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. 2003 Jan;69(1):373–382. doi: 10.1128/AEM.69.1.373-382.2003

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

Haitham A Hussain 1, John M Ward 1,*
PMCID: PMC152428  PMID: 12514018

Abstract

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.

Cytochrome P450 monooxygenase systems are encoded by a gene superfamily and play critical roles in the bioactivation and detoxification of a wide variety of xenobiotics. In humans they are a major class of biocatalyst involved in the oxidative metabolism of exogenous as well as endogenous compounds, including drugs, xenobiotics, fatty acids, bile acids, and steroids. These enzymes have considerable importance for determining the pharmacokinetic and toxicokinetic characteristics of drugs. It is sometimes difficult to chemically synthesize the same hydroxylated products that would occur in vivo in sufficient quantities to carry out toxicological studies on these drug derivatives. A biocatalytic approach to synthesizing large amounts of such hydroxylated compounds is thus attractive. Streptomyces species P450 enzymes are soluble, are not membrane bound like their human counterparts, and often have a broad range of substrate specificities. Thus recombinant Streptomyces enzymes could be used to prepare drug metabolites in quantity to assess their toxicological effects.

The soil bacterium Streptomyces griseolus ATCC 11796 expresses two distinct cytochrome P450 monooxygenases, designated cytochrome P450SU1 (CYP105A1) and cytochrome P450SU2 (CYP105B1) (37). These two enzymes are able to metabolize sulfonylurea herbicides such as chlorimuron-ethyl and xenobiotics such as phenobarbital (8, 30, 33). These two systems each consist of an inducible cytochrome P450, a ferredoxin, and a poorly characterized NAD(P)H:ferredoxin reductase (27). The two cytochrome P450-encoding genes and their adjacent ferredoxin-encoding genes have been cloned and sequenced (27, 31). The S. griseolus P450 and adjacent ferredoxin can be expressed both in bacteria and higher plants, but cells containing the P450 enzymes alone are unable to metabolize herbicides (29, 30). It has been found that genes for both P450s are cotranscribed with those for their ferredoxins, Fd1 and Fd2, demonstrating that there is a well-defined relationship between the P450 and its cognate ferredoxin and that these ferredoxins are necessary for P450 activity (30). The level of NAD(P)H:ferredoxin reductase in cell extracts is very low, and no reductase gene has yet been isolated from S. griseolus (28). However, in vitro studies show that the absence of significant S. griseolus reductase activity can be overcome by reconstitution with ferredoxin NADP reductase from spinach chloroplasts or putidaredoxin reductase from the Pseudomonas putida P450CAM system (27, 30). It has been shown by O'Keefe et al. (30) that there is a direct correlation between the P450/ferredoxin content and the in vivo metabolism rates, and these workers suggested that the overall limiting parameters in vivo are the levels of P450 and/or ferredoxin but not of the reductase. In contrast their in vitro results showed low levels of reductase activity in crude soluble protein extracts, and this implied that whole-cell P450 activity might be limited by reductase availability.

To study the structural and functional aspects of P450 enzymes, large amounts of purified protein are necessary, more than can generally be purified from wild-type bacteria or animal tissue (particularly that of humans) (15). Heterologous expression of cytochrome P450 in Escherichia coli has become a very useful tool in biomedical research and is widely used in the study of many mammalian cytochrome P450 monooxygenases. In this paper, S. griseolus cytochromes P450SU1 and P450SU2 were expressed in E. coli to high levels to study the activity of these proteins toward heterologous compounds as well as to investigate their application in biocatalysis. We show that catalytically active P450 levels in vivo are enhanced by cloning a ferredoxin reductase of Streptomyces coelicolor and expressing this alongside the P450 and ferredoxin in E. coli. We also show the expression of these proteins in Streptomyces lividans and efficient biotransformation with the recombinant S. lividans.

MATERIALS AND METHODS

Strains, plasmids, and culture conditions.

E. coli DH5α and TOP10 were used as hosts for transformation (Table 1). E. coli strains carrying plasmids were grown in Luria-Bertani medium at 37°C. Other standard microbial and recombinant techniques used throughout this work are as described by Sambrook et al. (39). Media were supplemented with 100 μg of ampicillin/ml and 50 μg of kanamycin/ml when required. Plasmid DNA was isolated from bacteria with Qiagen kits. DNA fragments for subcloning were isolated from agarose gels with a QIAEXII gel extraction kit (Qiagen). PCR products were cloned into the pCRII-TOPO vector by using the TOPO TA cloning kit (Invitrogen). Several E. coli host strains that are compatible with the pET expression systems from Novagen, Invitrogen, and Stratagene were used to overexpress P450 proteins.

TABLE 1.

