Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli

Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthe-sis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adreno-doxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins iden-tical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl- β -cy-clodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.


INTRODUCTION
Cytochromes P450 are ubiquitously distributed hemoproteins with broad field of catalytic activity towards various substances of exogenous and endogenous origin.As external monooxygenases, most of P450s functions as substrate binding terminal oxidases utilize external reductant, with electron transfer for oxygen activation and substrate conversion [1].
The objective challenges with the studies of steroidogenic P450-systems in mammalian organs, such as the presence in the cells of few P450 isoforms on different topogenesis stages, stimulate creating of modeling systems which are based on heterologous protein expression in microorganisms for in vitro and in vivo investigations.
loning and characterization of the individual steroido-C genic proteins in yeasts and bacteria were described earlier.Later on, the works were published on the improvement or modification of P450s features (e.g.membrane-binding, or substrate specificity change), as well as on the design of transgenic microorganisms with expression of multicomponent enzyme systems capable of performing few, or even cascade of mammalian steroidogenic reactions in one microorganism [4].
The strains of E. coli are often used as a host microorganism for expression of recombinant P450s since these bacteria do not contain their own P450s [5].Application of E. coli expression system often provides a high expression level of heterologous P450s, and in particular, that of steroidogenic P450s in their active forms.Besides, mature forms of mitochondrial and microsomal P450s lacking the N-terminal targeting sequence can be expressed [6].Such systems are especially suitable for investigation of P450s' structure/function by site-directed mutagenesis and protein engineering.
Applications of E. coli expression systems and purified recombinant steroidogenic proteins are known for analyses of their topogenesis [7], interactions with redox partners [8] and substrates [9], and study of structural characteristics [10,11], etc.Moreover, such systems can also be used for medical purposes (for example, [12,13]).Recently, the effect of different therapeutic agents on CYP11A1 activity has been evaluated using purified recombinant P40scc (CYP11A1) in the in vitro reconstituted system [14].
The expression of mature form of mitochondrial bovine P450scc (mP450scc) as a spectrophotometrically and catalytically active protein in E. coli was firstly reported in 1991 [6].The enzymatic activity of P450scc was estimated toward 25-hydroxycholesterol using solubilized membranes in the presence of purified bovine Adx and AdR.Similar results were later published for the mature form of P450scc from human placenta [15].As shown, recombinant cytochromes P40scc inserted into the cytoplasmic membrane were basically similar to the native proteins.[6,15,16].Besides, functional AdR [17,18] and Adx [15,19,20] of different origin were expressed in E. coli.
In our previous works, we have attempted to generate recombinant bacterial cells bearing heterologous СН/L system using two distinct approaches for P450scc coexpressing with Adx and AdR redox partners.One approach was based on the expression in E. coli of the fused side-chain cleavage system with catalytic domains being connected by short (2-5 amino acid residues) linkers [21].However, the in vitro activity of this system was lower as compared with the system built of the separate purified bovine constituents, probably due to the steric hindrance for the interaction of active centers of the particular domains.The second approach involved expression in E. coli of the mammalian CH/L system constituents based on the polycistron (tandem) plasmid [22].Cell-free homogenate was shown to transform cholesterol to pregnenolone, but the activity was again very low.It was possible that the low level of in vitro activity was related to the different origin of the co-expressed proteins-bovine P450scc and human Adx and AdR.The expression of bovine cholesterol hydroxylase system with the use of co-transformation by two plasmids was later described in [23], but no data on the activity, neither in vivo nor in vitro, were reported.
In this work, the vector including cDNA for all three proteins of bovine CH/L system (P450scc, Adx and AdR) was constructed and the in vivo activity of the recombinant E. coli strain generated was examined.
Statistically methylated β-cyclodextrin (MCD) was obtained from Wacker Chemie (Germany); electrophoresis reagents-from Bio-Rad (USA).Nutrient media (LB and TB [24]) for bacterial growth have been prepared using materials supplied by Difco (USA).Nitrocellulose filters Hybond-C extra were obtained from Amersham (USA).
All DNA modifying enzymes and DNA Extraction Kit have been purchased from MBI Fermentas (Lithuania).The reaction mixtures preparation, sample incubation and enzyme inactivation were carried out according to the manufacturer's instructions (Fermentas Catalogue & Product Application Guide).
Primary antibodies (IgG fraction) against bovine P450scc, AdR, and Adx were kindly provided by Prof. V. M. Shkumatov (Institute of Physico-Chemical Problems, Minsk State University, Belarus).

