Engineering the CYP101 system for in vivo oxidation of unnatural substrates

Stephen G. Bell, Charles F. Harford-Cross and Luet-Lok Wong,1

Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary and conclusions
 References
 
The protein engineering of CYP enzymes for structure–activity studies and the oxidation of unnatural substrates for biotechnological applications will be greatly facilitated by the availability of functional, whole-cell systems for substrate oxidation. We report the construction of a tricistronic plasmid that expresses the CYP101 monooxygenase from Pseudomonas putida, and its physiological electron transfer co-factor proteins putidaredoxin reductase and putidaredoxin in Escherichia coli, giving a functional in vivo catalytic system. Wild-type CYP101 expressed in this system efficiently transforms camphor to 5-exo-hydroxycamphor without further oxidation to 5-oxo-camphor until >95% of camphor has been consumed. CYP101 mutants with increased activity for the oxidation of diphenylmethane (the Y96F–I395G mutant), styrene and ethylbenzene (the Y96F–V247L mutant) have been engineered. In particular, the Y96F–V247L mutant shows coupling efficiency of approximately 60% for styrene and ethylbenzene oxidation, with substrate oxidation rates of approximately 100/min. Escherichia coli cells transformed with tricistronic plasmids expressing these mutants readily gave 100-mg quantities of 4-hydroxydiphenylmethane and 1-phenylethanol in 24–72 h. This new in vivo system can be used for preparative scale reactions for product characterization, and will greatly facilitate directed evolution of the CYP101 enzyme for enhanced activity and selectivity of substrate oxidation.

Keywords: CYP101/in vivo/oxidation/P450cam/pCW/putidaredoxin/putidaredoxin reductase/self-sufficient/whole-cell systems


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary and conclusions
 References
 
The cytochrome P450 (CYP) family of enzymes catalyse the oxidation of an astonishing range of organic compounds in many reactions of crucial importance in living organisms. The primary activity of CYP enzymes is the oxidation of chemically inert, often non-activated aliphatic C–H bonds to the alcohol functionality. This reaction has no equivalent in classical chemical synthesis and thus CYP enzymes have huge potential in biotransformation, e.g. for the preparation of key synthetic intermediates and the single-step synthesis of fine chemicals.

CYP101 (cytochrome P450cam) catalyses the oxidation of camphor to 5-exo-hydroxycamphor as the first step in camphor metabolism by Pseudomonas putida (Gunsalus and Wagner, 1978Go). The availability of crystal structures of wild-type CYP101 and mutants with a variety of bound substrates (Poulos et al., 1987Go; Raag and Poulos, 1991Go) make this enzyme an attractive target for protein engineering for biotransformation and other biotechnological applications. Active site mutants of CYP101 have been shown to oxidize a wide range of compounds (Loida and Sligar, 1993Go; Mueller et al, 1995Go; Wong et al., 1997Go; England et al., 1998Go; Bell et al., 2001Go; Jones et al., 2001Go). CYP101 activity requires two other proteins, the flavoprotein putidaredoxin reductase (PdR) and the iron–sulfur protein putidaredoxin (Pd), to mediate the transfer of electrons from the NADH co-factor to the CYP101 enzyme. Mammalian enzymes typically require a FAD/FMN dependent NADPH-P450 reductase instead of two separate electron transfer proteins. The feasibility of applying CYP101 and other CYP enzymes in biotransformation depends on maximizing the catalyst lifetime whilst minimizing or removing the need to use co-factors. In vitro co-factor recycling systems are available, or the NAD(P)H co-factor and the electron transfer proteins can be eliminated altogether by supplying the electrons from an electrode. Electrochemically-driven substrate oxidation by CYP101 (Kazlauskaite et al., 1996Go; Zhang et al., 1997Go; Lvov et al., 1998Go) and mammalian CYP enzymes have been described (Estabrook et al., 1996aGo) and preparative scale reactions reported (Estabrook et al., 1996bGo).

An alternative strategy to co-factor recycling and electrochemically driven turnover is a whole-cell system in which all the proteins in a CYP enzyme system are expressed in a single heterologous host to constitute a catalytically competent in vivo substrate oxidation system. CYP102 (P450BM-3) from Bacillus megaterium contains a monooxygenase and an electron transfer reductase in a single polypeptide chain (Fulco, 1991Go) and this self-sufficient system can readily support in vivo substrate turnover (Schneider et al., 1998aGo,bGo). Using CYP102 as a model, substrate oxidation systems have been developed in which a CYP enzyme and a NADPH-P450 reductase are expressed as catalytically active fusion proteins in yeast or E.coli (Fisher et al, 1996Go; Gillam et al., 1997Go; Parikh and Guengerich, 1997Go; Liu et al., 1998Go; Lacour and Ohkawa, 1999Go). Triple fusion enzymes have also been reported for CYP11A1 (Harikrishna et al., 1993Go) and CYP101 (Sibbesen et al., 1996Go). The latter system was shown to oxidize camphor to 5-exo-hydroxycamphor. In vitro studies showed the triple fusion protein to be significantly less active than the reconstituted system and the in vivo reaction produced the further oxidation product 5-oxo-camphor before all of the camphor had been consumed (Sibbesen et al., 1996Go).