Microorganisms and plasmids used in the study

Strain or plasmid Characteristics Reference or source
E. coli
    DH5α F−endA1 recA1 hsdR17(rk− mk+) supE44 λ−thi-1 gyrA(Na1) relA1 φ80 lacZΔM15Δ (lacZY A-argF) BRL
    TOP10 F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80 lacZΔM15 ΔlacX74 recA1 deoR araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG Invitrogen
    AD494(DE3) Δara-leu7697 ΔlacX74 ΔphoAPvuII phoR ΔmalF3 F′ [lac+ (lacIq) pro] trxB::kan (DE3) Novagen
    AD494(DE3)pLysS Δara-leu7697 ΔlacX74 ΔphoAPvuII phoR ΔmalF3 F′ [lac+ (lacIq) pro] trxB::kan (DE3)pLysS (Cm r) Novagen
    B834(DE3) F−ompT hsdSB (rB− mB−) gal dcm met (DE3) Novagen
    B834(DE3)pLysS F−ompT hsdSB (rB− mB−) gal dcm met (DE3)pLysS (Cmr) Novagen
    BL21(DE3) F−ompT hsdSB (rB− mB−) gal dcm (DE3) Novagen
    BL21(DE3)pLysS F−ompT hsdSB (rB− mB−) gal dcm (DE3)pLysS (Cmr) Novagen
    BL21trxB(DE3) F−ompT hsdSB (rB− mB−) gal dcm (DE3) trxB15::kan (DE3) Novagen
    BL21trxB(DE3)pLysS F−ompT hsdSB (rB− mB−) gal dcm (DE3) trxB15::kan (DE3)pLysS (CmR) Novagen
    HMS174(DE3) F−recA1 hsdR(rK12− mK12+) Rifr (DE3) Novagen
    HMS174(DE3)pLysS F−recA1 hsdR(rK12− mK12+) Rifr (DE3)pLysS (Cmr) Novagen
    Rosetta(DE3) F−ompT hsdSB (rB− mB−) gal dcm lacY1 (DE3) pRARE (Cmr) Novagen
    Rosetta(DE3)pLysS) F−ompT hsdSB (rB− mB−) gal dcm lacY1 (DE3) pLysSRARE (Cmr) Novagen
    Tuner(DE3) F−ompT hsdSB (rB− mB−) gal dcm lacY1 (DE3) Novagen
    Tuner(DE3)pLysS F−ompT hsdSB (rB− mB−) gal dcm lacY1 (DE3)pLysS (Cmr) Novagen
    BL21-CodonPlus(DE3)-RIL F−ompT hsdS (rB− mB−) dcm+ Tetrgal λ (DE3) endA Hte [argU ileY leuW Camr] Stratagene
    BL21-CodonPlus(DE3)-RP F−ompT hsdS (rB− mB−) dcm+ Tetrgal λ (DE3) endA Hte [argU proL Camr] Stratagene
    BL21 StarTM (DE3) F−ompT hsdSB (rB− mB−) gal dcm rnel31 (DE3) Invitrogen
    BL21 StarTM (DE3)pLysS F−ompT hsdSB (rB− mB−) gal dcm rnel31 (DE3)pLysS (Camr) Invitrogen
    ET12567 dam13::Tn9 dcm6 hsdM hsdR recF143 zjj201::Tn10 galK2 galT22 ara14 lacY1 xy15 leuB6 thil tonA31 rpsL136 hisG4 tsx78 mtli glnV44 F− 21
Streptomyces
    S. lividans 1326 Wild type
    S. coelicolor A3(2) Wild type John Innes Centrea
    S. griseolus ATCC 11796 Wild type John Innes Centre ATCCb
Plasmids
    pCRII-TOPO TOPO cloning vector; Ampr Kanr Invitrogen
    pET21a Overexpression vector; Ampr Novagen
    pIJ8600 Conjugative and integrative shuttle vector; Aprr Thior 42
    pCJR29 Streptomyces expression vector; Ampr Thior 38
    pBW160 Conjugative shuttle expression vector; Ampr Thior B. Rudd (personal communication)
    pALTER:EX2 Low-copy-number expression vector; Tetr Promega
    pQR270 pCRII-TOPO carrying PCR product of P450SU1 and Fd1 of S. griseolus This study
    pQR271 pCRII-TOPO carrying PCR product of P450SU2 and Fd2 of S. griseolus This study
    pQR273 NdeI/BamHI fragment of pQR270 into NdeI- and BamHI-cut pET21a This study
    pQR274 NdeI/BamHI fragment of pQR271 into NdeI- and BamHI-cut pET21a This study
    pQR291 NdeI/BamHI fragment of pQR270 into NdeI- and BamHI-cut pIJ8600 This study
    pQR292 NdeI/BamHI fragment of pQR271 into NdeI- and BamHI-cut pIJ8600 This study
    pQR293 BamHI/HindIII fragment deleted from pALTER. EX2 vector This study
    pQR294 pCRII-TOPO carrying PCR product of SC4B10 ferredoxin reductase gene of S. coelicolor This study
    pQR295 pCRII-TOPO carrying PCR product of SCF15A ferredoxin NADP reductase gene of S. coelicolor This study
    pQR296 NcoI/PstI fragment of pDR294 into NcoI- and PstI-cut of pQR293 This study
    pQR297 NcoI/PstI fragment of pDR295 into NcoI- and PstI-cut of pQR293 This study
    pQR355 NdeI/SpeI fragment of pQR270 into NdeI- and XbaI-cut pBW160 This study
    pQR356 NdeI/XbaI fragment of pQR271 into NdeI- and XbaI-cut pBW160 This study
    pQR367 EcoRI/NotI blunt end fragment of pQR297 into EcoRI- and HindII blunt end-cut pQR273 This study
    pQR368 EcoRI/NotI blunt end fragment of pQR297 into EcoRI and HindII blunt end-cut pQR274 This study
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a

United Kingdom.

b

ATCC, American Type Culture Collection.

Standard methods of culture for S. griseolus ATCC 11796, S. coelicolor A3 (2), and S. lividans 1326 and conditions for genomic DNA and plasmid DNA isolation and conjugation were as described previously (18). Thiostrepton, apramycin, and nalidixic acid were used at final concentrations of 10 μg, 1 mg, and 0.5 mg per ml, respectively. Streptomyces strains were grown on complete medium GY containing 3.3% glucose and 1.5% yeast extract or SFM containing 2% mannitol, 2% soy flour, and 1.6% agar.

Construction of expression vectors in E. coli.

