Microbial transformation of androst-4-ene-3,17-dione by three fungal species Absidia griseolla var. igachii, Circinella muscae and Trichoderma virens

Article abstract of DOI:10.1016/j.molcatb.2016.01.007

Microbial transformation of androst-4-ene-3,17-dione (AD;I) by three fungal species including Absidia griseolla var. igachii, Circinella muscae and Trichoderma virens was investigated for the first time. While A. griseolla and C. muscae carried out hydroxylation reactions, the third fungi performed reduction of the 17-carbonyl group in a chemoselective manner. Incubation of AD by A. griseolla yielded four metabolites 6β- (II), 7α- (III), 7β- (VI) and 14α-hydroxy-AD (V), among which 6β-hydroxy-AD (II) was identified as the major product. Furthermore, the metabolites produced during AD biotransformation by C. muscae were 6β- (II), 7β- (III) and 14α-hydroxy-AD (V). On the other hand, T. virens remarkably reduced AD into testosterone (VI) as the only product with 60% yield. These metabolites were purified by TLC and identified by ¹H NMR, ¹³C NMR and other spectroscopic data.

Full text of DOI:10.1016/j.molcatb.2016.01.007

Accepted Manuscript
Title: Microbial transformation of androst-4-ene-3,17-dione
by three fungal species Absidia griseolla var. igachii,
Circinella muscae and Trichoderma virens
Author: Marjan Heidary Zohreh Habibi
PII:
S1381-1177(16)30007-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.01.007
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Journal of Molecular Catalysis B: Enzymatic
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8-10-2015
12-1-2016
12-1-2016
Please cite this article as: Marjan Heidary, Zohreh Habibi, Microbial transformation
of androst-4-ene-3,17-dione by three fungal species Absidia griseolla var.igachii,
CircinellamuscaeandTrichodermavirens, JournalofMolecularCatalysisB:Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2016.01.007
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Microbial transformation of androst-4-ene-3,17-dione by three fungal species
Absidia griseolla var. igachii, Circinella muscae and Trichoderma virens
Marjan Heidarya, Zohreh Habibia* z_habibi@sbu.ac.ir
aDepartment of Pure Chemistry, Faculty of Chemistry, Shahid Beheshti University,
G.C., Tehran, Iran
*Corresponding author: Tel/Fax: +98-21-29903110.
1

Graphical Abstract
2

Highlights
 Biotransformation of androst-4-ene-3,17-dione (AD) was investigated.
 Three filamentous fungi were used for the biotransformation.
 A.griseolla produced 6β, 7α-, 7β-, and 14α-hydroxy-AD.
 C. muscae produced 6β-, 7β-, and 14α-hydroxy-AD.
 Testosterone as only product was obtained in biotransformation of AD by T.
virens.
3

Abstract
Microbial transformation of androst-4-ene-3,17-dione (AD; I) by three fungal species
including Absidia griseolla var. igachii, Circinella muscae and Trichoderma virens was
investigated for the first time. While A. griseolla and C. muscae carried out
hydroxylation reactions, the third fungi performed reduction of the 17-carbonyl group in
a chemoselective manner. Incubation of AD by A. griseolla yielded four metabolites 6β-
(II), 7α- (III), 7β- (VI) and 14α-hydroxy-AD (V), among which 6β-hydroxy-AD (II)
was identified as the major product. Furthermore, the metabolites produced during AD
biotransformation by C. muscae were 6β- (II), 7β- (III) and 14α-hydroxy-AD (V). On
the other hand, T. virens remarkably reduced AD into testosterone (VI) as the only
product with 60% yield. These metabolites were purified by TLC and identified by 1H
NMR, 13C NMR and other spectroscopic data.
Keywords: Absidia griseolla var. igachii; Androst-4-ene-3,17-dione;
Biotransformation; Circinella muscae; Trichoderma virens
4

