Biotransformation of dehydro-epi-androsterone by Aspergillus parasiticus: Metabolic evidences of BVMO activity

Article abstract of DOI:10.1016/j.steroids.2016.03.018

The research on the synthesis of steroids and its derivatives is of high interest due to their clinical applications. A particular focus is given to molecules bearing a D-ring lactone like testolactone because of its bioactivity. The Aspergillus genus has been used to perform steroid biotransformations since it offers a toolbox of redox enzymes. In this work, the use of growing cells of Aspergillus parasiticus to study the bioconversion of dehydro-epi-androsterone (DHEA) is described, emphasizing the metabolic steps leading to D-ring lactonization products. It was observed that A. parasiticus is not only capable of transforming bicyclo[3.2.0]hept-2-en-6-one, the standard Baeyer-Villiger monooxygenase (BVMO) substrate, but also yielded testololactone and the homo-lactone 3β-hydroxy-17a-oxa-d-homoandrost-5-en-17-one from DHEA. Moreover, the biocatalyst degraded the lateral chain of cortisone by an oxidative route suggesting the action of a BVMO, thus providing enough metabolic evidences denoting the presence of BVMO activity in A. parasiticus. Furthermore, since excellent biotransformation rates were observed, A. parasiticus is a promising candidate for the production of bioactive lactone-based compounds of steroidal origin in larger scales.

Full text of DOI:10.1016/j.steroids.2016.03.018

Steroids 109 (2016) 44–49
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Biotransformation of dehydro-epi-androsterone by Aspergillus
parasiticus: Metabolic evidences of BVMO activity
M. Laura Mascotti a,1, Martín A. Palazzolo a, Fabricio R. Bisogno b, Marcela Kurina-Sanz a,
⇑
a Area de Química Orgánica, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, INTEQUI-CONICET, San Luis 5700, Argentina
b Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, INFIQC-CONICET, Córdoba 5000, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
The research on the synthesis of steroids and its derivatives is of high interest due to their clinical
applications. A particular focus is given to molecules bearing a D-ring lactone like testolactone because
of its bioactivity. The Aspergillus genus has been used to perform steroid biotransformations since it offers
a toolbox of redox enzymes. In this work, the use of growing cells of Aspergillus parasiticus to study the
bioconversion of dehydro-epi-androsterone (DHEA) is described, emphasizing the metabolic steps lead-
ing to D-ring lactonization products. It was observed that A. parasiticus is not only capable of transforming
bicyclo[3.2.0]hept-2-en-6-one, the standard Baeyer-Villiger monooxygenase (BVMO) substrate, but also
Received 26 October 2015
Received in revised form 10 March 2016
Accepted 22 March 2016
Available online 26 March 2016
Keywords:
Steroids
yielded testololactone and the homo-lactone 3b-hydroxy-17a-oxa-D-homoandrost-5-en-17-one from
Dehydro-epi-androsterone
Cortisone
Biotransformation
Aspergillus parasiticus
Baeyer-Villiger monooxygenase
DHEA. Moreover, the biocatalyst degraded the lateral chain of cortisone by an oxidative route suggesting
the action of a BVMO, thus providing enough metabolic evidences denoting the presence of BVMO activity
in A. parasiticus. Furthermore, since excellent biotransformation rates were observed, A. parasiticus is a
promising candidate for the production of bioactive lactone-based compounds of steroidal origin in larger
scales.
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
[9–11]. In particular, the use of filamentous fungi to biotransform
androstan- and pregnan-based steroids has been comprehensively
Steroids are a large family of molecules widely distributed in
Nature. They are structurally diverse solid alcohols sharing a com-
mon four-ring nucleus [1]. These molecules exhibit vital roles in
animal, fungal and plant cells. Likewise, they are used to treat sev-
ere human health disorders such as cancer, diabetes and obesity
[2,3], among others. In particular, the discovery of the antineopla-
sic properties of testolactone in 1962 [4] triggered the search for
bioactive D-ring lactone-based compounds [5].
The generation of oxyfunctionalized steroidal products is a mul-
tistep, complex task to achieve by chemical means [6–8]. Alterna-
tively, the use of microbial catalysts represents a simpler and
greener strategy to perform regio- and stereoselective hydroxyla-
tions, regioselective C@C reductions and isomerizations and, to a
lesser extent, C@O reductions and Baeyer-Villiger oxidation
reactions on the D-ring and the lateral chain of these molecules
documented [12,13]. Among them, Fusarium [14] species have
been shown to display excellent dehydrogenation activities. On
the other hand, strains from Beauveria [15], Cunninghamella [16],
Mucor [17–19] as well as Fusarium [20] were reported to perform
regio- and stereoselective hydroxylations. Recently, Wu and
coworkers described the hydroxylation of dehydro-epi-andros-
terone (DHEA) by Colletotrichum lini [21]. Furthermore, the genus
Penicillium [22,23] has been employed to carry out D-ring lac-
tonizations. Nevertheless, Aspergillus strains might be the biocata-
lysts of choice to study the transformation profile of a particular
steroidal molecule, since this genus is capable of performing all
the aforementioned reactions, as extensively referred by several
authors [24–28].
It is demonstrated that the Aspergillus genus provides a toolbox
of redox enzymes [29,30]. Nowadays, the Baeyer-Villiger
monooxygenase (BVMO) activity is one of the most studied from
biomolecular as well as biotechnological perspectives [31–33]. In
this context, the purpose of this work was to find Aspergillus strains
capable of yielding steroidal D-ring lactonization products using
DHEA as an androstan-derived model compound.
⇑
Corresponding author.
E-mail address: marcelakurina@gmail.com (M. Kurina-Sanz).
Present address: Laboratorio de Biología Molecular, Facultad de Química,
1
Bioquímica y Farmacia, Universidad Nacional de San Luis, IMIBIO-SL CONICET, San
Luis 5700, Argentina.
http://dx.doi.org/10.1016/j.steroids.2016.03.018
0039-128X/Ó 2016 Elsevier Inc. All rights reserved.

M.L. Mascotti et al. / Steroids 109 (2016) 44–49
45
spraying the p-anisaldehyde reagent, as described elsewhere. To
assess the biotransformation profile of each substrate, samples
were analyzed by GC-FID using a Clarus 500 instrument (Perkin
Elmer) using pure analytic standards. Conversion was determined
using a 007 (methyl 5% phenyl silicone) capillary column (Quad-
rex) and calculated using relative areas. Product purifications were
done by column chromatography on silica gel 60 (230-400 mesh,
Merck). Mixtures of Hx: EtOAc were initially used as mobile
phases, changing the composition from 100:0 to 30:70 by a 10%
per column volume (Vc). Finally, two Vc of pure EtOAc followed
by two Vc of EtOAc: MeOH (90:10) were employed. The identity
of the isolated compounds was determined by NMR, HRMS-ESI
and GC/LRMS-EI. The 1H and 13C NMR spectra were recorded on
a AC-200 or AMX-400 spectrometer (at 200.13 or 400.16 MHz,
and at 50.23 or 100.62 MHz, respectively) (Bruker) using CDCl3
as solvent with TMS as internal standard. HRMS-ESI measurements
were done on a microTOF-Q II (Bruker) coupled to a UPLC-DAD
1200 (Agilent) at a resolution of 5000 (5% valley definition), by
70 eV electron ionization, at an accelerating voltage of 8 kV. GC/
LRMS-EI were performed at 70 eV using an ion trap (GCQ Plus)
with MSn (Finnigan, Thermo-Quest), operated at a fundamental
rf-drive of 1.03 MHz. Helium was used as the damping gas at an
uncorrected gauge reading of 6Á10À5 Torr. An OV-5 (5% diphenyl
95% dimethylpolysiloxane) capillary column (OVS) was used.
Compounds were analyzed by comparing their mass spectra with
the NIST spectral library (Wiley). Structural data is given in the
Supplementary Information.
2. Experimental
2.1. Chemicals and microorganisms
All the chemicals were purchased in analytical grade from
Sigma Aldrich or Merck and were used without further purifica-
tion. Culture media components were obtained from Britania.
The Aspergillus strains used comprised A. flavus, A. fumigatus and
A. japonicus, which were acquired from the collections of Universi-
dad de Buenos Aires (Argentina), Universidad Nacional de Rio
Cuarto (Argentina) and Colección de Cultivos Fúngicos IIB-INTECH
(Argentina), respectively, as well as A. candidus and A. parasiticus,
which were both obtained from Universidad Nacional del Litoral
(Argentina). Microorganisms stored at 4 °C in Czapek Yeast (CY)
agar slopes were inoculated to erlenmeyers containing CY liquid
medium, which composition per 1 L distilled H2O is 5 g yeast
extract, 30 g sucrose, 1 g K2HPO3, 0.3 g NaNO3, 0.5 g KCl, 0.5 g
MgSO4Á7H2O, 0.01 g FeSO4Á7H2O, 0.005 g CuSO4Á5H2O and 0.01 g
ZnSO4Á7H2O. Cultures were incubated at 28 °C and 150 rpm.
2.2. Microbial screening
To test the capability of the Aspergillus strains to transform the
model BVMO substrate rac-bicyclo[3.2.0]hept-2-en-6-one (1), bio-
catalysts were prepared according to previous reports [31,34].
Then, 1 (2.5 mM final concentration) was added to 50 mL erlen-
meyer flasks containing 500 mg wet cells (2.5% (p/v)) of an individ-
ual strain each in 20 mL of CY medium. Biotransformations were
run by duplicate –mean values are given- and incubated at 28 °C
and 150 rpm during 72 h. Controls without 1 and without biocata-
lyst were also run. Samples were taken as described in Section 2.3.1
and analyzed by GC-FID using a Clarus 500 instrument (Perkin
Elmer) and pure analytic standards. Conversion was determined
employing a Rt-bDEXse (Restek) chiral capillary column and calcu-
lated using relative areas of the substrate and the lactone-based
products only. The enantiomeric excesses were calculated as done
by Chen et al. [35]. See Supplementary Information for further
details.
3. Results and discussion
3.1. Screening of BVMO activity in Aspergillus strains
Considering previous reports in literature describing the poten-
tial of the Aspergillus genus as a biocatalyst to perform Baeyer-
Villiger oxidations of the D-ring in several steroids [36–39], we
selected a small collection of strains from this genus to screen
for this enzymatic activity by using rac-bicyclo[3.2.0]hept-2-
en-6-one (1) as model substrate [40]. Since redox cofactors are
needed, we employed growing cell cultures in order to display
self-sufficient transformations. Although all the strains assayed
were capable to oxidize the model substrate 1 to the corresponding
normal and/or abnormal lactones (Table 1, Supplementary
Information), A. parasiticus exhibited the highest regio- and enan-
tioselectivity by yielding mainly the normal (1S, 5R) and the abnor-
mal (1R, 5S) lactones from the enantiomers (1R, 5S) and (1S, 5R) of
substrate 1, respectively (Fig. 1). Since dehydrogenase activity is
ubiquitous and constitutive in these fungi, carbonyl reduction
products were observed as well (data not shown). Because of these
results, A. parasiticus was selected as the biocatalyst to perform the
transformation of DHEA (2).
2.3. Biotransformation of steroids
Biocatalysts were prepared as referred above. In the analytical
scale, 50 mL erlenmeyer flasks containing 500 mg wet cells (2.5%
(p/v)) of A. parasiticus in 20 mL CY medium were used. To start
the biotransformations, each substrate was dissolved in DMSO or
DMF (1% (v/v) final concentration) and added to the erlenmeyer
flasks. Biotransformations were incubated at 28 °C and 150 rpm
and were run by duplicate, mean values are shown. Controls with-
out biocatalyst and without substrate were also included. To per-
form time-course experiments, multiples batches were prepared
and samples were taken by withdrawing batches and controls in
duplicate at a time. In the semipreparative scale, the same proce-
dure described for the analytical scale was followed, but using a
biotransformation volume of 200 mL in a 500 mL erlenmeyer flask.
A total of five batches were run and combined after 96 h.