The entire coding regions of the genes encoding cytochrome P450SU1 and its adjacent ferredoxin, Fd1, and cytochrome P450SU2 and its adjacent ferredoxin, Fd2, were generated as PCR fragments from the published sequence (27, 31) of S. griseolus ATCC 11796 genomic DNA. The primers contain an NdeI site added on the 5′ end of the P450 gene and a BamHI site on the 3′ end of the ferredoxin gene. The following primers were used to amplify the P450SU1-Fd1 genes: forward primer, 5′-GTCATATGACCGATACCGCCACGACG-3′; reverse primer, 5′-CTGGATCCTATTCCGTGTCCTCGACG-3′. The following primers were used to amplify the P450SU2-Fd2 genes: forward primer, 5′-GTCATATGACGACCGCAGAACGCACC-3′; reverse primer, 5′-CAGGATCCTCAGTCGGTCACCGTGATC-3′. The PCRs were carried out as described in the Taq PCR Handbook (Qiagen) with Taq DNA polymerase and Q solution. The PCR mixture was heated at 95°C for 5 min and then subjected to 25 cycles of amplification (95°C, 30 s; 65°C, 1 min; 72°C, 2 min) followed by a final extension at 72°C for 10 min. The 1.5-kb PCR products were cloned first into the pCRII-TOPO vector to produce pQR270 for P450SU1-Fd1 and pQR271 for P450SU2-fd2. The DNA sequences of the PCR-amplified fragments were determined at this stage. The resulting PCR products were then digested with NdeI and BamHI and subcloned into those sites in the overexpression vector pET21a to produce pQR273 for P450SU1-Fd1 and pQR274 for P450SU2-Fd2. These constructs contain the complete P450-coding region and that encoding its ferredoxin under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible T7 RNA polymerase. The constructs pQR273 and pQR274 were then transformed into E. coli BL21(DE3)pLysS.

Heterologous expression and activity in E. coli.

E. coli BL21(DE3)pLysS was used as a host for the expression of S. griseolus P450 genes. The transformants were grown overnight at 37°C in Luria-Bertani medium containing ampicillin (100 μg per ml) and chloramphenicol (34 μg per ml). One hundred milliliters of Terrific broth (43) containing ampicillin and chloramphenicol was inoculated with 1 ml of the overnight culture and incubated at 37°C and 250 rpm. When the optical density (A600) reached 0.6 to 0.8, 1 mM α-aminolevulinic acid (ALA) and 0.5 mM FeCl3 were added to the culture. After further incubation for 20 min at 25°C, the expression was induced by the addition of 1 mM IPTG (41), and cells were grown for a further 24 to 48 h at 25°C and 180 rpm. For protein visualization, cells were isolated by centrifugation and resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and heated to 90°C. Proteins were separated by SDS-PAGE on 15% gels and stained with Coomassie blue. The amounts of overexpressed proteins were measured by densitometry.

For quantitation of recombinant cytochrome P450, cells were isolated by centrifugation at 4°C and resuspended in 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM EDTA, 20% glycerol, 1.5 mM dithiothreitol (DTT), and 0.4% Triton. Lysozyme (0.5 mg per ml) was added, and the suspension left on ice for 30 min. Phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM, and the mixture was sonicated on ice with an MSE Soniprep cell disrupter by using 10 pulses for 10 s with 30-s cooling intervals. Cell debris was removed by centrifugation, and the cytosol was isolated by centrifugation at 100,000 × g for 60 min. The soluble fraction was then used to measure the CO-reduced P450 absorption with a CARY3 UV-visible spectrophotometer (Varian). Extracts were diluted in 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM EDTA, 20% glycerol, 1.5 mM DTT, and 0.4% Triton, and the carbon monoxide-reduced difference spectra were measured as described by Omura and Sato (32) by using an extinction coefficient of 91 mM−1 cm−1. Protein content was measured by the method of Bradford (3).

Biotransformation of 7-ethoxycoumarin by P450s.

The side chain cleavage of 7-ethoxycoumarin was used in whole-cell biocatalysis assays. Both the substrate and product are fluorescent and could be readily detected by UV light (300 to 320 nm). This gives a sensitive qualitative method of assaying on thin-layer chromatography (TLC) plates. Recombinant cytochrome P450 monooxygenases in different E. coli strains, grown as described above, were induced as described above, and 1 mM 7-ethoxycoumarin was added 10 min after induction with IPTG in Terrific broth medium, followed by slow growth at 25°C for 48 h. One milliliter of the mixture was extracted with 0.5 ml of ethyl acetate, and the substrate and products were separated by silica gel TLC (TLC plate silica gel 60; Merck) using a solvent system of hexane-ethyl acetate (3:2 [vol/vol]) for the bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin.

Construction of expression vectors in Streptomyces and expression of P450 genes in S. lividans 1326.

A conjugative Streptomyces-E. coli shuttle expression vector (pBW160) constructed by B. Rudd (personal communication) was used. This vector contains oriT from the Inc-P group plasmid RP4 and requires transfer functions to be supplied in trans by the methylation-deficient E. coli ET12567 donor strain (21) carrying the nontransmissible pUZ8002. pBW160 is a derivative of pCJR29 (38) which has an SCP2* replication region and which has the act1 promoter for expression. The entire coding regions of the genes encoding cytochrome P450SU1 and P450SU2 with their ferredoxins were subcloned from pQR270 digested with NdeI and SpeI for P450SU1 and from pQR271 digested with NdeI and XbaI for P450SU2 into the NdeI/XbaI sites of the pBW160 shuttle expression vector. This allowed construction of pQR355 and pQR356, respectively. The expression plasmids (pQR355 and pQR356) were then transformed into the conjugative host strain E. coli ET12567(pUZ8002) and then transferred by conjugation into S. lividans 1326 as described by Flett et al. (7). The exconjugants were selected on thiostrepton.

The entire coding regions of the genes encoding cytochrome P450SU1 and P450SU2 with their ferredoxins were subcloned from pQR270 and pQR271 digested with NdeI and BamHI into the same sites of the conjugative shuttle vector pIJ8600. pIJ8600 contains the attP region of φC31 and allows the insertion of the gene as a single copy into the genome of S. lividans (42). pIJ8600 also has the tipA promoter, allowing controlled levels of expression by using thiostrepton. First the P450 genes and those of their ferredoxins were cloned into the pIJ8600 vector to produce pQR291 and pQR292 in E. coli, and then these constructs were transferred by conjugation into S. lividans 1326. The exconjugants were selected on apramycin and then tested by Southern blotting to confirm the insertion of the gene into the S. lividans genome.