1. Introduction
Microbial transformation of steroids, independently or integrated with the other partial
syntheses, has been commercially used in the pharmaceutical industry to produce
steroid derivatives [1−3]. Among microorganisms which act as the biocatalysts in
biotransformation of steroids, fungal species effectively utilize the groups of enzymes
for catalyzing different chemical reactions such as hydroxylation and reduction on
steroid substances. Fungi under mild condition can easily insert functional groups to
chemically inactive carbons with the high regio- and stereospecificity [4,5]. Regarding
the advantages of biotransformation, an important stream of researches has been
increasingly trying to identify precious steroids derivatives as well as to enhance the
yield of the biotransformation process [6−8].
Androst-4-en-3,17-dione (AD) which is a fundamental intermediate for invaluable
steroid drugs, belongs to the 17-keto steroid family and has been used in the industrial
synthesis of estradiol or estrone [9]. Bioreduction of AD to testosterone has been
performed by the enzyme 17β-hydroxysteroid dehydrogenase which is present in most
cells of the male organisms. Microbial hydroxylation of AD is broadly used in effective
synthesis of novel AD derivatives which is not obtainable through chemical synthesis
methods. Most positions in AD molecule have been hydroxylated by different microbial
strains. For example, biotransformation of AD by Beauveria bassiana was reported, in
which the separated products were obtained in the different pH media [10]. Moreover,
formation of various hydroxyl steroid derivatives from AD was demonstrated by the
filamentous fungus Mucor racemosus [11] and Neurospora crassa [12]. Additionally,
biotransformation of AD by Curvularia lunata resulted in four hydroxylation
derivatives and one reduction metabolite [13]. Most metabolites obtained from AD
bioconversion, either are biologically active compounds or can be applied as the
5

intermediate to produce invaluable steroid drugs. For instance, one important class of
metabolites are 14α-hydroxy-AD derivatives which prevent from aromatase activity and
has anti-tumor properties. In addition, 7α-hydroxy-AD has applications in the medical
industry and particularly in production of diuretics [9,14].
Fungi belonging to the Trichoderma and Circinella genera are widely available in the
nature and have been utilized in the efficient biotransformation of exogenous steroids
and demonstrated high catalytic activity in the hydroxylation, lactonization,
hydrogenation and ester bond hydrolysis of steroids [15−18]. However, to the best of
our knowledge, none of the Circinella species have been used in the bioconversion of
AD. In particular, neither C. muscae nor T. virens has been incorporated in the
biotransformation of any steroids. Beside this, in our previous studies, the application of
Absidia griseolla var. igachii in the biotransformation of progesterone and testosterone
was investigated which resulted in the corresponding 14-hydroxy derivatives as the
main products with the desirable yields [19−21]. Therefore, unrevealed potential of C.
muscae and T. virens in the biotransformation of steroid structures as well as the
desirable results which were gained from application of A. griseolla in the
biotransformation of mentioned steroids, provide a basis to examine the ability of new
microbial biocatalysts in the bioconversion of AD. In the present study, the ability of
mentioned three fungal species in biotransformation of AD was investigated which
resulted in hydroxylation on the position C-6β, C-7α, C-7β and C-14α as well as
reduction on the position of C-17β.
2. Materials and methods
2.1. Chemicals and analytical methods
6

AD was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The components
of culture media were purchased from Scharlau (Barcelona, Spain). Inorganic salts and
analytical grade solvents were procured from Merck (Darmstadt, Germany). Nuclear
magnetic resonance (NMR) spectra were determined on a Bruker Avance-300 with
tetramethylsilane (TMS) as internal standard in CDCl3. Chemical shifts (δ) are given in
parts per million (ppm) relative to TMS. Coupling constants (J) are given in hertz (Hz).
The EI-MS spectra were obtained with a Finnigan MAT TSQ-70 instrument. The IR
spectra were recorded on a Shimadzu IR-470 spectrometer. Column chromatography
was conducted with silica gel 60 mesh (Merck, Darmstadt, Germany). Preparative thin
layer chromatography (TLC) was performed on silica gel 60 mesh GF254 plates (20  20
cm).
2.2. Microorganisms and fermentation conditions
The fungal strains A. griseolla var. igachii, C. muscae and T. virens were obtained from
the Persian type culture collection (Iranian Research Organization for Science and
Technology). The strains were maintained on potato-dextrose agar plates (20 g glucose,
300 g diced potatoes, 15.0 g agar/L) at 4 °C and freshly sub-cultured before using in the
transformation experiment. For each fungus, the experiment was conducted in ten 250
mL Erlenmeyer flasks, each containing 100 mL of the sterilized autoclaved cultivation
medium which were inoculated with freshly obtained spores from agar slope cultures.
After cultivation at 24 °C for 24 h in a rotary shaker (120 rpm), 100 mg of AD dissolved
in 1 mL of absolute ethanol was added to each flask to achieve a final concentration of 1
mg mL-1. Every day, a sample was collected from the cultures and the progress of the
reaction was monitored by TLC. Due to study the catalytic efficiency of
7

