Microbial hydroxylation of 17β-estradiol by Penicillium brevicompactum

Article abstract of DOI:10.1080/10242422.2016.1247816

Microbial hydroxylation of 17β-estradiol (1) with Penicillium brevicompactum, a fungal species not used in biotransformation so far, yielded four metabolites: 1, 3, 5-estratriene-3, 15α-diol-17-one (2); 1, 3, 5-estratriene-3, 6α, 17β-triol (3); 1, 3, 5-estratriene-3, 15α, 17β-triol (4); and 1, 3, 5-estratriene-3, 6α, 15α-triol-17-one (5). All the products were determined by ¹H NMR, ¹³C NMR, two-dimensional NMR, and HRMS techniques. Compounds 3, 4, and 5 are reported for the first time via microbial transformation, and 5 is a new compound as far as we know. Possible metabolic pathway of 17β-estradiol via Penicillium brevicompactum was also proposed.

Full text of DOI:10.1080/10242422.2016.1247816

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Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Microbial hydroxylation of 17β-estradiol by
Penicillium brevicompactum
Lihong Shan, Yang Li, Yanjie Chen, Minghui Yin, Jiajia Huang, Zhenzhong
Zhang, Xiufang Shi & Hongmin Liu
To cite this article: Lihong Shan, Yang Li, Yanjie Chen, Minghui Yin, Jiajia Huang,
Zhenzhong Zhang, Xiufang Shi & Hongmin Liu (2016): Microbial hydroxylation of 17β-
estradiol by Penicillium brevicompactum, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2016.1247816
To link to this article: http://dx.doi.org/10.1080/10242422.2016.1247816
Published online: 18 Nov 2016.
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Date: 18 November 2016, At: 05:07

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Biocatalysis and Biotransformation, 2016; Early Online: 1–7
REVIEW ARTICLE
Microbial hydroxylation of 17b-estradiol by Penicillium
brevicompactum
LIHONG SHAN1,2, YANG LI3, YANJIE CHEN1,2, MINGHUI YIN1,2, JIAJIA HUANG1,2
ZHENZHONG ZHANG1,2, XIUFANG SHI1,2 & HONGMIN LIU1,2
,
2
1School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, PR China, Collaborative Innovation
3
Center of New Drug Research and Safety Evaluation, Zhengzhou, PR China, and The People’ Hospital of
Jiaozuo City, Jiaozuo, PR China
Abstract
Microbial hydroxylation of 17b-estradiol (1) with Penicillium brevicompactum, a fungal species not used in biotransformation
so far, yielded four metabolites: 1, 3, 5-estratriene-3, 15a-diol-17-one (2); 1, 3, 5-estratriene-3, 6a, 17b-triol (3); 1, 3, 5-
estratriene-3, 15a, 17b-triol (4); and 1, 3, 5-estratriene-3, 6a, 15a-triol-17-one (5). All the products were determined by 1H
NMR, 13C NMR, two-dimensional NMR, and HRMS techniques. Compounds 3, 4, and 5 are reported for the first time via
microbial transformation, and 5 is a new compound as far as we know. Possible metabolic pathway of 17b-estradiol via
Penicillium brevicompactum was also proposed.
Keywords: Biotransformation, 17ꢀ-estradiol, hydroxylation, Penicillium brevicompactum
Introduction
2-hydroxyestradiol and 4-hydroxyestradiol possess-
ing putative carcinogenic metabolites (Li et al.
2014). The CYP-450 isoforms are largely respon-
sible for the hydroxylation of estradiol in human
body (Federico et al. 1994; Anthony et al. 2003). So
far, microbial hydroxylation of 17b-estradiol mainly
appears in 2, 4, 6b-, 7a-, 7b-, 15a-, and 16a-
positions and some metabolites have certain bio-
logical activities (Laskin et al. 1964; Koshimura et al.
2010; Kurisu et al. 2010; Hussain et al. 2011). In
continuation of the work with microbial transform-
ation of steroids (Ge et al. 2008; Shan et al. 2009),
we screened local soil samples and identified a fungal
strain as Penicillium brevicompactum through 16S
rRNA sequencing with the help of Microbial ID
(Newark, DE). As far as we knew, the Penicillium sp.
used in the biotransformation of steroid mainly
resulted in the metabolism of D ring d-lactones
(Bartman´ska et al. 2005; Liu et al. 2006; Huang et al.
2010; Swizdor 2013; Yang et al. 2014), occasionally
resulted in hydrogenation (Miller and Hessler 1970;
Yildirim et al. 2010), side chain degradation
Microorganisms have been widely applied for bio-
transformation of steroids in order to obtain more
functionalized compounds due to high regio- and
stereoselectivity of these reactions (Wang et al.
2013). In particular, microbial hydroxylation of
steroids provides a useful mild synthetic method for
´
obtaining access to rare steroids (Bartmanska et al.
2005). Meanwhile, hydroxylation products of ster-
oids are important due to their physiological role in
mammalian organisms (Li and Bigelow 2010; Bhatti
and Khera 2012). Every site in the steroid molecule
is accessible for microbial hydroxylation and various
microbial strains may introduce hydroxyl group at
the same position in a steroid molecule.
Estrogens are gonadal steroidal hormones that
have important roles in reproduction (Salci and
Biryol 2002). 17b-Estradiol is the most potent and
naturally occurring estrogen in human beings fol-
lowed by estrone and estriol, and is the main isomer
that activates the estrogen receptor (Guo et al. 2010).
The hydroxylation of b-estradiol has been studied for
Correspondence Xiufang Shi; Hongmin Liu, School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Ke Xue Da Dao, Zhengzhou 450001, PR
China. Tel/fax: +86 371 67781739. E-mail: liuhm@zzu.edu.cn; xfshi@zzu.edu.cn
(Received 21 July 2015; revised 8 January 2016; accepted 18 August 2016)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2016 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2016.1247816

