Functional expression of mammalian NADPH-cytochrome P450 oxidoreductase on the cell surface of Escherichia coli.
To develop a whole-cell oxidoreductase system without the practical limitation of substrate/product transport, easy preparation, stability of enzymes, and low expression levels, we here report the development of a whole cell biocatalyst displaying rat NADPH-cytochrome P450 oxidoreductase (CPR, 77-kDa) on the surface of Escherichia coli by using ice-nucleation protein from Pseudomonas syringae. Surface localization and functionality of the CPR were verified by flow cytometry, electron microscopy, and measurements of enzyme activities. The results of this study comprise the first report of microbial cell-surface display of diflavin-containing mammalian enzymes. This system will allow us to select and develop oxidoreductases, containing bulky and complex prosthetic groups of FAD and FMN, into practically useful whole-cell biocatalysts for broad biological and biotechnological applications including the selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, and bio-chip development.
Yim
Sung-Kun
SK
Hormone Research Center and School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea.
Jung
Heung-Chae
HC
Pan
Jae-Gu
JG
Kang
Hyung-Sik
HS
Ahn
Taeho
T
Yun
Chul-Ho
CH
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein; D009251:NADPH-Ferrihemoprotein Reductase