Biotransformation of progesterone by the ascomycete Aspergillus niger N402

Article abstract of DOI:10.1134/S0006297918010030

The ability of the ascomyceteAspergillus niger N402 to transform exogenous progesterone was investigated. We found that this strain has steroid-hydroxylating activity and can introduce a hydroxyl group into the progesterone molecule mainly at positions C11(α) and C21 with predominant formation of 21-hydroxyprogesterone (deoxycortone). In addition, formation of 6β,11α-dihydroxyprogesterone was also observed. Studying the effects of the growth medium composition and temperature on progesterone conversion by A. niger N402 showed that the most intense accumulation of 21-hydroxyprogesterone occurred in minimal synthetic medium at 28°C. Increasing the cultivation temperature to 37°C resulted in almost complete inhibition of the hydroxylase activity in the minimal medium. In the complete medium, a similar increase in temperature inhibited 11α-hydroxylase activity and completely suppressed 6β-hydroxylase activity, but it produced no effect on 21-hydroxylating activity.

Full text of DOI:10.1134/S0006297918010030

ISSN 0006ꢀ2979, Biochemistry (Moscow), 2018, Vol. 83, No. 1, pp. 26ꢀ31. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © O. S. Savinova, P. N. Solyev, D. V. Vasina, T. V. Tyazhelova, T. V. Fedorova, T. S. Savinova, 2018, published in Biokhimiya, 2018, Vol. 83, No. 1,
pp. 71ꢀ77.
Originally published in Biochemistry (Moscow) OnꢀLine Papers in Press, as Manuscript BM17ꢀ361, November 20, 2017.
Biotransformation of Progesterone
by the Ascomycete Aspergillus niger N402
1
2
1
O. S. Savinova *, P. N. Solyev , D. V. Vasina ,
1
1
3
T. V. Tyazhelova , T. V. Fedorova , and T. S. Savinova
1
Bach Institute of Biochemistry, Biotechnology Research Center,
Russian Academy of Sciences, 119071 Moscow, Russia; Eꢀmail: savinova_os@rambler.ru
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
2
3
Lomonosov Moscow State University, Faculty of Chemistry, 119991 Moscow, Russia
Received August 4, 2017
Revision received September 15, 2017
Abstract—The ability of the ascomycete Aspergillus niger N402 to transform exogenous progesterone was investigated. We
found that this strain has steroidꢀhydroxylating activity and can introduce a hydroxyl group into the progesterone molecule
mainly at positions C11(α) and C21 with predominant formation of 21ꢀhydroxyprogesterone (deoxycortone). In addition,
formation of 6β,11αꢀdihydroxyprogesterone was also observed. Studying the effects of the growth medium composition and
temperature on progesterone conversion by A. niger N402 showed that the most intense accumulation of 21ꢀhydroxyproꢀ
gesterone occurred in minimal synthetic medium at 28°C. Increasing the cultivation temperature to 37°C resulted in almost
complete inhibition of the hydroxylase activity in the minimal medium. In the complete medium, a similar increase in temꢀ
perature inhibited 11αꢀhydroxylase activity and completely suppressed 6βꢀhydroxylase activity, but it produced no effect on
2
1ꢀhydroxylating activity.
DOI: 10.1134/S0006297918010030
Keywords: Aspergillus niger N402, progesterone, biotransformation, 21ꢀhydroxylation, 11αꢀhydroxylation, deoxycortone
Highly selective oneꢀstep hydroxylation of steroids tions [2]. Because of this, the search for new strains capaꢀ
can be achieved only with enzymatic systems of microorꢀ ble of selective biological transformation of progesterone
ganisms, mostly mycelial fungi, including those from the into 21ꢀhydroxyprogesterone is extremely important.
genus Aspergillus. Almost all Aspergillus fungi convert
The process of targeted progesterone hydroxylation
progesterone into its 11αꢀderivative. However, some strongly depends on the transforming strain, but also on
Aspergillus strains can introduce hydroxyl groups at other the cultivation conditions, such as medium composition
positions (i.e. position 21) of the progesterone molecule. and pH, presence of metal ions, and temperature regime.