3.2. Biotransformation of dehydro-epi-androsterone (DHEA)
When the biotransformation of 2 was assayed in analytical scale
(100 mg), five products were detected (Fig. 2) by GC/ LRMS-EI and
NMR analyses (see Supplementary Information). Among them,
three compounds are supposed to directly derive from the
substrate, being androstenediol (3) –a product of the reduction of
the carbonyl in the D-ring-, androstenedione (4) –obtained by
the oxidation of hydroxyl in position 3 and the subsequent isomer-
ization of the C@C from C5 to C4- and the homo-lactone 3b-
2.3.1. Work-up and analysis
Each batch was filtered in vacuo to remove cells and then reac-
tion mixtures were extracted three times with one volume of ethyl
acetate. Organic layers were combined, dried with sodium sulfate,
concentrated in vacuo and spotted on silica gel 60 F254 TLC plates
(Merck) to qualitatively assess the substrate consumption and the
appearance of biotransformation products. The chromatographic
analysis was done using the mixture n-hexane: ethyl acetate as
mobile phase and plates were revealed under UV light and by
hydroxy-17a-oxa-D-homoandrost-5-en-17-one (5), produced after
a D-ring lactonization reaction. Supported in literature reports, it
is possible to propose that these molecules were obtained by the
action of a dehydrogenase, a 3b-HSD/isomerase (E2) and a BVMO

46
M.L. Mascotti et al. / Steroids 109 (2016) 44–49
A. parasiticus
growing cells
A. parasiticus
growing cells
H
H
H
O
O
O
O
O
H
H
H
O
normal lactone
(1R, 5S)
abnormal lactone
(1S, 5R)
(1S, 5R)
(c = 2 %)
(c = 66 %)
A. parasiticus
growing cells
A. parasiticus
growing cells
H
O
H
H
O
O
H
H
H
O
normal lactone
(1R, 5S)
(c = 5 %)
(1S, 5R)
abnormal lactone
(1R, 5S)
(c = 25 %)
1
(racemic mixture)
Fig. 1. Biotransformation of rac-bicyclo[3.2.0]hept-2-en-6-one (1) by A. parasiticus growing cells. Conversions (c) were determined by GC-FID.
OH
OH
E2
HO
O
E1
E2
18
3
6
O
17
19
O
16
C
D
A
B
3
HO
4
6
O
2
4
E3
O
O
O
O
E2
O
HO
5
7
Fig. 2. Biotransformation route of DHEA (2) by A. parasiticus growing cells. The reaction sequences are given from time-course experiments (solid arrows). Enzymes E1 to E3
are proposed to catalyze each step on the basis of literature reports. Dashed arrows are given for possible although not detected reactions. E1: dehydrogenase, E2: 3b-HSD/
isomerase, E3: BVMO.
(E3) [41–43], respectively. Then, compounds 3 and 5 were further
metabolized to yield testosterone (6) and testololactone (7),
respectively. Again, a 3b-HSD/isomerase activity might have led
to these structures. Noteworthy, product 6 could have also been
obtained from compound 4 by the reduction of the carbonyl group
at position 17, as determined by Cvelbar et al. [44]. In addition, the
steroid 7 could have been produced from the D-ring lactonization
of the same molecule [36]. Besides, testosterone (6) would have
yielded testololactone (7) via 4, as reported by Yildirim et al. using
cultures of A. terreus [39] and A. tamarii [26]. However, from time-
course experiments the occurrence of these transformations was
not observed under the assayed conditions (Table 2, Supplemen-
tary Information). Our results are in agreement with early findings
of Mostafa et al. [36], which showed A. parasiticus is capable of
oxidizing progesterone to 6 and 7. Nevertheless, it should be noted
that, as far as we are concerned, the biotransformation profile of
DHEA by A. parasiticus as described herein was not reported previ-
ously. It should be addressed that during the preparation of this
manuscript, the first version of the genome of A. parasiticus SU-1
was released (genome ID: ASM95608v1). By performing a prelim-
inary in silico homology searching for BVMO-encoding sequences,
we found at least 17 open reading frames as putative BVMOs, thus
supporting our considerations based on metabolic observations.
