Development of a high-yielding bioprocess for 11-α hydroxylation of canrenone under conditions of oxygen-enriched air supply

Article abstract of DOI:10.1016/j.steroids.2016.09.013

A high yielding bioprocess for 11-α hydroxylation of canrenone (1a) using Aspergillus ochraceus ATCC 18500 was developed. The optimization of the biotransformation involved both fermentation (for achieving highly active mycelium of A. ochraceus) and biotransformation with the aim to obtain 11-α hydroxylation with high selectivity and yield. A medium based on sucrose as C-source resulted particularly suitable for conversion of canrenone into the corresponding 11-hydroxy derivative, whereas the use of O₂-enriched air and dimethyl sulfoxide (DMSO) as a co-solvent for increasing substrate solubility played a crucial role for obtaining high yields (>95%) of the desired product in high chemical purity starting from 30?mM (10.2?g/L) of substrate. The structure of the hydroxylated product was confirmed by a combination of two-dimensional NMR proton-proton correlation techniques.

Full text of DOI:10.1016/j.steroids.2016.09.013

Steroids 116 (2016) 1–4
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Development of a high-yielding bioprocess for 11-
a
hydroxylation of
canrenone under conditions of oxygen-enriched air supply
a
b
a
a
a
Martina Letizia Contente , Benedetta Guidi , Immacolata Serra , Valerio De Vitis , Diego Romano ,
c
d
e
a,
⇑
Andrea Pinto , Roberto Lenna , Ricardo Pinheiro de Souza Oliveira , Francesco Molinari
a
Department of Food, Environmental and Nutritional Science (DeFENS), University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
Department of Medical Biotechnology and Translational Medicine, University of Milan, Via Saldini 50, 20133 Milan, Italy
Department of Pharmaceutical Sciences (DISFARM), University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
Industriale Chimica, Via Grieg 13, 21047 Saronno (VA), Italy
b
c
d
e
Biochemical and Pharmaceutical Technology Department, Faculty of Pharmaceutical Sciences, University of São Paulo, Av Professor Lineu Prestes 580, São Paulo 05508-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2016
Received in revised form 12 September
A high yielding bioprocess for 11-a hydroxylation of canrenone (1a) using Aspergillus ochraceus ATCC
18500 was developed. The optimization of the biotransformation involved both fermentation (for achiev-
ing highly active mycelium of A. ochraceus) and biotransformation with the aim to obtain 11- hydrox-
a
2
016
ylation with high selectivity and yield. A medium based on sucrose as C-source resulted particularly
suitable for conversion of canrenone into the corresponding 11-hydroxy derivative, whereas the use of
Accepted 18 September 2016
Available online 22 September 2016
O
2
-enriched air and dimethyl sulfoxide (DMSO) as a co-solvent for increasing substrate solubility played
a crucial role for obtaining high yields (>95%) of the desired product in high chemical purity starting from
0 mM (10.2 g/L) of substrate. The structure of the hydroxylated product was confirmed by a combina-
tion of two-dimensional NMR proton-proton correlation techniques.
Ó 2016 Elsevier Inc. All rights reserved.