Mammalian CYP enzymes and NADPH-P450 reductases have also been co-expressed as separate entities from bicistronic constructs in E.coli, yeast and baculovirus systems (Blake et al., 1996Go; Chen et al., 1997Go), and shown to support catalytic substrate oxidation (Parikh et al., 1997Go; Pritchard et al., 1998Go). The in vivo activity of a fusion protein and a bicistronic system of rat NADPH-P450 reductase and CYPC17 for 17{alpha}-hydroxylation of progesterone and pregnelone have been compared (Shet et al., 1997Go). The bicistronic system had the higher activity and was used for the preparative scale oxidation of progesterone.

We report here the construction of a tricistronic plasmid containing the three genes of the CYP101 system. This plasmid enables E.coli cells to express the CYP101 enzyme and the two physiological electron transfer proteins for efficient substrate oxidation in vivo. Active site mutants of CYP101 with enhanced activity for the oxidation of diphenylmethane, styrene and ethylbenzene were engineered. The oxidation products of these compounds, in particular styrene oxide and 1-phenylethanol, are useful synthetic intermediates. The catalytic activity and applicability of the system for the in vivo oxidation of two classes of substrates—diphenylmethane, which is highly insoluble, and styrene and ethylbenzene, which have millimolar solubility in water—were investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary and conclusions
 References
 
General

The pCWori+ vector was provided by Richard Dalquist (University of Oregon, USA) and Roland Wolf (University of Dundee, UK). Enzymes for molecular biology were from New England Biolabs, UK. All chemicals were molecular biology grade or of the highest purity available and were from Sigma-Aldrich or Anachem, UK. General DNA manipulations followed standard methods (Sambrook et al., 1989Go). Site-directed mutagenesis was carried out by the Kunkel method (Kunkel, 1985Go). UV-vis spectra were recorded on a Varian CARY 1E spectrophotometer equipped with a Peltier temperature controller (±0.1°C). Gas chromatography (GC) analysis was carried out on a Fisons 8000 series instrument equipped with a flame ionization detector on a DB1 fused silica capillary column (0.25 mm i.d.x30 m) or a ß-DEX chiral phase column (0.5 mm i.d.x30 m).

DNA manipulations

The existing clone of PdR (pAW5301) had previously been constructed in our laboratory by amplifying the camA gene encoding PdR from the total cellular DNA of P.putida (ATCC 17453). The primer 5'-CAGAGATTAAGAATTCATAAACACATGGAGTGCGTGCCATATGAACGCAAAC-3' was used for binding to the 5' end of the camA gene to introduce an EcoRI site for cloning into the pGLW11 vector (a derivative of pKK223; Brosius and Holy, 1984Go), a ribosome-binding site (RBS) GGAG and a NdeI site at the start of the gene which also changed the start codon from GTG to ATG to increase the protein expression level (Peterson et al., 1990Go). Restriction enzyme recognition sequences and RBS are underlined. The primer 5'-AGCCTCCACGGATCCTCAGCTACTCAGGCAC-TACTCAG-3' was used at the 3' end to introduce a BamHI cloning site after the stop codon. The camA gene contained a HindIII site restriction site, which spanned amino acids 49–51 of PdR. This site was removed by introducing the silent mutation AAGCTT->AAGCCT (both GCT and GCC encode alanine at the 50 position).

The camB gene was amplified from P.putida using the primer 5'-AACAGAGATTAAGGATCCCTGAGTAGTGCCTGAAAT-3' which bound to the end of the camA gene in P.putida and introduced a BamHI cloning site. The amplified fragment therefore included the 70 bp region upstream of the RBS for the camB gene. The primer used for the 3' end, 5'-CGATGAAAGCTTCGATGCTCTAGACTCCACTCGCGCA-TAGATGGGGATTAGATATTCACCTACTTACCATTGCCT-ATCGGG-3', incorporated a 35 bp spacer between the end of the camB gene and the start of the camC to mimic the arrangement in P.putida (Unger et al., 1986Go). The RBS sequence GGAG to be used to initiate synthesis of the CYP101 enzyme was followed immediately by the XbaI and HindIII sites for the cloning of the camC gene encoding CYP101. The amplified fragment was then digested with BamHI and HindIII, purified by agarose gel electrophoresis and ligated with the similarly digested pAW5301 to generate the new plasmid pSGB. This plasmid was digested with NdeI and HindIII and the camAcamB fragment cloned into the expression vector pCWori+ to give the plasmid pCWSGB. The presence of the insert was confirmed by restriction digests and the insert was fully sequenced.

