Biotransformation of progesterone by Aspergillus nidulans VKPM F-1069 (wild type)

Article abstract of DOI:10.1016/j.steroids.2019.05.013

Biotechnological transformation of steroids using enzyme systems of microorganisms is often the only possible method to modify the molecule in the industrial production of steroid drugs. Filamentous fungus Aspergillus nidulans has been little studied as a steroid-transforming microorganism. We studied the ability of the A. nidulans VKPM F-1069 strain to transform progesterone (PG) for the first time. This strain converts PG into 3 main products: 11α-hydroxy-PG, 11α-acetoxy-PG and 6β,11α-dihydroxy-PG. It has been established that in the first stage, the hydroxylation of PG occurs into C11α position, then the formed 11α-hydroxy-PG is modified into 11α-acetoxy-PG and 6β,11α-dihydroxy-PG. It was found that changes in the composition of the growth medium, aeration and the duration of the mycelium cultivation do not affect the qualitative composition of PG transformation products, but their ratios have changed. Under conditions of limited aeration, the direction of secondary modification of 11α-hydroxy-PG is shifted towards the formation of 11α-acetoxy-PG.

Full text of DOI:10.1016/j.steroids.2019.05.013

Accepted Manuscript
Biotransformation of progesterone byAspergillus nidulansVKPM F-1069 (wild
type)
Olga S. Savinova, Pavel N. Solyev, Daria V. Vasina, Tatiana V. Tyazhelova,
Tatiana V. Fedorova, Tatiana S. Savinova
PII:
S0039-128X(19)30111-4
https://doi.org/10.1016/j.steroids.2019.05.013
STE 8421
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 December 2018
25 May 2019
29 May 2019
Please cite this article as: Savinova, O.S., Solyev, P.N., Vasina, D.V., Tyazhelova, T.V., Fedorova, T.V., Savinova,
T.S., Biotransformation of progesterone by Aspergillus nidulans VKPM F-1069 (wild type), Steroids (2019), doi:
https://doi.org/10.1016/j.steroids.2019.05.013
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Biotransformation of progesterone by Aspergillus nidulans VKPM F-1069 (wild type)
Olga S. Savinovaa,*, Pavel N. Solyevb, Daria V. Vasinaa, Tatiana V. Tyazhelovac, Tatiana V.
Fedorovaa, Tatiana S. Savinovad.
Authors’ affiliations
aBach Institute of Biochemistry, Federal Research Center “Fundamentals of Biotechnology”
Russian Academy of Sciences, Leninski prospect, 33, building 2, Moscow, Russian Federation,
119071;
bEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova st., 32,
Moscow, Russian Federation, 119991;
c N.I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119333 Russia
dFaculty of Chemistry, Lomonosov Moscow State University, Leninskiye gory, 1-3, Moscow,
Russian Federation, 119991
*Corresponding author: Olga S. Savinova, Bach Institute of Biochemistry, Federal Research
Center “Fundamentals of Biotechnology” Russian Academy of Sciences, Leninski prospect, 33,
building 2, Moscow, Russian Federation, 119071, e-mail: savinova_os@rambler.ru
Authors’ E-mail addresses:
*Olga S. Savinova: savinova_os@rambler.ru
Pavel N. Solyev: solyev@gmail.com
Daria V. Vasina: d.v.vasina@gmail.com
Tatiana V. Tyazhelova: t.tyazhelova@gmail.com
Tatiana V. Fedorova: fedorova_tv@mail.ru
Tatiana S. Savinova: tatiana.savinova@gmail.com
Abstract
Biotechnological transformation of steroids using enzyme systems of microorganisms is
often the only possible method to modify the molecule in the industrial production of steroid drugs.
Filamentous fungus Aspergillus nidulans has been little studied as a steroid-transforming
microorganism. We studied the ability of the A. nidulans VKPM F-1069 strain to transform
progesterone (PG) for the first time. This strain converts PG into 3 main products: 11α-hydroxy-PG,
11α-acetoxy-PG and 6β,11α-dihydroxy-PG. It has been established that in the first stage, the
hydroxylation of PG occurs into С11α position, then the formed 11α-hydroxy-PG is modified into
1

11α-acetoxy-PG and 6β,11α-dihydroxy-PG. It was found that changes in the composition of the
growth medium, aeration and the duration of the mycelium cultivation do not affect the qualitative
composition of PG transformation products, but their ratios have changed. Under conditions of
limited aeration, the direction of secondary modification of 11α-hydroxy-PG is shifted towards the
formation of 11α-acetoxy-PG.
Keywords: Aspergillus nidulans, biotransformation, progesterone, 11α-hydroxyprogesterone, 11α-
acetoxyprogesterone, 6β,11α-dihydroxyprogesterone
Highlights:
 A.nidulans F-1069 strain converts PG to 11α-OH-PG, 11α-OAc-PG and 6β,11α-diOH-PG
 The medium composition and the growth phase of fungus affect the ratio of products
 Limitation of aeration shifts the direction of 11α-OH-PG modification to acetylation
Abbreviations:
PG (I), progesterone, pregn-4-ene-3,20-dione; 11α-OH-PG (II), 11α-hydroxyprogesterone,
11α-hydroxypregn-4-ene-3,20-dione; 11α-OAc-PG (III), 11α-acetoxyprogesterone, 11α-
acetoxypregn-4-ene-3,20-dione; 6β,11α-diOH-PG (IV), 6β,11α-dihydroxyprogesterone, 6β,11α-
dihydroxypregn-4-ene-3,20-dione; CM, complete medium; MM, minimal synthetic medium;
DMSO, dimethyl sulfoxide; DCM, dichloromethane; ER, endoplasmic reticulum; ESI, electrospray
ionization; HRMS, high-resolution mass spectrometry; TLC, thin-layer chromatography
2