Since 21ꢀhydroxyprogesterone is a commonly used drug, ElꢀKady [3] demonstrated that Aspergillus niger cultures
the reaction of progesterone hydroxylation at position 21 hydroxylate progesterone at positions 6β and 11α. When
is of considerable practical interest. 21ꢀHydroxyprogesteꢀ 11αꢀhydroxyprogesterone was used as a substrate, the
rone (deoxycorticosterone, INN desoxycortone, CAS 6βꢀhydroxylase activity achieved a maximum at pH 6.5
2
+
2+
No. 64ꢀ85ꢀ7) is a natural hormone with mineralocortiꢀ and was inhibited by Co and Cd . The ability of A.
coid activity that is produced by the adrenal cortex. It is niger to transform progesterone was also found by Fouad
used in medicine in the form of its esters (acetate, et al. [4], who showed that A. niger converted progesꢀ
pivalate) for treatment of Addison’s disease, hypocortiꢀ terone into a mixture of 11αꢀhydroxyprogesterone and
cism, myasthenia, adynamia, and other disorders [1]. The 6β,11αꢀdihydroxyprogesterone at a 65.7 : 35.5 ratio.
use of chemical methods for hydroxyl group introduction Metwali [5] showed that A. niger (isolate No. 11/3) transꢀ
at position 21 is hindered by several undesirable side reacꢀ formed progesterone into 21ꢀhydroxyprogesterone, and
this reaction was significantly affected by the medium
composition and cultivation conditions of (pH and temꢀ
Abbreviations: CM, complete medium; HRMS, highꢀresolution
perature) – the maximal 21ꢀhydroxyprogesterone formaꢀ
tion being observed after 48 h of transformation at 28°C
and pH 6.5.
mass spectrometry; MM, minimal medium; NMR, nuclear
magnetic resonance; TLC, thinꢀlayer chromatography.
*
To whom correspondence should be addressed.
26

BIOTRANSFORMATION OF PROGESTERONE BY Aspergillus niger
27
In microorganisms, hydroxylation of steroids is catꢀ All experiments were carried out in triplicate; aliquots
alyzed by cytochrome P450ꢀdependent monooxygenases (10 ml) of the experimental and control cell suspensions
[
6]. So far, no data have been published on the ability of were withdrawn every 24 h (except the first aliquot that
cytochrome P450ꢀdependent monooxygenases from the was collected 8 h after addition of the steroid). The samꢀ
A. niger N402 to introduce a hydroxyl group into an ples of fungal cells were disintegrated with a Potter
exogenous steroid molecule, so we investigated the homogenizer, and the resulting homogenate was extractꢀ
capacity of this strain to biotransform progesterone and ed three times with an equal volume of ethyl acetate. The
studied the influence of the medium composition and combined extracts were washed twice with 5 ml of water
temperature regime on the directionality of progesterone each time and completely evaporated under vacuum. The
transformation.
