American Journal of Molecular Biology, 2013, 3, 173-182 AJMB http://dx.doi.org/10.4236/ajmb.2013.34023 Published Online October 2013 (http://www.scirp.org/journal/ajmb/) Functional reconstruction of bovine P450scc steroidogenic system in Escherichia coli Desislava S. Makeeva1, Dmitry V. Dovbnya2*, Marina V. Donova2, Ludmila A. Novikova1 1Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 2Institute of Biochemistry & Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia Email: *anagoge@rambler.ru Received 18 July 2013; revised 15 August 2013; accepted 3 September 2013 Copyright © 2013 Desislava S. Makeeva et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Mammalian cytochrome P450scc enzyme system cata- lyzes the initial step in steroid hormone biosynthe- sis—cholest ero l hy d roxy lat io n fo llo wed by clea vag e of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin re- ductase, AdR) and an iron-sulfur protein (adreno- doxin, Adx). In this work, the three-component elec- tron transport chain (AdR/Adx/CYP11A1) from bo- vine adrenal cortex has been implemented in Es- cherichia 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 mo- lecular weights and immuno-specificity. The experi- ments 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 pres- ence 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 stud- ies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts. Keywords: Cytochrome P450; CYP11A1; Adrenodoxin; Adrenodoxin Reductase; Steroid Hormone Biosynthesis; Heterologous Expression 1. INTRODUCTION Cytochromes P450 are ubiquitously distributed hemopro- teins with broad field of catalytic activity towards vari- ous substances of exogenous and endogenous origin. As external monooxygenases, most of P450s functions as substrate binding terminal oxidases utilize external re- ductant, with electron transfer for oxygen activation and substrate conversion [1]. Cytochrome P450scc (CYP11A1, EC 1.14.15.6) cata- lyzes the side-chain cleavage of cholesterol in bovine adrenal cortex mitochondria. The mechanism involves three sequential monooxygenation reactions—produc- tion of 22R-hydroxycholesterol (22HC), 20α, and 22R- dihydroxycholesterol followed by the cleavage of the C20-C22 bond [2]. Natural partners of P450scc are adrenodoxin (Adx) and adrenodoxin reductase (AdR). The former is a [2Fe-2S] ferredoxin, and the latter is NADPH-dependent flavine reductase (EC 1.18.1.2). These three proteins (CYP11A1, Adx, AdR) are from cholesterol hydroxylase/20,22-lyase system (CH/L) which catalyzes the initial step of steroidogenesis in mammals: cholesterol conversion to pregnenolone—the key pre- cursor of all steroid hormones (Figure 1). Further steps include pregnenolone modification with at least five P450s, 3β-hydroxysteroid dehydrogenase/ Δ5,4-isomerase and 17β-hydroxysteroid dehydrogenase in the endoplasmic reticulum (ER) (CYP17, CYP21, CYP19) and mitochondria (CYP11B1, CYP11B2) of ste- roidogenic mammalian cells thus resulting in the forma- tion of different steroid hormones [3]. The objective challenges with the studies of steroido- genic P450-systems in mammalian organs, such as the presence in the cells of few P450 isoforms on different topogenesis stages, stimulate creating of modeling sys- tems which are based on heterologous protein expression in microorganisms for in vitro and in vivo investigations. loning and characterization of the individual steroido- C *Corresponding author. OPEN ACCESS
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 174 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). Membrane- bound 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. genic proteins in yeasts and bacteria were described ear- lier. Later on, the works were published on the improve- ment or modification of P450s features (e.g. mem- brane-binding, or substrate specificity change), as well as on the design of transgenic microorganisms with expres- sion of multicomponent enzyme systems capable of per- forming few, or even cascade of mammalian steroido- genic reactions in one microorganism [4]. The strains of E. coli are often used as a host microor- ganism 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 ex- pression 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 ex- pressed [6]. Such systems are especially suitable for in- vestigation of P450s’ structure/function by site-directed mutagenesis and protein engineering. Applications of E. coli expression systems and puri- fied 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 reconsti- tuted system [14]. The expression of mature form of mitochondrial bo- vine P450scc (mP450scc) as a spectrophotometrically and catalytically active protein in E. coli was firstly re- ported in 1991 [6]. The enzymatic activity of P450scc was estimated toward 25-hydroxycholesterol using solu- bilized 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 ex- pressed 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 co- expressing with Adx and AdR redox partners. One ap- proach 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) link- ers [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 par- ticular domains. The second approach involved expres- sion in E. coli of the mammalian CH/L system constitu- ents based on the polycistron (tandem) plasmid [22]. Cell-free homogenate was shown to transform choles- terol 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 pro- teins—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 Copyright © 2013 SciRes. OPEN ACCESS
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 175 proteins of bovine CH/L system (P450scc, Adx and AdR) was constructed and the in vivo activity of the recombi- nant E. coli strain generated was examined. 2. MATERIALS AND METHODS 2.1. Materials The following reagents were used in the work: δ-ami- nolevulonic acid (δ-ALA), isopropyl-β-D-thiogalacto- pyranoside (IPTG), diaminobenzidine tetrachloride hy- drate, cholesterol oxidase and horse-radish peroxidase- conjugated anti-rabbit antibodies were purchased from Sigma (USA), Tween 80 was from Serva (Germany). Statistically methylated β-cyclodextrin (MCD) was ob- tained 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 ob- tained 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). Steroids: cholesterol (98% purity) was purchased from Serva (Germany); pregnenolone (pregn-5-ene-3-ol-20- one) and progesterone (pregn-4-ene-3,20-dione) were from Sigma (USA), and Steraloids (USA), respectively. Gradient-grade HPLC solvents have been purchased from Panreac (Spain). Other reagents were of analytical grade and have been purchased from domestic compa- nies (Russia). 2.2. Bacterial Strains and Plasmids Plasmid pTrc99A/P450scc [6] containing cDNA for ma- ture 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, Southwest- ern Medical Center, Dallas, TX, USA). The pBar_Twin plasmid was kindly provided by Prof. R. Bernhardt (University of Saarlandes, Saabrucken, Germany). Escherichia coli strain DH5αc (Gibco-BRL) and E. coli recombinant strains JM109/F2, JM109/D36, and JM109/E32 (collection names) were used in this work. E. coli strains JM109/F2, JM109/D36, and JM109/E32 have been generated earlier in our laboratory on the base of E. coli strain JM-109 (Promega, USA) as a result of trans- formation by pTrc99A/F2 (with artificial cDNA enco- ding for fusion CH/L system), and pTrc99A/P450scc/ AdR.Adx, or pTrc99A/P450scc/AdR/Adx (with cDNAs encoding for all separate proteins of CH/L), respectively [7,21,22]. The strains and plasmids used in this study are sum- marized 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 ri- bosome 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. Ex- traction of DNA was carried out using DNA Extraction Kit. Plasmid DNA for cloning procedures was isolated from bacteria by the alkaline lysis method [24]. Trans- formation of E. сoli cells was performed in accordance with known protocol [24] thus resulting in a recombinant E. coli DH5αc/Triple (Table 1). 2.3. 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 liq- uid LB (Luria-Bertani broth) medium containing am- picillin (100 μg/ml) aerobically on a rotary shaker GH- 4103 Bottmingen HT (Germany) (140 rpm) at 37˚С, di- luted 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). 2.4. Ds-Na-PAAG Electrophoresis and Western Immunoblotting The cells of E. coli DH5αc/Triple25 obtained as de- scribed above (as per 2.3.) were harvested by centrifuga- tion, re-suspended in sample buffer [26] and disrupted by eating for 2 minutes at 100˚C. Cell homogenates were h Copyright © 2013 SciRes. OPEN ACCESS
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 Copyright © 2013 SciRes. 176 OPEN ACCESS Table 1. Escherichia coli strains and plasmids used in this study. Recipient strain used for construction pBar_Triple Genotype Source DH5 c supE44 lac U169 ( 80 lacZ M15) hsdR17 recA1end A1 gyrA96 thi-1 relA1 Gibco-BRL Initial vectors used for construction pBar_Triplea Characteristics References pTrc99A/mP450scc bla (AmpR), trp/lac/trc promoter, cDNA for P450scc(b) [6] pBar_Twin bla (AmpR), tac1/tac2/lacUV5 promoter, cDNAs for AdR(b) and Adx(b) inserted in one expression cassette [25] Recombinant strains used for cholesterol conversion Expression vector Inserta References JM-109/F2 pTrc99A/F2 cDNA for P450scc(b)-AdR(h)-Adx(h) fusion [21] JM-109/D36 pTrc99A/P450/AdR/Adx cDNAs for P450scc(b) and AdR(h) inserted in the first expression cassette and cDNA for Adx(h) inserted in the second expression cassette [7] JM-109/E32 pTrc99A/P450/AdR.Adx cDNAs for P450scc(b), AdR(h), Adx(h) inserted in one expression cassette [22] DH5 c/Triple25 pBar_Triple cDNAs for P450scc(b), AdR(b), Adx(b) inserted in one expression cassette This study aThe plasmids indicated in the table contain cDNAs encoding the human (h) or bovine (b) mature forms of proteins. 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 anti- body conjugated with horseradish peroxidase. As it was shown earlier [16,22], the primary antibodies against bo- vine P450scc, AdR, and Adx used in this work bind ef- fectively with respective either native or recombinant proteins of P450scc system expressed in E. coli. Protein bands were visualized using diaminobenzidine tetrachlo- ride hydrate. Protein in homogenates was measured by the Lowry method [28]. 2.5. 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 de- scribed above (in 2.2) with some modifications. The overnight culture (1%, v/v) was inoculated in 50 ml TB- medium 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 solu- tions (each of 50 µl) were added. The microelement so- lutions were composed according to [25]. The microele- ment solution 1 contained (g/l): FeCl2·6H2O—4.07; CaCl2·2H2O—0.28; CoCl2·6H2O—0.28; ZnCl2·4H2O— 0.19; CuSO4·5H2O—0.26; H3BO4—0.07. Solution 2 contained 0.28 g/l Na2MoO4·2H2O. After the additions, А600 was ~1.5. The cells were further incubated for 2 h at 29˚С. The culture obtained was used for cholesterol bio- conversion. 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 ho- mogenized 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) expres- sion was not induced; b) no cholesterol was added; c) recipient E. coli strains were used. 2.6. Steroid Analyses The samples of cultivation broth (10 ml) were twice ex- tracted with ethyl acetate (firstly—with double, then with equal solvent volume), the organic phases were com- bined 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-pres- sure liquid chromatography (HPLC) on the HPLC sys- tem Series 1200 (Agilent, USA) at 50˚C and eluents flow rate of 1 ml/min. Components were separated on a Sym- metry 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) iso- cratic—in a system composed of 52% acetonitrile, 48% H2O and 0.01% acetic acid, 2) in a system containing 64% acetonitrile, 36% H2O 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. 2.7. Enzymatic Treatment of Extracted Steroids The evaporated organic extracts of cultivation broth sam-
