Microbial transformation of dehydroepiandrosterone (DHEA) by some fungi

Article abstract of DOI:10.1080/10242422.2020.1844191

In this work, biotransformations of dehydroepiandrosterone (DHEA) 1 by Ulocladium chartarum MRC 72584, Cladosporium sphaerospermum MRC 70266 and Cladosporium cladosporioides MRC 70282 have been reported. U. chartarum MRC 72584 mainly hydroxylated 1 at C-7α and C-7β, accompanied by a minor hydroxylation at C-4β, a minor epoxidation from the β-face and a minor oxidation at C-7 subsequent to its hydroxylations. 3β,7β-Dihydroxy-5β,6β-epoxyandrostan-17-one 6, 3β,4β,7α-trihydroxyandrost-5-en-17-one 7 and 3β,4β,7β-trihydroxyandrost-5-en-17-one 8 from this incubation were identified as new metabolites. C. sphaerospermum MRC 70266 converted some of 1 into a 3-keto-4-ene steroid and then hydroxylated at C-6α, C-6β and C-7α, accompanied a minor 5α-reduction and a minor oxidation at C-6 following its hydroxylations. C. sphaerospermum MRC 70266 also hydroxylated some of 1 at C-7α and C-7β. C. cladosporioides MRC 70282 converted almost half of 1 into a 3-keto-4-ene steroid and then hydroxylated at C-6α and C-6β. C. cladosporioides MRC 70282 also reduced some of 1 at C-17.

Full text of DOI:10.1080/10242422.2020.1844191

Biocatalysis and Biotransformation
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Microbial transformation of
dehydroepiandrosterone (DHEA) by some fungi
Kudret Yildirim , Ali Kuru , Semra Yılmazer Keskin & Sinan Ergin
To cite this article: Kudret Yildirim , Ali Kuru , Semra Yılmazer Keskin & Sinan Ergin (2020):
Microbial transformation of dehydroepiandrosterone (DHEA) by some fungi, Biocatalysis and
Biotransformation, DOI: 10.1080/10242422.2020.1844191
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2020.1844191
RESEARCH ARTICLE
Microbial transformation of dehydroepiandrosterone (DHEA) by some fungi
Kudret Yildirim , Ali Kuru , Semra Yılmazer Keskin
and Sinan Ergin
Department of Chemistry, Faculty of Arts and Sciences, Sakarya University, Sakarya, 54187, Turkey
ABSTRACT
ARTICLE HISTORY
Received 20 May 2020
Revised 10 October 2020
Accepted 26 October 2020
In this work, biotransformations of dehydroepiandrosterone (DHEA) 1 by Ulocladium chartarum
MRC 72584, Cladosporium sphaerospermum MRC 70266 and Cladosporium cladosporioides MRC
70282 have been reported. U. chartarum MRC 72584 mainly hydroxylated 1 at C-7a and C-7b,
accompanied by a minor hydroxylation at C-4b, a minor epoxidation from the b-face and a
minor oxidation at C-7 subsequent to its hydroxylations. 3b,7b-Dihydroxy-5b,6b-epoxyandrostan-
17-one 6, 3b,4b,7a-trihydroxyandrost-5-en-17-one 7 and 3b,4b,7b-trihydroxyandrost-5-en-17-one
8 from this incubation were identified as new metabolites. C. sphaerospermum MRC 70266 con-
verted some of 1 into a 3-keto-4-ene steroid and then hydroxylated at C-6a, C-6b and C-7a,
accompanied a minor 5a-reduction and a minor oxidation at C-6 following its hydroxylations. C.
sphaerospermum MRC 70266 also hydroxylated some of 1 at C-7a and C-7b. C. cladosporioides
MRC 70282 converted almost half of 1 into a 3-keto-4-ene steroid and then hydroxylated at C-
6a and C-6b. C. cladosporioides MRC 70282 also reduced some of 1 at C-17.
KEYWORDS
Ulocladium chartarum;
Cladosporium sphaerosper-
mum; Cladosporium
cladosporioides; dehydroe-
piandrosterone;
biotransformation
Introduction
also formed de novo in the brain and regarded as neu-
roactive steroids (Choudhary et al. 2012; Kozłowska
et al. 2017).
Steroids are biologically and pharmaceutically import-
ant natural products as they play vital roles in many
physiological activities. Steroidal drugs have been
widely used to treat and prevent different diseases
due to their anti-inflammatory, anti-microbial, anti-
tumour, anti-estrogenic, anti-diabetic, anti-HIV and
anti-convulsant properties (Kolet et al. 2013).
Preparing steroidal drugs by conventional synthetic
methods usually has some unfavourable outcomes,
such as being time consuming, more expensive and
environmentally unfriendly processes. However, micro-
bial steroid biotransformations are extensively used to
obtain the mentioned steroids as they can handle
these unfavourable outcomes due to their high regio-,
stereo- and chemoselectivities (Faber 2003; Bhatti and
Khera 2012; Donova and Egorova 2012; Kolet et al.
2013; Nassiri-Koopaei and Faramarzi 2015).
Dehydroepiandrosterone (DHEA) 1 is an important
mammalian hormone that is converted in vivo to
androgens and oestrogens. Most of DHEA 1 is formed
in the adrenal cortex although some of 1 is also
formed in testicles, ovaries and brain cells. DHEA 1
and its metabolites have been known as immune sys-
tem strengtheners in animals. These metabolites are
The fungal genus Ulocladium includes species with
diverse distributions and roles. Ulocladium species are
mostly saprotrophs. Some Ulocladium species are food
spoilers ruining nuts, fruit and cereals and other
Ulocladium species are plant pathogens destroying
some plants such as, cucumber, Kasahui potato and
oak. A few Ulocladium species have drawn attention
as potential pectinase, cellulose, glucanase and lipase
producers and bio-control agents especially against
some Botrytis species, such as B. aclada and B. cinerea
(Andersen and Hollensted 2008).
Cladosporium is
a
large fungal genus and
Cladosporium species are cosmopolitan in distribution.