Bacterial Strains and Plasmids
Plasmid pTrc99A/P450scc [6] containing cDNA for mature bovine cytochrome P450scc and plasmid pBar_Twin [25] containing cDNAs for mature forms of bovine AdR and Adx as a single expression cassette were used in this work.Plasmid pTrc99A/P450scc was kindly provided by Prof. M. R. Waterman (University of Texas, Southwestern Medical Center, Dallas, TX, USA).The pBar_Twin plasmid was kindly provided by Prof. R. Bernhardt (University of Saarlandes, Saabrucken, Germany).
The strains and plasmids used in this study are summarized in Table 1.

Construction of pBar_Triple Plasmid
To derive a suitable expression vector containing cDNA encoding for three cholesterol hydroxylase proteins, two initial plasmids-pTrc99A/P450scc and pBar_Twin, were used.The plasmid pBar_Twin was sequentially digested with restriction endonuclease EcoRI (the site located after the termination codon cDNA encoding for Adx), filled in with Klenow fragment, and treated with thermosensitive alkaline phosphatase.The DNA insert (1583 bp) encoding for mature bovine P450scc with ribosome binding site (RBS) in front of it was excised by MbiI and SalI from pTrc99A/P450scc plasmid, filled in using Klenow polymerase and ligated with linearized and blunt ended pBar_Twin, so that ribosome-binding site (RBS) and cytochrome P450scc cDNA were inserted into the expression cassette beyond cDNA for Adx.
The DNA fragments obtained by restriction were separated using electrophoresis in 1% agarose gel.Extraction of DNA was carried out using DNA Extraction Kit.Plasmid DNA for cloning procedures was isolated from bacteria by the alkaline lysis method [24].Transformation of E. сoli cells was performed in accordance with known protocol [24] thus resulting in a recombinant E. coli DH5αc/Triple (Table 1).

Expression of Recombinant Proteins in E. coli Cells
In order to express the recombinant proteins, the cells of individual colonies were grown overnight in 5 ml of liquid LB (Luria-Bertani broth) medium containing ampicillin (100 μg/ml) aerobically on a rotary shaker GH-4103 Bottmingen HT (Germany) (140 rpm) at 37˚С, diluted 1:200 with TB (Terrific Broth) medium, and again cultivated at 37˚C for 3 -4 h.Synthesis of recombinant proteins was then induced by an addition of IPTG to 0.5 mM, and the cell growth was continued in the presence of ampicillin (100 μg/ml) and δ-ALA (0.5 mM) for 48 h at 24 -28˚C with constant shaking (140 rpm).

Ds-Na-PAAG Electrophoresis and Western Immunoblotting
The cells of E. coli DH5αc/Triple25 obtained as described above (as per 2.3.) were harvested by centrifugation, re-suspended in sample buffer [26] and disrupted by eating for 2 minutes at 100˚C.Cell homogenates were h subjected to SDS-PAGE in 10, or 15% gel [26] and Western blotting [27].Upon SDS-PAGE and protein transfer from gel onto nitrocellulose membrane, the latter was consecutively treated by a primary antibody (IgG fraction) to P450scc, AdR, or Adx and a secondary antibody conjugated with horseradish peroxidase.As it was shown earlier [16,22], the primary antibodies against bovine P450scc, AdR, and Adx used in this work bind effectively with respective either native or recombinant proteins of P450scc system expressed in E. coli.Protein bands were visualized using diaminobenzidine tetrachloride hydrate.
Protein in homogenates was measured by the Lowry method [28].

In Vivo Activity of Cholesterol Hydroxylase/Lyase System
In order to determine the activity of bovine P450scc/ Adx/AdR system in recombinant E. coli cells in vivo, the bacteria were grown and expression was induced as described above (in 2.2) with some modifications.The overnight culture (1%, v/v) was inoculated in 50 ml TBmedium supplemented with 100 μg/ml ampicillin, and cultivated at 37˚С aerobically (160 rpm) for 4 h.Then, IPTG (1 mM), δ-ALA (0.5 mM) and microelement solutions (each of 50 µl) were added.The microelement solutions were composed according to [25].The microelement solution 1 contained (g/l): FeCl Cholesterol was added to the final concentration 0.5 mM (193 mg/l) in a form of 100-fold aqueous concen-trates: 1) as a solution in MCD (250 mM), or 2) as fine suspension stabilized with Tween 80 (100 g/l) and homogenized on ultrasonic bath (Cole-Parmer, USA) at 42 kHz, 100 Wt, for 5 min.After cholesterol addition, the cells were incubated at 24˚C and 180 rpm for 74 h.The samples were taken every 24 h since cholesterol addition.In controls, the following variants were used: a) expression was not induced; b) no cholesterol was added; c) recipient E. coli strains were used.

