title: Microbial expression of alkaloid biosynthetic enzymes for characterization of their properties.
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.
authors.affiliation: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Minami
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Ikezawa
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Sato
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
chemical_list: D000470:Alkaloids; D003577:Cytochrome P-450 Enzyme System