Microbial biotransformation of water-insoluble herbimycin A to 11-hydroxy-(11-demethoxy)-herbimycin C by Eupenicillium sp. SD017

Article abstract of DOI:10.1016/j.molcatb.2009.09.007

Herbimycin A, a typically water-insoluble anti-tumor drug and an analog of geldanamycin, was transformed by fungus Eupenicillium sp. SD017. The water solubility and anti-tumor activity of converted products were evaluated after separation, purification, a

Full text of DOI:10.1016/j.molcatb.2009.09.007

Journal of Molecular Catalysis B: Enzymatic 62 (2010) 76–80
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Microbial biotransformation of water-insoluble herbimycin A to
11-hydroxy-(11-demethoxy)-herbimycin C by Eupenicillium sp. SD017
Lianwu Xiea,b,∗, Guanghua Wangb, Hua Zhangc, Yongchang Ouyangb, Wei Sunb, Xiang Lib
a School of Sciences, Central South University of Forestry & Technology, 498 South Shaoshan Road, Changsha, Hunan 410004, PR China
b LMB-CAS, LMM-GD, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China
c New Drug Research & Development Center of North China Pharmaceutical Group Corporation, National Microbial Medicine Engineering & Research Center,
Shijiazhuang 050015, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2009
Received in revised form
10 September 2009
Accepted 10 September 2009
Available online 22 September 2009
Herbimycin A, a typically water-insoluble anti-tumor drug and an analog of geldanamycin, was trans-
formed by fungus Eupenicillium sp. SD017. The water solubility and anti-tumor activity of converted
products were evaluated after separation, purification, and structure elucidation. The results showed
that herbimycin A can be converted into herbimycin C 1 and 11-hydroxy-(11-demethoxy)-herbimycin
C 2 with water solubility increased. The latter is a novel compound, and its IC50 to A549, MCF-7, Ehrlich
and HeLa cells is 152.9, 0.46, 10.7 and 109.3 ␮g/ml, respectively, which are nearly in the range of those
between herbimycin A and herbimycin C. It is suggested that an O-demethylase or/and methyltransferase
may play an important role on biotransformation in this case. This study will firstly serve as a fundamen-
tal methodology for further structural alteration and functional improvement of the water-insoluble
anti-tumor drugs using microbial resources.
Keywords:
Herbimycin A
Biotransformation
Water-insoluble anti-tumor drug
O-Demethylase
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
has pointed to the need for more water-soluble and less hap-
atotoxic form of these drugs [10]. The apparent hepatoxicity of
The benzoquinonoid ansamycin, herbimycins including her-
bimycin A, B and C, as geldanamycin analogs, had been firstly
isolated from the fermentation broth of Streptomyces hygroscop-
icus AM-3672 by Omura and his colleagues from 1978 to 1986
[1–3]. Herbimycin possess a wide range of promising bioactivi-
ties, including herbicidal effect and the inhibition of radial tumor,
bacterial and fungal cell growth [4–6]. The reported IC50 values
of herbimycin and other geldanamycin derivates against several
tumor cell lines range from 1 ng/ml to 30 ␮g/ml, such as IC50 val-
ues of herbimycin A, B and C are 3.5 ␮g/ml, 3.2 ␮g/ml and 7.3 ␮g/ml
to HeLa cells, and 0.4 ␮g/ml, 0.8 ␮g/ml and 1.2 ␮g/ml to Ehrlich
cells, respectively [2]. Herbimycin A (Fig. 1), like geldanamycin and
macbecin, has been observed to reverse the morphology of can-
cer cells by reducing kinase activity and cellular phophotyrosine
content of various tyrosine kinases and receptors coding onco-
genes [7]. And now 17-allylamino-17-demethoxygeldanamycin
(17-AAG), one of geldanamycin derivates, is currently undergo-
ing phaseland II clinical trials [8,9]. However, experience with
the behavior of geldanamycin, 17-AAG in animals and humans
water-insoluble geldanamycin deviates could be attributed to the
reactivity of the quinine ring with cellular nucleophiles, low tar-
get binding affinity, and degradation of the molecular into a toxic
metabolite [11]. So, it is crucially necessary to modify the chemi-
cal structure of water-insoluble drug into more water-soluble and
less toxic derivate with high performance. Biotransformation is
an important tool in the structural modification of organic com-
pounds, such as natural products, due to its significant regio- and
stereo-selectivities [12,13] which is usually difficult to achieve by
chemical means. In this paper, we report the successfully microbial
biotransformation of herbimycin A into products with enhanced
bioactivity and increased polarity by the fungus cultures of Eupeni-
cillium sp. SD017, which can produce several bioactive products like
curvularin derivates we described before [14].
2. Materials and methods
2.1. General experimental procedures
Melting points were determined on a XRS-1 digital-melting
point apparatus, and the values were uncorrected. The 1H and
13C NMR data were collected on a Bruker AVANCE-500 (Bruker,
Switzerland) spectrometer at 500 MHz, and the chemical shifts
were recorded in ı (ppm) relative to Si(Me)4 with coupling con-
stants J in Hz. Electron ionization-mass spectrometry (EI-MS)
∗
Corresponding author at: School of Sciences, Central South University of
Forestry & Technology, 498 South Shaoshan Road, Changsha, Hunan 410004, PR
China. Tel.: +86 731 85623648; fax: +86 731 85623648.
E-mail address: xiecsu@126.com (L. Xie).
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.09.007

L. Xie et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 76–80
77
Fig. 1. The reaction formula for microbial biotransformation of herbimycin A by Eupenicillium sp. SD017.
was performed using a MAT95XP mass spectrometer (Thermo,
USA).
K2HPO4, 2 g NaCl, 0.3 g casein, 0.05 g MgSO4·7H2O, 0.02 g CaCO3,
0.01 g FeSO4·7H2O, and 18 g agar in 500 ml sterile distilled water
premixed with 500 ml filtered sea water.
2.2. Chemicals
The germination and growth of the marine fungal isolate was
undertaken on the Gauze’s No. 1 sea water medium (GSW), which
was composed of 20 g soluble starch, 1 g KNO3, 0.5 g K2HPO4,
0.5 g MgSO4·7H2O, 0.5 g NaCl, 0.01 g FeSO4·7H2O, and 30 g agar
in 500 ml sterile distilled water premixed with 500 ml filtered
sea water. All above inorganic chemicals were from Guangzhou
Chemical Reagent Factory, and the organic reagents from Amresco
Inc.
Herbimycin A was provided by New Drug Research & Devel-
opment Center of North China Pharmaceutical Group Corporation.
Silica gel (100–200 mesh) for open column chromatography was
produced by the Qingdao Marine Chemical Factory (Qingdao,
China). SephadexTM LH-20 was from GE Healthcare (GE, USA, made
in Sweden). All other chemicals used in this study were of analytical
grade.
2.3. Tumor cell lines
2.6. Preparative HPLC conditions
The anti-tumor screening for purified biotransformation prod-
The purification of converted products was mainly performed on
a HITCHI L-2000 preparative high performance liquid chromatog-
raphy (HITCHI, Japan) with one YMC semi-preparative ODS column
(5 mm, ˚ 10 mm × 250 mm) and MeOH–H2O solution as the mobile
phase. The flow rate was 1.5 ml/min, and the detection wavelength
was 271 nm.
ucts involved
4 types of tumor cell including human lung
adenocarcinoma epithelial cell line (A549), human Henrietta Lacks
cervical cancer cell line (HeLa), mice Ehrlich ascites carcinoma cell
line (Ehrlich) and human breast adenocarcinoma cell line (MCF-7).
2.4. Isolation and identification of strain Eupenicillium sp.
2.7. Biotransformation procedure
The fungal isolate was obtained by serial dilution method [15]
from sponge samples collected from South China Sea (18◦ 13ꢀ N;
109◦ 29ꢀ E) near Sanya, Hainan, China in July, 2005. The sponge
sample was later identified as Axinella sp. by Dr. K.J. Lee (Depart-
ment of Biology, Hannam University, 133 Ojungdong, Daedukgu,
Daejeon, Korea) through personal communication. The bioactive
fungal strain was identified to be Eupenicillium sp. by comparing its
morphological features with the reference description [16] and by
online BLAST analysis of its 18S rDNA sequence with those submit-
ted sequences on GenBank database.
The fresh mycelium grew on GSW agar at 28 ◦C for 5 days was
inoculated into 500 ml Erlenmeyer flasks containing 150 ml GSW
medium. After 2 days of incubation at 28 ◦C on rotary shaker at
200 rpm, a 10 ml seed culture liquid was transferred into each
500 ml Erlenmeyer flask containing 150 ml GSW medium. After 5
days of incubation, a total amount of 200 mg of herbimycin A dis-
solved in 20 ml of DMSO was distributed equally into 40 flasks.
The incubation was allowed to continue for additional 5 days on
the shaker. The cultures were then pooled and filtered in vacuo.