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2
L. Shan et al.
urban area of Zhengzhou city and identified by
morphologic observation and 16s rRNA sequencing.
The fungus was maintained on agar slopes composed
of peptone (1.2%), dextrose (3.0%), KH2PO4
(0.09%), yeast extract (0.1%), agar (3.0%), and
distilled water (pH 4.5) at 4 ꢂC and freshly sub-
cultured before using in the transformation experi-
ments. Liquid culture medium contained peptone
(1.2%), dextrose (3.0%), KH2PO4 (0.09%), yeast
extract (0.1%), and distilled water at pH 4.5.
´
(Bartmanska et al. 2005). The hydroxylation reaction
was also reported, which frequently occurs at 1a
(Tweit et al. 1962; Liu et al. 2011), 15a (Irrgang et al.
´
1992; Bartmanska et al. 2005), 15b, 11a (Capek and
Hanc 1962), and 12b-positions (El-Tayeb et al.
1964). But according to our view, the microbial
hydroxylation of 17b-estradiol by Penicillium brevi-
compactum has not been reported so far. In this paper,
the biotransformation mode of 17b-estradiol by
P. brevicompactum was studied, and four metabolites
were identified, which were mono- or dihydroxyl
products in C-6a, C-15a, and sometimes accompa-
nied with oxidization at C-17 position. Metabolite of
1, 3, 5-estratriene-3, 6a, 15a-triol-17-one (5) is
reported for the first time. The biotransformation
pathway of 17b-estradiol by P. brevicompactum to
main products is also discussed.
Culture conditions. For screening scale biotrans-
formation of 17b-estradiol, spores freshly obtained
from peptone dextrose agar slopes were transferred
aseptically into 250 mL Erlenmeyer flasks containing
50 mL of liquid sterile culture medium
(2 ꢁ 108 spores/mL) and were incubated on rotary
shakers for 2 days, operating at 210 r/min and 30 ꢂC.
Then, the substrate was diluted with acetone with a
concentration of 100 g/L, followed by 0.5 mL of the
solution added into every flask. The cultures were
continued and 3 mL of broth of every flask harvested
respectively every 24 h within 7 days was extracted
with the same volume of ethyl acetate, respectively.
Then, the solvent was evaporated under reduced
pressure to give reaction mixtures for TLC analysis.
For preparative scale of biotransformation of 17b-
estradiol by P. brevicompactum, spores freshly
obtained from peptone dextrose agar slopes were
transferred aseptically into every 500 mL Erlenmeyer
flasks with 100 mL of liquid sterile culture medium.
After 2 days incubation at 210 r/min and 30 ꢂC,
1500 mg of 17b-estradiol (1) dissolved in 15 mL of
acetone was evenly added to these 15 flasks. Further
culture lasted for 5 days, and the mycelium was
separated from the broth by filtration under vacuum,
and rinsed in ultrasonic with ethyl acetate
(3 ꢁ 200 mL) to ensure that all of the available
products were isolated from the mycelium. The
mycelial broth was extracted with the same volume
of ethyl acetate for three times. The organic extract
was combined, concentrated into about 200 mL, and
then washed with saturated aqueous NaHCO3, brine
and distilled water respectively for three times.
Subsequently, the organic extract dried over anhyd-
rous sodium sulfate and was concentrated in vacuum
to give crude extract.
Experiments
Instruments and materials
Sterilization was carried out in HVE-50 Hirayama
autoclave. Aseptic operation was carried out in Class
II A/B3 biological safety cabinet from Forma Scientific
(Marietta, OH). Incubation was carried out on HZQ-
Q orbital shaker. Thin layer chromatographic (TLC)
plates was visualized under ultraviolet light and by
spraying with ethanol–phosphomolybdic acid (20%)
reagent, followed by heating for color development.
Purifications were carried out by column chromatog-
raphy using silica gel (200–300 mesh) as the stationary
phase. Melting points (Mps) were measured on a
WC-1 melting-point apparatus and were uncor-
rected. Infrared (IR) spectra were recorded on a
Thermo Nicolet 200 spectrometer using KBr disks in
the 400–4000 cmꢀ1 region. High Resolution-Electron
Spray Mass Spectra (HR-EMS) were recorded on a
Waters Q-Tof MS System (Milford, MA) in positive
ion mode. The H and 13C nuclear magnetic reson-
1
ance (NMR) spectra were obtained using a Bruker
Avance DPX-400 spectrometer (Billerica, MA) at
400 and 100 MHz, respectively. Compounds were
dissolved in DMSO-d6 with tetramethylsilane (TMS)
as internal standard. High performance liquid chro-
matography (HPLC) analysis was performed on a
Waters HPLC system and the column was
Phenomenex C18 column (250 ꢁ 4.60 mm, 5 mm).
17b-Estradiol was purchased from Zhejiang Xianju
Pharmaceutical Co., Ltd. (Taizhou, China). Solvents
were analytical grade.
Time course of substrate and metabolic products
A time course experiment was conducted in order to
determine the metabolic pathway. Conditions were
identical to the screening scale biotransformation
experiment. The flask was harvested every 24 h within
6 days. Reaction mixtures extracted were dissolved in
CH3OH (3 mL), 20 mL of which was then analyzed by
Microorganism and culture mediums
The fungal strain Penicillium brevicompactum was
isolated from several collected soil samples of the