residue was dissolved in 5 ml of a dichloromethane/
methanol mixture (2 : 1 v/v) and analyzed for the bioꢀ
transformation products by quantitative TLC on Silica gel
60 F254 TLC plates (Merck, USA).
MATERIALS AND METHODS
Isolation of biotransformation products. To isolate
Materials. Progesterone (I) (CAS No. 57ꢀ83ꢀ0, the products of progesterone biotransformation, mycelial
C H O ) and 11αꢀhydroxyprogesterone (III) (CAS No. cells were disintegrated directly in the cultivation medium
2
1
30
2
8
0ꢀ75ꢀ1, C H O ) from Steraloids Inc. (USA), 21ꢀ with a Potter homogenizer, and the resulting homogenate
21 30 3
hydroxyprogesterone (II) (CAS No. 64ꢀ85ꢀ7, C H O ) was extracted three times with an equal volume of ethyl
2
1
30
3
and yeast extract from SigmaꢀAldrich (USA), inorganic acetate. The combined extracts were washed with water,
salts from Fluka (Germany) and Amresco (USA), pepꢀ dried with Na SO , and completely evaporated. The
2
4
tone from Bacto Difco (USA), and dimethyl sulfoxide residue was dissolved in 5 ml of dichloromethane/ethyl
99.0%) from Serva (USA) were used. All other reagents acetate mixture (2 : 1) and subjected to chromatography
and solvents (chemically pure grade and analytical grade) on a 16 × 650ꢀmm Silica gel 60 column (0.043ꢀ0.063 mm;
were purchased from Russian chemical companies. Merck) using 30× amount of the sorbent to the weight of
(
Microorganisms and cultivation. Aspergillus niger the dry residues. A mixture of dichloromethane and aceꢀ
N402 cspA genotype strain (Aspergillus niger van Tieghem tone (0ꢀ25%) was used as the eluent. The eluted fractions
ATCC 64974, anamorph (synonym FGSC A733)), a were analyzed for the content of biotransformation prodꢀ
derivative of the A. niger van Tieghem ATCC 9029 strain ucts by TLC on Silica gel 60 F254 TLC plates (Merck) in
with short conidiophores [7], was used in the study.
the dichloromethane/acetone solvent system (9 : 1 or 4 :
To obtain inoculum, the strain was grown on agar 1 v/v). The biotransformation products were visualized
medium containing 3 g/liter malt extract, 1 g/liter pepꢀ on the plates under UV light (254 nm); the plates were
tone, and 20 g/liter agar at 28°C for 7ꢀ10 days.
then sprayed with 1% vanillin solution in 10% aqueous
Further cultivation was performed in complete solution of HClO and developed at 100ꢀ120°C.
4
medium (CM) [8] containing (g/liter): glucose, 40;
Identification of biotransformation products. The
MgSO ⋅7H O, 1; KH PO , 0.74; peptone, 1; yeast structure and purity of the isolated compounds were conꢀ
4
2
2
4
1
extract, 1; Lꢀasparagine, 0.7; and in minimal synthetic firmed by TLC (in comparison to known standards), Hꢀ
medium (MM) containing (g/liter): glucose, 20; and CꢀNMR, and highꢀresolution mass spectrometry
1
3
MgSO ⋅7H O, 0.52; KH PO , 1.52; NaNO , 0.85; (HRMS).
4
2
2
4
3
1
13
CuSO ⋅5H O, 0.13; CaCl , 0.11; KCl, 0.52; plus (in
The Hꢀ and CꢀNMR spectra were registered with
4
2
2
mg/liter) MnSO ⋅5H O, 1; ZnSO ⋅7H O, 0.8; a Bruker Avanceꢀ400 spectrometer (Bruker BioSpin
4
2
4
2
Na MoO ⋅4H O, 0.8; FeSO ⋅5H O, 0.8; plus 5 μg/liter GmbH, USA) with the working frequencies of 400 MHz
2
4
2
4
2
1
13
H BO in 0.1 M phosphateꢀcitrate buffer (pH 6.6).
for HꢀNMR and 100.6 MHz for CꢀNMR (with supꢀ
The microorganisms were grown at 28 or 37°C in a pression of the carbon–proton interactions) and a Bruker
Brunswick Innova 44 shaker at 240ꢀ250 rpm (amplitude, Avance III spectrometer (Bruker BioSpin GmbH) with
3
3
1
5
cm).
