Surface display of heme- and diflavin-containing cytochrome P450 BM3 in Escherichia coli: a whole cell biocatalyst for oxidation.
Cytochrome P450 enzymes (P450s) are involved in the synthesis of a wide variety of valuable products and in the degradation of numerous toxic compounds. The P450 BM3 (CYP102A1) from Bacillus megaterium was the first P450 discovered to be fused to its redox partner, a mammalian-like diflavin reductase. Here, we report the development of a whole cell biocatalyst using ice-nucleation protein (Inp) from Pseudomonas syringae to display a heme- and diflavin-containing oxidoreductase, P450 BM3 (a single, 119-kDa polypeptide with domains of both an oxygenase and a reductase) on the surface of Escherichia coli. Surface localization and functionality of the fusion protein containing P450 BM3 were verified by flow cytometry and measurement of enzymatic activities. The results of this study comprise the first report of microbial cell-surface display of heme- and diflavin-containing enzyme. This system should allow us to select and develop oxidoreductases containing heme and/or flavins, into practically useful whole-cell biocatalysts for extensive biotechnological applications including selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, live vaccine development, and bio-chip development.
Yim
Sung-Kun
SK
School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Korea.
Kim
Dong-Hyun
DH
Jung
Heung-Chae
HC
Pan
Jae-Gu
JG
Kang
Hyung-Sik
HS
Ahn
Taeho
T
Yun
Chul-Ho
CH
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005415:Flavins / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D006418:Heme / Q000235:genetics / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005415:Flavins; C063113:ice nucleation protein; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases