Preparation of androsta-1,4-diene-3,17-dione from sterols using Mycobacterium neoaurum VKPM Ac-1656 strain

Article abstract of DOI:10.1134/S1068162007030132

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI₃ in situ. Nauka/Interperiodica 2007.

Full text of DOI:10.1134/S1068162007030132

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 3, pp. 354–358. © Pleiades Publishing, Inc., 2007.
Original Russian Text © M.A. Molchanova, V.A. Andryushina, T.S. Savinova, T.S. Stytsenko, N.V. Rodina, N.E. Voishvillo, 2007, published in Bioorganicheskaya Khimiya, 2007,
Vol. 33, No. 3, pp. 379–384.
Preparation of Androsta-1,4-diene-3,17-dione from Sterols Using
Mycobacterium neoaurum VKPM Ac-1656 Strain
1
M. A. Molchanovaa, , V. A. Andryushinaa, T. S. Savinovab, T. S. Stytsenkoa,
N. V. Rodinaa, N. E. Voishvilloa
a Bioengineering Center, Russian Academy of Sciences, pr. 60-letiya Oktyabrya 7/1, Moscow, 117312 Russia
b Faculty of Chemistry, Moscow State University, Leninskie gory, Moscow, 119992 Russia
Received September 4, 2006; in final form, October 15, 2006
Abstract—A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is
toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were
transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high
yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product.
Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of
M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation
of hydrocarbonate ion from NaNI3 in situ.
Key words: androstadienedione, cholesterol, Mycobacterium neoaurum, microbiological cleavage, phytoster-
ols, sorbents
DOI: 10.1134/S1068162007030132
INTRODUCTION
Androst-4-ene-3,17-dione, androsta-1,4-diene-
RESULTS AND DISCUSSION
The goal of this work was the finding of the transfor-
mation conditions of sterols at their concentration of no
less than 10 g/l using the M. neoaurum VKPM Ac-1656
culture [11]. The strain was obtained by the method of
conventional mutagenesis and selection. It differed
from the parent strain [12] by that it terminated the
transformation of 1–2 g/l of cholesterol at the stages of
the side chain cleavage and the conversion of ∆5-3β-
hydroxy group into ∆1,4-3-keto group. The bacterium
can transform sterols intoADD because it does not syn-
thesize 9α-hydroxylase, whose action consists in the
cleavage of the steroid backbone in the presence of
1,2-dehydrogenase in cells [2] (Scheme 1).
3,17-dione, and 9α-hydroxyandrost-4-ene-3,17-dione
are being used since the latter half of the last century as
key compounds in the synthesis of a number of pharma-
cologically active steroids [1].2 The mentioned steroids
are obtained by the selective cleavage of the C17-side
chain of plant and animal sterols with mutant bacterial
strains in which the biosynthesis of enzymes catalyzing
the steroid backbone degradation is inhibited. The
microorganisms containing no 9α-hydroxylase form
ADD [2]. They are mostly mutant strains of mycobac-
teria [2–9], e.g. Mycobacterium fortuitum [5] and
M. vaccae [7].
The microbiological approach to obtaining of ADD
causes problems due to its toxicity for microorganisms
at the concentrations in medium above 1.0 g/l [7, 10].
This makes impossible the reaction at a concentration
of the transformed substrate, Cho or PS, of more than
1.0 g/l. Moreover, ADD is formed, as a rule, in a mix-
ture with AD (sometimes at the ADD/AD ratio of 1 : 1),
which significantly complicates the purification of the
desired product and decreases its yield [4–10].