the working frequencies of 300 MHz for HꢀNMR and
1
3
Biotransformation of progesterone with A. niger 75.5 MHz for CꢀNMR (with suppression of the carꢀ
N402. Spore suspension was introduced into 750ꢀml culꢀ bon–proton interactions). The solvents used were deuterꢀ
ture flasks containing 100 ml of the cultivation medium, ated chloroform (99.8% D; SigmaꢀAldrich) or deuterated
and the fungus was grown in a shaker at 28 or 37°C for DMSO (99.9% D; SigmaꢀAldrich) relative to tetramꢀ
9
6 h. Progesterone solution in dimethyl sulfoxide ethylsilane (TMS) (NMR grade, ≥99,9%; Sigmaꢀ
(
DMSO) was them added to the cultivation medium to Aldrich) as an internal standard (δ, ppm).
the final concentration of 1 g/liter (DMSO concentration
in the cultivation medium was 4%, v/v).
Highꢀresolution mass spectra were registered with a
Bruker Daltonics micrOTOFꢀQ II triple quadrupole
Progesterone transformation was performed for timeꢀofꢀflight mass spectrometer using electrospray ionꢀ
92 h under the same cultivation conditions. Control ization (ESI); measurements were done for positively
1
samples were incubated in the absence of progesterone. charged ions. The voltage on the capillary was 4500 V;
BIOCHEMISTRY (Moscow) Vol. 83 No. 1 2018

28
SAVINOVA et al.
range of scanned masses, m/z 50ꢀ3000; external calibraꢀ (m, 4H, CHꢀ1β and CHꢀ17 and CH ꢀ2), 2.34ꢀ2.06 (m,
2
2
3
tion (Electrospray Calibrant Solution; Fluka, Germany); 2H, CH ꢀ6β and CHꢀ12β), 2.02 (td, J
13.4 Hz J
2
1a,1b
1α,2
spray pressure, 0.4 bar; flow rate, 3 μl/min; nitrogen as 3.5 Hz 1H, CHꢀ1α), 1.95ꢀ1.91 (m, 1H, CHꢀ7β), 1.87ꢀ
spray gas (6 liters/min); interface temperature, 180°C. 1.83 (m, 1H, CHꢀ15α), 1.79ꢀ1.29 (m, 10H, CHꢀ8 and
The samples were placed in the mass spectrometer spray CHꢀ15β and CH ꢀ16 and CHꢀ12α and CHꢀ1α and CHꢀ
2
chamber after HPLC on an Agilent 1260 chromatograph 6α and CHꢀ14 and CH ꢀ11), 1.18 (s, 3H, CH ꢀ19),
2
3
equipped with an Agilent Poroshell 120 ECꢀC18 column 1.22ꢀ0.93 (m, 2H, CHꢀ7α and CHꢀ9), 0.68 (s, 3H, CH3ꢀ
3.0 × 50 mm; 2.7 μm) and protective cartridge using an 18). 1
(
3
autosampler. The samples were in 50% acetonitrile (LCꢀ
CꢀNMR (100.6 MHz CDCl , δ): 210.24 (s, COꢀ
3
MS grade; Panreac, Spain) in water (MilliQꢀpurified; 20), 199.55 (s, COꢀ3), 170.86 (s, Cꢀ5), 124.04 (s, CHꢀ4),
Merck Millipore KGaA, Germany). The samples were 69.47 (s, CHꢀ21), 59.08 (s, CHꢀ17), 56.12 (s, CHꢀ14),
eluted from the column at a flow rate of 400 μl/min with 53.61 (s, CHꢀ9), 44.72 (s, Cꢀ13), 38.62 (s, Cꢀ10), 38.42
a gradient of acetonitrile concentration in water in the (s, CH ꢀ12), 35.75 (s, CH ꢀ1), 33.59 (s, CHꢀ8), 33.91 (s,
2
2
following regime: 0ꢀ15% acetonitrile for 6 min, 15ꢀ85% CH ꢀ2), 32.78 (s, CH ꢀ6), 31.92 (s, CH ꢀ7), 24.52 (s,
2
2
2
acetonitrile for 1.5 min, 85ꢀ0% acetonitrile for 0.1 min, CH ꢀ15), 22.99 (s, CH ꢀ16), 20.90 (s, CH ꢀ11), 17.42 (s,
2
2
2
and 0% acetonitrile for 2.4 min. The retention times CH ꢀ19), 13.52 (s, CH ꢀ18).