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 177 ples obtained as described above (2.6) were suspended in 0.5 ml of 0.05 M sodium phosphate buffer (pH 7.5) sup- plemented 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 recom- binant 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 de- scribed above (2.6). 3. RESULTS AND DISCUSSION 3.1. 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 con- trolled 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 con- structed using similar polycistrone plasmids for protein co-expression in bacterial cells [29-31]. In the present work, the vector was created for co-ex- pression 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 trans- formation of E. coli DH5 c cells and subsequent IPTG- controlled co-expression of the contained cDNAs for all constituents of the CH/L system. 3.2. 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 fol- lowed by Western-blotting with antibodies against P450scc, AdR or Adx. The immunodetection results are presented in Figure 3. According to Figures 3A-C, recombinant protein mo- lecular weights (mP450scc, 54 kD; mAdR, 53 kD; mAdx, 12 kD) and their immunospecificity corresponded to the mature forms of P450scc, AdR and Adx from bovine adrenal cortex. 3.3. 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 Figure 2. Structure of the tricistronic plas- mid pBar_Triple for co-expression of the cholesterol hydroxylase/lyase system pro- teins. 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 con- struction. An ampicillin resistance gene al- lows selection for plasmid uptake. Expres- sion of inserted cDNAs is driven by the tac1/tac2/lacUV5 promoter. Figure 3. Co-expression of P450scc, AdR and Adx in E. coli cells. A cellular homoge- nates (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 corre- sponds to homogenate of non-transformed cells, and lane 2 corresponds to homogenate E. coli DH5αc/pBar_Triple. Copyright © 2013 SciRes. OPEN ACCESS
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 Copyright © 2013 SciRes. 178 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. A— pregnenolone 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 induc- tion; D—E. coli DH5αc/Triple cells induced with 0.5 mM IPTG. 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. Dif- ferent 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 fa- cilitate cholesterol conversion in our case. The dependence of pregnenolone formation by recom- binant 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 forma- tion by IPTG-induced cells was 2 - 3 times higher than in the case of non-induced cells. Pregnenolone concentra- tion by IPTG-induced cells reached its maximal level for 24 h, while this period was no less than 48 h for non- induced cells (Figur e 5). Figure 5. Influence of IPTG-induction and cholesterol addi- tion mode (in aqueous MCD solution or as Tween 80—sta- bilized suspension) on pregnenolone formation by recom- binant E. coli DH5αc/Triple at different incubation times. The average values of three measurements are presented. (almost on the lowest level of reliable detection range) was observed when cholesterol was added as a suspen- sion 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 produc- tion by the non-induced cells, while very low activity OPEN ACCESS
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 179 reached 0.192 µmol/l, while more than 3-fold less preg- nenolone 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 recombi- nant cells. CD-mediated enhancement of microbial sterol side chain cleavage was reported earlier [34-36]. 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 hydropho- bic substances to and from microbial cells, thus func- tioning 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. 3.4. Comparison of E. coli DH5αc/Triple with Analogous Strains In our previous works, several recombinant strains have been created on the base of E. coli JM-109, bearing CH/L system proteins [7,21,22] (Tab le 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 opti- mized 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 (Ta- ble 1) cultured under conditions of induced expression of heterologous cDNAs. In order to provide reliable quanti- tative detection, the extracts were 10 - 30-fold concen- trated 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 cor- related 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 pregne- nolone formed, it was converted to more reliably