These fungi are usually isolated from air, food, plants,
paint and textiles (Bensch et al. 2012; Sandoval-Denis
et al. 2016). Cladosporium species are characteristic
endophytes, plant pathogens and even hyperparasites
of other fungi (Sandoval-Denis et al. 2016). Some
Cladosporium species are also known as pathogenic to
humans and animals (Sandoval-Denis et al. 2015).
A number of fungal species have been widely used
for the biotransformation of DHEA 1 (Mahato and
Garai 1997; Fernandes et al. 2003; Bhatti and Khera
CONTACT Kudret Yildirim
kudrety@sakarya.edu.tr
Department of Chemistry, Faculty of Arts and Sciences, Sakarya University, Sakarya,
54187, Turkey
Supplemental data for this article can be accessed here.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
K. YILDIRIM ET AL.
2012; Donova and Egorova 2012; Nassiri-Koopaei and
Faramarzi 2015). However, as far as the biotransform-
ation of DHEA 1 by Ulocladium and Cladosporium spe-
cies are concerned, there are no reports in the
literature. In this work, biotransformations of DHEA 1
by Ulocladium chartarum MRC 72584, Cladosporium
sphaerospermum MRC 70266 and Cladosporium clado-
sporioides MRC 70282 were carried out for 5 days in
order to investigate its metabolism by these fungi.
obtained from TUBITAK, Marmara Research Centre,
Food Science and Technology Research Institute,
Culture Collection Unit, Kocaeli, Turkey. Stock cultures
were maintained at on PDA slopes at 4 ꢀC.
The liquid medium (1 L) for all fungi was prepared
by mixing glucose (20 g), peptone (5 g) and yeast
extract (5 g) in distilled water (Shebany 2012). The
medium for each fungus was evenly distributed
among 10 culture flasks of 250 mL capacity (100 mL in
each) and autoclaved for 20 min at 121 ꢀC. Spores of
each fungus freshly obtained from PDA slopes were
separately transferred into one set of 10 flasks contain-
ing sterile medium in a biological safety cabinet. After
cultivation of each fungus at 28 ꢀC for 3 days on a
rotary shaker (160 rpm), DHEA 1 (1 g) dissolved in
10 ml of DMF was evenly distributed aseptically
among the flasks. The biotransformation of the sub-
strate by each fungus was carried out in 10 flasks for
5 days under the same conditions. Each biotransform-
ation experiment was run with a control flask contain-
ing non-inoculated sterile medium and the substrate.
After 5 days of incubation, each control was harvested
and analysed by TLC. No metabolites were detected in
the controls.
Materials and methods
Instrumental methods
1H NMR spectra were recorded in deuterochloroform
with tetramethylsilane as an internal standard refer-
ence at 300 MHz with a Varian Mercury 300 spectrom-
eter (Palo Alto, CA). 13C NMR spectra were recorded in
deuterochloroform at 75 MHz with a Varian Mercury
300 spectrometer (Palo Alto, CA). Chemical shifts are
given in ppm (d scale), coupling constants (J) are
given in Hz. Infra-red spectra were recorded using a
Perkin Elmer Spectrum Two spectrometer (Whaltam,
MA). Elemental analysis was performed using
a
Thermo Finnigan Flash EA 1112 elemental analyser
(Bremen, Germany). Melting points were determined
by an Electrothermal IA 9200 melting point apparatus
(Staffordshire, UK) and are uncorrected. Thin layer
chromatography (TLC) was carried out with 0.2 mm
thick Merck Kieselgel 60 F254 TLC plates (Istanbul,
Turkey) using ethyl acetate/n-hexane (1:1) as eluent.
TLC plates were dipped into a p-anisaldehyde/H2SO4
reagent and heated to 120 ꢀC for 3 min in order to
visualise the spots.
Isolation, identification and quantification of
metabolites
After incubation, each fungal mycelium was separated
from the broth by filtration under vacuum and rinsed
with ethyl acetate (500 ml). Each broth was then sep-
arately extracted three times with each 1 L of ethyl
acetate. The organic extracts from each incubation
was separately dried over anhydrous sodium sulphate
and then evaporated in vacuo to give a brown gum.
The steroids in each brown gum was separately chro-
matographed on silica gel 60 (Merck 107734), eluting
with increasing concentrations of ethyl acetate in n-
hexane. Steroid mixtures from the columns were then
separated by column chromatography on aluminium
oxide 90 active neutral (activity stage I, Merck 101077),
using smaller columns. The purified steroids were crys-
tallised in appropriate solvents and identified by using
melting points and a combination of NMR, IR and
elemental analysis. Quantification of metabolites was
given as conversion rate (Sripalakit et al. 2006).
Chemicals
DHEA (Mw: 288.414 g/mol) 1 was purchased from
Sigma-Aldrich (Istanbul, Turkey). Solvents were of ana-
lytical grade and were purchased from Merck
(Istanbul, Turkey). Potato dextrose agar (PDA) and agar
for PDA slopes and ingredients for liquid medium
were also purchased from Merck (Istanbul, Turkey).
Microorganisms and fermentation conditions
U. chartarum MRC 72584, C. sphaerospermum MRC
70266 and C. cladosporioides MRC 70282 were
½weight of metaboliteꢁ=½molecular weight of metaboliteꢁ
ð Þ
Conversion % ¼
ꢂ 100
½weight of DHEAꢁ=½molecular weight of DHEAꢁ

BIOCATALYSIS AND BIOTRANSFORMATION
3
Conversion of DHEA 1 into metabolites was estimated
as follows:
mp 181–182 ꢀC, lit mp 177–179 ꢀC (Holland and
Diakow 1979); IR mmax 3400, 1740, 1145, 1085 cmꢃ1
;
EA: Calculated for C19H28O4 (320.414 g/mol): C 71.22, H
8.81; found: C 71.14, H 8.63%; 1H NMR (CDCl3): see
Table 1; 13C NMR (CDCl3): see Table 2.