Expression and activity of cytochrome P450 in S. lividans 1326.

S. lividans carrying recombinant plasmids was grown in 50 ml of GY medium plus 5 μg of thiostrepton per ml in 250-ml baffled Erlenmeyer flasks containing a coiled spring. After incubation of the cultures at 28°C in a rotary shaker for 40 h, 1 mM ALA was added to the culture. Following further incubation for 20 min at 28°C, the substrate 7-ethoxycoumarin was added to a final concentration of 1 mM, and cultures were incubated for a further 1, 2, 3, or 4 days. For quantitation of recombinant cytochrome P450, cells were harvested, washed by centrifugation in 20% sucrose, and resuspended in 100 mM potassium phosphate buffer, pH 7.5, containing 2 mM EDTA, 20% glycerol, 1.5 mM DTT, and 0.4% Triton. Lysozyme at 2 mg per ml was added, and the suspension was left on ice for 30 min. Phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM, and the mixture was then broken in a French pressure cell at 15,000 lb/in2. The extracts were clarified by centrifuging cell debris and unbroken mycelia at 100,000 × g for 60 min (4°C). The supernatant was used to analyze the CO-reduced difference spectra as described by Omura and Sato (32). For substrate analysis, 1 ml of the cell culture was extracted with 0.5 ml of ethyl acetate and analyzed by TLC for the bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin.

Construction of a vector for coexpression of two proteins in E. coli.

New vector pQR293 for the coexpression of recombinant genes in E. coli was developed to enable efficient coexpression of more than one gene under the control of the T7 RNA polymerase. This was achieved by reconstruction of the pALTER.Ex2 expression vector (Promega). This plasmid has the low-copy-number p15a origin of replication, which is compatible with the ColE1 origin of replication, and has a tetracycline resistance marker for selection. pALTER.Ex2 vector DNA has two promoters (tac and T7), which lie opposite to each other. The tac promoter and its ribosome binding site have been removed by deleting the BamHI/HindIII fragment and removing the sticky ends by filling in with Klenow. The resulting plasmid has only the T7 promoter, which can be expressed in E. coli BL21(DE3) alongside other compatible vectors such as the pET series.

Cloning and expression of S. coelicolor ferredoxin reductases.

The entire coding regions of ferredoxin reductase SC4B10 and ferredoxin NADP reductase SCF15A genes were generated as PCR fragments from the published sequence (S. coelicolor genome project [http://www.sanger.ac.uk/Projects/S_coelicolor/]) of S. coelicolor genomic DNA. The primers pairs were designed to add an NcoI site at the 5′ end of the coding sequence and have a PstI site on the 3′ side of the gene, added by using, for the ferredoxin reductase SCF15A gene, forward primer 5′-GACCATGGCCCGCCCTCTGCGGGTAG-3′ and reverse primer 5′-GACTGCAGTCAGGCGCCGCTCTCGCG-3′ and, for the ferredoxin reductase SC4B10 gene, forward primer 5′-GTCCATGGCGCGTGCGAAGACGTTC-3′ and reverse primer 5′-GACTGCAGTCACGCCTGGTCTCCCG-3′. The PCR conditions were as described above. The 1.2-kb PCR product of the SC4B10 gene and the 1.3-kb PCR product of the SCF15A gene were first cloned into pCRII-TOPO vector to produce pQR294 (for SC4B10) and pQR295 (for SCF15A). The DNA sequence of the PCR-amplified fragment was determined at this stage. The resulting PCR products were then digested with NcoI and PstI and subcloned into those sites in the reconstructed pALTER.Ex2 expression vector (pQR293). This allowed construction of pQR293 derivatives containing the complete ferredoxin reductase-coding region under the control of the T7 promoter, producing pQR296 (for SC4B10) and pQR297 (for SCF15A). The expression plasmids were then transformed into E. coli BL21 Star(DE3) containing compatible plasmid pQR273 (P450SU1) or pQR274 (P450SU2). The addition of tetracycline at the normal concentration (12 μg per ml) to the medium to select for the pQR293 derivatives led to slow cell growth and reduced expression of P450 in E. coli. Therefore, the level of expression was enhanced by reducing the concentration of tetracycline to 5 μg per ml.

In addition to the above constructs, we also constructed operons containing the cytochrome P450, ferredoxin, and ferredoxin reductase genes, which are transcribed from the T7 promoter in one plasmid. The ferredoxin NADP reductase SCF15A gene was subcloned from the pALTER.Ex2 vector derivative, pQR297, into the expression vector pET21a derivatives containing the P450SU1 (pQR273) and P450SU2 (pQR274) genes and their adjacent ferredoxins. This was carried out by first digesting pQR296 and pQR297 with NotI and blunt ending with Klenow fragments. The plasmids were then digested with EcoRI to isolate fragments that contain the ribosome binding site, the T7 promoter, and the ferredoxin reductase gene. These fragments were then cloned downstream of each of the P450 genes and the associated ferredoxin genes in pQR273 and pQR274, which had been digested with HindIII and blunt ended with Klenow fragments, followed by EcoRI digestion, giving the gene order P450-ferredoxin-ferredoxin reductase. These new constructs (pQR367 and pQR368) were then transformed into E. coli BL21 Star(DE3)pLysS.

RESULTS

Expression of S. griseolus cytochrome P450s in E. coli.