3
3
were: 6β,11αꢀdihydroxyprogesterone – 4.2 min; 11αꢀ
hydroxyprogesterone – 5.2 min; 21ꢀhydroxyprogesꢀ (published mp, 244ꢀ246°C [11]).
terone – 5.6 min; progesterone – 6.7 min. HRMS spectrum C H O (m/z): calculated for
6β,11αꢀDihydroxyprogesterone: mp, 242ꢀ244°C
2
1
30
4
+
Melting points of the isolated compounds were [M+H] 347.2217, found 347.2212; calculated for
determined with a Melting Point Mꢀ565 instrument [M+Na] 364.2482, found 364.2482.
+
1
(
Büchi Labor Technik AG, Switzerland).
HꢀNMR (300 MHz DMSOꢀd ): 5.65 (s, 1H CHꢀ
6
Characterization of biotransformation products. 11αꢀ 4), 5.08 (wd. s, 1H, OH), 4.35 (wd. s, 1H, OH), 4.14
3
Hydroxyprogesterone: mp, 164ꢀ166°C (published mp, (wd. s, 1H, CHꢀ6), 3.86 (ddd (pseudoꢀdt), 1H, J11,9
3
3
1
64ꢀ165°C [9]).
10.4 Hz J1
4.6 Hz J
4.4 Hz CHꢀ11), 2.75 (ddd
1,12α
11,12β
2
HRMS spectrum C H O (m/z): calculated for (pseudoꢀtd of protons X in the ABXY system), 1H, J
2
1
30
3
1a,1b
+
3
3
[
[
M+H] 331.2268, found 331.2264; calculated for 13.7 Hz J1
3.6 Hz J
3.3 Hz CHꢀ1β), 2.61 (dd
a,2a
1a,2b
+
3
3
M+Na] 353.2087, found 353.2088.
(pseudoꢀt), 1H, J
HꢀNMR (400 MHz, CDCl , δ): 5.73 (s, 1H, CHꢀ 2.43 (ddd, 1H, J
8.5 Hz J
8.8 Hz CHꢀ17),
1
7,16a
17,16b
1
2
3
3
14.7 Hz J
4.5 Hz J
3.6 Hz
2a,1b
3
2a,2b
2a,1a
3
3
4
), 4.04 (ddd (pseudoꢀdt), J = 10.3 Hz J
4.8 Hz CHꢀ2β), 2.18 (m, 2H, CHꢀ2α and CHꢀ12β), 2.08
11,12α
3
1
1,9
3
2
J11,12β 4.6 Hz 1H, CHꢀ11β), 2.66 (dt, J1β,1α 13.7 Hz J
(wd. s, 4H, CH ꢀ21 and CHꢀ16β), 1.92ꢀ1.86 (m, 1H,
1β,2
3
3
4
.4 Hz 1H, CHꢀ1β), 2.55 (dd (pseudoꢀt), J
8.7 Hz CHꢀ8), 1.86ꢀ1.84 (m, 2H, CHꢀ1α and CHꢀ7β), 1.67ꢀ
J17,16β 9.1 Hz 1H, CHꢀ17), 2.48ꢀ2.26 (m, 5H, CH ꢀ2 and 1.57 (m, 2H, CHꢀ16α and CHꢀ15α), 1.47 (dd (pseudoꢀ
1
7,16α
3
2
3
3
CH ꢀ6 and CHꢀ12β), 2.22ꢀ2.10 (m, 1H, CHꢀ16β), 2.13 t), 1H, J
10.4 Hz J
11.6 Hz CHꢀ12α), 1.39 (s,
2
12α,11
12α,12β
2
3
(
s, 3H, CH ꢀ21), 2.02 (td, J
13.7 Hz J 4.5 Hz 1H, 3H, CH ꢀ19), 1.30ꢀ1.08 (m, 3H, CHꢀ7α and CHꢀ15β
3
1a,1b
1α,2
3
CHꢀ1α), 1.87ꢀ1.81 (m, 1H, CHꢀ7β), 1.78ꢀ1.64 (m, 3H, and CHꢀ14), 1.00 (m (pseudoꢀt), 1H, CHꢀ9), 0.60 (s,
CHꢀ8 and CHꢀ15α and CHꢀ16α), 1.58ꢀ1.47 (2H (t, 3H, CH ꢀ18).
