Biotransformation of 5-en-3β-ol steroids by Mucor circinelloides lusitanicus

Article abstract of DOI:10.3109/10242422.2015.1089865

In this work, we report the mode of biotransformation of 5-en-3β-ol steroids using Mucor circinelloides lusitanicus for the first time. Here, we selected seven 5-en-3β-ol steroids as substrates. The main characteristic of the fungus was to introduce a 7α-hydroxyl group into substrates 1--5. With substrate 2, 3β, 7α, 11α-trihydroxypregna-5-en-20-one (2b) was obtained as the final product in good yield (46.4%). All the metabolites were determined by infrared spectra, high-resolution mass spectrometry, proton nuclear magnetic resonance, and carbon-13 nuclear magnetic resonance.

Full text of DOI:10.3109/10242422.2015.1089865

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Biotransformation of 5-en-3β-ol steroids by Mucor
circinelloides lusitanicus
Lihong Shan, Kai Jiao, Minghui Yin, Jiajia Huang, Yanjie Chen, Shangshang
Qin & Hongmin Liu
To cite this article: Lihong Shan, Kai Jiao, Minghui Yin, Jiajia Huang, Yanjie Chen,
Shangshang Qin & Hongmin Liu (2016) Biotransformation of 5-en-3β-ol steroids by
Mucor circinelloides lusitanicus, Biocatalysis and Biotransformation, 34:3, 83-88, DOI:
10.3109/10242422.2015.1089865
To link to this article: http://dx.doi.org/10.3109/10242422.2015.1089865
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Date: 01 December 2016, At: 18:33

Biocatalysis and Biotransformation, 2016; 34(3):83–88
RESEARCH ARTICLE
Biotransformation of 5-en-3b-ol steroids by Mucor circinelloides
lusitanicus
1
,2
1
1
1
LIHONG SHAN , KAI JIAO , MINGHUI YIN , JIAJIA HUANG , YANJIE CHEN ,
1
1,2
1,2
SHANGSHANG QIN & HONGMIN LIU
1
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, People’s Republic of China and
Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou,
2
People’s Republic of China
Abstract
In this work, we report the mode of biotransformation of 5-en-3b-ol steroids using Mucor circinelloides lusitanicus for the first
time. Here, we selected seven 5-en-3b-ol steroids as substrates. The main characteristic of the fungus was to introduce a
7a-hydroxyl group into substrates 1–5. With substrate 2, 3b, 7a, 11a-trihydroxypregna-5-en-20-one (2b) was obtained as the
final product in good yield (46.4%). All the metabolites were determined by infrared spectra, high-resolution mass
spectrometry, proton nuclear magnetic resonance, and carbon-13 nuclear magnetic resonance.
Keywords: biotransformation, 5-en-3b-ol steroids, 7a-hydroxylation, 11a-hydroxylation, Mucor circinelloides
lusitanicus
Introduction
points to an important role for B-ring modification,
especially the 7a-hydroxylation. Although 7a-
hydroxylation is the most common reaction in the
biotransformation of steroids and there are a lot of
reports of organisms used in the 7a-hydroxylation of
steroids (Andrea et al. 2006; Schaaf & Dettner 2000;
Teresa 1999), few can be readily applied on a large
scale. Studies of the 7a-hydroxylation mechanism
are restricted because none of the 7a-hydroxylated
derivatives of 3-hydroxy-steroids is commercially
available. Therefore, there is still much remaining
to be learned about new biocatalytic microorganisms
and increasing yield. On the other hand, 11a-
hydroxylation is a commonly used reaction in the
steroid industry (Petri cˇ et al. 2010). It has been
reported for several species of filamentous fungi
including Cephalosporium aphidicola (James & Almaz
1996), Rhizopus spp., Aspergillus spp. (Petri cˇ et al.
2010), Curvularia spp. (Fernandes et al. 2003),
Cunninghamella spp. (Muhammad et al. 2005),
The biotransformation of steroids by microorgan-
isms has been studied extensively over recent dec-
ades (Agnieszka & Jadwiga 2007), with the most
important transformations being thiose catalyzed
by hydroxylases and dehydrogenases. To introduce
hydroxyl groups into the steroids structure is
very difficult by chemical synthesis except for the
17-position, which explains the extensive study of
hydroxylation of steroids by organism. Steroid
hydroxylation by microorganisms offers the possibil-
ity of obtaining both regioselective and stereoslective
reactions.