When concentration of Cho in medium was elevated
to 10 g/l, a significant inhibition of the culture growth
was observed: logarithmic growth phase decreased by
10 h as compared with the control, with an insignificant
increase of the number of cells, and the stationary
growth phase was practically absent. One can see in the
figure that the stationary growth phase continues up to
50–60 h upon the normal growth. We carried out the
cholesterol transformation in the presence of a resin
1
Corresponding author; phone: +7 (495) 135-3049; e-mail: adsorbing ∆4-3-ketosteroids [13] to exclude the contact
averakot@bk.ru
of formed ADD with microorganism cells. A
2
Abbreviations: AD, androst-4-ene-3,17-dione; ADD, androsta-
1,4-diene-3,17-dione; Cho, cholesterol; CFU, colony-forming
macroporous styrene–divinylbenzene copolymer was
used as such a sorbent for the obtaining of ADD from
unit; DHMP, 1-dehydrohydroxymethylpregnane; and PS, phy-
tosterols.
sterols with M. vaccae. The content ofADD in the reac-
354

PREPARATION OF ANDROSTA-1,4-DIENE-3,17-DIONE
355
CH
3
CH
3
O
CH
3
CH
CH
CH
3
3
3
3
2
3
CH
CH
CH
3
H C
3
H
3
3
1
O
2
OH
HO
O
HO
O
CH
3
XC
CH
3
OH
O
5
Scheme 1. Bacterial cleavage of steroid backbone of cholesterol catalyzed by enzymes: 1, 3β-hydroxy-∆ -steroid dehydrogenase [EC
1.1.1.145]; 2, steroid-9α-monooxygenase [EC 1.14.19.24]; 3, 3-ketoketosteroid-∆dehydrogenase [EC 1.3.99.4] according to [2].
tion mixture was 7.7 g/l, while the phytosterol load acryl ether, and methacrylic acid. These polymers and
(sitosterol/campesterol) was 15 g/l [7].
two overcrosslinked polystyrene resins MN-200 and
MN-202 that effectively adsorb aromatic compounds
from biological fluids [17] but not used earlier for the
isolation of steroids were tested for the ADD isolation
for the following parameters: non-toxicity, mechanical
strength, sorption capacity, selectivity of sorption of
∆4-3-ketosteroids, and ease of steroid desorption. The
sorbents under study had specific surface area of 500–
1000 m2/g and pore volume >1 cm3/g.
We chose a modified copolymer of ethylstyrene and
divinylbenzene, which efficiently adsorbs corticoster-
oids [13]. One can see in the figure that, in the presence
of this sorbent, the normal culture growth and almost
complete conversion of cholesterol (loading of 10 g/l)
took place; the accumulation of ADD achieved 5.5 g/l
after three days of incubation, with the formation of
ADD starting at the initial growth phase.
The transformation of phytosterols (10 g/l) was car-
ried out under the same conditions. The formation of
ADD from phytosterols occurs, however, very slowly
under the conditions suitable for cholesterol. The com-
plete conversion of phytosterols to ADD was not
achieved even after five days, with no balance of ste-
roids in cultural liquid and on the sorbent being
observed. A similar result has earlier been obtained
when phytosterols were substituted for cholesterol
transformed to AD with M. neoaurum An-1634 culture
[14].
The most appropriate sorbents for the process of
phytosterol transformation toADD under intensive stir-
ring were the polymers with a high mechanical strength
(i.e., the polymers not destructed under stirring) and
specific surface area of more than 800 m2/g. Sorbent
MN-200 was chosen among these polymers.
ADD, % of the calculated value
100
logN
9.5
9.0
8.5
8.0
7.5
7.0
6.5
90
80
70
60
50
40
30
20
10
0
2
1
It has earlier been demonstrated [6, 15, 16] that the
cleavage of the phytosterol side chain more branched
than that of cholesterol occurred with the participation
of HCO–3 ion. Therefore, we tried to saturate the reac-
tion mixture with hydrocarbonate ion by using a turbu-
lent stirring in the flasks supplied with baffles. Only in
this case the optimal concentration of HCO–3 ion was
3
4
maintained due to the presence of ë‡ëé3 in the
medium and carbon dioxide formed due to aerobic res-
piration of bacteria (Scheme 2).
18 23 29 42 45 50 53 56 60 66 96
Time, h
However, the rigorous stirring regime required a
special mechanical strength from the sorbent. The suit-
able sorbents were chosen from a series of highly
porous nonionic polymers obtained by polymerization
of divinylbenzene and ethylstyrene in the presence of
various modifying compounds, such as vinyl acetate,
(2, 3) M.neoaurum growth and (1, 4) accumulation of ADD
upon fermentation (1, 2) in the presence and (3, 4) in the
absence of sorbent. Solid and dotted arrows show bound-
aries of logarithmic growth phase in the presence and in the
absence of sorbent, respectively. N, the number of CFU per
one milliliter of culture fluid.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007

356
MOLCHANOVA et al.