1. Introduction
One of the main challenges of industrial corticosteroid synthesis is the introduction of oxygen
function at the C11 position of steroid molecule, which is difficult to achieve by chemical methods.
However, hydroxylation of the inactive C–H bond of the steroid nucleus in one step with high
selectivity is possible only using the enzymatic system of microorganisms [1]. Therefore, 11(/)-
hydroxylation of steroids by microorganisms is an economically significant method for the
production of pharmacologically more active derivatives, including those having a halogen atom at
position C9. In addition, biotechnological methods for modifying steroid compounds are more
environmentally friendly comparing to chemical methods. Therefore, the search for effective
hydroxylating microorganisms remains to be relevant.
Progesterone (pregn-4-ene-3,20-dione, PG) is a natural hormone that regulates reproductive
function. It is one of the model compounds for studying the specificity of the hydroxylation of
steroids in the series of pregnanes by microorganisms. Microorganisms, including filamentous
fungi, are able to hydroxylate PG, introducing a hydroxyl group in different positions of the
molecule [2-4]. The efficiency of these processes depends not only on the applied strain of the
microorganism but also on the conditions of transformation (medium composition, pH value,
aeration, temperature regime of cultivation) [5].
Filamentous fungi of the Aspergillus genus are applied in the industrial biotechnology for 11α-
hydroxylation of steroids, for example, A. ochraceus [6; 7]. Furthermore, it is known that the
direction of sterane skeleton hydroxylation by fungi of the genus Aspergillus may depend on the
strain used: for example, PG can undergo hydroxylation at positions С6β, C11α, C11β, С14α,
С17α, С21 [8, 9]. Herewith, it is possible to form not only the monohydroxylated product, but also
its mixtures with dihydroxylated and trihydroxylated products. Some species, for example,
A. terreus [10], А.tamarii [11], А. versicolor [12], А. flavus [13], A. sojae [14] transform PG not in
the direction of hydroxylation but in the direction of the pregnane side chain elimination with the
formation of the androstane series compounds. Thus, the problem of regio- and stereo-selective
microbiological oxidation is one of the most important in the chemistry of steroids [1].
It has been shown that in various fungi including the Aspergillus genus hydroxylation is
catalyzed by steroid-hydroxylation enzymes, which are a complex of cytochrome P450-dependent
monooxygenase with flavoprotein [15, 16]. These enzymes (e.g., progesterone-11-
monooxygenase (EC No. 1.14.99.14), steroid-21-monooxygenase (EC No. 1.14.99.10) et al. [17,
18]) are part of a multienzyme oxidative complex of mycelial fungi. Steroid hydroxylases are
highly stereospecific enzymes, often localized on the endoplasmic reticulum (ER) membrane and
3

are mainly induced by substrates [1]. Their sensitivity to various environmental factors
(temperature, pH, etc.) determines the direction of substrate transformation.
The mycelial fungus Aspergillus nidulans is one of the most well-known eukaryotic
systems, widely used as a model system for decoding cell cycle biology, pathogenicity, drug
resistance, human diseases, primary and secondary metabolism of other microorganisms. Despite
this, the A. nidulans strains are not sufficiently studied as steroid-transforming microorganisms.
However, there are reports that this species have 11-monooxygenase activity and are able to
transform PG with the formation of 11α-OH-PG predominantly. For example, M.J. Henry and H.D.
Sisler [19] reported that A. nidulans (Eidam) Winter fungus (strain 003) hydroxylates PG to form a
mixture containing 11α-OH-PG, 6-OH-PG and 6,11α-diOH-PG. The culture of the fungus
A. nidulans (from the collection of the Center of cultures of the laboratory of microbiological
chemistry, National Research Center, Cairo, local habitat) transformed PG not only into 11α-OH-
PG and 6,11α-diOH-PG, but also into 21-OH-PG [20, 21].
The purpose of this study was to evaluate the ability of the wild-type A. nidulans VKPM F-
1069 strain to transform PG, to study the effect of transformation conditions, namely, the
composition of the culture medium, the duration of the mycelium cultivation and the aeration
conditions, on its direction.
2. Experimental
2.1. Materials
Progesterone (I) (CAS No. 57-83-0, С21Н30О2, M.w. 314.46; Steraloids Inc., USA) and 11-
hydroxyprogesterone (II) (CAS No. 80-75-1, С21Н30О3, M.w. 330.46; Steraloids Inc., USA), were
used as initial substrates and as standards. 11α-Acetoxyprogesterone (III) (CAS № 2268-98-6,
С23Н32О4, M.w. 372.5; Steraloids Inc., USA) was used as a standard.
Inorganic salts was purchased from companies Fluka (Germany) and Amresco (USA). The
yeast extract (Sigma-Aldrich, USA), peptone (Bacto Difco, USA), dimethyl sulfoxide (99.0%;
Serva, USA) and agar (Difco Becton Dickinson and Company, Sparks, USA) were used.
Other materials and solvents (chemically pure grade and analytical grade) were purchased
from Russian commercial suppliers.
2.2 Microorganism and culture growth
The strain Aspergillus nidulans VKPM F-1069 wild type (synonym FGSC A4; ATCC 3863,
12996, 26451; CBS 112.46; NRRL 194) from Russian National Collection of Industrial
Microorganisms (VKPM) was used.
4

To obtain spore inoculation material, A. nidulans strain was grown on the agar medium
MPA (malt extract - 3 g/L, peptone - 1 g/L, agar - 20 g/L) at 37 °C for 7-10 days. Spores were
washed from the agar medium with sterile water. Aqueous suspension of spores was introduced into
the 750 ml shaker flasks containing 100 ml of the cultivation medium up to final concentration
1×105 spores/ml. Two media for further inoculation were used: complete medium (CM) or minimal
synthetic medium (MM).
The CM medium has the following composition (g/L): glucose, 40; MgSO4×7H2O, 1.0;
КН2РО4, 0.74; peptone, 1.0; yeast extract, 1.0; L-asparagine, 0.7 [22], 200 ml of 0.5 M phosphate
citrate buffer (pH 6.6), distilled water to 1 L.
The MM medium has the following composition (g/L): glucose, 20; MgSO4×7H2O, 0.52;
KH2PO4, 1.52; NaNO3, 0.85; CuSO4×5H2O, 0.13; CaCl2, 0.11; KCl, 0.52; mixture of
microelements, 1 ml; 200 ml of 0.5 M phosphate citrate buffer (pH 6.6); distilled water to 1 L.
Mixture of microelements consists of (mg/L): MnSO4×5H2O, 800; CuSO4×5H2O, 400;
ZnSO4×7H2O, 800; FeSO4×7H2O, 800; H3BO3, 50; (NH4)2MoO4×4H2O, 800 [23].
The cultivation of the fungus was carried out on an orbital shaker Brunswick™ Innova®
44/44R at 240–250 rpm (amplitude 5 cm) at 37 °C.
2.3. Biotransformation procedure
After inoculation was over, the initial substrate was loaded into each flask. In all
experiments, the substrates were introduced into the cultivation medium as a solution in dimethyl
sulfoxide (DMSO). Final DMSO concentration in medium was 4% (v/v) in all experiments. Blank
experiment was carried out in the same conditions without A. nidulans cells. Control samples were
incubated in the absence of substrate. All experiments were performed at 37 °C at least in triplicate.
2.3.1 PG transformation
The initial concentration of PG in the medium was 1 g/L in all experiments (i.e. 100 mg/1
flask). The transformation process was carried out for 66 hours by 96-h mycelium at 240–250 rpm
to identify the products of the PG transformation.
To determine the effect of the duration of the mycelium cultivation on the result of PG
transformation 24, 48, 96 and 192-h mycelium was used, the transformation process was carried out
for 66 hours at 240–250 rpm.
The substrate bioconversion by the A. nidulans strain was performed in conditions of
intensive aeration (240–250 rpm) or limited aeration (90–100 rpm) under the same cultivation
conditions for 216 h by 96-h mycelium to study the effect of aeration. The samples were taken after
5

