Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784

Abstract

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.

Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

“…Moreover the hybrid-fused enzyme constructed from the biooxygenating component of the mevastatin hydroxylase from S. carbophilus SANK 62585 and a FR-coding region from the selfsufficient P450RhF monooxygenase from Rhodococcus sp. NCIMB 9784 (Nodate et al 2006) were described (Klaassen et al 2010). …”

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

“…The SGR1085 gene was amplified by PCR using two primers, SGR1085_F (5=-TACCATATGAACTGC CCGCACACTGC-3=; the NdeI site is underlined, and the start codon is in boldface) and SGR1085_R (5=-TACGAATTCGCCCAGGAGGACCG-3=; the EcoRI site is underlined), with S. griseus chromosomal DNA (25 ng) as the template. The 1.2-kb fragment amplified was digested with NdeI and EcoRI and cloned between the NdeI and EcoRI sites of pRED (29) to construct pCYP154C3-RED. In this plasmid, the original stop codon (TAG) of the SGR1085 gene was removed, and the gene was fused in frame with the RhF gene of Rhodococcus species.…”

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum

“…Various publications describe the use of reductase domains from self-sufficient CYPs, such as the RhF from Rhodococcus sp. and BM3 from Bacillus megaterium, for activation of type I CYP enzymes (23)(24)(25). Such hybrid fusion enzymes may catalyze oxidation reactions in absence of other oxidoreductase/ferredoxin partners.…”