Sequence modifications and heterologous expression of eukaryotic cytochromes P450 in Escherichia coli.
The development of a heterologous expression system is often a principal step in biochemical and biotechnological studies on cytochromes P450 (P450s). However, heterologous expression of eukaryotic membrane-bound P450s in Escherichia coli is still a trial-and-error process because theoretical and systematical experimental procedures have not yet been established. In this study, we generated a series of chimeric variants of fungal P450s by replacing their N-terminal domains with the N-terminal domains of other P450s and explored their potentials for heterologous expression in E. coli. Large-scale screening demonstrated that a number of fungal P450s could be expressed when their N-terminal amino acid sequences were replaced with the corresponding domain of CYP5144C1, even when the expression of the non-chimeric sequence was unpromising. Furthermore, a comprehensive screening resulted in the identification of 64 different types of chimeric partners whose N-terminal domains could potentially be used to increase the expression levels of various P450s. These findings will help to elaborate experimental strategies for high-level heterologous expression of a variety of eukaryotic membrane-bound P450s in E. coli.
Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Electronic address: ichinose@agr.kyushu-u.ac.jp.
Hirofumi
H
Ichinose
Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Mayumi
M
Hatakeyama
Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Yukiyo
Y
Yamauchi
D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005658:Fungi / Q000201:enzymology* / Q000235:genetics*; D050506:Mutant Chimeric Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics
D050506:Mutant Chimeric Proteins; D003577:Cytochrome P-450 Enzyme System
Chimerization; Escherichia coli; Eukaryotic cytochrome P450; Heterologous expression; N-terminal domain; Sequence modification