It is worthy to highlight that when the biotransformation of 2
was performed in semipreparative scale –five times larger than
the analytical scale, 500 mg DHEA-, the homo-lactone 5 was
detected as the only product (166 mg, 33% isolated yield after col-
umn chromatography). The different results obtained from one
scale to the other could be attributed to a variation in the molecu-
lar oxygen availability, as observed by Kolek and coworkers
employing P. lilacinum as biocatalyst under similar conditions
[45]. Specifically, a more extensive surface between the biocatalyst
and the reaction medium might have considerably eased the O2
mass transfer, due to cells may be increased in number but not
in size. Moreover, since no further conversion of product 5 to 7
was evidenced it would be possible to assume that the isomeriza-
tion activities, as well as the reductive ones, might have been
suppressed by an increase in the molecular oxygen availability in
the cells. Hence, the metabolic fate of 2 could have been routed
to the BVMO-catalyzed D-ring lactonization by this effect.

M.L. Mascotti et al. / Steroids 109 (2016) 44–49
47
A. parasiticus
growingcells
O
O
8
9
A. parasiticus
growingcells
O
8 (c = 100%)
O
OH
HO
O
A. parasiticus
O
O
growingcells
O
O
10
11 (c = 100%)
Fig. 3. Transformation of 4-cholesten-3-one (8), 5-cholesten-3-one (9) and cortisone (10) biocatalyzed by A. parasiticus growing cells. Conversions (c) as well as product
identities were determined by GC/EI-LRMS.
O
HO
HO
OH
HO
O
OH
O
OH
O
O
O
O
E3
E4
spontaneus
O
O
O
O
O
H2O
10
11
OH
HO
O
Fig. 4. Suggested metabolic pathway in A. parasiticus for the biotransformation of cortisone (10) to adrenosterone (11). Compounds between brackets were not detected.
E3: BVMO, E4: hydrolase.
It should be emphasized that although the comprehensively
reported whole-cell biotransformations of steroids represent a
strong evidence of the presence of BVMOs in fungi [23,36,46] there
is no register in literature up to date of an isolated fungal BMVO
acting on this class of compounds. The available recombinant fun-
gal enzymes, CAMO [47], BVMOAf1 [48] and those from Aspergillus
flavus [49] are not capable of transforming them. For this reason,
these results represent a starting point to perform the cloning
and expression of new fungal BVMOs and its further biochemical
and biocatalytic characterization.
andrenosterone (11) –also known as the Reichstein’s substance G
[50]- was produced with complete conversion when cortisone
(10) was employed as substrate. The structures of the obtained
compounds were analyzed by GC/LRMS-EI (see Supplementary
Information). Interestingly, A. parasiticus did not exhibit BVMO nei-
ther reductive activities on carbonyl groups in rings A and/or C in
any of the assayed molecules. However, the biocatalyst was
capable of transforming the lateral chain of 10 in an oxidative fash-
ion so as to yield 11, as aforementioned. Noticeably, the pathway
leading to this molecule could involve an initial step that may be
catalyzed by a BVMO, in agreement with early observations made
by Brannon and coworkers for the bioconversion of progesterone
to testololactone by A. tamarii [51]. A possible mechanistic inter-
pretation of the route leading from compound 10 to product 11
by A. parasiticus is given in Fig. 4.
3.3. Biotransformation of other oxo-functionalized steroids susceptible
of BMVO transformation
To further assess the biocatalytic performance of A. parasiticus,
three other steroids structurally linked to 2 and susceptible of
BMVO transformation were also tested as substrates in analytical
scale (Fig. 3). The use of 4-cholesten-3-one (8) did not yield bio-
transformation products. On the other hand, when 5-cholesten-
3-one (9) was selected, compound 8 was quantitatively formed,
thus showing the occurrence of isomerization activity. Finally,
4. Conclusion
In initial experiments, we detected a novel biocatalyst from
the Aspergillus genus, named A. parasiticus, that transformed

48
M.L. Mascotti et al. / Steroids 109 (2016) 44–49
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Acknowledgements
This work was supported by Grants from Universidad Nacional
de San Luis PROCIO 2-1412, Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET) PIP 00360 and Agencia Nacional
de Promoción Científica y Tecnológica (ANPCyT) PICT 2011-1416.
MLM and MAP are postdoctoral CONICET fellows. FRB and MKS
are members of the Research Career of CONICET, Argentina.
Authors thank Dr. Carlos Ardanaz for performing the GC/LRMS-EI
analyses and Lic. Mónica Ferrari for technical assistance. The
authors declare no commercial or financial conflict of interest.
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2016.03.
018.
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