Microbial Expression of Alkaloid Biosynthetic Enzymes for Characterization of Their Properties

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


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 



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.).


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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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