Steroid Analyses
The samples of cultivation broth (10 ml) were twice extracted with ethyl acetate (firstly-with double, then with equal solvent volume), the organic phases were combined and vacuum-evaporated to dryness.The residue was re-dissolved in 1 ml of 50% aqueous acetonitrile and insolubles formed were separated by centrifugation at 5,000 × g, 15 min.Steroids were analyzed by high-pressure liquid chromatography (HPLC) on the HPLC system Series 1200 (Agilent, USA) at 50˚C and eluents flow rate of 1 ml/min.Components were separated on a Symmetry column (Waters, USA) 250 mm × 4.6 mm (with a guard column 20 mm × 3.9 mm) packed with reverse phase ODS (5 µm) by three different methods: 1) isocratic-in a system composed of 52% acetonitrile, 48% H 2 O and 0.01% acetic acid, 2) in a system containing 64% acetonitrile, 36% H 2 O and 0,01% acetic acid; 3) in a linear gradient of acetonitrile (50% from 0 to 10 min; 50% -88% from 10 to 20 min; 88% from 20 to 25 min).
Peak detection was carried out by UV absorbance at 200 and 240 nm.Identification of the peaks and the quantification of pregnenolone and progesterone were carried out using external standard technique.

Enzymatic Treatment of Extracted Steroids
The evaporated organic extracts of cultivation broth sam-ples obtained as described above (2.6) were suspended in 0.5 ml of 0.05 M sodium phosphate buffer (pH 7.5) supplemented with MCD (50 µM).In control, the same buffer containing 2 mg/l pregnenolone was used.The mixtures obtained were incubated with 8 U/ml of recombinant cholesterol oxidase (Sigma, USA) at 30˚C for 20 h.Then, the samples were diluted with equal volume of acetonitrile and centrifuged (5,000 × g, 15 min).The supernatants were applied for HPLC analyses as described above (2.6).

Plasmid Construction for Co-Expresssion of Three Bovine Proteins
For expression of more than one protein in bacteria, a plasmid which contains cDNAs encoding for different proteins and RBSs before the each of heterologous cDNA in a single transcription unit can be constructed.Therefore, the heterologous genes reading should be controlled by one promoter and one terminator.One fusion (hybrid) mRNA should be formed, with an independent translation of the individual proteins.Several functional monooxygenase systems were published to be constructed using similar polycistrone plasmids for protein co-expression in bacterial cells [29][30][31].
In the present work, the vector was created for co-expression in E. coli cells of three bovine system proteins, -CYP11A1/Adx/AdR.
To express cytochrome P450scc cDNA alongside with the cDNAs for AdR and Adx as a single transcription unit, DNA fragment encoding the P450scc and RBS in front of it was cloned into the pBar_Twin [25].The cells of E. coli were transformed with ligase mixture, and after restriction analysis of the recombinant plasmids the clones were isolated which did contain the vector where RBS and cDNA encoding for P450scc were situated downstream of Adx cDNA seguence.Selected plasmid contained the nucleotide sequences of the heterologous proteins and RBSs in a single expression cassette in the following order [RBS-AdR-RBS-Adx-RBS-P450scc].
The resulting tricistronic co-expressing vector was designated as pBar_Triple (Figure 2) and used for transformation of E. coli DH5c cells and subsequent IPTGcontrolled co-expression of the contained cDNAs for all constituents of the CH/L system.

Co-Expression of Bovine CH/L System Proteins in E. coli Cells
Co-expression of bovine P450scc, AdR and Adx in E. coli DH5c transformed with pBar_Triple was carried out using medium supplemented with ampicillin upon induction of transcription of heterologous cDNAs from the recombinant plasmid (as per 2.3).Recombinant pro-teins were identified in the cell homogenates (as per 2.4) using electrophoretic analysis in polyacrylamide gel followed by Western-blotting with antibodies against P450scc, AdR or Adx.The immunodetection results are presented in Figure 3.