The filtrate was extracted with 6 L of ethyl acetate for three times.
The organic extract was evaporated to dryness in a rotary evapo-
rator under reduced pressure at 50 ◦C to yield 1.3 g of a brownish
residue.
2.5. Culture media
The strain isolation was performed on the starch casein KNO3
Agar (SCKA) medium composed of 20 g starch, 2 g KNO3, 2 g

78
L. Xie et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 76–80
Table 1
NMR spectral data for biotransformation compound 1 and 2.
Carbon no.
Herbimycin A
13C
Compound 1
Compound 2
1H
13C
1H
13C
1H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
144.6
78.7
36.7
34.0
83.4
82.3
34.1
130.1
131.6
79.2
–
144.8
79.2
32.3
34.0
83.5
72.9
29.7
132.8
131.8
79.4
–
144.8
79.2
32.0
34.0
83.5
72.7
34.1
131.5
131.6
82.4
–
4.50 (1H, br s)
1.64 (1H, m)
1.75 (2H, m)
3.48 (1H, m)
5.50 1H, br s)
2.64 (1H, br s)
5.65 (1H, br s)
–
5.34 (1H, br s)
4.48 (1H, s)
5.86 (1H, t)
6.50 (1H, dd)
6.97 (1H, br d)
–
8.60 (1H, s)
–
7.34 (1H, d)
–
6.63 (1H, s)
–
4.50 (1H, br s)
1.65 (1H, m)
1.76 (2H, m)
3.53 (1H, m)
3.50 (1H, m)
2.62 (1H, br s)
5.67 (1H,br s)
–
5.33 (1H, s)
4.51 (1H, s)
5.87 (1H, t)
6.51 (1H, dd)
6.98 (1H, br d)
–
8.81 (1H, s)
–
7.35 (1H, d)
–
6.64 (1H, s)
–
3.35 (3H, s)
0.82 (3H, d)
3.32 (3H, s)
–
4.49 (1H, br s)
1.65 (1H, m)
1.75 (2H, br s)
3.53 (1H, m)
3.48 (1H, m)
2.64 (1H, br s)
5.65 (1H, br s)
–
5.34 (1H, s)
4.50 (1H, s)
5.86 (1H, t)
6.50 (1H, dd)
6.98 (1H, br d)
–
8.80 (1H, s)
–
7.34 (1H, d)
–
6.63 (1H, s)
–
3.35 (3H, s)
0.82 (3H, d)
3.31 (3H, s)
–
78.3
78.6
66.6
136.7
125.6
128.3
134.5
168.7
138.2
112.9
187.7
132.6
183.9
59.8
136.8
125.7
128.3
134.7
168.2
138.3
113.1
187.9
132.8
184.2
58.5
136.8
125.7
128.3
134.7
168.7
138.3
113.1
187.9
132.7
184.1
58.4
18
19
20
21
22
2-OCH3
3-CH3
5-OCH3
6-OCH3
6-OH
7-CH3
9-CH3
10-OCONH2
11-OCH3
11-OH
15-CH3
3.36 (3H, s)
0.81 (3H, d)
3.31 (3H, s)
3.53 (3H, s)
–
1.08 (3H, d)
1.65 (3H, br s)
–
13.6
57.6
58.4
13.7
57.7
–
13.6
57.7
–
2.63 (1H, s)
1.09 (3H, d)
1.63 (3H, br s)
–
3.35 (3H, s)
–
2.64 (1H, br s)
1.09(3H, d)
1.65 (3H, br s)
–
16.3
14.1
155.9
56.0
–
14.2
12.4
155.8
56.0
–
16.6
14.2
155.8
–
–
12.4
3.34 (3H, s)
–
2.03 (3H, s)
–
2.64 (1H br s)
2.01 (3H, s)
12.4
12.6
2.02 (3H, br s)
2.8. Separation and purification of biotransformation products
optimal temperature for growth is 28 ◦C. Eupenicillium sp. SD017
(CGMCC 3.5706) was later preserved in the China General Microbi-
ological Culture Collection Center.
As we described elsewhere [17], the obtained residue was first
fractionated by silica gel open column chromatography and eluted
with CHCl3–MeOH (95:5–75:25, v/v) to afford six fractions. Frac-
tion II was subjected to preparative HPLC eluted with MeOH–H2O
(65:35, v/v) and recrystallized from MeOH to yield 17.2 mg of
compound 1 as a yellowish amorphous powder. Fraction IV was
separated by preparative HPLC eluted with MeOH–H2O (55:45, v/v)
and further purified on a Sephadex LH-20 column (CHCl3–MeOH,
1:1) and then recrystallized from MeOH to yield 15.4 mg of com-
pound 2 as a yellowish amorphous powder. Purities of two products
were over 95%, determined by HPLC with UV detector.