The P450 genes and the genes encoding their ferredoxins were amplified from S. griseolus ATCC 11796 genomic DNA by PCR using primers from the DNA sequence of the P450SU1 (CYP105A1) and P450SU2 (CYP105B1) genes and cloned into the pCRII-TOPO vector. The sequences of both DNA strands were determined, and this revealed the expected DNA sequence of the genes encoding P450SU1 and P450SU2 and their ferredoxins, Fd1 and Fd2. The genes encoding P450 and their ferredoxins were then cloned into the pET21a vector and transformed into E. coli BL21(DE3)pLysS (in order to express the proteins in E. coli.) The molecular masses of P450SU1 and P450SU2 were both 44 kDa as confirmed by SDS-PAGE and are consistent with the predicted sequences. The P450SU1 protein showed a lower level of expression, representing about 13% ± 0.2% of the total cellular protein, compared to the high level of expression of the P450SU2 polypeptide, which represents about 25% ± 0.1% of the total protein as seen on SDS-PAGE gel (Fig. 1A and B). However, the amount of protein seen as a band on an SDS-PAGE gel does not show how much of the protein is correctly folded and active.

FIG. 1.

FIG. 1.

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(A) SDS-PAGE of different E. coli host strains containing extra copies of rare codons expressing P450SU1. Lane 1, molecular mass markers as shown on the left; lane 2, BL21 cells; lane 3, pQR273 in BL21 Star(DE3)pLysS; lane 4, pQR273 in Rossetta(DE3); lane 5, pQR273 in Rossetta(DE3)pLysS; lane 6, pQR273 in BL21-CodonPlus(DE3)-RP; lane 7, pQR273 in BL21-CodonPlus(DE3)-RIL. (B) pQR274 (P450SU2) in BL21 Star(DE3)pLysS. (C) CO difference spectra of P450SU2 expressed in E. coli BL21 Star(DE3)pLysS.

Evaluation of the levels of correctly folded recombinant P450 was carried out by determining the CO-reduced difference spectra (9, 40). The cells were grown in Terrific broth alone or with the heme precursor, ALA, or with ALA and FeCl3. P450SU2 showed only the distinctive peak at 450 nm (Fig. 1C), with the amounts of cytochrome P450, as determined by its CO-reduced spectra, being 13 nmol per g of protein for Terrrific broth alone, 95 nmol per g of protein for Terrific broth supplemented with 1 mM ALA, and 125 nmol per g of protein for Terrific broth supplemented with 1 mM ALA and 0.5 mM FeCl3. Thus supplementation with ALA and FeCl3 produced almost a 10-fold increase in active P450. In contrast the yield of active P450SU1 was not enhanced by the addition of ALA and FeCl3 to the growth medium.

Optimizing expression of P450 using different host strains and growth conditions.

Different conditions were used in an attempt to optimize the amount of active cytochrome P450SU1 and P450SU2 in E. coli. Initial studies were carried out with E. coli BL21(DE3)pLysS and were aimed at determining the optimal IPTG concentration for induction of P450SU1 and P450SU2. The optimal level of IPTG was 1 mM, and there was no difference between addition of IPTG at the beginning of growth and addition at mid-log phase (optical density at 600 nm, 0.8).

Time course studies revealed that the maximum production of P450SU2, as determined by evaluation of CO difference spectra, was obtained after 24 to 35 h of induction at 25°C, and this amount was reduced by one-half after 60 h. Accordingly all subsequent expression studies were carried out with an induction period of 35 h and growth at 25°C.

Although the BL21(DE3)pLysS E. coli strain is a convenient and effective host for protein expression, the production of the recombinant P450SU1 was limited. Only P450SU2 was successfully expressed in this E. coli strain. In an attempt to increase the levels of P450SU1 and P450SU2 expression in E. coli, P450SU1 and P450SU2 with their ferredoxins (encoded by pQR273 and pQR274, respectively) were transformed into various E. coli expression strains (Table 1). The cells were grown, induced, and harvested 24 and 35 h after induction. SDS-PAGE revealed slight increases in soluble-protein profiles in BL21 Star(DE3) and BL21 Star(DE3)pLysS E. coli strains transformed with pQR273 and pQR274. The P450SU1 construct was expressed at a low level and yielded a readily measurable cytochrome P450 absorption peak (450 nm) when evaluated on the basis of CO difference spectra for seven E. coli host strains (six of which contain the pLysS plasmid). Five of the strains failed to produce a detectable cytochrome P450 signal. The highest expression of P450SU1, determined by CO-reduced spectra, was obtained with BL21 Star(DE3)pLysS, with maximal CO-reduced measurable cytochrome P450 production of 35 nmol per g of protein (Fig. 2). The expression of active P450SU2 was always higher than that of P450SU1. The most efficient expression was obtained with BL21 Star(DE3)pLysS. The yield of active P450SU2 expressed in BL21Star(DE3)pLysS was 256 nmol per g of protein.

FIG. 2.

FIG. 2.

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Expression of active S. griseolus cytochrome P450s in different E. coli host strains calculated from the reduced-CO absorbance. Each bar is the mean of two induction and expression experiments. The two values were between 5 and 8% of each other. Bars: 1, BL21(DE3); 2, BL21(DE3)pLysS; 3, AD494(DE3); 4, AD494(DE3)pLysS; 5, B834(DE3); 6, B834(DE3)pLysS; 7, BL21trxB(DE3); 8, BL21trxB(DE3)pLysS; 9, Tuner(DE3); 10, Tuner(DE3)pLysS; 11, BL21Star(DE3); 12, BL21Star(DE3)pLysS.

The side chain cleavage of 7-ethoxycoumarin by recombinant cytochrome P450SUI and P450SU2 in these strains was tested. 7-Ethoxycoumarin was added to the cultures after induction with IPTG. TLC analysis showed an increase in bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin when the P450s were expressed in BL21 Star(DE3)pLysS compared to bioconversion in the other host strains (data not shown).

Optimizing expression of P450SU1 in various E. coli host strains containing rare-codon tRNAs.