Activity of Bovine CH/L System Reconstructed in Bacterial Cells
The recombinant strain E. coli DH5αc/Triple25 was tested for the activity towards cholesterol.As shown in Figure 4, pregnenolone was formed both   by IPTG-induced (A) and non-induced cells (B) thus evidencing that promoter which controls transcription of heterologous cDNA is not a "strongly inducible" one.
It is well-known that cholesterol is a poorly soluble substrate with an aqueous solubility of 2 -10 mg per liter [32].This extremely poor solubility may be a reason of low substrate availability to microbial cell enzymes.Different approaches are used in order to provide cholesterol availability to microbial cells (for review, see [33]).We assumed that cholesterol micronization with detergents, or its solubilization using cyclodextrins (CDs) are the most suitable modes of substrate addition which can facilitate cholesterol conversion in our case.
The dependence of pregnenolone formation by recombinant E. coli DH5αc/Triple on the mode of cholesterol addition and IPTG-induction is illustrated by Figure 5.As shown, the amount and rate of pregnenolone formation by IPTG-induced cells was 2 -3 times higher than in the case of non-induced cells.Pregnenolone concentration by IPTG-induced cells reached its maximal level for 24 h, while this period was no less than 48 h for noninduced cells (Figure 5).(almost on the lowest level of reliable detection range) was observed when cholesterol was added as a suspension with Tween 80.When using IPTG-induced culture and MCD-solubilized cholesterol, pregnenolone yield As shown in Figure 5, addition of cholesterol as a MCD solution resulted in higher pregnenolone production by the non-induced cells, while very low activity OPEN ACCESS reached 0.192 µmol/l, while more than 3-fold less pregnenolone concentration was detected when cholesterol micronization with Tween 80 was applied.
Thus, the results evidence that the mode of cholesterol addition is of importance for the activity of the recombinant cells.CD-mediated enhancement of microbial sterol side chain cleavage was reported earlier [34][35][36].
The enhancement effect can be mainly attributed to steroid solubilization by the formation of water-soluble inclusion complex of CDs with steroids.Besides, CDs may facilitate the transport of poorly soluble hydrophobic substances to and from microbial cells, thus functioning as effective substrate/product delivery systems.As reported, CDs themselves do not penetrate through bacterial cell membranes, but can disrupt the outermost cell wall layers of the gram-positive bacteria [36].The detail study of MCD effect on the cells of E. coli is out of the purposes of the current work, and can be investigated especially.

Comparison of E. coli DH5αc/Triple with Analogous Strains
In our previous works, several recombinant strains have been created on the of E. coli JM-109, bearing CH/L system proteins [7,21,22] (Table 1).Three genetic constructs were designed for co-expression of mature proteins (lacking of N-terminal addressing sequences) of the CH/L system and used at the construction of the strains.The first construct was pTrc99A/P450scc/AdR/ Adx containing (similar to pBar_Triple) P450scc, AdR, and Adx cDNA within the same expression cassette.The other construct was pTrc/P450scc/AdR.Adx containing P450scc and AdR cDNA within the same expression cassette, and the gene of Adx was inserted into the same plasmid within a separate transcription unit (regulated by its own promoter and terminator).The third plasmid-pTrc99A/F2, contained hybrid cDNA encoding the fu-sion protein P450scc-AdR-Adx.For re-construction of mammalian CH/L in these cases, cDNAs of different origin were applied-bovine P450scc, and human AdR and Adx.As evidenced by immune-enzyme analysis (ELISA), the cell-free homogenates of the recombinants containing these plasmids demonstrated in vitro hydroxylase/lyase activity towards 22(R)-hydroxy cholesterol [7,21,22].However, no in vivo activity was detected.In the current study, we compared cholesterol conversion by these strains with newly constructed E. coli DH5c/Triple25.The experiments were carried out at the conditions optimized for E. coli DH5c/Triple25 as described above.
Pregnenolone was detected in very low amounts at cholesterol incubation with the D36 and E32 strains (Table 1) cultured under conditions of induced expression of heterologous cDNAs.In order to provide reliable quantitative detection, the extracts were 10 -30-fold concentrated before analysis.The approximately two-fold higher level of pregnenolone production was observed in the case of D36 strain as compared to E32.It is well correlated with the expected higher level of Adx expression in D36 which evidently enhanced functioning of the whole system.
Besides, in order to confirm the identity of pregnenolone formed, it was converted to more reliably detected progesterone by commercial cholesterol oxidase (as per 2.6.2).The control experiment confirmed complete enzymatic conversion of pregnenolone to progesterone.Thus, both methods (as per 2.6 and 2.7) confirmed the formation of pregnenolone by the strains of D36 and E32.
Low efficiency of cholesterol conversion by E. coli JM-109/F2 strain which expressed fusion CH/L system did not allow reliable detection of pregnenolone.This result indicated that in vivo activity of the fused recombinant CH/L is much lower than that of the D36 and E32 strains which were transformed with plasmids, allowing simultaneous expression of individual CH/L system proteins.This is consistent with the data reported on in vitro activity of the recombinant CH/L systems.Probably, the catalytic centers of the fused domains in this CH/L were either unable to interact, or misfolded thus leading to low cholesterol side-chain cleavage activity [21].Much higher in vivo activity of E. coli DH5αc/ Triple25 synthesizes the three component bovine CH/L system, as compared with JM109 strain synthesizing the proteins of different origin (bovine P450scc and human AdR and Adx) evidence the preference of homologous P450scc system over its heterologous analog.But the reason of this difference is not fully clear: either heterologous system with proteins of different origin is less active, or the more optimal stoichiometry of the expressed proteins is provided in E. coli DH5αc/Triple25.The latter can be particularly caused by different location of cDNA-sequences of the component proteins in the expression cassette of the recombinant plasmids used.For instance, the order shift in the location of cDNAs encodes for cytochrome P45027B1, Adx and NADPH; cytochrome P450-reductase in the expression cassette of the tandem plasmids resulted in 3-5-fold change of protein content [29].Besides, pBar_Triple vector directs the synthesis of the truncated bovine Adx (4-108) [25].Different aggregation of foreign protein molecules also can not be excluded-the formation of inactive forms of the recombinant Р450scc, AdR and Adx, i.e., the so called "inclusion bodies", in E. coli was reported earlier [22].
In conclusion, the bovine CH/L system was firstly reconstructed in E. coli using pBar_Triple vector.The recombinant strain created is capable to produce up to 420 µg/l of pregnenolone for 24 h, and the level of the productivity was higher than hitherto reported for the similar E. coli recombinants.The strain can be applied as a modeling system in the basic research of mammalian steroidogenic system features.