3
3
13
J12α,12β 11.3 Hz 1H, CHꢀ12α), 1.31 (s, 3H, CH3ꢀ19),
CꢀNMR (300 MHz DMSOꢀd ): 208.14 (s, COꢀ
20), 199.63 (s, COꢀ3), 169.56 (s, Cꢀ5), 125.51 (s, CHꢀ4),
13.1 Hz 71.22 (s, CHOHꢀ6), 67.12 (s, CHOHꢀ11), 62.37 (s, CHꢀ
α,7β
6
3
1
.29ꢀ1.20 (m, 2H, CHꢀ12α and CHꢀ14), 1.14 (t J
9
,8
3
1
0.3 Hz 1H, CHꢀ9), 1.14ꢀ1.04 (2× ddd, J
7
3
3
J7,6 13.0 Hz J
3.8 Hz 2H, CH ꢀ7α), 0.69 (s, 3H, 17), 58.20 (s, CHꢀ9), 54.72 (s, CHꢀ14), 49.33 (s, CH2ꢀ
12), 43.61 (s, Cꢀ13), 38.87 (s, Cꢀ10), 38.55 (s, CH ꢀ1),
CꢀNMR (100.6 MHz CDCl , δ): 208.91 (s, COꢀ 37.98 (s, CH ꢀ7), 34.05 (s, CH ꢀ2), 30.92 (s, CH ꢀ21),
7α,8
2
CH3ꢀ18).
2
1
3
3
2
2
3
2
6
5
4
0), 200.23 (s, COꢀ3), 170.96 (c, Cꢀ5), 124.65 (s, CHꢀ4), 27.94 (s, CHꢀ8), 23.83 (s, CH ꢀ15), 22.25 (s, CH ꢀ16),
8.93 (s, CHꢀ11), 63.21 (s, CHꢀ17), 59.08 (s, CHꢀ9), 19.57 (s, CH ꢀ19), 14.16 (s, CH ꢀ18).
2
2
3
3
5.44 (s, CHꢀ14), 50.53 (s, CH ꢀ12), 44.19 (s, Cꢀ13),
2
0.02 (s, Cꢀ10), 37.59 (s, CH ꢀ1), 35.04 (s, CH ꢀ2), 34.23
2
2
(
s, CH ꢀ6), 33.65 (s, CHꢀ8), 31.65 (s, CH ꢀ7), 31.37 (s,
RESULTS AND DISCUSSION
2
2
CH ꢀ21), 24.31 (s, CH ꢀ15), 23.07 (s, CH ꢀ16), 18.40 (s,
3
2
2
CH ꢀ19), 14.55 (s, CH ꢀ18).
In this work we investigated the ability of A. niger
1ꢀHydroxyprogesterone: mp, 141ꢀ142°C (published strain N402 to transform progesterone. Progesterone is
mp, 142ꢀ144°C [10]). commonly used as a model compound in studies of
HRMS spectrum C H O (m/z): calculated for steroid hydroxylation by mycelial fungi. We also assessed
3
3
2
2
1
30
3
+
[
M+H] 331.2268, found 331.2268.