Examination of the codons present in the genes encoding cytochrome P450SU1 and its ferredoxin indicated that the coding sequences contain a large number of the least-used codons in E. coli, in particular CCC and CCT (proline), GGA (glycine), and CGG and CGA (arginine), which are very rarely used in E. coli genes. This may be the reason for the low-level protein expression of cytochrome P450SU1 and low-level expression of either of the cloned ferredoxins as assessed by SDS-PAGE. Comparison of the codon usage of P450SU1 and P450SU2 gene sequences indicated that the P450SU2 gene uses the more-prevalent E. coli codons.

The frequencies of rare codons in E. coli genes are as follows: CCC (Pro), 0.5%; CCT (Pro), 0.7%, GGA (Gly), 0.8%, CGG (Arg) 0.3%, CGA (Arg), 0.5% (23). In the P450SU1 gene the frequencies of these codons are as follows: CCC, 1.5%; CCT, 2.3%; GGA, 2.4%; CGG, 2.4%, CGA, 3%.

Table 2 shows the use of modified BL21(DE3) E. coli cells, namely, BL21-CodonPlus(DE3)-RP, BL21-CodonPlus(DE3)-RIL cells, or Rossetta(DE3) and Rossetta(DE3)pLysS. These expression hosts have been constructed to contain extra copies of the E. coli tRNA genes with rare codons for the expression of genes from organisms with codon usage different from that of E. coli. The most efficient expression of P450SU1 in these hosts was obtained with BL21-CodonPlus(DE3)-RP and BL21-CodonPlus(DE3)-RIL. P450SU1 represented about 18% of the total protein as seen on Coomassie blue-stained SDS-PAGE gel (the value for the expressed protein band in a duplicate induction experiment was within 0.2%). None of the ferredoxins (molecular mass, 7 kDa) were visible on SDS-PAGE gel when stained with Coomassie brilliant blue but were visible when Tricine SDS gels were used and were stained with silver when expressed in all of the expression strains used here (data not shown). The highest activity of P450SU1 was seen when BL21-CodonPlus(DE3)-RIL was used as the expression host. The yield for active cytochrome expressed from P450SU1 was 50 nmol per g of protein (Table 2).

TABLE 2.

Expression of active S. griseolus cytochrome P450SU1 in different E. coli host strains containing extra copies of E. coli rare-codon tRNA genes

E. coli strain Protein concna (g/liter) Level of active P450b in:
nmol/ liter nmol/g of protein % Soluble protein
P450SU1 (pQR273) in BL21 Star(DE3)pLysS 1.55 60 39 4.8
P450SU1 (pQR273) in Rossetta(DE3) 1.45 27 19 2.5
P450SU1 (pQR273) in Rossetta(DE3)pLysS 1.25 26 21 2.9
P450SU1 (pQR273) in BL21-CodonPlus(DE3)-RP 1.45 43 30 4.2
P450SU1 (pQR273) in BL21-CodonPlus(DE3)-RIL 1.50 75 50 6.5
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a

This is the total protein concentration in the cell extract after differential centrifugation.

b

Active P450 is calculated from the reduced-CO absorbance as described in Materials and Methods. The data are the means of two different expression experiments for each construct combination. The two values were between 4 and 7% of each other.

Optimizing expression of P450 in the presence of ferredoxin reductase.

The genes encoding the native S. griseolus ferredoxin reductases have not yet been cloned or characterized. Instead two of the ferredoxin reductase genes, which have been identified in the genomic DNA sequence of S. coelicolor A3 (2), were amplified and expressed alongside the recombinant P450SU1 and P450SU2 genes. Two forms of construct were made. The first used a coexpression system produced by cloning the ferredoxin reductase genes separately into the expression vector pQR293 (Table 1). Plasmids were then introduced into an E. coli expression host containing genes encoding P450SU1 or P450SU2 and their ferredoxins on a pET vector. This two-plasmid system allows expression of two different genes cloned under the control of T7 promoters and allowed different combinations of cloned genes to be rapidly set up by transforming one of the expression plasmids into a strain with an existing expression plasmid. These two plasmids contain compatible replication origins, which should yield approximately equal copy numbers of both vectors. The proteins were then expressed together in E. coli BL21 Star(DE3), and the CO-reduced activity was measured (Fig. 3A). This shows that for both P450SU1 and P450SU2 only the ferredoxin reductase SCF15A gives an increase in active P450.

FIG. 3.

FIG. 3.

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Expression of S. griseolus cytochrome P450s in different E. coli host strains containing different ferredoxin reductases. (A) Reduced-CO activity. Each column is an average of three experiments. (B) Reduced-CO difference spectrum of P450SU1 expressed in E. coli without ferredoxin reductase (i) and with ferredoxin reductase (ii). (C) TLC analysis showing the bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin. Lanes: 1, pQR273 in BL21 Star(DE3)pLysS; 2, pQR273 and pQR296 in BL21 Star(DE3); 3, pQR273 and pQR297 in BL21 Star(DE3); 4, pQR367 in BL21 Star(DE3)pLysS; 5, pQR274 in BL21 Star(DE3)pLysS; 6, pQR274 and pQR296 in BL21 Star(DE3); 7, pQR274 and pQR297 in BL21 Star(DE3); 8, pQR368 in BL21 Star(DE3)pLysS.

The second type of construct was an operon consisting of three genes in one expression plasmid, made by cloning the ferredoxin reductase SCF15A gene downstream of the genes encoding the P450s and their ferredoxins. The new constructs pQR367 and pQR368 were then expressed in E. coli BL21 Star(DE3)pLysS, and cells were grown with ampicillin and chloramphenicol.

The highest activity was seen with the constructs (pQR367 and pQR368) that contain a P450 operon and that express ferredoxin and ferredoxin NADP reductase SCF15A, with maximum levels of spectrally measurable cytochrome P450 production of 55 nmol per g of protein for P450SU1 and 413 nmol per g of protein for P450SU2 (Fig. 3A). Figure 3B shows the increased amount of the absorbing form of the cytochrome P450, P450SU1, when a ferredoxin reductase is coexpressed in the same cell. There is a shift in the ratio of P420 to P450 in the spectrum shown in Fig. 3B, ii, for the strain containing the ferredoxin reductase SCF15A from S. coelicolor.