Further elution with ethyl acetate in n-hexane (9:1)
yielded 3b,7b-dihydroxy-5b,6b-epoxyandrostan-17-one
Biotransformation of DHEA 1 by U. chartarum
MRC 72584
Incubation of DHEA 1 with U. chartarum MRC 72584
for 5 days yielded a brown gum (2127 mg), which was
then chromatographed on silica gel. Elution with ethyl
acetate in n-hexane (3:7) yielded the unreacted sub-
strate (197 mg), which was identified by comparison of
6
(34 mg, 3%); crystallised from ethyl acetate
as needles; mp 181–182 ꢀC; IR mmax 3352, 1740, 1140,
1090 cmꢃ1; EA: Calculated for C19H28O4 (320.414 g/
mol): C 71.22, H 8.81; found: C 71.08, H 8.67%; 1H
NMR (CDCl3): see Table 1; 13C NMR (CDCl3): see
Table 2.
1
its H and 13C NMR spectra with those of an authen-
tic sample.
Elution with ethyl acetate in n-hexane (3:2) yielded
Elution with pure ethyl acetate yielded 3b,4b,7a-tri-
hydroxyandrost-5-en-17-one 7 (33 mg, 3%); crystallised
from ethyl acetate as needles; mp 203–204 ꢀC; IR mmax
3b-hydroxyandrost-5-en-7,17-dione
2
(63 mg, 6%);
crystallised from methanol as prisms; mp 237–238 ꢀC,
lit mp 239 ꢀC (Kołek et al. 2011); IR mmax 3485, 1730,
1660 cmꢃ1; EA: Calculated for C19H26O3 (302.398 g/
mol): C 75.46, H 8.67; found: C 75.35, H 8.50%; 1H
NMR (CDCl3): see Table 1; 13C NMR (CDCl3): see
Table 2.
3430, 1740 cmꢃ1
; EA: Calculated for C19H28O4
(320.414 g/mol): C 71.22, H 8.81; found: C 71.10, H
1
8.65%; H NMR (CDCl3): see Table 1; 13C NMR (CDCl3):
see Table 2.
Further elution with pure ethyl acetate yielded
3b,4b,7b-trihydroxyandrost-5-en-17-one 8 (45 mg, 4%);
crystallised from ethyl acetate as needles; mp
203–204 ꢀC; IR mmax 3455, 1740 cmꢃ1; EA: Calculated
for C19H28O4 (320.414 g/mol): C 71.22, H 8.81; found: C
Elution with ethyl acetate in n-hexane (4:1) yielded
3b,7b-dihydroxyandrost-5-en-17-one 3 (170 mg, 16%);
crystallised from ethyl acetate-petroleum ether as nee-
dles; mp 212–213 ꢀC, lit mp 215–217 ꢀC (Kołek et al.
2011); IR mmax 3245, 1735 cmꢃ1; EA: Calculated for
C19H28O3 (304.414 g/mol): C 74.96, H 9.27; found: C
1
71.12, H 8.69%; H NMR (CDCl3): see Table 1; 13C NMR
(CDCl3): see Table 2.
1
74.79, H 9.10%; H NMR (CDCl3): see Table 1; 13C NMR
(CDCl3): see Table 2.
Biotransformation of DHEA 1 by C.
sphaerospermum MRC 70266
Further elution with ethyl acetate in n-hexane (4:1)
yielded
3b,7a-dihydroxyandrost-5-en-17-one
4
(127 mg, 12%); crystallised from ethyl acetate-petrol-
eum ether as needles; mp 179–180 ꢀC, lit mp
181–182 ꢀC (Kołek et al. 2011); IR mmax 3360, 1740
cmꢃ1; EA: Calculated for C19H28O3 (304.414 g/mol): C
74.96, H 9.27; found: C 74.81, H 9.12%; 1H NMR
(CDCl3): see Table 1; 13C NMR (CDCl3): see Table 2.
Elution with ethyl acetate in n-hexane (9:1) yielded
Incubation of DHEA 1 with C. sphaerospermum MRC
70266 for 5 days yielded a brown gum (1943 mg),
which was then chromatographed on silica gel.
Elution with ethyl acetate in n-hexane (3:7) yielded the
unreacted substrate (180 mg), which was identified by
1
comparison of its H and 13C NMR spectra with those
of an authentic sample.
3b,7a-dihydroxy-5b,6b-epoxyandrostan-17-one
(22 mg, 2%); crystallised from ethyl acetate as needles;
5
Elution with ethyl acetate in n-hexane (2:3) yielded
5a-androstan-3,6,17-trione 9 (42 mg, 4%); crystallised
Table 1. 1H NMR data determined in CDCl3 for compounds 1–13.
Compound
18-H
19-H
Other significant resonances
1
2
3
4
5
6
7
8
0.87
0.86
0.87
0.86
0.84
0.85
0.89
0.90
0.89
0.91
0.93
0.91
0.75
1.01
1.18
1.05
0.98
1.01
1.03
1.21
1.26
1.00
1.37
1.22
1.22
1.01
3.46 tt, J ¼ 5.0 and 12.0 Hz (3a-H), 5.35 d, J ¼ 5.0 Hz (6-H)
3.64 m (3a-H), 5.70 bs (6-H)
3.52 m (3a-H), 3.91 m (7a-H), 5.28 bs (6-H)
3.54 m (3a-H), 3.94 bs (7b-H) 5.60 d, J ¼ 5.0 Hz (6-H)
3.12 d, J ¼ 3.2 Hz (6a-H), 3.73 bs (3a-H), 4.17 bs (7b-H)
3.18 bs (6a-H), 3.64 t, J ¼ 9.5 Hz (7a-H), 3.72 m (3a-H)
3.62 m (3a-H), 4.06 bs (7b-H), 4.18 d, J ¼ 3.2 Hz (4a-H), 5.90 d, J ¼ 5.0 Hz (6-H)
3.60 m (3a-H), 3.96 d, J ¼ 7.9 Hz (7a-H), 4.17 d, J ¼ 3.2 Hz (4a-H), 5.62 d, J ¼ 2.1 Hz (6-H)
–
9
10
11
12
13
4.36 bs (6a-H), 5.81 s (4-H)
4.37 m (6b-H), 6.19 d, J ¼ 1.8 Hz (4-H)
4.10 bs (7b-H), 5.83 s (4-H)
3.52 m (3a-H), 3.64 t, J ¼ 8.5 Hz (17a-H), 5.34 d, J ¼ 5.0 Hz (6-H)

4
K. YILDIRIM ET AL.
Table 2. 13C NMR data for DHEA 1 and its metabolites determined in CDCl3.