Keywords:
3
Steroid hydroxylation
Aspergillus ochraceus
Biotransformation
Biocatalysis
Monooxygenase
Canrenone
1
. Introduction
hydroxylation mostly depends on the substrate employed. a-
Hydroxylation is preferred with progesterone [6–10], 2-oxatestos-
The regio- and stereoselective hydroxylation of steroids by fun-
terone [11], 13b-ethyl-gona-4-en-3,17-dione [12], canrenone [13],
gal strains is a well-known method for the preparation of corticos-
teroids and progestogens having an oxygen substituent at C-11
position [1,2]. To overcome synthetic laborious steps, different spe-
cies of eumycetes (i.e., Rhizopus and Aspergillus) are employed to
perform the 11-hydroxylation of steroids because of the high
regio- and stereoselective specificity of their oxygenating enzymes
estr-4-en-3,17-dione [13], 16a, 17-epoxyprogesterone [14], 5a-
androstan-3-one [15], whereas b-hydroxylation was found with
cortexolone [16]. Generally, the bioconversion yield and selectivity
is affected by different parameters, such as substrate concentra-
tion, aeration, and agitation; the chosen strain can be also impor-
tant for regio- and enantioselectivity [2], as observed for other
steroid biotransformation as well [17]. High substrate concentra-
tions are needed to achieve industrially relevant biotransforma-
tions, but often dramatically limit conversion and selectivity. In
[
3]. Although microbial oxidation is a good method for the simpli-
fication of the steroid drugs production, large-scale biotransforma-
tions are sometimes limited by low yields because of the poor
solubility of substrates in the reaction medium and formation of
different oxygenated by-products, which sometimes are very-diffi-
cult to separate [4,5]. Another problem is that many fungal species
are very specific in their nutritional needs, so the use of cultural
growing media is drastically limited. Microbial 11-hydroxylation
of steroids using Aspergillus ochraceus is a well-known process
and applicable on different substrates; the stereoselectivity of the
the case of 11a-hydroxylation of canrenone, good yields and selec-
tivity were obtained only when pure substrate concentration was
kept below 5–6 g/L (14.7–17.6 mM) [13,18]; higher substrate con-
centrations not only limited overall conversions, but also yielded
undesired by-products due to hydroxylation of different positions
[19].
In this study we have investigated an efficient, economical and
industrially scalable method for the conversion of canrenone into
the corresponding 11
a-canrenone using Aspergillus ochraceus ATCC
⇑
Corresponding author.
E-mail address: francesco.molinari@unimi.it (F. Molinari).
1
8500 as the biocatalyst. This microorganism is able to grow on
http://dx.doi.org/10.1016/j.steroids.2016.09.013
039-128X/Ó 2016 Elsevier Inc. All rights reserved.
0

2
M.L. Contente et al. / Steroids 116 (2016) 1–4
different inexpensive nutrient media and to be stored, after
lyophilisation, without losing its oxygenating skills [6,7]. Aspergil-
lus ochraceus ATCC 18500 was employed for the preparation of
at 299 nm for 1a, and at 240 nm for 2a. The mobility of substrate
and product was: 1a = 16.3 min, 2a = 7.9 min.
NMR spectroscopy. H NMR and 13C NMR spectra were recorded
1
11a-hydroxy-canrenone, a key intermediate for the preparation
of eplerenone (an important potassium sparing diuretic useful in
on a Varian Gemini 300 MHz spectrometer using the residual sig-
1
13
nal of the deuterated solvent as internal standard. H and
C
the heart failure treatment) [20].
chemical shifts (d) are expressed in ppm, and coupling constants
(
J) in hertz (Hz).
Two-dimensional proton-proton NOESY experiments were per-
formed using standard pulse sequences, present in the spectrome-
2
. Materials and methods
ter library.
2.1. Materials
1
2
a H NMR (300 MHz,CDCl
3
): d = 1.05 (s, 3H), 1.25 (s, 3H), 1.29–
1
2
.59 (m, 4H), 1.62 (s, br, OH), 1.86–2.06 (m, 5H), 2.28–2.46 (m, 4H),
All the solvents and reagents were purchased from Sigma-
Aldrich, whereas canrenone and reference products were kindly
furnished by Industriale Chimica (Saronno, Varese, Italy).
.5–2.65 (m, 4H), 4.12 (sest, J = 4.8 Hz, 1H), 5.7 (s, 1H), 6.03 (dd,
13
J = 9.7, 1.8 Hz, 1H), 6.03 (dd, J = 9.7, 2.2 Hz, 1H) ppm; C NMR
300 MHz, CDCl ): d = 15.6, 17.2, 22.5, 29.1, 31.1, 34.2, 35.5, 35.5,
5.8, 36.3, 37.7, 43.8, 46.6, 55.7, 67.9, 94.8, 124.7, 128.8, 138.0,
62.9, 176.4, 199.9 ppm.