The camC gene was then cloned from the pCHC–camC plasmid (Westlake et al., 1999Go) into the pCWSGB plasmid using the XbaI and HindIII restriction sites to give the tricistronic plasmid pCWSGB–camC.

Expression of the enzymes in E.coli

The CYP101 enzymes and the electron transfer proteins (Pd and PdR) were expressed in E.coli and purified according to literature methods (Peterson et al., 1990Go; Yasukochi et al., 1994Go; Westlake et al., 1999Go). Escherichia coli DH5{alpha} cells were transformed with the pCWSGB and pCWSGB–camC plasmids and plated on LB agar plates containing 50 µg/ml ampicillin. Transformants were streaked to single colonies which were used to inoculate 10 ml of LB broth containing 50 µg/ml ampicillin and grown at 30°C overnight. This culture was used to inoculate 1 l of LB broth. Protein expression was induced by the addition of 0.4 mM IPTG and the culture incubated for 20 h at 30°C.

In vivo substrate oxidation

The cells were harvested by centrifugation at 5000 g for 10 min and washed in E.coli minimal medium [EMM, per litre: 7 g of K2HPO4, 3 g of KH2PO4, 0.5 g of Na3citrate, 0.1 g of MgSO4, 1 g of (NH4)2SO4, 5 ml of 40% (w/v) glucose, 50 ml of 1 M Tris pH 7.4]. After centrifugation the cells were resuspended in 1 l of EMM and 500 ml aliquots were placed in 2 l Erlenmeyer flasks. Hexadecane was added to 2% (v/v) to act as the second phase in a two-phase system to protect the bacterium from the build-up of substrates and products to toxic levels (Preusting et al., 1993Go; Desai and Banat, 1997Go). The reactions were started by the addition of substrate from a 1 M stock in ethanol (2 ml of camphor, 1 ml of styrene and ethylbenzene and 300 µl of diphenylmethane). The reaction was monitored by removing 1 ml of the mixture, adding an internal standard and extracting with 400 µl of ethyl acetate. The organic phase was separated by centrifugation and analysed by GC.

For camphor the DB-1 column temperature was held at 60°C for 6 min then increased to 120°C at 50°C/min and held at this temperature for 15 min. The retention times were: camphor, 11.4 min; 5-exo-hydroxycamphor, 19.1 min; 5-oxo-camphor, 14.0 min. For diphenylmethane the temperature was increased from 150 to 230°C at 5°C/min and held at this temperature for 6 min. The retention times were: diphenylmethane, 5.6 min; 4-hydroxydiphenylmethane, 10.0 min. For styrene and ethylbenzene the column was held at 45°C for 2 min and then the temperature was increased to 100°C at 4°C/min. The retention times were: styrene, 8.3 min; styrene oxide, 14.3 min; ethylbenzene, 7.4 min; 1-phenylethanol, 14.3 min. In order to determine the rate of product formation, calibration plots were obtained for (1R,2R,5R)-(+)-2-hydroxy-3-pinanone (an isomer of 5-exo-hydroxycamphor), styrene oxide, 1-phenylethanol and 4-hydroxydiphenylmethane by extracting solutions containing buffer, product and internal standards and analysing the extracts as described above.

In vitro activity assays

Incubation mixtures (1.5 ml) contained 40 mM phosphate buffer pH 7.4, 1 µM CYP101, 10 µM Pd (1 µM for comparison between in vivo and in vitro oxidation of camphor), 1 µM PdR and 200 mM KCl. Substrates were added as a 1 M stock in ethanol to 1 mM final concentration. The mixture was incubated at 30°C for 2 min and the reaction started by the addition of 350 µM of NADH. The absorbance at 340 nm was monitored and the rates were calculated using {varepsilon}340 = 6.22/mM/cm. In calculating the substrate-dependent NADH turnover rate the background rate of NADH consumed by air oxidation of the reduced forms of the electron transfer proteins was taken into account (Nickerson et al., 1997Go). After the addition of an internal standard, to 1 ml of this incubation mixture, organics were extracted with 400 µl of ethyl acetate and the products analysed by GC as above. We found that it was necessary to obtain separate calibration plots for in vitro incubations, presumably because the proteins in the in vitro assays had different effects on the organic extraction steps compared to E.coli cells in the in vivo reactions. The coupling efficiency is defined as the proportion (expressed as a percentage) of NADH consumed that is utilized for product formation.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary and conclusions
 References
 