14 and 24 h, and then every 24 hours to study the kinetics of products accumulation. The content of
steroids was estimated in mol%, based on the initial substrate.
2.3.2 11α-OH-PG transformation
The transformation of 11α-OH-PG was performed under conditions of intensive aeration
with initial concentration of substrate in the medium 0.5 g/L (i.e. 50 mg/1 flask) by 96-h mycelium.
Two experiments with duration of 24 and 216 h transformation were performed. Upon completion
of the processes isolation and identification of the products were carried out.
2.4 Isolation of biotransformation products
To isolate the products of biotransformation, mycelium cells were disintegrated directly in
the cultivation medium with a Potter-Elvehjem homogenizer in portions of 10-35 ml, and the
resulting homogenate was extracted three times with an equal volume of ethyl acetate. The
combined extracts were washed with water, dried with Na2SO4, and completely evaporated. The
residue was dissolved in 5 ml of dichloromethane (DCM)/ethyl acetate mixture (2:1) and subjected
to chromatography on a 16×650-mm Silica gel 60 column (0.043-0.063 mm; Merck) using 30 times
the amount of the sorbent to the weight of the dry residue. A mixture of DCM and acetone (0-25%)
was used as the eluent. The eluted fractions were analyzed for the content of biotransformation
products by thin-layer chromatography (TLC) on Silica gel 60 F254 TLC plates (Merck) in the DCM
/acetone solvent systems A (9:1 v/v) or B (4:1 v/v). The biotransformation products were visualized
on the plates under UV light (254 nm); the plates were then sprayed with 1% vanillin solution in
10% aqueous solution of HClO4 and developed at 100-120 °C. The fractions showing the
simultaneous presence of few metabolites were subjected to preparative TLC on the same plates.
Individual substances were eluted from the plates with DCM-acetone mixture (1:1 v/v) and the
solvent was evaporated in vacuo to dryness. The compounds were crystallized from diethyl ether.
Chromatographic purity of the substances was monitored by TLC.
2.5 Identification of biotransformation products
The structure and purity of the isolated compounds were confirmed by TLC and HPLC (in
1
13
comparison to known standards), H NMR and C NMR, and high-resolution mass spectrometry
(HRMS).
1
13
The H NMR and C NMR spectra were registered on a Bruker Avance-400 spectrometer
1
13
(Bruker, USA) with the working frequencies of 400 MHz for H-NMR and 100.6 MHz for C-
NMR (with complete proton decoupling) and a Bruker Avance III spectrometer (Bruker, USA) with
1
13
the working frequencies of 300 MHz for H NMR and 75.5 MHz for C NMR (with complete
6

proton decoupling) at 300K. Chemical shifts in ppm were measured relative to the residual solvent
1
13
1
signals as internal standards (CDCl3, δ H: 7.26 ppm, C: 77.1 ppm; DMSO-d6, δ H: 2.50 ppm,
13C: 39.5 ppm) and relative to tetramethylsilane, spin–spin coupling constants (J) are given in Hz.
High-resolution mass spectra (HRMS) were registered on a Bruker Daltonics micrOTOF-Q
II hybrid quadrupole time-of-flight mass spectrometer using electrospray ionization (ESI);
measurements were done for positively charged ions. The voltage on the capillary was 4500 V;
range of scanned masses, m/z 50-3000; external calibration (Electrospray Calibrant Solution; Fluka,
Germany); nebulizer pressure: 0.4 bar; flow rate: 3 μl/min; nitrogen as dry gas (6 liters/min);
interface temperature: 180 °C. The samples were injected into the mass spectrometer chamber from
the Agilent 1260 HPLC system equipped with an Agilent Poroshell 120 EC-C18 column (3.0×50
mm; 2.7 μm) and an identically packed security guard using an autosampler. The samples were in
50% acetonitrile (LC-MS grade; Panreac, Spain) in water (MilliQ ultrapure water; Merck Millipore
KGaA, Germany). The column was eluted with a gradient of acetonitrile (A) concentrations in
water (B) with a flow rate of 400 μl/min in the following gradient parameters: 0-15% A for 6 min,
15-85% A for 1.5 min, 85-0% A for 0.1 min, and 0% A for 2.4 min. The retention times were:
6β,11α-diOH-PG – 4.2 min; 11α-OH-PG – 5.2 min; 11α-OAc-PG – 6.0 min, PG – 6.7 min.
Melting points of the isolated compounds were determined with a Melting Point M-565
instrument (Büchi Labor Technik AG, Switzerland).
2.6 Preparation of 11-acetoxyprogesterone by chemical method
A solution of 11α-OH-PG (34 mg) in acetic anhydride (0.2 ml) containing one drop of 60%
HClO4 was kept at room temperature for 1 h. After completion of the reaction, the reaction mixture
was added dropwise in 5 ml of water containing 1 ml of 25% ammonia solution, and stirred for 30
min (pH ~ 7.5). Then the reaction mass was extracted with DCM three times, the extract was
washed with water until neutral, evaporated to dryness. A dry residue (39 mg) was obtained, and
11α-OAc-PG (27 mg) was recovered by preparative chromatography. The yield was 70.5%, mp.
173-174 С (published mp. 175-177 С [24]).
2.7 Characterization of biotransformation products
11-Hydroxypregn-4-ene-3,20-dione (11-hydroxyprogesterone, 11α-OH-PG, II),
Mp. 164-166 С (published mp. 164-165 С [25]). M.w. 330.46.
HRMS spectrum C21H30O3 (m/z): calculated for [M+H]+ 331.2268, found 331.2264;
calculated for [M+Na]+ 353.2087, found 353.2088.
1H NMR (400 MHz, CDCl3, ): 5.73 (s, 1H, CH-4), 4.04 (ddd (pseudo-dt), 3J11, 9 = 10,3 Hz
3J11, 12α 4.8 Hz 3J11, 12β 4.6 Hz 1H, CH-11), 2.66 (dt, 2J1β, 1α 13.7 Hz 3J1β, 2 4.4 Hz 1H, CH-1β), 2.55
7

