American Journal of Molecular Biology, 2013, 3, 173-182 AJMB Published Online October 2013 (
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: *
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.
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
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 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
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.
D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182
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
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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.
2.1. 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
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
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
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D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182
Copyright © 2013 SciRes.
Table 1. Escherichia coli strains and plasmids used in this study.
Recipient strain used for
construction pBar_Triple Genotype Source
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]
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-
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).
3.1. 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, -
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.
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D. S. Makeeva et al. / American Journal of Molecular Biology 3 (2013) 173-182
Copyright © 2013 SciRes.
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
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
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
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.
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
+ Tween 80 Tracesb Tracesb 72
+ MCD 60.5 ± 3 0.192 ± 0.009 72
+ Tween 80 18.35 ± 3.5 0.058 ± 0.011 72
+ MCD 30.8 ± 7 0.097 72
+ Tween 80 0 0 72
+ MCD Tracesb Tracesb 72
+ 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
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.
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.
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