The pCWSGB plasmid

The genes encoding the three proteins of the CYP101 system were cloned into the pCWori+ vector in the order camA (PdR), camB (Pd) and camC (CYP101) downstream of the promoter and the RBS in the vector (Figure 1Go). This vector was chosen for the present system because it has two powerful tac promoters in tandem and it had been successfully used to express the triple fusion CYP101 system (Sibbesen et al., 1996Go) as well as numerous mammalian CYP enzymes. The restriction sites used for the construction of the in vivo CYP101 system were NdeI at the 5' end of camA, BamHI at the 3' end of camA and before camB, XbaI at the end of camB followed by HindIII. The new plasmid pCWSGB contained the camA and camB genes followed by a RBS and then the XbaI and HindIII restriction sites for the cloning of mutant camC genes into the plasmid. The resulting tricistronic pCWSGB–camC plasmid will express all three proteins in a single E.coli host. Gene transcription gives a single mRNA molecule and translation of each gene is initiated by its own RBS positioned at the optimal distance upstream of each ATG start codon.



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Fig. 1. Construction of the pSGB, pCWSGB and the pCWSGB-camC plasmids. (i) Cloning of the camB PCR product into pAW5301 using the BamHI and HindIII restriction sites. (ii) Cloning of camA and camB from pSGB into pCW using the NdeI and HindIII restriction sites. (iii) Cloning of the camC gene from pCHC-camC into pCWSGB using the XbaI and Hind III restriction sites.

 
The first step in the construction of the pCWSGB plasmid was to remove the HindIII restriction site present within the camA gene by introducing a silent mutation. The mutant PdR thus obtained was expressed in E.coli and purified. Its spectroscopic and catalytic properties were identical to those of the wild-type protein (data not shown). The camB gene was then amplified from the total cellular DNA of P.putida. One oligonucleotide primer was designed to retain a 70 bp sequence found naturally between the camA and camB genes in P.putida (Unger et al., 1986Go). The other primer contained a 35 bp sequence between camB and camC (Unger et al., 1986Go) and also introduced a RBS sequence (GGAG) for the expression of the camC gene which was to be inserted between the unique restriction sites XbaI and HindIII just downstream of this RBS. After restriction enzyme digestion and ligation into pAW5301, the camA–camB gene block was excised and cloned into the pCWori+ vector to generate the pCWSGB plasmid (Figure 1Go).

The pCWSGB plasmid was transformed into E.coli and used to express PdR and Pd. SDS–PAGE analysis showed two new bands that corresponded to the molecular weights of the two proteins (data not shown). The levels of expression were below those observed with E.coli cells expressing just one of these proteins. The cells were lysed by sonication and the supernatant was able to support in vitro camphor oxidation when the CYP101 system was reconstituted by adding wild-type CYP101 and NADH. We conclude that both PdR and Pd were functionally expressed and in their soluble forms. GC analysis revealed that 5-exo-hydroxycamphor was the only product (data not shown).

In vivo camphor oxidation

The camC gene encoding wild-type (WT) CYP101 was excised from the pCHC–WT plasmid (Westlake et al., 1999Go) with the restriction enzymes XbaI and HindIII and cloned into the pCWSGB plasmid. Escherichia coli cells containing the new pCWSGB–WT plasmid were grown in LB media and then induced with IPTG. The presence of active CYP101 enzyme in the cells was detected by the Fe2+-CO versus Fe2+ UV-vis difference spectrum (Omura and Sato, 1964Go). A small increase in the absorbance at 450 nm was observed which corresponded to a nominal CYP101 concentration of 50–75 nM. This expression level is significantly lower than that observed when CYP101 was expressed on its own in E.coli.

The E.coli cells were resuspended in EMM, camphor was added and the culture returned to the orbital shaker incubator. A 500 ml suspension of 2.5 g wet weight of cells was found to oxidize 4 mM (approximately 300 mg) of camphor completely within 4 h. Time-course analysis by GC showed that the rate of 5-exo-hydroxycamphor formation was 4460 ± 220 nmol/g wet cells/min. Assuming the expression level estimated from the difference spectrum, this activity corresponded to a camphor oxidation rate of approximately 150 nmol/nmol CYP101/min, which compared favourably with the activity of the in vitro reconstituted system containing the three proteins, PdR, Pd and CYP101, in a 1:1:1 ratio (220/min). The further oxidation of 5-exo-hydroxycamphor to 5-oxo-camphor, which was greater than expected in the in vivo oxidation of camphor by triple fusion proteins (Sibbesen et al., 1996Go), was found to occur with the pCWSGB–WT tricistronic system only after >95% of the camphor had been consumed (by GC). Even then this oxidation was found to be slow compared to camphor oxidation. Addition of further aliquots of camphor together with more carbon and nitrogen sources revealed that the system was active for at least another 48 h.