The hydroxylation of 7-ethoxycoumarin by recombinant P450SU1 and P450SU2 in several E. coli strains was tested. TLC analysis of extracts shows a qualitative increase in bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin by the construct that contains P450SU1 or P450SU2 with their ferredoxins and the ferredoxin reductase (Fig. 3C).

Expression of S. griseolus P450s in S. lividans 1326.

Our first attempt to introduce P450SU1 and P450SU2 into S. lividans used plasmids (pQR291 and pQR292) in which the P450SU1 and P450SU2 genes were positioned downstream of the thiostrepton-inducible tipA promoter. Reduced-carbon monoxide difference spectra of the soluble extract of S. lividans carrying one copy of P450SU1 or -SU2 following the addition of ALA in GY medium, with growth at 28°C after induction of the tipA promoter, were investigated. Identical parallel cultures of S. lividans were used as the negative control. The induced cultures show a distinctive peak at 420 nm for the inactive form of P450 in the strains with the inserted P450SU1 and -SU2 but no 450-nm peak. The effect of addition of FeCl3 to the medium was investigated, and again only the 420-nm absorbance was found.

The S. griseolus P450 genes were then cloned into the low-copy-number conjugative Streptomyces-E. coli shuttle expression vector pBW160, in which the genes encoding P450SU1 and P450SU2 with their ferredoxins were positioned downstream of the actI promoter. The resulting E. coli clones, pQR355 and pQR356, were conjugated into S. lividans 1326. S. lividans carrying recombinant plasmids pQR355, pQR356, and pBW160 as the negative control was grown in GY medium at 28°C followed by the addition of ALA. Cells were grown for 2, 3, and 4 days, and the cell extract was analyzed for the presence of P450SU1 and P450SU2 as measured by their CO difference spectra. S. lividans 1326 containing pQR355 or pQR356 showed the 450-nm absorbance characteristic of P450s, and this was not observed in extracts of the control culture, S. lividans 1326(pBW160), in spite of the probable presence—because we assume that the S. lividans and S. coelicolor genomes have the same structural and genetic organizations—of 20 cytochrome P450 genes in the genomic DNA of S. lividans (S. coelicolor genome project [http://www.sanger.ac.uk/Projects/S_coelicolor/]) (20).

Reduced-carbon monoxide difference spectra of the soluble fractions of S. lividans 1326 strains containing pQR355 (P450SU1) or pQR356 (P450SU2) showed a peak absorbance at 450 nm, which was not observed in extracts of the control culture, S. lividans 1326(pBW160). The yield for active cytochrome expressed from P450SU1 was 27 nmol per g of protein; for P450SU2 the yield was 28 nmol per g of soluble protein.

The catalytic activities of P450SU1 and P450SU2 were investigated with the model substrate 7-ethoxycoumarin following growth at 28°C on GY medium containing ALA for 1, 2, 3, or 4 days. TLC analysis of extracts from cells containing P450SU1 and P450SU2 revealed the ο-dealkylation of 7-ethoxycoumarin to 7-hydroxycoumarin (Fig. 4), not observed in extract of the control culture, S. lividans 1326(pBW160). The TLC analysis showed a greater ο-dealkylation of 7-ethoxycoumarin by P450SU1 than by P450SU2.

FIG. 4.

FIG. 4.

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TLC analysis showing the bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin by S. griseolus cytochrome P450SU1 and P450SU2 in S. lividans.

DISCUSSION

S. griseolus ATCC 11796 contains two cytochrome P450 proteins, P450SU1 and -SU2, which can hydroxylate a variety of sulfonylurea herbicides (33). These are distinct gene products with different spectroscopic, chromatographic, and antigenic properties (25, 26, 31). The cytochrome P450SU1 and P450SU2 genes have been expressed previously in E. coli, but the amount of measurable cytochrome P450 production was low as calculated from the CO difference spectrum (31). Our first expression constructs of P450SU1 and -SU2 show a P450 band of about 13% of total cell protein for P450SU1 and 25% of total cell protein for P450SU2 on SDS-PAGE gel, but this is not matched by the amount of active P450 as calculated from the CO-reduced absorbance. We have, therefore, attempted to develop an efficient expression system for P450SU1 and P450SU2 to allow production of active, correctly folded enzyme.

A series of modifications were required to improve expression of P450SU1 and P450SU2 in E. coli. The changes required were in growth conditions and the choice of E. coli host strain. Adding ALA and FeCl3 to the growth medium increased the amount of spectrally measurable cytochrome P450SU2 expression. In other systems, the addition of ALA to the culture medium stimulates heme biosynthesis to match the increase in P450 polypeptide synthesis (15). It has been observed previously that such an increase of P450 suggests that the rate-limiting step in the synthesis of the hemoprotein lies in the synthesis of the heme prosthetic group (16, 35). This fact, together with the increases above in the amount of CO-reduced P450, suggests that it is not only the protein expression level but also the final formation of correctly folded holoenzyme with the heme group in its proper protein environment which increase when ALA and FeCl3 are added. Significantly, the difference spectra of P450SU2 showed no absorbance at 420 nm, indicating no conversion of the cytochrome to the inactive, low-spin, P420 form when ALA and FeCl3 were added. The amount of spectrally measurable cytochrome P450SU1 expression was not enhanced by the addition of ALA and FeCl3 to the growth medium. The observation of P450-to-P420 conversion was first described by Omura and Sato (32), and subsequently this transition has been induced by using various conditions and chemicals (2, 12, 13). Healy et al. (10) explained that the reason that they were unable to observe the 450-nm reduced-CO difference spectrum with cytochrome P450 TxtC may be improper protein folding or improper incorporation of the heme group into the apoenzyme in E. coli.