(
3
1
3
2
.2. Microorganism and cultivation
Aspergillus ochraceus ATCC 18500 was routinely maintained on
M5YE agar (malt broth, yeast extract 0.5%, agar 1.5% pH 5.6).
Growth in shake flasks: Erlenmeyer flasks (1 L) containing
3. Results
1
00 mL of liquid medium (malt extract 3%, sucrose 2%, yeast
3.1. Optimization of the growth conditions in shake flasks
extract 0.3%, pH 5.6) were inoculated from slants prepared with
M5YE-agar medium. Each flask was inoculated with one slant, sus-
pending the culture with 5 mL of sterile water. The flasks were
incubated on a rotatory shaker at 28 °C, 150 rpm, for 48 h. Culture
media were prepared using different combinations of the following
products:
Firstly, an optimization of the growth conditions of A. ochraceus
in shake flasks was performed for obtaining high and selective
activity towards canrenone 1a. The substrate (15 mM, 5.1 g/L)
was added after 48 h of growth. Yields of 11a-canrenone (2a)
(expressed as g/L gmycelium of 2a produced after 24 h) and chemical
purity of 2a produced (evaluated by HPLC) were used as response
parameters. It is known that hydroxylation of different position of
C-sources: glucose (1–5%), sucrose (1–5%), lactose (1–5%), glyc-
erol (1–5%), maltose (1–5%), soluble starch (1–5%).
C- and N-sources: malt extract (0–1%), yeast extract (0–1%),
corn steep liquor (0–1%).
1a may decrease the selectivity of 11-a hydroxylation with A.
ochraceus [19]. Simultaneous evaluation of different combinations
and concentrations of C-sources and N-sources (glucose, sucrose,
lactose, glycerol, maltose, hydrolysed starch, malt extract, yeast
extract, corn steep) were evaluated using the Multisimplex exper-
imental design, already used for optimizing fermentations and bio-
transformations [21,22]. The best hydroxylation conditions were
found when A. ochraceus was grown for 48 h at 28 °C, 300 rpm in
a medium composed with 2% sucrose, 3% malt extract, 0.3% yeast
extract at pH 5.6, giving the highest molar conversion (68–70%,
analytical yield) of 1a into 2a (chemical purity 99.5%), starting
from 15 mM substrate concentration.
Growth in stirred tank reactor: inoculum was prepared using
two 1 L baffled Erlenmeyer flasks containing 100 mL of liquid med-
ium (malt extract 3%, sucrose 2%, yeast extract 0.3%, pH 5.6) pre-
inoculated from slants prepared with M5YE-agar medium. Each
flask was inoculated with one slant, suspending the culture with
5 mL of sterile water and incubated on a rotatory shaker at 28 °C,
150 rpm, for 48 h. A 20 L stirred-tank bioreactor Applikon Biobench
20 L (Applikon Biotechnology B.V.) equipped with an on-line data
acquisition, control system, gas mixer, and pH and O
2
electrodes
was employed as stirred tank reactor (STR). The STR was filled with
5
0
L of liquid medium (malt extract 3%, sucrose 2%, yeast extract
.3%, pH 5.6). The bioreactor was inoculated with 200 mL of inocu-
3
.2. Optimization of 11-
a hydroxylation of canrenone in stirred tank
lum and incubated at 28 °C for 48 h with air inlet 300 vvm, stirring
00 rpm, pH maintained at 5.6. Dry weight was determined after
filtration of the mycelium with a Buchner funnel and dried for
(
bio)reactor
3
Once found the best conditions of growth in shake flask, the fer-
2
4 h at 110 °C. Results were an average of five replicates.