During analysis of the incubation mixture added with her-
bimycin A, two new peaks with bigger area were observed in
HPLC, which were not found in control tests. One peak showed UV
absorption maxima at 269 nm and the other at 271 nm, which is
characteristic adsorption wavelength for the benzoquinone-ringed
ansamycin. Thus, they should be the biotransformed products of
herbimycin A. In the preparative biotransformation, a total amount
of 200 mg of herbimycin A was fed to the microbial cultures. After
5 days incubation, the products were obtained by extracting with
ethyl acetate for three times. The obtained extract was subjected to
silica gel open column chromatography, Sephadex LH-20 column
chromatography and preparative HPLC to afford two pure prod-
ucts. The products were structurally characterized by comparing
their IR, EI-MS data and NMR spectra including 1H NMR, 13C NMR,
DEPT, HMQC, HMBC, 1H–1H COSY and NOESY with the substrate
and related compounds. Compound 1 is yellowish amorphous pow-
der, [˛]2D5 + 210.4 (c 0.5, CHCl3); IR ꢀmax (KBr): 3530, 3410, 3350,
1730, 1695, 1660, 1645, 1600, 1075 cm−1; EI-MS (m/z): 583 (M+Na,
56), 562 (M+2, 9), 560 (M+, 5), 517 (100). Compound 2 is yellow-
ish amorphous powder, [˛]2D5 + 231.6 (c 0.5, CHCl3); IR ꢀmax (KBr):
3534, 3410, 3350, 1725, 1690, 1655, 1645, 1605, 1082 cm−1; EI-BMS
(m/z): 569 (M+Na, 34), 548 (M+2, 17), 546 (M+, 9), 503 (100). The
NMR data of compound 1 and 2 with substrate are given in Table 1.
The results showed that the molecular backbone of products was
not destroyed but only demethylation occurred on C-6 and/or C-
11. Thereby, these two products were structurally identified as
herbimycin C 1 and 11-hydroxy-(11-demethoxy)-herbimycin C 2
(Fig. 1), and the latter is a novel compound.
2.9. Bioassay
Using above anti-tumor screening cells, IC50 values of com-
pound 1 and 2 (in DMSO) against the 4 types of tumor cell lines
was tested in triplicate according to the MTT method described in
the literature [18] with DMSO as a negative control and taxolTM as
a positive control. And the bioassay was carried out at the Institute
of Nutrisciences and Health, National Research Council of Canada.
3. Results and discussion
In screening of the most active strain for biotransformation
of herbimycin A, it was found that herbimycin A was completely
metabolized in the cultures of one isolate SD017 from marine
sponge Axinella sp. collected in South China Sea near Sanya, later
identified as fungus Eupenicillium sp. Its main morphological char-
acteristics are its tough, dense penicilli bearing long, broad columns
of conidia, and its smooth-walled, unflanged ascospores which are
produced within 14 days of inoculation onto GSW medium, the
In optimizing the conditions for biotransformation, the growth
curve for Eupenicillium sp. SD017 cultures was recorded (data not

L. Xie et al. / Journal of Molecular Catalysis B: Enzymatic 62 (2010) 76–80
79
Table 2
The IC50 values (in ␮g/ml) of biotransformed products against tumor cell lines.
Compounds
Tumor cell lines
A549a
MCF-7
Ehrlich
HeLa
83.6 3.8
153.8 7.3
109.3 10.8
7.4 0.4
Herbimycin A
Compound 1
Compound 2
Taxol
90.4 4.9b
219.3 16.7
152.9 11.9
4.5 0.4
0.03 0.001
0.87 0.06
0.46 0.03
3.2 0.2
1.8 0.06
5.4 0.2
10.7 0.6
8.6 0.5
a
A549, human lung adenocarcinoma epithelial cell line; HeLa, human Henrietta
Lacks cervical cancer cell line; Ehrlich, mice Ehrlich ascites carcinoma cell line; MCF-
7, human breast adenocarcinoma cell line.
b
Data with standard deviations under bioassay in triplicate according to the MTT
method with taxol as a positive control.