Although E. coli has a remarkable capacity to produce a large quantity of protein, there are limits when the codon usage in the mRNA for the recombinant gene differs from that of the E. coli host cells (5, 17). It is known that an excess of any of these rare codons creates problems during translation, leading to a reduction in the quantity of the protein synthesized. Moreover, the cellular levels of certain tRNAs may fluctuate with growth conditions (4, 14). This was demonstrated previously by Kleber-Janke and Becker (19), who increased expression of several recombinant peanut allergens in E. coli. The expression of the P450SU1 protein was enhanced from 13% ± 0.2% to 18% ± 0.2% of the total protein (from densitometry of stained bands on SDS-PAGE gel) with these modified cells, in particular BL21-CodonPlus(DE3)-RP and BL21-CodonPlus(DE3)-RIL. The amount of spectrally measurable cytochrome P450SU1 was also increased by the same ratio, from 35 to 50 nmol per g of protein. The levels of active P450SU1 are not as high as those of P450SU2; this is probably due to the presence of other rare codons in the P450SU1 gene which are not provided by these CodonPlus strains. These two strains have extra copies of only the tRNAs for argU (AGA and AGG) and proL (CCC) in BL21-CodonPlus(DE3)-RP and argU (AGA and AGG), ileY (AUA), and leuW (CUA) in BL21-CodonPlus(DE3)-RIL but do not have cloned tRNA genes with CCT (Pro), GGA (Gly), and CGG (Arg) and CGA codons, which are present in the P450SU1 gene.

Prokaryotic cytochrome P450 systems are usually multicomponent systems and require the presence of a ferredoxin reductase and a ferredoxin to couple electron flow from NAD(P)H to the terminal cytochrome P450 component. Microbial cytochrome P450s can be one of two different classes: class I (or B class) P450, receiving electrons from a two-component reductase system, ferredoxin, and NADH ferredoxin reductase, such as in P450CAM, (22), or class II (or E class) P450, receiving electrons from NADPH-cytochrome P450 reductase fused to the P450 in one continuous polypeptide, such as in P450BM3 (24) and P450Rhf (36). Ferredoxin reductase is an essential component of the cytochrome P450 monooxygenase complex. Only a few ferredoxin reductases of prokaryotic P450 systems have been purified and characterized because of their unstable nature and relatively low levels of expression (1, 34).

The active level of cytochrome P450SU2 was improved, and that of P450SU1 was slightly improved, in the strains with the rare-codon tRNA as calculated on the basis of the reduced-CO spectrum, so we tested an additional way of improving activity. In E. coli the yield of correctly folded P450 was further improved by cloning the S. coelicolor ferredoxin reductases SCF15A and SC4B10 and coexpressing these ferredoxin reductases in the same cell from a recombinant plasmid (Fig. 3A and B) and cloning the gene encoding ferredoxin reductase SCF15A downstream of the genes encoding P450 and their ferredoxins. This shifts the ratio of P420 to P450 in the direction of P450. It appears that simply coexpressing the ferredoxin reductase alongside the P450 and ferredoxin stabilizes the folded, active form of the P450, as shown by the reduced-CO spectrum when no substrate is present and no electron flow is needed. It may be that this indicates an in vivo association of these proteins which stabilizes the P450. Because of this increased active P450 form, a greater whole-cell biotransformation is seen, and this may be further enhanced by the ferredoxin reductase, which provides the correct electron transfer pathway. This shows that the P450-ferredoxin pair functions in vivo, that S. griseolus ferredoxins can accept electrons from these two S. coelicolor ferredoxin reductases, and that S. coelicolor ferredoxin reductase has a broad specificity to donate electrons to nonnative ferredoxins.

The initial failure to enhance the amount of spectrally measurably cytochrome P450 production of P450SU1 in E. coli prompted us to look at a suitable alternative host. S. lividans was selected because it is the closest relative to S. griseolus for which well-developed expression vectors exist and hence the most homologous expression background that was readily available for expressing recombinant S. griseolus P450 genes. Expression of another cytochrome P450 in S. lividans has been successfully achieved previously (6, 11) although this was done to investigate the role of P450 in antibiotic biosynthesis. An SCP2-based plasmid expression system was used to express P450SU1 and P450SU2 in S. lividans. The level of recombinant P450SU1 and P450SU2 activity obtained in S. lividans was lower than the activity obtained from the E. coli strain. This is probably because of the lower-copy-number expression vector, pBW160, in S. lividans and the use of the actI promoter, which is presumably weaker than the T7 promoter. The level of active P450 might be further enhanced in S. lividans by the expression of the ferredoxin reductase SCF15A gene of S. coelicolor cloned downstream of the P450 genes, similar what has been achieved in E. coli to enhance expression of P450SU1 and P450SU2 monooxygenases. These activities will probably also be enhanced once the indigenous electron transport component ferredoxin reductase of the S. griseolus cytochrome P450 system is cloned. However, TLC analysis showed that the bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin by the recombinant P450SU1 and P450SU2 in S. lividans was two- to threefold higher than that obtained from the recombinant E. coli strain. This indicates that the Streptomyces host might be more robust in the face of prolonged contact with the biotransformation substrate than E. coli.

The strategy for increasing the activity of these two bacterial cytochrome P450s, namely, the inclusion of ferredoxin reductase to both stabilize the P450 and then provide an optimal electron transfer system, may be a general phenomenon which could be applied to the enhancement of other cloned P450 enzymes.

Acknowledgments

This work was sponsored by the BBSRC LINK Applied Biocatalysis program grant no. 31/ABC11433. This link grant has support from GSK, Pfizer, and Ultrafine Chemicals, United Kingdom.

We are very grateful for the comments and advice of B. Henderson on the manuscript.

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