(dd (pseudo-t), 3J17, 16α 8.7 Hz 3J17, 16β 9.1 Hz 1H, CH-17), 2.48-2.26 (m, 5H, CH2-2 and CH2-6 and
2
3
CH-12β), 2.22-2.10 (m, 1H, CH-16β), 2.13 (s, 3H, CH3-21), 2.02 (td, J1a, 1b 13.7 Hz J1α, 2 4.5 Hz
1H, CH-1α), 1.87-1.81 (m, 1H, CH-7β), 1.78-1.64 (m, 3H, CH-8 and CH-15α and CH-16α), 1.58-
3
1.47 (2H (t, J12α, 12β 11.3 Hz 1H, CH-12α), 1.31 (s, 3H, CH3-19), 1.29-1.20 (m, 2H, CH-12α and
CH-14), 1.14 (t 3J9, 8 10.3 Hz 1H, CH-9), 1.14-1.04 (2×ddd, 3J7α, 7β 13.1 Hz 3J7, 6 13.0 Hz 3J7α, 8 3.8
Hz 2H, CH2-7α), 0.69 (s, 3H, CH3-18).
13C NMR (100.6 МHz CDCl3, ): 208.91 (s, CO-20), 200.23 (s, CO-3), 170.96 (s, C-5),
124.65 (s, CH-4), 68.93 (s, CH-11), 63.21 (s, CH-17), 59.08 (s, CH-9), 55.44 (s, CH-14), 50.53 (s,
CH2-12), 44.19 (s, C-13), 40.02 (s, C-10), 37.59 (s, CH2-1), 35.04 (s, CH2-2), 34.23 (s, CH2-6),
33.65 (s, CH-8), 31.65 (s, CH2-7), 31.37 (s, CH3-21), 24.31 (s, CH2-15), 23.07 (s, CH2-16), 18.40
(s, CH3-19), 14.55 (s, CH3-18).
The 1H NMR and 13C NMR spectrums were identical to the standard sample.
11-Acetoxypregn-4-ene-3,20-dione (11-acetoxyprogesterone, 11α-OAc-PG, III)
Mp. 172-174 С (published mp. 175-177 С [24]. M.w. 372.5;
HRMS spectrum C23H32O4 (m/z): calculated for [M+H]+ 373.2373, found 373.2361;
calculated for [M+Na]+ 395.2193, found 395.2183.
1H NMR (300 MHz, DMSO-d6) , δ: 5.67 (s, 1H, CH-4), 5.14 (dt, 3J11, 9 = 10.6 Hz, 3J11, 10 5.0
Hz, 1H, CH-11), 2,62 (dd (pseudo-t), 3J17, 16α 8.9 Hz, 3J17, 16β 9.1 Hz, 1H, CH-17), 2.47-2.35 (m, 2H,
CH-2β and CH-6β),2.29-2.12 (m, 3H, CH-6α and CH-12β and CH-2α), 2.10-1.96 (m, 1H, CH-16β),
2.05 (s, 3H, CH3-21), 2.01 (s, 3H, CH3-23), 1.91-1.78 (m, 3H, CH2-1 and CH-7α), 1.72-1.60 (m,
3H, CH-16α and CH-15α and CH-8), 1.50 (t, 3J12α, 12β 11.6 Hz, 1H, CH-12α), 1.44 (t, 3J9, 8 10.6 Hz,
1H, CH-9), 1.39-1.29 (ddd, 3J14, 8 10.7 Hz, 3J14, 15β 6.4 Hz, 3J14, 15α 5.6 Hz, 1H, CH-14), 1.29 (wd.s,
3
3
3
3
4H, CH3-19 and CH-15β), 1.17-1.00 (2×ddd, J7α, 7β 12.6 Hz, J7, 6α ~ J7, 6β 12.2 Hz, J7α, 8 3.1 Hz,
2H, CH2-7), 0.64 (s, 3H, CH3-18).
13C NMR (300 MHz, DMSO-d6) , δ: 208.61 (с, CO-20), 198.40 (с, CO-3), 170.14 (с, C-5),
170.10 (с, CO-22) 124.48 (с, CH-4), 70.58 (с, CHOH-11), 62.30 (с, CH-17), 55.34 (с, CH-9), 54.39
(с, CH-14), 45.02 (с, CH2-12), 43.54 (с, C-13), 39.75 (с, C-10), 36.79 (с, CH2-1), 34.72 (с, CH-8)
34.19 (с, CH2-2), 33.03 (с, CH2-6), 31.67 (с, CH2-7), 31.37 (с, CH3-21), 24.16 (с, CH2-15), 22.96
(с, CH2-16), 22.02 (с, CH3-23), 18.20 (с, CH3-19), 14.24 (с, CH3-18).
The 1H NMR and 13C NMR spectrums were identical to the standard sample, as well as the
spectra of the sample obtained by the chemical method of 11-OH-PG acetylation.
6,11-Dihydroxypregn-4-ene-3,20-dione (6,11-dihydroxyprogesterone, 6β,11α-di-OH-PG,
IV)
8

Mp. 242-244 С (published mp. 244-246 С [26]. M.w. 346.46.
HRMS spectrum C21H30O4 (m/z): calculated for [M+H]+ 347.2217, found 347.2212; calculated for
[M+NH4]+ 364.2482, found 364.2482.
1H NMR (300 МHz DMSO-d6): 5.65 (s, 1H CH-4), 5.08 (br. s, 1H, OH), 4.35 (wd. s, 1H,
OH), 4.14 (br. s, 1H, CH-6), 3.86 (ddd (pseudo-dt), 3J11, 9 10.4 Hz 3J11, 12α 4.6 Hz 3J11, 12β 4.4 Hz 1H,
CH-11), 2.75 (ddd (pseudo-td of protons X in the ABXY system), 2J1a, 1b 13.7 Hz 3J1a, 2a 3.6 Hz 3J1a,
3
3
3.3 Hz, 1H, CH-1β), 2.61 (dd (pseudo-t), J17, 16a 8.5 Hz J17, 16b 8.8 Hz,1H, CH-17), 2.43 (ddd,
2b
3
3
2J2a, 2b 14.7 Hz J2a, 1a 4.5 Hz J2a, 1b 3.6 Hz, 1H, CH-2β), 2.18 (m, 2H, CH-2α and CH-12β), 2.08
(br. s, 4H, CH3-21 and CH-16β), 1.92-1.86 (m, 1H, CH-8), 1.86-1.84 (m, 2H, CH-1α and CH-7β),
1.67-1.57 (m, 2H, CH-16α and CH-15α), 1.47 (dd (pseudo-t), 3J12α, 11 10.4 Hz 3J12α, 12β 11.6 Hz, 1H,
CH-12α), 1.39 (s, 3H, CH3-19), 1.30-1.08 (m, 3H, CH-7α and CH-15β and CH-14), 1.00 (m
(pseudo-t), 1H, CH-9), 0.60 (s, 3H, CH3-18).
13C NMR (300 МHz DMSO-d6): 208.14 (s, CO-20), 199.63 (s, CO-3), 169.56 (s, C-5),
125.51 (s, CH-4), 71.22 (s, CHOH-6), 67.12 (s, CHOH-11), 62.37 (s, CH-17), 58.20 (s, CH-9),
54.72 (s, CH-14), 49.33 (s, CH2-12), 43.61 (s, C-13), 38.87 (s, C-10), 38.55 (s, CH2-1), 37.98 (s,
CH2-7), 34.05 (s, CH2-2), 30.92 (s, CH3-21), 27.94 (s, CH-8), 23.83 (s, CH2-15), 22.25 (s, CH2-16),
19.57 (s, CH3-19), 14.16 (s, CH3-18).
3. Results and discussion
It is known that the steroid-hydroxylation activity of the mycelial fungus may depend on the
duration of mycelium cultivation before substrate loading [27]. The kinetics of A. nidulans F-1069
mycelium growth in CM medium was studied. This medium was previously described by A.H. El-
refai and K.M. Ghanem [20], as the most favorable for the conversion of PG to 11-hydroxy-
derivative by A. nidulans culture. The kinetics of fungal biomass accumulation in the process of
growing is presented (Figure 1).
Figure 1
On 96 h of cultivation the A. nidulans F-1069 reached the end of the logarithmic growth
phase accompanied with the significant biomass production. Therefore, a 96-h mycelium was used
for identification of the PG transformation products.
3.1. Biotransformation of PG
9