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Microbial hydroxylation of 17ꢀ-estradiol
3
HPLC. Each analytical determination was performed
in triplicate. The mobile phases were CH3CN/water
gradient (26:74, 55:45, v/v) over 30 min, where the
mobile phases were held for 16 and 14 min, respect-
ively. The flow rate was 1.0 mL/min and the detection
wavelength was 205 nm.
(Burrows et al. 1973); IR (KBr) ꢁmax: 3358 (br),
2930, 1659, 1612, 1584, 1501, 1453, 1376, 1346,
1252, 1167, 1134, 1050,1013, 969, 866, 820, 796,
774, 623 cmꢀ1; 1H NMR (DMSO-d6) ꢂ (ppm): 7.06
(1H, d, J¼ 8.5 Hz, H-1), 6.73 (1H, d, J¼ 2.6 Hz, H-
4), 6.61 (1H, dd, J¼ 8.4, 2.6 Hz, H-2), 4.47 (1H, t,
J¼ 4.9 Hz, H-6b), 3.56–3.50 (tt, J¼ 9.2, 4.6 Hz, H-
17a), 0.69 (3H, s, H-18); 13C NMR data (shown in
Table 1); MS: m/z: 271.1697 [M ꢀ H2O + H]+
(calcd. 271.1698).
Product isolation and analysis
The crude extract (1570 mg) was subjected to an
open silica gel column and eluted with chloroform/
methanol gradient (chloroform/methanol, 30:1,
20:1, 10:1, v/v). Purification of the crude extract
using chloroform/methanol (30:1, v/v) afforded
compound 2 (321 mg). Elution with chloroform/
methanol (20:1, v/v) gave two compounds, 3
(267 mg) and 4 (360 mg) sequentially. From chloro-
form/methanol (10:1, v/v) elution, compound 5
(310 mg) was obtained. All the metabolites were
identified by Mps and a combination of IR, MS, and
two-dimensional NMR. The solvents used for prod-
ucts crystallization was ethyl acetate/petroleum ether
(5:1, v/v).
1, 3, 5-Estratriene-3, 15a, 17b-triol (4), white
powder, Yield: 21.6%; mp: 225.4–230.9 ꢂC (248–
250 ꢂC) (Laskin and Fried 1964); IR (KBr) ꢁmax
:
3346 (br), 2926, 2868, 1609, 1588, 1502, 1445,
1240, 1135, 1054, 966, 929, 871, 786, 619,
;
567 cmꢀ1 1H NMR (DMSO-d6) ꢂ (ppm): 7.04
(1H, d, J¼ 8.5 Hz, H-1), 6.50 (1H, dd, J¼ 8.4,
2.6 Hz, H-2), 6.43 (1H, d, J¼ 2.5 Hz, H-4), 3.96–
3.81 (1H, m, H-15b), 3.70–3.66 (1H, dd, J¼ 8.6,
6.1 Hz, H-17a), 0.66 (3H, s, H-18); 13C NMR data
(shown in Table 1); MS: m/z: 271.1700
[M ꢀ H2O + H]+ (calcd.271.1698).
1, 3, 5-Estratriene-3, 6a, 15a-triol-17-one (5),
colorless needles; Yield: 17.8%; mp: 217–219 ꢂC; IR
(KBr) ꢁmax: 3373 (br), 2929, 1727, 1612, 1502,
1453, 1379, 1348, 1288, 1249, 1053, 1019, 971,
1, 3, 5-Estratriene-3, 15a-diol-17-one (2), white
powder; Yield: 19.4%; mp: 240–242 ꢂC (226–