microorganisms, control reactions consisting of cultivation medium without
microorganism were similarly incubated.
2.3. Product isolation and purification
At the end of incubation, the fermentation media were extracted three times with
chloroform. The organic extract was evaporated under reduced pressure on a rotary
evaporator which was then loaded on preparative TLC with n-hexane/ethyl acetate.
Incubation of AD (1g) for 4 days with the fungus A. griseolla gave products namely 6β-
hydroxy-AD (II; 475 mg, 45%) as major metabolite and also 14α-hydroxy-AD (V; 221
mg, 21%), 7α-hydroxy-AD (III; 147 mg, 14%) and 7β-hydroxy-AD (IV; 63 mg, 6%).
The hydroxylated metabolites of AD obtained after 4 days incubation of C. muscae
were purified and identified through their spectroscopic data. Chromatographic
purification of the products resulted in the isolation of three metabolites namely 6β-
hydroxy-AD (II; 412 mg, 39%), 14α-hydroxy-AD (V; 380 mg, 36%) and 7β-hydroxy-
AD (IV; 127 mg, 12%). Incubation of AD with T. virens produced testosterone (VI;
637 mg) as the only metabolite in the desired yields 60%. The yields of each product
were calculated based on the percentage of dry weight of each compound. The sites of
hydroxylation of the metabolites were determined according to the interpretation of 1H
NMR, 13C NMR spectral data. Supplementary evidence for identification of the
compounds was obtained by comparing optical rotations and melting points of
metabolites with literature. The molecular structures of metabolites are shown in Fig. 1.
2.4. Time course study of the metabolites
In order to investigate time course study of the metabolites, sampling was carried out
every 12 h. For this purpose, in each sampling, 20 mL of the broth was taken and
8

extracted with chloroform (3 × 20 mL), then the combined organic phases were dried
over Na2SO4 and the solvent was evaporated under reduced pressure. Afterwards, the
crude product was redissolved in 1 mL HPLC grade acetonitrile and injected to HPLC
(20 μL) at room temperature. The mobile phase consisted of A (water–acetonitrile,
45:55) and B, 100% acetonitrile. The HPLC system was operated at a flow rate of 0.5
mL/min with a linear gradient of 100% A to 100% B over 20 min, and then held at
100% B for 15 min.
3. Results and discussion
3.1. 6β-Hydroxy-AD (II)
Colorless crystals; mp 190–191 °C; (lit [22] 190–192 °C), [α]D20 + 117 ° (CHCl3, c =
1.0), (lit [23] [α]D20 + 122 ° (CHCl3, c = 1.0)); IR (KBr); νmax: 3470, 1721, 1668, 1615
cm−1; MS m/z (%) 302 (25) (M+, C19H26O3), 284 (21), 140 (52), 112 (62), 57 (100); 1H
NMR (300.13 MHz, CDCl3) δ (ppm): 0.96 (3H, s, H-18), 1.42 (3H, s, H-19), 4.41 (1H,
brs, H-6α), 5.84 (1H, s, H-4); 13C NMR (75.47 MHz, CDCl3, see table 1); Rf in n-
hexane/ethyl acetate (1:3 (v/v)): 0.57.
The mass spectrum of compound II showed the molecular ion peak at m/z 302,
corresponding to the formula C19H26O3. IR spectrum showed a strong hydroxyl band at
3470 cm-1. Additionally, the appearance of a new downfield signal at δ 4.34 ppm (1H,
brs) in the 1H NMR spectrum and a carbon resonance at δ 72.8 ppm in the 13C NMR
spectrum of II indicated insertion of a hydroxyl group added to a secondary carbon.
Comparison of the 1H NMR, 13C NMR, optical rotation and melting point of metabolite
II with literature was consistent with hydroxylation at the 6β-position [22,23].
3.2. 7α-Hydroxy-AD (III)
9