Engineering CYP101 for the oxidation of target unnatural substrates

Wild-type CYP101 does not oxidize diphenylmethane (Fowler et al., 1994Go), but shows low activity for the oxidation of styrene and ethylbenzene (Filipovic et al., 1992Go; Fruetel et al., 1992Go). The active site mutant Y96A has been reported to oxidize diphenylmethane to 4-hydroxydiphenylmethane (Fowler et al., 1994Go), but notably the Y96F mutant showed no activity (Bell et al., 1997Go). This was attributed to the accommodation of one phenyl group of diphenylmethane in the space generated by the Y96A mutation. The other phenyl group of the substrate would then point towards the haem. Pursuing this rationale we sought to increase the activity by altering the binding orientation of the substrate by targeting the I395 residue for mutagenesis. I395 is at approximately the same height above the haem as Y96, and its side-chain adopts a conformation to maximize van der Waals interactions with the phenyl side-chain of F87 (Figure 2Go). The I395G mutation should create space to accommodate one phenyl ring of diphenylmethane while orientating the other phenyl group in relation to the haem differently from the Y96A mutant. The double mutant Y96F–I395G was prepared; the Y96F mutation was used to increase the active site hydrophobicity to promote substrate binding. As shown in Table IGo, the activity of this double mutant was 2.5 times that of Y96A single-site mutant and both the NADH turnover rate and coupling efficiency were increased. This result strongly suggests that CYP101 can be engineered to oxidize compounds substantially larger than camphor by manipulating the active site volume and topology as so to manoeuvre the substrate binding orientation.



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Fig. 2. The active site structure of CYP101 with bound camphor. The two views, from the side (left) and the top (right) show that I395 is above V295 and at the height above the haem as F87 and Y96, while V247 is above L244 and T101.

 

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Table I. The NADH turnover and, in brackets, substrate oxidation rate (both given as nmol/nmol CYP101/min) and coupling efficiency (% of NADH consumed and utilized for product formation) for the oxidation of target unnatural substrates by wild-type CYP101 and active site mutants
 
We have shown previously that both the Y96A and Y96F mutations increased the styrene oxidation activity of CYP101 (Nickerson et al., 1997Go). Both mutations accelerated NADH turnover but the Y96F mutant had the higher styrene oxidation rate by virtue of a higher coupling efficiency. It was proposed that the larger phenyl side chain of the Y96F restricted styrene closer to the haem for more efficient substrate oxidation. In order to increase the styrene oxidation activity further we introduced the V247L mutation to reduce the space available in the upper part of the CYP101 substrate-binding pocket (Figure 2Go). The data in Table IGo show that the Y96F–V247L mutant had a slightly lower NADH turnover rate than the Y96F single-site mutant but the styrene oxidation activity was 50% higher because the coupling efficiency was nearly doubled by the V247L mutation. As for the WT and Y96F mutant, styrene oxide was the only product from the Y96F–V247L mutant.

The ethylbenzene oxidation activity of CYP101 has been investigated by site-directed mutagenesis of various active site residues, resulting in a 6-fold increase in activity over the wild-type (Loida and Sligar, 1993Go). Since ethylbenzene is structurally similar to styrene, we also examined its oxidation by the Y96F and Y96F–V247L mutants. 1-Phenylethanol was the only product. The data in Table IGo show that, compared with styrene, wild-type CYP101 had a 5-fold higher NADH oxidation rate but lower coupling efficiency with ethylbenzene. The Y96F and Y96F–V247L mutations increased the NADH turnover rate by approximately 30%, but the coupling efficiencies were increased by an order of magnitude. As with styrene, the Y96F–V247L double mutant was the most active (134 versus 3.3/min for the wild-type), primarily due to the very high coupling efficiency. For comparison, the rate of camphor oxidation by wild-type CYP101 under identical conditions is 1000/min. Overall, the data are entirely consistent with the rationale that the V247L mutation constrains styrene and ethylbenzene closer to the haem than in the wild-type and the Y96F mutant, thus giving rise to faster and more efficient oxidation of these two unnatural substrates.