HꢀNMR (400 MHz CDCl , δ): 5.73 (s, 1H, CHꢀ4), perature on the hydroxylation activity of A. niger and
.18 (m, 2H, CH ꢀ21). 3,26 (wd. s., 1H, OH), 2.48ꢀ2.35 directionality of progesterone hydroxylation.
the effects of medium composition and cultivation temꢀ
1
3
4
2
BIOCHEMISTRY (Moscow) Vol. 83 No. 1 2018

BIOTRANSFORMATION OF PROGESTERONE BY Aspergillus niger
29
1
3
II CM
CꢀNMR spectroscopies and chromatography coupled
2
1
1
00
80
60
II MM
III CM
III MM
with highꢀresolution mass spectrometry. Figure 1 shows
the kinetics of accumulation of progesterone biotransforꢀ
mation products in CM and MM at 28°C.
After 8 h of cultivation, no biotransformation prodꢀ
ucts were identified in MM, whereas CM contained only
small amounts of III (Fig. 1). After 24 h of cultivation,
the concentration of III in CM increased two times; the
medium also contained trace quantities of II. In contrast,
in MM the content of II was higher than the content of
III. Both media lacked the secondary biotransformation
product IV; however, it appeared in CM when A. niger
N402 was cultivated for more than 24 h. After 72 h of culꢀ
tivation, the content of IV in CM increased almost two
times; only trace amounts of IV were found in MM.
However, accumulation of II in this medium was more
intensive than in CM. ElꢀRefai et al. [12] observed forꢀ
mation of IV during progesterone biotransformation by A.
nidulans under similar conditions and concluded that at
pH close to neutral, 6βꢀhydroxylation is a secondary
process and 6β,11αꢀdihydroxyprogesterone is formed
from the previously synthesized 11αꢀhydroxyprogesꢀ
terone.
IV CM
IV MM
140
120
100
8
0
0
0
0
6
4
2
0
24
48 72 96 120 144 168 192
Transformation time, h
Fig. 1. Accumulation of progesterone biotransformation products
in MM (solid line) and CM (dashed line) for 192 h at 28°C:
rhombi, 21ꢀhydroxyprogesterone (II); circles, 11αꢀhydroxyproꢀ
gesterone (III); triangles, 6β,11αꢀdihydroxyprogesterone (IV).
The effect of the cultivation medium composition on
Therefore, in both tested media progesterone bioꢀ
the targeted hydroxylation of progesterone was studied at transformation by A. niger N402 at 28°C was shifted
two different temperatures (28 and 37°C) in two different toward 21ꢀhydroxylation. Compound IV appeared in CM
media: complete medium (CM) that has been described in trace amounts only after 24 h of biotransformation, in
by ElꢀRefai et al. [12] as the most favorable for progesꢀ MM – after 48 h, which may have resulted from the inhiꢀ
terone conversion into its 11αꢀhydroxyderivative by bition of 6βꢀmonooxygenase activity in MM. It should be
Aspergillus nidulans, and minimal medium (MM) that noted that the maximum accumulation of III was
contained 0.5 mM CuSO . Cultivation of A. niger and the observed only after 48 h of transformation irrespectively
4
process of progesterone transformation were studied in of the medium composition and then stayed at the same
the same temperature regime.
level. On the contrary, accumulation of II continued up to
We found that at 28°C, A. niger N402 exhibited 168 h of incubation in both media.
hydroxylating activity and introduced a hydroxyl group
Apparently, the reactions of 11αꢀ and 21ꢀhydroxylaꢀ
into the progesterone molecule at positions C21 and tion of progesterone are catalyzed by different enzymes.
C11(α) in both CM and MM. The byꢀproducts of this In some Aspergillus fungi, these processes are catalyzed by
biotransformation were 6β,11αꢀdihydroxyprogesterone cytochrome P450ꢀdependent monooxygenases that
and four minor hydroxylation products that were syntheꢀ belong to a multienzymatic oxidative complex, namely
sized in amounts insufficient for their identification. The progesterone 11αꢀmonooxygenase (EC 1.14.99.14) and
formation of 21ꢀhydroxyprogesterone (II), 11αꢀhydroxyꢀ steroid 21ꢀmonooxygenase (EC 1.14.99.10) [13, 14].
progesterone (III), and 6β,11αꢀdihydroxyprogesterone
IV) by A. niger N402 was confirmed by HꢀNMR and formation by A. niger N402.