Carbon atom
1
2
3
4
5
6
7
8
9
10
11
12
13
1
2
3
4
5
6
7
8
36.93
31.19
71.01
41.81
140.92
120.44
31.13
31.19
49.92
36.36
20.07
30.50
47.30
51.44
21.61
35.59
221.32
13.27
19.18
38.32
30.88
70.03
41.74
166.55
125.65
201.20
45.57
49.92
36.17
20.43
30.55
47.78
44.18
24.04
35.54
220.60
13.63
17.31
36.79
31.11
71.07
41.52
143.48
125.49
72.66
40.23
48.13
36.56
20.29
31.33
47.73
51.10
24.11
35.93
221.50
13.51
19.09
36.86
30.96
70.98
42.47
146.35
123.46
64.13
37.42
41.84
37.10
19.97
31.15
47.06
44.83
21.82
35.74
221.38
13.20
18.19
36.58
30.50
68.89
41.44
63.72
64.62
65.95
34.14
41.90
34.28
20.95
30.89
46.96
44.48
20.97
35.45
220.89
13.02
16.99
36.84
30.69
68.78
41.49
67.41
67.56
74.24
37.66
49.73
34.34
21.20
31.12
47.75
50.58
24.95
35.86
221.50
13.50
16.85
36.55
24.77
71.86
76.68
146.97
129.21
64.16
37.05
42.82
37.14
19.42
30.84
47.06
44.76
21.90
35.73
221.33
13.22
19.32
36.18
25.06
72.05
76.13
144.96
131.08
72.62
40.36
48.85
36.55
19.86
31.04
47.70
51.18
24.09
35.95
221.22
13.53
20.63
37.35
36.82
210.90
41.10
57.39
208.22
45.22
37.21
53.32
37.86
20.84
30.95
48.01
51.38
21.56
35.54
219.65
13.72
12.53
36.89
34.03
200.58
126.20
168.35
72.40
37.11
29.29
53.50
37.96
20.14
31.12
47.56
50.75
21.59
35.69
220.81
13.67
19.40
36.19
33.68
199.48
119.96
171.10
68.19
39.89
29.64
53.69
39.00
20.25
31.07
47.51
50.50
21.70
35.67
220.14
13.65
18.20
35.30
33.82
199.33
126.95
166.96
39.25
66.93
40.95
45.22
38.48
20.07
30.87
47.25
45.54
21.19
35.66
220.92
13.43
16.94
37.24
31.46
71.70
42.22
140.82
121.37
30.46
31.92
50.19
36.54
20.64
36.54
42.69
51.28
23.41
29.68
81.87
10.94
19.42
9
10
11
12
13
14
15
16
17
18
19
from ethyl acetate as plates; mp 191–192 ꢀC, lit mp
196–197 ꢀC (Numazawa et al. 1988); IR mmax 1740,
Further elution with ethyl acetate in n-hexane (4:1)
yielded 3b,7a-dihydroxyandrost-5-en-17-one
4
1720, 1700 cmꢃ1
; EA: Calculated for C19H26O3
(211 mg, 20%); which was identified by comparison of
1
its H and 13C NMR spectra with those of the previ-
(302.398 g/mol): C 75.46, H 8.67; found: C 75.28, H
1
8.47%; H NMR (CDCl3): see Table 1; 13C NMR (CDCl3):
ously isolated metabolite.
see Table 2.
Elution with ethyl acetate in n-hexane (1:1) yielded
6b-hydroxyandrost-4-en-3,17-dione 10 (157 mg, 15%);
crystallised from acetone as prisms; mp 189–190 ꢀC, lit
mp 190–193 ꢀC (Hanson et al. 1996); IR mmax 3445,
Biotransformation of DHEA 1 by C.
cladosporioides MRC 70282
1740, 1670 cmꢃ1
; EA: Calculated for C19H26O3
Incubation of DHEA 1 with C. cladosporioides MRC
70282 for 5 days yielded a brown gum (2077 mg),
which was then chromatographed on silica gel.
Elution with ethyl acetate in n-hexane (3:7) yielded the
unreacted substrate (221 mg), which was identified by
(302.398 g/mol): C 75.46, H 8.67; found: C 75.36, H
1
8.55%; H NMR (CDCl3): see Table 1; 13C NMR (CDCl3):
see Table 2.
Further elution with ethyl acetate in n-hexane (1:1)
yielded 6a-hydroxyandrost-4-en-3,17-dione 11 (53 mg,
5%); crystallised from acetone as needles; mp
224–225 ꢀC, lit mp 229–230 ꢀC (Balant and Ehrenstein
1952); IR mmax 3500, 1735, 1655 cmꢃ1; EA: Calculated
for C19H26O3 (302.398 g/mol): C 75.46, H 8.67; found: C
1
comparison of its H and 13C NMR spectra with those
of an authentic sample.
Elution with ethyl acetate in n-hexane (1:1) yielded
3b,17b-dihydroxyandrost-5-ene 13 (81 mg, 8%); crystal-
lised from acetone as prisms; mp 178-179 ꢀC, lit mp
180–183 ꢀC (Choudhary et al. 2003); IR mmax 3470, 1660
cmꢃ1; EA: Calculated for C19H30O2 (290.430 g/mol): C
78.57, H 10.41; found: C 78.40, H 10.33%; 1H NMR
(CDCl3): see Table 1; 13C NMR (CDCl3): see Table 2.