de- tected progesterone by commercial cholesterol oxidase (as per 2.6.2). The control experiment confirmed com- plete enzymatic conversion of pregnenolone to proges- terone. Thus, both methods (as per 2.6 and 2.7) con- firmed the formation of pregnenolone by the strains of D36 and E32. The strain DH5αc/Triple25 produced up to 420 µg/ml pregnenolone, thus demonstrating 7 - 13-fold higher ac- tivity as compared with D36 and E32 strains, corre- spondingly (Table 2). Low efficiency of cholesterol conversion by E. coli Table 2. Conversion of cholesterol to pregnenolone by recombinant E. coli strains. Pregnenolone Recombinant E. coli straina IPTG induction Mode of substrate additionµg/l μM Time, h + MCD 420 ± 15 1.33 ± 0.047 24 - MCD 168 ± 14 0.53 ± 0.044 48 DH5αc/Triple25 + Tween 80 Tracesb Tracesb 72 + MCD 60.5 ± 3 0.192 ± 0.009 72 JM-109/D36 + Tween 80 18.35 ± 3.5 0.058 ± 0.011 72 + MCD 30.8 ± 7 0.097 72 JM-109/E32 + Tween 80 0 0 72 + MCD Tracesb Tracesb 72 JM-109/F2 + Tween 80 0 0 72 The average values of three measurements are presented. aThe plasmids indicated in the table contain cDNAs encoding the human (h) or bovine (b) mature orms of proteins; bPregnenolone amount was lower than detection reliability. f Copyright © 2013 SciRes. OPEN ACCESS
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 180 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 recom- binant 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 pro- teins. 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 het- erologous system with proteins of different origin is less active, or the more optimal stoichiometry of the ex- pressed 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 pro- tein content [29]. Besides, pBar_Triple vector directs the synthesis of the truncated bovine Adx (4-108) [25]. Dif- ferent 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 re- constructed in E. coli using pBar_Triple vector. The re- combinant strain created is capable to produce up to 420 µg/l of pregnenolone for 24 h, and the level of the pro- ductivity was higher than hitherto reported for the similar E. coli recombinants. The strain can be applied as a mod- eling system in the basic research of mammalian steroi- dogenic system features. 4. ACKNOWLEDGEMENTS The authors are grateful to Prof. M. R. Waterman for providing the plasmid pTrc99A/P450scc. Prof. R. Bernhardt and Dr. F. Hannemann are kindly acknowledged for providing the plasmid pBar_Twin. The work was supported by Russian Foundation for Basic Research (RFBR) Grant N 12-08-00895-а. REFERENCES [1] Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J., Feyereisen, R., Waxman, D., Waterman, M.R., Gotoh, O., Coon, M.J., Estabrook, R.W., Gunsulus, I.C. and Nebert, D.W. (1996) P450 superfamily: Update on new sequ- ences, gene mapping, accession numbers and nomencla- ture. Pharmacogenetics, 6, 1-42. http://dx.doi.org/10.1097/00008571-199602000-00002 [2] Orme-Johnson, N.R. (1990) Distinctive properties of adrenal cortex mitochondria. Biochimica et Biophysica Acta, 1020, 213-231. http://dx.doi.org/10.1016/0005-2728(90)90151-S [3] Miller, W.L. (2008) Steroidogenic enzymes. Endocrine Development, 13, 1-18. [4] Szczebara, F.M., Chandelier, C., Villeret, C., Masurel, A., Bourot, S., Duport, C., Blanchard, S., Groisillier, A., Te- stet, E., Costaglioli, P., Cauet, G., Degryse, E., Balbuena, D., Winter, J., Achstetter, T., Spagnoli, R., Pompon, D. and Dumas, B. (2003) Total biosynthesis of hydrocorti- sone from a simple carbon source in yeast. Nature Bio- technology, 21, 143-149. http://dx.doi.org/10.1038/nbt775 [5] Nelson, D.R., Kamataki, T., Waxman, D.J., Guengerich, F.P., Estabrook, R.W., Feyereisen, R., Gonzalez, F.J., Coon, M.J., Gunsulus, I.C., Gotoh, O.,Okuda, K. and Ne- bert, D.W. (1993) The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early triv- ial names of enzymes, and nomenclature. DNA and Cell Biology, 12, 1-51. http://dx.doi.org/10.1089/dna.1993.12.1 [6] Wada, A., Mathew, P.A., Barnes, H.J., Sanders, D., Es- tabrook, R.W. and Waterman, M.R. (1991) Expression of functional bovine cholesterol side-chain cleavage