mentation and biotransformation were carried out in a conven-
tional stirred tank reactor (STR). A. ochraceus was grown in the
STR employing the medium optimized in shake flasks (2% sucrose,
2.3. Biotransformations
3
% malt extract, 0.3% yeast extract at pH 5.6 rpm); the first exper-
iment was carried out with an agitation of 400 rpm and aeration of
.0 vvm. Biotransformation was started by addition of 15 mM of 1a
after 48 h of growth, furnishing a molar conversion of 82% into 2a
chemical purity 99.5%). The growth and biotransformation in STR
After 48 h of growth, the substrate (neat or solubilized in sol-
vents) was added to the whole culture to start the biotransforma-
tion. Pure air and different air/O ratios (5/1, 3/1, and 1/1) were
2
2
used during the biotransformation. The reaction was followed by
HPLC. When the bioconversion was over, mycelium was filtered
and washed with dichloromethane; the supernatant was extracted
(
allowed for a remarkable increase of the yield of 2a with respect to
what obtained in shake flasks (maximum yield 70% from 15 mM
substrate concentration). This first positive result in STR led us to
study different conditions of aeration and agitation during the pro-
3
times with dichloromethane and the organic extracts collected,
dried over Na SO and the solvent was removed; the crude product
was purified by flash chromatography (n-hexane/EtOAc 1:1).
2
4
cess of growth and biotransformation. O
2
is poorly soluble in water
(
7.5 mg/L at 1 atm and 30 °C [23]) and, therefore, transfer of O
2
2
.4. Analyticals
from the gas to the liquid phase is often the limiting step in aerobic
bioprocesses, including enzymatic hydroxylations; different condi-
tions of aeration and agitation may have a strong impact on dis-
solved oxygen. Neat substrate (15 mM) was added after 48 h and
pH maintained at 5.6 (Table 1).
Molar conversions were evaluated by HPLC (Purospher Star
RP18e 250 * 4.6 mm, 5
mobile phase: CH CN/water 60:40, flow rate 0.5 mL/min, detection
lm column (Merck, Darmstadt, Germany),
3

M.L. Contente et al. / Steroids 116 (2016) 1–4
3
Table 1
Effect of aeration and agitation on 11-
a hydroxylation of canrenone (1a) with
Aspergillus ochraceus ATCC 18500 in stirred tank reactor. Biotransformation was
started by adding 15 mM of neat substrate. *DOT = Dissolved oxygen tension.
Entry
Agitation (rpm)
Aeration (vvm)
Molar conversion (%)
1
2
3
4
5
6
7
8
9
200
200
200
400
400
400
600
600
600
1.0
1.5
2.0
1.0
1.5
2.0
1.0
1.5
2.0
75
78
78
76
80
82
82
84
84
Table 2
Effect of substrate concentration on 11-
in hot DMSO (4% v/v).
a
hydroxylation of canrenone (1a) dissolved
Fig. 2. 11-
a
Hydroxylation of canrenone (1a, 30 mM dissolved in hot DMSO) with
Aspergillus ochraceus ATCC 18500 performed in STR: aeration with pure air (red) or
2
5% -enriched air (black). Circles refer to dissolved oxygen tension (DOT)
O
2
Entry Substrate
mM)
Molar conversion
(%)
Chemical purity of 2a Time
registered during the biotransformation.
(
(%)
(h)
1
2
3
4
5
7.5
>97
95
85
72
30
>99.5
>99.5
>99.5
99.4
36
48
48
72
72
15.0
22.5
30.0
37.5
The use of hot DMSO (4% v/v) gave the highest yield (95%) starting
from 15 mM 1a (under the same conditions, the addition of neat
substrate allowed for 82% yield). These conditions were hence
applied for the biotransformation of different concentrations of
98.7
1
a in STR (Table 2).
Yields and hydroxylation rates tended to decrease by increasing
Table 3
Effect of substrate concentration on 11-
in hot DMSO (4% v/v).
a
hydroxylation of canrenone (1a) dissolved
substrate concentrations (entries 2–5), and, for substrate concen-
trations above 22.5 mM (entries 4 and 5), the chemical purity of
the product decreased as well.