It was found that the enzymes are extracellular and constitu-
tive, since strain broth in vitro could convert the exogenous
substrates to the two isolated products. It was suggested that
an O-demethylase or/and methyltransferase may play an impor-
tant role on biotransformation. Further work will focus on cDNA
cloning and enzymatic expression to characterize the enzyme(s)
involved. If the enzymes’ properties and additional informa-
tion were unearthed, the biotransformation rate and yield could
be optimized through enhancing their expression and activities.
Subsequently, the enzymes could be extracted, purified and immo-
bilized for large-scale production of the desired products.
It is possible to obtain more water-soluble derivate(s) of her-
bimycin A because the demethylation reaction took place at the
methoxyl moiety connected with C-6 or/and C-11. The water sol-
ubility of the compound 2, the modified compound with new
structure, has been increased about 10 times after microbial bio-
transformation through water resolve assay (data not shown). In
addition, the water solubility of another product was much stronger
than those of the substrate herbimycin A. Most importantly, the
IC50 of 11-hydroxy-(11-demethoxy)-herbimycin C to the A549 cells
is 152.9 ␮g/ml, to MCF-7 cells is 0.46 ␮g/ml, to Ehrlich cells is
10.7 ␮g/ml, and to HeLa cells is 109.3 ␮g/ml, which are nearly in the
range of those between herbimycin A and herbimycin C (Table 2).
In conclusion, we have obtained a powerful method for prepa-
ration of C-6/C-11 demethylated herbimycin A derivatives by
Eupenicillium sp. SD017 cultures. This might provide a useful
tool to prepare bioactive geldanamycin derivates. This study will
firstly serve as a fundamental methodology for further structural
alteration and functional improvement of the water-insoluble anti-
tumor drugs using microbial resources.
Fig. 2. The biotransformation curves for herbimycin A.
shown). The results showed that the optimal time for substrate
addition was at the late logarithmic-phase to the early stationary-
phase (5th day) during the strain growth period. Substrate addition
at this period of time resulted in an efficient bioconversion of sub-
strate and yield of products; for example, the yields for 1 and 2
were 2-fold more than those of substrate addition at the beginning
of the strain growth period.
In general, the concentration of substrate in fermentation broth
affects the yield of converted products in the biotransformation
process. Thus, the effects of six different concentrations of sub-
strates (10 mg/l, 20 mg/l, 30 mg/l, 40 mg/l, 50 mg/l and 60 mg/l) on
the substrate bioconversion were investigated and the results indi-
cated that the optimal amount of substrate addition was 30 mg/l. At
this concentration, the substrate was efficiently converted (almost
100%), and simultaneously the products (1 and 2) reached the high-
est yields (data not shown).
The biotransformation curves, namely the plot for the percent-
age of HPLC peak area of herbimycin A, compound 1 and 2 versus
incubation time under 28 ◦C on rotary shaker at 200 rpm is given
in Fig. 2. The results indicate the percentage of peak area (i.e. rel-
ative amount) of herbimycin A decreases along with the lapse of
incubation time, and the relative amount of compound 1 increase
until reaching a maximal value at 3th day before dropping down
to an equilibrium value, while the concentration of compound 2
steadily increased until reaching an equilibrium value. It was sug-
gested that the initiate biotransformed product was compound
1 whereas compound 2 was biosynthesized from compound 1
through a demethylation process catalyzed by an O-demethylase
or/and methyltransferase which are occasionally found in biotrans-
formation and biodegradation study. It was reported that dicamba
O-demethylase from Pseudomonas maltophilia can catalyze the con-
version of the herbicide dicamba to 3,6-dichlorosalicylic acid [19].
And Kaufmann et al. [20] isolated the O-demethylase from one
homoacetogenic strain and proved the O-demethylase is an ether-
cleaving enzyme system. Therefore, a proposed reaction formula
for biotransformation of herbimycin A by Eupenicillium sp. SD017
is given as Fig. 1.
According to HPLC analysis on the incubation cultures, although
the biotransformation yields for 1 and 2 were determined to be 42%
and 53%, respectively, the recovery rate of isolated biotransforma-
tion products is relative less comparing with the amount of the
substrate due to the loss in the separation and purification process.
To understand whether the extracellular enzyme or endo-
cellular one, whether the constitutive enzyme or inducible one
contributes the biotransformation, a series of experiments were
designed to characterize the enzymes through the methods of
cell-free culture and substrate/product concentration analysis.
Acknowledgements
This research was partially funded by National Supportive
Plan Project of Science and Technology (2006BAB19B02), and the
Research Foundation of Science and Technology Plan Project in
Guangdong Province (2008A030203004), and also financially sup-
ported by the Doctoral Fund and Youth Fund of Central South
University of Forestry & Technology.
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