Isolation and functional analysis of cytochrome P450 CYP153A genes from various environments.
The cytochrome P450 CYP153 family is thought to mediate the terminal hydroxylation reactions of n-alkanes. We isolated 16 new P450 CYP153A genes (central region) from various environments such as petroleum-contaminated soil and groundwater, as well as one from the n-alkane-degrading bacterium Alcanivorax borkumensis SK2 (designated P450balk). The sequences of the new P450 genes were extended by PCR to generate full-length chimeric P450 genes, using the N- and C-terminal domains of P450balk. A differential CO-reduced P450 spectral analysis indicated that 8 P450 genes among the 16 chimeric genes were expressed in Escherichia coli to generate a soluble and functional enzyme. The several functional chimeric P450s and P450balk were further fused to the reductase domain of the self-sufficient P450 monooxygenase (P450RhF) at the C-terminus. E. coli cells expressing these self-sufficient P450 chimeric genes converted n-alkanes, cyclohexane, 1-octene, n-butylbenzene, and 4-phenyl-1-butene into 1-alkanols, cyclohexanol, 1,2-epoxyoctane, 1-phenyl-4-butanol, and 2-phenethyl-oxirane, respectively.
Kubota
Mitsutoshi
M
Marine Biotechnology Institute, Iwate, Japan. norihiko.misawa@mbio.jp
Nodate
Miho
M
Yasumoto-Hirose
Mina
M
Uchiyama
Taku
T
Kagami
Osamu
O
Shizuri
Yoshikazu
Y
Misawa
Norihiko
N
D000473:Alkanes / Q000737:chemistry / Q000378:metabolism; D000475:Alkenes / Q000737:chemistry / Q000378:metabolism; D001555:Benzene Derivatives / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004777:Environment; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D010578:Petroleum; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012988:Soil Microbiology; D012989:Soil Pollutants; D014871:Water Microbiology; D014874:Water Pollutants, Chemical
D000473:Alkanes; D000475:Alkenes; D001555:Benzene Derivatives; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D010578:Petroleum; D012989:Soil Pollutants; D014874:Water Pollutants, Chemical; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; C037690:1-octene; D006899:Mixed Function Oxygenases; C435228:cytochrome P-450 CYP153 (Acinetobacter); C053761:n-butylbenzene