Colorless crystals; mp 240–242 °C; (lit [12] 245–248 °C), [α]D20 + 155 ° (CHCl3, c =
1.0), (lit [24] [α]D20 + 143 ° (CHCl3, c = 1.0)); IR (KBr); νmax: 3421, 1733, 1661 and
1611 cm−1; MS m/z (%) 302 (28) (M+, C19H26O3), 284 (14), 136 (58), 109 (62), 57
(100); 1H NMR (300.13 MHz, CDCl3) δ (ppm): 0.90 (3H, s, H-18), 1.25 (3H, s, H-19),
4.13 (1H, brs, H-7β), 5.85 (1H, s, H-4); 13C NMR (75.47 MHz, CDCl3, see table 1); Rf
in n-hexane/ethyl acetate (1:3 (v/v)): 0.21.
The mass spectrum showed the molecular ion peak at m/z 302. Analysis of the NMR
spectra indicated the appearance of a broad singlet resonance at δ 4.13 ppm (1H, brs)
and a carbon resonance at δ 67.1.
3.3. 7β-Hydroxy-AD (IV)
Colorless crystals; mp 214–216 °C; (lit [22] 215–217 °C), [α]D20 + 182 ° (CHCl3, c =
1.0), (lit [22] [α]D20 + 178 ° (CHCl3, c = 1.0)); IR (KBr); νmax: 3464, 1689 and 1620
cm−1; MS m/z (%) 302 (71) (M+, C19H26O3), 282 (10), 134 (50), 109 (100); 1H NMR
(300.13 MHz, CDCl3) δ (ppm): 0.94 (3H, s, H-18), 1.26 (3H, s, H-19), 3.51 (1H, brs, H-
7β), 5.80 (1H, s, H-4); 13C NMR (75.47 MHz, CDCl3, see table 1); Rf in n-hexane/ethyl
acetate (1:3 (v/v)): 0.34.
A new downfield methine proton appeared at δ 3.51 ppm (1H, brs) in the 1H NMR
spectrum. The 13C NMR spectrum also supported the presence of a hydroxyl group (δ
75.1 ppm). Definitive confirmation of the structure of the compound was given by
comparison with NMR data previously reported for 7β-hydroxy-AD [22].
3.4. 14α-Hydroxy-AD (V)
Colorless crystals; mp 258–260 °C; (lit [24] 259–262 °C), [α]D20 + 160 ° (CHCl3, c =
1.0), (lit [24] [α]D20 + 155 ° (CHCl3, c = 1.0)); IR (KBr); νmax: 3420, 1721, 1670, 1607,
10