CH
CH
3
3
H C
3
CH
H C
CH
3
3
3
3
H C
CH
H
3 H
CH
CH
3
3
H C
3
H
H
H C
3
H
H
CH OH
COOH
COOH
H
H
2
HO
O
β-Sitosterol
+H*CO3–
*CO–
*CO–
2
2
COOH
COOH
–CH3COOH
–C2H5*COOH
–C2H5COOH
O
COOH
–C2H5COOH
O
O
CH
CH
H
3
3
CaCO3 + CO2 + H2O
Ca2+ + 2HCO3–
H C
3
H
H C
3
H
+
H
H
H
O
O
AD
ADD
Scheme 2. Degradation of the side chain of β-sitosterol according to [6, 15, 16].
The time of addition of the sorbent to the medium tion), the yield of ADD was twofold reduced and biom-
ass density was less by one order of magnitude. When
the fermentation was carried out without a sorbent like
in the experiments with cholesterol, the number of bac-
teria did not increase and the total amount of ADD did
not exceed 1.0 g/l.
was of great importance for the phytosterol transforma-
tion toADD. If sorbent MN-200 was added either along
with the inoculum (control) or at the end of the lag
phase (20 h after the beginning of incubation), ADD
was formed in almost the same yield; however, in the
latter case, the biomass density determined as the num-
ber of colony-forming units (CFU) per ml of cultural
liquid was two times less than in control (the table). The
addition of the sorbent at the beginning of the logarith-
Under the optimal fermentation conditions (sorbent
IN-200 was introduced into the medium simulta-
neously with inoculum; the sorbent/phytosterol ratio
was 1 : 10; intensive stirring of the cultural fluid was
mic growth phase (30 h after the beginning of incuba- used), the total conversion of soy phytosterols (10 g/l)
Growth of M. neoaurum VKPM Ac-1656 culture with the formation of ADD upon fermentation in the presence of sorbent
Amount of steroids after 60 h
Transformation variant*
CFU/ml
PS, g/l
ADD, %
Fermentation for 60 h without sorbent
Fermentation for 60 h with sorbent
5 × 107
4.8 × 109
4.7 × 108
4.0–5.0
traces
10–16
67–70
30–35
Sorbent was added 30 h
after the beginning of fermentation
2.5–3.0
Sorbent was added 20 h
after the beginning of fermentation
2.1 × 109
0.5–1.0
60–65
7
Note: * The content of cells and phytosterols in the reaction mixture after inoculation was 3 × 10 CFU/ml and 9.8 g/l, respectively.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007

PREPARATION OF ANDROSTA-1,4-DIENE-3,17-DIONE
357
was completed for 55–60 h, with the sum of the result-
Cholesterol (98%) or soybean phytosterols (sito-
ing products being in balance.The content of ADD in sterol, 42%; stigmasterol, 28%; campesterol, 24%)
the mixture of the products was 65–70% and the were ground to particles of less than 15 µm in size and
amount of side products, DHMP and AD, did not
exceed 20%. The mixture did not contain the steroids
with the reduced 17-keto group (1,2-dehydrotestoster-
one and testosterone) and the ADD : AD ratio in the
mixture was 10/1. ADD containing less than 3% AD
was obtained after the separation of DHMP from the
mixture of the resulting products by the treatment with
ë‡Cl2 in aqueous isopropanol followed by crystalliza-
tion of the residue.
introduced in the form of a suspension in a medium
containing 0.3% Tween-80. Sterols and sorbents (10 ml
per 100 ml of medium) were added to the nutrient
medium (100 ml), which was then sterilized for 30 min
at a steam pressure of 0.8 atm.
Monitoring the transformation process. The
dynamics of ADD accumulation and mycobacterium
growth was carried out in 250-ml Erlenmeyer flasks
provided with the baffles and containing the medium
(30 ml) and the sorbent (5 ml). Every six hours after
inoculation, three flasks were taken off from the shaker,
culture fluid was extracted with ethyl acetate, and the
amount of the residual sterol was determined by TLC.
