Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784.
Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.
Nodate
Miho
M
Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate, 026-0001, Japan.
Kubota
Mitsutoshi
M
Misawa
Norihiko
N
D020003:1-Octanol / Q000378:metabolism; D002164:Camphor / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D062385:Hydroxybenzoates / Q000378:metabolism; D009795:Octanes / Q000378:metabolism; D010226:Parabens / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics
D062385:Hydroxybenzoates; D009795:Octanes; D010226:Parabens; C009091:protocatechuic acid; D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; C038193:4-hydroxybenzoic acid; D020003:1-Octanol; C026728:octane