It has been reported that many 7a-hydroxylated
steroids show important activities such as upregulat-
ing immunity (Morfin 2002), improving memory
(
(
Yau et al. 2006), being neuronal activators
Matsunaga et al. 2004), and treating adolescent
obesity (M a´ cˇ ov a´ et al. 2014). Therefore, recent work
Correspondence: S. Qin and H. Liu, School of Pharmaceutical Sciences of Zhengzhou University, Zhengzhou 450001, People’s Republic of China.
Tel/Fax: +86 371 67781739. E-mail: liuhm@zzu.edu.cn (H.L); qinshangshang@126.com (S.Q.)
(Received 4 December 2014; revised 13 May 2015; accepted 31 August 2015)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2016 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.3109/10242422.2015.1089865

84
W. Shan et al.
O
O
O
1
1
7
7
HO
7
HO
O
HO
1
1
1
2
3
a 7α−ΟΗ
b 7β−ΟΗ
2a 7α−ΟΗ
2b 7α,11α−ΟΗ
3a 7α−ΟΗ
OH
O
7
7
HO
HO
5
4
4
a
7α−ΟΗ
Figure 1. Structures of the substrates (1–5) and the metabolites (1a–4a).
Trichoderma hamatum (Agnieszka & Jadwiga 2007),
and Cochliobolus lunatus (Marija et al. 1990) can
introduce hydroxyl groups into the 11-position of the
steroid skeleton.
O
O
HO
Fungi are widely used in steroid biotransform-
ations for their versatile enzymatic repertoire.
Although fungi belonging to the genus Mucor have
been applied to transform steroids (Andrew et al.
HO
6
7
Figure 2. The structures of cholesterol (6) and diosgenin (7).
2
007; Faramarzi et al. 2008; Ge et al. 2008; Wang
which are difficult to synthesize by chemical methods
(Shan et al. 2009, 2013).
et al. 2013), no studies have been carried out using
Mucor circinelloides lusitanicus to transform 5-en-3b-
ol steroids to date. M. circinelloides lusitanicus is
widely distributed in nature and has considerable
economic importance (Mar ´ı a et al. 2005; Mirosława
et al. 2006; Saha 2004; Tadeusz et al. 2002).
We have found that the fungus Mucor racemosus
could hydroxylate 5-en-3b-ol steroids at 7a, 7b, and
Materials and methods
Instrumental methods
Sterilization was carried out in an HVE-50 Hirayama
autoclave. Aseptic operations were performed in a
Class II A/B3 Biological Safety Cabinet from Forma
Scientific. Incubation was carried out on a HZQ-Q
orbital shaker. Transformations were monitored by
thin layer chromatography (TLC) on pre-coated
silica gel GF254 (0.5 mm, Qingdao Hailang,
Qingdao city, China). Components were visualized
by heating. Flash column chromatography (FCC)
was carried out on silica gel (100ꢀ200 mesh,
Shanghai, China). Infrared (IR) spectra were rec-
orded on a Thermo Nicolet 200 spectrometer using
11a-positions (Ge et al. 2008), but the yield of
the 11a-hydroxylated product was only 19.6%. Now
we have employed M. circinelloides lusitanicus to
transform 5-en-3b-ol steroids in order to explore
the potential hydroxylation utility of this fungus.