The culture fluid was removed from the sorbent and
successively extracted with ethanol and ethyl acetate.
The extracts were combined and evaporated, and the
dry residue was dissolved in ethanol. The amounts of
ADD and side products were determined by TLC and
HPLC. The yield of ADD was estimated in grams per
liter of the culture fluid and in percents relative to the
amount calculated for the average molecular mass of
the studied sterols (the figure shows the data averaged
for every three flasks).
Thus, the technique of the culture fluid saturation
with hydrocarbonate ion upon transforming phytoster-
ols was demonstrated to be advantageous. This ion is
formed in situ as the result of interaction of the medium
components, ëé2 formed by the aerobic bacteria respi-
ration and CaCO3; all of them were highly dispersed
due to the turbulent stirring. The transformation of phy-
tosterols (10 g/l) in the same medium under conven-
tional laminar stirring was completed only after five
days rather than for 55–60 h as in the case of turbulent
stirring.
EXPERIMENTAL
Dynamics of the culture growth. The accumula-
tion of biomass was determined as the amount of CFU
in 3- to 6-h intervals of fermentation. Culture fluid was
removed from the sorbent and inoculated on agar
medium in Petri dishes after series of standard dilutions
[14]. Since the studied culture is characterized by its
ability to form multicellular conglomerates, the first
dilution was achieved using water containing 0.01%
Tween-80, followed by homogenization of the suspen-
sion for 2–3 min. The colonies were counted after incu-
bation of dishes in a thermostat at 30°ë for seven days.
Analysis of steroid compounds. The quantitative
analysis of the transformed sterols and reaction prod-
ucts was carried out by TLC and HPLC. AD, testoster-
one, 1-dehydrotestosterone, and DHMP were used as
reference compounds along with the transformed ste-
rols and ADD. Plates Sorbfil (Krasnodar, Russia) were
used for TLC in a 1 : 3 benzene–acetone mixture.
HPLC was carried out at room temperature on a Gilson
chromatograph (United States) at 254 nm using col-
umns (4.0 × 250 mm) with Silasorb C18 (10 µm).
Culture growth and transformation conditions.
The culture of M. neoaurum VKPM Ac-1656 [11] was
stored on agar slant containing (g/l) soybean meal (5.0),
glucose (10.0), citric acid (2.2), urea (0.5), NH4Cl (1.0),
KH2PO4 (0.5), MgSO4 (0.5), FeSO4 (0.05), and CaCO3
(1.5), pH 6.8–7.1. This culture incubated for no more
than 20 days was used for transformation. Growth in
flasks and transformation processes were carried out at
30°ë on a rotary shaker at 220 rpm. Biomass was trans-
ferred from agar into 750-ml flasks containing 100 ml
of the mixture indicated above (without agar) and incu-
bated for four days. The aliquot of the resulting inocu-
lum (10% v/v) was transferred into 100 ml of the
medium containing (g/l) defatted soybean meal (15.0),
not defatted soybean meal (10.0), glucose (10.0), citric
acid (2.2), urea (0.5), NH4Cl (1.0), NH4H2PO4 (1.5),
MgSO4 (0.5), FeSO4 (0.05), CaCO3 (1.5–2.0), and Cho
or PS (10.0), pH 6.8–7.2. Common 750-ml Ehrlen-
Isolation of ADD. After the completion of fermen-
tation of mixture of phytosterols (10 g) containing 94%
of the transformed sterols, sorbent (100 ml) was sepa-
rated from the culture fluid and washed with water at
30–35°C. The complete extraction of steroids was car-
ried out with acetone (3–4 times), and the extracts were
combined and evaporated. The resulting aqueous emul-
sion containingADD was diluted with ethyl acetate, the
aqueous layer was removed, and the organic layer was
concentrated. The residual oil (6.8 g) containing ADD
(66%), AD (8.6%), and DHMP (8.8%) according to
HPLC was dissolved in ethyl acetate (70 ml). A suspen-
sion consisting of CaCl2 (5 g), isopropanol (40 ml), and
water (0.2 ml) was added to the oil, and the resulting
mixture was kept at shaking for two h at room temper-
ature, followed by the removal of precipitate. The
supernatant was dried by Na2SO4, the solvent was evap-
meier flasks and the same flasks provided with one baf- orated in a vacuum, and ethyl ether was added to the
fle were used for the transformation of cholesterol and residue. Filtration of the resulting precipitate gaveADD
phytosterols, respectively.