The stereoselectivity of the CYP101 enzymes in the formation of the chiral oxidation products styrene oxide and 1-phenylethanol was investigated by chiral phase GC analysis. We reported previously that the Y96F mutation did not alter the selectivity of styrene oxide formation, with both the wild-type and this mutant showing an R:S ratio of 15:85 (Nickerson et al., 1997Go). Addition of the V247L mutation reduced the selectivity marginally to R:S = 17:83. The selectivity of 1-phenylethanol was decreased from R:S = 79:21 for the wild-type to R:S = 65:35 in the Y96F mutant. As with styrene, the V247L mutation decreased the selectivity, to R:S = 57:43 for 1-phenylethanol. The data clearly indicate that the binding orientation of both substrates is altered by the mutations. Since V247 is located high up in the active site (Figure 2Go), we believe that the selectivity can be further manipulated by mutations closer to the haem.

In vivo oxidation of unnatural substrates

The camC genes encoding the Y96F–V247L and Y96F–I395G double mutants were cloned into the pCWSGB plasmid. The two resulting pCWSGB–camC plasmids were transformed into E.coli to investigate if this tricistronic system could oxidize diphenylmethane, styrene and ethylbenzene in vivo. Escherichia coli cells transformed with either plasmid turned blue after induction, indicating indigo formation observed previously with other in vivo CYP systems (Gillam et al., 1999Go). We note that wild-type CYP101 did not catalyse indigo formation, which is another indication of the possibility of broadening the CYP101 substrate range by mutagenesis.

After resuspension in EMM, hexadecane was added to 2% (v/v) to form an inert organic phase to help protect the E.coli cells from poisoning by organic substrates and products (Kawakami and Nakahara, 1994Go; Wubbolts et al., 1996Go). On the basis of the substrate oxidation rates (Table IGo), the Y96F–V247L mutant was chosen for studying the in vivo oxidation of styrene and ethylbenzene, while the Y96F–I395G mutant was used to carry out diphenylmethane oxidation. Substrates were added to approximately 1 mM concentration. GC analysis showed that the in vivo system gave the same products as those in vitro and that the Y96F–V247L mutant oxidized 1 mM styrene and ethylbenzene within 12 h. Quantitative analysis showed that the amount of 1-phenylethanol correlated well with the amount of ethylbenzene oxidized, but the styrene oxide concentration was significantly below what was expected from the amount of styrene consumed. When more substrate was added, the amount of styrene oxide increased very slowly, but 100-mg quantities of 1-phenylethanol could be routinely obtained after 24 h. Control experiments, in which styrene oxide was added to a culture of E.coli host cells transformed with the pCW vector lacking any insert, showed that styrene oxide was transformed by the E.coli cells in the absence of any CYP101. As expected from the in vitro activity data, the in vivo oxidation of diphenylmethane by the Y96F–I395G mutant was much slower. However, the system was sufficiently long-lived that 100-mg quantities of 4-hydroxydiphenylmethane could also be obtained after 48–72 h.

Control reactions were carried out with E.coli cells containing the pCWSGB plasmid or with wild-type CYP101 expressed on its own. Neither system showed any oxidation activity with the substrates. These results show conclusively that the pCWSGB–camC system catalyses the in vivo oxidation of both soluble and highly insoluble hydrophobic organic compounds. The relative ease with which 100-mg quantities of substrate oxidation products can be obtained is important because it opens up the possibility of isolating and then characterizing the oxidation products of even complex molecules by NMR and mass spectroscopy. For these compounds the potential oxidation products are often not available or difficult to synthesize by chemical methods.


    Summary and conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary and conclusions
 References
 
The pCWSGB plasmid allows the straightforward cloning of genes encoding CYP101 mutants for the co-expression of these mutants with the electron transfer proteins PdR and Pd. Escherichia coli cells harbouring a variety of pCWSGB–camC plasmids have been shown to oxidize camphor and a number of unnatural substrates in vivo. Application of simple rationales enabled the engineering of CYP101 to improve the activity for the oxidation of diphenylmethane, styrene and ethylbenzene. The in vivo system was shown to be viable for the preparation of substrate oxidation products on a 100-mg scale within 24–72 h. Styrene oxide was further transformed by the E.coli strain employed in the present work, but other micro-organisms which have been successfully utilized for larger scale preparation styrene oxide could be used as the host (Furuhashi et al., 1986Go; Wubbolts et al., 1994Go, 1996Go).

The work of Sibbeson et al. showed that triple fusion proteins containing CYP101, Pd and PdR functioned as a self-sufficient catalytic systems in E.coli (Sibbeson et al., 1996). Analysis of these fusion proteins revealed a suboptimal interaction between the Pd and CYP101 domains of the fusion proteins. The tricistronic system constructed here overcomes this suboptimal interaction by expressing the proteins of the CYP101 system separately such that the in vivo catalytic activity of the system is comparable to the in vitro system.