Further elution with ethyl acetate in n-hexane (1:1)
1
75.23, H 8.51%; H NMR (CDCl3): see Table 1; 13C NMR
(CDCl3): see Table 2.
Elution with ethyl acetate in n-hexane (3:2) yielded
7a-hydroxyandrost-4-en-3,17-dione 12 (63 mg, 6%);
crystallised from methanol as needles; mp 232–233 ꢀC,
lit mp 227–230 ꢀC (Holland and Thomas 1982); IR mmax
3400, 1740, 1660, 1615 cmꢃ1; EA: Calculated for
C19H26O3 (302.398 g/mol): C 75.46, H 8.67; found: C
yielded
6b-hydroxyandrost-4-en-3,17-dione
10
(387 mg, 37%), which was identified by comparison of
1
its H and 13C NMR spectra with those of the previ-
1
75.25, H 8.49%; H NMR (CDCl3): see Table 1; 13C NMR
ously isolated metabolite.
(CDCl3): see Table 2.
Further elution with ethyl acetate in n-hexane (1:1)
Elution with ethyl acetate in n-hexane (4:1) yielded
yielded
6a-hydroxyandrost-4-en-3,17-dione
11
3b,7b-dihydroxyandrost-5-en-17-one
3 (43 mg, 4%);
1
which was identified by comparison of its H and 13C
NMR spectra with those of the previously iso-
lated metabolite.
(105 mg, 10%), which was identified by comparison of
1
its H and 13C NMR spectra with those of the previ-
ously isolated metabolite.

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 1. Biotransformation of DHEA 1 by U. chartarum MRC 72584.
6.61 ppm). These shifts further indicated that a 7a-
hydroxylation had taken place. NMR data of 4 were
consistent with prior literature (Kołek et al. 2011).
Results and discussion
Incubation of DHEA 1 with U. chartarum MRC 72584
for 5 days yielded seven metabolites (Figure 1). The
first metabolite was identified as 3b-hydroxyandrost-5-
en-7,17-dione 2. The 13C NMR spectrum of 2 lacked
the C-7 resonance of 1 at dC 31.13 ppm and had a
new carbon atom resonance at dC 201.20 ppm, sug-
gesting the presence of a new carbonyl group at C-7
(see Supplemental data). The 13C NMR spectrum of 2
also showed a downfield shift for C-8 (DdC 14.38 ppm)
and a c-gauche upfield shift for C-14 (DdC 7.26 ppm),
further supporting the presence of a new carbonyl
group at C-7. NMR data of 2 were in accordance with
literature (Kołek et al. 2011).
The fourth metabolite was identified as 3b,7a-dihy-
droxy-5b,6b-epoxyandrostan-17-one 5. The 1H NMR
spectrum of 5 lacked 6-H resonance of 1 at dH
5.35 ppm (1H, d, J ¼ 5.0 Hz) and showed a new reson-
ance at dH 3.12 ppm (1H, d, J ¼ 3.2 Hz), showing the
presence of an epoxy group. The 13C NMR spectrum
of 5 showed typical C-1 resonance at dC 36.58 ppm
due to the presence of two oxygen bearing C atoms
(respectively, at dC 63.72 and dC 64.62 ppm) in close
proximity, indicating that an epoxydation had taken
place from the b-face (Poza et al. 2008). NMR spectra
of 5 showed two typical resonances at dH 4.17 ppm
(1H, bs) and dC 65.95 ppm, suggesting the presence of
a 7a-hydroxyl group next to a b-epoxy group (Poza
et al. 2008). 1H NMR data of 5 were in accordance
with previous literature (Holland and Diakow 1979).
The fifth metabolite was identified as 3b,7b-dihy-
droxy-5b,6b-epoxyandrostan-17-one 6. The 1H NMR
spectrum of 6 lacked 6-H resonance of 1 at dH
5.35 ppm (1H, d, J ¼ 5.0 Hz) and had a new resonance
at dH 3.18 ppm (1H, bs), indicating the presence of an
epoxy group. The 13C NMR spectrum of 6 had charac-
teristic C-1 resonance at dC 36.84 because of the pres-
ence of two oxygen bearing C atoms (respectively, at
dC 67.41 and dC 67.56 ppm) close to it, suggesting that
an epoxydation had taken place from the b-face (Poza
et al. 2008). NMR spectra of 6 had two characteristic
resonances at dH 3.64 ppm (1H, t, J ¼ 9.5 Hz) and dC
74.24 ppm, suggesting the presence of a 7b-hydroxyl
group in close proximity with a b-epoxy group (Poza
et al. 2008). The presence of two methyl, seven methy-
lene, six methine and four quaternary carbons in the
DEPT spectra of 6 were in agreement with the pro-
posed structure of the metabolite. 3b,7b-Dihydroxy-
The second metabolite was identified as 3b,7b-dihy-
droxyandrost-5-en-17-one 3. The H NMR spectrum of
1
the metabolite had a characteristic resonance at dH
3.91 ppm (1H, m) and experienced an upfield shift for
the 6-H resonance (DdH 0.07 ppm), which was consist-
ent with the presence of a 7b-hydroxyl group (Kołek
et al. 2011). The 13C NMR spectrum of 3, on compari-
son to 1, showed downfield shifts for C-6 (DdC
5.05 ppm) and C-8 (DdC 9.04 ppm) whereas it showed
c-gauche upfield shifts for C-9 (DdC 1.79 ppm) and C-
14 (DdC 0.34 ppm), which were further consistent with
the presence of a 7b-hydroxyl group. NMR data of 3
were in accordance with previous literature (Kołek
et al. 2011).