In the present study, seven 5-en-3b-ol steroids
(
1-5, shown in Figure 1 and 6–7 shown in Figure 2)
were selected as substrates. Substrates 1-5 can be
transformed to their 7a-hydroxylated derivatives. It
is interesting that the main product from 2 was 3b,
ꢁ
1
7a, 11a-trihydroxypregna-5-en-20-one (2b) which
KBr disks in the 400ꢀ4000 cm
region. Melting
points were measured on a WC-1 melting-point
apparatus and are uncorrected. proton nuclear mag-
could be easily crystallized from the mixture with a
relatively high yield (46%). This makes M. circinel-
loides lusitanicus different from its’ other congeneric
organism used in the transformation of 5-en-3b-ol
steroids (Andrew et al. 2007; Ge et al. 2008;
Madyastha & Joseph 1995, Wang et al. 2013). This
allows us to prepare some new steroid compounds
1
netic resonance ( H NMR), carbon-13 nuclear
13
magnetic resonance ( C NMR) spectra were
obtained on a Bruker DPX400 instrument with
Me Si as internal standard, and the chemical shifts
4
are given in ꢀ values. High resolution–electron spray

Biotransformation of 5-en-3ꢁ-ol steroids 85
mass spectra were recorded on a Waters Q-Tof MS
System in positive ion mode. Starting materials were
obtained commercially (Hanzhong Hanjiang-
Zhenhua Bio-Tec. LTD., Hanzhong, China).
Optical rotations were determined on a Perkin
(250 ml) containing 50 ml liquid sterile fermentation
medium and incubated on a rotary shaker at 210 rpm
ꢂ
and 30 C for 2 days. Then, 150 mg of substrate 2 was
dissolved in DMSO and added to each fermentation
flask. The fermentations were continued and 3 ml
broth of every flask harvested every 24 h over 7 days
and extracted with the same volume of ethyl acetate.
Then, the solvent was evaporated under vacuum to
give the reaction mixtures.
ꢂ
Elmer 341 polarimeter at 20 C in MeOH. Single
crystal structure was carried out on a Rigaku
R-AXIS-IV area detector.
Extracted reaction mixtures were dissolved in
CH OH (3 ml) and then analyzed by high-perform-
Microorganism
3
ance liquid chromatography (20 ml). Each analytical
determination was performed in triplicate. The
The organism used in the present study was isolated
from soil of the north suburb of Zhengzhou, People’s
Republic of China, and identified as M. circinelloides
lusitanicus by the company of Microbial ID (Newark,
NJ). The fungus was maintained on potato dextrose
mobile phase was CH OH/water (70:30, v/v) at a
3
flow rate of 1 ml/min, run over 15 min. UV detection
was determined at 205.0 nm.
ꢂ
agar (PDA) slants at 4 C.
Results
Conditions and transformation
Identification of metabolites of 1
3ꢁ, 7ꢂ-Dihydroxyandrost-5-en-17-one (1a)
Colorless crystals (crystallized from methanol); Yield:
The optimized liquid medium consisted of maltose
4
.3%, peptone 2.3%, yeast extract 0.1%, K HPO4
2
0
.9%, MgSO ꢃ7H O 0.9% and pH 6.0. Flasks (500
4
2
ml) containing 100 ml medium were inoculated with
ꢂ
4
3.6%;
mp:
ꢂ
179.3ꢀ181.2 C
(literature
ꢂ
8
2
0
spore suspension (1.0 ml, 2.2 ꢄ 10 counts) from a
1
81.5ꢀ183.5 C) (Crabb et al. 1980); ½ꢂꢅ -71
D
c.0.21, MeOH); IR (KBr)ꢃ : 3431, 2933, 1730,
2
-day-old culture grown on PDA slants and incu-
(
max
ꢁ1
ꢂ
bated at 28 C on a rotary shaker (210 rpm) for 48 h.
Then, 100 mg of a 5-en-3b-ol steroid substrate
dissolved in 1 ml acetone or dimethyl sulfoxide was
added to the flask. Incubation was continued for
1
657, 1621, 1375, 1057 cm ; H NMR (MeOD):
1
0
.89 (3H, s, H-18), 1.02 (3H, s, H-19), 3.46 (1H, m,
2
H-3), 3.89 (1H, t, H-7, J ¼ 7.2 Hz), 5.57 (1H, dd, H-
13
, J ¼ 1.4, 5.2 Hz); C NMR data (shown in Table 1);
6
4
days.
+
MS: m/z: 305.2112 [M + H] (calcd. 305.2117)
Supplement Figures HNMR-1a-1 and HNMR-1a).