(4.3 g) containing 3% of AD.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007

358
MOLCHANOVA et al.
11. Voishvillo, N.E., Andryushina V.A., Molchanova, M.A.,
REFERENCES
Stytsenko T.S., and Skryabin, K.G., Claim for RF Patent
200555117587/13, positive decision: September 26,
2006.
1. Fernandes, P., Cruz, A., Angelova, B., Pinheiro, H.M.,
and Cabral, J.M.S., Enzyme and Microbial Technology,
2003, vol. 32, pp. 688–705.
2. Akhrem, A.A. and Titov, Yu.A., Steroidy i mikroorga-
nizmy (Steroids and Microorganisms), Moscow: Nauka,
1970.
3. Donova, M.V., Dovbnya, D.V., Kalinichenko, A.N.,
Arinbasarova, A.Yu., Vagabova, L.M., Morozova, Z.V.,
and Koshcheenko, K.A., RF Patent 2 039 824, Byull. Izo-
bret., 1995, no. 20.
4. Gutierrez, M.S., Rojas, N., Perez, I., Borrego, S., and
Espinosa, M., Rev. CENIC, Cienc. Biol., 1999, vol. 30,
pp. 12–16.
5. Wovcha, M.G., Biggs, C.B., and Pyke, T.R., US Patent
4345033, 1982.
6. Sih, C.J., US Patent 4755463, 1988.
7. Gottschaldt, B., Grosse, H.H., Horhold, C., Wetzker, M.,
Naumann, H., Heller, I., Burke, M., and Plonka, G., DD
Patent 301740, 1993.
12. Voishvillo, N.E., Andryushina, V.A., Savinova, T.S.,
Stytsenko, T.S., Vasil’eva, N.A., Turova, T.P., Kolga-
nova, T.V., and Skryabin, K.G., Prikl. Biokhim. Mikro-
biol., 2003, vol. 39, pp. 173–179.
13. Andryushina V.A., Stytsenko, T.S., Baklashova, T.G.,
Koshcheenko, K.A., Grinenko, T.S., Pashkin, I.I., Arin-
basarova, A.Yu., and Sukhodol’skaya G.V., RF Patent
2 093518, Byull. Izobret., 1997, no. 29.
14. Rodina, N.V., Molchanova, M.A., Voishvillo, N.E.,
Andryushina, V.A., Stytsenko, T.S., Savinova, T.S., and
Skryabin, K.G., Biotecnology 2005: State of the Art and
Prospects of Development, JSC Expo Biochem-Technol-
ogies, 2005, Nova Science Publishers, NewYork: Bioth-
echnology, Agriculture and the Food Industry, 2006,
pp. 145–155.
15. Fujimoto,Y., Chen, C.S., Szeleczky, Z., Tullio, D.D., and
Sih, C.J., J. Am. Chem. Soc., 1983, vol. 104,
pp. 4718−4720.
8. Weber, A. and Kennecke, M., US Patent 5516649, 1996.
16. Fujimoto, Y., Chen, C.S., Gopolan, A.S., and Sih, C.J.,
9. Kutney, J.P., Milanova, R.R., Vassilev, C.D., Ste-
fanov, S.S., and Nedelcheva, N.V., US Patent 6071714,
1999.
J. Am. Chem. Soc., 1983, vol. 104, pp. 4720–4722.
17. Medvedeva, O.M., Kuzakina, V.S., Dmitrienko, S.G.,
Tikhomirova, T.I., and Shpigin, O.A., J. Anal. Chem.,
2004, vol. 59, p. 669.
10. Srivastava, S.K., Srivastava, R.A.K., and Mathur, S.N.,
J. Appl. Bacteriol., 1985, vol. 59, pp. 399–402.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 3 2007