The third metabolite was identified as 3b,7a-dihy-
droxyandrost-5-en-17-one 4. The metabolite showed a
typical resonance (1H, bs) at dH 3.94 ppm and a down-
field shift (DdH 0.25 ppm) for the 6-H resonance of 1,
indicating the presence of a 7a-hydroxyl group (Kołek
et al. 2011). The 13C NMR spectrum of the metabolite
showed downfield shifts for C-6 (DdC 3.02 ppm) and
C-8 (DdC 6.23 ppm) whereas it showed c-gauche
upfield shifts for C-9 (DdC 8.08 ppm) and C-14 (DdC

6
K. YILDIRIM ET AL.
Figure 2. Biotransformation of DHEA 1 by C. sphaerospermum MRC 70266.
5b,6b-epoxyandrostan-17-one 6 was determined as a
new metabolite.
downfield shift for C-8 (DdC 9.17 ppm) whilst it
showed c-gauche upfield shift for C-9 (DdC
a
The sixth metabolite was identified as 3b,4b,7a-tri-
hydroxyandrost-5-en-17-one 7. The metabolite showed
two characteristic resonances at dH 4.18 ppm (1H, d,
J ¼ 3.2 Hz) and dC 76.68 ppm, indicating the presence a
4b-hydroxyl group adjacent to a 3b-hydroxyl group of
1.07 ppm), further indicating the presence of a 7b-
hydroxyl group. The presence of two methyl, six
methylene, seven methine and four quaternary car-
bons in the DEPT spectra of 8 were in agreement with
the proposed structure of the metabolite. 3b,4b,7b-
Trihydroxyandrost-5-en-17-one 8 was determined as a
new metabolite.
1
1 (Ruddock et al. 1998). The H NMR spectrum of 7
had a downfield shift (DdH 0.16 ppm) for the 3a-H res-
onance (1H, tt, J ¼ 5.0 and 12.0 Hz) of 1, further indi-
cating the presence of a 4b-hydroxyl group. The 13C
NMR spectrum of 7 showed a typical resonance at dC
64.16 ppm, suggesting the presence of a 7a-hydroxyl
group (Kołek et al. 2011). The 13C NMR spectrum of 7
had a downfield shift for C-8 (DdC 5.86 ppm) whilst it
had a c-gauche upfield shift for C-9 (DdC 7.10 ppm),
further suggesting the presence of a 7a-hydroxyl
group. The presence of two methyl, six methylene,
seven methine and four quaternary carbons in the
DEPT spectra of 7 were in accordance with the
Incubation of DHEA 1 with C. sphaerospermum MRC
70266 for 5 days yielded six metabolites (Figure 2).
The first metabolite was identified as 5a-androstan-
1
3,6,17-trione 9. The H NMR spectra of 9 lacked char-
acteristic the 3a-H resonance (1H, tt, J ¼ 5.0 and 12 Hz)
at dH 3.46 ppm and the 6-H resonance (1H, d,
J ¼ 5.0 Hz) at dH 5.35 ppm of 1, suggesting that the
conversion of the 3b-hydroxyl group into the 3-oxo
group and the hydrogenation of the double bond
took place. A comparison of the 13C NMR spectrum of
9 with that in literature indicated that the hydrogen-
ation of the double bond had taken place from the
a-face (Al-Awadi et al. 2005). The 13C NMR spectrum
of 9 lacked C-6 resonance of 1 at dC 120.44 ppm and
had a new resonance at dC 208.22 ppm, indicating
that an oxidation following its hydroxylations at C-6
had taken place. NMR data of 9 were consistent with
prior literature (Al-Awadi et al. 2005).
proposed
structure
of
the
triol.
3b,4b,7a-
Trihydroxyandrost-5-en-17-one 7 was identified as a
new metabolite.
The seventh metabolite was identified as 3b,4b,7b-
trihydroxyandrost-5-en-17-one 8. The metabolite
showed two characteristic resonances at dH 4.17 ppm
(1H, d, J ¼ 3.2 Hz) and dC 76.13 ppm, suggesting the
presence a 4b-hydroxyl group in close proximity with
a 3b-hydroxyl group of 1 (Ruddock et al. 1998). The
1H NMR spectrum of 8 had a downfield shift (DdH
0.14 ppm) for the 3a-H resonance (1H, tt, J ¼ 5.0 and
12.0 Hz) of 1, further suggesting the presence of a 4b-
hydroxyl group. The 13C NMR spectrum of 8 showed a
characteristic resonance at dC 72.62 ppm, indicating
the presence of a 7b-hydroxyl group (Kołek et al.
The second metabolite was identified as 6b-hydrox-
1
yandrost-4-en-3,17-dione 10. The H NMR spectrum of
10 lacked the 3a-H resonance of starting material at
dH 3.46 ppm (1H, tt, J ¼ 5.0 and 12.0 Hz) and showed
important downfield shifts for the double bond reson-
ance (DdH 0.46 ppm) and the 19-methyl group reson-
ance (DdH 0.36 ppm) of 1, indicating that the 5-en-3b-
hydroxyl moiety of 1was converted into a 4-en-3-keto
moiety. The 1H NMR spectrum of 10 also showed a
2011). The 13C NMR spectrum of 8 showed
a

BIOCATALYSIS AND BIOTRANSFORMATION
7
Figure 3. Biotransformation of DHEA 1 by C. cladosporioides MRC 70282.
characteristic resonance at dH 4.36 ppm (1H, bs), sug-
The fifth metabolite was determined as 3b,7b-dihy-
gesting the presence of a 6b-hydroxyl group (Hanson
et al. 1996). The 13C NMR spectrum of 10 had a down-
field shift for C-7 (DdC 5.98 ppm) whilst it had a
c-gauche upfield shift for C-8 (DdC 1.90 ppm), further
suggesting the presence of a 6b-hydroxyl group. NMR
data of 10 were in agreement with previous literature
(Hanson et al. 1996).
droxyandrost-5-en-17-one 3 by comparison of its NMR
spectra with those of the previously isolated metabolite.
The sixth metabolite was identified as 3b,7a-dihy-
droxyandrost-5-en-17-one 4 by comparison of its NMR
spectra
with
those
of
the
previously
isolated metabolite.
