Modification of small molecules by using cytochrome P450 expressed in Escherichia coli.
We developed a system for bioconverting diverse compounds using P450s produced in Escherichia coli. Vectors for the expressing various P450 cDNAs quickly and easily in E. coli were developed by using several restriction enzyme sites. Three types of P450 (2C2, 2C29, and 2D22) were produced using these plasmids. Substrates were directly added to the incubation medium and metabolized. To obtain pure product from the medium, we first tried production of P450 in synthetic medium. The amount of another P450 2C43 produced in the synthetic medium was similar to the amount produced in Luria broth (LB) medium. Next, estradiol, a steroid, was added as a substrate, incubated, and the metabolite was extracted and analyzed by high-performance liquid chromatography. The metabolite extracted from synthetic medium was purer than that obtained from LB medium. Three P450s (2C29, 2C2, and 2A4) metabolized testosterone at different positions. P450 2C29 metabolized 7-ethoxycoumarin, androstendione, and dehydroepiandrosterone in this medium. P450s produced in the synthetic medium may be useful for producing various modified compounds for high-throughput screening.
Uno
Tomohide
T
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Nakao
Atsushi
A
Masuda
Satoko
S
Taniguchi
Yuuki
Y
Kanamaru
Kengo
K
Yamagata
Hiroshi
H
Nakamura
Masahiko
M
Imaishi
Hiromasa
H
Oono
Kiyoharu
K
D000595:Amino Acid Sequence; D001711:Biotransformation; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D013256:Steroids / Q000378:metabolism*
D003470:Culture Media; D013256:Steroids; D003577:Cytochrome P-450 Enzyme System