1151 cm−1; MS m/z (%) 302 (25) (M+, C19H26O3), 220 (15), 116 (21), 78 (33), 69 (40),
57 (100); 1H NMR (300.13 MHz, CDCl3) δ (ppm): 1.06 (3H, s, H-18), 1.23 (3H, s, H-
19), 5.76 (1H, s, H-4); 13C NMR (75.47 MHz, CDCl3, see table 1); Rf in n-hexane/ethyl
acetate (1:3 (v/v)) 0.67.
The 13C NMR displayed the presence of a new oxygen-bearing methine carbon signal at
δ 80.7, but no downfield proton resonance was observed in 1H NMR spectrum in the
range of δ 3.5–4.5 ppm. The data obtained confirmed that the hydroxyl group should be
attached to a tertiary carbon. Finally, assignment of 1H and 13C NMR signals by
comparison of the shift data obtained with that reported was consistent with
hydroxylation at the 14α-position [24].
3.5. Testosterone (VI)
Colorless crystals; mp 151–152 °C; (lit [24] 154–156 °C), [α]D20 +105 ° (MeOH, c =
1.0) (lit [22] [α]D20 +106 ° (MeOH, c = 1.0)); IR (KBr); νmax: 3441, 1668, 1612; MS m/z
(%) 288 (47) (M+, C19H28O2), 246 (35), 167 (38), 55 (100);1H NMR (300.13 MHz,
CDCl3) δ (ppm): 0.81 (3H, s, H-18), 1.20 (3H, s, H-19), 3.66 (1H, t, J = 8.5 Hz, H-17α),
5.74 (1H, s, H-4); 13C NMR (75.47 MHz, CDCl3, see table 1); Rf in n-hexane/ethyl
acetate (1:2): 0.40.
The mass spectrum of metabolite had a molecular ion peak at m/z 288, two mass units
higher than the molecular ion of substrate (286). The 13C NMR spectrum for this
metabolite showed 19 carbon signals, that signal at δ 220 ppm corresponding to 17-
carbonyl had been disappeared and an oxygenated carbon at δ 81.6 ppm was observed.
The 1H NMR spectrum showed downfield methylene protons resonating at δ 3.66 ppm
(t, J = 8.5 Hz). The NMR data were in agreement with those reported in literature [22].
11

3.6. Time course study
A time course study was separately performed for each microorganism and the results
indicated that there were four products including II−V from bioconversion of AD by A.
griseolla which 6β-, 7β- and 14α-hydroxylation appeared in the broth from the first day
while 7α-hydroxy-AD was produced from the second day. The biotransformation yield
increased gradually and reaches the maximum amount after 4 days of incubation. After
4 days, the amount of metabolites appeared to be constant with further incubation time
(Fig. 2a). In incubation of AD by C. muscae showed that all the products were
accumulated from the 12 hour of fermentation (Fig. 2b). The last time course study was
constructed to investigate the bioconversion of AD by T. virens. The obtained results
indicated that AD was not hydroxylated and only testosterone was obtained (Fig. 2c).
The metabolite was quickly established in the very first 12 hours and was increased
until the day 3 in which the metabolite reached its maximum amount. Afterward, there
was not any meaningful change in the amount of the metabolite.
Although due to the useful biological activity of AD derivatives, different
microorganisms have been applied in the bioconversion process of AD (see table 2), the
endeavor for identifying more economical microorganisms are still in progress with the
aim of producing metabolites with the higher yields as well as more stereo- and
regiospecificity [2,5].
17β-reduction of AD is one of the key reactions for synthesizing testosterone, however
in the most microbial transformation of AD, the oxidated products have also been
observed beside the reduction of carbonyl group (see table2 - entries 2, 6 and 14).
Faramarzi et al. through applying Nostoc muscorum have reported the selective
reduction of AD to testosterone in which in order to increasing the yields of reaction,
microalgal cell immobilization and organic solvent have been used (see table 2, entry
12

12). In the current study, T. virens acted as the regio-selective biocatalyst in the
biotransformation of AD and merely produced testosterone with the desirable yield of
60% which nominated T. virens as the industrial candidate of reducing steroid
compounds.
Furthermore, the biotransformation of AD by A. griseolla and C. muscae has been
resulted in hydroxylation at some specific sites in the molecule of AD, which the major
products of both strains were 6β- and 14α-hydroxy-AD. It is necessary to mention that
the obtained result for A. griseolla is consistent with our previous studies in which A.
griseolla was effectively used in the biotransformation of other steroids [19−21].
4. Conclusion
Examining different microorganisms in biotransformation of steroid derivatives has
become an interesting field of research. In this study, the potential of three fungi
species, A. griseolla, C. muscae and T. virens in biotransformation of AD was
examined. Microbial hydroxylation of AD by A. griseolla effectively resulted in the
formation of products II, III, IV and V. Furthermore, biotransformation of AD by C.
muscae produced metabolites II, IV and V. The above fungi showed the high ability in
stereoselective hydroxylation. In contrast with the two mentioned fungi, no
hydroxylation took place in the biotransformation of AD by T. virens, but this strain
was able to reduce a ketone in C-17 to the corresponding hydroxyl group. To the best of
our knowledge this is the first report on biotransformation of AD by these fungal
species.
Acknowledgement
13