After growing mycelium for 96 h, the PG was loaded as a solution in DMSO into the growth
medium without mycelium separation. DMSO not only increases the substrate solubility in aqueous
medium [28] but also leads to an increase in the yield of 11-hydroxylated product [29].
It has been found that the strain A. nidulans VKPM F-1069 has steroid-hydroxylation
activity. PG (I) was converted to three major metabolites: 11-OH-PG (II), 11-OAc-PG (III) and
6,11-diOH-PG (IV) with the following Rf values 0.43, 0.84 and 0.24, respectively, (PG Rf -
0.89) in system B (eluted twice) (Figure S1). The products were identified via HPLC-HRMS
(Figure 2).
Figure 2
The formation of monohydroxylated product (II) was supported by accurate mass
determination of C21H30O3 in ESI HRMS, m/z: [M+H]+ found 331.2264 (calcd. 331.2268) and
1
[M+Na]+ found 353.2088 (calcd. 353.2087). 11α-OH-PG (II) was identified by its H NMR and
13C NMR spectra and in comparison with that of the PG (I) and the spectra of the 11α-OH-PG
sample (II), obtained by us earlier [4] (spectra can be found in the Supplementary material, S2-S4).
The structure of the acetylated product (III) was determined by several analytical methods.
Formation of III was supported by accurate mass determination of C23H32O4 in ESI HRMS, m/z:
[M+H]+ found 373.2361 (calcd. 373.2373) and [M+Na]+ found 395.2183 (calcd. 395.2193). ). NMR
spectra of the obtained 11-OAc-PG (III) were identical to the spectra of the commercial standard
sample and the chemically synthesized one (section 2.6 of this article). In addition, 11-OAc-PG
(III) obtained during the PG transformation was hydrolyzed by a known method of chemical
solvolysis under base catalysis conditions: by KOH in methanol medium [30] to form 11-OH-PG.
(spectra can be found in the Supplementary material, S5-S13).
1
13
The formation of dihydroxylated poduct (IV) was confirmed by the data of Н NMR, С
NMR, 2D NMR spectroscopy and HPLC-HRMS, and matched with 6,11-diOH-PG sample
obtained by us earlier [4] (spectra can be found in the Supplementary material, S14-S22). There are
peculiarities in the observed signals that haven’t been discussed earlier in literature. For example,
the proton at CH-6 is expected as a part of the ABX system, nevertheless, it appears unresolved as a
broadened singlet. This can be explained by the long-range coupling interaction 4JCH-6, CH-4 through
space, since these hydrogen atoms are coplanar, via the so called W-pathways. The effect is
additionally confirmed in HMBC spectrum where H-4 / C-6 cross-peak demonstrates a pronounced
interaction (as well as H-4/C-2 or H-6/C-8 cross-peak). Several CH2-signals are found in ABXY
patterns in 1H NMR, e.g. CH2-1, CH2-7, CH2-12, as well as in ABX patterns, e.g. CH2-15 and CH2-
16 groups. Their assignment is challenging; due to the conformational restrictions they are vastly
splitted in 1H NMR with the second-order effects, overlap with other signals and may be misplaced
10

in the attribution. We carried out an independent thorough investigation with 2D NMR experiments
in DMSO-d6 and compared our assignment with previous studies on NMR of the steroid systems
13
[31]. Our data confirm the shifts assignments of earlier reports of C NMR of 6β,11α-dihydroxy-
PG [32], and clarify the missing characteristic CH2-7 and CH2-10 signals of earlier papers [26], as
well as previous lack of fine structure description of 1H NMR spectrum.
1
13
All the H, C, HSQC and HMBC NMR spectra, HRMS spectral data as well as HPLC
profiles of the formed progesterone derivatives can be found in the Supporting information.
3.2. The effect of the duration of mycelium cultivation on the transformation products
accumulation
To determine the effect of the duration of the mycelium cultivation on the result of PG
transformation, we used the mycelium at the beginning, middle and at the end of the logarithmic
growth phase (24, 48 and 96 h, respectively), as well as mycelium at the stationary growth phase
(192 h). The processes were performed with intensive aeration under the conditions described above
in section 2.3.1.
Regardless of the duration of the fungus cultivation, the component composition of the
major products did not change, however their ratios have changed (Figure 3, Supplementary
material Table S23).
Figure 3
With an increase of the duration of the mycelium cultivation up to 96 h, the PG conversion
increased. The most intensive accumulation of products III and IV occurred when using 96-h
mycelium (peak of the logarithmic growth phase). The transformation of PG (I) by stationary
growth phase mycelium (192 h) resulted in lower PG conversion with reduced quantities of
products III and IV, and increased content of (II) comparing with 96 h mycelium. Reduction of 6β-
hydroxylating activity of A. nidulans strain with the increase of the duration of the mycelium
cultivation was observed earlier [20].
3.3. The kinetics of products accumulation during the PG biotransformation
Some authors note that at pH values close to neutral, 6β-hydroxylation is a secondary
process, and 6,11α-diOH-PG is produced from the initially formed 11α-OH-PG [20, 33, 34]. Here
we assume that 11α-OAc-PG is also a product of its secondary modification. To confirm this, we
studied the kinetics of products accumulation during PG transformation process. The
biotransformation was performed under the conditions described above (section 3.1) for 216 hours.
The kinetics of products accumulation is shown in Figure 4 (solid lines).
11