In conclusion, the whole-cell system offers a simple and effective method of synthesizing 100-mg quantities of substrate oxidation products. The total yield of products could be greatly improved, with the ultimate aim of synthetic scale reactions, by altering the fermentation conditions and further engineering of the system to enhance the expression levels. Finally, this in vivo system will also allow rapid screening of the activity of CYP101 mutants in protein engineering studies, including forced evolution experiments.


    Notes
 
1 To whom correspondence should be addressed. E-mail: luet.wong{at}chem.ox.ac.uk Back


    Acknowledgments
 
This work was supported by the Higher Education Funding Council for England (HEFCE). The generosity of Professors R.Dalquist and C.R.Wolf is gratefully acknowledged.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Summary and conclusions
 References
 
Bell,S.G., Rouch,D.A. and Wong,L.-L. (1997) J. Mol. Catal. B: Enzym., 3, 293–302.[ISI]

Bell,S.G., Sowden,R.J. and Wong,L.-L. (2001) Chem. Commun., 635–636.

Blake,J.A.R., Pritchard,M., Ding,S., Smith,G.C.M., Burchell,B., Wolf,C.R. and Friedberg,T. (1996) FEBS Lett., 397, 210–214.[ISI][Medline]

Brosius,J. and Holy,A. (1984) Proc. Natl Acad. Sci. USA, 81, 6929–6933[Abstract]

Chen,L., Buters,J.T.M., Hardwick,J.P., Tamura,S., Penman,B.W., Gonzalez,F.J. and Crespi,C.L. (1997) Drug. Metab. Dispos., 25, 399–405.[Abstract/Free Full Text]

Desai, J.D. and Banat,I.M. (1997) Microbiol. Mol. Biol. Rev., 61, 47–64.[Abstract]

England,P.A., Harford-Cross,C.F., Stevenson,J.-A., Rouch,D.A. and Wong,L.-L. (1998) FEBS Lett., 424, 271–274.[ISI][Medline]

Estabrook,R.W., Faulkner,K.M., Shet,M.S. and Fisher,C.W. (1996a) Methods Enzymol., 272, 44–51.[ISI][Medline]

Estabrook,R.W., Shet,M.S., Faulkner,K. and Fisher,C.W. (1996b) Endocr. Res., 22, 665–671.[ISI][Medline]

Fisher,M.T. and Sligar,S.G. (1985) J. Am. Chem. Soc., 107, 5018–5019.[ISI]

Fisher,C.W., Shet,M.S. and Estabrook,R.W. (1996) Methods Enzymol., 272, 15–25.[ISI][Medline]

Filipovic,D., Paulsen,M.D., Loida,P.J., Sligar,S.G. and Ornstein,R.L. (1992) Biochem. Biophys. Res. Commun., 189, 488–495.[ISI][Medline]

Fowler,S.M., England,P.A., Westlake,A.C.G., Rouch,D.A., Nickerson,D.P., Blunt,C., Braybrook,D., West,S., Wong,L.-L. and Flitsch,S.L. (1994) Chem. Commun., 2761–2762.

Fruetel,J.A., Collins,J.R., Camper,D.L., Loew,G.H. and Ortiz de Montellano,P.R. (1992) J. Am. Chem. Soc., 114, 6987–6993.[ISI]

Fulco,A.J. (1991) Annu. Rev. Pharmacol. Toxicol., 31, 177–203.[ISI][Medline]

Furuhashi,K., Shintani,M. and Takagi,M. (1986) Appl. Microbiol. Biotechnol., 23, 218–223.[ISI]

Gillam,E.M.J., Wunsch,R.M., Ueng,Y.-F., Shimada,T., Reilly,P.E.B., Kamataki,T. and Guengerich,F.P. (1997) Arch. Biochem. Biophys., 346, 81–90.[ISI][Medline]

Gillam,E.M.J., Aguinaldo,A.M.A., Notley,L.M., Kim,D., Mundkowski,R.G., Volkov,A.A., Arnold,F.H., Soucek,P., DeVoss, J.J. and Guengerich,F.P. (1999) Biochem. Biophys. Res. Commun., 265, 469–472.[ISI][Medline]

Gunsalus,I.C. and Wagner,G.C. (1978) Methods Enzymol., 52, 166–188.[Medline]

Harikrishna,J.A., Black,S.M., Szklarz,G.D. and Miller,W.L. (1993) DNA Cell Biol., 12, 371–379.[ISI][Medline]

Jones,J.P., O'Hare,E.J. and Wong, L.-L. (2001) Chem. Commun., 247–248.