Entry
O
2
fraction
Substrate
(mM)
Molar
conversion (%)
Chemical purity of
2a (%)
(%)
Under the hypothesis that the diffusion of O
overall yield of the biotransformation, we evaluated the effect of
enriching the inlet air with pure O . The dissolved oxygen tension
2
was limiting the
1
2
3
4
5
6
7
8
9
15
15
15
25
25
25
35
35
35
15.0
30.0
37.5
15.0
30.0
37.5
15.0
30.0
37.5
>97
93
76
>97
>97
84
>97
>97
85
>99.5
>99.5
99.4
>99.5
>99.5
99.3
>99.5
>99.5
99.1
2
(DOT) can be increased in bioprocesses with filamentous fungi by
applying different oxygen concentrations in the inlet gas [24].
We investigated the effect of different oxygen fraction in the inlet
air, ranging from 15 to 35%, on the biotransformation of 1a with A.
ochraceus (Table 3). It was observed that an O
allowed for accumulation of 2a up to yield >97% starting from
0 mM of 1a, with chemical purity of the recovered 2a >99.5%. At
2
fraction of 25%
3
higher substrate concentration, yields in the range of 76–85% were
obtained with less chemical purity, no matter the concentration of
O applied. No significant increase in the biotransformation yield
2
The application of different levels of agitation and aeration had
no dramatic effect on the monooxygenase activity of the mycelium
of A. ochraceus; in fact, the dissolved oxygen tension (DOT) mea-
sured during these experiments resulted to be low and in a narrow
could be observed by increasing the O
Fig. 1).
2
fraction up to 35%. (See
range (3–5%), indicating that O
2
was largely consumed and only
These experiments showed that the use of air enriched with O
2
low concentrations of O
oxygenation.
2
were available as substrate for the steroid
was crucial for obtaining higher yields in the hydroxylation catal-
ysed by mycelium-bound monooxygenase of Aspergillus ochraceus
ATCC 18500 with the possibility to totally convert 30 mM 1a into
chemically pure 2a, whereas only 72% yield was obtained from
30 mM 1a in the absence of added O (see Table 2). Fig. 2 shows
2
the different profile of the biotransformation of 1a (30 mM,
One of the main problem in steroids biotransformation is the
low solubility of substrates in aqueous media. Different method-
ologies for improving their solubility were investigated such as
the use of different amounts of co-solvents (acetone, ethanol,
DMSO, DMF), the use of micronized powders, and sonicated sus-
pensions (1/10 in deionized water, sonication: 3 cycle for 7 min).
10.2 g/L) and the correspondent registered dissolved oxygen ten-
sion (DOT) using pure air and O -enriched air.
2
Fig. 1. 11-a Hydroxylation of canrenone (1a) with Aspergillus ochraceus ATCC 18500.

4
M.L. Contente et al. / Steroids 116 (2016) 1–4
NOE
Acknowledgement
21
O
22
This work was supported with a PhD grant by Industriale Chim-
ica SRL, Saronno, Italy.
H
18
20
O
HO
NOE
17
12
1
1
13
References
19
16
15
[
1] H.L. Holland, Steroids 64 (1999) 178–186.
1
4
9
14
[2] P. Fernandes, A. Cruz, B. Angelova, H.M. Pinheiro, J.M.S. Cabral, Enzyme Microb.
Technol. 32 (2003) 688–705.
2
3
10
8
[
3] S. Petric, T. Hakki, R. Bernhardt, D. Zigon, B. Cresnar, J. Biotechnol. 150 (2010)
428–437.
7
O
5
[4] S.B. Mahato, S. Garai, Steroids 62 (1997) 332–345.
[5] H.N. Bhatti, R.A. Khera, Steroids 77 (2012) 1267–1290.
[6] C. Vezina, S.N. Sehgal, K. Singh, Appl. Environ. Microbiol. 11 (1963) 50–57.
6
2
a
[7] S.N. Sehgal, K. Singh, C. Vezina, Can. J. Microbiol. 14 (1968) 529–532.
[
8] M. Shibahara, J.A. Moody, L.L. Smith, Biochim. Biophys. Acta 202 (1970) 172–
79.
9] J.Y. Houng, W.P. Chiang, K.C. Chen, C. Tiu, Enzyme Microb. Technol. 16 (1994)
85–491.