Figure 4
It is important to note, that TLC and HPLC chromatograms of the sample taken after 14 h of
transformation showed the presence of untransformed PG (98.5% of loaded) and 11-OAc-PG (0.8
mol %). During further transformation the formation of 6,11-diOH-PG and 11α-OH-PG was
observed.
After 216 h of transformation from 300 mg of loaded PG (3 flasks) three main products
were observed. The dry residue after the evaporation of the extract contained 123 mg (34.6 mol %)
of 11-OAc-PG, 6.3 mg (2.0 mol %) of 11α-OH-PG, and 150 mg (45.4 mol %) 6β,11α-diOH-PG
аccording to HPLC analysis. The untransformed PG was absent.
3.4. Transformation of 11α-OH-PG
Additionally, experiments on the transformation of 11α-OH-PG under similar conditions
were performed. 200 mg of 11-OH-PG was loaded for the transformation (4 flasks).
After 24 hours of transformation 178 mg (88.0 mol %) of untransformed 11α-OH-PG and
19.8 mg (8.8 mol %) of 11-OAc-PG were found in the dry residue after evaporation of the extract
according to TLC and HPLC data.
After 216 hours of transformation the dry residue contained 192 mg (85.2 mol %) of 11-
OAc-PG and 26 mg (12.4 mol %) of 6β,11α-diOH-PG. According to HPLC data 11-OH-PG was
practically absent. The yield of crystalline 11-OAc-PG was 180 mg (80%, counting on the loaded
11-OH-PG).
Thus, the complete conversion both PG and 11-OH-PG was achieved after 216 hours of
transformation. However, the selectivity of 11-OAc-PG formation in the first case was 34.6 mol%,
and in the second it reached 85 mol%. Consequently, products III and IV are competitive and can
be obtained both from PG and from 11α-hydroxy-PG. However, the appearance of 6β,11α-diOH-
PG occurs earlier when using PG as an initial substrate (Figure 4B). This may be due to the
presence of PG, which is known to be an inducer of 6β-hydroxylase [20]. The scheme of the studied
transformations is shown in Figure 5.
Figure 5
In the first stage, the hydroxylation of PG occurs into C11α position, and then the formed
11α-hydroxy-PG is modified into 11α-acetoxy-PG and 6β,11α-diOH-PG.
It is known that some microorganisms can perform acetylation of secondary alcohols,
including steroid compounds. For example, the ability of yeast and yeast-like organisms
(Saccharomyces fragilis, S. lactis, Candida pseudotropicalis and Torulopsis sphaerica) to acetylate
testosterone in the CM medium was previously described [22]. However, acetylation occurred only
12

with 17β-hydroxy derivative, whereas the corresponding 17α-epimer remained intact. In addition,
the authors noted that under these conditions acetylation of the steroid molecule with the hydroxy
group at the 11α, 11β, 20β and 21 positions does not occur. Therefore, acetylation of the secondary
hydroxyl groups is usually carried out chemically, using, for example, acetic anhydride as the
acetylating agent. For example, 11-OAc-PG is obtained from 11-OH-PG by the chemical
method using acetic anhydride in pyridine medium [24].The ability of mycelial fungi, in particular,
the Aspergillus genus, to acetylate steroid alcohols has not been described previously. Here we
report about the ability of the A. nidulans VKPM F-1069 strain to acetylate 11-OH-PG for the first
time.
It is known that esters are the products of an enzyme-catalyzed condensation reaction
between acyl-CoA and alcohols. The biochemical esterification reaction is catalyzed by an ester
synthase and the ester synthesis rate is determined by the concentration of the available substrate
and the total enzymatic activity [35].
Moreover, fungal steroid hydroxylases are often localized on the ER membrane [1, 16]. The
synthesis of fatty acids with the participation of acetyl-coenzyme A (acetyl-CoA) occurs on the
membranes of the ER in the cytoplasm as well [36]. Acetyl-CoA is the sole donor of acetyl groups
for acetylation [37, 38]. It is known to be also involved in O-acetylation of polysaccharides,
alkaloids, anthocyanins, isoprenoids and phenols in plants [39].
It can be assumed that acetylation of 11-OH-PG occurs with the participation of acetyl-CoA,
which can act as a donor of the acetyl group. However, the amount of 11-OAc-PG depends not
only on the concentration of 11-OH-PG (i.e. 11-hydroxylation activity of a strain) but also on the
activity of the enzyme 6β-hydroxylase, under the action of which 11-OH-PG is converted into
6β,11α-diOH-PG - a product of the competitive direction of its transformation. It is known that the
efficiency of hydroxylation processes depends on the aeration [40].
Therefore, we have assumed that the direction of secondary modification of the primary formed
11-OH-PG can be dependent on the aeration intensity.
3.5. The aeration effect on the accumulation of PG transformation products
The aeration effect on the PG biotransformation was studied in shake flasks with varying
agitation regimes: intensive aeration and limited aeration. The substrate was loaded after 96 hours
of strain cultivation in CM. Figure 4A shows the dependence of PG conversion rate on the agitation
regime, and Figure 4B shows the kinetics of the products accumulation under different aeration.
As can be seen from Figure 4A, the PG conversion rate decreases when aeration is limited.
At the same time, during the transformation under limited aeration conditions, there is a decrease in
the accumulation of compound IV, and an increase in the content of the monohydroxylated product
13

(II) in comparison with the conditions of intensive aeration (Figure 4B). The maximum amount of
product IV is reached by 96 h of transformation under the intensive aeration (45.4 mol %), and by
120 h under limited aeration (36.3 mol %), and does not change further. It should also be noted that
the aeration regime has little effect on the accumulation rate and the amount of 11-OAc-PG. After
the 6β-hydroxylation process has stopped, the process of 11-OH-PG acetylation continues in both
cases, as can be seen from Figure 4B. Moreover, it proceeds more intensively under conditions of
limited aeration.
3.6. The medium composition effect on the accumulation of transformation products
To evaluate the effect of medium composition on the result of PG transformation we used
CM and MM media. The diagram of PG biotransformation products accumulation by 96-h culture
of the fungus on different media under conditions of intensive aeration is shown in Figure 6.
Figure 6
The biotransformation proceeds to form the same composition of products with both media
for inoculation and PG transformation. Herewith, PG conversion in MM medium slows down more
than twice as compared with the CM medium. However, the quantity of the monohydroxylated
product (II) on the MM medium is twice higher, due to the secondary transformation suppression:
6β-hydroxylating activity is halved, and acetylating activity is 6 times lower. A similar decrease in
the activity of 6β-hydroxylase during the PG transformation by the A. niger strain in MM medium
at 37 °C was observed earlier by us [4].
Conclusion
In this study, we demonstrated that filamentous fungus A. nidulans VKPM F-1069
hydroxylates the PG at the position C11α with subsequent 6β-hydroxylation and acetylation of the
formed 11α-OH-PG in situ. The possibility of biocatalytic acetylation of the steroid secondary
hydroxyl group by filamentous fungus is shown by us for the first time. It has been found that
changes in the duration of the mycelium cultivation and the composition of the growth medium do
not affect the qualitative composition of PG transformation products but changes their ratio. Under
limited aeration, the direction of secondary modification of 11α-OH-PG was shifted towards the
formation of 11α-OAc-PG by reducing the 6-hydroxylase activity.
One of the advantages of the A. nidulans strain used is the absence of the side-chain
cleavage at the C17-C20 bond with the formation of the androstane series compounds
(androstenedione, testosterone and its acetate) during the transformation of PG.
14

