1998) Expression of catalytically active human cytochrome P450scc in Escherichia coli and mutagenesis of isoleucine-462. Archives of Biochemistry and Biophysics, 353, 109-115
http://dx.doi.org/10.1006/abbi.1998.0621

[16]   Shkumatov, V.M., Radyuk, V.G., Faletrov, Y.V., Vinogradova, A.A., Luzikov, V.N. and Novikova, L.A. (2006) Expression of cytochrome P450scc in Escherichia coli cells: Intracellular location and interaction with bacterial redox proteins. Biochemistry (Moscow), 71, 884-892.
http://dx.doi.org/10.1134/S0006297906080104

[17]   Brandt, M.E. and Vickery, L.E. (1992) Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli. Archives of Biochemistry and Biophysics, 294, 735-740.
http://dx.doi.org/10.1016/0003-9861(92)90749-M

[18]   Sagara, Y., Wada, A., Takata, Y., Waterman, M.R., Sekimizu, K. and Horiuchi, T. (1993) Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization. Biological & Pharmaceutical Bulletin, 7, 627-630.
http://dx.doi.org/10.1248/bpb.16.627

[19]   Uhlmann, H., Beckert, V., Schwarz, D. and Bernhardt, R. (1992) Expression of bovine adrenodoxin and site-directed mutagenesis of [2Fe-2S] cluster ligands. Biochemical and Biophysical Research Communications, 188, 1131-1138.
http://dx.doi.org/10.1016/0006-291X(92)91349-U

[20]   Sagara, Y. and Aramaki, H. (1998) Overproduction in Escherichia coli and characterization of the precise mature form of rat adrenodoxin. Biological & Pharmaceutical Bulletin, 21, 1106-1109.
http://dx.doi.org/10.1248/bpb.21.1106

[21]   Nazarov, P.A., Drutsa, V.L., Miller, W.L., Shkumatov, V.M., Luzikov, V.N. and Novikova, L.A. (2003) Some features of formation and functioning of fused cholesterol hydroxylase/lyase. DNA and Cell Biology, 22, 243-252. http://dx.doi.org/10.1089/104454903321908638

[22]   Shashkova, T.V., Luzikov, V.N. and Novikova, L.A. (2006) Coexpression of all constituents of the cholesterol hydroxylase/lyase system in Escherichia coli cells. Biochemistry (Moscow), 71, 810-814.
http://dx.doi.org/10.1134/S0006297906070145

[23]   Kеmbaren, R.F. and Janssen, D.B. (2008) Coexpression of three-component cytochrome P450scc in E. coli. In: 9th International symposium on Cytochrome P450 Biodiversity and Biotechnology, Abstracts, Nice, June 8-12, 2008, p. 70.

[24]   Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[25]   Hannemann, F., Virus, C. and Bernhardt, R. (2006) Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases. Journal of Biotechnology, 124, 172-181. http://dx.doi.org/10.1016/j.jbiotec.2006.01.009

[26]   Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.
http://dx.doi.org/10.1038/227680a0

[27]   Sidhu, R.S. and Bollon, A.P. (1987) Analysis of alphafactor secretion signals by fusing with acid phosphatase of yeast. Gene, 54, 175-184.
http://dx.doi.org/10.1016/0378-1119(87)90485-9

[28]   Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randal, R.J. (1951) Protein measurement with folin-phenol reagent. The Journal of Biological Chemistry, 193, 265-275.

[29]   Sawada, N., Sakaki, T., Kitanaka, S., Takeyama, K., Kato, S. and Inouye, K. (1999) Enzymatic properties of human 25-hydroxyvitamin D3 1α-hydroxylase. European Journal of Biochemistry, 265, 950-956.
http://dx.doi.org/10.1046/j.1432-1327.1999.00794.x

[30]   Salamanca-Pinzón, S.G. and Guengerich, F.P. (2011) A tricistronic human adrenodoxin reductase-adrenodoxincytochrome P450 27A1 vector system for substrate hydroxylation in Escherichia coli. Protein Expression and Purification, 79, 231-236.
http://dx.doi.org/10.1016/j.pep.2011.05.008

[31]   Ringle, M., Khatri, Y., Zapp, J., Hannemann, F. and Bernhardt, R. (2012) Application of a new versatile electron transfer system for cytochrome P450-based Escherichia coli whole-cell bioconversions. Applied Microbiology and Biotechnology, 97, 7741-7754.
http://dx.doi.org/10.1007/s00253-012-4612-0

[32]   Haberland, M.E. and Reynolds, J.A. (1973) Self-association of cholesterol in aqueous solution. Proceedings of the National Academy of Sciences of USA, 70, 2313-2316.
http://dx.doi.org/10.1073/pnas.70.8.2313

[33]   Donova, M.V. and Egorova, O.V. (2012) Microbial steroid transformations: Current state and prospects. Applied Microbiology and Biotechnology, 94, 1423-1447.
http://dx.doi.org/10.1007/s00253-012-4078-0

[34]   Hesselink, P.G.M., van Vliet, S., de Vries, H. and Witholt, B. (1989) Optimization of steroid side-chain cleavage by Mycobacterium sp. in the presence of cyclodextrins. Enzyme and Microbial Technology, 11, 398-404.
http://dx.doi.org/10.1016/0141-0229(89)90133-6

[35]   Jadoun, J. and Bar, R. (1993) Microbial transformations in a cyclodextrin medium. Part 4. Enzyme vs microbial oxidation of cholesterol. Applied Microbiology and Biotechnology, 40, 477-482.
http://dx.doi.org/10.1007/BF00175734

[36]   Donova, M.V., Nikolayeva, V.M., Dovbnya, D.V., Gulevskaya, S.A. and Suzina, N.E. (2007) Methyl-β-cyclodextrin alters growth, activity and cell envelope features of sterol transforming mycobacteria. Microbiology, 153, 1981-1992.
http://dx.doi.org/10.1099/mic.0.2006/001636-0

 
 
Top