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Microbial Expression of Alkaloid Biosynthetic Enzymes for Characterization of Their Properties

  • Hiromichi Minami
  • Nobuhiro Ikezawa
  • Fumihiko Sato
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 643)

Abstract

A wide variety of secondary metabolites are produced in higher plants. These metabolites are synthesized in specific organs/cells at certain developmental stages and/or under specific environmental conditions. Since these biosynthetic activities are rather restricted and difficult to detect, the biochemical characterization of biosynthetic enzymes involved in secondary metabolism has been limited compared to those involved in primary metabolism. Recently, however, progress in tissue culture and molecular biology has made it easier to study biosynthetic enzymes. Here we describe protocols for expressing some biosynthetic enzymes in Escherichia coli expression systems, since this system is both efficient and cost-effective. First, we describe a standard system for expressing biosynthetic enzymes as a soluble protein under the T7 promoter of the pET expression system in E. coli. In addition, the successful expression of cytochrome P450 in E. coli in an active soluble form with N-terminal modification is discussed, since P450 is the critical enzyme in secondary metabolite biosynthesis.

Key words

Alkaloid microbial expression system Escherichia coli pET system cytochrome P450 bioconversion 

Notes

Acknowledgments

This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, and Science and Technology (21248013 to F. S. and 20200036 to H. M.).

References

  1. 1.
    Sato, F., Inui, T., and Takemura, T. (2007) Metabolic engineering in isoquinoline alkaloid biosynthesis. Curr. Pharm. Biotechnol. 8, 211–218.PubMedCrossRefGoogle Scholar
  2. 2.
    Rathbone, D. A., and Bruce, N. C. (2002) Microbial transformation of alkaloids. Curr. Opin. Microbiol. 5, 274–281.PubMedCrossRefGoogle Scholar
  3. 3.
    Ro, D. K., Paradise, E. M., Ouellet, M., Fisher, K. J., Newman, K. L., Ndungu, J. M., Ho, K. A., Eachus, R. A., Ham, T. S., Kirby, J., Chang, M. C., Withers, S. T., Shiba, Y., Sarpong, R., and Keasling, J. D. (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943.PubMedCrossRefGoogle Scholar
  4. 4.
    Minami, H., Kim, J.S., Ikezawa, N., Takemura, T., Katayama, T., Kumagai, H., and Sato, F. (2008) Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl. Acad. Sci. USA 105, 7393–7398.PubMedCrossRefGoogle Scholar
  5. 5.
    Hawkins, K. M. and Smolke, C. D. (2008) Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat. Chem. Biol. 4, 564–573.PubMedCrossRefGoogle Scholar
  6. 6.
    Muchmore, D. C., McIntosh, L.P., Russell, C. B., Anderson, D. E., and Dahlquist, F. W. (1989) Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol. 177, 44–73.PubMedCrossRefGoogle Scholar
  7. 7.
    Gegner, J. A., and Dahlquist, F. W. (1991) Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA. Proc. Natl. Acad. Sci. USA 88, 750–754.PubMedCrossRefGoogle Scholar
  8. 8.
    Minami, H., Dubouzet, E., Iwasa, K., and Sato, F. (2007) Functional analysis of norcoclaurine synthase in Coptis japonica. J. Biol. Chem. 282, 6274–6282.PubMedCrossRefGoogle Scholar
  9. 9.
    Monier, S., Van Luc, P., Kreibich, G., Sabatini, D. D., and Adesnik, M. (1988) Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane. J. Cell Biol. 107, 457–470.PubMedCrossRefGoogle Scholar
  10. 10.
    Kagawa, N., Kusano, K., and Nonaka, Y. (2000) Heterologous expression of mammalian cytochrome P450 as active forms in Escherichia coli. Seibutsu-kogaku Kaishi 78, 82–93.Google Scholar
  11. 11.
    Fujita, K., and Kamataki, T. (2002) Genetically engineered bacterial cells co-expressing human cytochrome P450 with NADPH-cytochrome P450 reductase: prediction of metabolism and toxicity of drugs in humans. Drug Metab. Pharmacokinet. 17, 1–22.PubMedCrossRefGoogle Scholar
  12. 12.
    Ikezawa, N., Iwasa, K., and Sato, F. (2008) Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells. J. Biol. Chem. 283, 8810–8821.PubMedCrossRefGoogle Scholar
  13. 13.
    Richardson, T. H., Jung, F., Griffin, K. J., Wester, M., Raucy, J. L., Kemper, B., Bornheim, L. M., Hassett, C., Omiecinski, C. J., and Johnson, E. F. (1995) A universal approach to the expression of human and rabbit cytochrome P450 s of the 2C subfamily in Escherichia coli. Arch. Biochem. Biophys. 323, 87–96.PubMedCrossRefGoogle Scholar
  14. 14.
    Looman, A. C., Bodlaender, J., Comstock, L. J., Easton, D., Jhurani, P., de Boer, H. A., and van Knippenberg, P. H. (1987) Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli. EMBO J. 6, 2489–2492.PubMedGoogle Scholar
  15. 15.
    Barnes, H. J., Arlotto, M. P., and Waterman, M. R. (1991) Expression and enzymatic activity of recombinant cytochrome P450 17α-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. USA 88, 5597–5601.PubMedCrossRefGoogle Scholar
  16. 16.
    Morishige, T., Choi, K.B., and Sato, F. (2004) In vivo bioconversion of tetrahydroisoquinoline by recombinant coclaurine N-methyltransferase. Biosci. Biotechnol. Biochem. 68, 939–941.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Hiromichi Minami
    • 1
  • Nobuhiro Ikezawa
    • 2
  • Fumihiko Sato
    • 3
  1. 1.Research Institute for Bioresources and BiotechnologyIshikawa Prefectural UniversityIshikawaJapan
  2. 2.Department of Biological SciencesUniversity of CalgaryCalgaryCanada
  3. 3.Division of Integrated Life Science, Graduate School of BiostudiesKyoto UniversityKyotoJapan

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