(
Isolation of transformation products
3
Colorless crystals (crystallized from methanol);
ꢁ, 7ꢁ-Dihydroxyandrost-5-en-17-one (1b)
After incubation, the fermentation media and the
mycelium were extracted separately with ethyl acet-
ate. Since the mycelial extract contained only the
added substrate, it was not processed further. The
media extracts were washed three times each by
ꢂ
Yield: 12.6%; mp: 208.6ꢀ209.8 C (literature
ꢂ
20
D
ꢂ
2
15ꢀ216 C) (Crabb et al. 1980); ½ꢂꢅ -93 (c.0.29,
MeOH); IR (KBr)ꢃ : 3364, 2930, 1730, 1660,
629, 1380, 1056 cm ; H NMR (DMSO-d ): 0.78
max
ꢁ1
1
1
6
saturated NaHCO , brine, and distilled water,
3
(3H, s, H-18), 0.97 (3H, s, H-19), 3.26 (1H, m, H-
respectively and dried with anhydrous sodium sul-
fate. After evaporation of the solvent, the mixed
products were analyzed by TLC in ethyl acetate/
chloroform (1:1) and separated by silica gel
chromatography using the same solvents as eluant.
Product 2b could be obtained by recrystallizing
the extract mixture with methanol and chloroform
3
13
), 3.70 (1H, d, H-7, J ¼ 8.0 Hz), 5.18 (1H, s, H-6);
C NMR data (shown in Table 1); MS: m/z:
+
05.2115 [M + H] (calcd. 305.2117) (Supplement
3
Figures HNMR-1b-1 and HNMR-1b).
Identification of metabolites of 2
(
1:3).
3
Colorless crystals (crystallized from methanol);
ꢁ, 7ꢂ-Dihydroxypregna-5-en-20-one (2a)
Time course experiment
ꢂ
Yield: 24.4%; mp: 178.2ꢀ180.7 C (literature
ꢂ
20
180ꢀ182 C) (Baulieu et al. 2004); ½ꢂꢅ -49
ꢂ
The time course of the experiment was conducted as a
guide to the sequence of reactions. Freshly obtained
spores from peptone dextrose agar slopes were
transferred aseptically into three Erlenmeyer flasks
D
(c.0.20, MeOH); IR (KBr)ꢃ : 3420, 2935, 1698,
max
ꢁ
1
1661, 1358, 1054 cm ; H NMR (CDCl ): 0.64
1
3
(3H, s, H-18), 1.00 (3H, s, H-19), 2.14 (3H, s,

86
W. Shan et al.
1
3
ꢂ
Table I. C NMR spectral data for 1aꢀ4a (100.6 M Hz, 25 C,
Identification of metabolites of 4
TMS).
a
c
b
c
c
c
3ꢁ, 7ꢂ-Dihydroxypregna-16ꢂ, 17ꢂ-epoxy -5-en -20-
one (4a)
Colorless crystals (crystallized from methanol);
Carbon
1a
1b
2a
2b
3a
4a
1
2
3
4
5
6
7
8
9
38.6
32.1
71.9
42.9
146.8
124.6
64.9
38.6
46.4
38.0
21.2
32.4
43.8
49.6
22.7
36.6
223.9
13.7
18.6
36.8
31.3
71.3
41.9
141.6
127.1
70.0
39.3
48.2
36.3
20.2
31.7
47.3
50.9
24.2
35.7
220.5
13.4
19.0
37.0
31.6
71.3
42.2
146.3
123.7
65.2
37.5
41.9
37.4
20.7
38.2
43.8
49.7
24.4
22.9
63.5
13.0
18.2
209.6
31.3
38.4
31.6
70.2
42.8
144.6
124.3
63.5
37.1
47.8
38.5
67.2
49.1
43.6
49.5
23.7
22.6
62.8
14.1
17.7
208.7
31.3
36.3
31.2
69.6
42.0
143.9
124.2
63.4
35.4
41.5
36.8
19.8
34.1
45.2
49.5
31.7
145.7
154.2
15.4
17.7
196.1
31.3
36.9
31.5
70.2
42.5
144.4
124.7
63.6
35.8
39.4
37.4
20.2
31.7
41.2
42.1
27.2
60.8
70.8
15.2
18.2
205.3
26.2
ꢂ
Yield: 14.7%; mp: 185ꢀ186 C (literature
ꢂ
20
85ꢀ186 C) (Ge et al. 2008); ½ꢂꢅ -64 (c.0.20,
ꢂ
1
MeOH); IR (KBr)ꢃ : 3430, 2955, 1720, 1644,
D
max
ꢁ
1 1
1376 cm ; H NMR (DMSO-d ): 1.01 (3H, s, H-
6
1
8), 1.06 (3H, s, H-19), 2.08 (3H, s, H-21), 3.18
1H, m, 3-H), 3.68 (1H, s, 7-H), 4.02 (1H, s, 16-H),
(
1
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
4
5
6
7
8
9
0
1
13
5.51 (1H, d, H-6, J ¼ 4.8 Hz); C NMR data
+
(
shown in Table I); MS: m/z: 369.2036 [M + Na]
calcd. 369.2042) (Supplement Figure HNMR-4a).