Incubation of DHEA 1 with C. cladosporioides MRC
70282 for 5 days yielded three metabolites (Figure 3).
The first metabolite was identified as 3b,17b-dihydrox-
The third metabolite was identified as 6a-hydrox-
1
yandrost-4-en-3,17-dione 11. The H NMR spectrum of
1
11 lacked the 3a-H resonance of starting material at
dH 3.46 ppm (1H, tt, J ¼ 5.0 and 12.0 Hz) and had sig-
nificant downfield shifts for the double bond reson-
ance (DdH 0.84 ppm) and the 19-methyl group
resonance (DdH 0.21 ppm), suggesting that the 5-en-
3b-hydroxyl moiety of 1 was changed into a 4-en-3-
keto moiety. NMR spectra of 11 had typical resonan-
ces at dH 4.37 ppm (1H, m) and dC 68.19 ppm, indicat-
ing the presence of a 6a-hydroxyl group (Kollerov
et al. 2008). The 13C NMR spectrum of 11 showed a
downfield shift for C-7 (DdC 8.76 ppm) whilst it
yandrost-5-ene 13. The H NMR spectrum of the first
metabolite had a new resonance (1H, t, J ¼ 8.5 Hz) at
dH 3.64 ppm and showed an upfield shift (DdH
0.12 ppm) for the 18-methyl resonance of 1. The 13C
NMR spectrum of the metabolite lacked the C-17 res-
onance of 1 at dC 221.32 ppm and had a new carbon
atom resonance at dC 81.87 ppm. All these results sug-
gested that a reduction at C-17 had taken place. NMR
data of 13 were consistent with literature (Choudhary
et al. 2003).
The second metabolite was identified as 6b-hydrox-
yandrost-4-en-3,17-dione 10 by comparison of its
NMR spectra with those of the previously iso-
lated metabolite.
showed
a c-gauche upfield shift for C-8 (DdC
1.55 ppm), further indicating the presence of a 6a-
hydroxyl group. NMR data of 11 were in accordance
with previous literature (Kollerov et al. 2008).
The third metabolite was determined as 6a-hydrox-
yandrost-4-en-3,17-dione 11 by comparison of its
NMR spectra with those of the previously iso-
lated metabolite.
The fourth metabolite was identified as 7a-hydrox-
1
yandrost-4-en-3,17-dione 12. The H NMR spectrum of
12 lacked the 3a-H resonance of starting material at
dH 3.46 ppm (1H, tt, J ¼ 5.0 and 12.0 Hz) and showed
important downfield shifts for the double bond reson-
ance (DdH 0.48 ppm) and the 19-methyl group reson-
ance (DdH 0.21 ppm), suggesting that the 5-en-3b-
hydroxyl moiety of 1 was converted into a 4-en-3-keto
moiety or the double bond isomerisation and an oxi-
In this work, as can also be seen from Table 3, all
fungi metabolised 1 in different ways. During the incu-
bation with U. chartarum MRC 72584, 1 was mainly
hydroxylated at C-7a and C-7b, accompanied by a
minor hydroxylation at C-4b, a minor epoxidation
from the b-face and a minor oxidation at C-7 following
its hydroxylations. 3b,7b-Dihydroxy-5b,6b-epoxyandro-
stan-17-one 6, 3b,4b,7a-trihydroxyandrost-5-en-17-one
7 and 3b,4b,7b-trihydroxyandrost-5-en-17-one 8 from
the incubation of 1 with this fungus were determined
as new metabolites. During the incubation with C.
sphaerospermum MRC 70266, 1 may have been con-
verted into a 3-keto-4-ene steroid and hydroxylated at
C-6a, C-6b and C-7a, accompanied by a minor 5a-
reduction and a minor oxidation at C-6 subsequent to
1
dation at C-3 took place. The H NMR spectrum of 12
showed a characteristic resonance at dH 4.10 ppm (1H,
bs), suggesting the presence of a 7a-hydroxyl group
(Holland and Thomas 1982). The 13C NMR spectrum of
12 had a downfield shift for C-8 (DdC 9.76 ppm) whilst
it had a c-gauche upfield shift for C-9 (DdC 4.70 ppm),
further suggesting the presence of a 7a-hydroxyl
group. NMR data of 12 were in agreement with prior
literature (Holland and Thomas 1982).

8
K. YILDIRIM ET AL.
Table 3. Conversion rates for metabolites following chromatography.
Fungus
Metabolite
% Conversion
U. chartarum MRC 72584
3b-Hydroxyandrost-5-en-7,17-dione 2
3b,7b-Dihydroxyandrost-5-en-17-one 3
3b,7a-Dihydroxyandrost-5-en-17-one 4
3b,7a-Dihydroxy-5b,6b-epoxyandrostan-17-one 5
3b,7b-Dihydroxy-5b,6b-epoxyandrostan-17-one 6
3b,4b,7a-Trihydroxyandrost-5-en-17-one 7
3b,4b,7b-Trihydroxyandrost-5-en-17-one 8
5a-Androstan-3,6,17-trione 9
6b-Hydroxyandrost-4-en-3,17-dione 10
6a-Hydroxyandrost-4-en-3,17-dione 11
7a-Hydroxyandrost-4-en-3,17-dione 12
3b,7b-Dihydroxyandrost-5-en-17-one 3
3b,7a-Dihydroxyandrost-5-en-17-one 4
3b,17b-Dihydroxyandrost-5-ene 13
6
16
12
2
3
3
4
4
C. sphaerospermum MRC 70266
C. cladosporioides MRC 70282
15
5
6
4
20
8
6b-Hydroxyandrost-4-en-3,17-dione 10
6a-Hydroxyandrost-4-en-3,17-dione 11
37
10
its hydroxylations to form 9, 10 and 11. Some of 1
was only hydroxylated at C-7a and C-7b by the same
fungus. The presence of metabolites 9-11 with an oxy-
gen function C-6 further suggested that some of 1
may have been converted into a 3-keto-4-ene steroid,
hydroxylated at C-6a and C-6b and then oxidised at
C-6. The lower conversion rate for 9, compared to
those of 10 and 11, may have also suggested that 9
was occurred from both 10 and 11 or only one of
them, following their 5a-reduction and oxidations at
C-6. The lower conversion rate for 12, compared to
that of 4, may have suggested that 12 was occurred
from 4 by a 3b-hydroxysteroid dehydrogenase/D5-D4
isomerase activity. There is also another possibility
that 12 mihgt have been occurred from 4 by oxida-
tion at C-3 and the isomerisation of the double bond.