The obtained data can serve as a basis for the development of the medical drugs synthesis
technology alternative to chemical synthesis, where A. nidulans strain can be used as a biocatalyst
for biotechnological modification of steroids. Compounds II and III obtained by biocatalytic
transformation are of interest not only as precursors of highly active steroid preparations but also as
independent drugs with gestagenic and antiandrogenic activity [41-43].
Acknowledgement
This work was supported by the Russian Foundation for Basic Research, grant №18-34-00653.
15

Figures
Figure 1 – Kinetics of fungal biomass accumulation
Figure 2 – HPLC profile of PG biotransformation major metabolites. HPLC conditions: Agilent
Poroshell 120 EC-C18 (3.0  50 mm; 2,7 µm) column; flow rate 400 µL/min in a gradient of
concentrations of A (CH3CN) in B (H2O): 0-15% A for 6.0 min, 15%-85% A for 1.5 min, 85%-0%
A for 0.1 min, 0% A for 2.4 min.
Figure 3 – Effect of the duration of the mycelium cultivation on the ratio of PG transformation
products
Figure 4 – Kinetics of the PG conversion under different aeration: A- content of the residual
amount of PG, B- content of transformation products. Designations: solid line –intensive aeration,
dashed line – limited aeration; ■- PG (I), ▲ – 11-OH-PG (II), ● – 11α-OAc-PG (III), X– 6β,11α-
diOH-PG (IV).
Figure 5 – Scheme of PG transformation
Figure 6 – Effect of medium composition on the ratio of PG transformation products
Appendix A. Supplementary data
Supplementary data to this article can be found online at ….
References
[1] M.V. Donova, O.V. Egorova, Microbial steroid transformations: Current state and prospects.
Appl. Microbiol. Biotechnol. 94 (2012) 1423-1447. https://doi.org/10.1007/s00253-012-4078-0
[2] P. Fernandes, A. Cruz, B. Angelova, H.M. Pinheiro, J.M.S. Cabral, Microbial conversion of
steroid compounds: recent developments, Enz. Microb. Technol. 32 (2003) 688–705.
https://doi.org/10.1016/S0141-0229(03)00029-2
[3] M. Vitas, K. Smith, D. Rozman, R. Komel, Progesterone Metabolism by the Filamentous
Fungus Cochliobolus lunatus. J. Steroid Biochem. Molec. Biol. 49(1) (1994) 87-92.
https://doi.org/10.1016/0960-0760(94)90305-0
[4] A.A. Zohri, Progesterone Transformation as a Biochemical Aid in Classification of the Genus
Emericella, Folia Microbiol. 45(5) (2000) 391-396. PMID:11347266
16

[5] O.S. Savinova, P.N. Solyev, D.V. Vasina, T.V. Tyazhelova, T.V. Fedorova, T.S. Savinova,
Biotransformation of progesterone by ascomycete Aspergillus niger N402, Biochemistry
(Moscow), 83(1) (2018) 26-31. https://doi.org/10.1134/S0006297918010030
[6] T.K. Dutta, T.B. Samanta. Bioconversion of progesterone by the activated immobilized conidia
of Aspergillus ochraceus TS. Curr. Microbiol. 39(6) (1999) 309-312. DOI:10.1007/s002849900464
[7] P. Somal, C.L. Chopra. Microbial conversion of steroids. III: 11a-hydroxylation by fungal
mycelium. Appl. Microbiol. Biotechnol. 21(5) (1985) 267-269. https://doi.org/10.1007/bf00252701
[8] C.M. Hull, A.G.S. Warrilow, N.J. Rolley, C.L. Price, I.S. Donnison, D.E. Kelly, S.L. Kelly, Co-
production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal
steroid hydroxylases. Biotechnol Biofuels 10(1) (2017) 226. https://doi.org/10.1186/s13068-017-
0904-z
[9] T. Hosseinabadi, H. Vahidi, B. Nickavar, F. Kobarfard, Biotransformation of progesterone by
whole cells of filamentous fungi Aspergillus brasiliensis, Iran J. Pharm. Res. 14 (2015) 919-924.
PMCID:PMC4518121
[10] K. Yildirim, A. Uzuner, and E.Y. Gulcuoglu, Biotransformation of some steroids by
Aspergillus terreus MRC 200365, Collect. Czech. Chem. Commun. 75(6) (2010) 665–673.
https://doi.org/10.1135/cccc2009545
[11] K. Yildirim, A. Uzuner, and E.Y. Gulcuoglu, Baeyer-Villiger oxidation of some steroids by
Aspergillus tamarii MRC 72400, Collect. Czech. Chem. Commun. 76(6) (2011) 743–754.
https://doi.org/10.1135/cccc2011008
[12] H.Y. Yang, H.L. Su, G. Du, G.J. Shen, J.X. Sun, and H.Y. Chen. Progesterone side-chain
cleavage by Aspergillus versicolor. Advanced Materials Research. 781-784 (2013) 1164-1167.
https://doi.org/10.4028/www.scientific.net/AMR.781-784.1164
[13] M. Eman Mostafa, A.A. Zohri. Progesterone side-chain degradation by some species of
Aspergillus
flavus
group.
Folia
Microbiol
(Praha).
45(3)
(2000)
243-247.
https://doi.org/10.1007/bf02908952
[14] M. Javid, B. Nickavar, H.Vahidi, M.A. Faramarzi, Baeyer-Villiger oxidation of progesterone
by Aspergillus sojae PTCC 5196, Steroids 140 (2018) 52-57.
https://doi.org/10.1016/j.steroids.2018.07.008
[15] P. Durairaj, J.-S. Hur, H. Yun, Versatile biocatalysis of fungal cytochrome P450
monooxygenases, Microb. Cell. Fact.15 (2016) 125. https://doi.org/10.1186/s12934-016-0523-6
[16] K. Kristan, T.L. Rižner, Steroid-transforming enzymes in fungi. The Journal of Steroid
Biochemistry
and
Molecular
Biology,
129(1-2)
(2012)
79–91.
https://doi.org/10.1016/j.jsbmb.2011.08.012
17