(
Discussion
All products of the transformations were identified
1
13
through IR, H NMR, C NMR, and high-reso-
lution mass spectrometry analysis. The positions and
configurations of the hydroxyl group introduced
were confirmed mainly from the ꢀ changes in
NMR spectra relative to the starting material and
data reported in the literature. X-Ray diffraction
analysis was carried out to establish the axial (a)
orientation of C7 and C11 hydroxyl group in
compound 2b shown in Figure 3.
a
MeOD.
b
3
CDCl .
c
6
DMSO-d .
2
H-21), 2.59 (1H, t, H-17, J ¼ 9.2 Hz), 3.59 (1H, m,
H-3), 3.87 (1H, bs, H-7), 5.62 (1H, dd, H-6, J ¼ 1.2
1
.2 Hz); C NMR data (shown in Table I); MS: m/
3
5
+
z:
333.2424
Supplement Figures HNMR-2a).
[M + H]
(calcd.
333.2430)
To clarify whether the microorganism had the
ability to hydroxylate 5-en-3b-ol steroids, two repre-
sentative compounds, i.e. dehydroepiandrosterone
(
3ꢁ, 7ꢂ, 11ꢂ-Trihydroxypregna-5-en-20-one (2b)
Colorless needle crystals (crystallized from methanol);
(
1) and pregnenolone (2) were used as substrates. By
biotransformation, 7a, 7b, or 11a monohydroxy or
dihydroxy products were obtained. 3b, 7a-
Dihydroxyandrost-5-en-17-one (1a) and 3b, 7a,
ꢂ
Yield: 46.4%; mp: 242.8ꢀ243.8 C (literature
ꢂ
20
D
ꢂ
2
55ꢀ257 C) (Baulieu et al. 2004); ½ꢂꢅ -40.2
(
c.0.99, MeOH); IR (KBr)ꢃ : 3328, 2969, 2936,
max
1
1a-trihydroxypregna-5-en-20-one (2b) were the
ꢁ
700, 1357, 1046 cm ; H NMR (DMSO-d ): 0.50
6
1 1
1
main products of substrates 1 and 2, respectively.
These results indicated that 7a-hydroxylation was a
characteristic feature of M. circinelloides lusitanicus in
transforming 5-en-3b-ol steroids. We utilized the
microorganism to transform 3b-Hydroxypregna-5,
(
3H, s, H-18), 1.00 (3H, s, H-19), 2.07 (3H, s, H-21),
2
2
3
5
.59 (1H, t, H-17, J ¼ 9.1 Hz), 3.34 (1H, m, H-3),
.57 (1H, d, H-7, J ¼ 8.5 Hz), 3.80 (1H, m, H-11),
13
.44 (1H, d, H-6, J ¼ 5.3 Hz); C NMR data (shown
+
in Table I); MS: m/z: 371.2208 [M + Na] (calcd.
71.2198). For X-ray diffraction analysis is shown in
Figure 3(Supplement Figure HNMR-2b).