Figure 1 .
Figure 1.The general organization and function of mammalian cholesterol hydroxylase/lyase system.The original system is located in adrenocortical mitochondria and includes cytochrome Р450scc (CYP11A1) and its redox partners: Adx (adrenodoxin, a member of [2Fe-2S] ferredoxins family) and AdR (adrenodoxin reductase, a NADPH-dependent flavin reductase).Membranebound cytochrome P450scc catalyzes cholesterol conversion to pregnenolone in three sequential steps including hydroxylation in positions C-22 and C-20 and C-C cleavage of the formed diol.

Figure 2 .
Figure 2. Structure of the tricistronic plasmid pBar_Triple for co-expression of the cholesterol hydroxylase/lyase system proteins.pBar_Triple (8.287 kb) contains cDNAs for bovine cytochrome P450scc, AdR and Adx in a single expression cassette.The unique EcoRI site was used during the construction.An ampicillin resistance gene allows selection for plasmid uptake.Expression of inserted cDNAs is driven by the tac1/tac2/lacUV5 promoter.

Figure 3 .
Figure 3. Co-expression of P450scc, AdR and Adx in E. coli cells.A cellular homogenates (60 µg of total protein each), from E. coli culture were subjected to SDS-PAGE (15% (A) or 10% (B and C) acrylamide) and Western-blotting.Recombinant proteins were detected with antisera to Adx (A), AdR (B) and P450scc (C).In each case, lane 1 corresponds to homogenate of non-transformed cells, and lane 2 corresponds to homogenate E. coli DH5αc/pBar_Triple.

Figure 4 .
Figure 4. Pregnenolone formation from cholesterol by E. coli DH5αc/Triple.The cells were grown as described in 2.3 and incubated during 48 h with 0.5 mM cholesterol which was added as an aqueous MCD-solution.Reversed-phase HPLC profiles of cultivation broth extracts were obtained in a linear gradient of acetonitrile as described in 2.6, method 3 at 200 nm.Retention time of pregnenolone (17.15 min) is indicated by arrows.Apregnenolone external standard injection (4.5 mg/l); B-control profile (at incubation of recipient E. coli DH5αc strain with cholesterol); C-E. coli DH5αc/Triple without induction; D-E. coli DH5αc/Triple cells induced with 0.5 mM IPTG.

Figure 5 .
Figure 5. Influence of IPTG-induction and cholesterol addition mode (in aqueous MCD solution or as Tween 80-stabilized suspension) on pregnenolone formation by recombinant E. coli DH5αc/Triple at different incubation times.The average values of three measurements are presented.

Table 1 .
Escherichia coli strains and plasmids used in this study.
a The plasmids indicated in the table contain cDNAs encoding the human (h) or bovine (b) mature forms of proteins.