[17] D. Schomburg, and I. Schomburg, Class 1 -Oxidoreductases XII. Vol. 27. Springer Handbook
of Enzymes. Springer Verlag, Berlin, Second ed., 2006, p.302
[18] T.B. Samanta, D.K. Ghosh, Characterization of progesterone 11 alpha-hydroxylase of
Aspergillus ochraceus TS: a cytochrome P-450 linked monooxygenase, J. Steroid. Biochem. 28
(1987) 327-332. https://doi.org/10.1016/0022-4731(87)91026-0
[19] M.J. Henry, H.D. Sisler, Effects of sterol biosynthesis-inhibiting (SBI) fungicides on
cytochrome P-450 oxygenations in fungi. Pesticide Biochemistry and Physiology, 22(3) (1984)
262-275. https://doi.org/10.1016/0048-3575(84)90019-1
[20] A.H. El-Refai, K.M. Ghanem. Some physiological relations of progesterone conversion by
Aspergillus nidulans. Egyptian Journal of Microbiology (EJM), 22(2) (1987) 327-338
[21] A.H. El-Refai, K.M. Ghanem, Microbial response to steroids, Egyptian Journal of
Microbiology (EJM). 23(1) (1989) 1-11
[22] A. Čapek, M. Tadra, J. Tůma, Microbial transformations of steroids XXIV. Separation of
androstane 17-hydroxy-epimers,
https://doi.org/10.1007/BF02874190
Folia Microbiol (Praha). 9(6) (1964) 380–382.
[23] A.M. Chulkin, D.S. Loginov, E.A. Vavilova, A.R. Abyanova, I.N. Zorov, S.A. Kurzeev, O.V.
Koroleva, S.V. Benevolenskii, Enzymological properties of endo-(1-4)-β-glucanase Eg12p of
Penicillium canescens and characteristics of structural gene egl2, Biochemistry (Moscow), 74 (6)
(2009) 655–652. https://doi.org/10.1134/S0006297909060108
[24] D.H. Peterson, H.C. Murray, S.H. Eppstein, L.M. Reineke, A. Weintraub, P.D. Meister, H.M.
Leigh. Microbiological Transformations of Steroids. I. Introduction of Oxygen at Carbon-11 of
Progesterone. J. Am. Chem. Soc. 74(23) (1952) 5933–5936. https://doi.org/10.1021/ja01143a033
[25] K. Yildirim, A. Kuru. Biotransformation of some steroids by Aspergillus candidus. Journal of
Chemical Research. 39(9) (2015) 546-549. https://doi.org/10.3184/174751915x14403454824263
[26] Z. Habibi, M.Yousefi, Sh. Ghanian, M. Mohammadi, S. Ghasemi, Biotransformation of
progesterone by Absidia griseolla var. igachii and Rhizomucor pusillus, Steroids, 77 (2012) 1446–
1449. https://doi.org/10.1016/j.steroids.2012.08.010
[27] S.D. Shuvalova, K. N. Gabinskaya, Microbiological Transformation of Cortexolone Into
Hydrocortisone. Pharm. Chem. J. 36 (2002) 664-666. https://doi.org/10.1023/A:1023461729540
[28] S.H. Yalkowsky, Y. He, P. Jain, Handbook of aqueous solubility data. Second edition. CRC
Press. Tailor & Francis Group. 2010. P.1254. ISBN 13: 978-1-4398-0246-5
[29] M.L. Contente, B. Guidi, I. Serra, V. De Vitis, D. Romano, A. Pinto, R. Lenna, R. P. de Souza
Oliveira, F. Molinaria. Development of a high-yielding bioprocess for 11α-hydroxylation of
canrenone under conditions of oxygen-enriched air supply. Steroids. 116 (2016) 1-4.
https://doi.org/10.1016/j.steroids.2016.09.013
18

[30] T. Kubota, F. Hayashi. Studies on A-norsteroids – VI: Directing effects of the C11-substituents
on the addition of osmium tetroxide to steroidal 1,4 -3-ketones. Tetrahedron. 23(2) (1967) 995-
1006. https://doi.org/10.1016/0040-4020(67)85047-6
[31] M. Jaeger, R.L.E.G. Aspers, Steroids and NMR. Annual Reports on NMR Spectroscopy, 77
(2012) 115-258. https://doi.org/10.1016/b978-0-12-397020-6.00003-9
[32] S. Al-Awadi, M. Afzal & S. Oommen. Studies on Geobacillus stearothermophilus – Part V:
Transformation of 11α-hydroxyprogesterone. Biocatal. Biotransform. 23(5) (2005) 323-328.
https://doi.org/10.1080/10242420500285710
[33] S.P. Kolet, S. Niloferjahan, S. Haldar, R. Gonnade, H.V. Thulasiram, Biocatalyst mediated
production of 6,11-dihydroxy derivatives of 4-ene-3-one steroids, Steroids, 78 (2013) 1152–
1158. https://doi.org/10.1016/j.steroids.2013.08.004
[34] W.A. Fouad, I.H. Abbas, K.M. Elwan, M.A. Swellum, Kh.A. El-Dougdoug, Biotransformation
of progesterone by microbial steroids, J. Appl. Sci. Res. 35 (2009) 137–143
[35] M. Yilmaztekin, T. Cabaroglu, H. Erten, Effects of Fermentation Temperature and Aeration on
Production of Natural Isoamyl Acetate by Williopsis saturnus var. saturnus, BioMed Res. Int.
(2013) 1-6. https://doi.org/10.1155/2013/870802
[36] V. Passoth. Lipids of Yeasts and Filamentous Fungi and Their Importance for Biotechnology.
In eBook Biotechnology of Yeasts and Filamentous Fungi. Ed.: Andriy A. Sibirny. Springer
International Publishing AG. (2017) 149-204. https://doi.org/10.1007/978-3-319-58829-2_6
[37] D.L. Nelson, E.E. Cox. Lehninger principles of biochemistry. W. H. Freeman and Company,
(2013).1334.
[38] F. Pietrocola, L. Galluzzi, J. M.Bravo-San Pedro, F. Madeo, G. Kroemer, Acetyl Coenzyme
A: A Central Metabolite and Second Messenger, Cell Metabolism 21(6) (2015) 805-821.
https://doi.org/10.1016/j.cmet.2015.05.014
[39] M. Pauly, V. Ramirez. New Insights Into Wall Polysaccharide O-Acetylation. Front. Plant Sci.
9 (2018). https://doi.org/10.3389/fpls.2018.01210
[40] S.S. Mohamed, M.S. Shafei, R.F. Allam, A.M. Elazzazy, M.M. Abo Elsoud, H.A. El-Meno.
Effect of Aeration Rate on the Biotransformation of Cortexolone Using Cunninghamella elegans in
a Laboratory Scale Bioreactor. World Applied Sciences Journal. 25(2) (2013) 176-183.
https://doi.org/10.5829/idosi.wasj.2013.25.02.1331
19

[41] J. Tamm. Hair and skin cosmetics - contg. 11-alpha-hydroxy-progesterone. Patent DE
2757024 (1979)
[42] E. Rossel-Barranko. Pharmaceutical composition for treatment of skin diseases. Patent RU
2432952 (2011)
[43] P.Y. Savechenkov, D.C. Chiara, R. Desai, A.T. Stern, X.Zhou, A.M. Ziemba, A.L. Szabo,
Y.Zhang, J.B. Cohen, S.A. Forman, K.W. Miller, K.S. Bruzik. Synthesis and pharmacological
evaluation of neurosteroid photoaffinity ligands. European Journal of Medicinal Chemistry. 136
(2017) 334–347. https://doi.org/10.1016/j.ejmech.2017.04.043
20