230 ꢂC) (Laskin and Fried 1964); IR (KBr) ꢁmax
:
1
864, 824, 750, 626 cmꢀ1; H NMR (DMSO-d6) ꢂ
3369, 2924, 1726, 1619, 1508, 1448, 1286, 1241,
1054, 1017, 980, 852, 819, 747, 623 cmꢀ1; 1H NMR
(DMSO-d6) ꢂ (ppm): 7.05 (1H, d, J¼ 8.5 Hz, H-1),
6.52 (1H, dd, J¼ 8.4, 2.6 Hz, H-2), 6.45 (1H, d,
J¼ 2.5 Hz, H-4), 4.29–4.22 (td, J¼ 14.3, 6.6 Hz, H-
15b), 0.85 (3H, s, H-18); 13C NMR data (shown in
Table 1); MS: m/z: 269.1538 [M ꢀ H2O + H]+
(calcd. 269.1542).
(ppm): 7.07 (1H, d, J¼ 8.5 Hz, H-1), 6.72 (1H, d,
J¼ 2.5 Hz, H-4), 6.62 (1H, dd, J¼ 8.4, 2.7 Hz, H-2),
4.48(1H, s, H-6b), 4.26–4.19 (1H, m, H-15b), 0.87
(3H, s, H-18); 13C NMR data (shown in Table 1);
MS: m/z: 285.1490 [M ꢀ H2O + H]+ (calcd.
285.1491).
1, 3, 5-Estratriene-3, 6a, 17b-triol (3), white pow-
der; Yield: 16.1%; mp: 240–242 ꢂC (230–235 ꢂC)
Results and discussions
The fungus of Penicillium brevicompactum was
screened from the north-east of the Zhengzhou
urban area for its potential biotransformation ability
of steroid substrates. The PCR product was
sequenced and BLAST analysis of 16S rRNA gene
was performed. Sequences having maximum simila-
rities were retrieved, aligned with that of isolate
C28030-1-4-1. The alignment was subjected to
neighbor-joining analysis indicated the fungus as
Penicillium brevicompactum (shown in Figure 1).
When 17b-estradiol was fed to the mature myce-
lium of P. brevicompactum, four new plots were
observed on the TLC plate, and thus was selected for
scale up study. The isolate mixture after 4 days
incubation with 17b-estradiol yielded four metabol-
ites 2–5, and the structure of the transformation
products is shown in Figure 2.
Table 1. 13C NMR data for compounds 1–5 determined in
DMSO-d6 (ꢂ ppm).
Compounds
Carbon atom
1
2
3
4
5
1
2
3
4
5
6
7
8
126.5
113.2
155.3
115.4
137.6
29.6
27.4
39.2
44.3
130.9
26.6
37.1
43.3
50.0
23.3
30.4
80.5
11.7
126.7
113.3
155.4
115.4
137.7
29.8
27.6
38.8
44.1
130.3
26.2
32.0
50.5
56.7
68.8
46.5
217.0
15.5
125.9
114.8
155.5
116.7
140.6
66.0
33.2
37.1
44.0
130.9
26.2
36.9
43.5
49.8
23.2
30.4
80.6
11.8
126.7
113.2
155.3
115.3
137.8
29.9
27.9
39.4
44.5
130.8
26.7
37.5
44.3
57.5
71.1
43.1
78.0
13.3
125.7
114.4
155.0
116.3
140.0
65.4
31.3
32.0
43.4
129.7
25.2
36.3
50.0
55.9
68.3
46.0
216.4
15.0
9
10
11
12
13
14
15
16
17
18
Product 2, Rf¼0.43 (chloroform/methanol ¼30:1,
v/v), was obtained as white powder with 19.4% yield