cyto- chrome P450 (P450scc) in E. coli. Archives of Biochem- istry and Biophysics, 290, 376-380. http://dx.doi.org/10.1016/0003-9861(91)90554-V [7] Novikova, L.A., Faletrov, Y.V., Kovaleva, I.E., Mau- ersberger, S., Luzikov, V.N. and Shkumatov, V.M. (2009) From structure and functions of steroidogenic enzymes to new technologies of gene engineering. Biochemistry (Mo- scow), 74, 1482-1504. http://dx.doi.org/10.1134/S0006297909130057 [8] Heinz, A., Hannemann, F., Müller, J.J., Heinemann, U. and Bernhardt, R. (2005) The interaction domain of the redox protein adrenodoxin is mandatory for binding of the electron acceptor CYP11A1, but is not required for binding of the electron donor adrenodoxin reductase. Biochemical and Biophysical Research Communications, 338, 491-498. http://dx.doi.org/10.1016/j.bbrc.2005.08.077 [9] Pikuleva, I.A., Mackman, R.L., Kagawa, N., Waterman, M.R. and Ortiz de Montellano, P.R. (1995) Active-site topology of bovine cholesterol side-chain cleavage cyto- chrome P450 (P450scc) and evidence for interaction of tyrosine 94 with the side chain of cholesterol. Archives of Biochemistry and Biophysics, 322, 189-197. http://dx.doi.org/10.1006/abbi.1995.1451 [10] Headlam, M. J., Wilce, M. C. and Tuckey, R. C. (2003) The F-G loop region of cytochrome P450scc (CYP11A1) interacts with the phospholipid membrane. Biochimica et Biophysica Acta, 1617, 96-108. http://dx.doi.org/10.1016/j.bbamem.2003.09.007 Copyright © 2013 SciRes. OPEN ACCESS
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 181 [11] Strushkevich, N., MacKenzie, F., Cherkesova, T., Gra- bovec, I., Usanov, S. and Park, H.W. (2011) Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system. Proceedings of the National Academy of Sciences of USA, 108, 10139-10143. http://dx.doi.org/10.1073/pnas.1019441108 [12] Shet, M.S., Fisher, C.W., Tremblay, Y., Belanger, A., Conley, A.J., Mason, J.I. and Estabrook, R.W. (2007) Comparison of the 17 alpha-hydroxylase/C17,20-lyase activities of porcine, guinea pig and bovine P450c17 us- ing purified recombinant fusion proteins containing P450c17 linked to NADPH-P450 reductase. Drug Me- tabolism Reviews, 39, 289-307. http://dx.doi.org/10.1080/03602530701468391 [13] Oyama, T., Kagawa, N., Sugio, K., Uramoto, H., Hatano, O., Harada, N., Kaneko, K., Kawamoto, T. and Yasumoto, K. (2009) Expression of aromatase CYP19 and its relationship with parameters in NSCLC. Frontiers in Bioscience, 14, 2285-2292. http://dx.doi.org/10.2741/3379 [14] Mast, N., Linger, M. and Pikuleva, I.A. (2012) Inhibition and stimulation of activity of purified recombinant CYP11A1 by therapeutic agents. Molecular and Cellular Endocrinology, 371, 100-106. http://dx.doi.org/10.1016/j.mce.2012.10.013 [15] Woods, S.T., Sadleir, J., Downs, T., Triantopoulos, T., Headlam, M.J. and Tuckey, R.C. (1998) Expression of catalytically active human cytochrome P450scc in Es- cherichia coli and mutagenesis of isoleucine-462. Ar- chives of Biochemistry and Biophysics, 353, 109-115. http://dx.doi.org/10.1006/abbi.1998.0621 [16] Shkumatov, V.M., Radyuk, V.G., Faletrov, Y.V., Vino- gradova, A.A., Luzikov, V.N. and Novikova, L.A. (2006) Expression of cytochrome P450scc in Escherichia coli cells: Intracellular location and interaction with bacterial redox proteins. Biochemistry (Moscow), 71, 884-892. http://dx.doi.org/10.1134/S0006297906080104 [17] Brandt, M.E. and Vickery, L.E. (1992) Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli. Archives of Biochemistry and Biophysics, 294, 735-740. http://dx.doi.org/10.1016/0003-9861(92)90749-M [18] Sagara, Y., Wada, A., Takata, Y., Waterman, M.R., Se- kimizu, K. and Horiuchi, T. (1993) Direct expression of adrenodoxin reductase in Escherichia coli and the func- tional characterization. Biological & Pharmaceutical Bulletin, 7, 627-630. http://dx.doi.org/10.1248/bpb.16.627 [19] Uhlmann, H., Beckert, V., Schwarz, D. and Bernhardt, R. (1992) Expression of bovine adrenodoxin and site-di- rected mutagenesis of [2Fe-2S] cluster ligands. Bio- chemical and Biophysical Research Communications, 188, 1131-1138. http://dx.doi.org/10.1016/0006-291X(92)91349-U [20] Sagara, Y. and Aramaki, H. (1998) Overproduction in Escherichia coli and characterization of the precise ma- ture form of rat adrenodoxin. Biological & Pharmaceuti- cal Bulletin, 21, 1106-1109. http://dx.doi.org/10.1248/bpb.21.1106 [21] Nazarov, P.A., Drutsa, V.L., Miller, W.L., Shkumatov, V.M., Luzikov, V.N. and Novikova, L.A. (2003) Some features of formation and functioning of fused cholesterol hydroxylase/lyase. DNA and Cell Biology, 22, 243-252. http://dx.doi.org/10.1089/104454903321908638 [22] Shashkova, T.V., Luzikov, V.N. and Novikova, L.A. (2006) Coexpression of all constituents of the cholesterol hydroxylase/lyase system in Escherichia coli cells. Bio- chemistry (Moscow), 71, 810-814. http://dx.doi.org/10.1134/S0006297906070145 [23] Kеmbaren, R.F. and Janssen, D.B. (2008) Coexpression of three-component cytochrome P450scc in E. coli. In: 9th International symposium on Cytochrome P450 Bio- diversity and Biotechnology, Abstracts, Nice, June 8-12, 2008, p. 70. [24] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Mo- lecular Cloning: A Laboratory Manual, Cold Spring Har- bor Laboratory Press, New York. [25] Hannemann, F., Virus, C. and Bernhardt, R. (2006) De- sign of an Escherichia coli system for whole cell medi- ated steroid synthesis and molecular evolution of steroid hydroxylases. Journal of Biotechnology, 124, 172-181. http://dx.doi.org/10.1016/j.jbiotec.2006.01.009 [26] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. http://dx.doi.org/10.1038/227680a0 [27] Sidhu, R.S. and Bollon, A.P. (1987) Analysis of alpha- factor secretion signals by fusing with acid phosphatase of yeast. Gene, 54, 175-184. http://dx.doi.org/10.1016/0378-1119(87)90485-9 [28] Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randal, R.J. (1951) Protein measurement with folin-phenol re- agent. The Journal of Biological Chemistry, 193, 265- 275. [29] Sawada, N., Sakaki, T., Kitanaka, S., Takeyama, K., Kato, S. and Inouye, K. (1999) Enzymatic properties of human 25-hydroxyvitamin D3 1α-hydroxylase. European Jour- nal of Biochemistry, 265, 950-956. http://dx.doi.org/10.1046/j.1432-1327.1999.00794.x [30] Salamanca-Pinzón, S.G. and Guengerich, F.P. (2011) A tricistronic human adrenodoxin reductase-adrenodoxin- cytochrome P450 27A1 vector system for substrate hy- droxylation in Escherichia coli. Protein Expression and Purification, 79, 231-236. http://dx.doi.org/10.1016/j.pep.2011.05.008 [31] Ringle, M., Khatri, Y., Zapp, J., Hannemann, F. and Bernhardt, R. (2012) Application of a new versatile elec- tron transfer system for cytochrome P450-based Es- cherichia coli whole-cell bioconversions. Applied Micro- biology and Biotechnology, 97, 7741-7754. http://dx.doi.org/10.1007/s00253-012-4612-0 [32] Haberland, M.E. and Reynolds, J.A. (1973) Self-asso- ciation of cholesterol in aqueous solution. Proceedings of the National Academy of Sciences of USA, 70, 2313-2316. http://dx.doi.org/10.1073/pnas.70.8.2313 [33] Donova, M.V. and Egorova, O.V. (2012) Microbial ster- oid transformations: Current state and prospects. Applied Microbiology and Biotechnology, 94, 1423-1447. Copyright © 2013 SciRes. OPEN ACCESS
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182 Copyright © 2013 SciRes. 182 OPEN ACCESS http://dx.doi.org/10.1007/s00253-012-4078-0 [34] Hesselink, P.G.M., van Vliet, S., de Vries, H. and Witholt, B. (1989) Optimization of steroid side-chain cleavage by Mycobacterium sp. in the presence of cyclodextrins. En- zyme and Microbial Technology, 11, 398-404. http://dx.doi.org/10.1016/0141-0229(89)90133-6 [35] Jadoun, J. and Bar, R. (1993) Microbial transformations in a cyclodextrin medium. Part 4. Enzyme vs microbial oxidation of cholesterol. Applied Microbiology and Bio- technology, 40, 477-482. http://dx.doi.org/10.1007/BF00175734 [36] Donova, M.V., Nikolayeva, V.M., Dovbnya, D.V., Gu- levskaya, S.A. and Suzina, N.E. (2007) Methyl-β-cyc- lodextrin alters growth, activity and cell envelope fea- tures of sterol transforming mycobacteria. Microbiology, 153, 1981-1992. http://dx.doi.org/10.1099/mic.0.2006/001636-0
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