Figure 2 shows the reactions of progesterone transꢀ
1
(
Fig. 2. Transformation of progesterone by A. niger N402: I, progesterone; II, 21ꢀhydroxyprogesterone; III, 11αꢀhydroxyprogesterone;
IV, 6β,11αꢀdihydroxyprogesterone.
BIOCHEMISTRY (Moscow) Vol. 83 No. 1 2018

3
0
SAVINOVA et al.
50°C) on the content of progesterone transformation
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
products by Aspergillus phoenicis and showed that accuꢀ
mulation of 11αꢀhydroxyprogesterone (III) and 6β,11αꢀ
dihydroxyprogesterone (IV) was maximal at 28°C after
20 h. When A. phoenicis was incubated for the same time
at 40°C, the content of 11αꢀdihydroxyprogesterone (III)
decreased three times, while a 6β,11αꢀdihydroxyprogesꢀ
terone (IV) was completely absent.
II
III
IV
Therefore, we demonstrated that A. niger N402
exhibits steroidꢀhydroxylating activity and modifies the
progesterone molecule at two positions (C11(α) and C21)
with subsequent 6βꢀhydroxylation of 11αꢀhydroxyproꢀ
gesterone. In both tested media, the main product of
hydroxylation was 21ꢀhydroxyprogesterone. When bioꢀ
transformation was carried out at 28°C for 168 h, the
amount of 21ꢀhydroxyprogesterone in MM was 33%
higher than in CM, and formation of 11αꢀhydroxyproꢀ
gesterone was significantly inhibited. Increasing the temꢀ
perature to 37°C did not affect 21ꢀhydroxylating activity
in CM but suppressed 11αꢀhydroxylating activity and
0
2
8
37
Temperature regime, °С
Fig. 3. Accumulation of progesterone biotransformation products
in CM after incubation for 72 h at 28 and 37°C.
Earlier research showed that the presence of copper completely inhibited 6βꢀhydroxylating activity, which
ions in the growth medium inhibits steroid С21ꢀ indicates different temperature sensitivity of the correꢀ
monooxygenase (EC 1.14.99.10) [13]. However, our sponding enzymes. We believe the results of this study to
experiments demonstrated that 0.5 mM CuSO in the be of a great practical significance because they would
4
growth MM did not suppress steroid C21ꢀmonooxygeꢀ allow control of the hydroxylation process and its selecꢀ
nase activity of A. niger N402. On the contrary, the conꢀ tivity to preferentially synthesize required hydroxy derivꢀ
tent of II in the copperꢀcontaining MM was considerably ative to facilitate purification of the target product.
2
+
higher than in CM containing no Cu probably due to
the activation of 21ꢀmonooxygenase in the presence of
2
+
Cu . The activity of 11αꢀmonooxygenase was supꢀ Acknowledgments
pressed, thereby indicating different sensitivity of these
two enzymes to metal ions. 6βꢀHydroxylase was also
The authors thank R. A. Novikov, Engelhardt
inhibited in MM, which correlates with the results of Elꢀ Institute of Molecular Biology, for registering NMR specꢀ
Kady [3] who related this inhibition to the presence of tra.
2
+
2+
Co and Cd in the medium.
This work was supported by the Russian Foundation
Another factor that determines the yield of biotransꢀ for Basic Research (project No. 17ꢀ04ꢀ00536ꢀa; Identifiꢀ
formation products is the cultivation conditions (e.g. cation of biotransformation products by chromatoꢀmassꢀ
temperature) [12]. Here we studied the effect of temperaꢀ spectrometry).
ture increase to 37°C on the quantitative and qualitative
content of progesterone biotransformation products after
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