During the incubation with C. cladosporioides MRC
70282, almost half of 1 was converted into a 3-keto-4-
ene steroid and then hydroxylated at C-6a and C-6b.
Some of 1 was only reduced at C-17 by the
same fungus.
As stated above, these three fungi metabolised 1 in
different ways. In some recent works, U. chartarum
MRC 72584 and C. sphaerospermum MRC 70266 also
metabolised androstenedione and testosterone, struc-
turally very similar two 3-keto-4-ene steroids, in some
different ways. U. chartarum MRC 72584 hydroxylated
androstenedione at C-6b, C-7a, C-7b and C-14a,
accompanied by an oxidation at C-6 following its
hydroxylation, a reduction at C-17 and a 5a-reduction
(Yildirim et al. 2020). However, U. chartarum MRC
72584 hydroxylated testosterone at C-6b, C-7b, C-12b
and C-14a, accompanied by oxidations at C-6 and at
C-17, and a 5a-reduction (Yildirim et al. 2018b). C.
sphaerospermum MRC 70266 mainly hydroxylated
androstenedione at C-6b, accompanied by a hydroxyl-
ation at C-15a, a reduction at C-17, a 5a-reduction
and oxidations at C-6 and C-16 following their hydrox-
ylations (Yildirim et al. 2020). However, C. sphaerosper-
mum MRC 70266 mainly hydroxylated testosterone at
C-6b, accompanied by some minor hydroxylations at
C-7b, C-12b, C-15a and C-16b (Yildirim et al. 2019). U.
chartarum MRC 72584 and C. sphaerospermum MRC
70266 may have metabolised androstenedione and
testosterone in some different ways due to the lack of
a 17b-hydroxyl group in androstenedione.
In another recent work, however, C. cladosporioides
MRC 70282 metabolised androstenedione and testos-
terone in a similar way. C. cladosporioides MRC 70282
reduced androstenedione at C-17 and hydroxylated at
C-16b, and then oxidised to a ketone at C-16, accom-
panied by a minor epimerisation at C-17. C. cladospor-
ioides MRC 70282 hydroxylated testosterone at C-16b
and then oxidised at C-16, accompanied by a minor
independent oxidation at C-17 and epimerisation at
C-17 (Yildirim et al. 2018a). These results may suggest
that the lack of a 17b-hydroxyl group in androstene-
dione could not change the way C. cladosporioides
MRC 70282 metabolised androstenedione and
testosterone.
In the present work, although C. sphaerospermum
MRC 70266 and C. cladosporioides MRC 70282 con-
verted some of 1 into 3-keto-4-ene steroids, U. charta-
rum MRC 72584 did not convert 1 into any 3-keto-4-
ene steroids. C. sphaerospermum MRC 70266 and C.
cladosporioides MRC 70282 then metabolised these 3-
keto-4-ene steroids in different ways than they did on
androstenedione and testosterone (Yildirim et al.
2018a, 2018b, 2019, 2020).
A number of fungal strains belonging genera, such
as Rhizopus, Mucor, Fusarium, Penicillium, Rhizomucor
and Aspergillus have been previously incubated with
DHEA 1. These incubations have given interesting
results, such as microbial hydroxylations (especially at

BIOCATALYSIS AND BIOTRANSFORMATION
9
C-7a, C-7b and C-15a), Baeyer-Villiger oxidations, 5a-
reduction, dehydrogenation and reduction at C-17
(Mahato and Garai 1997; Fernandes et al. 2003; Bhatti
and Khera 2012; Donova and Egorova 2012; Nassiri-
Koopaei and Faramarzi 2015).
ORCID
Kudret Yildirim
Ali Kuru
Semra Yılmazer Keskin
9467-3171
http://orcid.org/0000-0002-7889-5903
http://orcid.org/0000-0002-8300-1572
http://orcid.org/0000-0002-
Sinan Ergin
http://orcid.org/0000-0002-0600-2213
Reports of fungal biotransformations on 1 stating a
5a-reductase activity and hydroxylase activity at C-6b
following a 3b-hydroxysteroid dehydrogenase/D5-D4
isomerase activity, hydroxylase activities at C-7a and
C-7b, a 17-ketoreductase activity and some oxidore-
ductase activities subsequent to hydroxylase activities
at different carbon atoms are very common (Mahato
and Garai 1997; Fernandes et al. 2003; Bhatti and
Khera 2012; Donova and Egorova 2012; Nassiri-
Koopaei and Faramarzi 2015). However, a fungal
hydroxylase activity at C-4b and epoxidase activity
from the b-face on 1 are rare. For example, in one
work, 1 was hydroxylated by Metarrhizium velutinum
M6 at C-4b (Uszycka-Horawa et al. 1986). In another
work, 1 was epoxidised by Cephalosporium aphidicola
from the b-face (Rahman et al. 2000). As far as fungal
steroid biotransformations on 1 are concerned, the
6a-hyroxylase activities following 3b-hydroxysteroid
dehydrogenase/D5-D4 isomerase activities were first
shown by C. sphaerospermum MRC 70266 and C. cla-
dosporioides MRC 70282.
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1
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Disclosure statement
Kolet SP, Niloferjahan S, Haldar S, Gonnade R, Thulasiram
HV. 2013. Biocatalyst mediated production of 6b,11a-dihy-
droxy derivatives of 4-ene-3-one steroids. Steroids. 78(11):
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The authors report no decleration of interest. The authors
alone are responsible for the content and writing of
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