1
6-diene-20-one (3) and 3b-Hydroxypregna-16a,
7a-epoxy-5-en-20-one (4), and found that they
3
1
were also hydroxylated at the 7a-position. However,
the yields of 3a and 4a were much lower than that of
1a. The double bond and the epoxy group seemed to
Identification of metabolites of 3
hinder the activity of the hydroxylase. If androstene-
diol (5) was added to the medium of the matured
fungi, the products 1a and 1b could be obtained with
yields of 8.9% and 11.1% respectively, which were
much lower than that of 1. This revealed that M.
circinelloides lusitanicus could be induced to produce a
17-oxidase, but the complex transformation meant
the yield of 1a and 1b was very low. When choles-
terol (6) and diosgenin (7) (Figure 2) were fed to the
broth of the mature organism separately, the original
material was unchanged. We speculate that the large
3
ꢁ, 7ꢂ-Dihydroxypregna-5, 16-diene-20-one (3a)
ꢂ
White powder; Yield: 10.6%; mp: 189ꢀ191 C;
2
D
0
ꢂ
½
ꢂꢅ -74.1 (c.0.50, MeOH) (Gao et al. 2011); IR
(
KBr)ꢃ
:
3480, 2934, 1660, 1588, 1372,
max
ꢁ
055 cm ; H NMR (DMSO-d ): 0.83 (3H, s, H-
6
1 1
1
1
8), 0.93 (3H, s, H-19), 2.21 (3H, s, H-21), 3.33
(
1H, m, 3-H), 3.69 (1H, bs, 7-H), 5.43 (1H, d, H-6,
13
J ¼ 4.9 Hz), 6.91 (1H, s, 16-H); C NMR data
+
(
shown in Table I); MS: m/z: 353.2087 [M + Na]
calcd. 353.2093) (Supplement Figure HNMR-3a).
(

Biotransformation of 5-en-3ꢁ-ol steroids 87
Figure 3. The X-ray diffraction analysis diagram of 2b.
and the medium components to improve the yield of
b. With the optimized conditions, 2b could be
2
obtained in much higher yield (46.4%) than reported
for M. racemosus (19.6%) (Ge et al. 2008) and
M. piriformis (35.0%) (Madyastha & Joseph 1995).
The time course of the biotransformation of
pregnenolone (2) was also studied (shown in
Figure 4). From the time course, we concluded
that the 7a-hydroxylation took place rapidly within
2
4 h, then the 11a-hydroxyl group was introduced
subsequently. After 36 h, the concentration of
monohydroxylated metabolite had declined and the
dihydroxylated metabolite accumulated to a max-
imum at 48 h then slightly declined to around 46% at
9
6ꢀ108 h. Feeding 2a to mature fungi, produced 2b
in the fermentation broth after 12h, further confirm-
ing that 2a is an intermediate and 2b is the finally
product.
Figure 4. Time course for the biotransformation of pregnenolone
2) by Mucor circinelloides lusitanicus.
(
It is known that compound 1 is a major hormone
in human nerve tissues, and its’ therapeutic role in
repairing neurons is well documented. Recent
studies indicated that hydroxylated metabolites of 1
have many profound activities (Matsunaga et al.
2004; Yau et al. 2006). So, using M. circinelloides
lusitanicus, we have generated some structural modi-
fication and evaluated bioactivity, which showed that
some of the benzylidene derivatives of 1a and 2b
have remarkable activity against EC109 cells (Shan
et al. 2009, 2013). This needs to be further studied.
group at C17 in the structures of 6 and 7 made them
unsuitable for the active site of the hydroxylase (Peart
et al. 2011). Some investigations have reported that
some Mucor sp. can isomerize 5-en-3-ol steroids to
4-en-3-one structures or cleave the C17 side-chain.
M. circinelloides lusitanicus appears to lack the isom-
erase and could not cleave the side-chain of the
selected substrates 2–4 and 6-7 (Hunter et al. 2009).
The ability of M. circinelloides lusitanicus to trans-
form 5-en-3b-ol steroids is similar to M. racemosus
and Mucor piriformis, but M. circinelloides lusitanicus is
more useful in transforming 2. Substrates 1 and 3–5
could not be dihydroxylated over the same trans-
forming period, which revealed that the specific
structure of 2 was more suitable for the hydroxylase
of M. circinelloides lusitanicus. The product 2b is not
commercially available, hindering further studies on
structural modification and evaluation of activity. So
we optimized the original transformation conditions
Declaration of interest
The authors report no declarations of interest. The
authors alone are responsible for the content and
writing of the paper.
Authors express their appreciation to the company
of Microbial ID (USA) for the identification of the
fungus. This research work was supported by
National Natural Science Foundations of China

88
W. Shan et al.
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