Kawakami,K. and Nakahara,T. (1994) Biotechnol. Bioeng., 43, 918–924.[ISI]

Kazlauskaite,J. Westlake,A.C.G., Wong,L.-L. and Hill,H.A.O. (1996) Chem. Commun., 2189–2190.

Kunkel,T.A. (1985) Proc. Natl Acad. Sci. USA, 82, 488–492.[Abstract]

Lacour,T. and Ohkawa,H. (1999) Biochim. Biophys. Acta, 1433, 87–102.[ISI][Medline]

Liu,Y., Kondo,A., Ohkawa,H., Shiota,N. and Fukuda,H. (1998) Biochem. Eng. J., 2, 229–235.[ISI]

Loida,P.J. and Sligar,S.G. (1993) Biochemistry, 32, 11530–11538.[ISI][Medline]

Lvov,Y.M., Lu,Z., Schenkman,J.B., Zu,X. and Rusling,J.F. (1998) J. Am. Chem. Soc., 120, 4073–4080.[ISI]

Mueller,E.J., Loida,P.J. and Sligar,S.G. (1995) In Ortiz de Montellano,P.R. (ed.), Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Press, New York, pp. 83–124.

Nickerson,D.P., Harford-Cross,C.F. and Wong,L.-L. (1997) FEBS Lett., 405, 153–156.[ISI][Medline]

Omura,T. and Sato,R. (1964) J. Biol. Chem., 239, 2370–2378.[Free Full Text]

Parikh,A. and Guengerich,F.P. (1997) Protein Expr. Purif., 9, 346–354.[ISI][Medline]

Parikh,A., Gillam,E.M.J. and Guengerich,F.P. (1997) Nat. Biotechnol., 15, 784–788.[ISI][Medline]

Peterson,J.A., Lorence,M.C. and Amarneh,B. (1990) J. Biol. Chem., 265, 6066–6073.[Abstract/Free Full Text]

Poulos,T.L., Finzel,B.C. and Howard,A.J. (1987) J. Mol. Biol., 195, 687–699.[ISI][Medline]

Preusting,H., Halzenberg,W. and Witholt,B. (1993) Enzyme Microb. Technol., 15, 311–316.[ISI]

Pritchard,M.P., Glancey,M.J., Blake,J.A.R., Gilham,D.E., Burchell,B., Wolf,C.R. and Friedberg,T. (1998) Pharmacogenetics, 8, 33–42.[ISI][Medline]

Raag,R. and Poulos,T.L. (1991) Biochemistry, 30, 2674–2684.[ISI][Medline]

Sambrook,J., Fritsch,E.F. and Maniatas,T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Schneider,S., Wubbolts,M.G., Sanglard,D. and Witholt,B. (1998a) Appl. Environ. Microbiol., 64, 3784–3790.[Abstract/Free Full Text]

Schneider,S., Wubbolts,M.G., Sanglard,D. and Witholt,B. (1998b) Tetrahedron: Asymm., 9, 2833–2844.[ISI]

Shet,M.S., Fisher,C.W. and Estabrook,R.W. (1997) Arch. Biochem. Biophys., 339, 218–225.[ISI][Medline]

Sibbesen,O., De Voss,J.J. and Ortiz de Montellano,P.R. (1996) J. Biol. Chem., 271, 22462–22469.[Abstract/Free Full Text]

Unger,B.P., Gunsalus,I.C. and Sligar,S.G. (1986) J. Biol. Chem., 261, 1158–1163.[Abstract/Free Full Text]

Westlake,A.C.G., Harford-Cross,C.F., Donovan,J. and Wong,L.-L. (1999) Eur. J. Biochem., 265, 929–935.[Abstract/Free Full Text]

Wong,L.-L., Westlake,A.C.G. and Nickerson,D.P. (1997) Struct. Bonding, 88, 175–207.[ISI]

Wubbolts,M.G., Hoven,J., Melgert,B. and Witholt,B. (1994) Enzyme Microb. Technol., 16, 887–894.[ISI]

Wubbolts,M.G., Favre-Bulle,A.J. and Witholt,B. (1996) Biotechnol. Bioeng., 52, 301–308.[ISI]

Yasukochi,T., Okada,O., Hara,T., Sagara,Y., Sekimizu,K. and Horiuchi,T. (1994) Biochim. Biophys. Acta, 1204, 84–90.[ISI][Medline]

Zhang,Z., Nassar,A.-E.F., Lu,Z., Schenkman,J.B. and Rusling,J.F. (1997) J. Chem. Soc., Faraday Trans., 93, 1769–1774.[ISI]

Received May 10, 2001; revised July 19, 2001; accepted July 31, 2001.





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