This work was supported by the Research Council of Shahid Beheshti University
Tehran, Iran for which the authors are thankful.
14

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17

Figure Captions
Fig. 1. The structure of AD and its metabolites.
18

(a)
(b)
(c)
19

Fig. 2. Time course profile of AD biotransformation by (a) A. griseolla, (b) C. Muscae
and (c) T. virens.
20

Tables
Table 1. 13C NMR data for AD and its metabolites (δ in ppm downfield from TMS,
in CDCl3).
Carbon
atom
1
I
II
III
IV
V
VI
35.6
33.8
199.1
124.0
170.2
32.4
31.1
35.0
53.7
38.5
20.2
30.6
47.4
50.7
21.6
35.6
220.0
13.6
17.2
37.1
34.2
200.2
126.6
167.8
72.8
35.8
29.4
53.6
38.1
20.2
31.3
47.7
50.9
21.7
37.2
220.1
13.8
19.6
35.4
33.9
198.0
127.2
166.7
41.0
67.1
39.3
45.4
38.5
20.1
30.9
47.3
45.6
21.2
35.7
220.3
13.5
17.0
36.6
34.2
202.1
125.2
171.2
39.5
75.1
43.5
52.1
39.5
21.2
32.8
49.4
51.8
25.6
36.6
224.2
14.6
17.6
35.7
33.1
199.5
124.2
170.0
33.1
25.5
37.9
46.8
38.6
19.1
24.5
52.5
80.7
30.2
33.9
218.3
17.9
17.3
35.6
35.5
199.6
123.8
171.3
33.9
32.7
35.7
53.9
38.6
20.6
36.4
42.8
50.4
23.3
31.5
81.6
11.0
17.4
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
21

Table 2. Microbial transformation of AD by various microorganisms
Entry Microorganism
Products
References
[25]
[10]
1
2
Aspergillus ochraceus
Beauveria bassiana
11α-Hydroxy-AD
11α-Hydroxy-AD
Testosterone
11α,14α-Dihydroxy-AD
6β,11α,17β-Trihydroxy-AD
3α,11α,17β-Trihydroxy-5α-AD
Androsta-1,4-diene-3,17-dione (ADD)
9α-Hydroxy-AD
3-OH-SADT
6β,7β-Dihydroxy-AD
6β-Hydroxy-AD
3
4
Bordetella sp.
[26]
[27]
Botryosphaeria obtusa
7α-Hydroxy-AD
15α-Hydroxy-AD
15α-Hydroxy-AD
11α,15α-Dihydroxy-AD
ADD
5
6
Colletotrichum lini
Curvularia lunata
[28]
[13]
Testosterone
11α-Hydroxy-AD
11α,17β-Dihydroxy-AD
7α-Hydroxy-AD
14α-Hydroxy-AD
14α,17β-Dihydroxy-AD
6β-Hydroxy-AD
7
8
Mucor griseocyanus
Mucor (M881)
[29]
[22]
11α-Hydroxy-AD
7β-Hydroxy-AD
6β,11α-Dihydroxy-AD
14α-Hydroxy-AD
7α-Hydroxy-AD
9
Mucor racemosus
[11]
14α,17β-Dihydroxy-AD
6β,14α-Dihydroxy-AD
6β,11α,17β-Trihydroxy-AD
6,14α-dihydroxyandrost-4-ene-3-one
6β,14α-Dihydroxy-AD
6β,9α-Dihydroxy-AD
7α-Hydroxy-AD
10
11
Mucor sp.
Neurospora crassa
[30]
[12]
9α-Hydroxy-AD
14α-Hydroxy-AD
Androst-4,6-dien-3,17-dione
Testosterone
12
13
Nostoc muscorum
Rhizopous nigricans
[31]
[32]
7α-Hydroxy-AD
11α-Hydroxy-AD
Testosterone
14
Thamnostylum piriforme
[33]
6β-Hydroxy-AD
14α-Hydroxy-AD
6β-Hydroxy-AD
7α-Hydroxy-AD
22