1
Fig. 3. Assignment of 11-hydroxyl group configuration using NOESY experiments.
[
4
[
10] K.C. Chen, W.S. Yin, C. Tiu, J.Y. Houng, Enzyme Microb. Technol. 16 (1994) 551–
555.
3
.3. Determination of the hydroxylation positions and relative
[11] H.L. Holland, G. Lakshmaiah, J. Mol. Catal. B: Enzym. 6 (1999) 83–88.
[12] P. Grisenti, F. Pecora, E. Verza, M. Leoni, L. Bossi, U.S. Patent 7667056 B2, 2010.
[13] M. Wiersma, P. van der Meijden, WO Patent 9721830, 1997.
configurations by NMR
The structure of the hydroxylation products of steroids can be
determined by combination of NMR techniques [25]. In the case
of the derivative 2a, the observation of NOESY correlations from
H-19 to H-11, and from H-11 to H-18, indicated that the H-11
and both the methyl groups are in syn configuration (Fig. 3).
[14] S. Mao, B. Hua, N. Wang, X. Hu, Z. Ge, Y. Li, S. Liu, F. Lu, J. Chem. Technol.
Biotechnol. 88 (2013) 287–292.
[
15] A.M. Bell, J.W. Browne, W.A. Denny, E.R.H. Jones, A. Kasal, G.D. Meakins, J.
Chem. Soc., Perkin Trans. 1 (1972) 2930–2936.
[16] F. Naghibi, P. Ataie, M. Mosaddegh, Int. J. Pharm. Pharm. Sci. 4 (2012) 402–403.
[
17] D. Romano, V. Ferrario, D. Mora, R. Lenna, F. Molinari, Steroids 73 (2008) 112–
15.
18] X. Zhang, S. Rong, B.M. Ding, Chin. J. Pharm. 43 (2012) 17–20.
1
[
4
. Conclusions
[19] S.F. Rong, X.L. Zhang, B.M. Ding, Y.F. Wang, X.H. Pan, J. Zhang, Y. Shen, Chin.
Chem. Lett. 23 (2012) 313–316.
[
20] A.J. Reyes, W.P. Leary, G. Crippa, M.F.C. Maranhao, R. Hernandez-Hernandez,
Eur. J. Intern. Med. 16 (2005) 3–11.
The hydroxylation of canrenone (1a) by Aspergillus ochraceus
ATCC 18500 has been optimized in terms of yields and selectivity.
The optimization was carried out in a stirred tank (bio)reactor,
showing that the most crucial parameters for obtaining high-yield-
ing conversion of the substrates were the use of a suited co-solvent
[21] M.L. Contente, F. Molinari, I. Serra, A. Pinto, D. Romano, Eur. J. Org. Chem. 2016
2016) 1260–1263.
22] D. Romano, R. Gandolfi, S. Guglielmetti, F. Molinari, Food Chem. 124 (2011)
096–1098.
[23] P.E. Liley, G.H. Thomson, D.G. Friend, T.E. Daubert, E. Buck, Physical and
chemical data, in: R.H. Perry, D.W. Green (Eds.), Perry’s Chemical Engineers’
Handbook, McGraw-Hill, New York, 1999.
(
[
1
(
hot DMSO 4% vol/vol) and in particular the employment of O
enriched air for providing high dissolved oxygen tension. Specifi-
cally, the use of an inlet air containing 25% of O allowed for high
yields of the desired 11 -derivative (starting from 30 mM in of 1a)
2
-
[
24] Y.Q. Cui, R.G.J.M. van der Lans, K.C.A.M. Luyben, Biotechnol. Bioeng. 57 (1998)
409–419.
2
[
25] A. Romano, D. Romano, E. Ragg, F. Costantino, R. Lenna, R. Gandolfi, F. Molinari,
Steroids 71 (2006) 429–434.
a
in high chemical purity (>99.5% by HPLC). The structure and rela-
tive configuration of the hydroxylated product were confirmed
by a two-dimensional NMR proton-proton correlation techniques
(
NOESY).